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Jean-Yves Sgro
Inst. for Mol.Virology
731B Bock Labs
1525 Linden Drive Madison, WI 53706

Table of Contents for this page:

  • Current Issue
  • Advanced Online Publications Articles
  • Current Issue of Journal of Virology

    JVI Current Issue

  • Editorial Board [Masthead]

  • Articles of Significant Interest Selected from This Issue by the Editors [Spotlight]

  • Defective Viral Genomes: Critical Danger Signals of Viral Infections [Gem]

  • Viruses efficiently block the host antiviral response in order to replicate and spread before host intervention. The mechanism initiating antiviral immunity during stealth viral replication is unknown, but recent data demonstrate that defective viral genomes generated at peak virus replication are critical for this process in vivo. This article summarizes the supporting evidence and highlights gaps in our understanding of the mechanisms and impact of immunostimulatory defective viral genomes generated during natural infections.

  • Binding of the Kaposi's Sarcoma-Associated Herpesvirus to the Ephrin Binding Surface of the EphA2 Receptor and Its Inhibition by a Small Molecule [Vaccines and Antiviral Agents]

  • The ephrin receptor tyrosine kinase A2 (EphA2) is an entry receptor for Kaposi's sarcoma-associated herpesvirus (KSHV) that is engaged by the virus through its gH/gL glycoprotein complex. We describe here that natural ephrin ligands inhibit the gH/gL-EphA2 interaction. The effects of point mutations within EphA2 demonstrated that KSHV gH/gL interacts with EphA2 through a restricted set of the same residues that mediate binding of A-type ephrins. Two previously described inhibitors of the EphA2 interaction with ephrin A5 also inhibited binding of KSHV gH/gL to EphA2. The more potent of the two compounds inhibited KSHV infection of blood vessel and lymphatic endothelial cells in the micromolar concentration range. Our results demonstrate that interaction of KSHV with EphA2 occurs in a fashion similar to that of the natural ephrin ligands. Our data further indicate a new avenue for drug development against KSHV.

    IMPORTANCE Our study reports two important findings. First, we show that KSHV engages its receptor, the receptor tyrosine kinase EphA2, at a site that overlaps the binding site of the natural ephrin ligands. Second, we demonstrate that KSHV infection of target cells can be blocked by a small-molecule inhibitor of the viral glycoprotein-EphA2 interaction. These findings represent a novel avenue for the development of strategies to treat KSHV-associated diseases.

  • PB2 Mutations D701N and S714R Promote Adaptation of an Influenza H5N1 Virus to a Mammalian Host [Virus-Cell Interactions]

  • Mutation D701N in the PB2 protein is known to play a prominent role in the adaptation of avian influenza A viruses to mammalian hosts. In contrast, little is known about the nearby mutations S714I and S714R, which have been observed in some avian influenza viruses highly pathogenic for mammals. We have generated recombinant H5N1 viruses with PB2 displaying the avian signature 701D or the mammalian signature 701N and serine, isoleucine, and arginine at position 714 and compared them for polymerase activity and virus growth in avian and mammalian cells, as well as for pathogenicity in mice. Mutation D701N led to an increase in polymerase activity and replication efficiency in mammalian cells and in mouse pathogenicity, and this increase was significantly enhanced when mutation D701N was combined with mutation S714R. Stimulation by mutation S714I was less distinct. These observations indicate that PB2 mutation S714R, in combination with the mammalian signature at position 701, has the potential to promote the adaptation of an H5N1 virus to a mammalian host.

    IMPORTANCE Influenza A/H5N1 viruses are avian pathogens that have pandemic potential, since they are spread over large parts of Asia, Africa, and Europe and are occasionally transmitted to humans. It is therefore of high scientific interest to understand the mechanisms that determine the host specificity and pathogenicity of these viruses. It is well known that the PB2 subunit of the viral polymerase is an important host range determinant and that PB2 mutation D701N plays an important role in virus adaptation to mammalian cells. In the present study, we show that mutation S714R is also involved in adaptation and that it cooperates with D701N in exposing a nuclear localization signal that mediates importin-aalpha; binding and entry of PB2 into the nucleus, where virus replication and transcription take place.

  • A Single Amino Acid in EBNA-2 Determines Superior B Lymphoblastoid Cell Line Growth Maintenance by Epstein-Barr Virus Type 1 EBNA-2 [Genome Replication and Regulation of Viral Gene Expression]

  • Sequence differences in the EBNA-2 protein mediate the superior ability of type 1 Epstein-Barr virus (EBV) to transform human B cells into lymphoblastoid cell lines compared to that of type 2 EBV. Here we show that changing a single amino acid (S442D) from serine in type 2 EBNA-2 to the aspartate found in type 1 EBNA-2 confers a type 1 growth phenotype in a lymphoblastoid cell line growth maintenance assay. This amino acid lies in the transactivation domain of EBNA-2, and the S442D change increases activity in a transactivation domain assay. The superior growth properties of type 1 EBNA-2 correlate with the greater induction of EBV LMP-1 and about 10 cell genes, including CXCR7. In chromatin immunoprecipitation assays, type 1 EBNA-2 is shown to associate more strongly with EBNA-2 binding sites near the LMP-1 and CXCR7 genes. Unbiased motif searching of the EBNA-2 binding regions of the differentially regulated cell genes identified an ETS-interferon regulatory factor composite element motif that closely corresponds to the sequences known to mediate EBNA-2 regulation of the LMP-1 promoter. It appears that the superior induction by type 1 EBNA-2 of the cell genes contributing to cell growth is due to their being regulated in a manner different from that for most EBNA-2-responsive genes and in a way similar to that for the LMP-1 gene.

    IMPORTANCE The EBNA-2 transcription factor plays a key role in B cell transformation by EBV and defines the two EBV types. Here we identify a single amino acid (Ser in type 1 EBV, Asp in type 2 EBV) of EBNA-2 that determines the superior ability of type 1 EBNA-2 to induce a key group of cell genes and the EBV LMP-1 gene, which mediate the growth advantage of B cells infected with type 1 EBV. The EBNA-2 binding sites in these cell genes have a sequence motif similar to the sequence known to mediate regulation of the EBV LMP-1 promoter. Further detailed analysis of transactivation and promoter binding provides new insight into the physiological regulation of cell genes by EBNA-2.

  • Phosphoacceptors Threonine 162 and Serines 170 and 178 within the Carboxyl-Terminal RRRS/T Motif of the Hepatitis B Virus Core Protein Make Multiple Contributions to Hepatitis B Virus Replication [Genome Replication and Regulation of Viral Gene Expression]

  • Phosphorylation of serines 157, 164, and 172 within the carboxyl-terminal SPRRR motif of the hepatitis B virus (HBV) core (C) protein modulates HBV replication at multiple stages. Threonine 162 and serines 170 and 178, located within the carboxyl-terminal conserved RRRS/T motif of HBV C protein, have been proposed to be protein kinase A phosphorylation sites. However, in vivo phosphorylation of these residues has never been observed, and their contribution to HBV replication remains unknown. In this study, [32P]orthophosphate labeling of cells expressing C proteins followed by immunoprecipitation with anti-HBc antibody revealed that threonine 162 and serines 170 and 178 are phosphoacceptor residues. A triple-alanine-substituted mutant, mimicking dephosphorylation of all three residues, drastically decreased pregenomic RNA (pgRNA) encapsidation, thereby decreasing HBV DNA synthesis. In contrast, a triple-glutamate-substituted mutant, mimicking phosphorylation of these residues, decreased DNA synthesis without significantly decreasing encapsidation. Neither triple mutant affected C protein expression or core particle assembly. Individual alanine substitution of threonine 162 significantly decreased minus-strand, plus-strand, and relaxed-circular DNA synthesis, demonstrating that this residue plays multiple roles in HBV DNA synthesis. Double-alanine substitution of serines 170 and 178 reduced HBV replication at multiple stages, indicating that these residues also contribute to HBV replication. Thus, in addition to serines 157, 164, and 172, threonine 162 and serines 170 and 178 of HBV C protein are also phosphorylated in cells, and phosphorylation and dephosphorylation of these residues play multiple roles in modulation of HBV replication.

    IMPORTANCE Threonine 162, within the carboxyl-terminal end of the hepatitis B virus (HBV adw) core (C) protein, has long been ignored as a phosphoacceptor, even though it is highly conserved among mammalian hepadnaviruses and in the overlapping consensus RxxS/T, RRxS/T, and TP motifs. Here we show, for the first time, that in addition to the well-known phosphoacceptor serines 157, 164, and 172 in SPRRR motifs, threonine 162 and serines 170 and 178 in the RRRS/T motif are phosphorylated in cells. We also show that, like serines 157, 164, and 172, phosphorylated and dephosphorylated threonine 162 and serines 170 and 178 contribute to multiple steps of HBV replication, including pgRNA encapsidation, minus-strand and plus-strand DNA synthesis, and relaxed-circular DNA synthesis. Of these residues, threonine 162 is the most important. Furthermore, we show that phosphorylation of C protein is required for efficient completion of HBV replication.

  • Deep Sequencing of HIV-Infected Cells: Insights into Nascent Transcription and Host-Directed Therapy [Virus-Cell Interactions]

  • Polyadenylated mature mRNAs are the focus of standard transcriptome analyses. However, the profiling of nascent transcripts, which often include nonpolyadenylated RNAs, can unveil novel insights into transcriptional regulation. Here, we separately sequenced total RNAs (Total RNAseq) and mRNAs (mRNAseq) from the same HIV-1-infected human CD4+ T cells. We found that many nonpolyadenylated RNAs were differentially expressed upon HIV-1 infection, and we identified 8 times more differentially expressed genes at 12 h postinfection by Total RNAseq than by mRNAseq. These expression changes were also evident by concurrent changes in introns and were recapitulated by later mRNA changes, revealing an unexpectedly significant delay between transcriptional initiation and mature mRNA production early after HIV-1 infection. We computationally derived and validated the underlying regulatory programs, and we predicted drugs capable of reversing these HIV-1-induced expression changes followed by experimental confirmation. Our results show that combined total and mRNA transcriptome analysis is essential for fully capturing the early host response to virus infection and provide a framework for identifying candidate drugs for host-directed therapy against HIV/AIDS.

    IMPORTANCE In this study, we used mass sequencing to identify genes differentially expressed in CD4+ T cells during HIV-1 infection. To our surprise, we found many differentially expressed genes early after infection by analyzing both newly transcribed unprocessed pre-mRNAs and fully processed mRNAs, but not by analyzing mRNAs alone, indicating a significant delay between transcription initiation and mRNA production early after HIV-1 infection. These results also show that important findings could be missed by the standard practice of analyzing mRNAs alone. We then derived the regulatory mechanisms driving the observed expression changes using integrative computational analyses. Further, we predicted drugs that could reverse the observed expression changes induced by HIV-1 infection and showed that one of the predicted drugs indeed potently inhibited HIV-1 infection. This shows that it is possible to identify candidate drugs for host-directed therapy against HIV/AIDS using our genomics-based approach.

  • Discovery and Evolution of Bunyavirids in Arctic Phantom Midges and Ancient Bunyavirid-Like Sequences in Insect Genomes [Genetic Diversity and Evolution]

  • Bunyaviridae is a large family of RNA viruses chiefly comprised of vertebrate and plant pathogens. We discovered novel bunyavirids that are approximately equally divergent from each of the five known genera. We characterized novel genome sequences for two bunyavirids, namely, Kigluaik phantom virus (KIGV), from tundra-native phantom midges (Chaoborus), and Nome phantom virus (NOMV), from tundra-invading phantom midges, and demonstrated that these bunyavirid-like sequences belong to an infectious virus by passaging KIGV in mosquito cell culture, although the infection does not seem to be well sustained beyond a few passages. Virus and host gene sequences from individuals collected on opposite ends of North America, a region spanning 4,000 km, support a long-term, vertically transmitted infection of KIGV in Chaoborus trivittatus. KIGV-like sequences ranging from single genes to full genomes are present in transcriptomes and genomes of insects belonging to six taxonomic orders, suggesting an ancient association of this clade with insect hosts. In Drosophila, endogenous virus genes have been coopted, forming an orthologous tandem gene family that has been maintained by selection during the radiation of the host genus. Our findings indicate that bunyavirid-host interactions in nonbloodsucking arthropods have been much more extensive than previously thought.

    IMPORTANCE Very little is known about the viral diversity in polar freshwater ponds, and perhaps less is known about the effects that climate-induced habitat changes in these regions will have on virus-host interactions in the coming years. Our results show that at the tundra-boreal boundary, a hidden viral landscape is being altered as infected boreal phantom midges colonize tundra ponds. Likewise, relatively little is known of the deeper evolutionary history of bunyavirids that has led to the stark lifestyle contrasts between some genera. The discovery of this novel bunyavirid group suggests that ancient and highly divergent bunyavirid lineages remain undetected in nature and may offer fresh insight into host reservoirs, potential sources of emerging disease, and major lifestyle shifts in the evolutionary history of viruses in the family Bunyaviridae.

  • Functional Annotation of Cotesia congregata Bracovirus: Identification of Viral Genes Expressed in Parasitized Host Immune Tissues [Genome Replication and Regulation of Viral Gene Expression]

  • Bracoviruses (BVs) from the Polydnaviridae family are symbiotic viruses used as biological weapons by parasitoid wasps to manipulate lepidopteran host physiology and induce parasitism success. BV particles are produced by wasp ovaries and injected along with the eggs into the caterpillar host body, where viral gene expression is necessary for wasp development. Recent sequencing of the proviral genome of Cotesia congregata BV (CcBV) identified 222 predicted virulence genes present on 35 proviral segments integrated into the wasp genome. To date, the expressions of only a few selected candidate virulence genes have been studied in the caterpillar host, and we lacked a global vision of viral gene expression. In this study, a large-scale transcriptomic analysis by 454 sequencing of two immune tissues (fat body and hemocytes) of parasitized Manduca sexta caterpillar hosts allowed the detection of expression of 88 CcBV genes expressed 24 h after the onset of parasitism. We linked the expression profiles of these genes to several factors, showing that different regulatory mechanisms control viral gene expression in the host. These factors include the presence of signal peptides in encoded proteins, diversification of promoter regions, and, more surprisingly, gene position on the proviral genome. Indeed, most genes for which expression could be detected are localized in particular proviral regions globally producing higher numbers of circles. Moreover, this polydnavirus (PDV) transcriptomic analysis also reveals that a majority of CcBV genes possess at least one intron and an arthropod transcription start site, consistent with an insect origin of these virulence genes.

    IMPORTANCE Bracoviruses (BVs) are symbiotic polydnaviruses used by parasitoid wasps to manipulate lepidopteran host physiology, ensuring wasp offspring survival. To date, the expressions of only a few selected candidate BV virulence genes have been studied in caterpillar hosts. We performed a large-scale analysis of BV gene expression in two immune tissues of Manduca sexta caterpillars parasitized by Cotesia congregata wasps. Genes for which expression could be detected corresponded to genes localized in particular regions of the viral genome globally producing higher numbers of circles. Our study thus brings an original global vision of viral gene expression and paves the way to the determination of the regulatory mechanisms enabling the expression of BV genes in targeted organisms, such as major insect pests. In addition, we identify sequence features suggesting that most BV virulence genes were acquired from insect genomes.

  • Virus Particle Release from Glycosphingolipid-Enriched Microdomains Is Essential for Dendritic Cell-Mediated Capture and Transfer of HIV-1 and Henipavirus [Virus-Cell Interactions]

  • Human immunodeficiency virus type 1 (HIV-1) exploits dendritic cells (DCs) to promote its transmission to T cells. We recently reported that the capture of HIV-1 by mature dendritic cells (MDCs) is mediated by an interaction between the glycosphingolipid (GSL) GM3 on virus particles and CD169/Siglec-1 on MDCs. Since HIV-1 preferentially buds from GSL-enriched lipid microdomains on the plasma membrane, we hypothesized that the virus assembly and budding site determines the ability of HIV-1 to interact with MDCs. In support of this hypothesis, mutations in the N-terminal basic domain (29/31KE) or deletion of the membrane-targeting domain of the HIV-1 matrix (MA) protein that altered the virus assembly and budding site to CD63+/Lamp-1-positive intracellular compartments resulted in lower levels of virion incorporation of GM3 and attenuation of virus capture by MDCs. Furthermore, MDC-mediated capture and transmission of MA mutant viruses to T cells were decreased, suggesting that HIV-1 acquires GSLs via budding from the plasma membrane to access the MDC-dependent trans infection pathway. Interestingly, MDC-mediated capture of Nipah and Hendra virus (recently emerged zoonotic paramyxoviruses) M (matrix) protein-derived virus-like particles that bud from GSL-enriched plasma membrane microdomains was also dependent on interactions between virion-incorporated GSLs and CD169. Moreover, capture and transfer of Nipah virus envelope glycoprotein-pseudotyped lentivirus particles by MDCs were severely attenuated upon depletion of GSLs from virus particles. These results suggest that GSL incorporation into virions is critical for the interaction of diverse enveloped RNA viruses with DCs and that the GSL-CD169 recognition nexus might be a conserved viral mechanism of parasitization of DC functions for systemic virus dissemination.

    IMPORTANCE Dendritic cells (DCs) can capture HIV-1 particles and transfer captured virus particles to T cells without establishing productive infection in DCs, a mechanism of HIV-1 trans infection. We have recently identified CD169-mediated recognition of GM3, a host-derived glycosphingolipid (GSL) incorporated into the virus particle membrane, as the receptor and ligand for the DC-HIV trans infection pathway. In this study, we have identified the matrix (MA) domain of Gag to be the viral determinant that governs incorporation of GM3 into HIV-1 particles, a previously unappreciated function of the HIV-1 MA. In addition, we demonstrate that the GSL-CD169-dependent trans infection pathway is also utilized as a dissemination mechanism by henipaviruses. GSL incorporation in henipaviruses was also dependent on the viral capsid (M) protein-directed assembly and budding from GSL-enriched lipid microdomains. These findings provide evidence of a conserved mechanism of retrovirus and henipavirus parasitization of cell-to-cell recognition pathways for systemic virus dissemination.

  • Particle Conformation Regulates Antibody Access to a Conserved GII.4 Norovirus Blockade Epitope [Pathogenesis and Immunity]

  • GII.4 noroviruses (NoVs) are the primary cause of epidemic viral acute gastroenteritis. One primary obstacle to successful NoV vaccination is the extensive degree of antigenic diversity among strains. The major capsid protein of GII.4 strains is evolving rapidly, resulting in the emergence of new strains with altered blockade epitopes. In addition to characterizing these evolving blockade epitopes, we have identified monoclonal antibodies (MAbs) that recognize a blockade epitope conserved across time-ordered GII.4 strains. Uniquely, the blockade potencies of MAbs that recognize the conserved GII.4 blockade epitope were temperature sensitive, suggesting that particle conformation may regulate functional access to conserved blockade non-surface-exposed epitopes. To map conformation-regulating motifs, we used bioinformatics tools to predict conserved motifs within the protruding domain of the capsid and designed mutant VLPs to test the impacts of substitutions in these motifs on antibody cross-GII.4 blockade. Charge substitutions at residues 310, 316, 484, and 493 impacted the blockade potential of cross-GII.4 blockade MAbs with minimal impact on the blockade of MAbs targeting other, separately evolving blockade epitopes. Specifically, residue 310 modulated antibody blockade temperature sensitivity in the tested strains. These data suggest access to the conserved GII.4 blockade antibody epitope is regulated by particle conformation, temperature, and amino acid residues positioned outside the antibody binding site. The regulating motif is under limited selective pressure by the host immune response and may provide a robust target for broadly reactive NoV therapeutics and protective vaccines.

    IMPORTANCE In this study, we explored the factors that govern norovirus (NoV) cross-strain antibody blockade. We found that access to the conserved GII.4 blockade epitope is regulated by temperature and distal residues outside the antibody binding site. These data are most consistent with a model of NoV particle conformation plasticity that regulates antibody binding to a distally conserved blockade epitope. Further, antibody "locking" of the particle into an epitope-accessible conformation prevents ligand binding, providing a potential target for broadly effective drugs. These observations open lines of inquiry into the mechanisms of human NoV entry and uncoating, fundamental biological questions that are currently unanswerable for these noncultivatable pathogens.

  • Activation of c-jun N-Terminal Kinase upon Influenza A Virus (IAV) Infection Is Independent of Pathogen-Related Receptors but Dependent on Amino Acid Sequence Variations of IAV NS1 [Virus-Cell Interactions]

  • A hallmark cell response to influenza A virus (IAV) infections is the phosphorylation and activation of c-jun N-terminal kinase (JNK). However, so far it is not fully clear which molecules are involved in the activation of JNK upon IAV infection. Here, we report that the transfection of influenza viral-RNA induces JNK in a retinoic acid-inducible gene I (RIG-I)-dependent manner. However, neither RIG-I-like receptors nor MyD88-dependent Toll-like receptors were found to be involved in the activation of JNK upon IAV infection. Viral JNK activation may be blocked by addition of cycloheximide and heat shock protein inhibitors during infection, suggesting that the expression of an IAV-encoded protein is responsible for JNK activation. Indeed, the overexpression of nonstructural protein 1 (NS1) of certain IAV subtypes activated JNK, whereas those of some other subtypes failed to activate JNK. Site-directed mutagenesis experiments using NS1 of the IAV H7N7, H5N1, and H3N2 subtypes identified the amino acid residue phenylalanine (F) at position 103 to be decisive for JNK activation. Cleavage- and polyadenylation-specific factor 30 (CPSF30), whose binding to NS1 is stabilized by the amino acids F103 and M106, is not involved in JNK activation. Conclusively, subtype-specific sequence variations in the IAV NS1 protein result in subtype-specific differences in JNK signaling upon IAV infection.

    IMPORTANCE Influenza A virus (IAV) infection leads to the activation or modulation of multiple signaling pathways. Here, we demonstrate for the first time that the c-jun N-terminal kinase (JNK), a long-known stress-activated mitogen-activated protein (MAP) kinase, is activated by RIG-I when cells are treated with IAV RNA. However, at the same time, nonstructural protein 1 (NS1) of IAV has an intrinsic JNK-activating property that is dependent on IAV subtype-specific amino acid variations around position 103. Our findings identify two different and independent pathways that result in the activation of JNK in the course of an IAV infection.

  • Progression From IgD+ IgM+ to Isotype-Switched B Cells Is Site Specific during Coronavirus-Induced Encephalomyelitis [Pathogenesis and Immunity]

  • Various infections in the central nervous system (CNS) trigger B cell accumulation; however, the relative dynamics between viral replication and alterations in distinct B cell subsets are largely unknown. Using a glia-tropic coronavirus infection, which is initiated in the brain but rapidly spreads to and predominantly persists in the spinal cord, this study characterizes longitudinal changes in B cell subsets at both infected anatomical sites. The phase of T cell-dependent, antibody-independent control of infectious virus was associated with a similar recruitment of naive/early-activated IgD+ IgM+ B cells into both the brain and spinal cord. This population was progressively replaced by CD138nndash; IgDnndash; IgM+ B cells, isotype-switched CD138nndash; IgDnndash; IgMnndash; memory B cells (Bmem), and CD138+ antibody-secreting cells (ASC). A more rapid transition to Bmem and ASC in spinal cord than in brain was associated with higher levels of persisting viral RNA and transcripts encoding factors promoting B cell migration, differentiation, and survival. The results demonstrate that naive/early-activated B cells are recruited early during coronavirus CNS infection but are subsequently replaced by more differentiated B cells. Furthermore, viral persistence, even at low levels, is a driving force for accumulation of isotype-switched Bmem and ASC.

    IMPORTANCE Acute and chronic human CNS infections are associated with an accumulation of heterogeneous B cell subsets; however, their influence on viral load and disease is unclear. Using a glia-tropic coronavirus model, we demonstrate that the accumulation of B cells ranging from early-activated to isotype-switched differentiation stages is both temporally and spatially orchestrated. Acutely infected brains and spinal cords indiscriminately recruit a homogeneous population of early-activated B cells, which is progressively replaced by diverse, more differentiated subsets. The latter process is accelerated by elevated proinflammatory responses associated with viral persistence. The results imply that early-recruited B cells do not have antiviral function but may contribute to the inflammatory environment or act as antigen-presenting cells. Moreover, CNS viral persistence is a driving force promoting differentiated B cells with protective potential.

  • Dependence of Coronavirus RNA Replication on an NH2-Terminal Partial Nonstructural Protein 1 in cis [Genome Replication and Regulation of Viral Gene Expression]

  • Genomes of positive (+)-strand RNA viruses use cis-acting signals to direct both translation and replication. Here we examine two 5'-proximal cis-replication signals of different character in a defective interfering (DI) RNA of the bovine coronavirus (BCoV) that map within a 322-nucleotide (nt) sequence (136 nt from the genomic 5' untranslated region and 186 nt from the nonstructural protein 1 [nsp1]-coding region) not found in the otherwise-identical nonreplicating subgenomic mRNA7 (sgmRNA7). The natural DI RNA is structurally a fusion of the two ends of the BCoV genome that results in a single open reading frame between a partial nsp1-coding region and the entire N gene. (i) In the first examination, mutation analyses of a recently discovered long-range RNA-RNA base-paired structure between the 5' untranslated region and the partial nsp1-coding region showed that it, possibly in concert with adjacent stem-loops, is a cis-acting replication signal in the (+) strand. We postulate that the higher-order structure promotes (+)-strand synthesis. (ii) In the second examination, analyses of multiple frame shifts, truncations, and point mutations within the partial nsp1-coding region showed that synthesis of a PEFP core amino acid sequence within a group A lineage betacoronavirus-conserved NH2-proximal WAPEFPWM domain is required in cis for DI RNA replication. We postulate that the nascent protein, as part of an RNA-associated translating complex, acts to direct the DI RNA to a critical site, enabling RNA replication. We suggest that these results have implications for viral genome replication and explain, in part, why coronavirus sgmRNAs fail to replicate.

    IMPORTANCE cis-Acting RNA and protein structures that regulate (+)-strand RNA virus genome synthesis are potential sites for blocking virus replication. Here we describe two: a previously suspected 5'-proximal long-range higher-order RNA structure and a novel nascent NH2-terminal protein component of nsp1 that are common among betacoronaviruses of group A lineage.

  • Uracil DNA Glycosylase BKRF3 Contributes to Epstein-Barr Virus DNA Replication through Physical Interactions with Proteins in Viral DNA Replication Complex [Genome Replication and Regulation of Viral Gene Expression]

  • Epstein-Barr virus (EBV) BKRF3 shares sequence homology with members of the uracil-N-glycosylase (UNG) protein family and has DNA glycosylase activity. Here, we explored how BKRF3 participates in the DNA replication complex and contributes to viral DNA replication. Exogenously expressed Flag-BKRF3 was distributed mostly in the cytoplasm, whereas BKRF3 was translocated into the nucleus and colocalized with the EBV DNA polymerase BALF5 in the replication compartment during EBV lytic replication. The expression level of BKRF3 increased gradually during viral replication, coupled with a decrease of cellular UNG2, suggesting BKRF3 enzyme activity compensates for UNG2 and ensures the fidelity of viral DNA replication. In immunoprecipitation-Western blotting, BKRF3 was coimmunoprecipitated with BALF5, the polymerase processivity factor BMRF1, and the immediate-early transactivator Rta. Coexpression of BMRF1 appeared to facilitate the nuclear targeting of BKRF3 in immunofluorescence staining. Residues 164 to 255 of BKRF3 were required for interaction with Rta and BALF5, whereas residues 81 to 166 of BKRF3 were critical for BMRF1 interaction in glutathione S-transferase (GST) pulldown experiments. Viral DNA replication was defective in cells harboring BKRF3 knockout EBV bacmids. In complementation assays, the catalytic mutant BKRF3(Q90L,D91N) restored viral DNA replication, whereas the leucine loop mutant BKRF3(H213L) only partially rescued viral DNA replication, coupled with a reduced ability to interact with the viral DNA polymerase and Rta. Our data suggest that BKRF3 plays a critical role in viral DNA synthesis predominantly through its interactions with viral proteins in the DNA replication compartment, while its enzymatic activity may be supplementary for uracil DNA glycosylase (UDG) function during virus replication.

    IMPORTANCE Catalytic activities of both cellular UDG UNG2 and viral UDGs contribute to herpesviral DNA replication. To ensure that the enzyme activity executes at the right time and the right place in DNA replication forks, complex formation with other components in the DNA replication machinery provides an important regulation for UDG function. In this study, we provide the mechanism for EBV UDG BKRF3 nuclear targeting and the interacting domains of BKRF3 with viral DNA replication proteins. Through knockout and complementation approaches, we further demonstrate that in addition to UDG activity, the interaction of BKRF3 with viral proteins in the replication compartment is crucial for efficient viral DNA replication.

  • Cryo-Electron Microscopy Structure of Lactococcal Siphophage 1358 Virion [Structure and Assembly]

  • Lactococcus lactis, a Gram+ lactic acid-producing bacterium used for the manufacture of several fermented dairy products, is subject to infection by diverse virulent tailed phages, leading to industrial fermentation failures. This constant viral risk has led to a sustained interest in the study of their biology, diversity, and evolution. Lactococcal phages now constitute a wide ensemble of at least 10 distinct genotypes within the Caudovirales order, many of them belonging to the Siphoviridae family. Lactococcal siphophage 1358, currently the only member of its group, displays a noticeably high genomic similarity to some Listeria phages as well as a host range limited to a few L. lactis strains. These genomic and functional characteristics stimulated our interest in this phage. Here, we report the cryo-electron microscopy structure of the complete 1358 virion. Phage 1358 exhibits noteworthy features, such as a capsid with dextro handedness and protruding decorations on its capsid and tail. Observations of the baseplate of virion particles revealed at least two conformations, a closed and an open, activated form. Functional assays uncovered that the adsorption of phage 1358 to its host is Ca2+ independent, but this cation is necessary to complete its lytic cycle. Taken together, our results provide the complete structural picture of a unique lactococcal phage and expand our knowledge on the complex baseplate of phages of the Siphoviridae family.

    IMPORTANCE Phages of Lactococcus lactis are investigated mainly because they are sources of milk fermentation failures in the dairy industry. Despite the availability of several antiphage measures, new phages keep emerging in this ecosystem. In this study, we provide the cryo-electron microscopy reconstruction of a unique lactococcal phage that possesses genomic similarity to particular Listeria phages and has a host range restricted to only a minority of L. lactis strains. The capsid of phage 1358 displays the almost unique characteristic of being dextro handed. Its capsid and tail exhibit decorations that we assigned to nonspecific sugar binding modules. We observed the baseplate of 1358 in two conformations, a closed and an open form. We also found that the adsorption to its host, but not infection, is Ca2+ independent. Overall, this study advances our understanding of the adhesion mechanisms of siphophages.

  • Restriction of HIV-1 by Rhesus TRIM5{alpha} Is Governed by Alpha Helices in the Linker2 Region [Virus-Cell Interactions]

  • TRIM5aalpha; proteins are a potent barrier to the cross-species transmission of retroviruses. TRIM5aalpha; proteins exhibit an ability to self-associate at many levels, ultimately leading to the formation of protein assemblies with hexagonal symmetry in vitro and cytoplasmic assemblies when expressed in cells. However, the role of these assemblies in restriction, the determinants that mediate their formation, and the organization of TRIM5aalpha; molecules within these assemblies have remained unclear. Here we show that aalpha;-helical elements within the Linker2 region of rhesus macaque TRIM5aalpha; govern the ability to form cytoplasmic assemblies in cells and restrict HIV-1 infection. Mutations that reduce aalpha;-helix formation by the Linker2 region disrupt assembly and restriction. More importantly, mutations that enhance the aalpha;-helical content of the Linker2 region, relative to the wild-type protein, also exhibit an increased ability to form cytoplasmic assemblies and restrict HIV-1 infection. Molecular modeling of the TRIM5aalpha; dimer suggests a model in which aalpha;-helical elements within the Linker2 region dock to aalpha;-helices of the coiled-coil domain, likely establishing proper orientation and spacing of protein domains necessary for assembly and restriction. Collectively, these studies provide critical insight into the determinants governing TRIM5aalpha; assembly and restriction and demonstrate that the antiviral potency of TRIM5aalpha; proteins can be significantly increased without altering the affinity of SPRY/capsid binding.

    IMPORTANCE Many members of the tripartite motif (TRIM) family of proteins act as restriction factors that directly inhibit viral infection and activate innate immune signaling pathways. Another common feature of TRIM proteins is the ability to form protein assemblies in the nucleus or the cytoplasm. However, the determinants in TRIM proteins required for assembly and the degree to which assembly affects TRIM protein function have been poorly understood. Here we show that alpha helices in the Linker2 (L2) region of rhesus TRIM5aalpha; govern assembly and restriction of HIV-1 infection. Helix-disrupting mutations disrupt the assembly and restriction of HIV-1, while helix-stabilizing mutations enhance assembly and restriction relative to the wild-type protein. Circular dichroism analysis suggests that that the formation of this helical structure is supported by intermolecular interactions with the coiled-coil (CC) domain in the CCL2 dimer. These studies reveal a novel mechanism by which the antiviral activity of TRIM5aalpha; proteins can be regulated and provide detailed insight into the assembly determinants of TRIM family proteins.

  • Regulation of the Human Endogenous Retrovirus K (HML-2) Transcriptome by the HIV-1 Tat Protein [Cellular Response to Infection]

  • Approximately 8% of the human genome is made up of endogenous retroviral sequences. As the HIV-1 Tat protein activates the overall expression of the human endogenous retrovirus type K (HERV-K) (HML-2), we used next-generation sequencing to determine which of the 91 currently annotated HERV-K (HML-2) proviruses are regulated by Tat. Transcriptome sequencing of total RNA isolated from Tat- and vehicle-treated peripheral blood lymphocytes from a healthy donor showed that Tat significantly activates expression of 26 unique HERV-K (HML-2) proviruses, silences 12, and does not significantly alter the expression of the remaining proviruses. Quantitative reverse transcription-PCR validation of the sequencing data was performed on Tat-treated PBLs of seven donors using provirus-specific primers and corroborated the results with a substantial degree of quantitative similarity.

    IMPORTANCE The expression of HERV-K (HML-2) is tightly regulated but becomes markedly increased following infection with HIV-1, in part due to the HIV-1 Tat protein. The findings reported here demonstrate the complexity of the genome-wide regulation of HERV-K (HML-2) expression by Tat. This work also demonstrates that although HERV-K (HML-2) proviruses in the human genome are highly similar in terms of DNA sequence, modulation of the expression of specific proviruses in a given biological situation can be ascertained using next-generation sequencing and bioinformatics analysis.

  • Porcine Epidemic Diarrhea Virus Nucleocapsid Protein Antagonizes Beta Interferon Production by Sequestering the Interaction between IRF3 and TBK1 [Virus-Cell Interactions]

  • Porcine epidemic diarrhea virus (PEDV), a porcine enteropathogenic coronavirus, causes lethal watery diarrhea in piglets and results in large economic losses in many Asian and European countries. A large-scale outbreak of porcine epidemic diarrhea occurred in China in 2010, and the virus emerged in the United States in 2013 and spread rapidly, posing significant economic and public health concerns. Previous studies have shown that PEDV infection inhibits the synthesis of type I interferon (IFN), and viral papain-like protease 2 has been identified as an IFN antagonist. In this study, we found that the PEDV-encoded nucleocapsid (N) protein also inhibits Sendai virus-induced IFN-bbeta; production, IFN-stimulated gene expression, and activation of the transcription factors IFN regulatory factor 3 (IRF3) and NF-B. We also found that N protein significantly impedes the activation of the IFN-bbeta; promoter stimulated by TBK1 or its upstream molecules (RIG-I, MDA5, IPS-1, and TRAF3) but does not counteract its activation by IRF3. A detailed analysis revealed that the PEDV N protein targets TBK1 by direct interaction and that this binding sequesters the association between TBK1 and IRF3, which in turn inhibits both IRF3 activation and type I IFN production. Together, our findings demonstrate a new mechanism evolved by PEDV to circumvent the host's antiviral immunity.

    IMPORTANCE PEDV has received increasing attention since the emergence of a PEDV variant in China and the United States. Here, we identify nucleocapsid (N) protein as a novel PEDV-encoded interferon (IFN) antagonist and demonstrate that N protein antagonizes IFN production by sequestering the interaction between IRF3 and TBK1, a critical step in type I IFN signaling. This adds another layer of complexity to the immune evasion strategies evolved by this economically important viral pathogen. An understanding of its immune evasion mechanism may direct us to novel therapeutic targets and more effective vaccines against PEDV infection.

  • Nucleotide Oligomerization and Binding Domain 2-Dependent Dendritic Cell Activation Is Necessary for Innate Immunity and Optimal CD8+ T Cell Responses to Influenza A Virus Infection [Pathogenesis and Immunity]

  • Nucleotide oligomerization and binding domain (NOD)-like receptors (NLRs) are important in the innate immune response to viral infection. Recent findings have implicated NLRP3, NOD2, and NLRX1 as important players in the innate antiviral response, but their roles in the generation of adaptive immunity to viruses are less clear. We demonstrate here that NOD2 is critical for both innate and adaptive immune responses necessary for controlling viral replication and survival during influenza A virus (IAV) infection. Nod2nndash;/nndash; mice have reduced beta interferon (IFN-bbeta;) levels and fewer activated dendritic cells (DCs), and the DCs are more prone to cell death in the lungs of Nod2nndash;/nndash; mice during IAV infection. In agreement with the role for DCs in priming adaptive immunity, the generation of virus-specific CD8+ T cells and their activation and production of IFN- were lower in Nod2nndash;/nndash; mice. Furthermore, Nod2nndash;/nndash; DCs, when cocultured with T cells in vitro, have a lower costimulatory capacity. Thus, Nod2nndash;/nndash; DCs are unable to efficiently prime CD8+ T cells. These findings demonstrate that Nod2 is critical for the generation of both innate and adaptive immune responses necessary for controlling IAV infection.

    IMPORTANCE The innate immune system is the host's first line of defense against invading pathogens and is also necessary for alerting and activating T and B cells to initiate the adaptive immune response. We demonstrate here that the innate immune receptor NOD2 is required for the production of antiviral type I interferons and the activation and survival of dendritic cells that, in turn, alert T cells to the presence of influenza A virus infection. In mice that are missing NOD2, interferon levels are lower, and the CD8+ T cell response is impaired. As a result, the animals cannot control virus replication in their lungs as efficiently. This discovery helps us understand how the body naturally responds to virus infection and may help in the development of vaccines that use NOD2 to stimulate the CD8+ T cell response, thus providing better protection against influenza A virus infection.

  • Human T-Cell Leukemia Virus Type 3 (HTLV-3) and HTLV-4 Antisense-Transcript-Encoded Proteins Interact and Transactivate Jun Family-Dependent Transcription via Their Atypical bZIP Motif [Virus-Cell Interactions]

  • Human T-cell leukemia virus types 3 and 4 (HTLV-3 and HTLV-4) are recently isolated retroviruses. We have previously characterized HTLV-3- and HTLV-4-encoded antisense genes, termed APH-3 and APH-4, respectively, which, in contrast to HBZ, the HTLV-1 homologue, do not contain a typical bZIP domain (M. Larocque EEacute; Halin, S. Landry, S. J. Marriott, W. M. Switzer, and B. Barbeau, J. Virol. 85:12673nndash;12685, 2011, doi:10.1128/JVI.05296-11). As HBZ differentially modulates the transactivation potential of various Jun family members, the effect of APH-3 and APH-4 on JunD-, c-Jun-, and JunB-mediated transcriptional activation was investigated. We first showed that APH-3 and APH-4 upregulated the transactivation potential of all tested Jun family members. Using an human telomerase catalytic subunit (hTERT) promoter construct, our results also highlighted that, unlike HBZ, which solely modulates hTERT expression via JunD, both APH-3 and APH-4 acted positively on the transactivation of the hTERT promoter mediated by tested Jun factors. Coimmunoprecipitation experiments demonstrated that these Jun proteins interacted with APH-3 and APH-4. Although no activation domain was identified for APH proteins, the activation domain of c-Jun was very important in the observed upregulation of its activation potential. We further showed that APH-3 and APH-4 required their putative bZIP-like domains and corresponding leucine residues for interaction and modulation of the transactivation potential of Jun factors. Our results demonstrate that HTLV-encoded antisense proteins behave differently, and that the bZIP-like domains of both APH-3 and APH-4 have retained their interaction potential for Jun members. These studies are important in assessing the differences between HBZ and other antisense proteins, which might further contribute to determining the role of HBZ in HTLV-1-associated diseases.

    IMPORTANCE HBZ, the antisense transcript-encoded protein from HTLV-1, is now well recognized as a potential factor for adult T-cell leukemia/lymphoma development. In order to better appreciate the mechanism of action of HBZ, comparison to antisense proteins from other HTLV viruses is important. Little is known in relation to the seemingly nonpathogenic HTLV-3 and HTLV-4 viruses, and studies of their antisense proteins are limited to our previously reported study (M. Larocque EEacute; Halin, S. Landry, S. J. Marriott, W. M. Switzer, and B. Barbeau, J. Virol. 85:12673nndash;12685, 2011, doi:10.1128/JVI.05296-11). Here, we demonstrate that Jun transcription factors are differently affected by APH-3 and APH-4 compared to HBZ. These intriguing findings suggest that these proteins act differently on viral replication but also on cellular gene expression, and that highlighting their differences of action might lead to important information allowing us to understand the link between HTLV-1 HBZ and ATL in infected individuals.

  • Influenza Virus PB1 and Neuraminidase Gene Segments Can Cosegregate during Vaccine Reassortment Driven by Interactions in the PB1 Coding Region [Structure and Assembly]

  • Egg-grown influenza vaccine yields are maximized by infection with a seed virus produced by "classical reassortment" of a seasonal isolate with a highly egg-adapted strain. Seed viruses are selected based on a high-growth phenotype and the presence of the seasonal hemagglutinin (HA) and neuraminidase (NA) surface antigens. Retrospective analysis of H3N2 vaccine seed viruses indicated that, unlike other internal proteins that were predominantly derived from the high-growth parent A/Puerto Rico/8/34 (PR8), the polymerase subunit PB1 could be derived from either parent depending on the seasonal strain. We have recently shown that A/Udorn/307/72 (Udorn) models a seasonal isolate that yields reassortants bearing the seasonal PB1 gene. This is despite the fact that the reverse genetics-derived virus that includes Udorn PB1 with Udorn HA and NA on a PR8 background has inferior growth compared to the corresponding virus with PR8 PB1. Here we use competitive plasmid transfections to investigate the mechanisms driving selection of a less fit virus and show that the Udorn PB1 gene segment cosegregates with the Udorn NA gene segment. Analysis of chimeric PB1 genes revealed that the coselection of NA and PB1 segments was not directed through the previously identified packaging sequences but through interactions involving the internal coding region of the PB1 gene. This study identifies associations between viral genes that can direct selection in classical reassortment for vaccine production and which may also be of relevance to the gene constellations observed in past antigenic shift events where creation of a pandemic virus has involved reassortment.

    IMPORTANCE Influenza vaccine must be produced and administered in a timely manner in order to provide protection during the winter season, and poor-growing vaccine seed viruses can compromise this process. To maximize vaccine yields, manufacturers create hybrid influenza viruses with gene segments encoding the surface antigens from a seasonal virus isolate, important for immunity, and others from a virus with high growth properties. This involves coinfection of cells with both parent viruses and selection of dominant progeny bearing the seasonal antigens. We show that this method of creating hybrid viruses does not necessarily select for the best yielding virus because preferential pairing of gene segments when progeny viruses are produced determines the genetic makeup of the hybrids. This not only has implications for how hybrid viruses are selected for vaccine production but also sheds light on what drives and limits hybrid gene combinations that arise in nature, leading to pandemics.

  • Disease Severity Is Associated with Differential Gene Expression at the Early and Late Phases of Infection in Nonhuman Primates Infected with Different H5N1 Highly Pathogenic Avian Influenza Viruses [Cellular Response to Infection]

  • Occasional transmission of highly pathogenic avian H5N1 influenza viruses to humans causes severe pneumonia with high mortality. To better understand the mechanisms via which H5N1 viruses induce severe disease in humans, we infected cynomolgus macaques with six different H5N1 strains isolated from human patients and compared their pathogenicity and the global host responses to the virus infection. Although all H5N1 viruses replicated in the respiratory tract, there was substantial heterogeneity in their replicative ability and in the disease severity induced, which ranged from asymptomatic to fatal. A comparison of global gene expression between severe and mild disease cases indicated that interferon-induced upregulation of genes related to innate immunity, apoptosis, and antigen processing/presentation in the early phase of infection was limited in severe disease cases, although interferon expression was upregulated in both severe and mild cases. Furthermore, coexpression analysis of microarray data, which reveals the dynamics of host responses during the infection, demonstrated that the limited expression of these genes early in infection led to a failure to suppress virus replication and to the hyperinduction of genes related to immunity, inflammation, coagulation, and homeostasis in the late phase of infection, resulting in a more severe disease. Our data suggest that the attenuated interferon-induced activation of innate immunity, apoptosis, and antigen presentation in the early phase of H5N1 virus infection leads to subsequent severe disease outcome.

    IMPORTANCE Highly pathogenic avian H5N1 influenza viruses sometimes transmit to humans and cause severe pneumonia with ca. 60% lethality. The continued circulation of these viruses poses a pandemic threat; however, their pathogenesis in mammals is not fully understood. We, therefore, investigated the pathogenicity of six H5N1 viruses and compared the host responses of cynomolgus macaques to the virus infection. We identified differences in the viral replicative ability of and in disease severity caused by these H5N1 viruses. A comparison of global host responses between severe and mild disease cases identified the limited upregulation of interferon-stimulated genes early in infection in severe cases. The dynamics of the host responses indicated that the limited response early in infection failed to suppress virus replication and led to hyperinduction of pathological condition-related genes late in infection. These findings provide insight into the pathogenesis of H5N1 viruses in mammals.

  • Crucial Roles of Interleukin-7 in the Development of T Follicular Helper Cells and in the Induction of Humoral Immunity [Vaccines and Antiviral Agents]

  • T follicular helper (Tfh) cells are specialized providers of cognate B cell help, which is important in promoting the induction of high-affinity antibody production in germinal centers (GCs). Interleukin-6 (IL-6) and IL-21 have been known to play important roles in Tfh cell differentiation. Here, we demonstrate that IL-7 plays a pivotal role in Tfh generation and GC formation in vivo, as treatment with anti-IL-7 neutralizing antibody markedly impaired the development of Tfh cells and IgG responses. Moreover, codelivery of mouse Fc-fused IL-7 (IL-7-mFc) with a vaccine enhanced the generation of GC B cells as well as Tfh cells but not other lineages of T helper cells, including Th1, Th2, and Th17 cells. Interestingly, a 6-fold-lower dose of an influenza virus vaccine codelivered with Fc-fused IL-7 induced higher antigen-specific and cross-reactive IgG titers than the vaccine alone in both mice and monkeys and led to markedly enhanced protection against heterologous influenza virus challenge in mice. Enhanced generation of Tfh cells by IL-7-mFc treatment was not significantly affected by the neutralization of IL-6 and IL-21, indicating an independent role of IL-7 on Tfh differentiation. Thus, IL-7 holds promise as a critical cytokine for selectively inducing Tfh cell generation and enhancing protective IgG responses.

    IMPORTANCE Here, we demonstrate for the first time that codelivery of Fc-fused IL-7 significantly increased influenza virus vaccine-induced antibody responses, accompanied by robust expansion of Tfh cells and GC B cells as well as enhanced GC formation. Furthermore, IL-7-mFc induced earlier and cross-reactive IgG responses, leading to striking protection against heterologous influenza virus challenge. These results suggest that Fc-fused IL-7 could be used for inducing strong and cross-protective humoral immunity against highly mutable viruses, such as HIV and hepatitis C virus, as well as influenza viruses.

  • The Pulmonary Localization of Virus-Specific T Lymphocytes Is Governed by the Tissue Tropism of Infection [Pathogenesis and Immunity]

  • The migration of pathogen-specific T cells into nonlymphoid tissues, such as the lung, is critical to control peripheral infections. Use of in vivo intravascular labeling of leukocytes has allowed for improved discrimination between cells located in the blood from cells present within peripheral tissues, such as the lung. This is particularly important in the lung, which is comprised of an intricate network of blood vessels that harbors a large proportion of the total blood volume at any given time. Recent work has demonstrated that ggt;80% of antigen-specific effector CD8 T cells remain in the pulmonary vasculature following an intratracheal infection with a systemic viral pathogen. However, it remains unclear what proportion of effector CD8 T cells are located within lung tissue following a localized respiratory viral infection. We confirm that most effector and memory CD8 T cells are found in the vasculature after an intranasal infection with the systemic pathogens lymphocytic choriomeningitis virus (LCMV) or vaccinia virus (VACV). In contrast, following pulmonary viral infections with either respiratory syncytial virus (RSV) or influenza A virus (IAV), 80 to 90% of the antigen-specific effector CD8 T cells were located within lung tissue. Similarly, the majority of antigen-specific CD4 T cells were present within lung tissue during a pulmonary viral infection. Furthermore, a greater proportion of gamma interferon-positive (IFN-+) effector CD8 and CD4 T cells were located within lung tissue following a localized respiratory viral infection. Our results indicate that T cells exhibit significantly altered distribution patterns dependent upon the tissue tropism of the infection.

    IMPORTANCE The migration of T cells to nonlymphoid sites, such as the lung, is critical to mediate clearance of viral infections. The highly vascularized lung holds up to 40% of blood, and thus, the T cell response may be a reflection of lymphocytes localized to the pulmonary vasculature instead of lung tissue. We examined the localization of T cell responses within the lung following either a localized or systemic viral infection. We demonstrate that following intranasal infection with a systemic pathogen, most T cells are localized to the pulmonary vasculature. In contrast, T cells are primarily localized to lung tissue following a respiratory viral infection. Our results demonstrate vast differences in the localization of T cell responses within the lung parenchyma between pathogens that can replicate locally versus systemically and that intravascular antibody labeling can be utilized to assess the localization patterns of T cell responses in nonlymphoid organs.

  • Transfer of the Amino-Terminal Nuclear Envelope Targeting Domain of Human MX2 Converts MX1 into an HIV-1 Resistance Factor [Virus-Cell Interactions]

  • The myxovirus resistance 2 (MX2) protein of humans has been identified recently as an interferon (IFN)-inducible inhibitor of human immunodeficiency virus type 1 (HIV-1) that acts at a late postentry step of infection to prevent the nuclear accumulation of viral cDNA (C. Goujon et al., Nature 502:559nndash;562, 2013,; M. Kane et al., Nature 502:563nndash;566, 2013,; Z. Liu et al., Cell Host Microbe 14:398nndash;410, 2013, In contrast, the closely related human MX1 protein, which suppresses infection by a range of RNA and DNA viruses (such as influenza A virus [FluAV]), is ineffective against HIV-1. Using a panel of engineered chimeric MX1/2 proteins, we demonstrate that the amino-terminal 91-amino-acid domain of MX2 confers full anti-HIV-1 function when transferred to the amino terminus of MX1, and that this fusion protein retains full anti-FluAV activity. Confocal microscopy experiments further show that this MX1/2 fusion, similar to MX2 but not MX1, can localize to the nuclear envelope (NE), linking HIV-1 inhibition with MX accumulation at the NE. MX proteins are dynamin-like GTPases, and while MX1 antiviral function requires GTPase activity, neither MX2 nor MX1/2 chimeras require this attribute to inhibit HIV-1. This key discrepancy between the characteristics of MX1- and MX2-mediated viral resistance, together with previous observations showing that the L4 loop of the stalk domain of MX1 is a critical determinant of viral substrate specificity, presumably reflect fundamental differences in the mechanisms of antiviral suppression. Accordingly, we propose that further comparative studies of MX proteins will help illuminate the molecular basis and subcellular localization requirements for implementing the noted diversity of virus inhibition by MX proteins.

    IMPORTANCE Interferon (IFN) elicits an antiviral state in cells through the induction of hundreds of IFN-stimulated genes (ISGs). The human MX2 protein has been identified as a key effector in the suppression of HIV-1 infection by IFN. Here, we describe a molecular genetic approach, using a collection of chimeric MX proteins, to identify protein domains of MX2 that specify HIV-1 inhibition. The amino-terminal 91-amino-acid domain of human MX2 confers HIV-1 suppressor capabilities upon human and mouse MX proteins and also promotes protein accumulation at the nuclear envelope. Therefore, these studies correlate the cellular location of MX proteins with anti-HIV-1 function and help establish a framework for future mechanistic analyses of MX-mediated virus control.

  • MicroRNA miR-BART20-5p Stabilizes Epstein-Barr Virus Latency by Directly Targeting BZLF1 and BRLF1 [Genome Replication and Regulation of Viral Gene Expression]

  • Epstein-Barr virus (EBV) is a human herpesvirus associated with various tumors. Rather than going through the lytic cycle, EBV maintains latency by limiting the expression of viral genes in tumors. Viral microRNAs (miRNAs) of some herpesviruses have been reported to directly target immediate early genes and suppress lytic induction. In this study, we investigated whether BamHI-A rightward transcript (BART) miRNAs targeted two EBV immediate early genes, BZLF1 and BRLF1. Bioinformatic analysis predicted that 12 different BART miRNAs would target BRLF1. Of these, the results of a luciferase reporter assay indicated that only one interacted with the 3' untranslated region (UTR) of BRLF1: miR-BART20-5p. miR-BART20-5p's effect on gene expression involved two putative seed match sites in the BRLF1 3' UTR, but a mutant version of the miRNA, miR-BART20-5pm, had no effect on expression. As expected from the fact that the entire 3' UTR of BZLF1 resides within the 3' UTR of BRLF1, miR-BART20-5p interacted with the 3' UTR of BZLF1 as well. BZLF1 and BRLF1 mRNA and protein expression was suppressed in cells of an AGS cell line infected with the recombinant Akata strain of EBV (AGS-EBV) transfected with a miR-BART20-5p mimic. The expression of various EBV early proteins was also suppressed by the miR-BART20-5p mimic. In contrast, BZLF1 and BRLF1 expression in AGS-EBV cells transfected with a miR-BART20-5p inhibitor was enhanced. Furthermore, progeny virus production was suppressed by the miR-BART20-5p mimic and enhanced by the miR-BART20-5p inhibitor in AGS-EBV cells induced for the lytic cycle. Our data suggest that miR-BART20-5p plays a key role in latency maintenance in EBV-associated tumors by directly targeting immediate early genes.

    IMPORTANCE Herpesviruses maintain latency using various mechanisms and establish lifelong infection in the host. From time to time, herpesviruses are reactivated and express immediate early genes which trigger a lytic cascade, leading to the production of progeny viruses. Recently, some herpesviruses have been shown to use their own microRNAs (miRNAs) to downregulate immediate early genes to inhibit the lytic cycle. This study presents evidence that EBV also downregulates two immediate early genes by miR-BART20-5p to suppress the lytic cycle and progeny virus production. Overall, this is the first study to report the direct regulation of EBV immediate early genes by an EBV miRNA, implying its likely importance in latency maintenance in EBV-associated tumors.

  • Quantitative Proteomic Analysis of the Influenza A Virus Nonstructural Proteins NS1 and NS2 during Natural Cell Infection Identifies PACT as an NS1 Target Protein and Antiviral Host Factor [Virus-Cell Interactions]

  • Influenza A virus (IAV) replication depends on the interaction of virus proteins with host factors. The viral nonstructural protein 1 (NS1) is essential in this process by targeting diverse cellular functions, including mRNA splicing and translation, cell survival, and immune defense, in particular the type I interferon (IFN-I) response. In order to identify host proteins targeted by NS1, we established a replication-competent recombinant IAV that expresses epitope-tagged forms of NS1 and NS2, which are encoded by the same gene segment, allowing purification of NS proteins during natural cell infection and analysis of interacting proteins by quantitative mass spectrometry. We identified known NS1- and NS2-interacting proteins but also uncharacterized proteins, including PACT, an important cofactor for the IFN-I response triggered by the viral RNA-sensor RIG-I. We show here that NS1 binds PACT during virus replication and blocks PACT/RIG-I-mediated activation of IFN-I, which represents a critical event for the host defense. Protein interaction and interference with IFN-I activation depended on the functional integrity of the highly conserved RNA binding domain of NS1. A mutant virus with deletion of NS1 induced high levels of IFN-I in control cells, as expected; in contrast, shRNA-mediated knockdown of PACT compromised IFN-I activation by the mutant virus, but not wild-type virus, a finding consistent with the interpretation that PACT (i) is essential for IAV recognition and (ii) is functionally compromised by NS1. Together, our data describe a novel approach to identify virus-host protein interactions and demonstrate that NS1 interferes with PACT, whose function is critical for robust IFN-I production.

    IMPORTANCE Influenza A virus (IAV) is an important human pathogen that is responsible for annual epidemics and occasional devastating pandemics. Viral replication and pathogenicity depends on the interference of viral factors with components of the host defense system, particularly the type I interferon (IFN-I) response. The viral NS1 protein is known to counteract virus recognition and IFN-I production, but the molecular mechanism is only partially defined. We used a novel proteomic approach to identify host proteins that are bound by NS1 during virus replication and identified the protein PACT, which had previously been shown to be involved in virus-mediated IFN-I activation. We find that NS1 prevents PACT from interacting with an essential component of the virus recognition pathway, RIG-I, thereby disabling efficient IFN-I production. These observations provide an important piece of information on how IAV efficiently counteracts the host immune defense.

  • Modification of the Hepatitis B Virus Envelope Protein Glycosylation Pattern Interferes with Secretion of Viral Particles, Infectivity, and Susceptibility to Neutralizing Antibodies [Virus-Cell Interactions]

  • The envelope proteins of hepatitis B virus (HBV) bear an N-linked glycosylation site at N146 within the immunodominant a-determinant in the antigenic loop (AGL) region. This glycosylation site is never fully functional, leading to a nearly 1/1 ratio of glycosylated/nonglycosylated isoforms in the viral envelope. Here we investigated the requirement for a precise positioning of N-linked glycan at amino acid 146 and the functions associated with the glycosylated and nonglycosylated isoforms. We observed that the removal of the N146 glycosylation site by mutagenesis was permissive to envelope protein synthesis and stability and to secretion of subviral particles (SVPs) and hepatitis delta virus (HDV) virions, but it was detrimental to HBV virion production. Several positions in the AGL could substitute for position 146 as the glycosylation acceptor site. At position 146, neither a glycan chain nor asparagine was absolutely required for infectivity, but there was a preference for a polar residue. Envelope proteins bearing 5 AGL glycosylation sites became hyperglycosylated, leading to an increased capacity for SVP secretion at the expense of HBV and HDV virion secretion. Infectivity-compatible N-glycosylation sites could be inserted at 3 positions (positions 115, 129, and 136), but when all three positions were glycosylated, the hyperglycosylated mutant was substantially attenuated at viral entry, while it acquired resistance to neutralizing antibodies. Taken together, these findings suggest that the nonglycosylated N146 is essential for infectivity, while the glycosylated form, in addition to its importance for HBV virion secretion, is instrumental in shielding the a-determinant from neutralizing antibodies.

    IMPORTANCE At the surface of HBV particles, the immunodominant a-determinant is the main target of neutralizing antibodies and an essential determinant of infectivity. It contains an N-glycosylation site at position 146, which is functional on only half of the envelope proteins. Our data suggest that the coexistence of nonglycosylated and glycosylated N146 at the surface of HBV reflects the dual function of this determinant in infectivity and immune escape. Hence, a modification of the HBV glycosylation pattern affects not only virion assembly and infectivity but also immune escape.

  • Absence of Genetic Differences among G10P[11] Rotaviruses Associated with Asymptomatic and Symptomatic Neonatal Infections in Vellore, India [Genetic Diversity and Evolution]

  • Rotaviruses (RVs) are leading causes of severe diarrhea and vomiting in infants and young children. RVs with G10P[11] genotype specificity have been associated with symptomatic and asymptomatic neonatal infections in Vellore, India. To identify possible viral genetic determinants responsible for differences in symptomology, the genome sequences of G10P[11] RVs in stool samples of 19 neonates with symptomatic infections and 20 neonates with asymptomatic infections were determined by Sanger and next-generation sequencing. The data showed that all 39 viruses had identical genotype constellations (G10-P[11]-I2-R2-C2-M2-A1-N1-T1-E2-H3), the same as those of the previously characterized symptomatic N155 Vellore isolate. The data also showed that the RNA and deduced protein sequences of all the Vellore G10P[11] viruses were nearly identical; no nucleotide or amino acid differences were found that correlated with symptomatic versus asymptomatic infection. Next-generation sequencing data revealed that some stool samples, both from neonates with symptomatic infections and from neonates with asymptomatic infections, also contained one or more positive-strand RNA viruses (Aichi virus, astrovirus, or salivirus/klassevirus) suspected of being potential causes of pediatric gastroenteritis. However, none of the positive-strand RNA viruses could be causally associated with the development of symptoms. These results indicate that the diversity of clinical symptoms in Vellore neonates does not result from genetic differences among G10P[11] RVs; instead, other undefined factors appear to influence whether neonates develop gastrointestinal disease symptoms.

    IMPORTANCE Rotavirus (RV) strains have been identified that preferentially replicate in neonates, in some cases, without causing gastrointestinal disease. Surveillance studies have established that G10P[11] RVs are a major cause of neonatal infection in Vellore, India, with half of infected neonates exhibiting symptoms. We used Sanger and next-generation sequencing technologies to contrast G10P[11] RVs recovered from symptomatic and asymptomatic neonates. Remarkably, the data showed that the RNA genomes of the viruses were virtually indistinguishable and lacked any differences that could explain the diversity of clinical outcomes among infected Vellore neonates. The sequencing results also indicated that some symptomatic and some asymptomatic Vellore neonates were infected with other enteric viruses (Aichi virus, astrovirus, salvirus/klassevirus); however, none could be correlated with the presence of symptoms in neonates. Together, our findings suggest that other poorly defined factors, not connected to the genetic makeup of the Vellore G10P[11] viruses, influence whether neonates develop gastrointestinal disease symptoms.

  • Predicted Structure and Domain Organization of Rotavirus Capping Enzyme and Innate Immune Antagonist VP3 [Structure and Assembly]

  • Rotaviruses and orbiviruses are nonturreted Reoviridae members. The rotavirus VP3 protein is a multifunctional capping enzyme and antagonist of the interferon-induced cellular oligoadenylate synthetase-RNase L pathway. Despite mediating important processes, VP3 is the sole protein component of the rotavirus virion whose structure remains unknown. In the current study, we used sequence alignment and homology modeling to identify features common to nonturreted Reoviridae capping enzymes and to predict the domain organization, structure, and active sites of rotavirus VP3. Our results suggest that orbivirus and rotavirus capping enzymes share a domain arrangement similar to that of the bluetongue virus capping enzyme. Sequence alignments revealed conserved motifs and suggested that rotavirus and orbivirus capping enzymes contain a variable N-terminal domain, a central guanine-N7-methyltransferase domain that contains an additional inserted domain, and a C-terminal guanylyltransferase and RNA 5'-triphosphatase domain. Sequence conservation and homology modeling suggested that the insertion in the guanine-N7-methyltransferase domain is a ribose-2'-O-methyltransferase domain for most rotavirus species. Our analyses permitted putative identification of rotavirus VP3 active-site residues, including those that form the ribose-2'-O-methyltransferase catalytic tetrad, interact with S-adenosyl-l-methionine, and contribute to autoguanylation. Previous reports have indicated that group A rotavirus VP3 contains a C-terminal 2H-phosphodiesterase domain that can cleave 2'-5' oligoadenylates, thereby preventing RNase L activation. Our results suggest that a C-terminal phosphodiesterase domain is present in the capping enzymes from two additional rotavirus species. Together, these findings provide insight into a poorly understood area of rotavirus biology and are a springboard for future biochemical and structural studies of VP3.

    IMPORTANCE Rotaviruses are an important cause of severe diarrheal disease. The rotavirus VP3 protein caps viral mRNAs and helps combat cellular innate antiviral defenses, but little is known about its structure or enzymatic mechanisms. In this study, we used sequence- and structure-based alignments with related proteins to predict the structure of VP3 and identify enzymatic domains and active sites therein. This work provides insight into the mechanisms of rotavirus transcription and evasion of host innate immune defenses. An improved understanding of these processes may aid our ability to develop rotavirus vaccines and therapeutics.

  • Identification of Human Cytomegalovirus Genes Important for Biogenesis of the Cytoplasmic Virion Assembly Complex [Virus-Cell Interactions]

  • Human cytomegalovirus (HCMV) has many effects on cells, including remodeling the cytoplasm to form the cytoplasmic virion assembly complex (cVAC), the site of final virion assembly. Viral tegument, envelope, and some nonstructural proteins localize to the cVAC, and cytoskeletal filaments radiate from a microtubule organizing center in the cVAC. The endoplasmic reticulum (ER)-to-Golgi intermediate compartment, Golgi apparatus, and trans-Golgi network form a ring that outlines the cVAC. The center of the cVAC ring is occupied by numerous vesicles that share properties with recycling endosomes. In prior studies, we described the three-dimensional structure and the extensive remodeling of the cytoplasm and shifts in organelle identity that occur during development of the cVAC. The objective of this work was to identify HCMV proteins that regulate cVAC biogenesis. Because the cVAC does not form in the absence of viral DNA synthesis, we employed HCMV-infected cells transfected with synthetic small interfering RNAs (siRNAs) that targeted 26 candidate early-late and late protein-coding genes required for efficient virus replication. We identified three HCMV genes (UL48, UL94, and UL103) whose silencing had major effects on cVAC development, including failure to form the Golgi ring and dispersal of markers of early and recycling endosomes. To confirm and extend the siRNA results, we constructed recombinant viruses in which pUL48 and pUL103 are fused with a regulatable protein destabilization domain (dd-FKBP). In the presence of a stabilizing ligand (Shield-1), the cVAC appeared to develop normally. In its absence, cVAC development was abrogated, verifying roles for pUL48 and pUL103 in cVAC biogenesis.

    IMPORTANCE Human cytomegalovirus (HCMV) is an important human pathogen that causes disease and disability in immunocompromised individuals and in children infected before birth. Few drugs are available for treatment of HCMV infections. HCMV remodels the interior of infected cells to build a factory for assembling new infectious particles (virions), the cytoplasmic virion assembly complex (cVAC). Here, we identified three HCMV genes (UL48, UL94, and UL103) as important contributors to cVAC development. In addition, we found that mutant viruses that express an unstable form of the UL103 protein have defects in cVAC development and production of infectious virions and produce small plaques and intracellular virions with aberrant appearances. Of these, only the reduced production of infectious virions is not eliminated by chemically stabilizing the protein. In addition to identifying new functions for these HCMV genes, this work is a necessary prelude to developing novel antivirals that would block cVAC development.

  • Extraribosomal L13a Is a Specific Innate Immune Factor for Antiviral Defense [Cellular Response to Infection]

  • We report a novel extraribosomal innate immune function of mammalian ribosomal protein L13a, whereby it acts as an antiviral agent. We found that L13a is released from the 60S ribosomal subunit in response to infection by respiratory syncytial virus (RSV), an RNA virus of the Pneumovirus genus and a serious lung pathogen. Unexpectedly, the growth of RSV was highly enhanced in L13a-knocked-down cells of various lineages as well as in L13a knockout macrophages from mice. In all L13a-deficient cells tested, translation of RSV matrix (M) protein was specifically stimulated, as judged by a greater abundance of M protein and greater association of the M mRNA with polyribosomes, while general translation was unaffected. In silico RNA folding analysis and translational reporter assays revealed a putative hairpin in the 3'untranslated region (UTR) of M mRNA with significant structural similarity to the cellular GAIT (gamma-activated inhibitor of translation) RNA hairpin, previously shown to be responsible for assembling a large, L13a-containing ribonucleoprotein complex that promoted translational silencing in gamma interferon (IFN-)-activated myeloid cells. However, RNA-protein interaction studies revealed that this complex, which we named VAIT (respiratory syncytial virus-activated inhibitor of translation) is functionally different from the GAIT complex. VAIT is the first report of an extraribosomal L13a-mediated, IFN--independent innate antiviral complex triggered in response to virus infection. We provide a model in which the VAIT complex strongly hinders RSV replication by inhibiting the translation of the rate-limiting viral M protein, which is a new paradigm in antiviral defense.

    IMPORTANCE The innate immune mechanisms of host cells are diverse in nature and act as a broad-spectrum cellular defense against viruses. Here, we report a novel innate immune mechanism functioning against respiratory syncytial virus (RSV), in which the cellular ribosomal protein L13a is released from the large ribosomal subunit soon after infection and inhibits the translation of a specific viral mRNA, namely, that of the matrix protein M. Regarding its mechanism, we show that the recognition of a specific secondary structure in the 3' untranslated region of the M mRNA leads to translational arrest of the mRNA. We also show that the level of M protein in the infected cell is rate limiting for viral morphogenesis, providing a rationale for L13a to target the M mRNA for suppression of RSV growth. Translational silencing of a viral mRNA by a deployed ribosomal protein is a new paradigm in innate immunity.

  • Maturation and Vesicle-Mediated Egress of Primate Gammaherpesvirus Rhesus Monkey Rhadinovirus Require Inner Tegument Protein ORF52 [Structure and Assembly]

  • The tegument layer of herpesviruses comprises a collection of proteins that is unique to each viral species. In rhesus monkey rhadinovirus (RRV), a close relative of the human oncogenic pathogen Kaposi's sarcoma-associated herpesvirus, ORF52 is a highly abundant tegument protein tightly associated with the capsid. We now report that ORF52 knockdown during RRV infection of rhesus fibroblasts led to a greater than 300-fold reduction in the viral titer by 48 h but had little effect on the number of released particles and caused only modest reductions in the levels of intracellular viral genomic DNA and no appreciable change in viral DNA packaging into capsids. These data suggested that the lack of ORF52 resulted in the production and release of defective particles. In support of this interpretation, transmission electron microscopy (TEM) revealed that without ORF52, capsid-like particles accumulated in the cytoplasm and were unable to enter egress vesicles, where final tegumentation and envelopment normally occur. TEM also demonstrated defective particles in the medium that closely resembled the accumulating intracellular particles, having neither a full tegument nor an envelope. The disruption in tegument formation from ORF52 suppression, therefore, prevented the incorporation of ORF45, restricting its subcellular localization to the nucleus and appearing, by confocal microscopy, to inhibit particle transport toward the periphery. Ectopic expression of small interfering RNA (siRNA)-resistant ORF52 was able to partially rescue all of these phenotypic changes. In sum, our results indicate that efficient egress of maturing virions and, in agreement with studies on murine gammaherpesvirus 68 (MHV-68), complete tegumentation and secondary envelopment are dependent on intact ORF52.

    IMPORTANCE The tegument, or middle layer, of herpesviruses comprises both viral and cellular proteins that play key roles in the viral life cycle. A subset of these proteins is present only within members of one of the three subfamilies (alphaherpesviruses, betaherpesviruses, or gammaherpesviruses) of Herpesviridae. In this report, we show that the gammaherpesvirus-specific tegument protein ORF52 is critical for maturation of RRV, the closest relative of Kaposi's sarcoma-associated herpesvirus (KSHV) (a human cancer-causing pathogen) that has undergone this type of analysis. Without ORF52, the nascent subviral particles are essentially stuck in maturation limbo, unable to acquire the tegument or outer (envelope) layers. This greatly attenuates infectivity. Our data, together with earlier work on a murine homolog, as well as a more distantly related human homolog, provide a more complete understanding of how early protein interactions involving virus-encoded tegument proteins are critical for virus assembly and are also, therefore, potentially attractive therapeutic targets.

  • Functional Analyses of the Three Simian Hemorrhagic Fever Virus Nonstructural Protein 1 Papain-Like Proteases [Genome Replication and Regulation of Viral Gene Expression]

  • The N-terminal region of simian hemorrhagic fever virus (SHFV) nonstructural polyprotein 1a is predicted to encode three papain-like proteases (PLP1aalpha;, PLP1bbeta;, and PLP1). Catalytic residues and cleavage sites for each of the SHFV PLP1s were predicted by alignment of the SHFV PLP1 region sequences with each other as well as with those of other arteriviruses, and the predicted catalytic residues were shown to be proximal by homology modeling of the SHFV nsp1s on porcine respiratory and reproductive syndrome virus (PRRSV) nsp1 crystal structures. The functionality of the predicted catalytic Cys residues and cleavage sites was tested by analysis of the autoproteolytic products generated in in vitro transcription/translation reactions done with wild-type or mutant SHFV nsp1 constructs. Cleavage sites were also analyzed by mass spectroscopy analysis of selected immunoprecipitated cleavage products. The data showed that each of the three SHFV PLP1s is an active protease. Cys63 was identified as the catalytic Cys of SHFV PLP1aalpha; and is adjacent to an Ala instead of the canonical Tyr observed in other arterivirus PLP1s. SHFV PLP1 is able to cleave at both downstream and upstream nsp1 junction sites. Although intermediate precursor polyproteins as well as alternative products generated by each of the SHFV PLP1s cleaving at sites within the N-terminal region of nsp1bbeta; were produced in the in vitro reactions, Western blotting of SHFV-infected, MA104 cell lysates with SHFV nsp1 protein-specific antibodies detected only the three mature nsp1 proteins.

    IMPORTANCE SHFV is unique among arteriviruses in having three N-terminal papain-like protease 1 (PLP1) domains. Other arteriviruses encode one or two active PLP1s. This is the first functional study of the SHFV PLP1s. Analysis of the products of in vitro autoprocessing of an N-terminal SHFV nonstructural 1a polypeptide fragment showed that each of the three SHFV PLP1s is active, and the predicted catalytic Cys residues and cleavage sites for each PLP1 were confirmed by testing mutant constructs. Several unique features of the SHFV PLP1s were discovered. The SHFV PLP1aalpha; catalytic Cys63 is unique among arterivirus PLP1s in being adjacent to an Ala instead of a Trp. Other arterivirus PLP1s cleave only in cis at a single downstream site, but SHFV PLP1 can cleave at both the downstream nsp1-nsp2 and upstream nsp1bbeta;-nsp1 junctions. The three mature nsp1 proteins were produced both in the in vitro reactions and in infected cells.

  • HIV-1 Vpr Redirects Host Ubiquitination Pathway [Virus-Cell Interactions]

  • HIV-1 modulates key host cellular pathways for successful replication and pathogenesis through viral proteins. By evaluating the hijacking of the host ubiquitination pathway by HIV-1 at the whole-cell level, we now show major perturbations in the ubiquitinated pool of the host proteins post-HIV-1 infection. Our overexpression- and infection-based studies of T cells with wild-type and mutant HIV-1 proviral constructs showed that Vpr is necessary and sufficient for reducing whole-cell ubiquitination. Mutagenic analysis revealed that the three leucine-rich helical regions of Vpr are critical for this novel function of Vpr, which was independent of its other known cellular functions. We also validated that this effect of Vpr was conserved among different subtypes (subtypes B and C) and circulating recombinants from Northern India. Finally, we establish that this phenomenon is involved in HIV-1-mediated diversion of host ubiquitination machinery specifically toward the degradation of various restriction factors during viral pathogenesis.

    IMPORTANCE HIV-1 is known to rely heavily on modulation of the host ubiquitin pathway, particularly for counteraction of antiretroviral restriction factors, i.e., APOBEC3G, UNG2, and BST-2, etc.; viral assembly; and release. Reports to date have focused on the molecular hijacking of the ubiquitin machinery by HIV-1 at the level of E3 ligases. Interaction of a viral protein with an E3 ligase alters its specificity to bring about selective protein ubiquitination. However, in the case of infection, multiple viral proteins can interact with this multienzyme pathway at various levels, making it much more complicated. Here, we have addressed the manipulation of ubiquitination at the whole-cell level post-HIV-1 infection. Our results show that HIV-1 Vpr is necessary and sufficient to bring about the redirection of the host ubiquitin pathway toward HIV-1-specific outcomes. We also show that the three leucine-rich helical regions of Vpr are critical for this effect and that this ability of Vpr is conserved across circulating recombinants. Our work, the first of its kind, provides novel insight into the regulation of the ubiquitin system at the whole-cell level by HIV-1.

  • Avian Influenza H7N9/13 and H7N7/13: a Comparative Virulence Study in Chickens, Pigeons, and Ferrets [Pathogenesis and Immunity]

  • Human influenza cases caused by a novel avian H7N9 virus in China emphasize the zoonotic potential of that subtype. We compared the infectivity and pathogenicity of the novel H7N9 virus with those of a recent European avian H7N7 strain in chickens, pigeons, and ferrets. Neither virus induced signs of disease despite substantial replication in inoculated chickens and rapid transmission to contact chickens. Evidence of the replication of both viruses in pigeons, albeit at lower levels of RNA excretion, was also detected. No clear-cut differences between the two H7 isolates emerged regarding replication and antibody development in avian hosts. In ferrets, in contrast, greater replication of the avian H7N9 virus than of the H7N7 strain was observed with significant differences in viral presence, e.g., in nasal wash, lung, and cerebellum samples. Importantly, both viruses showed the potential to spread to the mammal brain. We conclude that efficient asymptomatic viral replication and shedding, as shown in chickens, facilitate the spread of H7 viruses that may harbor zoonotic potential. Biosafety measures are required for the handling of poultry infected with avian influenza viruses of the H7 subtype, independently of their pathogenicity for gallinaceous poultry.

    IMPORTANCE This study is important to the field since it provides data about the behavior of the novel H7N9 avian influenza virus in chickens, pigeons, and ferrets in comparison with that of a recent low-pathogenicity H7N7 strain isolated from poultry. We clearly show that chickens, but not pigeons, are highly permissive hosts of both H7 viruses, allowing high-titer replication and virus shedding without any relevant clinical signs. In the ferret model, the potential of both viruses to infect mammals could be demonstrated, including infection of the brain. However, the replication efficiency of the H7N9 virus in ferrets was higher than that of the H7N7 strain. In conclusion, valuable data for the risk analysis of low-pathogenicity avian influenza viruses of the H7 subtype are provided that could also be used for the risk assessment of zoonotic potentials and necessary biosafety measures.

  • Asthma Increases Susceptibility to Heterologous but Not Homologous Secondary Influenza [Pathogenesis and Immunity]

  • Asthma was the most common comorbidity observed among patients hospitalized with influenza A virus during the 2009 pandemic. However, little remains known about how the asthmatic phenotype influences protective immune responses against respiratory viral pathogens. Using the ovalbumin-induced allergic lung inflammation model, we found that asthmatic mice, unlike nonasthmatic mice, were highly susceptible to secondary heterologous virus challenge. While primary virus infection generated protective memory immune responses against homologous secondary virus challenge in both asthmatic and nonasthmatic mice, full protection against heterologous A/California/04/2009 (CA04) viral infection was observed only in nonasthmatic mice. Significant reductions in CA04-specific IgA, IgG, and IgM levels and in CA04-neutralizing activity of bronchoalveolar lavage fluid (BALF) was observed following secondary CA04 challenge of PR8-immunized asthmatic mice. Furthermore, transfer of immune BALF obtained from nonasthmatic, but not asthmatic, donors following secondary viral infection generated protection against CA04 in naive recipients. Nonspecific B-cell activation by CpG inoculation restored protection in PR8-immunized, CA04-challenged asthmatic mice. These results demonstrate a causal link between defective mucosal antibody responses and the heightened susceptibility of asthmatic mice to influenza infection and provide a mechanistic explanation for the observation that asthma was a major risk factor during the 2009 influenza pandemic.

    IMPORTANCE The prevalence of asthma worldwide is increasing each year. Unfortunately, there is no cure for asthma. Asthmatic individuals not only suffer from consistent wheezing and coughing but are also believed to be more prone to serious lung infections that result in bronchitis and pneumonia. However, little is known about the influence of asthma on host mucosal immunity. Here we show that antibody responses during secondary heterologous influenza infections are suboptimal and that this is responsible for the increased mortality in asthmatic mice from viral infections. Understanding the mechanism of increased susceptibility will aid in developing new antiviral therapies for asthmatic patients.

  • An Alphavirus-Based Adjuvant Enhances Serum and Mucosal Antibodies, T Cells, and Protective Immunity to Influenza Virus in Neonatal Mice [Vaccines and Antiviral Agents]

  • Neonatal immune responses to infection and vaccination are biased toward TH2 at the cost of proinflammatory TH1 responses needed to combat intracellular pathogens. However, upon appropriate stimulation, the neonatal immune system can induce adult-like TH1 responses. Here we report that a new class of vaccine adjuvant is especially well suited to enhance early life immunity. The GVI3000 adjuvant is a safe, nonpropagating, truncated derivative of Venezuelan equine encephalitis virus that targets dendritic cells (DCs) in the draining lymph node (DLN) and produces intracellular viral RNA without propagating to other cells. RNA synthesis strongly activates the innate immune response so that in adult animals, codelivery of soluble protein antigens induces robust humoral, cellular, and mucosal responses. The adjuvant properties of GVI3000 were tested in a neonatal BALB/c mouse model using inactivated influenza virus (iFlu). After a single immunization, mice immunized with iFlu with the GVI3000 adjuvant (GVI3000-adjuvanted iFlu) had significantly higher and sustained influenza virus-specific IgG antibodies, mainly IgG2a (TH1), compared to the mice immunized with antigen only. GVI3000 significantly increased antigen-specific CD4+ and CD8+ T cells, primed mucosal immune responses, and enhanced protection from lethal challenge. As seen in adult mice, the GVI3000 adjuvant increased the DC population in the DLNs, caused activation and maturation of DCs, and induced proinflammatory cytokines and chemokines in the DLNs soon after immunization, including gamma interferon (IFN-), tumor necrosis factor alpha (TNF-aalpha;), granulocyte colony-stimulating factor (G-CSF), and interleukin 6 (IL-6). In summary, the GVI3000 adjuvant induced an adult-like adjuvant effect with an influenza vaccine and has the potential to improve the immunogenicity and protective efficacy of new and existing neonatal vaccines.

    IMPORTANCE The suboptimal immune responses in early life constitute a significant challenge for vaccine design. Here we report that a new class of adjuvant is safe and effective for early life immunization and demonstrate its ability to significantly improve the protective efficacy of an inactivated influenza virus vaccine in a neonatal mouse model. The GVI3000 adjuvant delivers a truncated, self-replicating viral RNA into dendritic cells in the draining lymph node. Intracellular RNA replication activates a strong innate immune response that significantly enhances adaptive antibody and cellular immune responses to codelivered antigens. A significant increase in protection results from a single immunization. Importantly, this adjuvant also primed a mucosal IgA response, which is likely to be critical for protection during many early life infections.

  • Structure of Influenza Virus N7: the Last Piece of the Neuraminidase "Jigsaw" Puzzle [Structure and Assembly]

  • There are nine subtypes of influenza A virus neuraminidase (NA), N1 to N9. In addition, influenza B virus also contains NA, and there are two influenza virus NA-like molecules, N10 and N11, which were recently identified from bats. Crystal structures for all of these proteins have been solved, with the exception of N7, and there is no published report of N6, although a structure has been deposited in the Protein Data Bank. Here, we present the N7 and N6 structures at 2.1 AAring; and 1.8 AAring;, respectively. Structural comparison of all NA subtypes shows that both N7 and N6 highly resemble typical group 2 NA structures with some special characteristics, including an additional cavity adjacent to their active sites formed by novel 340-loop conformations. Comparative analysis also revealed new structural insights into the N-glycosylation, calcium binding, and second sialic acid binding site of influenza virus NA. This comprehensive study is critical for understanding the complexity of the most successful influenza drug target and for the structure-based design of novel influenza inhibitors.

    IMPORTANCE Influenza viruses impose a great burden on society, by the human-adapted seasonal types as well as by variants that occasionally jump from the avian reservoir to infect humans. The surface glycoprotein neuraminidase (NA) is essential for the propagation of the virus and currently the most successfully drug-targeted molecule. Therefore, the structural and functional analysis of NA is critical for the prevention and control of influenza infections. There are nine subtypes of influenza A virus NA (N1 to N9). In addition, influenza B virus also contains NA, and there are two influenza NA-like molecules, N10 and N11, which were recently identified in bats. Crystal structures for all of these proteins have been solved and reported with the exception of N7 and N6. The structural analysis of influenza virus N7 and N6 presented in this study therefore completes the puzzle and adds to a comprehensive understanding of influenza virus NA.

  • Infection and Pathogenesis of Canine, Equine, and Human Influenza Viruses in Canine Tracheas [Pathogenesis and Immunity]

  • Influenza A viruses (IAVs) can jump species barriers and occasionally cause epidemics, epizootics, pandemics, and panzootics. Characterizing the infection dynamics at the target tissues of natural hosts is central to understanding the mechanisms that control host range, tropism, and virulence. Canine influenza virus (CIV; H3N8) originated after the transfer of an equine influenza virus (EIV) into dogs. Thus, comparing CIV and EIV isolates provides an opportunity to study the determinants of influenza virus emergence. Here we characterize the replication of canine, equine, and human IAVs in the trachea of the dog, a species to which humans are heavily exposed. We define a phenotype of infection for CIV, which is characterized by high levels of virus replication and extensive tissue damage. CIV was compared to evolutionarily distinct EIVs, and the early EIV isolates showed an impaired ability to infect dog tracheas, while EIVs that circulated near the time of CIV emergence exhibited a CIV-like infection phenotype. Inoculating dog tracheas with various human IAVs (hIAVs) showed that they infected the tracheal epithelium with various efficiencies depending on the virus tested. Finally, we show that reassortant viruses carrying gene segments of CIV and hIAV are viable and that addition of the hemagglutinin (HA) and neuraminidase (NA) of CIV to the 2009 human pandemic virus results in a virus that replicates at high levels and causes significant lesions. This provides important insights into the role of evolution on viral emergence and on the role of HA and NA as determinants of pathogenicity.

    IMPORTANCE Influenza A viruses (IAVs) have entered new host species in recent history, sometimes with devastating consequences. Canine influenza virus (CIV) H3N8 originated from a direct transfer of an equine influenza virus (EIV) in the early 2000s. We studied the infection patterns of IAVs that circulate in dogs or to which dogs are commonly exposed and showed that CIV emergence was likely caused by an adaptive driver, as evolutionarily distinct EIVs display distinct infection phenotypes. We also showed that many human viruses can infect dog tracheas and that reassortment with CIV results in viable viruses. Finally, we showed that the hemagglutinin and neuraminidase of CIV act as virulence factors. Our findings have significant implications because they show that dogs might act as "mixing vessels" in which novel viruses with pandemic potential could emerge and also provide experimental evidence supporting the role of viral evolution in influenza virus emergence.

  • Host Species Restriction of Middle East Respiratory Syndrome Coronavirus through Its Receptor, Dipeptidyl Peptidase 4 [Virus-Cell Interactions]

  • Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012. Recently, the MERS-CoV receptor dipeptidyl peptidase 4 (DPP4) was identified and the specific interaction of the receptor-binding domain (RBD) of MERS-CoV spike protein and DPP4 was determined by crystallography. Animal studies identified rhesus macaques but not hamsters, ferrets, or mice to be susceptible for MERS-CoV. Here, we investigated the role of DPP4 in this observed species tropism. Cell lines of human and nonhuman primate origin were permissive of MERS-CoV, whereas hamster, ferret, or mouse cell lines were not, despite the presence of DPP4. Expression of human DPP4 in nonsusceptible BHK and ferret cells enabled MERS-CoV replication, whereas expression of hamster or ferret DPP4 did not. Modeling the binding energies of MERS-CoV spike protein RBD to DPP4 of human (susceptible) or hamster (nonsusceptible) identified five amino acid residues involved in the DPP4-RBD interaction. Expression of hamster DPP4 containing the five human DPP4 amino acids rendered BHK cells susceptible to MERS-CoV, whereas expression of human DPP4 containing the five hamster DPP4 amino acids did not. Using the same approach, the potential of MERS-CoV to utilize the DPP4s of common Middle Eastern livestock was investigated. Modeling of the DPP4 and MERS-CoV RBD interaction predicted the ability of MERS-CoV to bind the DPP4s of camel, goat, cow, and sheep. Expression of the DPP4s of these species on BHK cells supported MERS-CoV replication. This suggests, together with the abundant DPP4 presence in the respiratory tract, that these species might be able to function as a MERS-CoV intermediate reservoir.

    IMPORTANCE The ongoing outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) has caused 701 laboratory-confirmed cases to date, with 249 fatalities. Although bats and dromedary camels have been identified as potential MERS-CoV hosts, the virus has so far not been isolated from any species other than humans. The inability of MERS-CoV to infect commonly used animal models, such as hamster, mice, and ferrets, indicates the presence of a species barrier. We show that the MERS-CoV receptor DPP4 plays a pivotal role in the observed species tropism of MERS-CoV and subsequently identified the amino acids in DPP4 responsible for this restriction. Using a combined modeling and experimental approach, we predict that, based on the ability of MERS-CoV to utilize the DPP4 of common Middle East livestock species, such as camels, goats, sheep, and cows, these form a potential MERS-CoV intermediate host reservoir species.

  • Hepatitis C Virus Entry Is Impaired by Claudin-1 Downregulation in Diacylglycerol Acyltransferase-1-Deficient Cells [Pathogenesis and Immunity]

  • Diacylglycerol acyltransferase-1 (DGAT1) is involved in the assembly of hepatitis C virus (HCV) by facilitating the trafficking of the HCV core protein to the lipid droplet. Here, we abrogated DGAT1 expression in Huh-7.5 cells by using either the transcription activator-like effector nuclease (TALEN) or lentivirus vector short hairpin RNA (shRNA) and achieved complete long-term silencing of DGAT1. HCV entry was severely impaired in DGAT1-silenced Huh-7.5 cell lines, which showed markedly diminished claudin-1 (CLDN1) expression. In DGAT1-silenced cell lines, the forced expression of CLDN1 restored HCV entry, implying that the downregulation of CLDN1 is a critical factor underlying defective HCV entry. The expression of the gene coding for hepatocyte nuclear factor 4aalpha; (HNF4aalpha;) and other hepatocyte-specific genes was also reduced in DGAT1-silenced cell lines. After DGAT1 gene rescue, CLDN1 expression was preserved, and HCV entry was restored. Strikingly, after DGAT1 silencing, CLDN1 expression and HCV entry were also restored by low-dose palmitic acid treatment, indicating that the downregulation of CLDN1 was associated with altered fatty acid homeostasis in the absence of DGAT1. Our findings provide novel insight into the role of DGAT1 in the life cycle of HCV.

    IMPORTANCE In this study, we report the novel effect of complete silencing of DGAT1 on the entry of HCV. DGAT1 was recently reported as a host factor of HCV, involved in the assembly of HCV by facilitating the trafficking of the HCV core protein to lipid droplets. We achieved complete and long-term silencing of DGAT1 by either TALEN or repeated transduction of lentivirus shRNA. We found that HCV entry was severely impaired in DGAT1-silenced cell lines. The impairment of HCV entry was caused by CLDN1 downregulation, and the expression of HNF4aalpha; and other hepatocyte-specific genes was also downregulated in DGAT1-silenced cell lines. Our results suggest new roles of DGAT1 in human liver-derived cells: maintaining intracellular lipid homeostasis and affecting HCV entry by modulating CLDN1 expression.

  • The Gammaherpesviruses Kaposi's Sarcoma-Associated Herpesvirus and Murine Gammaherpesvirus 68 Modulate the Toll-Like Receptor-Induced Proinflammatory Cytokine Response [Virus-Cell Interactions]

  • The human pathogen Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease, establishes lifelong latency upon infection. Murine gammaherpesvirus 68 (MHV68) is a well-established model for KSHV. Toll-like receptors (TLRs) play a crucial role for the innate immune response to pathogens. Although KSHV and MHV68 are detected by TLRs, studies suggest they modulate TLR4 and TLR9 signaling, respectively. In this study, we show that in bone marrow-derived macrophages (BMDMs), MHV68 did not induce a detectable proinflammatory cytokine response. Furthermore, MHV68 abrogated the response to TLR2, -4, -7, and -9 agonists in BMDMs. Similarly to observations with MHV68, infection with KSHV efficiently inhibited TLR2 signaling in THP-1 monocytes. Using a KSHV open reading frame (ORF) library, we found that K4.2, ORF21, ORF31, and the replication and transcription activator protein (RTA)/ORF50 inhibited TLR2-dependent nuclear factor kappa B (NF-B) activation in HEK293 TLR2-yellow fluorescent protein (YFP)- and Flag-TLR2-transfected HEK293T cells. Of the identified ORFs, RTA/ORF50 strongly downregulated TLR2 and TLR4 signaling by reducing TLR2 and TLR4 protein expression. Confocal microscopy revealed that TLR2 and TLR4 were no longer localized to the plasma membrane in cells expressing RTA/ORF50. In this study, we have shown that the gammaherpesviruses MHV68 and KSHV efficiently downmodulate TLR signaling in macrophages and have identified a novel function of RTA/ORF50 in modulation of the innate immune response.

    IMPORTANCE The Toll-like receptors (TLRs) are an important class of pattern recognition receptors of the innate immune system. They induce a potent proinflammatory cytokine response upon detection of a variety of pathogens. In this study, we found that the gammaherpesviruses murine gammaherpesvirus 68 (MHV68) and Kaposi's sarcoma-associated herpesvirus (KSHV) efficiently inhibit the TLR-mediated innate immune response. We further identified the KSHV-encoded replication and transcription activator protein (RTA) as a novel modulator of TLR signaling. Our data suggest that the gammaherpesviruses MHV68 and KSHV prevent activation of the innate immune response by targeting TLR signaling.

  • Western Equine Encephalitis Virus: Evolutionary Analysis of a Declining Alphavirus Based on Complete Genome Sequences [Genetic Diversity and Evolution]

  • Western equine encephalitis virus (WEEV) is an arbovirus from the genus Alphavirus, family Togaviridae, which circulates in North America between birds and mosquitoes, occasionally causing disease in humans and equids. In recent decades, human infection has decreased dramatically; the last documented human case in North America occurred in 1994, and the virus has not been detected in mosquito pools since 2008. Because limited information exists regarding the evolution of WEEV, we analyzed the genomic sequences of 33 low-passage-number strains with diverse geographic and temporal distributions and performed comprehensive phylogenetic analyses. Our results indicated that WEEV is a highly conserved alphavirus with only approximately 5% divergence in its most variable genes. We confirmed the presence of the previously determined group A and B lineages and further resolved group B into three sublineages. We also observed an increase in relative genetic diversity during the mid-20th century, which correlates with the emergence and cocirculation of several group B sublineages. The estimated WEEV population size dropped in the 1990s, with only the group B3 lineage being sampled in the past 20 years. Structural mapping showed that the majority of substitutions in the envelope glycoproteins occurred at the E2-E2 interface. We hypothesize that an event occurred in the mid-20th century that resulted in the increased genetic diversity of WEEV in North America, followed by genetic constriction due to either competitive displacement by the B3 sublineage or stochastic events resulting from a population decline.

    IMPORTANCE Western equine encephalitis virus (WEEV) has caused several epidemics that resulted in the deaths of thousands of humans and hundreds of thousands of equids during the past century. During recent decades, human infection decreased drastically and the virus has not been found in mosquito pools since 2008. Because limited information exists regarding the evolution of WEEV, we analyzed 33 complete genome sequences and conducted comprehensive phylogenetic analyses. We confirmed the presence of two major lineages, one of which diverged into three sublineages. Currently, only one of those sublineages is found circulating in nature. Understanding the evolution of WEEV over the past century provides a unique opportunity to observe an arbovirus that is in decline and to better understand what factors can cause said decline.

  • Altered Viral Fitness and Drug Susceptibility in HIV-1 Carrying Mutations That Confer Resistance to Nonnucleoside Reverse Transcriptase and Integrase Strand Transfer Inhibitors [Vaccines and Antiviral Agents]

  • Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTI) and integrase (IN) strand transfer inhibitors (INSTI) are key components of antiretroviral regimens. To explore potential interactions between NNRTI and INSTI resistance mutations, we investigated the combined effects of these mutations on drug susceptibility and fitness of human immunodeficiency virus type 1 (HIV-1). In the absence of drug, single-mutant viruses were less fit than the wild type; viruses carrying multiple mutations were less fit than single-mutant viruses. These findings were explained in part by the observation that mutant viruses carrying NNRTI plus INSTI resistance mutations had reduced amounts of virion-associated RT and/or IN protein. In the presence of efavirenz (EFV), a virus carrying RT-K103N together with IN-G140S and IN-Q148H (here termed IN-G140S/Q148H) mutations was fitter than a virus with a RT-K103N mutation alone. Similarly, in the presence of EFV, the RT-E138K plus IN-G140S/Q148H mutant virus was fitter than one with the RT-E138K mutation alone. No effect of INSTI resistance mutations on the fitness of RT-Y181C mutant viruses was observed. Conversely, RT-E138K and -Y181C mutations improved the fitness of the IN-G140S/Q148H mutant virus in the presence of raltegravir (RAL); the RT-K103N mutation had no effect. The NNRTI resistance mutations had no effect on RAL susceptibility. Likewise, the IN-G140S/Q148H mutations had no effect on EFV or RPV susceptibility. However, both the RT-K103N plus IN-G140S/Q148H and the RT-E138K plus IN-G140S/Q148H mutant viruses had significantly greater fold increases in 50% inhibitory concentration (IC50) of EFV than viruses carrying a single NNRTI mutation. Likewise, the RT-E138K plus IN-G140S/Q148H mutant virus had significantly greater fold increases in RAL IC50 than that of the IN-G140S/Q148H mutant virus. These results suggest that interactions between RT and IN mutations are important for NNRTI and INSTI resistance and viral fitness.

    IMPORTANCE Nonnucleoside reverse transcriptase inhibitors and integrase inhibitors are used to treat infection with HIV-1. Mutations that confer resistance to these drugs reduce the ability of HIV-1 to reproduce (that is, they decrease viral fitness). It is known that reverse transcriptase and integrase interact and that some mutations can disrupt their interaction, which is necessary for proper functioning of these two enzymes. To determine whether resistance mutations in these enzymes interact, we investigated their effects on drug sensitivity and viral fitness. Although individual drug resistance mutations usually reduced viral fitness, certain combinations of mutations increased fitness. When present in certain combinations, some integrase inhibitor resistance mutations increased resistance to nonnucleoside reverse transcriptase inhibitors and vice versa. Because these drugs are sometimes used together in the treatment of HIV-1 infection, these interactions could make viruses more resistant to both drugs, further limiting their clinical benefit.

  • ISG15 Functions as an Interferon-Mediated Antiviral Effector Early in the Murine Norovirus Life Cycle [Pathogenesis and Immunity]

  • Human noroviruses (HuNoV) are the leading cause of nonbacterial gastroenteritis worldwide. Similar to HuNoV, murine noroviruses (MNV) are enteric pathogens spread via the fecal-oral route and have been isolated from numerous mouse facilities worldwide. Type I and type II interferons (IFN) restrict MNV-1 replication; however, the antiviral effectors impacting MNV-1 downstream of IFN signaling are largely unknown. Studies using dendritic cells, macrophages, and mice deficient in free and conjugated forms of interferon-stimulated gene 15 (ISG15) revealed that ISG15 conjugation contributes to protection against MNV-1 both in vitro and in vivo. ISG15 inhibited a step early in the viral life cycle upstream of viral genome transcription. Directly transfecting MNV-1 RNA into IFN-stimulated mouse embryonic fibroblasts (MEFs) and bone marrow-derived dendritic cells (BMDC) lacking ISG15 conjugates bypassed the antiviral activity of ISG15, further suggesting that ISG15 conjugates restrict the MNV-1 life cycle at the viral entry/uncoating step. These results identify ISG15 as the first type I IFN effector regulating MNV-1 infection both in vitro and in vivo and for the first time implicate the ISG15 pathway in the regulation of early stages of MNV-1 replication.

    IMPORTANCE Type I IFNs are important in controlling murine norovirus 1 (MNV-1) infections; however, the proteins induced by IFNs that restrict viral growth are largely unknown. This report reveals that interferon-stimulated gene 15 (ISG15) mitigates MNV-1 replication both in vitro and in vivo. In addition, it shows that ISG15 inhibits MNV-1 replication by targeting an early step in the viral life cycle, MNV-1 entry and/or uncoating. These results identify ISG15 as the first type I IFN effector regulating MNV-1 infection both in vitro and in vivo and for the first time implicate the ISG15 pathway in the regulation of viral entry/uncoating.

  • Isolation and Characterization of the DNA and Protein Binding Activities of Adenovirus Core Protein V [Structure and Assembly]

  • The structure of adenovirus outer capsid was revealed recently at 3- to 4-AAring; resolution (V. Reddy, S. Natchiar, P. Stewart, and G. Nemerow, Science 329:1071nndash;1075, 2010,; however, precise details on the function and biochemical and structural features for the inner core still are lacking. Protein V is one the most important components of the adenovirus core, as it links the outer capsid via association with protein VI with the inner DNA core. Protein V is a highly basic protein that strongly binds to DNA in a nonspecific manner. We report the expression of a soluble protein V that exists in monomer-dimer equilibrium. Using reversible cross-linking affinity purification in combination with mass spectrometry, we found that protein V contains multiple DNA binding sites. The binding sites from protein V mediate heat-stable nucleic acid associations, with some of the binding sites possibly masked in the virus by other core proteins. We also demonstrate direct interaction between soluble proteins V and VI, thereby revealing the bridging of the inner DNA core with the outer capsid proteins. These findings are consistent with a model of nucleosome-like structures proposed for the adenovirus core and encapsidated DNA. They also suggest an additional role for protein V in linking the inner nucleic acid core with protein VI on the inner capsid shell.

    IMPORTANCE Scant knowledge exists of how the inner core of adenovirus containing its double-stranded DNA (dsDNA) genome and associated proteins is organized. Here, we report a purification scheme for a recombinant form of protein V that allowed analysis of its interactions with the nucleic acid core region. We demonstrate that protein V exhibits stable associations with dsDNA due to the presence of multiple nucleic acid binding sites identified both in the isolated recombinant protein and in virus particles. As protein V also binds to the membrane lytic protein VI molecules, this core protein may serve as a bridge from the inner dsDNA core to the inner capsid shell.

  • Identification of B Cells as a Major Site for Cyprinid Herpesvirus 3 Latency [Virus-Cell Interactions]

  • Cyprinid herpesvirus 3 (CyHV-3), commonly known as koi herpesvirus (KHV), is a member of the Alloherpesviridae, and is a recently discovered emerging herpesvirus that is highly pathogenic for koi and common carp. Our previous study demonstrated that CyHV-3 becomes latent in peripheral white blood cells (WBC). In this study, CyHV-3 latency was further investigated in IgM+ WBC. The presence of the CyHV-3 genome in IgM+ WBC was about 20-fold greater than in IgMnndash; WBC. To determine whether CyHV-3 expressed genes during latency, transcription from all eight open reading frames (ORFs) in the terminal repeat was investigated in IgM+ WBC from koi with latent CyHV-3 infection. Only a spliced ORF6 transcript was found to be abundantly expressed in IgM+ WBC from CyHV-3 latently infected koi. The spliced ORF6 transcript was also detected in vitro during productive infection as early as 1 day postinfection. The ORF6 transcript from in vitro infection begins at nndash;127 bp upstream of the ATG codon and ends +188 bp downstream of the stop codon, +20 bp downstream of the polyadenylation signal. The hypothetical protein of ORF6 contains a consensus sequence with homology to a conserved domain of EBNA-3B and ICP4 from Epstein-Barr virus and herpes simplex virus 1, respectively, both members of the Herpesviridae. This is the first report of latent CyHV-3 in B cells and identification of gene transcription during latency for a member of the Alloherpesviridae.

    IMPORTANCE This is the first demonstration that a member of the Alloherpesviridae, cyprinid herpesvirus 3 (CyHV-3), establishes a latent infection in the B cells of its host, Cyprinus carpio. In addition, this is the first report of identification of gene transcription during latency for a member of Herpesvirales outside Herpesviridae. This is also the first report that the hypothetical protein of latent transcript of CyHV-3 contains a consensus sequence with homology to a conserved domain of EBNA-3B from Epstein-Barr virus and ICP4 from herpes simplex virus 1, which are genes important for latency. These strongly suggest that latency is evolutionally conserved across vertebrates.

  • Limited Dissemination and Shedding of the UL128 Complex-Intact, UL/b'-Defective Rhesus Cytomegalovirus Strain 180.92 [Pathogenesis and Immunity]

  • The UL128 complex of human cytomegalovirus (CMV) is a major determinant of viral entry into epithelial and endothelial cells and a target for vaccine development. The UL/b' region of rhesus CMV contains several open reading frames, including orthologs of the UL128 complex. We recently showed that the coding content of the rhesus CMV (RhCMV) UL/b' region predicts acute endothelial tropism and long-term shedding in vivo in the rhesus macaque model of CMV infection. The laboratory-passaged RhCMV 180.92 strain has a truncated UL/b' region but an intact UL128 complex. To investigate whether the presence of the UL128 complex alone was sufficient to confer endothelial and epithelial tropism in vivo, we investigated tissue dissemination and viral excretion following experimental RhCMV 180.92 inoculation of RhCMV-seronegative rhesus macaques. We show the presence of at least two virus variants in the RhCMV 180.92 infectious virus stock. A rare variant noted for a nontruncated wild-type-virus-like UL/b' region, rapidly emerged during in vivo replication and showed high-level replication in blood and tissues and excretion in urine and saliva, features similar to those previously reported in naturally occurring wild-type RhCMV infection. In contrast, the predominant truncated version of RhCMV 180.92 showed significantly lower plasma DNAemia and limited tissue dissemination and viral shedding. These data demonstrate that the truncated RhCMV 180.92 variant is attenuated in vivo and suggest that additional UL/b' genes, besides the UL128 complex, are required for optimal in vivo CMV replication and dissemination.

    IMPORTANCE An effective vaccine against human CMV infection will need to target genes that are essential for virus propagation and transmission. The human CMV UL128 complex represents one such candidate antigen since it is essential for endothelial and epithelial cell tropism, and is a target for neutralizing antibodies in CMV-infected individuals. In this study, we used the rhesus macaque animal model of CMV infection to investigate the in vivo function of the UL128 complex. Using experimental infection of rhesus macaques with a rhesus CMV virus variant that contained an intact UL128 complex but was missing several other genes, we show that the presence of the UL128 complex alone is not sufficient for widespread tissue dissemination and virus excretion. These data highlight the importance of in vivo studies in evaluating human CMV gene function and suggest that additional UL/b' genes are required for optimal CMV dissemination and transmission.

  • Oncolytic Activity of Avian Influenza Virus in Human Pancreatic Ductal Adenocarcinoma Cell Lines [Virus-Cell Interactions]

  • Pancreatic ductal adenocarcinoma (PDA) is the most lethal form of human cancer, with dismal survival rates due to late-stage diagnoses and a lack of efficacious therapies. Building on the observation that avian influenza A viruses (IAVs) have a tropism for the pancreas in vivo, the present study was aimed at testing the efficacy of IAVs as oncolytic agents for killing human PDA cell lines. Receptor characterization confirmed that human PDA cell lines express the alpha-2,3- and the alpha-2,6-linked glycan receptor for avian and human IAVs, respectively. PDA cell lines were sensitive to infection by human and avian IAV isolates, which is consistent with this finding. Growth kinetic experiments showed preferential virus replication in PDA cells over that in a nontransformed pancreatic ductal cell line. Finally, at early time points posttreatment, infection with IAVs caused higher levels of apoptosis in PDA cells than gemcitabine and cisplatin, which are the cornerstone of current therapies for PDA. In the BxPC-3 PDA cell line, apoptosis resulted from the engagement of the intrinsic mitochondrial pathway. Importantly, IAVs did not induce apoptosis in nontransformed pancreatic ductal HPDE6 cells. Using a model based on the growth of a PDA cell line as a xenograft in SCID mice, we also show that a slightly pathogenic avian IAV significantly inhibited tumor growth following intratumoral injection. Taken together, these results are the first to suggest that IAVs may hold promise as future agents of oncolytic virotherapy against pancreatic ductal adenocarcinomas.

    IMPORTANCE Despite intensive studies aimed at designing new therapeutic approaches, PDA still retains the most dismal prognosis among human cancers. In the present study, we provide the first evidence indicating that avian IAVs of low pathogenicity display a tropism for human PDA cells, resulting in viral RNA replication and a potent induction of apoptosis in vitro and antitumor effects in vivo. These results suggest that slightly pathogenic IAVs may prove to be effective for oncolytic virotherapy of PDA and provide grounds for further studies to develop specific and targeted viruses, with the aim of testing their efficacy in clinical contexts.

  • Kaposi's Sarcoma-Associated Herpesvirus K3 and K5 Ubiquitin E3 Ligases Have Stage-Specific Immune Evasion Roles during Lytic Replication [Virus-Cell Interactions]

  • The downregulation of immune synapse components such as major histocompatibility complex class I (MHC-I) and ICAM-1 is a common viral immune evasion strategy that protects infected cells from targeted elimination by cytolytic effector functions of the immune system. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two membrane-bound ubiquitin E3 ligases, called K3 and K5, which share the ability to induce internalization and degradation of MHC-I molecules. Although individual functions of K3 and K5 outside the viral genome are well characterized, their roles during the KSHV life cycle are still unclear. In this study, we individually introduced the amino acid-coding sequences of K3 or K5 into a K3 K5 recombinant virus, at either original or interchanged genomic positions. Recombinants harboring coding sequences within the K5 locus showed higher K3 and K5 protein expression levels and more rapid surface receptor downregulation than cognate recombinants in which coding sequences were introduced into the K3 locus. To identify infected cells undergoing K3-mediated downregulation of MHC-I, we employed a novel reporter virus, called red-green-blue-BAC16 (RGB-BAC16), which was engineered to harbor three fluorescent protein expression cassettes: EF1aalpha;-monomeric red fluorescent protein 1 (mRFP1), polyadenylated nuclear RNA promoter (pPAN)-enhanced green fluorescent protein (EGFP), and pK8.1-monomeric blue fluorescent protein (tagBFP), marking latent, immediate early, and late viral gene expression, respectively. Analysis of RGB-derived K3 and K5 deletion mutants showed that while the K5-mediated downregulation of MHC-I was concomitant with pPAN induction, the reduction of MHC-I surface expression by K3 was evident in cells that were enriched for pPAN-driven EGFPhigh and pK8.1-driven blue fluorescent protein-positive (BFP+) populations. These data support the notion that immunoreceptor downregulation occurs by a sequential process wherein K5 is critical during the immediately early phase and K3 plays a significant role during later stages.

    IMPORTANCE Although the roles of K3 and K5 outside the viral genome are well characterized, the function of these proteins in the context of the KSHV life cycle has remained unclear, particularly in the case of K3. This study examined the relative contributions of K3 and K5 to the downregulation of MHC-I during the lytic replication of KSHV. We show that while K5 acts immediately upon entry into the lytic phase, K3-mediated downregulation of MHC-I was evident during later stages of lytic replication. The identification of distinctly timed K3 and K5 activities significantly advances our understanding of KSHV-mediated immune evasion. Crucial to this study was the development of a novel recombinant KSHV, called RGB-BAC16, which facilitated the delineation of stage-specific phenotypes.

  • Limited Type I Interferons and Plasmacytoid Dendritic Cells during Neonatal Respiratory Syncytial Virus Infection Permit Immunopathogenesis upon Reinfection [Pathogenesis and Immunity]

  • Respiratory syncytial virus (RSV) infection is the number one cause of bronchiolitis in infants, yet no vaccines are available because of a lack of knowledge of the infant immune system. Using a neonatal mouse model, we previously revealed that mice initially infected with RSV as neonates develop Th2-biased immunopathophysiologies during reinfection, and we demonstrated a role for enhanced interleukin-4 receptor aalpha; (IL-4Raalpha;) expression on T helper cells in these responses. Here we show that RSV infection in neonates induced limited type I interferon (IFN) and plasmacytoid dendritic cell (pDC) responses. IFN alpha (IFN-aalpha;) treatment or adoptive transfer of adult pDCs capable of inducing IFN-aalpha; prior to neonatal RSV infection decreased Th2-biased immunopathogenesis during reinfection. A reduced viral load and downregulation of IL-4Raalpha; on Th2 cells were observed in IFN-aalpha;-treated neonatal mice, suggesting dual mechanisms of action.

    IMPORTANCE Respiratory syncytial virus (RSV) is the most significant cause of lower respiratory tract infection in infancy worldwide. Despite the dire need, we have failed to produce efficacious RSV vaccines or therapeutics. Part of the reason for this failure is our lack of understanding of how RSV interacts with the infant immune system to suppress the development of protective immunity. In the study described in the present paper, we used a neonatal mouse model, which more closely mimics human infants, to study the role of the innate immune system, particularly type I interferons (IFNs) and plasmacytoid dendritic cells (pDCs), in the pathogenesis of RSV infection. RSV infection in neonates induced limited type I IFN and pDC responses. IFN-aalpha; treatment or adoptive transfer of adult pDCs capable of producing IFN-aalpha; prior to neonatal RSV infection decreased Th2-biased immunopathogenesis during reinfection. These data suggest that IFN-aalpha; is a promising target for future RSV vaccine design.

  • The Hop-Like Stress-Induced Protein 1 Cochaperone Is a Novel Cell-Intrinsic Restriction Factor for Mitochondrial Tombusvirus Replication [Virus-Cell Interactions]

  • Recent genome-wide screens reveal that the host cells express an arsenal of proteins that inhibit replication of plus-stranded RNA viruses by functioning as cell-intrinsic restriction factors of viral infections. One group of cell-intrinsic restriction factors against tombusviruses contains tetratricopeptide repeat (TPR) domains that directly interact with the viral replication proteins. In this paper, we find that the TPR domain-containing Hop-like stress-inducible protein 1 (Sti1p) cochaperone selectively inhibits the mitochondrial membrane-based replication of Carnation Italian ringspot tombusvirus (CIRV). In contrast, Sti1/Hop does not inhibit the peroxisome membrane-based replication of the closely related Tomato bushy stunt virus (TBSV) or Cucumber necrosis virus (CNV) in a yeast model or in plants. Deletion of STI1 in yeast leads to up to a 4-fold increase in CIRV replication, and knockdown of the orthologous Hop cochaperone in plants results in a 3-fold increase in CIRV accumulation. Overexpression of Sti1p derivatives in yeast reveals that the inhibitory function depends on the TPR1 domain known to interact with heat shock protein 70 (Hsp70), but not on the TPR2 domain interacting with Hsp90. In vitro CIRV replication studies based on isolated mitochondrial preparations and purified recombinant proteins has confirmed that Sti1p, similar to the TPR-containing Cyp40-like Cpr7p cyclophilin and the Ttc4 oncogene-like Cns1 cochaperone, is a strong inhibitor of CIRV replication. Sti1p interacts and colocalizes with the CIRV replication proteins in yeast. Our findings indicate that the TPR-containing Hop/Sti1 cochaperone could act as a cell-intrinsic virus restriction factor of the mitochondrial CIRV, but not against the peroxisomal tombusviruses in yeast and plants.

    IMPORTANCE The host cells express various cell-intrinsic restriction factors that inhibit the replication of plus-stranded RNA viruses. In this paper, the authors find that the Hop-like stress-inducible protein 1 (Sti1p) cochaperone selectively inhibits the mitochondrial membrane-based replication of Carnation Italian ringspot tombusvirus (CIRV) in yeast. Deletion of STI1 in yeast or knockdown of the orthologous Hop cochaperone in plants leads to increased CIRV replication. In addition, overexpression of Sti1p derivatives in yeast reveals that the inhibitory function depends on the TPR1 domain known to interact with heat shock protein 70 (Hsp70), but not on the TPR2 domain interacting with Hsp90. In vitro CIRV replication studies based on isolated mitochondrial preparations and purified recombinant proteins have confirmed that Sti1p is a strong inhibitor of CIRV replication. The authors' findings reveal that the Hop/Sti1 cochaperone could act as a cell-intrinsic restriction factor against the mitochondrial CIRV, but not against the related peroxisomal tombusviruses.

  • Sumoylation of Influenza A Virus Nucleoprotein Is Essential for Intracellular Trafficking and Virus Growth [Virus-Cell Interactions]

  • Viruses take advantage of host posttranslational modifications for their own benefit. It was recently reported that influenza A virus proteins interact extensively with the host sumoylation system. Thereby, several viral proteins, including NS1 and M1, are sumoylated to facilitate viral replication. However, to what extent sumoylation is exploited by influenza A virus is not fully understood. In this study, we found that influenza A virus nucleoprotein (NP) is a bona fide target of sumoylation in both NP-transfected cells and virus-infected cells. We further found that NP is sumoylated at the two most N-terminal residues, lysines 4 and 7, and that sumoylation at lysine 7 of NP is highly conserved across different influenza A virus subtypes and strains, including the recently emerged human H7N9 virus. While NP stability and polymerase activity are little affected by sumoylation, the NP sumoylation-defective WSN-NPK4,7R virus exhibited early cytoplasmic localization of NP. The growth of the WSN-NPK4,7R virus was highly attenuated compared to that of the wild-type WSN virus, and the lysine residue at position 7 is indispensable for the virus's survival, as illustrated by the rapid emergence of revertant viruses. Thus, sumoylation of influenza A virus NP is essential for intracellular trafficking of NP and for virus growth, illustrating sumoylation as a crucial strategy extensively exploited by influenza A virus for survival in its host.

    IMPORTANCE Host posttranslational modifications are heavily targeted by viruses for their own benefit. We and others previously reported that influenza A virus interacts extensively with the host sumoylation system. However, the functional outcomes of viral sumoylation are not fully understood. Here we found that influenza A virus nucleoprotein (NP), an essential component for virus replication, is a new target of SUMO. This is the first study to find that NP from different influenza A viruses, including recently emerged H7N9, is sumoylated at conserved lysine 7. Our data further illustrated that sumoylation of influenza A virus NP is essential for intracellular trafficking of NP and virus growth, indicating that influenza A virus relies deeply on sumoylation to survive in host cells. Strategies to downregulate viral sumoylation could thus be a potential antiviral treatment.

  • Human Cytomegalovirus Modulates Monocyte-Mediated Innate Immune Responses during Short-Term Experimental Latency In Vitro [Pathogenesis and Immunity]

  • The ability of human cytomegalovirus (HCMV) to establish lifelong persistence and reactivate from latency is critical to its success as a pathogen. Here we describe a short-term in vitro model representing the events surrounding HCMV latency and reactivation in circulating peripheral blood monocytes that was developed in order to study the immunological consequence of latent virus carriage. Infection of human CD14+ monocytes by HCMV resulted in the immediate establishment of latency, as evidenced by the absence of particular lytic gene expression, the transcription of latency-associated mRNAs, and the maintenance of viral genomes. Latent HCMV induced cellular differentiation to a macrophage lineage, causing production of selective proinflammatory cytokines and myeloid-cell chemoattractants that most likely play a role in virus dissemination in the host. Analysis of global cellular gene expression revealed activation of innate immune responses and the modulation of protein and lipid synthesis to accommodate latent HCMV infection. Remarkably, monocytes harboring latent virus exhibited selective responses to secondary stimuli known to induce an antiviral state. Furthermore, when challenged with type I and II interferon, latently infected cells demonstrated a blockade of signaling at the level of STAT1 phosphorylation. The data demonstrate that HCMV reprograms specific cellular pathways in monocytes, most notably innate immune responses, which may play a role in the establishment of, maintenance of, and reactivation from latency. The modulation of innate immune responses is likely a viral evasion strategy contributing to viral dissemination and pathogenesis in the host.

    IMPORTANCE HCMV has the ability to establish a lifelong infection within the host, a phenomenon termed latency. We have established a short-term model system in human peripheral blood monocytes to study the immunological relevance of latent virus carriage. Infection of CD14+ monocytes by HCMV results in the generation of latency-specific transcripts, maintenance of viral genomes, and the capacity to reenter the lytic cycle. During short-term latency in monocytes the virus initiates a program of differentiation to inflammatory macrophages that coincides with the modulation of cytokine secretion and specific cellular processes. HCMV-infected monocytes are hindered in their capacity to exert normal immunoprotective mechanisms. Additionally, latent virus disrupts type I and II interferon signaling at the level of STAT1 phosphorylation. This in vitro model system can significantly contribute to our understanding of the molecular and inflammatory factors that initiate HCMV reactivation in the host and allow the development of strategies to eradicate virus persistence.

  • Vaccine-Induced HIV-1 Envelope gp120 Constant Region 1-Specific Antibodies Expose a CD4-Inducible Epitope and Block the Interaction of HIV-1 gp140 with Galactosylceramide [Virus-Cell Interactions]

  • Mucosal epithelial cell surface galactosylceramide (Galcer) has been postulated to be a receptor for HIV-1 envelope (Env) interactions with mucosal epithelial cells. Disruption of the HIV-1 Env interaction with such alternate receptors could be one strategy to prevent HIV-1 entry through the mucosal barrier. To study antibody modulation of HIV-1 Env-Galcer interactions, we used Galcer-containing liposomes to assess whether natural- and vaccine-induced monoclonal antibodies can block HIV-1 Env binding to Galcer. HIV-1 Env gp140 proteins bound to Galcer liposomes with Kds (dissociation constants) in the nanomolar range. Several HIV-1 ALVAC/AIDSVAX vaccinee-derived monoclonal antibodies (MAbs) specific for the gp120 first constant (C1) region blocked Galcer binding of a transmitted/founder HIV-1 Env gp140. Among the C1-specific MAbs that showed Galcer blocking, the antibody-dependent cellular cytotoxicity-mediating CH38 IgG and its natural IgA isotype were the most potent blocking antibodies. C1-specific IgG monoclonal antibodies that blocked Env binding to Galcer induced upregulation of the gp120 CD4-inducible (CD4i) epitope bound by MAb 17B, demonstrating that a conformational change in gp120 may be required for Galcer blocking. However, the MAb 17B itself did not block Env-Galcer binding, suggesting that the C1 antibody-induced gp120 conformational changes resulted in alteration in a Galcer binding site distant from the CD4i 17B MAb binding site.

    IMPORTANCE Galactosyl ceramide, a glycosphingolipid, has been postulated to be a receptor for the HIV-1 envelope glycoprotein (Env) interaction with mucosal epithelial cells. Here, we have mimicked this interaction by using an artificial membrane containing synthetic Galcer and recombinant HIV-1 Env proteins to identify antibodies that would block the HIV-1 Env-Galcer interaction. Our study revealed that a class of vaccine-induced human antibodies potently blocks HIV-1 Env-Galcer binding by perturbing the HIV-1 Env conformation.

  • Human and Host Species Transferrin Receptor 1 Use by North American Arenaviruses [Virus-Cell Interactions]

  • At least five New World (NW) arenaviruses cause hemorrhagic fevers in South America. These pathogenic clade B viruses, as well as nonpathogenic arenaviruses of the same clade, use transferrin receptor 1 (TfR1) of their host species to enter cells. Pathogenic viruses are distinguished from closely related nonpathogenic ones by their additional ability to utilize human TfR1 (hTfR1). Here, we investigate the receptor usage of North American arenaviruses, whose entry proteins share greatest similarity with those of the clade B viruses. We show that all six North American arenaviruses investigated utilize host species TfR1 orthologs and present evidence consistent with arenavirus-mediated selection pressure on the TfR1 of the North American arenavirus host species. Notably, one of these viruses, AV96010151, closely related to the prototype Whitewater Arroyo virus (WWAV), entered cells using hTfR1, consistent with a role for a WWAV-like virus in three fatal human infections whose causative agent has not been identified. In addition, modest changes were sufficient to convert hTfR1 into a functional receptor for most of these viruses, suggesting that a minor alteration in virus entry protein may allow these viruses to use hTfR1. Our data establish TfR1 as a cellular receptor for North American arenaviruses, highlight an "arms race" between these viruses and their host species, support the association of North American arenavirus with fatal human infections, and suggest that these viruses have a higher potential to emerge and cause human diseases than has previously been appreciated.

    IMPORTANCE hTfR1 use is a key determinant for a NW arenavirus to cause hemorrhagic fevers in humans. All known pathogenic NW arenaviruses are transmitted in South America by their host rodents. North American arenaviruses are generally considered nonpathogenic, but some of these viruses have been tentatively implicated in human fatalities. We show that these North American arenaviruses use the TfR1 orthologs of their rodent host species and identify TfR1 polymorphisms suggesting an ongoing "arms race" between these viruses and their hosts. We also show that a close relative of a North American arenavirus suggested to have caused human fatalities, the Whitewater Arroyo species complex virus AV96010151, uses human TfR1. Moreover, we present data that imply that modest changes in other North American arenaviruses might allow these viruses to infect humans. Collectively, our data suggest that North American arenaviruses have a higher potential to cause human disease than previously assumed.

  • Modulation of Kaposi's Sarcoma-Associated Herpesvirus Interleukin-6 Function by Hypoxia-Upregulated Protein 1 [Virus-Cell Interactions]

  • Kaposi's sarcoma-associated herpesvirus (KSHV, also called human herpesvirus 8) is linked to the development of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). KSHV expresses several proteins that modulate host cell signaling pathways. One of these proteins is viral interleukin-6 (vIL-6), which is a homolog of human IL-6 (hIL-6). vIL-6 is able to prevent apoptosis and promote proinflammatory signaling, angiogenesis, and cell proliferation. Although it can be secreted, vIL-6 is mainly an intracellular protein that is retained in the endoplasmic reticulum (ER). We performed affinity purification and mass spectrometry to identify novel vIL-6 binding partners and found that a cellular ER chaperone, hypoxia-upregulated protein 1 (HYOU1), interacts with vIL-6. Immunohistochemical staining reveals that both PEL and KS tumor tissues express significant amounts of HYOU1. We also show that HYOU1 increases endogenous vIL-6 protein levels and that HYOU1 facilitates vIL-6-induced JAK/STAT signaling, migration, and survival in endothelial cells. Furthermore, our data suggest that HYOU1 also modulates vIL-6's ability to induce CCL2, a chemokine involved in cell migration. Finally, we investigated the impact of HYOU1 on cellular hIL-6 signaling. Collectively, our data indicate that HYOU1 is important for vIL-6 function and may play a role in the pathogenesis of KSHV-associated cancers.

    IMPORTANCE KSHV vIL-6 is detectable in all KSHV-associated malignancies and promotes tumorigenesis and inflammation. We identified a cellular protein, called hypoxia-upregulated protein 1 (HYOU1), that interacts with KSHV vIL-6 and is present in KSHV-infected tumors. Our data suggest that HYOU1 facilitates the vIL-6-induced signaling, migration, and survival of endothelial cells.

  • Cytokine/Chemokine Responses in Activated CD4+ and CD8+ T Cells Isolated from Peripheral Blood, Bone Marrow, and Axillary Lymph Nodes during Acute Simian Immunodeficiency Virus Infection [Cellular Response to Infection]

  • Understanding the cytokine/chemokine networks in CD4+ and CD8+ T cells during the acute phase of infection is crucial to design therapies for the control of early human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) replication. Here, we measured early changes in CD4+ and CD8+ T cells in the peripheral blood (PB), bone marrow (BM), and axillary lymph node (ALN) tissue of rhesus macaques infected with SIVMAC251. At 21 days after infection, all tissues showed a statistically significant loss of CD4+ T cells along with immune activation of CD8+ T cells in PB and ALN tissue. Twenty-eight different cytokines/chemokines were quantified in either anti-CD3/28 antibody- or staphylococcal enterotoxin B-stimulated single-positive CD4+ and CD8+ T cells. PB CD4+ T cells produced predominantly interleukin-2 (IL-2), whereas CD4+ and CD8+ T-cell subsets in tissues produced bbeta;-chemokines both before and 21 days after SIV infection. Tissues generally exhibited massive upregulation of many cytokines/chemokines following infection, possibly in an attempt to mitigate the loss of CD4+ T cells. There was no evidence of a T-helper 1 (TH1)-to-TH2 shift in CD4+ T cells or a T-cytotoxic 1 (TC1)-to-TC2 cytokine shift in CD8+ T cells in PB, BM, and ALN T-cell subsets during the acute phase of SIV infection. Despite the upregulation of several important effector cytokines/chemokines (IL-2, IL-12, IL-17, gamma interferon, granulocyte-macrophage colony-stimulating factor) by CD4+ and CD8+ T cells, upregulation of bbeta;-chemokines (CCL2 and CCL22), basic fibroblast growth factor (FGF-basic), hepatocyte growth factor (HGF), and migration inhibition factor (MIF) may provide a poor prognosis either by inducing increased virus replication or by other unknown mechanisms. Therefore, drugs targeting bbeta;-chemokines (CCL2 and CCL22), FGF-basic, HGF, or MIF might be important for developing effective vaccines and therapeutics against HIV.

    IMPORTANCE Human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection results in early depletion of CD4+ T cells and dysregulation of protective immune responses. Therefore, understanding the cytokine/chemokine networks in CD4+ and CD8+ T cells in different tissues during the acute phase of infection is crucial to the design of therapies for the control of early viral replication. Here, we measured early changes in CD4+ and CD8+ T cells in peripheral blood (PB), bone marrow (BM), and axillary lymph node (ALN) tissue of rhesus macaques infected with SIVMAC251. There was no evidence of a T-helper 1 (TH1)-to-TH2 shift in CD4+ T cells or a T-cytotoxic 1 (TC1)-to-TC2 cytokine shift in CD8+ T cells in PB, BM, and ALN T-cell subsets during the acute phase of SIV infection. Despite the upregulation of several important effector cytokines/chemokines by CD4+ and CD8+ T cells, upregulation of bbeta;-chemokines, fibroblast growth factor-basic, hepatocyte growth factor, and migration inhibition factor may provide a poor prognosis.

  • West Nile Virus-Induced Activation of Mammalian Target of Rapamycin Complex 1 Supports Viral Growth and Viral Protein Expression [Virus-Cell Interactions]

  • Since its introduction in New York City, NY, in 1999, West Nile virus (WNV) has spread to all 48 contiguous states of the United States and is now the leading cause of epidemic encephalitis in North America. As a member of the family Flaviviridae, WNV is part of a group of clinically important human pathogens, including dengue virus and Japanese encephalitis virus. The members of this family of positive-sense, single-stranded RNA viruses have limited coding capacity and are therefore obligated to co-opt a significant amount of cellular factors to translate their genomes effectively. Our previous work has shown that WNV growth was independent of macroautophagy activation, but the role of the evolutionarily conserved mammalian target of rapamycin (mTOR) pathway during WNV infection was not well understood. mTOR is a serine/threonine kinase that acts as a central cellular censor of nutrient status and exercises control of vital anabolic and catabolic cellular responses such as protein synthesis and autophagy, respectively. We now show that WNV activates mTOR and cognate downstream activators of cap-dependent protein synthesis at early time points postinfection and that pharmacologic inhibition of mTOR (KU0063794) significantly reduced WNV growth. We used an inducible Raptor and Rictor knockout mouse embryonic fibroblast (MEF) system to further define the role of mTOR complexes 1 and 2 in WNV growth and viral protein synthesis. Following inducible genetic knockout of the major mTOR cofactors raptor (TOR complex 1 [TORC1]) and rictor (TORC2), we now show that TORC1 supports flavivirus protein synthesis via cap-dependent protein synthesis pathways and supports subsequent WNV growth.

    IMPORTANCE Since its introduction in New York City, NY, in 1999, West Nile virus (WNV) has spread to all 48 contiguous states in the United States and is now the leading cause of epidemic encephalitis in North America. Currently, the mechanism by which flaviviruses such as WNV translate their genomes in host cells is incompletely understood. Elucidation of the host mechanisms required to support WNV genome translation will provide broad understanding for the basic mechanisms required to translate capped viral RNAs. We now show that WNV activates mTOR and cognate downstream activators of cap-dependent protein synthesis at early time points postinfection. Following inducible genetic knockout of the major mTOR complex cofactors raptor (TORC1) and rictor (TORC2), we now show that TORC1 supports WNV growth and protein synthesis. This study demonstrates the requirement for TORC1 function in support of WNV RNA translation and provides insight into the mechanisms underlying flaviviral RNA translation in mammalian cells.

  • Epithelial Immunization Induces Polyfunctional CD8+ T Cells and Optimal Mousepox Protection [Vaccines and Antiviral Agents]

  • We assessed several routes of immunization with vaccinia virus (VACV) in protecting mice against ectromelia virus (ECTV). By a wide margin, skin scarification provided the greatest protection. Humoral immunity and resident-memory T cells notwithstanding, several approaches revealed that circulating, memory CD8+ T cells primed via scarification were functionally superior and conferred enhanced virus control. Immunization via the epithelial route warrants further investigation, as it may also provide enhanced defense against other infectious agents.

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    JVI Accepts: Articles Published Ahead of Print

  • Comparison of Genotypes I and III in Japanese encephalitis virus reveal distinct differences in their genetic and host diversity [PublishAheadOfPrint]

  • Japanese encephalitis (JE) is an arthropod-borne disease associated with the majority of viral encephalitis cases in the Asia-Pacific region. The causative agent, Japanese encephalitis virus (JEV), has been phylogenetically divided into five genotypes. Recent surveillance data indicates that genotype I (GI) is gradually replacing genotype III (GIII) as the dominant genotype.

    To investigate the mechanism behind the genotype shift and the potential consequences in terms of vaccine efficacy, human cases and virus dissemination, we collected (i) all full length and partial JEV molecular sequences and (ii) associated genotype and host information comprising a dataset of 873 sequences. We then examined differences between the two genotypes at the genetic and epidemiological level by investigating amino acid mutations, positive selection and host range.

    We found that although GI is dominant, it has fewer sites predicted to be under positive selection, a narrower host range and significantly fewer human isolates. For the E protein, the sites under positive selection define a haplotype set for each genotype that shows striking differences in their composition and diversity, with GIII showing significantly more variety than GI. Our results suggest that GI has displaced GIII by achieving a replication cycle that is more efficient but is also more restricted in its host range.

    Importance Japanese encephalitis is an arthropod-borne disease associated with the majority of viral encephalitis cases in the Asia-Pacific region. The causative agent, Japanese encephalitis virus (JEV), has been divided into five genotypes based on sequence similarity. Recent data indicates that genotype I (GI) is gradually replacing genotype III (GIII) as the dominant genotype. Understanding the reasons behind this shift and the potential consequences in terms of vaccine efficacy, human cases and virus dissemination are important for controlling the spread of the virus and reducing human fatalities. We collected all available full length and partial JEV molecular sequences and associated genotype and host information. We then examined differences between the two genotypes at the genetic and epidemiological level by investigating amino acid mutations, positive selection and host range. Our results suggest that GI has displaced GIII by achieving a replication cycle that is more efficient but more restricted in host range

  • Inhibition of pSTAT1 Nuclear Translocation and Antiviral Protein Expression in Human Brain Vascular Adventitial Fibroblasts Infected with Varicella Zoster Virus [PublishAheadOfPrint]

  • In VZV-infected primary human brain vascular adventitial fibroblasts (BRAFs), IFN-bbeta;, STAT1 and STAT2 transcripts, as well as STAT1 and STAT2 protein were decreased. IFN-aalpha; transcripts were increased but not secreted IFN-aalpha; protein. Compared to IFN-aalpha;-treated controls, in VZV-infected BRAFs, phosphorylated STAT1 did not translocate to the nucleus, resulting in impaired downstream expression of interferon-inducible antiviral Mx1. Overall, VZV interference with the type I interferon pathway may promote virus persistence in cerebral arteries.

  • Careers in Virology: Teaching at a Primarily Undergraduate Institution [PublishAheadOfPrint]

  • A faculty position at a primarily undergraduate institution requires working with undergraduates both in the classroom and research lab. Graduate students and postdoctoral fellows who are interested in such a career should understand that faculty at these institutions need to teach broadly and devise research questions that can be addressed safely and with limited resources compared to a Research I university. Aspects of, and ways to prepare for, this career will be reviewed herein.

  • Virome analysis of Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis ticks reveals novel highly divergent vertebrate and invertebrate viruses [PublishAheadOfPrint]

  • A wide range of bacterial pathogens have been described in ticks, yet the diversity of viruses in ticks is largely unexplored. In the United States, Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis are among the principal tick species associated with pathogen transmission. We used high-throughput sequencing to characterize the viromes of these tick species and identified the presence of Powassan virus and eight novel viruses. These included the most divergent nairovirus described to date, two new clades of tick-borne phleboviruses, a mononegavirus, and viruses with similarity to plant and insect viruses. Our analysis revealed that ticks are reservoirs to a wide range of viruses and suggests that discovery and characterization of tick-borne viruses will have implications on viral taxonomy and may provide insight into tick-transmitted diseases.

    Importance Ticks are implicated as vectors of a wide array of human and animal pathogens. To better understand the extent of tick-borne diseases, it is crucial to uncover the full range of microbial agents associated with ticks. Our current knowledge of the diversity of tick-associated viruses is limited, in part due to the lack of investigation of tick viromes. In this study we examined the virome of three tick species from the United States. We found that ticks are hosts to highly divergent viruses across several taxa, including ones previously associated with human disease. Our data underscore the diversity of tick-associated viruses and provides the foundation for further studies into viral etiology of tick-borne diseases.

  • Short Self-interacting N-terminal Region of Rubella Virus Capsid Protein Is Essential for Cooperative Actions of Capsid and Nonstructural p150 Proteins [PublishAheadOfPrint]

  • Nucleocapsid formation is a primary function of the rubella virus capsid protein, which also promotes viral RNA synthesis via an unknown mechanism. The present study demonstrates that in infected cells, the capsid protein is associated with the nonstructural p150 protein via the short self-interacting N-terminal region of the capsid protein. Mutational analyses indicated that hydrophobic amino acids in this N-terminal region are essential for its N-terminal self-interaction, which is critical for the capsidmmdash;p150 association. An analysis based on a subgenomic replicon system demonstrated that the self-interacting N-terminal region of the capsid protein plays a key role in promoting viral gene expression. Analyses using a virus-like particle (VLP) system also showed that the self-interacting N-terminal region of the capsid protein is not essential for VLP production, but is critical for VLP infectivity. These results demonstrate that the close cooperative actions of the capsid protein and p150 require the short self-interacting N-terminal region of the capsid protein during the life cycle of the rubella virus.

    IMPORTANCE The capsid protein of rubella virus promotes viral RNA replication via an unknown mechanism. This protein interacts with the nonstructural protein p150, but the importance of this interaction is also unclear. In this study, we demonstrate that the short N-terminal region of the capsid protein forms a homo-oligomer that is critical for the capsidmmdash;p150 interaction. These interactions are required for the viral-gene-expression-promoting activity of the capsid protein, allowing efficient viral growth. These findings provide information about the mechanisms underlying the regulation of rubella virus RNA replication via the cooperative actions of the capsid protein and p150.

  • Characterization of the mode-of-action of a potent Dengue virus capsid inhibitor [PublishAheadOfPrint]

  • Dengue viruses (DV) represent a significant global health burden, with up to 400 million infections every year and around 500,000 infected individuals developing life-threatening disease. In spite of attempts in developing vaccine candidates and anti-viral drugs, there is currently a lack of approved therapeutics for the treatment of DV infection. We have previously reported the identification of ST-148, a small molecule inhibitor exhibiting a broad and potent antiviral activity against DV in vitro and in vivo. In the present study, we investigated the mode-of-action of this promising compound by using a combination of biochemical, virological and imaging-based techniques. We confirmed that ST-148 targets the capsid protein and obtained evidence of a bi-modal antiviral activity affecting both assembly/release and entry of infectious DV particles. Importantly, by using a robust bioluminescence resonance energy transfer-based assay we observed a ST-148-dependent increase of capsid self-interaction. These results were corroborated by molecular modeling studies that also revealed a plausible model for compound binding to capsid protein and inhibition by a distinct resistance mutation. These results suggest that ST-148-enhanced capsid protein self-interaction perturbs assembly and disassembly of DV nucleocapsids probably by inducing structural rigidity. Thus, as previously reported for other enveloped viruses, stabilization of capsid protein structure is an attractive therapeutic concept also applicable for flaviviruses.

    Importance Dengue viruses (DV) are arthropod-borne viruses representing a significant global health burden. They infect up to 400 million people and are endemic in subtropical and tropical areas of the world. Currently, there are neither vaccines nor approved therapeutics for the prophylaxis or the treatment of DV infections, respectively. This study reports the characterization of the mode-of-action of ST-148, a small-molecule capsid inhibitor with potent antiviral activity against all DV serotypes. Our results demonstrate that ST-148 stabilizes capsid protein self-interaction, thereby likely perturbing assembly and disassembly of viral nucleocapsids by inducing structural rigidity. This, in turn, might interfere with the release of viral RNA from incoming nucleocapsids (llsquo;uncoatingrrsquo;) as well as assembly of progeny virus particles. As previously reported for other enveloped viruses, we propose capsid as a novel tractable target for flavivirus inhibitors.

  • Antiviral Regulation in Porcine Monocytic Cells at Different Activation States [PublishAheadOfPrint]

  • Monocytic cells, including macrophages and dendritic cells, exist in different activation states that are critical to the regulation of antimicrobial immunity. Many pandemic viruses are monocytotropic, including porcine reproductive and respiratory syndrome virus (PRRSV), which directly infects subsets of monocytic cells and interferes in antiviral responses. To study antiviral responses in PRRSV-infected monocytic cells, we characterized inflammatory cytokine responses and genome-wide profiled signature genes to investigate response pathways in uninfected and PRRSV-infected monocytic cells at different activation states. Our findings showed suppressed interferon (IFN) production in macrophages at non-antiviral states and an arrest of lipid metabolic pathways in macrophages at antiviral states. Importantly, porcine monocytic cells at different activation states were susceptible to PRRSV and responded differently to viral infection. Based on gene ontology analysis, two approaches were used to potentiate antiviral activity: 1) pharmaceutical modulation of cellular lipid metabolism and 2) in situ PRRSV replication-competent expression of IFN-aalpha;; both approaches significantly suppressed exogenous viral infection in monocytic cells. In particular, the engineered IFN-expressing PRRSV strain eliminated exogenous virus infection and sustained cell viability at 4 days post infection in macrophages. These findings suggest an intricate interaction of viral infection with activation status of porcine monocytic cells. An understanding and integration of antiviral infection with activation status of monocytic cells may provide a means of potentiating antiviral immunity.

    IMPORTANCE: Activation statuses of monocytic cells including monocytes, macrophages (Ms) and dendritic cells (DCs) are critically important for antiviral immunity. Unfortunately, the activation status of porcine monocytic cells or how cell activation status functionally interacts with antiviral immunity remains largely unknown. This is a significant omission because many economically important porcine viruses are monocytotropic including our focus PRRSV, which alone causes near $800M economic loss annually in the US swine industries. PRRSV is ideal for deciphering how monocytic cell activation statuses interact with antiviral immunity, because it directly infects subsets of monocytic cells and subverts overall immune responses. In this study, we systematically investigate the activation status of porcine monocytic cells to determine intricate interaction of viral infection with activation statuses, and functionally regulate antiviral immunity within the framework of the activation paradigm. Our findings may provide a means of potentiating antiviral immunity and leading to novel vaccines for PRRS prevention.

  • Exosomes from Human Immunodeficiency virus type-1 (HIV-1)-infected cells license quiescent CD4+ T lymphocytes to replicate HIV-1 through a Nef- and ADAM17-dependent mechanism [PublishAheadOfPrint]

  • Resting CD4+ T lymphocytes resist HIV infection. Here, we provide evidence that exosomes from HIV-1 infected cells render resting human primary CD4+ T lymphocytes permissive to HIV-1 replication. These results were obtained with trans-well co-cultures of HIV-1 infected cells with quiescent CD4+ T lymphocytes in the presence of inhibitors of exosome release, and confirmed using exosomes purified from supernatants of HIV-1 infected primary CD4+ T lymphocytes. We found that the expression of HIV-1 Nef in exosome-producing cells is both necessary and sufficient for cell activation as well as HIV-1 replication in target CD4+ T lymphocytes. We also identified a Nef domain important for the effects we observed, i.e., the 62EEEE65 acidic cluster domain. In addition, we observed that ADAM17, i.e., a disintegrin and metalloprotease converting pro-TNFaalpha; in its mature form, associates with exosomes from HIV-1 infected cells, and plays a key role in the HIV-1 replication in quiescent CD4+ T lymphocytes. Treatment with an inhibitor of ADAM17 abolished both activation and HIV-1 replication in resting CD4+ T lymphocytes. TNFaalpha; is the downstream effector of ADAM17 since the treatment of resting lymphocytes with anti-TNFaalpha; antibodies blocked the HIV-1 replication. The here presented data are consistent with a model where Nef induces intercellular communication through exosomes to activate bystander quiescent CD4+ T lymphocytes thus stimulating viral spread.

    Importance Overall, our findings support the idea that HIV evolved to usurp the exosome-based intercellular communication network to favor its spread in infected host.

  • Stability determinants of Murine Cytomegalovirus long non-coding RNA7.2 [PublishAheadOfPrint]

  • Cytomegalovirus is a ubiquitous herpesvirus that persistently replicates in glandular epithelial tissue. Murine Cytomegalovirus expresses a 7.2 kb long non-coding RNA, (RNA7.2) that is a determinant of viral persistence in the salivary gland. RNA7.2 is an extremely long-lived intron, yet the basis of its stability is unknown. We present data that localize key sequence determinants of RNA stability to the 3rrsquo; end of RNA7.2, and suggest that stability is a result of sustained lariat conformation.

  • HIV-1 infection leads to increased transcription of HERV-K (HML-2) proviruses in vivo but not to increased virion production. [PublishAheadOfPrint]

  • Recent studies suggest that human endogenous retrovirus group K (HERV-K) provirus expression plays a role in the pathogenesis of HIV-1 infection. In particular, RNA from the HML-2 subgroup of HERV-K proviruses has been reported to be highly expressed at the cellular level and detectable in the plasma of HIV-1 infected patients, suggestive of virion production and, perhaps, replication. In this study, we developed an HML-2 specific quantitative PCR assay, which detects 51 of the 89 known HML-2 proviruses in the human genome. Plasma and peripheral blood mononuclear cells (PBMCs) from HIV-negative controls and HIV-1 infected patients were collected for analysis of HML-2 RNA expression. Contrary to previous reports, we did not detect high levels of HML-2 RNA in the plasma of HIV-1 infected patients, but did observe a significant increase of HML-2 RNA in total PBMCs as compared to HIV-negative controls. The level of HML-2 expression in PBMCs does not appear to be related to patient use of antiretrovirals or to HIV-1 plasma RNA, cellular RNA or cellular DNA levels. To investigate the source of HML-2 RNA expression, patient PBMCs were sorted into CD3+ CD4+, CD3+CD8+, CD3-CD14+ and CD3-CD20+ cell subsets and then analyzed for HML-2 RNA levels. No single cell subset was enriched for HML-2 RNA expression in HIV-1 infected patients, but there appears to be substantial variability in the level of HML-2 expression dependent on the cell type.

    Importance Here, we report that HERV-K (HML-2) proviruses are expressed at significantly higher levels in PBMCs from patients with HIV-1 infection compared to uninfected individuals. However, contrary to previous reports, this expression did not lead to detectable virions in the plasma of these patients. In addition, we found that HML-2 proviruses were expressed in multiple blood cell types from HIV-1 infected individuals, and the magnitude of HML-2 expression was not related to HIV-1 disease markers in this patient cohort. These findings may have implications for HML-2 based therapies targeting HIV-1 infection.

  • Virus-specific memory CD8 T cells provide substantial protection from lethal SARS-CoV infection [PublishAheadOfPrint]

  • The Severe Acute Respiratory Syndrome-coronavirus (SARS-CoV) caused an acute human respiratory illness with high morbidity and mortality in 2002-2003. Several studies have demonstrated the role of neutralizing antibodies induced by the spike (S) glycoprotein in protecting susceptible hosts from lethal infection. However, the anti-SARS-CoV antibody response is short-lived in SARS-recovered patients making it critical to develop additional vaccine strategies. SARS-CoV-specific memory CD8 T cells persisted for up to six years after SARS-CoV infection, a time at which memory B cells and anti-virus antibodies were undetectable in SARS-recovered individuals. Here, we assessed the ability of virus-specific memory CD8 T cells to mediate protection against infection in the absence of SARS-CoV-specific memory CD4 T or B cells. We demonstrate that memory CD8 T cells specific for a single immunodominant epitope (S436 or S525) substantially protected 8-10 month old mice from lethal SARS-CoV infection. Intravenous immunization with peptide-loaded dendritic cells (DCs) followed by intranasal boosting with recombinant vaccinia virus (rVV) encoding S436 or S525 resulted in accumulation of virus-specific memory CD8 T cells in alveolar lavage fluid (BAL), lungs and spleen. Upon challenge with a lethal dose of SARS-CoV, virus-specific memory CD8 T cells efficiently produced multiple effector cytokines (IFN-, TNF-aalpha; and IL-2) and cytolytic molecules (granzyme B) and reduced lung viral loads. Overall, our results show that SARS-CoV-specific memory CD8 T cells protect susceptible hosts from lethal SARS-CoV infection, but also suggest that SARS-CoV specific CD4 T cell and antibody responses are necessary for complete protection.

    IMPORTANCE: Virus-specific CD8 T cells are required for pathogen clearance following primary SARS-CoV infection. However, the role of SARS-CoV-specific memory CD8 T cells in mediating protection after SARS-CoV challenge has not been previously investigated. Here, using a prime-boost immunization approach, we show that virus-specific CD8 T cells protect susceptible 8-10 month old mice from lethal SARS-CoV challenge. Thus, future vaccines against emerging coronaviruses should emphasize the generation of a memory CD8 T cell response for optimal protection.

  • Evasion of siRNA-directed antiviral silencing in Musa acuminata persistently infected with six distinct banana streak pararetroviruses [PublishAheadOfPrint]

  • Vegetatively-propagated crop plants often suffer from infections with persistent RNA and DNA viruses. Such viruses appear to evade the plant defenses that normally restrict viral replication and spread. The major antiviral defense mechanism is based on RNA silencing generating viral short interfering RNAs (siRNAs) that can potentially repress viral genes post-transcriptionally through RNA cleavage and transcriptionally through DNA cytosine methylation. Here we examined the RNA silencing machinery of banana plants persistently infected with six pararetroviruses after many years of vegetative propagation. Using deep sequencing, we reconstructed consensus master genomes of the viruses and characterized virus-derived and endogenous small RNAs. Consistent with the presence of endogenous siRNAs that can potentially establish and maintain DNA methylation, the banana genomic DNA was extensively methylated in both healthy and virus-infected plants. A novel class of abundant 20-nucleotide (nt) endogenous small RNAs with 5rrsquo; -terminal guanosine was identified. In all virus-infected plants, 21-24 nt viral siRNAs accumulated at relatively high levels (up to 22% of total small RNA population) and covered the entire circular viral DNA genomes in both orientations. The hotspots of 21-nt and 22-nt siRNAs occurred within ORFs I, II and 5rrsquo; -portion of ORF III, while 24-nt siRNAs were more evenly distributed along the viral genome. Despite the presence of abundant viral siRNAs of different size-classes, the viral DNA was largely free of cytosine methylation. Thus, the virus is able to evade siRNA-directed DNA methylation and thereby avoid transcriptional silencing. This evasion of silencing likely contributes to the persistency of pararetroviruses in banana plants.

    Importance We report that DNA pararetroviruses in Musa acuminata banana plants are able to evade DNA cytosine methylation and transcriptional gene silencing, despite being targeted by the host silencing machinery generating abundant 21-24 nucleotide short interfering RNAs. At the same time the banana genomic DNA is extensively methylated in both healthy and virus-infected plants. Our findings shed light on the siRNA-generating gene silencing machinery of banana and provide possible explanation why episomal pararetroviruses can persist in plants, while true retroviruses having an obligatory genome-integration step in their replication cycle do not exist in plants.

  • Downregulation of miR-526a by Enterovirus Inhibits RIG-I-Dependent Innate Immune Response [PublishAheadOfPrint]

  • Retinoic acid-inducible gene I (RIG-I) is an intracellular RNA virus sensor that induces type I interferon-mediated host protective innate immunity against viral infection. Although cylindromatosis (CYLD) has been shown to negatively regulate innate antiviral response by removing K-63-linked polyubiquitin from RIG-I, the regulation of its expression and the underlying regulatory mechanisms are still incompletely understood. Here we show that RIG-I activity is regulated by miR-526a-mediated inhibition of CYLD expression. We found that viral infection specifically upregulates miR-526a expression in macrophages via IRF-dependent mechanisms. In turn, miR-526a positively regulates virus-triggered type I Interferon (IFN-I) production, thus suppressing viral replication, the underlying mechanism of which is the enhancement of RIG-I K63-linked ubiquitination by miR-526a via suppressing the expression of CYLD. Remarkably, viral-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein, while ectopic miR-526a expression inhibits the replication of EV71 virus. The collective results of this study suggest a novel mechanism of the regulation of RIG-I activity during RNA virus infection by miR-526a and propose a novel mechanism for the evasion of innate immune response controlled by EV71.

    Importance RNA virus infection upregulates the expression of miR-526a in macrophages through IRF-dependent pathways. In turn, miR-526a positively regulates virus-triggered type I IFN production and inhibits viral replication, the underlying mechanism of which is the enhancement of RIG-I K-63 ubiquitination by miR-526a via suppressing the expression of CYLD. Remarkably, viral-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein, cells with overexpressed miR-526a were highly resistant to EV71 infection. The collective results of this study suggest a novel mechanism of the regulation of RIG-I activity during RNA virus infection by miR-526a and propose a novel mechanism for the evasion of innate immune response controlled by EV71.

  • New insights into the non-conserved non-coding region of the subtype-determinant HA and NA segments of influenza A viruses [PublishAheadOfPrint]

  • The non-coding regions (NCRs) of the eight RNA segments of influenza A virus consist of the highly conserved promoter region and the non-conserved segment-specific NCRs at both the 3' and 5' ends. The roles of the segment-specific NCRs of the eight segments have been extensively studied. However, the diversities in the same region of the two subtype-determinant HA and NA segments received little attention. In this report, we bioinformatically analyzed all available NCRs of HA and NA vRNAs of influenza A viruses and found that nucleotides in the segment-specific NCRs of HA and NA vRNAs are subtype-specific and varied significantly in sequence and length at both the 3' and 5' ends among different subtypes. We then systematically studied the biological significance of the HA subtype-specific NCRs (HA ssNCRs) of those common HA subtypes (H1-H7 and H9) in the context of WSN (H1N1) reverse genetics system. We found that the HA ssNCRs play a critical role in HA vRNA virion incorporation. Upon the HA vRNA incorporation, the 3' end HA ssNCR plays a more critical role than the 5' end HA ssNCR and no stringent compatibility between the two ends is required. Furthermore, our data imply that, in addition to particular nucleotide(s), the length of the HA ssNCR is involved in regulating HA vRNA incorporation efficiency. These results provide new insights into the HA segment virion incorporation that is critical for the emergence of epidemic and pandemic influenza A virus strains.

    IMPORTANCE The non-conserved non-coding regions (NCRs) of the eight segments of influenza A virus have been extensively studied whereas the diversities in the non-conserved NCRs of the two subtype-determinant segments HA and NA received little attention. In this report, we bioinformatically analyzed all available NCRs of HA and NA vRNAs and discovered that HA and NA vRNAs contain key subtype signatures in the NCR. Our functional studies of the HA ssNCRs of those common HA subtypes in the context of WSN virus (H1N1) demonstrated that the HA ssNCR modulates virus replication efficiency by influencing HA segment virion incorporation. Moreover, we revealed important features of the HA ssNCR in determining HA vRNA incorporation efficiency. These data not only show new genetic characteristics to influenza A viruses, but also provide further evidence in understanding the selective genome packaging of influenza virus required for the emergence of epidemic and pandemic influenza virus strains.

  • Deep sequencing of norovirus genomes defines evolutionary patterns in an urban tropical setting [PublishAheadOfPrint]

  • Norovirus is a highly transmissible infectious agent that causes epidemic gastroenteritis in susceptible children and adults. Norovirus infections can be severe and can be initiated from an exceptionally small number of viral particles. Detailed genome sequence data are useful for tracking norovirus transmission and evolution. To address this need we have developed a whole-genome deep sequencing method that generates entire genome sequences from small amounts of clinical specimens. This novel approach employs an algorithm for reverse transcription and PCR amplification primer design using all publically-available norovirus sequence data. Deep sequencing and de novo assembly were used to generate norovirus genomes from a large set of diarrheal patients attending 3 hospitals in Ho Chi Minh City, Vietnam, over a 2.5-year period. Positive selection analysis and direct examination of protein changes in the virus over time identified codons in the regions encoding proteins VP1, p48 (NS1-2) and p22 (NS4) under positive selection and expands the known targets of norovirus evolutionary pressure.

    Importance The high transmissibility and rapid evolution rate of norovirus, combined with short-lived host immune responses, are thought to be responsible for the virus causing a majority of pediatric viral diarrhea cases. The evolutionary patterns of this RNA virus have only been described in detail for a portion of the virus genome and never from a detailed urban tropical setting. We have developed robust deep sequencing methods for generating complete genome sequences directly from small amounts of patient fecal material. We use this method to provide a detailed sequence description of the noroviruses circulating in three Ho Chi Minh City hospitals over a 2.5-year period. The study identified patterns of virus change in known sites of host immune response and identified three additional regions of the virus genome under selection that were not previously recognized. In addition, the methods described here provide a robust full-genome sequencing platform for community-based virus surveillance.

  • Contribution of silent mutations to thermal adaptation of RNA bacteriophage Q{beta} [PublishAheadOfPrint]

  • Changes in protein function and other biological properties, such as RNA structure, are crucial for adaptation of organisms to novel or inhibitory environments. To investigate how mutations that do not alter amino acid sequence may be positively selected, we performed a thermal adaptation experiment using the single-stranded RNA bacteriophage Qbbeta; where the culture temperature was increased from 37.2ddeg;C to 41.2ddeg;C and finally to an inhibitory temperature of 43.6ddeg;C in a stepwise manner in three independent lines. Whole-genome analysis revealed 31 mutations, including 14 mutations that did not result in amino acid sequence alterations, in this thermal adaptation. Eight of the 31 mutations were observed in all three lines. Reconstruction and fitness analyses of Qbbeta; strains containing only mutations observed in all three lines indicated that five mutations that did not result in amino acid sequence changes but increased the amplification ratio appeared in the course of adaptation to growth at 41.2ddeg;C. Moreover, these mutations provided a suitable genetic background for subsequent mutations, altering the fitness contribution from deleterious to beneficial. These results clearly showed that mutations that do not alter the amino acid sequence play important roles in adaptation of this single-stranded RNA virus to elevated temperature.

    Importance Recent studies using whole-genome analysis technology suggested the importance of mutations that do not alter the amino acid sequence for adaptation of organisms to novel environmental conditions. It is necessary to investigate how these mutations may be positively selected and to determine to what degree such mutations that do not alter amino acid sequences contribute to adaptive evolution. Here, we report the roles of these silent mutations in thermal adaptation of RNA bacteriophage Qbbeta; based on experimental evolution where Qbbeta; showed adaptation to growth at an inhibitory temperature. Intriguingly, four synonymous mutations and one mutation in the untranslated region that spread widely in the Qbbeta; population during the adaptation process at moderately high temperature provided a suitable genetic background to alter the fitness contribution of subsequent mutations from deleterious to beneficial at a higher temperature.

  • Immunodominance Changes as a Function of the Infecting Dengue Virus Serotype and Primary versus Secondary Infection [PublishAheadOfPrint]

  • Dengue virus (DENV) is the causative agent of dengue fever (DF). Disease can be caused by any of four DENV serotypes (DENV1-4), sharing 67-75% sequence homology with one another. The effect of subsequent infections with different serotypes on the T cell repertoire is not fully understood. We utilized mice transgenic for human leukocyte antigens (HLA) lacking the IFN -aalpha;/bbeta; receptor to study responses to heterologous DENV infection. First, we defined the primary T cell response to DENV3 in the context of a wide range of HLA molecules. The primary DENV3 immune response recognized epitopes derived from all 10 DENV proteins, with a significant fraction of the response specific for structural proteins. This is in contrast to primary DENV2 infection where structural proteins are a minor component of the response, suggesting differential antigen immunodominance as a function of the infecting serotype. We next investigated the effect of secondary heterologous DENV infection on the T cell repertoire. In the case of both DENV2/3 and DENV3/2 heterologous infections, recognition of conserved/cross-reactive epitopes was either constant or expanded, as compared to homologous infection. Furthermore, in heterologous infection, previous infection with a different serotype impaired the development of responses directed to serotype specific but not conserved epitopes. Thus, a detrimental effect of previous heterotypic responses might not be due to dysfunctional and weakly crossreactive epitopes dominating the response. Rather, responses to the original serotype might limit the magnitude of responses directed against epitopes that are either crossreactive or specific for the most recently infecting serotype.

    Importance DENV transmission occurs in more than 100 countries and is an increasing public health problem in tropical and sub-tropical regions. At present, no effective antiviral therapy or licensed vaccine exists, and treatment is largely supportive in nature. Disease can be caused by any of the four DENV serotypes (DENV1-4), which share a high degree of sequence homology with one another. In this study we have addressed the question how the T cell repertoire changes as a function of infections with different serotypes and of subsequent heterologous secondary infections. This is of particular interest in the field of dengue viruses (DENV) where secondary infections with a different DENV serotypes increase the risk of severe disease. Our results on the evolution of the immune response after primary and secondary infection provides new insights into HLA-restricted T cell responses against DENV relevant for vaccine design against DENV

  • Genome-wide DNA methylation as an epigenetic consequence of Epstein-Barr virus infection of immortalized keratinocytes [PublishAheadOfPrint]

  • The oral cavity is a persistent reservoir for Epstein-Barr virus (EBV) with lifelong infection of resident epithelial and B cells. Infection of these cell types results in distinct EBV gene expression patterns regulated by epigenetic modifications involving DNA methylation and chromatin structure. Regulation of EBV gene expression relies on viral manipulation of the host epigenetic machinery that may result in long-lasting host epigenetic reprogramming. To identify epigenetic events following EBV infection, a transient infection model was established to map epigenetic changes in telomerase-immortalized oral keratinocytes. EBV-infected oral keratinocytes exhibited a predominantly latent viral gene expression program with some lytic or abortive replication. Calcium and methylcellulose-induced differentiation was delayed in EBV-positive and in clones that lost EBV compared to uninfected controls, indicating a functional consequence of EBV epigenetic modifications. Analysis of global cellular DNA methylation identified over 13,000 differentially methylated CpG residues in cells exposed to EBV compared to uninfected controls, with CpG island hypermethylation observed at several cellular genes. Although the vast majority of the DNA methylation changes were silent, 65 cellular genes that acquired CpG methylation showed altered transcript levels. Genes with increased transcript levels frequently acquired DNA methylation within the gene body while those with decreased transcript levels acquired DNA methylation near the transcription start site. Treatment with the DNA methyltransferase inhibitor, decitabine, restored expression of some hypermethylated genes in EBV-infected and EBV-negative transiently-infected clones. Overall, these observations suggested that EBV infection of keratinocytes leaves a lasting epigenetic imprint that can enhance the tumorigenic phenotype of infected cells.

    Importance Here we show that EBV infection of oral keratinocytes led to CpG island hypermethylation as an epigenetic scar of prior EBV infection that was retained after loss of the virus. Such EBV-induced epigenetic modification recapitulated the hypermethylated CpG island methylator phenotype (CIMP) observed in EBV-associated carcinomas. These epigenetic alterations not only impacted gene expression but resulted in delayed calcium and methylcellulose-induced keratinocyte differentiation. Importantly, these epigenetic changes occurred in cells that were not as genetically unstable as carcinoma cells indicating that EBV infection induced an epigenetic mutator phenotype. The impact of this work is that we have provided a mechanistic framework for how a tumor virus using the epigenetic machinery can act in a "hit-and-run fashion" with retention of epigenetic alterations after loss of the virus. Unlike genetic alterations, these virally-induced epigenetic changes can be reversed pharmacologically providing therapeutic interventions to EBV-associated malignancies.

  • Rational design of human metapneumovirus live attenuated vaccine candidates by inhibiting viral messenger RNA cap methyltransferase [PublishAheadOfPrint]

  • The paramyxoviruses, human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), and human parainfluenza virus 3 (hPIV3) are responsible for the majority of pediatric respiratory diseases and inflict significant economic loss, health costs, and emotional burdens. Despite major efforts, there are no vaccines available for these viruses. The conserved region VI (CR-VI) of the large (L) polymerase proteins of paramyxoviruses catalyzes methyltransferase (MTase) activities that typically methylate viral mRNAs at positions guanine N-7 (G-N-7) and ribose 2rrsquo; -O. In this study, we generated a panel of recombinant hMPVs carrying mutations in the S-adenosyl methionine (SAM) binding site in the CR-VI of L protein. These recombinant viruses were specifically defective in ribose 2rrsquo; -O, but not G-N-7 methylation, and were genetically stable and highly attenuated in cell culture and viral replication in the upper and lower respiratory tracts of cotton rats. Importantly, cotton rats vaccinated with these MTase-defective rhMPVs triggered a high level of neutralizing antibody and were completely protected from challenge with wild-type rhMPV. Collectively, our results indicate that (i) amino acid residues in the SAM binding site in hMPV L protein are essential for 2rrsquo; -O methylation, and (ii) inhibition of mRNA cap. MTase can serve as a novel target to rationally design live attenuated vaccines for hMPV, and perhaps other paramyxoviruses such as RSV and PIV3.

    Importance Human paramyxoviruses including RSV, hMPV, and PIV3 cause the majority of acute upper and lower respiratory tract infection in humans, particularly in infants, children, the elderly, and immunocompromised individuals. Currently, there is no licensed vaccine available. A formalin-inactivated vaccine is not suitable for these viruses because it causes enhanced lung damage upon reinfection with the same virus. A live attenuated vaccine is the most promising vaccine strategy for human paramyxoviruses. However, it remains a challenge to identify an attenuated virus strain that has an optimal balance between attenuation and immunogenicity. Using reverse genetics, we generated a panel of recombinant hMPVs that were specifically defective in ribose 2rrsquo; -O, but not G-N-7 methyltransferase (MTase). These MTase-defective hMPVs were genetically stable and sufficiently attenuated but retained high immunogenicity. This work highlights a critical role of 2rrsquo; -O MTase in paramyxovirus replication and pathogenesis, and a new avenue for the development of safe and efficacy live attenuated vaccines for hMPV and other human paramyxoviruses.

  • Kaposi's sarcoma-associated herpesvirus ORF18 and ORF30 are essential for late gene expression during lytic replication [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several human malignances. As saliva is likely the major vehicle for KSHV transmission, we studied in vitro KSHV infection of oral epithelial cells. Through infecting two types of oral epithelial cells, normal human oral keratinocytes (NHOKs) and papilloma-immortalized human oral keratinocyte (HOK16B) cells, we found that KSHV can undergo robust lytic replication in oral epithelial cells. By employing de novo lytic infection of HOK16B cells, we studied the function of two previously uncharacterized genes, ORF18 and ORF30, during the KSHV lytic cycle. For this purpose, an ORF18-deficient virus and an ORF30-deficient virus were generated using a mutagenesis strategy based on bacterial artificial chromosome (BAC) technology. We found that neither ORF18 nor ORF30 is required for immediately early, early gene expression, or viral DNA replication, but each is essential for late gene expression during both de novo lytic replication and reactivation. This critical role of ORF18 and ORF30 in late gene expression was also observed during KSHV reactivation. In addition, global analysis of viral transcripts by RNA-sequencing indicated that ORF18 and ORF30 control the same set of viral genes. Therefore, we suggest that these two viral ORFs are involved in the same mechanism or pathway that co-regulates the viral late genes as a group.

    Importance While KSHV can infect multiple cell types in vitro, only a few can support a full lytic replication cycle with progeny virions produced. Consequently, KSHV lytic replication is mostly studied through reactivation, which requires chemicals to induce the lytic cycle or over-expression of the viral transcriptional activator, RTA. In this study, we presented a robust de novo lytic infection system based on oral epithelial cells. Using this system, we demonstrated the role of two viral ORFs, ORF18 and ORF30, in regulating the viral gene expression during KSHV lytic replication. As the major route of KSHV transmission is thought to be via saliva, this new KSHV lytic replication system will have important utility in the field.

  • Plasmacytoid dendritic cells mediate synergistic effects of HIV and LPS on CD27+IgD- memory B cell apoptosis [PublishAheadOfPrint]

  • Background: The effects of heightened microbial translocation on B cells during HIV infection are unknown.

    Methods: We examined the in vitro effects of HIV and LPS on apoptosis of CD27+IgDnndash; memory B (mB) cell from healthy controls. In vivo analysis was conducted on a cohort of 82 HIV+ donors and 60 healthy controls.

    Results: In vitro exposure of PBMCs to LPS and HIV led to mB cell death via the Fas/Fas ligand (FasL) pathway. Plasmacytoid dendritic cells (pDCs) produced FasL in response to HIV via binding to CD4 and chemokine co-receptors. HIV and LPS increased Fas expression on mB cells in PBMCs which was dependent on the presence of pDCs and monocytes. Furthermore, mB cells purified from PBMCs and pretreated with both HIV and LPS were more sensitive to apoptosis when co-cultured with HIV-treated pDCs. Blocking the interferon receptor (IFNR) prevented HIV-stimulated FasL production in pDCs, HIV+LPS-induced Fas expression, and apoptosis of mB cells. In vivo or ex vivo, HIV+ donors have higher levels of plasma LPS, Fas expression on mB cells, and mB cell apoptosis compared to controls. Correspondingly, in HIV+ donors but not in controls, a positive correlation was found between plasma levels of FasL and HIV RNA, and between Fas expression on mB cells and plasma levels of LPS.

    Conclusion: This work reveals a novel mechanism of mB cell apoptosis mediated by LPS and HIV through the Fas/FasL pathway, with key involvement of pDCs and type I IFN, suggesting a role for microbial translocation in HIV pathogenesis.

    Importance: This study demonstrates that LPS and type I IFN, play an important role in memory B cell apoptosis in HIV infection. It reveals a previously unrecognized role of microbial translocation in HIV pathogenesis.

  • Inducible gene-expression in tumors colonized by modified oncolytic vaccinia virus strains [PublishAheadOfPrint]

  • Exogenous gene induction of therapeutic, diagnostic and safety mechanism could be a considerable improvement in oncolytic virotherapy. Here, we introduced a doxycycline-inducible promoter system (comprised of a tetracycline-repressor, several promoter constructs and a tet-operator sequence) into oncolytic recombinant vaccinia viruses (rVACV), which was further characterized in detail. Experiments in cell culture as well as in tumor bearing mice were analyzed to determine the role of the inducible system components. To accomplish this, we took advantage of the optical reporter construct, which resulted in the production of click-beetle luciferase as well as a red fluorescent protein. The results indicated that each of the system components could be used to optimize the induction rates and had influence on the background expression levels. Depending on a given gene to be induced in rVACV colonized tumors of patients, we discuss the doxycycline-inducible promoter system adjustment and further optimization.

    IMPORTANCE Oncolytic virotherapy of cancer can greatly benefit from the expression of heterologous genes. It is reasonable that some of those heterologous gene products could have detrimental effects on either the cancer patient or on the oncolytic virus itself if expressed at the wrong time or if the expression levels are too high. Therefore, exogenous control of gene expression levels by administration of a non-toxic inducer will have positive effects on the safety as well as the therapeutic outcome of oncolytic virotherapy. In addition, it paves the way for introduction of new therapeutic genes into the genome of oncolytic viruses that could not have been tested otherwise.

  • Molecular Characterization of a Lizard Adenovirus Reveals The First Atadenovirus with two Fiber Genes, and the First Adenovirus with Either One Short or Three Long Fibers per Penton [PublishAheadOfPrint]

  • Although adenoviruses have been found in a wide variety of reptiles including numerous squamate species, turtles and crocodiles, the number of reptilian adenovirus isolates is still scarce. The only fully sequenced reptilian adenovirus, snake adenovirus 1 (SnAdV-1), belongs to the Atadenovirus genus. Recently, two new atadenoviruses were isolated from a captive Gila monster (Heloderma suspectum) and Mexican beaded lizards (H. horridum). Here we report the full genomic and proteomic characterization of the latter, designated as lizard adenovirus 2 (LAdV-2). The dsDNA genome of LAdV-2 is 32,965 bp long with an average G+C content of 44.16%. The overall arrangement and gene content of the LAdV-2 genome was largely concordant with those in other atadenoviruses, except for four novel ORFs at the right end of the genome. Phylogeny reconstructions and plesiomorphic traits, shared with SnAdV-1, further supported the assignment of LAdV-2 to the Atadenovirus genus. Surprisingly, two fiber genes were found for the first time in an atadenovirus. After optimizing the production of LAdV-2 in cell culture, we determined the protein composition of the virions. The two fiber genes produce two fiber proteins of different size that are incorporated into the viral particles. Interestingly, the two different fiber proteins assemble as either one short or three long fiber projections per vertex. Stoichiometry estimations indicate that the long fiber triplet is present at only one or two vertices per virion. Neither triple fibers, nor a mixed number of fibers per vertex, had previously been reported for adenoviruses, or any other virus.

    IMPORTANCE Here we show that a lizard adenovirus, LAdV-2, has a penton architecture never observed before. LAdV-2 expresses two fiber proteins, one short and one long. In the virion, most vertices have one short fiber, but a few of them have three long fibers attached to the same penton base. This observation raises new intriguing questions on virus structure. How can the triple fiber attach to a pentameric vertex? What determines the number and location of each vertex type in the icosahedral particle? Since fibers are responsible for primary attachment to the host, this novel architecture also suggests a novel mode of cell entry for LAdV-2. Adenoviruses have a recognized potential in nanobiomedicine, but only a few of the more than 200 types found so far in nature have been characterized in detail. Exploring the taxonomic wealth of adenoviruses should improve our chances to successfully use them as therapeutic tools.

  • Phosphorylation of hepatitis C virus RNA polymerase Ser29 and Ser42 by PRK2 regulates viral RNA replication [PublishAheadOfPrint]

  • The hepatitis C virus (HCV) nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase (RdRp), is the key enzyme for HCV RNA replication. We previously showed that HCV RdRp is phosphorylated by protein kinase C-related kinase 2 (PRK2). In the present study, we used biochemical and reverse genetic approaches to demonstrate that HCV NS5B phosphorylation is crucial for viral RNA replication in cell culture. Two-dimensional phosphoamino acid analysis revealed that PRK2 phosphorylates NS5B exclusively at its serine residues in vitro and in vivo. Using in vitro kinase assays and mass spectrometry, we identified two phosphorylation sites, Ser29 and Ser42, on the 1 finger loop region that interacts with the thumb subdomain of NS5B. Colony-forming assays using drug-selectable HCV subgenomic RNA replicons revealed that preventing phosphorylation by Ala substitution at either Ser29 or Ser42 impairs HCV RNA replication. Furthermore, reverse genetic studies using HCV infectious clones encoding phosphorylation-defective NS5B confirmed the crucial role of these PRK2 phosphorylation sites in viral RNA replication. Molecular modeling studies predicted that the phosphorylation of NS5B stabilizes the interactions between its 1 loop and thumb subdomain, which are required for the formation of the closed conformation of NS5B known to be important for de novo RNA synthesis. Collectively, our results provide evidence that HCV NS5B phosphorylation has a positive regulatory role in HCV RNA replication.

    IMPORTANCE While the role of RdRps in viral RNA replication is clear, little is known about their functional regulation by phosphorylation. In this study, we addressed several important questions about the function and structure of phosphorylated HCV NS5B protein. Reverse genetic studies with HCV replicons encoding phosphorylation-defective NS5B mutants and analysis of their RdRp activity revealed previously unidentified NS5B protein features related to HCV replication and NS5B phosphorylation. These attributes most likely reflect potential structural changes induced by phosphorylation at the 1 finger loop region of NS5B with two identified phosphate acceptor sites, Ser29 and Ser42, which may transiently affect the closed conformation of NS5B. Elucidating the effects of dynamic changes in NS5B phosphorylation status during viral replication and its impact on RNA synthesis will improve our understanding of the molecular mechanisms of NS5B phosphorylation-mediated regulation of HCV replication.

  • The Epstein-Barr Virus EBNA1 Protein Regulates Viral latency Through Effects on Let-7 microRNA and Dicer [PublishAheadOfPrint]

  • The EBNA1 protein of Epstein-Barr virus (EBV) plays multiple roles in EBV latent infection including altering cellular pathways relevant for cancer. Here we have used microRNA cloning coupled with high throughput sequencing to identify the effects of EBNA1 on cellular miRNAs in two nasopharyngeal carcinoma cell lines. EBNA1 affected a small percentage of cellular miRNAs in both cell lines, in particular up-regulating multiple let-7 family miRNAs, including let-7a. EBNA1 effects on let-7a were verified by demonstrating that EBNA1 silencing in multiple EBV-positive carcinomas down-regulated let-7a. Accordingly, the let-7a target, Dicer, was found to be partially down-regulated by EBNA1 expression (at mRNA and protein levels) and up-regulated by EBNA1 silencing in EBV-positive cells. Reporter assays based on the Dicer 3rrsquo; UTR with and without let-7a target sites indicated that the effects of EBNA1 on Dicer were mediated by let-7a. EBNA1 was also found to induce the expression of let-7a primary RNAs in a manner dependent on the EBNA1 transcriptional activation region, suggesting that EBNA1 induces let-7a by transactivating the expression of its primary transcripts. Consistent with previous reports that Dicer promotes EBV reactivation, we found that a let-7a mimic inhibited EBV reactivation to the lytic cycle while a let-7 sponge increased reactivation. The results provide a mechanism by which EBNA1 could promote EBV latency by inducing let-7 miRNAs.

    IMPORTANCE The EBNA1 protein of Epstein-Barr virus (EBV) contributes in multiple ways to the latent mode of EBV infection that leads to life-long infection. In this study we identify a mechanism by which EBNA1 helps to maintain EBV infection in a latent state. This involves induction of a family of microRNAs (let-7 miRNA) that in turn decrease the level of the cellular protein Dicer. We demonstrate that let-7 miRNAs inhibit the reactivation of latent EBV, providing an explanation for our previous observation that EBNA1 promotes latency. In addition, since decreased levels of Dicer have been associated with metastatic potential, EBNA1 may increase metastases by down-regulating Dicer.

  • Dynamic regulation of host restriction factor expression over the course of HIV-1 infection in vivo [PublishAheadOfPrint]

  • In this study, we investigated the expression levels of host restriction factors in six untreated HIV-1-positive patients over the course of infection. We found that the host restriction factor gene expression profile consistently increased over time and significantly associated with CD4+ T cell activation and viral load. Our data are among the first to demonstrate the dynamic nature of host restriction factors in vivo over time.

  • Structural insights into the human metapneumovirus Glycoprotein ectodomain [PublishAheadOfPrint]

  • Human metapneumovirus is a major cause of respiratory tract infections worldwide. Previous reports have shown that the viral glycoprotein (G) modulates innate and adaptive immune responses, leading to incomplete immunity and promoting reinfection. Using bioinformatics analyses, static light scattering and small-angle x-ray scattering, we show that the extracellular region of G behaves as a heavily glycosylated, instrinsically disordered polymer. We discuss potential implications of these findings for the modulation of immune responses by G.

  • Novel indole-2-carboxamide compounds are potent broad spectrum antivirals active against western equine encephalitis virus in vivo [PublishAheadOfPrint]

  • Neurotropic alphaviruses, including western, eastern, and Venezuela equine encephalitis viruses, cause serious and potentially fatal central nervous system infections in humans, for which no currently approved therapies exist. We previously identified a series of thieno[3,2-b]pyrrole derivatives as novel inhibitors of neurotropic alphavirus replication using a cell-based phenotypic assay, and subsequently developed second and third generation indole-2-carboxamide derivatives with improved potency, solubility, and metabolic stability. In this report, we describe the antiviral activity of the most promising third generation lead compound, CCG205432, and closely related analogs, CCG206381 and CCG209023. These compounds have half maximal inhibitory concentrations ~1 mmu;M and selectivity indices ggt;100 in cell-based assays using western equine encephalitis virus replicons. Furthermore, CCG205432 retains similar potency against fully infectious virus in cultured human neuronal cells. These compounds show broad inhibitory activity against a range of RNA viruses in culture, including members of the Togaviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae families. Although their exact molecular target remains unknown, mechanism of action studies reveal that these novel indole-based compounds target a host factor that modulates cap-dependent translation. Finally, we demonstrate that both CCG205432 and CCG209023 dampen clinical disease severity and enhance survival of mice given a lethal western equine encephalitis virus challenge. These studies demonstrate that indole-2-carboxamide compounds are viable candidates for continued preclinical development as inhibitors of neurotropic alphaviruses, and potentially other RNA viruses.

    IMPORTANCE There are currently no approved drugs to treat infections with alphaviruses. We previously identified a novel series of compounds with activity against these potentially devastating pathogens. We have now produced third generation compounds with enhanced potency, and this manuscript provides detailed information on the antiviral activity of these advanced generation compounds, including activity in an animal model. The results of this study represent a notable achievement in the continued development of this novel class of antiviral inhibitors.

  • Herpes simplex virus gE/gI extracellular domains promote axonal transport and spread from neurons to epithelial cells. [PublishAheadOfPrint]

  • Following reactivation from latency, there are two distinct steps in the spread of herpes simplex virus (HSV) from infected neurons to epithelial cells: i) anterograde axonal transport of virus particles from neuron cell bodies to axon tips and ii) exocytosis and spread of extracellular virions across cell junctions into adjacent epithelial cells. HSV heterodimeric glycoprotein gE/gI is important for anterograde axonal transport and gE/gI cytoplasmic domains play important roles in sorting of virus particles into axons. However, the roles of the large (~400 residue) gE/gI extracellular (ET) domains in both axonal transport and neuron-to-epithelial cell spread have not been characterized. Two gE mutants: gE-277 and gE-348 contain small insertions in the gE ET domain, fold normally, form gE/gI heterodimers and are incorporated into virions. Both gE-277 and gE-348 did not function in anterograde axonal transport, there was markedly reduced numbers of viral capsids and glycoproteins, compared with wild type HSV. The defects in axonal transport were manifest in neuronal cell bodies involving missorting of HSV capsids before entry into proximal axons. Although there were diminished numbers of mutant gE-348 capsids and glycoproteins in distal axons, there was efficient spread to adjacent epithelial cells, similar to wild type HSV. By contrast, virus particles produced by HSV gE-277 spread poorly to epithelial cells, despite similar numbers of virus particles as gE-348. These results genetically separate the two steps in HSV spread from neurons to epithelial cells and demonstrate that the gE/gI ET domains functions in both processes.

    IMPORTANCE An essential phase of the life cycle of herpes simplex virus (HSV) and other aalpha;-herpesviruses is the capacity to reactivate from latency then spread from infected neurons to epithelial tissues. This spread involves at least two steps: i) anterograde transport to axon tips, followed by ii) exocytosis and extracellular spread from axons to epithelial cells. HSV gE/gI is a glycoprotein that facilitates this virus spread, although by poorly understood mechanisms. Here, we show that the extracellular (ET) domains of gE/gI promote the sorting of viral structural proteins into proximal axons to begin axonal transport. However, the gE/gI ET domains also participate in the extracellular spread from axon tips across cell junctions to epithelial cells. Understanding the molecular mechanisms involved in gE/gI-mediated sorting of virus particles into axons and extracellular spread to adjacent cells is fundamentally important in terms of identifying novel targets to reduce aalpha;-herpesvirus disease.

  • In Vivo Reactivation of Latent Herpes Simplex Virus 1 in Mice Can Occur in the Brain before in the Trigeminal Ganglion [PublishAheadOfPrint]

  • Herpes simplex virus 1 (HSV-1) establishes latency in neurons of brains and sensory ganglia of humans and experimentally infected mice. The latent virus can reactivate to cause recurrent infection. Both primary and recurrent infections can induce diseases, such as encephalitis. In humans, the majority of encephalitis cases occur as a recurrent infection. However, in the past, numerous mouse studies documented that viral reactivation occurs efficiently in the ganglion, but extremely rarely in the brain, when assessed ex vivo by cultivating minced tissue explants. Here we compare the brains and the trigeminal ganglia of mice latently infected with HSV-1 (strain 294.1 or McKrae) for levels of viral genomes and in vivo reactivation. The amounts of 294.1 and McKrae genomes in the brain stem were significantly higher than those in the trigeminal ganglion. Most importantly, 294.1 and McKrae reactivation was detected in the brain stem before in the trigeminal ganglion of mice treated with hyperthermia to reactivate latent virus in vivo. In addition, the brain stem yielded reactivated virus with a high frequency when compared with the trigeminal ganglion, especially in mice latently infected with 294.1 after hyperthermia treatment. These results provide evidence that the recurrent brain infection can be induced by the reactivation of latent virus in the brain in situ.

    IMPORTANCE HSV-1 establishes latency in neurons of brains and sensory ganglia of humans and experimentally infected mice. The latent virus can reactivate to cause recurrent infection. In the past, studies of viral reactivation focused on the ganglion, because efficient viral reactivation was detected in the ganglion, but not in the brain, when assessed ex vivo by cultivating mouse tissue explants. In this study, we report that the brain contains more viral genomes than the trigeminal ganglion of latently infected mice. Notably, the brain yields reactivated virus early and efficiently when compared with the trigeminal ganglion after mice are stimulated to reactivate latent virus. Our findings raise the potential importance of HSV-1 latent infection and reactivation in the brain.

  • A viral protein mediates superinfection exclusion at the whole organism level while is not required for exclusion at the cellular level [PublishAheadOfPrint]

  • Superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by the same or closely related virus, has been described for different viruses, including important pathogens of humans, animals, and plants. Citrus tristeza virus (CTV), a positive-sense RNA virus, represents a valuable model system for studying SIE due to the existence of several phylogenetically distinct strains. Furthermore, CTV allows for the possibility of examining SIE at the whole organism level. Previously we demonstrated that SIE by CTV is a virus-controlled function that requires the viral protein p33. In this study, we show that p33 mediates SIE at the whole organism level while it is not required for exclusion at the cellular level. Primary infection of a host with a fluorescent protein-tagged CTV variant lacking p33 did not interfere with the establishment of a secondary infection by the same virus labeled with a different fluorescent protein. However, cellular coinfection by both viruses was rare. The obtained observations along with estimates of the cellular multiplicity of infection (MOI) and MOI model selection suggested that low levels of cellular coinfection appear to be best explained by exclusion at the cellular level. Based on these results, we propose that SIE by CTV is operated at two levels mmdash; the cellular and the whole organism levels mmdash; by two distinct mechanisms that could function independently. This novel aspect of viral SIE highlights the intriguing complexity of this phenomenon further understanding of which may open up new avenues to manage virus diseases.

    Importance Many viruses exhibit superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by related viruses. SIE plays an important role in pathogenesis and evolution of virus populations. The observations described here suggest that SIE could be controlled independently at different levels of the host: the whole organism or individual cells. The p33 protein encoded by Citrus tristeza virus (CTV), an RNA virus, was shown to mediate SIE at the whole organism level, while it appeared not to be required for exclusion at the cellular level. SIE by CTV is, therefore, highly complex and appears to use different mechanisms than those proposed for other viruses. A better understanding of this phenomenon may lead to the development of new strategies for controlling viral diseases in human populations and agro-ecosystems.

  • Seed-sequence matched controls reveal limitations of siRNA knockdown in functional and structural studies of HCV NS5A-MOBKL1B interaction [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) is a widespread human pathogen causing liver cirrhosis and cancer. Similar to other viruses, HCV depends on host and viral factors to complete its life cycle. We used proteomic and yeast two-hybrid approaches to elucidate host factors involved in HCV nonstructural protein NS5A function and found that MOBKL1B interacts with NS5A. Initial experiments with siRNA knockdown suggesting a role in HCV replication led us to examine the interaction using biochemical and structural approaches. As revealed by a co-crystal structure of a core MOBKL1Bmmdash;NS5A peptide complex at 1.95 AAring;, NS5A binds to a hydrophobic patch on the MOBKL1B surface. Biosensor binding assays identified a highly conserved, 18 amino acid binding site in domain II of NS5A, which encompasses residues implicated in cyclophilin A (CypA)-dependent HCV RNA replication. However, a CypA-independent HCV variant had reduced replication in MOBKL1B knockdown cells, even though its NS5A does not interact with MOBKL1B. These discordant results prompted more extensive studies of MOBKL1B gene knockdowns, which included additional siRNAs and specifically matched seed-sequence siRNA controls. We found that reduced virus replication after treating cells with MOBKL1B siRNA was actually due to off-target inhibition, and indicated that the initial finding of virus replication dependence on the MOBKL1B-NS5A interaction was incorrect. Ultimately, using several approaches we found no relationship of the MOBKL1B-NS5A interaction to virus replication. These findings collectively serve as a reminder to investigators and scientific reviewers of the pervasive impact of siRNA off-target effects on interpretation of biological data.

    Importance Our study illustrates an underappreciated shortcoming of siRNA gene knockdown technology. We initially identified a cellular protein, MOBKL1B, as a binding partner with the NS5A protein of hepatitis C virus (HCV). MOBKL1B siRNA, but not irrelevant RNA treatment was associated with both reduced virus replication and the absence of MOBKL1B. Believing that HCV replication depended on the MOBKL1B-NS5A interaction, we carried out structural and biochemical analyses. Unexpectedly, an HCV variant lacking the MOBKL1B-NS5A interaction could not replicate after cells were treated with MOBKL1B siRNA. By repeating the MOBKL1B siRNA knockdowns, and including seed sequence-matched siRNA instead of irrelevant siRNA as a control, we found that the MOBKL1B siRNAs utilized had off-target inhibitory effects on virus replication. Collectively, our results suggest that stricter controls must be utilized in all RNAi-mediated gene knockdown experiments to ensure sound conclusions and a reliable scientific knowledge database.

  • Amino acid substitutions in PB1 of avian influenza viruses influence pathogenicity and transmissibility in chickens [PublishAheadOfPrint]

  • Amino acid substitutions in PB1 were introduced to characterize the interaction between polymerase activity and pathogenicity. Previously, we demonstrated that pathogenicity of the highly pathogenic avian influenza virus (HPAIV) H5N1 in chickens is regulated by the PB1 gene, using recombinant viruses containing the hemagglutinin (HA) and neuraminidase genes from an H5N1 strain, and other internal genes from two low-pathogenicity avian influenza viruses isolated from chicken (LP) and wild bird (WB) hosts (Y. Uchida et al., J. Virol. 86: 2686nndash;2695, 2012, doi: 10.1128/JVI.06374-11). In this study, we introduced a C38Y substitution into PB1 of WB and demonstrated that this substitution increased polymerase activity in vitro in DF-1 cells, as well as pathogenicity of the recombinant viruses in chickens. The V14A substitution in PB1 of LP reduced polymerase activity, but did not affect pathogenicity in chickens. Interestingly, the V14A substitution reduced viral shedding and transmissibility. These studies demonstrate that increasing polymerase activity directly correlates with enhanced pathogenicity, while decreased polymerase activity does not always correlate with pathogenicity and requires further analysis.

    IMPORTANCE We identified 2 novel amino acid substitutions in the PB1 gene of avian influenza virus that affect viral characteristics of highly pathogenic avian influenza virus (HPAIV) H5N1, such as viral replication and polymerase activity in vitro and pathogenicity and transmissibly in chickens. Amino acid substitution at residue 38 in PB1 directly affected pathogenicity in chickens, and was associated with changes in polymerase activity in vitro. Substitution at residue 14 reduced polymerase activity in vitro, while its effects on pathogenicity and transmissibility depended on the constellation of the internal genes.

  • Interferon regulatory factor 5 (IRF5)-dependent immune responses in the draining lymph node protect against West Nile virus infection [PublishAheadOfPrint]

  • Upon activation of Toll-like and RIG-I-like receptor signaling pathways, the transcription factor IRF5 translocates to the nucleus and induces antiviral immune programs. The recent discovery of a homozygous mutation in the immunoregulatory gene guanine exchange factor dedicator of cytokinesis 2 (Dock2mu/mu) in several Irf5-/- mouse colonies has complicated interpretation of immune functions previously ascribed to IRF5. To define the antiviral functions of IRF5 in vivo, we infected backcrossed Irf5-/- x Dock2wt/wt mice (hereafter called Irf5-/- mice) and independently-generated CMV-Cre Irf5fl/fl mice with West Nile virus (WNV), a pathogenic neurotropic flavivirus. Compared to congenic wild-type animals, Irf5-/- and CMV-Cre Irf5fl/fl mice were more vulnerable to WNV infection and this phenotype was associated with increased infection in peripheral organs, which resulted in higher viral titers in the central nervous system. The loss of IRF5, however, was associated with only small differences in the type I IFN response systemically and in the draining lymph node during WNV infection. Instead, lower levels of several other proinflammatory cytokines and chemokines, as well as fewer and less activated immune cells, were detected in the draining lymph node two days after WNV infection. WNV-specific antibody responses in Irf5-/- mice also were blunted in the context of live or inactivated virus infection and this was associated with fewer antigen-specific memory B cells and long-lived plasma cells. Our results with Irf5-/- mice establish a key role for IRF5 in shaping the early innate immune response in the draining lymph node, which impacts the spread of virus infection, optimal B cell immunity, and disease pathogenesis.

    IMPORTANCE Although the roles of IRF3 and IRF7 in orchestrating innate and adaptive immunity after viral infection are established, the function of the related transcription factor IRF5 remains less certain. Prior studies in Irf5-/- mice reported conflicting results as to the contribution of IRF5 in regulating type I IFN and adaptive immune responses. The lack of clarity may stem from a recently discovered homozygous loss-of-function mutation of the immunoregulatory gene Dock2 in several colonies of Irf5-/- mice. Here, using a mouse model with a deficiency in IRF5 and wild-type Dock2 alleles, we investigated how IRF5 modulates West Nile virus (WNV) pathogenesis and host immune responses. Our in vivo studies indicate that IRF5 has a key role in shaping the early pro-inflammatory cytokine response in the draining lymph node, which impacts immunity and control of WNV infection.

  • High risk HPV E6 protein promotes reprogramming of Fanconi Anemia patient cells through repression of p53 but does not allow for sustained growth of iPSC [PublishAheadOfPrint]

  • DNA repair plays a crucial role in embryonic and somatic stem cell biology and cell reprogramming. The Fanconi Anemia pathway, which promotes error-free repair of DNA double strand breaks, is required for somatic cell reprogramming to iPSC. Thus, cells from Fanconi Anemia patients, which lack this critical pathway, fail to reprogram to iPSC under standard conditions unless the defective FA gene is complemented. In this study, we utilized the oncogenes of high risk human papillomavirus type 16 to overcome the resistance to reprogramming of FA patient cells. We found that E6, but not E7, recovers FA iPS colony formation, and furthermore, that p53 inhibition is necessary and sufficient for this activity. The iPS colonies resulting from each of these approaches stained positive for alkaline-phosphatase, NANOG, and Tra-1-60, indicating that they were fully reprogrammed into pluripotent cells. However, FA iPSC were incapable of outgrowth into stable iPSC lines regardless of p53 suppression, whereas their FA-complemented counterparts grew efficiently. Thus, we conclude that the FA pathway is required for the growth of iPSC beyond reprogramming, and that p53-independent mechanisms are involved.

    Importance: A novel approach is described whereby HPV oncogenes are used as tools to uncover DNA repair-related molecular mechanisms affecting somatic cell reprogramming. The findings indicate that p53-dependent mechanisms block FA cells from reprogramming but also uncover a previously unrecognized defect in FA iPSC proliferation independent of p53.

  • Rooting the phylogenetic tree of MERS-Coronavirus by characterization of a conspecific virus from an African Bat [PublishAheadOfPrint]

  • The emerging Middle East respiratory syndrome-coronavirus (MERS-CoV) causes lethal respiratory infections mainly on the Arabian Peninsula. The evolutionary origins of MERS-CoV are unknown. We determined the full genome sequence of a CoV directly from fecal material obtained from a South African Neoromicia capensis bat (NeoCoV). NeoCoV shared essential details of genome architecture with MERS-CoV. 85% of the NeoCoV genome was identical to MERS-CoV on nucleotide level. Based on taxonomic criteria, NeoCoV and MERS-CoV belonged to one viral species. Presence of a genetically divergent S1 subunit within the NeoCoV Spike gene indicated that intra-Spike recombination events may have been involved in the emergence of MERS-CoV. NeoCoV constitutes a sister taxon to MERS-CoV, placing the MERS-CoV root between a recently-described virus from African camels and all other viruses. This suggests a higher viral diversity in camels than in humans. Together with serologic evidence for widespread MERS-CoV infection in camelids sampled up to 20 years back in Africa and the Arabian Peninsula, the genetic data indicates that camels act as sources of virus for humans rather than vice versa. The majority of camels on the Arabian Peninsula is imported from the greater Horn of Africa, where several Neoromicia species occur. The acquisition of MERS-CoV by camels from bats might have taken place in Sub-Saharan Africa. Camelids may represent mixing vessels for MERS-CoV and other mammalian CoVs.

    Importance It is unclear how, when and where the highly pathogenic MERS-CoV emerged. We characterized the full genome of an African bat virus closely related to MERS-CoV and show that human, camel and bat viruses belong to the same viral species. The bat virus roots the phylogenetic tree of MERS-CoV, providing evidence for an evolution of MERS-CoV in camels that preceded that in humans. The revised tree suggests that humans are infected by camels rather than vice versa. Although MERS-CoV cases occur mainly on the Arabian Peninsula, the data from this study together with serologic and molecular investigations of African camels indicate that the initial host switch from bats may have taken place in Africa. The emergence of MERS-CoV likely involved exchange of genetic elements between different viral ancestors. These exchanges may have taken place either in bat ancestors or camels acting as mixing vessels for viruses from different hosts.

  • Potent Neutralization of Vaccinia Virus by Divergent Murine Antibodies Targeting a Common Site of Vulnerability in L1 Protein [PublishAheadOfPrint]

  • Vaccinia virus (VACV) L1 is an important target for viral neutralization and has been included in multicomponent DNA or protein vaccines against orthopoxviruses. To further understand the protective mechanism of the anti-L1 antibodies, we generated five murine anti-L1 monoclonal antibodies (mAbs), which clustered into 3 distinct epitope groups. While two groups of anti-L1 failed to neutralize, one group of 3 mAbs potently neutralized VACV in an isotype- and complement-independent manner. This is in contrast to neutralizing antibodies against major VACV envelope proteins such as H3, D8 or A27, which failed to completely neutralize VACV unless the antibodies are of complement-fixing isotypes and complement is present. Compared to non-neutralizing anti-L1 mAbs, the neutralization antibodies bound to the recombinant L1 protein with a significantly higher affinity and could also bind to virions. By using a variety of techniques including the isolation of neutralization escape mutants, hydrogen/deuterium exchange mass spectrometry, and X-ray crystallography, the epitope of the neutralizing antibodies was mapped to a conformational epitope with Asp35 as the key residue. This epitope is similar to the epitope of 7D11, a previously described potent VACV neutralizing antibody. The epitope was recognized mainly by CDR1 and CDR2 of the heavy chain, which are highly conserved among antibodies recognizing the epitope. These antibodies, however, had divergent light chain and heavy chain CDR3 sequences. Our study demonstrates that the conformational L1 epitope with Asp35 is a common site of vulnerability for potent neutralization by a divergent group of antibodies.

    Importance Vaccinia virus, the live vaccine for smallpox, is one of the most successful vaccines in human history but presents a level of risk that has become unacceptable for the current population. Studying the immune protection mechanism of smallpox vaccine is important for understanding the basic principle of successful vaccines and the development of next generation, safer vaccines for highly pathogenic orthopoxviruses. We studied antibody targets in smallpox vaccine by developing potent neutralizing antibodies against vaccinia virus and comprehensively characterizing their epitopes. We found a site in vaccinia virus L1 protein as the target of a group of highly potent murine neutralizing antibodies. The analysis of antibody:antigen complex structure and the sequences of the antibody genes shed light on how these potent neutralizing antibodies are elicited from immunized mice.

  • Virulent bioluminescent and fluorescent dual reporter Marek's Disease virus (MDV) unveils an alternative MDV's spreading pathway in addition to cell-to-cell contact. [PublishAheadOfPrint]

  • Marek disease virus (MDV) is a growing threat for the poultry industry. Unfortunately, despite successful vaccination against the disease, MDV remains in circulation within vaccinated flocks, leading to the selection of increasingly virulent pathotypes. Detailed knowledge of the virus biology and the host/virus interaction is required to improve the vaccine efficiency. In the present study we have engineered an original, dual reporter MDV virus to track and quantify virus replication in vitro and in vivo.

  • Annexin V incorporated into influenza virus particles inhibits interferon-{gamma} signaling and promotes viral replication [PublishAheadOfPrint]

  • During the budding process, influenza A viruses (IAVs) incorporate multiple host cell membrane proteins. However, for most of them, their significance in viral morphogenesis and infectivity remains unknown. Here we demonstrate that the expression of annexin V (A5) is up-regulated at the cell surface upon IAV infection and that a substantial proportion of the protein is present in lipid rafts, the site of virus budding. Western blotting and immunogold analysis of highly purified IAV particles showed the presence of A5 in the virion. Significantly, interferon- (IFN-) induced stat-phosphorylation and IFN--induced protein 10 kDa (IP-10) production in macrophage-derived THP-1 cells was inhibited by purified IAV particles. Disruption of the IFN- signaling pathway was A5 dependent as down-regulation of its expression or its blockage reversed the inhibition and resulted in decreased viral replication, in vitro. The functional significance of these results was also observed in vivo. Thus, IAVs can subvert the IFN- antiviral immune response by incorporating A5 in their envelope during the budding process.

    IMPORTANCE Many enveloped viruses including influenza A viruses bud from the plasma membrane of their host cell and incorporate cellular surface proteins into viral particles. However, for the vast majority of these proteins, only the observation of their incorporation has been reported. In this manuscript, we demonstrated that the host protein annexin 5 is specifically incorporated into influenza virus particles during the budding process. Importantly, we showed that packaged annexin 5 counteracted the antiviral activity of interferon-gamma in vitro and in vivo. Thus, these results showed that Annexin 5 incorporated in the viral envelope of influenza viruses allow viral escape from immune surveillance. Understanding the role of host incorporated protein into virions may reveal how enveloped RNA viruses hijack the host cell machinery for their own purposes.

  • AAV Type 2 Wild-Type and Vector-Mediated Genomic Integration Profiles in Human Diploid Fibroblasts Analyzed by 3rd Generation PacBio DNA Sequencing [PublishAheadOfPrint]

  • Genome-wide analysis of adeno-associated virus (AAV) type 2 integration in HeLa cells has shown that wild-type AAV integrates at numerous genomic sites, including AAVS1 on chromosome 19q13.42. Multiple GAGY/C repeats, resembling consensus AAV Rep-binding sites are preferred, whereas rep-deficient AAV vectors (rAAV) regularly show a random integration profile. This study is the first study to analyze wild-type AAV integration in diploid human fibroblasts. Applying high-throughput 3rd generation PacBio-based DNA sequencing, integration profiles of wild-type AAV and rAAV are compared side by side. Bioinformatic analysis reveals that both, wild-type AAV and rAAV prefer open chromatin regions. Although genomic features of AAV integration largely reproduce previous findings, the pattern of integration hotspots differs from that described in HeLa cells before. DNase-Seq data for human fibroblasts and for HeLa cells reveal variant chromatin accessibility at preferred AAV integration hotspots that correlates with variant hotspot preferences. DNase-Seq patterns of these sites in human tissues including liver, muscle, heart, brain, skin and embryonic stem cells further underline variant chromatin accessibility. In summary, AAV integration is dependent on cell-type-specific, variant chromatin accessibility leading to random integration profiles for rAAV, whereas wild-type AAV integration sites cluster near GAGY/C repeats.

    Importance Adeno-associated virus type 2 (AAV) is assumed to establish latency by chromosomal integration of its DNA. This is the first genome-wide analysis of wild-type AAV2 integration in diploid human cells and the first to compare wild-type to recombinant AAV vector integration side by side under identical experimental conditions. Major determinants of wild-type AAV integration represent open chromatin regions with accessible consensus AAV Rep-binding sites. The variant chromatin accessibility of different human tissues or cell types will have impact on vector targeting to be considered during gene therapy.

  • Up to Four Distinct Polypeptides are produced from the {gamma}34.5 Open Reading Frame of Herpes Simplex Virus 2 [PublishAheadOfPrint]

  • The HSV-1 ICP34.5 protein strongly influences neurovirulence and regulates several cellular antiviral responses. Despite the clinical importance of HSV-2, relatively little is known about its ICP34.5 ortholog. We have found that HSV-2 produces up to four distinct forms of ICP34.5 in infected cells: full-length protein, one shorter form sharing the N-terminus, and two shorter forms sharing the C-terminus. These forms appeared with similar kinetics and accumulated in cells over much of the replication cycle. We confirmed that the N-terminal form is translated from the primary unspliced transcript to a stop codon within the intron unique to HSV-2 34.5. We found that the N-terminal form was produced in a variety of cell types, and by 9 of 10 clinical isolates. ICP27 influenced but was not required for expression of the N-terminal form. Western blot and reverse transcription PCR indicated the C-terminal forms did not contain the N-terminus, and were not products of alternative splicing or internal transcript initiation. Expression plasmids encoding methionine at amino acids 56 and 70 generated products which co-migrated in SDS-PAGE with the C1 and C2 forms, respectively, and mutation of these sites abolished C1 and C2. Using a recombinant HSV-2 encoding HA-tagged ICP34.5, we demonstrated that the C-terminal forms were also produced during infection of many human and mouse cell types, but were not detectable in mouse primary neurons. The protein diversity generated from the HSV-2 34.5 open reading frame implies additional layers of cellular regulation through potential independent activities associated with the various forms of ICP34.5.

    Importance The HSV-1 protein ICP34.5, encoded by the 34.5 gene, interferes with several host defense mechanisms by binding cellular proteins that would otherwise stimulate the cell's autophagic, translational arrest, and type I interferon responses to virus infection. ICP34.5 also plays a crucial role in determining the severity of nervous system infections with HSV-1 and HSV-2. The HSV-2 34.5 gene contains an intron not present in HSV-1 34.5. A shorter amino-terminal form of HSV-2 ICP34.5 can be translated from the unspliced 34.5 mRNA. Here we show that two additional forms consisting of the carboxy-terminal portion of ICP34.5 are generated in infected cells. Production of these N- and C-terminal forms is highly conserved among HSV-2 strains including many clinical isolates, and they are broadly expressed in several cell types but not mouse primary neurons. Multiple ICP34.5 polypeptides add additional complexity to potential functional interactions influencing HSV-2 neurovirulence.

  • P2X-selective purinergic antagonists are strong inhibitors of HIV-1 fusion during both cell-to-cell and cell-free infection [PublishAheadOfPrint]

  • Human Immunodeficiency Virus (HIV-1) is a chronic and incurable infection. Antiretroviral drugs effectively suppress replication; however, persistent activation of inflammatory pathways remains a key cause of morbidity. Recent studies proposed that purinergic signaling is required for HIV-1 viral infection. Purinergic receptors are distributed through a wide variety of tissue types that detect extracellular ATP as a danger signal released from dying cells. We have explored how these pathways are involved in transmission of HIV-1 from cell-to-cell through virological synapses. Infection of CD4+ T lymphocytes with HIV-1 in the presence of an inhibitor of P2X receptors effectively inhibited HIV-1 infection through both cell-free and cell-to-cell contact in a dose-dependent manner. Inhibition of direct cell-to-cell infection does not affect the formation of virological synapses or the subsequent cell-to-cell transfer of HIV-1. During both cell-free and cell-to-cell CD4+ T lymphocyte infection, purinergic antagonists blocked infection at the level of viral membrane fusion. During cell-to-cell transmission, within target lymphocytes we observed CXCR4 colocalization with the newly internalized virus particles, and found that the purinergic antagonists did not impair the recruitment of coreceptor CXCR4 to the site of Gag internalization in the target cell. In a screen of a library of purinergic antagonists, we found that the most potent inhibitors of HIV-1 fusion were those that target P2X receptors, while P2Y receptor antagonists or adenosine receptor antagonists were ineffective. Our results suggest that P2X receptors may provide a therapeutic target with potent antiviral activity against infection of CD4+ T lymphocytes by both cell-free and cell-to-cell infection.

    Importance This study identifies purinergic antagonists as potent inhibitors of HIV-1 cell-free and cell-to-cell mediated infection and provides stepwise determination of these compounds as inhibitors of HIV-1 viral membrane fusion. These data provide a rationale for the development of novel antiretroviral therapies that have a dual role in both direct anti-viral activity as well as HIV-associated inflammation. Purinergic antagonists are shown here to have equivalent efficacy in inhibiting HIV infection via cell-free and cell-to-cell infection and could provide an attractive therapeutic anti-HIV target that might avoid resistance by targeting a host signaling pathway that potently regulates HIV-infection. The high throughput screen of HIV-1 fusion inhibitors further defines P2X-selective compounds among purinergic compounds as being the most potent HIV entry inhibitors. Clinical studies on these drugs for other inflammatory indications suggest they are safe and thus, if developed for as an anti-HIV, they could reduce both HIV replication and HIV-related inflammation.

  • Inhibition of Antiviral Innate Immunity by Birnavirus VP3 Protein via Blockage of Viral dsRNA Binding to the Host Cytoplasmic RNA Detector MDA5 [PublishAheadOfPrint]

  • Chicken MDA5 (chMDA5), the sole known pattern recognition receptor for cytoplasmic viral RNA in chickens, initiates type I interferon (IFN) production. Infectious bursal disease virus (IBDV) evades host innate immunity but the mechanism is unclear. We report here that IBDV inhibited antiviral innate immunity via the chMDA5-dependent signaling pathway. IBDV infection did not induce efficient type I IFN production but antagonized the antiviral activity of IFN-bbeta; in DF-1 cells pretreated with IFN-aalpha;/bbeta;. Dual-luciferase assays and inducible expression systems demonstrated that IBDV protein VP3 significantly inhibited IFN-bbeta; expression stimulated by naked IBDV genomic dsRNA. VP3 protein competed strongly with chMDA5 to bind IBDV genomic dsRNA in vitro and in vivo, and VP3 from other birnaviruses also bound dsRNA. Site-directed mutagenesis confirmed that deletion of the VP3 dsRNA binding domain restored IFN-bbeta; expression. Our data demonstrate that VP3 inhibits antiviral innate immunity by blocking binding of viral genomic dsRNA to MDA5.

    IMPORTANCE MDA5, a known pattern recognition receptor and cytoplasmic viral RNA sensor, plays a critical role in host antiviral innate immunity. Many pathogens escape or inhibit the host antiviral immune response, but the mechanisms involved are unclear for most pathogens. We report here that birnaviruses inhibit host antiviral innate immunity via the MDA5-dependent signaling pathway. The antiviral innate immune system involving IFN-bbeta; did not function effectively during birnavirus infection, and the viral protein VP3 significantly inhibited IFN-bbeta; expression stimulated by naked viral genomic dsRNA. We also showed that VP3 blocked MDA5 binding to viral genomic dsRNA in vitro and in vivo. Our data reveal that birnavirus-encoded viral protein VP3 is an inhibitor of antiviral innate immune response and inhibits the antiviral innate immune response via the MDA5-dependent signaling pathway.

  • A Substitution in the Transmembrane Region of the Glycoprotein Leads to an Unstable Attenuation of Machupo Virus [PublishAheadOfPrint]

  • Machupo virus (MACV) is the etiologic agent of Bolivian hemorrhagic fever (BHF). Utilizing the reverse genetics system recently developed, we report the rescue of a rationally modified, recombinant MACV containing a single mutation in the transmembrane region of the glycoprotein. Following challenge of susceptible mice we identified a significant reduction in virulence in the novel virus. We also identified an instability leading to reversion of the single mutation to a wild type genotype.

  • Prolidase (PEPD) is required for early trafficking events during influenza A virus entry [PublishAheadOfPrint]

  • Influenza A virus (IAV) entry is a multi-step process that requires the interaction of the virus with numerous host factors. In this study, we demonstrate that prolidase (PEPD) is a cellular factor required by IAV for successful entry into target cells. PEPD was selected as a candidate during an entry screen performed on non-validated primary hits from previously published genome-wide siRNA screens. siRNA-mediated depletion of PEPD resulted in decreased growth of IAV during mono- and multi-cycle growth. This growth defect was independent of cell type or virus strain. Furthermore, IAV restriction was apparent as early as 3h post-infection and experiments in the absence of protein biosynthesis revealed that nuclear import of viral ribonucleoprotein complexes (vRNPs) was already blocked in the absence of PEPD. These results led us to investigate which step during entry was affected. Receptor expression, IAV attachment or internalization were not dependent on the presence of PEPD. However, when looking at the distribution of incoming IAV particles in PEPD knockdown cells, we found a localization pattern that differed compared to control cells: IAV mostly localized to the cell periphery and consequently, viral particles displayed reduced co-localization with early and late endosome markers and fusion between viral and endosomal membranes was strongly reduced. Finally, experiments using a competitive inhibitor of PEPD catalytic activity suggest that the enzymatic function of the dipeptidase is required for its proviral effect on IAV entry. In sum, this study establishes PEPD as a novel entry factor required for early endosomal trafficking of IAV.

    Importance Influenza A virus (IAV) continues to be a constant threat to public health. As IAV relies on its host cell for replication the identification of host factors required by the virus is of importance: First, such studies often reveal novel functions of cellular factors and can extend our knowledge of cellular processes. Second, we can further our understanding of processes that are required for entry of IAV into target cells. Third, the identification of host factors that contribute to IAV entry will enlarge the number of potential targets for the development of novel antiviral drugs that are of urgent need. Our study identifies prolidase (PEPD) as a novel entry factor of IAV required for correct routing within the endosomal compartment following virus internalization. Thereby, we link PEPD which has been shown to play a role during collagen recycling and growth factor signaling, to early events of viral infection.

  • Histone deacetylase 6 inhibits influenza A virus release by downregulating the trafficking of viral components to the plasma membrane via its substrate acetylated microtubules [PublishAheadOfPrint]

  • Mammalian cells produce many proteins such as IFITM3, ISG15, MxA, and viperin that inhibit influenza A virus (IAV) infection. Here we show that a new class of host protein, histone deacetylase 6 (HDAC6) inhibits IAV infection. We found that the HDAC6-overexpressing cells release about 3-fold less IAV progeny, whereas the HDAC6-depleted cells release about 6-fold more IAV progeny. The deacetylase activity of HDAC6 played a role in its anti-IAV function as tubacin, a specific small-molecule inhibitor of HDAC6, increased the release of IAV progeny in a dose-dependent manner. Further, as visualized by the electron microscopy, tubacin-treated cells showed an increase in IAV budding at the plasma membrane, the site of IAV assembly. Tubacin is a domain-specific inhibitor and binds to one of the two HDAC6 catalytic domains possessing the tubulin deacetylase activity. This indicated the potential involvement of acetylated microtubules in the trafficking of viral components to the plasma membrane. Indeed, as quantified by flow cytometry, there was about 2.0 to 2.5-fold increase and about 2-fold decrease in the amount of viral envelope protein hemagglutinin present on the plasma membrane of tubacin-treated, HDAC6-depleted, and HDAC6-overexpressing cells, respectively. In addition, the viral ribonucleoprotein complex was co-localized with acetylated microtubule filaments and viral nucleoprotein co-immunoprecipitated with acetylated tubulin. Together, our findings indicate that HDAC6 is an anti-IAV host factor and exerts its anti-IAV function by negatively regulating the trafficking of viral components to the host cell plasma membrane via its substrate acetylated microtubules.

    Importance Host cells produce many proteins that have the natural ability to restrict the influenza virus infection. Herein, we discovered that another host protein, histone deacetylase 6 (HDAC6) inhibits the influenza virus infection. We demonstrate that HDAC6 exerts its anti-influenza function by negatively regulating the trafficking of viral components to the site of influenza virus assembly via its substrate acetylated microtubules. HDAC6 is a multi-substrate enzyme and regulates multiple cellular pathways including the ones leading to various cancers, neurodegenerative diseases, and inflammatory disorders. Therefore, several drugs targeting HDAC6 are under clinical development for the treatment of wide range of diseases. Influenza virus continues to be a major global public health problem due to regular emergence of drug-resistant and novel influenza strains in humans. As an alternative antiviral strategy, HDAC6 modulators could be employed to stimulate the anti-influenza potential of endogenous HDAC6 to inhibit influenza virus infection.

  • Expression of porcine fusion protein IRF7/3(5D) efficiently controls foot-and-mouth disease virus replication [PublishAheadOfPrint]

  • Several studies have demonstrated that administration of type I, II, or III interferons (IFNs) delivered using a replication-defective human adenovirus 5 (Ad5) vector can effectively control foot-and-mouth disease (FMD) in cattle and swine during experimental infections. However, relatively high doses are required to achieve protection. In this study, we identified the functional properties of a porcine fusion protein, poIRF7/3(5D), as a biotherapeutic and enhancer of IFN activity against FMD virus (FMDV). We showed that poIRF7/3(5D) is a potent inducer of type I IFNs including IFNaalpha;, bbeta;, and but not type III IFN (IL28B), without inducing cytotoxicity. Expression of poIRF7/3(5D) significantly and steadily reduced FMDV viral titers by up to 6 log10 in swine and bovine cell lines. Treatment with an IFN receptor inhibitor (B18R) combined with an anti-IFNaalpha; antibody neutralized the antiviral activity in the supernatants of Ad5-poIRF7/3(5D) transduced cells. However, several transcripts with known antiviral function, and including type I IFNs, were still highly up-regulated (ranging from 8 to over 500 fold increase) by poIRF7/3(5D) in the presence of B18R. Furthermore, mice treated with Ad5-poIRF7/3(5D) showed antiviral activity in sera that was associated with high induction of IFNaalpha; and resulted in complete protection against FMDV challenge at 6, 24 or 48 hours post-treatment. This study, highlights for the first time, the antiviral potential of Ad5-poIRF7/3(5D) in vitro and in vivo against FMDV.

    Importance: FMD remains one of the most devastating diseases that affect livestock worldwide. Effective vaccine formulations are available but are serotype specific and require approximately 7 days for protective immunity. We have shown that vector-delivered IFN is an option to protect animals against many FMDV serotypes as soon as 24 h and for about 4 days post administration. Here we demonstrate that delivery of a constitutively active transcription factor that induces the production of endogenous IFNs and potentially other antiviral genes is a viable strategy to protect against FMD.

  • Envelope Glycoprotein Binding to the {alpha}4{beta}7 Integrin Complex is not a General Property of Most HIV-1 Variants [PublishAheadOfPrint]

  • The HIV-1 surface gp120 has been reported to bind and signal through aalpha;4bbeta;7 by means of a tripeptide motif in the V2 loop that mimics structures present in the natural ligands for aalpha;4bbeta;7, suggesting that aalpha;4bbeta;7 may facilitate HIV-1 infection of CD4+ T cells in the gut. Furthermore, immune correlates in the RV144 vaccine efficacy trial generated the hypothesis that V1V2 antibodies to an epitope near the putative aalpha;4bbeta;7 binding motif may play a role in protection against HIV-1 infection. In the interest of developing an assay to detect antibodies that block gp120 binding to aalpha;4bbeta;7, we used retinoic acid (RA)-activated human peripheral blood mononuclear cells (PBMC) and transfected 293T cells expressing the integrin complex to study the aalpha;4bbeta;7 binding properties of sixteen HIV-1 envelope glycoproteins. The natural ligand for aalpha;4bbeta;7, MAdCAM-1, bound efficiently to RA-PBMC and transfected 293T cells, and this binding was blocked by antibodies to aalpha;4. Gp120 from multiple HIV-1 subtypes bound to RA-PBMC from three donors in a CD4-dependent manner but little or no aalpha;4bbeta;7 binding was detected. Similarly, little or no binding to aalpha;4bbeta;7 on transfected 293T cells was detected with multiple gp120s and gp140s, including gp120s from transmitted/founder strains, and when gp120 was produced in CHO, 293T and 293S/GnT1-/- cells. Finally, we found no evidence that infectious HIV-1 virions produced in either PBMC or 293T cells could bind aalpha;4bbeta;7 on transfected 293T cells. Infectious HIV-1 virions and most gp120s/gp140s appear to be poor ligands for the aalpha;4bbeta;7 integrin complex under the conditions tested here.

    IMPORTANCE Certain HIV-1 gp120 envelope glycoproteins have been shown to bind the gut homing receptor aalpha;4bbeta;7, and it has been suggested that this binding facilitates mucosal transmission and virus replication in the gut mucosa. Additional evidence has generated the hypothesis that antibodies that bind near the putative aalpha;4bbeta;7 binding motif in the V2 loop of gp120, possibly disrupting gp120- aalpha;4bbeta;7 binding, may be important for HIV-1 vaccines. Our evidence indicates that infectious HIV-1 virions and many gp120s lack detectable aalpha;4bbeta;7 binding activity, suggesting that this homing receptor may play a limited role in HIV-1 transmission and pathogenesis.

  • TRIM13 is a negative regulator of MDA5-mediated type I IFN production [PublishAheadOfPrint]

  • Retinoic-acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are essential intracellular detectors of viral RNA. They contribute to the type I interferon (IFN) response that is crucial for host defense against viral infections. Given the potent anti-viral and pro-inflammatory activities elicited by the type I IFNs, its induction is tightly regulated. Members of the tripartite motif (TRIM) family of proteins have recently emerged as key regulators of anti-viral immunity. We show that TRIM13, an E3 ubiquitin ligase, is expressed in immune cells and is upregulated in bone marrow-derived macrophages upon stimulation with inducers of type I IFN. TRIM13 interacts with MDA5 and negatively regulates MDA5-mediated type I IFN production in vitro, acting upstream of IFN regulatory factor 3. We generated Trim13-/- mice and show that upon lethal challenge with encephalomyocarditis virus (EMCV), which is sensed by MDA5, Trim13-/- mice produce increased amounts of type I IFNs and survive longer than wild type mice. Trim13-/- murine embryonic fibroblasts (MEFs) challenged with EMCV or poly I:C also showed a significant increase in IFNbbeta; levels, but in contrast, IFNbbeta; responses to the RIG-I-detected Sendai virus were diminished, suggesting that TRIM13 may play a role in positively regulating RIG-I function. Conversely, we demonstrate that TRIM13 regulates the type I IFN response through inhibition of MDA5 activity, and that it functions non-redundantly to modulate MDA5 during EMCV infection.

    IMPORTANCE: The type I interferon (IFN) response is crucial for host defense against viral infections, and proper regulation of this pathway contributes to maintaining immune homeostasis. Retinoic-acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are intracellular detectors of viral RNA that induce to the type I IFN response. In this study, we show that the tripartite motif 13 (Trim13) expression is upregulated in response to inducers of type I IFN and that TRIM13 interacts with both MDA5 and RIG-I in vitro. Through the use of multiple in vitro and in vivo model systems, we show that TRIM13 is negative regulator of MDA5-mediated type I IFN production, and may also impact RIG-I mediated type I IFN production by enhancing RIG-I activity. This places TRIM13 at a key junction within the viral response pathway, and identifies it as one of the few known modulators of MDA5 activity.

  • Type III Interferon Attenuates a Vesicular Stomatitis Virus Based Vaccine Vector [PublishAheadOfPrint]

  • Vesicular stomatitis virus (VSV) has been extensively studied as a vaccine vector and oncolytic agent. Nevertheless, safety concerns have limited its widespread use in humans. The type III interferon (IFN-) family of cytokines shares common signaling pathways with the IFN-aalpha;/bbeta; family, and thus evokes similar antiviral activities. However, IFN- signals through a distinct receptor complex that is expressed in a cell type-specific manner, which restricts its activity to epithelial barriers, particularly the respiratory and gastrointestinal tracts. In this study, we determined how IFN- expression from recombinant VSV would influence vector replication, spread, and immunogenicity. We demonstrate that IFN- expression severely attenuates VSV in cell culture. In vivo, IFN- limits VSV replication in the mouse lung after intranasal administration and reduces virus spread to other organs. Despite this attenuation however, the vector retains its capacity to induce protective CD8 T cell and antibody responses after a single immunization. These findings demonstrate a novel method of viral vector attenuation that could be used in both vaccine and oncolytic virus applications.

    IMPORTANCE Viruses such as VSV that are used as vaccine vectors can induce protective T cell and antibody responses after a single dose. Additionally, IFN- is a potent antiviral agent that has certain advantages for clinical use compared to IFN-aalpha;/bbeta;, such as fewer patient side effects. Here, we demonstrate that IFN- attenuates VSV replication and spread following intranasal virus delivery, but does not reduce the ability of VSV to induce potent protective immune responses. These findings demonstrate that the type III IFN family may have widespread applicability for improving the safety and efficacy of viral vaccine and oncolytic vectors.

  • New Small Molecule Inhibitors Effectively Blocking Picornavirus Replication [PublishAheadOfPrint]

  • Few drugs targeting picornaviruses are available, making the discovery of antivirals a high priority. Here, we identified and characterized three compounds from a library of kinase inhibitors that block replication of poliovirus, Coxsackie virus B3 and encephalomyocarditis virus. Using an in vitro translation-replication system, we showed that these drugs inhibit different stages of the poliovirus lifecycle. A4 (1) inhibited both formation and functioning of the replication complexes while E5 (1) and E7 (2) were most effective during the formation but not the functioning step. Neither of the compounds significantly inhibited VPg uridylylation. Poliovirus resistant to E7 (2) had a mutation G5318A in the 3A protein. This mutation was previously found to confer resistance to enviroxime-like compounds which target a PI4KIIIbbeta;-dependent step in the viral replication. Analysis of host proteins recruitment showed that E7 (2) reduced amount of GBF1on the replication complexes, however the level of PI4KIIIbbeta; remained intact. E7 (2) as well as another enviroxime-like compound GW5074 interfered with the viral polyprotein processing affecting both 3C and 2A-dependent cleavages, and the resistant mutation G5318A partially rescued this defect. Moreover E7 (2) induced abnormal recruitment to membranes of the viral proteins, thus enviroxime-like compounds likely severely compromise interaction of the viral polyprotein with membranes. A4 (1) demonstrated partial protection from paralysis in a murine model of poliomyelitis. Multiple attempts to isolate resistant mutants in the presence of A4 (1) or E5 (1) were unsuccessful, showing that effective broad-spectrum antivirals could be developed on the basis of these compounds.

    Importance Diverse picornaviruses can trigger multiple human maladies, yet currently only hepatitis A virus and poliovirus can be controlled with vaccination. The development of anti-picornaviral therapeutics is also facing significant difficulties because these viruses readily generate resistance to compounds targeting either viral or cellular factors. Here we describe three novel compounds that effectively block replication of distant picornaviruses with minimal toxicity to the cells. The compounds prevent viral RNA replication after the synthesis of the uridylylated VPg primer. Importantly, two of the inhibitors are strongly refractory to the emergence of resistant mutants making them promising candidates for further broad-spectrum therapeutic development. Evaluation of one of the compounds in an in vivo model of poliomyelitis demonstrated partial protection from the onset of paralysis.

  • Age-dependent myeloid dendritic cell responses mediate resistance to La Crosse Virus induced neurological disease. [PublishAheadOfPrint]

  • La Crosse Virus (LACV) is the major cause of pediatric viral encephalitis in the USA; however, the mechanisms responsible for age-related susceptibility in the pediatric population are not well understood. Our current studies in a mouse model of LACV infection indicated that differences in myeloid dendritic cell (mDC) responses between weanling and adult mice accounted for susceptibility to LACV-induced neurological disease. We found that type I interferon (IFN) responses were significantly stronger in adult versus weanling mice. Production of these IFNs required both endosomal toll-like receptors (TLR) and cytoplasmic RIG-I like receptors (RLR). Surprisingly, IFN expression was not dependent on plasmacytoid DCs (pDCs) but rather mDCs, which were found in greater number and induced stronger IFN responses in adults than weanlings. Inhibition of these IFN responses in adults resulted in susceptibility to LACV-induced neurological disease, whereas post-infection treatment with type I IFN provided protection in young mice. These studies provide a definitive mechanism for age-related susceptibility to LACV encephalitis, where mDCs in young mice are insufficiently activated to control peripheral virus replication, thereby allowing virus to persist and eventually cause CNS disease.

    IMPORTANCE La Crosse Virus (LACV) is the primary cause of pediatric viral encephalitis in the United States. Although the virus infects both adults and children, over 80% of the reported neurological disease cases are in children. To understand why LACV primarily causes neurological disease in young animals, we used a mouse model where weanling mice, but not adult mice, develop neurological disease following virus infection. We found that an early immune response cell type, myeloid dendritic cells, was critical for protection in adult animals, and that these cells were reduced in young animals. Activation of these cells during virus infection or post-treatment with type I interferon in young animals provided protection from LACV. Thus, this study demonstrates a reason for susceptibility to LACV infection in young animals and shows that early therapeutic treatment in young animals can prevent neurological disease.

  • The effect of vaginal immunization in women with HIVgp140 and HSP70 on HIV-1 replication, innate and T cell adaptive immunity [PublishAheadOfPrint]

  • The international effort to prevent HIV-1 infection by vaccination has failed to develop an effective vaccine. The aim of this vaccine trial in women was to administer by the vaginal mucosal route a vaccine consisting of HIV-1gp140 linked to the chaperone 70kD heat shock protein (HSP70). The primary objective was to determine safety of the vaccine. The secondary objective was to examine HIV-1 infectivity ex-vivo and innate and adaptive immunity to HIV-1. Protocol defined female volunteers were recruited. HIV-1 CN54gp140 linked to HSP70 was administered by the vaginal route. Significant adverse reactions were not detected. HIV-1 was significantly inhibited ex vivo in the post- compared with the pre-immunization CD4+ T cells. The innate anti-viral restrictive factor APOBEC3G was significantly upregulated, as were CC-chemokines, which induce down-regulation of CCR5 in CD4+ T cells. Indeed, significant inverse correlation was found between the proportion of CCR5+ T cells and the concentration of CCL-3 or CCL-5. Importantly, APOBEC3G showed a significant inverse correlation, whereas CCR5 exhibited a trend to correlating with inhibition of HIV-1 infection (r=0.51). Furthermore, specific CD4+ and CD8+ T cell proliferative responses were significantly increased and CD4+ T cells showed a trend to inverse correlation with the viral load (r=-0.60). However, HIVgp140 specific IgG or IgA antibodies were not detected. The results provide proof of concept that an innate mechanism consisting of CC-chemokines, and APOBEC 3G, and adaptive immunity by CD4 and CD8 T cells might be involved in controlling HIV-1 infectivity following vaginal mucosal immunization in women.

    Importance Vaginal immunization of women with a vaccine consisting of HIVgp140 linked to the 70kD heat shock protein (HSP70) elicited ex vivo significant inhibition of HIV-1 replication in the post- compared with pre-immunization PBMC. There were no significant adverse events. The vaccine induced significant upregulation of CC chemokines and downmodulation of CCR5 expression in CD4+ T cells, as well as an inverse correlation between them. Furthermore, CCR5 was directly correlated with the viral load, consistent with the protective mechanism of a decrease in CCR5 molecules on CD4+ T cells decreasing HIV-1 envelope binding. The anti-viral restriction factor APOBEC3G was inversely correlated with the viral load, suggesting that it may inhibit intracellular HIV-1 replication. Both CD4+ and CD8+ T cells showed HIVgp140 and HSP70 specific proliferation. A strong inverse correlation was found between the CC-chemokine modulated CCR5 expressing CD4+ T cells and CD4+ or CD8+ T cells stimulated proliferation with HIVgp140, demonstrating a significant interaction between innate and adaptive immunity.

  • Impact of sequence variation in a dominant HLA-A*02-restricted epitope in HCV on priming and cross-reactivity of CD8+ T cells [PublishAheadOfPrint]

  • CD8+ T cells are an essential component of successful adaptive immune responses against hepatitis C virus (HCV). A major obstacle to vaccine design against HCV is the inherent viral sequence diversity. Here, we test the hypothesis, if different sequence variants of an immunodominant CD8+ T cell epitope all binding with high affinity to HLA class I target different T cell receptor repertoires and thereby influence the quality of the CD8+ T cell response. The impact of sequence differences in the HLA-A*02-restricted HCV NS31406-1415 epitope on in vitro priming of naiiuml;ve CD8+ T cells from seronegative donors and cross-reactivity of primed T cells with other epitope variants were characterized. Despite that the six epitope variants tested were all high-affinity binders to HLA-A*02:01, substantial differences in priming and cross-reactivity of CD8+ T cells were observed. The variant associated with the most reproducible priming and induction of T cells with broad cross-reactivity was a genotype 1b variant (KLSALGLNAV) that is more common in HCV isolates collected in Asia but is rare in sequences from Europe and North America. The superior immunogenicity and cross-reactivity of this relatively rare epitope variant was confirmed in HCV-specific memory CD8+ T cells from people who inject drugs, frequently exposed to HCV. Collectively, the data suggest that sequence differences at the epitope level between HCV isolates substantially impact CD8+ T cell priming and the degree of cross-reactivity with other epitope variants.

    Importance: The results have important implications for vaccine design against highly variable pathogens and suggest that evidence-based selection of the vaccine antigen sequence may improve immunogenicity and T cell cross-reactivity. Cross-reactive CD8+ T cells are likely beneficial for immune control of transmitted viruses carrying epitope variants and for prevention of immune escape during acute infection. To this end, rare epitope variants and potentially even altered epitope sequences associated with priming of broadly cross-reactive T cell receptors should be considered for vaccine design and need further testing.

  • A Mutation in the DNA Polymerase Accessory Factor of Herpes Simplex Virus 1 Restores Viral DNA Replication in the Presence of Raltegravir [PublishAheadOfPrint]

  • Previous reports showed that Raltegravir, a recently approved antiviral compound that targets HIV integrase, can inhibit the nuclease function of human cytomegalovirus (HCMV) in vitro. In this study, subtoxic levels of Raltegravir were shown to inhibit the replication of four different herpesviruses: HSV-1, HSV-2, HCMV and MCMV (mouse cytomegalovirus) by 30-700 fold depending on the dose and virus tested. Southern blot and qPCR revealed that Raltegravir inhibits DNA replication of HSV-1 rather than cleavage of viral DNA. A Raltegravir-resistant HSV-1 mutant was generated by repeated passage in the presence of 200 mmu;M Raltegravir. The genomic sequence of the resistant virus, designated clone 7, contains mutations in 16 open reading frames. Of these, mutations in UL15 (F198S, encoding the large terminase subunit), UL32 (A374V, required for DNA cleavage and packaging), UL42 (V296I, encoding the DNA polymerase accessory factor), and UL54 (A224S, encoding ICP27, an important transcriptional regulator) were introduced independently into the wild type HSV-1(F) genome and the recombinant viruses were tested for Raltegravir resistance. Viruses bearing both the UL15 and UL32 mutations inserted within the genome of the UL42 mutant were also tested. While the UL15, UL32 and UL54 mutant viruses were fully susceptible to Raltegravir, any virus bearing the UL42 mutation was as resistant to Raltegravir as clone 7. Overall, these results suggest that Raltegravir may be a valuable therapeutic agent against herpesviruses, and the antiviral activity targets the DNA polymerase accessory factor rather than the nuclease activity of the terminase.

    Importance This paper is important because it shows that Raltegravir, and anti-retrovirus drug targeting integrase, is effective against various herpesviruses. Drug resistance mapped to the herpesvirus DNA polymerase accessory factor which was an unexpected finding.

  • Chemokine (C-C motif) receptor 1 (CCR1) is required for efficient recruitment of neutrophils during respiratory infection with Modified Vaccinia virus Ankara [PublishAheadOfPrint]

  • Modified Vaccinia virus Ankara (MVA) serves as a versatile platform in vaccine development. This highly attenuated orthopoxvirus, which cannot replicate in mammalian cells, triggers strong innate immune responses including cell migration. Previously we have shown that induction of chemokine (C-C motif) ligand 2 (CCL2) by MVA is necessary for the recruitment of monocytes and T cells but not neutrophils to the lung. Here we identified neutrophil-attracting chemokines produced by MVA infected primary murine lung fibroblasts and murine bone marrow derived macrophages. We demonstrate that MVA but not vaccinia virus (VACV) strain WR induces chemokine expression, which is independent of Toll-like receptor 2 (TLR2) signaling. Additionally, we show that both chemokine (C-C motif) receptor 1 (CCR1) and chemokine (C-X-C motif) receptor 2 (CXCR2) are involved in MVA induced neutrophil chemotaxis in vitro. Finally, intranasal infection of Ccr1-/- mice with MVA as well as application of the CCR1 antagonist J-113863 revealed a role for CCR1 in leukocyte recruitment including neutrophils into the lung.

    IMPORTANCE Rapid attraction of leukocytes to the site of inoculation is unique to MVA in comparison to other VACV strains. The findings here extend current knowledge about the regulation of MVA induced leukocyte migration, particularly regarding neutrophils, that could potentially be exploited to improve other VACV strains currently in development as oncolytic viruses and viral vectors. Additionally, the data presented here indicate that the inflammatory response may vary depending on the cell type infected by MVA, highlighting the importance of the site of vaccine application. Moreover, the rapid recruitment of neutrophils and other leukocytes can directly contribute to the induction of adaptive immune responses elicited by MVA inoculation. Thus, a better understanding of leukocyte migration upon MVA infection is particularly relevant for further development and use of MVA-based vaccines and vectors.

  • Differential Responses of Plasmacytoid Dendritic Cells to Influenza and Distinct Viral Pathogens [PublishAheadOfPrint]

  • Plasmacytoid dendritic cells (pDCs) are key components of the innate immune response capable of synthesizing and rapidly releasing vast amounts of type I interferons (IFNs), particularly IFN-aalpha;. Here we investigated whether pDCs, often regarded as a mere source of IFN, discriminate between various functionally discrete stimuli, and to what extent this reflects differences in pDC responses other than IFN-aalpha; release. To examine the ability of pDCs to differentially respond to various doses of intact and infectious HIV, HCV, and H1N1 viruses, whole genome gene expression analysis, ELISA, and flow cytometry were applied to interrogate pDC responses at the transcriptional, protein, and cellular level. Our data demonstrates that pDCs differentially respond to various viral stimuli with significant changes in gene expression including those involved in pDC activation, migration, viral endocytosis, survival or apoptosis. In some cases, the expression of these genes were induced even at levels comparable to that of IFN-aalpha;. Interestingly, we also found that depending on the viral entity and viral titer used for stimulation, induction of IFN-aalpha; gene expression and the actual release of IFN-aalpha; are not necessarily temporally coordinated. In addition, our data suggests that high titer Influenza A (H1N1) virus infection can stimulate rapid pDC apoptosis.

    Importance Section: Plasmacytoid Dendritic Cells are key players of the viral immune response. With the host response to viral infection being dependent on specific virus characteristics, a thorough examination and comparison of dendritic cell response to various viruses at various titers is beneficial for the field of virology. Our study illustrates that pDC infection with Influenza, HIV, and HCV, results in a unique and differential response to each virus. These results have implications for future virology research, vaccine development, and the study and virology as a whole.

  • Diverse specificity, phenotype and anti-viral activity of cytomegalovirus specific CD8+ T cells. [PublishAheadOfPrint]

  • CD8+ T cells specific for pp65, IE1 and IE2 are present at high frequencies in HCMV seropositive individuals and these have been shown to have phenotypes associated with terminal differentiation, as well as both cytokine and proliferative dysfunctions, especially in the elderly. However, more recently, T cell responses to many other HCMV proteins have been described but little is known about their phenotype and function. Consequently, in this study, we chose to determine the diversity of HCMV specific CD8+ T cell responses to eleven HCMV ORFs in a cohort of donors aged 20 nndash; 80 years old as well as their ability to secrete IFN. Finally, we also tested their functional anti-viral capacity using a novel viral dissemination assay.

    We identified substantial CD8+ T cell responses by IFN ELISPOT assays to all eleven of these HCMV proteins and, across the cohort, individuals displayed a range of responses from the tightly focused to highly diverse which were stable over time. CD8+ T cell responses to the HCMV ORFs were highly differentiated and predominantly CD45RA+, CD57+ and CD28-, across the cohort. These highly differentiated cells had the ability to inhibit viral spread even following direct ex-vivo isolation. Taken together, our data argue that HCMV specific CD8+ T cells have effective anti-viral activity irrespective of the viral protein recognized across the whole cohort and despite viral immune evasion.

    IMPORTANCE Human cytomegalovirus (HCMV) is normally carried without clinical symptoms and is widely prevalent in the population, however, it often causes severe clinical disease in individuals with compromised immune responses. HCMV is never cleared after primary infection but persists in the host for life. In HCMV carriers, the immune response to HCMV includes large numbers of virus-specific immune cells and the virus has evolved many mechanisms to evade the immune response. While this immune response seems to protect healthy people from subsequent disease the virus is never eliminated. It has been suggested that this continuous surveillance by the immune system may have deleterious effects in later life. The data presented in this paper examines immune responses from a cohort of donors and shows that these immune cells are effective at controlling the virus and can overcome the viruses lytic cycle immunevasion mechanisms.

  • A detailed study of the interaction between human herpesvirus-6B glycoprotein complex and its cellular receptor, human CD134 [PublishAheadOfPrint]

  • Recently, we identified a novel receptor, CD134, which interacts with the human herpesvirus 6B (HHV-6B) glycoprotein (g)H/gL/gQ1/gQ2 complex and plays a key role in the entry of HHV-6B into target cells. However, details of the interaction between the HHV-6B gH/gL/gQ1/gQ2 complex and CD134 were unknown. In this study, we identified a cysteine-rich domain (CRD)2 of CD134 that is critical for binding to the HHV-6B glycoprotein complex and HHV-6B infection. Furthermore, we found that the expression of HHV-6B gQ1 and gQ2 subunits was sufficient for CD134 binding, which is different from the binding of human herpesvirus-6A (HHV-6A) to its receptor, CD46. Finally, we identified a region in gQ1 critical for HHV-6B gQ1 function. These results contribute much to our understanding of the interaction between this ligand and receptor pair.

    Importance We identified the domain in HHV-6B entry receptor, CD134 and the components in the HHV-6B gH/gL/gQ1/gQ2 complex required for ligand-receptor binding during HHV-6B infection. Furthermore, we identified domains in gQ1s of HHV-6A and -6B and a key amino acid residue in HHV-6B gQ1 required for its function. These data should be basis for further investigation of ligand-receptor interaction in HHV-6A and -6B study.

  • Influenza A and B intertypic reassortment through compatible viral packaging signals [PublishAheadOfPrint]

  • Influenza A and B viruses co-circulate in humans, and together cause disease and seasonal epidemics. These two types of influenza virus are evolutionarily divergent, and exchange of genetic segments inside co-infected cells occurs frequently within types, but never between influenza A and B viruses. Possible mechanisms inhibiting the intertypic reassortment of genetic segments could be due to incompatible protein functions of segment homologs, a lack of processing of heterotypic segments by influenza RNA-dependent RNA polymerase, an inhibitory effect of viral proteins on heterotypic virus function, or an inability to specifically incorporate heterotypic segments into budding virions. Here, we demonstrate that full-length influenza hemagglutinin (HA) of prototype B viruses can complement the function of multiple influenza A viruses. We show that viral non-coding regions were sufficient to drive gene expression for either type A or B influenza virus with its cognate or heterotypic polymerase. The native influenza B HA segment can not be incorporated into influenza A virions. However, by adding the influenza A packaging signals to full length influenza B glycoproteins, we rescued influenza A viruses that possessed HA, NA or both HA and NA of influenza B virus. Furthermore, we show that, similar to single-cycle infectious influenza A virus, influenza B virus cannot incorporate heterotypic transgenes due to packaging signal incompatibilities. Altogether, these results demonstrate that the lack of influenza A and B virus reassortants can be at least in part attributed to incompatibilities in the viral-specific packaging signals required for effective segment incorporation into nascent virions.

    IMPORTANCE Reassortment of influenza A or B viruses provides an evolutionary strategy leading to unique genotypes, which can spawn influenza A viruses with pandemic potential. However, the mechanism preventing intertypic reassortment, or gene exchange between influenza A and B viruses, is not well understood. Nucleotides comprising the coding termini of each influenza A gene segment are required for specific segment incorporation during budding. Whether influenza B shares a similar selective packaging strategy, or if packaging signals prevent intertypic reassortment, remain unknown. Here, we provide evidence suggesting a similar mechanism of influenza B genome packaging. Furthermore, by appending influenza A packaging signals onto influenza B segments, we rescued recombinant A/B viruses that can reassort in vitro with another influenza A virus. These findings suggest that divergent evolution of packaging signals aid in the speciation of influenza A and B virus and are in part responsible for the lack of intertypic viral reassortment.

  • Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 4 Antagonizes IFN{beta} Expression by Targeting NEMO [PublishAheadOfPrint]

  • Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly infectious pathogen that causes severe diseases in pigs and great economic losses to swine industry worldwide. Type I interferons (IFNs) play a crucial role in antiviral immunity. In the present study, we demonstrated that HP-PRRSV strain JXwn06 infection antagonized type I IFN expression induced by poly(I:C) in both porcine alveolar macrophages (PAMs) and blood monocyte-derived macrophages (BMo). Subsequently, we showed that the inhibition of poly(I:C)-induced IFNbbeta; production by PRRSV was dependent on the blocking of NF-B signaling pathways. By screening PRRSV nonstructural and structural proteins, we demonstrated nonstructural protein 4 (nsp4), a viral 3C-like serine protease, significantly suppressed IFNbbeta; expression. Moreover, we verified that nsp4 inhibited NF-B activation induced by the signaling molecules including RIG-I, VISA, TRIF, and IKKbbeta;. Nsp4 was shown to target NF-B essential modulator (NEMO) at the E349-S350 site to mediate its cleavage. Importantly, nsp4 mutants with defective protease activity abolished its ability to cleave NEMO and inhibit IFNbbeta; production. These findings might have implications for us to understand PRRSV pathogenesis and its mechanisms to evade host immune response.

    Importance Porcine reproductive and respiratory syndrome virus (PRRSV) is a major agent of respiratory diseases in pigs. Like many other viruses, PRRSV has evolved a variety of strategies to evade host antiviral innate immunity for survival and propagation. In this study, we show that PRRSV nsp4 is a novel antagonist of NF-B signaling pathway, which is responsible for regulating the expression of type I interferons and other crucial cytokines. We then investigated the underlying mechanism for nsp4 to suppress NF-B mediated IFNbbeta; production. We found that nsp4 interfered with NF-B signaling pathway through the cleavage of NEMO (a key regulator of NF-B signaling) at the site of E349-S350, leading to the down-regulation of IFNbbeta; production induced by poly(I:C). The data presented here may help us for better understanding of PRRSV pathogenesis.

  • The interferon-induced protein, Ifit2, protects mice from infection of the peripheral nervous system by Vesicular stomatitis virus [PublishAheadOfPrint]

  • The interferon system provides the first line of host defense against virus infection. Mouse pathogenesis studies have revealed the importance of specific interferon-induced proteins in providing protection against specific viruses. We have previously reported that one such protein, Ifit2, protects neurons of the central nervous system from intranasal infection by the neurotropic rhabdovirus, vesicular stomatitis virus (VSV). Here, we demonstrate that Ifit2 protects the peripheral nervous system from VSV infection as well. In Ifit2-/- mice, VSV, injected subcutaneously into the footpad, entered the proximal lymph node where it replicated and infected the nodal nerve endings. The infection spread to the sciatic nerve, the spinal cord and the brain, causing paralysis. In contrast, in the wild-type mice, although VSV replicated equally well in the lymph node, infection of the sciatic nerve and the rest of the nervous system was impaired, thus preventing paralysis. Ifit2 protected only the nervous system from VSV infection; other tissues were well protected even in Ifit2-/- mice. These results indicate that Ifit2 is the interferon-induced protein that prevents VSV infection of neurons of both the peripheral and the central nervous systems, thus inhibiting the consequent neuropathy; but it is dispensable for protecting the cells of other tissues from VSV infection.

    IMPORTANCE Although viral infection is quite common, the immune system effectively protects us from viral diseases. A major part of this protection is mediated by interferon, the antiviral cytokine secreted by virus-infected cells. To empower the neighboring uninfected cells in combating the oncoming infection, interferon induces the synthesis of more than 200 new proteins, many of which have antiviral activities. The virus studied here, vesicular stomatitis virus (VSV), like its relative, rabies virus, can cause neuropathy in mice, if it enters the peripheral nervous system through skin lesions; however, interferon can protect neurons from VSV infection. In this study, we have identified a specific interferon-induced protein, Ifit2, as the protein that protects neurons from VSV infection. Surprisingly, Ifit2 was not needed to protect other cell types from VSV. Our results indicate that the effector antiviral proteins of the interferon system have highly specialized functions.

  • Expansion of genotypic diversity and establishment of 2009 H1N1 pandemic-origin internal genes in pigs in China [PublishAheadOfPrint]

  • llsquo;Two-way' transmission of influenza viruses between humans and swine has been frequently observed and the occurrence of the 2009 H1N1 pandemic influenza (pdm/09) demonstrated that swine-origin viruses could facilitate the genesis of a pandemic strain. Although multiple introductions to and reassortment in swine of the pdm/09 virus have been repeatedly reported in both Eurasia and the Americas, its long-term impact on the development of swine influenza viruses (SIVs) has not been systematically explored. Our comprehensive evolutionary studies on the complete genomes of 387 SIVs obtained from 2009 to 2012 in influenza surveillance in China revealed 17 reassortant genotypes with pdm/09-origin genes. Even though the entire 2009 pandemic virus and its surface genes cannot persist, its internal genes have becoming established and are now the predominant lineages in pigs in the region. The main persistent pdm/09-origin reassortant forms had at least 5 pdm/09-origin internal genes and their surface genes primarily of European avian-like (EA) or human H3N2-like SIV origin. These findings represent a marked change to the evolutionary patterns and ecosystem of SIVs in China. It is possible that the pdm/09-origin internal genes may be in the process of replacing EA- or triple reassortant-like internal genes. These alterations to the SIV gene pool need to be continually monitored to assess changes in the potential for SIVs to transmit to humans.

    Importance Shortly after the emergence of the 2009 pandemic H1N1 (pdm/09) influenza virus, it was transmitted from humans to pigs and this continues to occur around the world. Many reassortants between pdm/09-origin viruses and enzootic swine influenza viruses (SIVs) have been detected. However, the long-term impact of pdm/09-origin viruses on the SIV gene pool, which could lead to the generation of influenza viruses with the potential to infect humans, has not been systematically examined. From extensive surveillance of SIVs over a 38-month period in southern China, it was found that, although neither complete pdm/09 viruses nor their surface genes could persist in pigs, their internal genes did persist. Over the survey period, these internal genes became predominant, potentially replacing those of the enzootic SIV lineages. The altered diversity of the SIV gene pool needs to be closely monitored for changes in the potential of SIVs to transmit to humans.

  • Human cytomegalovirus upregulates expression of the lectin, galectin-9, via induction of interferon-{beta} [PublishAheadOfPrint]

  • Regulation of the lectin, Galectin-9 (Gal-9), was investigated for the first time during Human Cytomegalovirus (HCMV) infection. Gal-9 transcription was significantly upregulated in transplant recipients with reactivated HCMV in vivo. In vitro Gal-9 was potently upregulated by HCMV independently of viral gene expression with interferon-bbeta; (IFN-bbeta;) identified as the mediator of this effect. This study defines an immunoregulatory protein potently increased by HCMV infection and a novel mechanism to control Gal-9 through IFN-bbeta; induction.

  • Dual modulation of type I interferon response by Bluetongue virus [PublishAheadOfPrint]

  • Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus that causes an economically important disease in ruminants. BTV infection is a strong inducer of type I interferon (IFN-I) in multiple cell types. It has been shown recently that BTV and more specifically the non-structural protein NS3 of BTV are able to modulate the IFN-I synthesis pathway. However, nothing is known about the ability of BTV to counteract IFN-I signaling. Here, we investigated the effect of BTV on the IFN-I response pathway and more particularly the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription protein (STAT) signaling pathway. We found that BTV infection triggered the expression of IFN-stimulated genes (ISGs) in A549 cells. However, upon stimulation with external IFN-I, we showed that activation of the IFN-stimulated response element (ISRE) promoter and expression of ISGs were inhibited in BTV-infected cells. We found that this inhibition involved two different mechanisms dependent on the time of infection. After overnight infection, BTV blocked specifically the phosphorylation and nuclear translocation of STAT1. This inhibition correlated with the re-distribution of STAT1 in regions adjacent to the nucleus. At a later time point of infection, BTV was found to interfere with the activation of other key components of the JAK/STAT pathway and to induce the downregulation of JAK1 and TYK2 protein expression. Overall, our study indicates for the first time that BTV is able to interfere with the JAK/STAT pathway to modulate the IFN-I response.

    Importance Bluetongue virus (BTV) causes a severe disease in ruminants and has an important impact on the livestock economy in endemic areas such as Africa. The emergence of strains, such as serotype 8 in Europe in 2006, can lead to important economic losses due to commercial restrictions and prophylactic measures. It has been known for many years that BTV is a strong inducer of type I interferon (IFN-I) in vitro and in vivo in multiple cell types. However, the ability of BTV to interact with the IFN-I system remains unclear. Here, we report that BTV is able to modulate the IFN-I response by interfering with the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription protein (STAT) signaling pathway. These findings contribute to the knowledge on how BTV infection interferes with the host's innate immune response and becomes pathogenic. This will also be important for the design of efficacious vaccine candidates.

  • Autographa californica Multiple Nucleopolyhedrovirus DNA Polymerase C-terminus is Required for Nuclear Localization and Viral DNA Replication [PublishAheadOfPrint]

  • The DNA polymerase (DNApol) of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is essential for viral DNA replication. The DNApol exonuclease and polymerase domains are highly conserved and are considered to be functional in DNA replication. However the role of the DNApol C-terminus has not yet been characterized. To identify whether only the exonuclease and polymerase domains are sufficient for viral DNA replication, several DNApol C-terminal truncations were cloned into a dnapol-null AcMNPV bacmid with a GFP reporter. Surprisingly most of the truncation constructs, despite containing both exonuclease and polymerase domains could not rescue viral DNA replication and viral production in bacmid transfected Sf21 cells. Moreover GFP fusions of these same truncations failed to localize to the nucleus. Truncation of the C-terminal amino acids 950 to 984 showed nuclear localization but allowed for only a limited and delayed viral spread. The C-terminus contains a typical bipartite NLS motif at residues 804 to 827 and a monopartite NLS motif at residues 939 to 948, respectively. Either NLS, as a GFP fusion peptide, localized to the nucleus but both NLSs were required for nuclear localization of DNApol. Alanine substitutions of a highly conserved baculovirus DNApol sequence at AcMNPV DNApol amino acids 972 to 981 demonstrated its importance for virus production and DNA replication. Collectively the data indicated that the C-terminus of AcMNPV DNApol contains two NLSs and a conserved motif all of which are required for nuclear localization of DNApol, viral DNA synthesis and viral production.

    Importance The baculovirus DNA polymerase (DNApol) is a highly specific polymerase allowing for viral DNA synthesis and hence virus replication in infected insect cells. We demonstrated that the exonuclease and polymerase domains of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) alone are insufficient for viral DNA synthesis and virus replication. Rather we identified three features, including two nuclear localization signals and a highly conserved 10 aa sequence in the AcMNPV DNApol C terminus, all three of which are important for both nuclear localization of DNApol and for DNApol activity as measured by viral DNA synthesis and virus replication.

  • Impact of HLA-selection pressure on HIV fitness at a population level [PublishAheadOfPrint]

  • Previous studies have demonstrated that effective CTL responses drive selection of escape mutations that reduce viral replication capacity (VRC). Escape mutations, including those with reduced VRC, can be transmitted and accumulate in a population. Here we compared two anti-retroviral therapy (ART)-naiiuml;ve HIV B-clade infected cohorts, in Mexico and Barbados, in which the most protective HLA alleles (HLA-B*27/57/58:01/81:01) are differentially expressed, at 8% and 34%, respectively. Viral loads were significantly higher in Mexico compared to Barbados (median 40,774 versus 14,200, p=llt;0.0001), and absolute CD4+T-cell counts somewhat lower (median 380/mm3 vs 403/mm3, p=0.007). We tested the hypothesis that the disparate frequencies of these protective HLA alleles would be associated with a higher VRC at the population level in Mexico. Analysis of VRC in subjects in each cohort, matched for CD4+T-cell count, revealed that the VRC was indeed higher in the Mexican cohort (mean 1.13 versus 1.03, p=0.0025). Although CD4 counts were matched, viral loads remained significantly higher in the Mexican subjects (p=0.04). This VRC difference was reflected by a significantly higher frequency in the Barbados cohort of HLA-B*27/57/58:01/81:01-associated Gag escape mutations previously shown to incur a fitness cost on the virus (p=0.004), a difference between the two cohorts that remained statistically significant even in subjects not expressing these protective alleles (p=0.01). These data suggest that viral setpoint and disease progression rates at the population level may be significantly influenced by the prevalence of protective HLA alleles such as HLA-B*27/57/58:01/81:01, and that CD4 count-based guidelines to initiate antiretroviral therapy (ART) may need to be modified accordingly, to optimize the effectiveness of treatment-for-prevention strategies and reduce HIV transmission rates to the absolute minimum.

    IMPORTANCE Immune control of HIV at an individual level is strongly influenced by HLA class I genotype. HLA class I molecules mediating effective immune control, such as HLA-B*27 and HLA-B*57, are associated with the selection of escape mutants that reduce viral replicative capacity. The escape mutants selected in infected patients can be transmitted and affect viral load and CD4 count in the recipient. These findings prompt the hypothesis that the frequency of protective alleles in a population may affect viral setpoints and rates of disease progression in that population. These studies of Mexico and Barbados, where the prevalence of protective HLA alleles is 8% and 34%, respectively, support this hypothesis. These data suggest that antiretroviral therapy (ART) treatment-for-prevention strategies will be less successful in populations such as Mexico where viral loads are higher for a given CD4 count. Consideration may therefore usefully be given to ART initiation at higher absolute CD4 counts in such populations to optimize the impact of ART for prevention.

  • Development of a Gaussia luciferase-based human norovirus protease reporter system: cell type-specific profile of Norwalk virus protease precursors and evaluation of inhibitors [PublishAheadOfPrint]

  • Norwalk virus (NV) is the prototype strain of human noroviruses (HuNoVs), a group of positive-strand RNA viruses in the Caliciviridae family and leading cause of epidemic gastroenteritis worldwide. Investigation of HuNoV replication and development of antiviral therapeutics in cell culture remain challenging tasks. Here we present NoroGLuc, a HuNoV protease reporter system based on a fusion of NV p41 protein with a naturally secreted Gaussia luciferase (GLuc), linked by the p41/p22 cleavage site for NV protease (Pro). Trans cleavage of NoroGLuc by NV Pro or Pro precursors results in release and secretion of an active GLuc. Using this system, we observed a cell type-specific activity profile of NV Pro and Pro precursors, suggesting that the activity of NV Pro is modulated by other viral proteins in the precursor forms and strongly influenced by cellular factors. NoroGLuc was also cleaved by Pro and Pro precursors generated from replication of NV stool RNA in transfected cells, resulting in a measurable increase of secreted GLuc. Truncation analysis revealed that the N-terminal membrane association domain of NV p41 is critical for NoroGLuc activity. Although designed for NV, a genogroup GI.1 norovirus, NoroGLuc also efficiently detects Pro activities from GII.3 and GII.4 noroviruses. At non-cytotoxic concentrations, protease inhibitors ZnCl2 and TLCK exhibited dose-dependent inhibitory effects on a GII.4 Pro by NoroGLuc assay. These results establish NoroGLuc as a pan-genogroup HuNoV protease reporter system that can be used for the study of HuNoV proteases and precursors, monitoring of viral RNA replication, and evaluation of antiviral agents.

    IMPORTANCE Human noroviruses are leading cause of epidemic gastroenteritis worldwide. Currently there are no vaccines or antiviral drugs available to counter these highly contagious viruses. These viruses are currently non-cultivatable in cell culture. Here we report the development of a novel cell-based reporter system called NoroGLuc that can be used for studying norovirus replication and also for screening/evaluation of antiviral agents. This system is based on the fusion between viral protein p41 and a naturally secreted Gaussia luciferase (GLuc) with a cleavage site that can be recognized by the viral protease. Cleavage of this fusion protein by the viral protease results in the release and secretion of an active GLuc. Using NoroGLuc we demonstrated a cell type-specific activity profile of the viral protease and its precursors and dose-dependent inhibitory effects of two protease inhibitors. This novel reporter system should be useful in probing norovirus replication and evaluating antiviral agents.

  • Structure Analysis of the Major Capsid Proteins of the Human Polyomavirus 6 and 7 Reveals an Obstructed Sialic Acid Binding Site [PublishAheadOfPrint]

  • Human polyomaviruses 6 and 7 (HPyV6 and HPyV7, respectively) are commonly found on human skin. We have determined X-ray structures of their major capsid proteins, VP1 at resolutions of 1.8 and 1.7 AAring;, respectively. In polyomaviruses, VP1 commonly determines antigenicity as well as cell-surface receptor specificity, and the protein is therefore linked to attachment, tropism, and ultimately viral pathogenicity. The structures of HPyV6 and HPyV7 VP1 reveal uniquely elongated loops that cover the bulk of the outer virion surfaces, obstructing a groove that binds sialylated glycan receptors in many other polyomaviruses. In support of this structural observation, interactions of VP1 with aalpha;2,3- and aalpha;2,6-linked sialic acids could not be detected in solution by Nuclear Magnetic Resonance spectroscopy. Single-cell binding studies indicate that sialylated glycans are likely not required for initial attachment to cultured human cells. Our findings establish distinct antigenic properties of HPyV6 and HPyV7 capsids and indicate that these two viruses engage non-sialylated receptors.

    Importance (150) Eleven new human polyomaviruses, including the skin viruses HPyV6 and HPyV7, have been identified during the last decade. In contrast to better-studied polyomaviruses, routes of infection, cell tropism and entry pathways of many of these new viruses remain largely mysterious.

    Our high-resolution X-ray structures of major capsid proteins VP1 from HPyV6 and HPyV7 reveal critical differences in surface morphology in comparison to all other known polyomavirus structures. A groove that engages specific sialic acid-containing glycan receptors in related polyomaviruses is obstructed and HPyV6 and HPyV7 VP1 do not interact with sialylated compounds in solution or on cultured human cells. A comprehensive comparison with other structurally characterized polyomavirus VP1 proteins enhances our understanding of molecular determinants that underlie receptor-specificity, antigenicity and ultimately pathogenicity within the polyomavirus family and highlight the need for structure-based analysis to better define phylogenetic relationships within the growing polyomavirus family and perhaps also other viruses.

  • Genogroup IV and VI Canine Noroviruses Interact with Histo-Blood Group Antigens [PublishAheadOfPrint]

  • Human noroviruses (HuNV) are a significant cause of viral gastroenteritis in man worldwide. HuNV attaches to cell surface carbohydrate structures known as histo-blood group antigens (HBGAs) prior to internalization, and HBGA polymorphism amongst human populations is closely linked to susceptibility to HuNV. Noroviruses are divided into 6 genogroups, with human strains grouped into genogroups I, II and IV. Canine norovirus (CNV) is a recently discovered pathogen in dogs, with strains classified into genogroups IV and VI. Whereas it is known that GI-GIII noroviruses bind to HBGAs and GV noroviruses recognize terminal sialic acid residues, the attachment factors for GIV or GVI noroviruses have not been reported. This study sought to determine the carbohydrate binding specificity of CNV, and compare this to the binding specificity of noroviruses from other genogroups. A panel of synthetic oligosaccharides were used to assess the binding specificity of CNV virus-like particles (VLPs), and identified aalpha;1,2 fucose as a key attachment factor. CNV VLP binding to canine saliva and tissue samples using ELISAs and immunohistochemistry confirmed that aalpha;1,2 fucose-containing H and A antigens of the HBGA family were recognized by CNV. Phenotyping studies demonstrated expression of these antigens in a population of dogs. The virus-ligand interaction was further characterized using blockade studies, cell lines expressing HBGAs and enzymatic removal of candidate carbohydrates from tissue sections. Recognition of HBGAs by CNV provides new insights into evolution of noroviruses and raises concerns regarding the potential for zoonotic transmission of CNV to humans.

    Importance Infections with human norovirus cause acute gastroenteritis in millions of people each year worldwide. Noroviruses can also affect non-human species, and are divided into 6 different groups based on their capsid sequences. Human noroviruses in genogroups I and II interact with histo-blood group antigen carbohydrates, bovine noroviruses (genogroup III) interact with alphaGal carbohydrates, and murine norovirus (genogroup V) recognizes sialic acids. The canine-specific strains of norovirus are grouped into genogroups IV and VI, and this study is the first to characterize which carbohydrate structures they can recognize. Using canine norovirus virus-like particles, this work shows that representative genogroup IV and VI viruses can interact with histo-blood groups antigens. The binding specificity of canine noroviruses is therefore very similar to the human norovirus strains classified into genogroups I and II. This raises interesting questions about the evolution of noroviruses, and suggests it may be possible for canine norovirus to infect man.

  • Creation of Rift Valley fever viruses with four-segmented genomes reveals flexibility in bunyavirus genome packaging [PublishAheadOfPrint]

  • Bunyavirus genomes comprise a small (S), medium (M) and a large (L) RNA segment of negative polarity. Although the untranslated regions (UTRs) have been shown to comprise signals required for transcription, replication and encapsidation, the mechanisms that drive the packaging of at least one S, M and L segment into a single virion to generate infectious virus are largely unknown. One of the most important members of the Bunyaviridae family that causes devastating disease in ruminants and occasionally humans is the Rift Valley fever virus (RVFV). Here we studied the flexibility of RVFV genome packaging by splitting the glycoprotein precursor gene, encoding the (NSm)GnGc polyprotein, into two individual genes encoding either (NSm)Gn or Gc. Using reverse-genetics, six viruses with a segmented glycoprotein precursor gene were rescued, varying from a virus comprising 2 S-type segments in the absence of an M-type segment to a virus consisting of 4 segments (RVFV-4s) of which 3 are M-type. Despite that all virus variants were able to grow in mammalian cell lines, they were unable to spread efficiently in cells of mosquito origin. Moreover, in vivo studies demonstrated that RVFV-4s is unable to cause disseminated infection and disease in mice, even in the presence of the main virulence factor NSs, but induced a protective immune response against a lethal challenge with wild-type virus. In summary, splitting bunyavirus glycoprotein precursor genes provides new opportunities to study bunyavirus genome packaging and offers new methods to develop next-generation live-attenuated bunyavirus vaccines.

    Importance Rift Valley fever virus (RVFV) causes devastating disease in ruminants and occasionally humans. Virions capable of productive infection comprise at least one copy of the small (S), medium (M) and large (L) RNA genome segments. The M segment encodes a glycoprotein precursor (GPC) protein that is co-translationally cleaved into Gn and Gc, which are required for virus entry and fusion. Here, we studied the flexibility of RVFV genome packaging and developed experimental live-attenuated vaccines by applying a unique strategy based on the splitting of the GnGc open reading frame. Several RVFV variants, varying from viruses comprising 2 S-type segments to viruses consisting of 4 segments (RVFV-4s) of which 3 are M-type could be rescued and were shown to induce a rapid protective immune response. Altogether, the segmentation of bunyavirus GPCs provides a new method for studying bunyavirus genome packaging and facilitates the development of novel live-attenuated bunyavirus vaccines.

  • Rapid expansion of CD8+ T cells in wildtype and type I interferon receptor deficient mice correlates with protection after low-dose emergency immunization with Modified Vaccinia virus Ankara [PublishAheadOfPrint]

  • Immunization with Modified Vaccinia virus Ankara (MVA) can rapidly protect mice against lethal ectromelia virus (ECTV) infection, serving as an experimental model for severe systemic infections. Importantly, this early protective capacity of MVA vaccination completely depends on virus-specific cytotoxic CD8+ T cell responses. We used MVA vaccination in the mousepox challenge model using ECTV infection to investigate the previously unknown factors required to elicit rapid protective T cell immunity in normal C57BL/6 mice and in mice lacking the interferon alpha/beta receptor (IFNAR-/-). We found a minimal dose of 105 plaque-forming units of MVA vaccine fully sufficient to allow robust protection against lethal mousepox, as assessed by the absence of disease symptoms and failure to detect ECTV in organs from vaccinated animals. Moreover, MVA immunization at low dosage also protected IFNAR-/- mice, indicating efficient activation of cellular immunity even in the absence of type I interferon signaling. When monitoring for virus specific CD8+T cell responses in mice vaccinated with the minimal protective dose of MVA, we found significantly enhanced levels of antigen specific T cells in animals that were MVA vaccinated and ECTV challenged compared to mice that were only vaccinated. The initial priming of naiiuml;ve CD8+ T cells by MVA immunization appears highly efficient, and even at low doses, mediates a rapid in vivo burst of pathogen-specific T cells upon challenge. Our findings define striking requirements for protective emergency immunization against severe systemic infections with orthopoxviruses.

    IMPORTANCE We demonstrate that single-shot low dose immunizations with vaccinia virus MVA can rapidly induce T cell mediated protective immunity against lethal orthopoxvirus infections. Our data provides new evidence for an efficient protective capacity of vaccination with replication deficient MVA. These data are of important practical relevance for public health as the effectiveness of a safety-tested next-generation smallpox vaccine based on MVA is still debated. Furthermore, producing sufficient amounts of vaccine is considered a major challenge should an outbreak occur. Moreover, prevention of other infections may require rapidly protective immunization, hence MVA could be an extremely useful vaccine for delivering heterologous T cell antigens, particularly for infectious diseases that fit a scenario of emergency vaccination.

  • Spatial localization of the Ebola glycoprotein mucin-like domain using cryo-electron tomography [PublishAheadOfPrint]

  • The Ebola virus glycoprotein mucin-like domain (MLD) is implicated in Ebola cell entry and immune evasion. Using cryo-electron tomography of Ebola virus-like particles, we determined a three-dimensional structure for the full-length glycoprotein in a near-native state and compared it to that of a glycoprotein lacking the MLD. Our results, which show that the MLD is located at the apex and the sides of each glycoprotein monomer, provide a structural template for analysis of MLD function.

  • Virus-like Particle Secretion and Genotype-dependent Immunogenicity of Dengue Virus Serotype 2 DNA Vaccine [PublishAheadOfPrint]

  • Dengue virus (DENV), composed of four distinct serotypes, is the most important and fast emerging arthropod-borne pathogen imposing a great deal of economic and public health burden. We constructed five genotypes of dengue virus serotype 2 (DENV-2) DNA vaccine candidates and evaluated immunogenicity, neutralizing (Nt) activity of elicited antibodies and the protective efficacy elicited in mice immunized with the vaccine candidates. We observed a significant correlation between the level of in vitro VLP secretion, the elicited antibody response and protective efficacy by different genotypic DNA vaccines in immunized mice. However, higher total IgG antibodies did not always translate into higher Nt antibodies against homologous and heterologous viruses. We also found that in contrast to previous reports, more than fifty percent of the total IgG are targeting EDIII of E protein and a substantial fraction of this population are interdomain antibodies that are highly neutralizing flavivirus sub-group cross-reactive, such as monoclonal antibody 1B7-5. In addition, the lack of critical epitope(s) in sylvatic virus recognized by interdomain antibodies, could be the major cause of poor protection of pVD2-Asian 1 genotype vaccinated mice from the lethal challenge of sylvatic virus. In conclusion, although pVD2-Asian 1 is immunogenic, elicits sufficient Nt antibody titers against all DENV-2 genotypes, and provides 100% protection against homologous Asian 1 and heterologous Cosmopolitan genotype virus challenge, it is critical to monitor the potential emergence of sylvatic genotype viruses since current vaccine candidates under development may not protect vaccinated humans from these viruses.

    IMPORTANCE Five genotype-specific dengue virus serotype 2 (DENV-2) DNA vaccine candidates were evaluated for their immunogenicity, homologous and heterologous neutralizing (Nt) antibody titers and cross-genotype protection in murine model. Protective immunity elicited by our prototype vaccine candidate (Asian 1, strain 16681) in mice was protective against other genotype viruses but not against the sylvatic genotype virus, whose emergence and potential risk after introduction into human population have previously been demonstrated. The underlying mechanism of lack of protection elicited by the prototype vaccine may at least be contributed by the absence of a flavivirus sub-group cross-reactive, highly neutralizing 1B7-5-like epitope in the sylvatic DENV-2. DENV DNA vaccine directs the synthesis and assembly of virus-like particles (VLP), induces immune responses similar to live-attenuated vaccines; and its flexibility permits the fast deployment of vaccine to combat emerging viruses such as sylvatic genotype viruses. Enhanced VLP secretion by the replacement of EDI-II of pVD2-Cosmopolitan with Asian 1 EDI-II has elicited significantly higher total IgG and Nt antibodies and suggests a novel approach to enhance immunogenicity of DNA vaccine. A DENV vaccine capable of eliciting protective immunity against existing and emerging genotype viruses should be the focus of future DENV vaccine development.

  • Sequence of events in measles virus replication: role of phosphoprotein-nucleocapsid interactions [PublishAheadOfPrint]

  • The genome of non-segmented negative strand RNA viruses is tightly embedded within a nucleocapsid made of a nucleoprotein (N) homopolymer. To ensure processive RNA synthesis, the viral polymerase L in complex with its co-factor phosphoprotein (P) binds the nucleocapsid that constitutes the functional template. Measles virus P and N interact through two binding sites. While binding of the P amino-terminus with the core of N (NCORE) prevents illegitimate encapsidation of cellular RNA, the interaction between their C-terminal domains, PXD and NTAIL is required for viral RNA synthesis. To investigate the binding dynamics between the two latter domains, the PXD F497 residue that makes multiple hydrophobic intramolecular interactions was mutated. Using a quantitative mammalian protein complementation assay, and recombinant viruses, we found that an increase in PXD to NTAIL binding strength is associated with a slower transcript accumulation rate and that abolishing the interaction renders the polymerase non-functional. The use of a newly developed system allowing conditional expression of wild type or mutated P genes, revealed that the loss of the PXD-NTAIL interaction results in reduced transcription by preformed transcriptases suggesting reduced engagement on the genomic template. These intracellular data indicate that the viral polymerase entry into and progression along its genomic template relies on a protein-protein interaction that serves as a tightly controlled dynamic anchor.

    Importance: Mononegavirales have a unique machinery to replicate RNA. Processivity of their polymerase is only achieved when the genome template is entirely embedded into a helical homopolymer of nucleoproteins that constitutes the nucleocapsid. The polymerase binds to the nucleocapsid template through the phosphoprotein. How the polymerase complex enters and travels along the nucleocapsid template to ensure uninterrupted synthesis of up to ~6700 nucleotide long messenger RNAs from 6 to 10 consecutive genes is unknown. Using a quantitative protein complementation assay and a biGene-biSilencing system allowing conditional expression of two P genes copies, the role of the P to N interaction in polymerase function was further characterized. We report here a dynamic protein anchoring mechanism that differs from all other known polymerases that rely only onto a sustained and direct binding to their nucleic acid template.

  • The histone deacetylase inhibitor vorinostat (SAHA) increases the susceptibility of uninfected CD4+ T cells to HIV by increasing the kinetics and efficiency of post-entry viral events [PublishAheadOfPrint]

  • Latently infected cells remain a primary barrier to eradication of HIV-1. Over the past decade, a better understanding of the molecular mechanisms by which latency is established and maintained has led to the discovery of a number of compounds that selectively reactivate latent proviruses without inducing polyclonal T cell activation. Recently, the histone deacetylase (HDAC) inhibitor vorinostat has been demonstrated to induce HIV transcription from latently infected cells when administered to patients. While vorinostat would be given in the context of antiretroviral therapy, infection of new cells by induced virus remains a clinical concern. Here, we demonstrate that vorinostat significantly increases the susceptibility of CD4+ T cells to infection by HIV in a dose- and time-dependent manner that is independent of receptor and coreceptor usage. Vorinostat does not enhance viral fusion with cells, but rather enhances the kinetics and efficiency of post-entry viral events including reverse transcription, nuclear import, and integration and enhances viral production in a spreading infection assay. Selective inhibition of the cytoplasmic class IIb histone deacetylase (HDAC)-6 with tubacin recapitulated the effect of vorinostat. These findings reveal a previously unknown, cytoplasmic effect of HDAC inhibitors promoting productive infection of CD4+ T cells that is distinct from their well-characterized effects on nuclear histone acetylation and LTR transcription. Our results indicate that careful monitoring of patients and ART intensification are warranted during vorinostat treatment and indicate that HDAC inhibitors that selectively target nuclear class I HDACs could reactivate latent HIV without increasing the susceptibility of uninfected cells to HIV.

    Importance HDAC inhibitors, particularly vorinostat, are currently being investigated clinically as a part of a llsquo;shock and kill' strategy to purge latent reservoirs of HIV. We demonstrate here that vorinostat increases the susceptibility of uninfected CD4+ T cells to infection with HIV, raising clinical concerns that vorinostat may reseed the viral reservoirs it is meant to purge, particularly during conditions of suboptimal drug exposure. We demonstrate that vorinostat acts following viral fusion and enhances the kinetics and efficiency of reverse transcription, nuclear import, and integration. The effect of vorinostat was recapitulated using the cytoplasmic HDAC6 inhibitor tubacin, revealing a novel and previously unknown cytoplasmic mechanism of HDAC inhibitors on HIV replication that is distinct from their well-characterized effects of LTR-driven gene expression. Moreover, our results suggest that treatment of patients with class I-specific HDAC inhibitors could induce latent viruses without increasing the susceptibility of uninfected cells to HIV.


  • Dendritic cells (DCs) are fundamental for the initiation of immune responses and are important players in AIDS immunopathogenesis. The modulation of DC functional activities represents a strategic mechanism for HIV-1 to evade immune surveillance. Impairment of DC function may result from bystander effects of HIV-1 envelope proteins independently of direct HIV-1 infection. In this study, we report that exposure of immature monocyte-derived DCs (MDDCs) to HIV-1 R5 gp120 resulted in the CCR5-dependent production of IL-6 via MAPK/NF-kB pathways. IL-6 in turn activated STAT3 by an autocrine loop. Concomitantly, gp120 promoted an early activation of STAT3 that further contributed to IL-6 induction. This activation paralleled a concomitant up-regulation of the STAT3 inhibitor PIAS3. Notably, STAT3/IL-6 pathway activation was not affected by the CCR5 specific ligand CCL4.

    These results identify STAT3 as a key signaling intermediate activated by gp120 in MDDCs and highlight the existence of a viral-induced dysregulation of the IL-6/STAT3 axis. HIV-1 gp120 signaling through STAT3 may provide an explanation for the impairment of DC function observed upon HIV exposure.

    Importance This study provides new evidence for the molecular mechanisms and signaling pathways triggered by HIV-1 gp120 in human DCs, in the absence of productive infection, emphasizing a role of aberrant signaling in early virus-host interaction, contributing to viral pathogenesis. We identified STAT3 as a key component in the gp120-mediated signaling cascade involving MAPK and NF-kB components, and ultimately leading to IL-6 secretion.

    STAT3 is now recognized as a key regulator of DC functions. Thus, the identification of this transcription factor as a signaling molecules mediating some of the gp120 biological effects, unveils a new mechanism by which HIV-1 may deregulate DC functions and contribute to AIDS pathogenesis.

  • A non-SUMOylated Tax protein is still functional for NF-{kappa}B pathway activation [PublishAheadOfPrint]

  • Whether NF-B promoter transactivation by the HTLV-1 Tax protein requires Tax SUMOylation is still a matter of debate. In this study, we revisited the role of Tax SUMOylation using a strategy based on the targeting of Ubc9, the unique E2 SUMO conjugating enzyme. We show that either a catalytically inactive form of Ubc9 (Ubc9-C93S) or Ubc9 siRNA dramatically reduce Tax conjugation to endogenous SUMO-1 or SUMO-2/3, demonstrating that as expected, Tax SUMOylation is under the control of the catalytic activity of Ubc9. We further report that a non-SUMOylated Tax protein produced in 293T cells is still able to activate either a transfected or an integrated NF-B reporter promoter and to induce expression of a NF-B-regulated endogenous gene. Importantly, blocking Ubc9 activity in T cells also results in the production of a non-SUMOylated Tax still fully functional for the activation of a NF-B promoter. These results provide the definitive evidence that Tax SUMOylation is not required for NF-B-driven gene induction.

    IMPORTANCE Human T-cell leukemia virus type 1 is able to transform CD4+ T lymphocytes. The viral oncoprotein Tax plays a key role in this process by promoting cell proliferation and survival, mainly through permanent activation of the NF-B pathway. Elucidating the molecular mechanisms involved in NF-B pathway activation by Tax is therefore a key issue to understand HTLV-1-mediated transformation. Tax SUMOylation was initially proposed to be critical for Tax-induced NF-B promoter activation, which was challenged by our later observation that a low SUMOylated Tax mutant was still functional for activation of NF-B promoters. To clarify the role of Tax SUMOylation, we set up a new approach based on the inhibition of the SUMOylation machinery in Tax-expressing cells. We show that blocking the SUMO conjugating enzyme Ubc9 abolishes Tax SUMOylation and that a non-SUMOylated Tax still activates NF-B promoters in either adherent cells or T cells.

  • SGHBV Envelope and Capsid Proteins Independently Contribute to its Ability to Package Capsids Containing ssDNA in Virions [PublishAheadOfPrint]

  • Hepadnaviruses selectively package capsids containing mature dsDNA genomes in virions. Snow goose hepatitis B virus (SGHBV) is the only known hepadnavirus that packages capsids containing ssDNA in virions. We found that cells replicating SGHBV produce virions containing ssDNA as efficiently as virions containing mature dsDNA. We determined that SGHBV capsid and envelope proteins independently contribute to the production of virions containing ssDNA; with the capsid protein (Cp) making a larger contribution. We identified that amino acid residues 74 and 107 of SGHBV Cp contribute to this feature of SGHBV. When we changed these residues in duck hepatitis B virus (DHBV) Cp, capsids containing immature ssDNA were packaged in virions. This result suggests that residues 74 and 107 contribute to the appearance of the "capsid packaging signal" on the surface of capsids and interact with the envelope proteins during virion formation. We also found that cells replicating SGHBV package a larger fraction of the total dsDNA they synthesize into virions compared to DHBV. We determined that the SGHBV envelope proteins are responsible for this property of SGHBV. Determining if the ability of SGHBV envelope proteins to cause the formation of virions containing ssDNA is related to its ability to support high levels of virion production or if these two properties are mechanistically distinct will provide insights into virion morphogenesis.

    Importance Cells replicating hepadnaviruses contain cytoplasmic capsids that contain mature and immature genomes. However, only capsids containing mature dsDNA genomes are packaged in virions. A mechanistic understanding of this phenomenon, which is currently lacking, is critical to understanding the process of hepadnaviral virion morphogenesis. In this study, we determined that the envelope proteins contribute to the ability of hepadnaviruses to selectively produce virions containing mature dsDNA genomes. Our finding sheds new light on the mechanisms underlying virion morphogenesis and challenges the dogma that "capsid maturation", and therefore the capsid protein (Cp), is solely responsible for the selective production of virions containing mature dsDNA genomes. Further, we identified amino acid residues of Cp that contribute to its ability to cause the selective production of virions containing mature dsDNA genomes. Future studies on the role of these residues in selective secretion will broaden our understanding of this poorly understood aspect of virus replication.

  • Genomic diversity of Epstein-Barr virus genomes isolated from primary nasopharyngeal carcinoma biopsies [PublishAheadOfPrint]

  • Undifferentiated nasopharyngeal carcinoma (NPC) has 100% association with Epstein-Barr virus (EBV). However, only three EBV genomes isolated from NPC patients have been sequenced to date and the role of EBV genomic variations in the pathogenesis of NPC is unclear. Our project aimed to obtain the sequences of EBV genomes in multiple NPC biopsies in the same geographic location in order to reveal their sequence diversity. Three published EBV (B95-8, C666-1, and HKNPC1) genomes were first re-sequenced using the sequencing workflow of target enrichment of EBV DNA by hybridization followed by next generation sequencing, de novo assembly and joining of contigs by Sanger sequencing. The sequences of eight NPC biopsy-derived EBV genomes, designated HKNPC2 to -9, were then determined. They harbored 1736 variations in total, including 1601 substitutions, 64 insertions and 71 deletions, in comparison with the reference EBV. Furthermore, genes encoding latent, early lytic, tegument and glyco-proteins were found to contain non-synonymous mutations of potential biological significance. Phylogenetic analysis showed that HKNPC6 and -7 genomes, which were isolated from tumor biopsies of advanced metastatic NPC cases, to be distinct from the other six NPC-EBV genomes, suggesting the presence of at least two parental lineages of EBV among the NPC-EBV genomes. In conclusion, much greater- sequence diversity among EBV isolates derived from NPC biopsies is demonstrated on a whole-genome level through a complete sequencing workflow. Large-scale sequencing and comparison of EBV genomes isolated from NPC and normal subjects should be performed to assess whether EBV genomic variations contribute to NPC pathogenesis.

    IMPORTANCE This study established a sequencing workflow from EBV DNA capture and sequencing to de novo assembly and contig joining. We reported eight newly sequenced EBV genomes isolated from primary NPC biopsies and revealed the sequence diversity on a whole genome level among these EBV isolates. At least two lineages of EBV strains are observed and recombination among these lineages is inferred. Our study has demonstrated the value of, and provided a platform for, genome sequencing of EBV.

  • Virus and Host Factors Affecting the Clinical Outcome of Bluetongue Virus Infection [PublishAheadOfPrint]

  • Bluetongue is a major infectious disease of ruminants caused by bluetongue virus (BTV), an arbovirus transmitted by Culicoides. Here, we assessed virus and host factors influencing the clinical outcome of BTV infection using a single experimental framework. We investigated how mammalian host species, breed, age, BTV serotypes, and strains within a serotype, affect the clinical course of bluetongue. Results obtained indicate that in small ruminants there is a marked difference in the susceptibility to clinical disease induced by BTV at the host species level, but less so at the breed level. No major differences in virulence were found between divergent serotypes (BTV-8 and BTV-2). However, we observed striking differences in virulence between closely related strains of the same serotype collected towards the beginning and the end of the European BTV-8 outbreak. As observed previously, differences in disease severity were also observed when animals were infected with either blood from a BTV-infected animal or from the same virus isolated in cell culture. Interestingly, with the exception of two silent mutations, full viral genome sequencing showed identical consensus sequences of the virus before and after cell culture isolation. However, deep sequencing analysis revealed a marked decrease in the genetic diversity of the viral population after passaging in mammalian cells. In contrast, passaging in Culicoides cells increased the overall number of low frequency variants compared to virus never passaged in cell culture. Thus, Culicoides might be a source of new viral variants and viral population diversity can be another factor influencing BTV virulence.

    IMPORTANCE Bluetongue is one of the major infectious diseases of ruminants. It is caused by an arbovirus known as Bluetongue virus (BTV). The clinical outcome of BTV infection is extremely variable. We show that there are clear links between the severity of bluetongue and the mammalian host species infected, while at the breed level differences were less evident. No differences were observed in the virulence of two different BTV serotypes (BTV-8 and BTV-2). In contrast, we show that the European BTV-8 strain isolated at the beginning of the bluetongue outbreak in 2006 was more virulent than a strain isolated towards the end of the outbreak. In addition, we show that there is a link between the variability of the BTV population as a whole and virulence and our data also suggest that Culicoides cells might function as an "incubator" of viral variants.

  • The UL12 Protein of Herpes Simplex Virus 1 Is Regulated by Tyrosine Phosphorylation [PublishAheadOfPrint]

  • The herpes simplex virus 1 UL12 protein (pUL12) is a nuclease that is critical for viral replication in vitro and neurovirulence in vivo. In this study, mass spectrometric analysis of pUL12 and phosphate-affinity SDS-polyacrylamide gel electrophoresis analysis identified tyrosine at pUL12 residue 371 (Tyr-371) as a pUL12 phosphorylation site: Tyr-371 is conserved in pUL12 homologs in herpesviruses in all Herpesviridae subfamilies. Replacement of Tyr-371 with phenylalanine (Y371F) in pUL12 (i) abolished its exonuclease activity in HSV-1-infected Vero, HEL and A549 cells; (ii) reduced viral replication and cell-cell spread, and expression of pUL12 in infected cells in a cell type-dependent manner; (iii) led to aberrant subcellular localization of pUL12 in infected cells in a cell type-dependent manner; and (iv) reduced HSV-1 neurovirulence in mice. The effects of the pUL12 Y371F mutation in cell cultures and mice were similar to those of a nuclease-dead double mutation in pUL12, although the Y371F mutation reduced viral replication several-fold more than the nuclease-dead double mutation in a cell type- and multiplicity of infection-dependent manner. Replacement of Tyr-371 with glutamic acid, which mimics constitutive phosphorylation, restored the wild-type phenotype in cell cultures and mice. These results suggested that phosphorylation of pUL12 Tyr-371 was essential for pUL12 to express its nuclease activity in HSV-1-infected cells, and that this phosphorylation promoted viral replication and cell-cell spread in cell cultures and nerurovirulence in mice mainly by up-regulating pUL12 nuclease activity and, in part, by regulating subcellular localization and expression of pUL12 in HSV-1-infected cells.

    IMPORTANCE Herpesviruses encode a considerable number of enzymes for their replication. Like cellular enzymes, the viral enzymes need to be properly regulated in infected cells. Although the functional aspects of herpesvirus enzymes have gradually been clarified, there is a lack of information on how most of these enzymes are regulated in infected cells. In the present study, we have reported that the enzymatic activity of herpes simplex virus 1 alkaline nuclease pUL12 was regulated by phosphorylation of pUL12 Tyr-371 in infected cells, and that this phosphorylation promoted viral replication and cell-cell spread in cell cultures and nerurovirulence in mice, mainly by up-regulating pUL12 nuclease activity. Interestingly, pUL12 and tyrosine at pUL12 residue 371 appeared to be conserved in all herpesviruses in the family Herpesviridae, raising the possibility that the herpesvirus pUL12 homologs may also be regulated by phosphorylation of the conserved tyrosine residue.

  • Prophylaxis with a respiratory syncytial virus (RSV) anti-G protein monoclonal antibody shifts the adaptive immune response to RSV rA2-line19F infection from Th2 to Th1 in BALB/c mice [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. In the present study, we investigated the effect of prophylactic treatment with the intact and F(ab')2 forms of an anti-G protein monoclonal antibody (mAb), 131-2G, on the humoral and cellular adaptive immune response to RSV rA2-line19F (r19F) challenge in BALB/c mice. The F(ab')2 form of 131-2G does not decrease virus replication but intact 131-2G does. The serum specimens for antibodies and spleen cells for memory T cell responses to RSV antigens were analyzed at 30, 45, 75 and 95 p.i. with/without prior treatment with 131-2G. The ratios of Th2/Th1 antibody isotypes at each time p.i indicated that both forms of mAb 131-2G shifted the subclass response from a Th2 (IgG1 and IgG2b) to a Th1 (IgG2A) bias. The ratio of IgG1/IgG2A antibody titer was 3-fold to 10-fold higher for untreated than mAb treated mice. There was also some increase in IgG (22%pplusmn;13 increase) and neutralization (32% increase) in antibodies with mAb 131-2G prophylaxis at 75 days p.i. Treatment with 131-2G significantly (plle;0.001) decreased the percent of IL-4 positive CD4 and CD8 in RSV stimulated spleen cells at all times p.i. while percent of IFN- T cells significantly (plle;0.001) increased gge;75 days p.i. The shift from a Th2 to a Th1 biased T cell response in treated compared to untreated mice likely was directed by the much higher levels of T-box transcription factor (Tbet) (gge;45% vs llt;10%) in CD4 and CD8 T cells and lower levels of Gata-3 (lle;2% vs gge;6%) in CD4 T cells in peptide stimulated, day 75 p.i. spleen cells. These data show that the RSV G protein affects both humoral and cellular adaptive immune responses and induction of 131-2G-like antibodies might improve the safety and long term efficacy of an RSV vaccine.

    Importance The data in this report suggest that the RSV G protein not only contributes to disease but also dampens the host immune response to infection. Both effects of G likely contribute to difficulties in achieving an effective vaccine. The ability of mAb 131-2G to block these effects of G suggests that inducing antibodies similar to 131-2G should prevent disease and enhance the adaptive immune response with later RSV infection. The fact that 131-2G binds to the 13 aa region conserved among all strains and flanking sequences are conserved within Group A or within Group B strains, simplifies the task of developing a vaccine to induce 131-2G-like antibodies. If our findings in mice apply to humans, then including the 131-2G binding region of G in a vaccine should improve its safety and efficacy.

  • Comprehensive RNA-seq analysis reveals contamination of multiple nasopharyngeal carcinoma cell lines with HeLa cell genomes. [PublishAheadOfPrint]

  • In an attempt to explore infectious agents associated with nasopharyngeal carcinomas (NPC), we employed our RNA-seq analysis pipeline, RNA CoMPASS to investigate the presence of ectopic organisms within a number of NPC cell lines commonly used by NPC/EBV researchers. Sequencing datasets from both CNE1 and HONE1 were found to contain reads for human papillomavirus (HPV-18). Subsequent real-time RT-PCR analysis on a panel of NPC cell lines identified HPV-18 in CNE1 and HONE1 as well as three additional NPC cell lines (CNE2, AdAH, and NPC-KT). Further analysis of the chromosomal integration arrangement of HPV-18 in NPCs revealed identical patterns to those observed in HeLa cells. Clustering based on human single nucleotide variation (SNV) analysis of two separate HeLa cell lines and several NPC cell lines demonstrated two distinct clusters with CNE1, and HONE1 clustering with the two HeLa cell lines. In addition, duplex-PCR based genotyping showed that CNE1, CNE2, and HONE1 do not have a HeLa cell specific L1 retrotransposon insertion, suggesting these three HPV-18+ NPC lines are likely products of a somatic hybridization with HeLa cells, which is also consistent with our RNA-seq based gene level SNV analysis. Taken together, we conclude a widespread HeLa contamination may exist in many NPC cell lines and authentication of these cell lines is recommended. Finally, we provide a proof of concept for the utility of an RNA-seq based approach for cell authentication.

    IMPORTANCE Nasopharyngeal carcinoma (NPC) cell lines are important model systems for analyzing the complex life cycle and pathogenesis of the Epstein-Barr virus (EBV). Using an RNA-seq based approach, we found HeLa cell contamination in several NPC cell lines that are commonly used in the EBV and related fields. Our data support the notion that contamination resulted from somatic hybridization with HeLa cells, likely occurring at the point of cell line establishment. Given the rarity of NPCs, the long history of NPC cell lines, and lack of rigorous cell line authentication, it is likely that the actual prevalence and impact of HeLa cell contamination on the EBV field might be greater. We therefore recommend cell line authentication prior to performing experiments using NPC cell lines to avoid inaccurate conclusions. The novel RNA-seq based cell authentication approach reported here can serve as a comprehensive method for validating cell lines.

  • The PI3K isoform dependence of tumor formation is determined by the genetic mode of PI3K pathway activation rather than by tissue type. [PublishAheadOfPrint]

  • Previous work has shown that prostate cancer in a Pten null murine model is dependent on the p110bbeta; isoform of PI3K, while breast cancer driven by either polyoma middle T antigen (MT) or HER2 is p110aalpha; dependent. Whether these differences in isoform dependence arise from tissue specificity, or from the nature of the oncogenic signal activating the PI3K pathway, is important given increasing interest in using isoform specific PI3K inhibitors in cancer therapy. To approach this question, we studied the PI3K isoform dependence of our recently constructed prostate cancer model driven by MT. Since MT activates a number of signaling pathways, we first confirmed that the MT prostate model was actually dependent on PI3K. A newly-generated transgenic prostate line expressing a MT allele (Y315F), known to be defective for PI3K binding, displayed a markedly reduced ability to drive tumor formation. We next selectively ablated expression of either p110aalpha; or p110bbeta; in mice expressing wild type MT in the prostate. We found that tumor formation driven by MT was significantly delayed by loss of p110aalpha; expression, while ablation of p110bbeta; had no effect. Since tumor formation by MT is p110aalpha; dependent in prostate as well as in mammary gland, our data suggest that PI3K isoform dependence is driven by the mode of PI3K pathway activation rather than by tissue type.

    Importance: Middle T antigen, the oncogene of polyoma virus, can drive tumor formation in a variety of cell types and tissues. Interestingly MT has no intrinsic enzymatic activity but instead functions by binding and activating cellular signaling proteins. One of the most important of these is the lipid kinase PI3 kinase, which was first studied in MT immunoprecipitates. Ubiquitously expressed PI3K comes in two major isoforms: p110aalpha; and p110bbeta;. Previous work in animal models showed that p110aalpha; was the key isoform in breast tumors driven by oncogenes including MT and HER2 while p110bbeta; was key in prostate tumors driven by Pten loss. We asked the simple question of whether a prostate tumor driven by MT would depend on p110aalpha;, suggesting that mode of activation determines p110 isoform dependence, or p110bbeta;, suggesting that tissue type determines isoform dependence. The clear answer is that MT depends on p110aalpha; in both prostate and breast.

  • Identification of conserved residues in HCV envelope glycoprotein E2 modulating virus dependence on CD81 and SRB1 entry factors [PublishAheadOfPrint]

  • In spite of the high variability of its sequence, Hepatitis C virus (HCV) envelope glycoprotein E2 contains several conserved regions. In this study, we explored the structural and functional features of the highly conserved E2 aa502-520 segment that had been proposed as a fusion peptide and that has been shown to strongly overlap with a potential conserved neutralizing epitope. For this purpose, we used reverse genetics to introduce point mutations within this region, and we characterized the phenotype of these mutants in the light of the recently published structure of E2. The functional analyses showed that their phenotype is in agreement with the position of the corresponding residues in E2 crystal structure. In contrast, our data ruled out the involvement of this region in membrane fusion and they indicate that alternative conformations would be necessary to expose the potential neutralizing epitope present in this segment. Of particular interest, we identified three specific mutations (Y507L, V514A and V515A) located within this neutralizing epitope, which only mildly reduced infectivity and showed no assembly defect. These mutations modulated HCV dependence on the viral receptor SRB1 and/or they also modulated virion sensitivity to neutralizing antibodies. Importantly, their characterization also showed that amino acids Y507, V514 and V515 contribute to E2 interaction with HCV receptor CD81. In conclusion, our data show that the highly conserved E2 aa502-520 segment plays a key role in cell entry by influencing the association of the viral particle with co-receptors and neutralizing antibodies.

    Importance Hepatitis C virus (HCV) envelope proteins E1 and E2 exhibit sequence variability. However some segments of the envelope proteins are highly conserved suggesting that these sequences play a key role at some steps of the HCV life cycle. In this work, we characterized the function and structure of a highly conserved E2 region that is targeted by neutralizing antibodies and had been proposed as a fusion peptide. Our data ruled out the involvement of this region in membrane fusion but allowed for the identification of new residues modulating the interaction of the virus with entry factors and its sensitivity to neutralizing antibodies. Moreover structural data suggest that alternative conformations could exist for E2, which would explain the presence of a partially masked neutralizing epitope in this segment in the currently available E2 structure. Overall our findings highlight the importance of conserved regions in the sequences of HCV envelope proteins.

  • Contribution of Glutamine Residues in the Helix 4-5 Loop to Capsid-capsid Interactions in Simian Immunodeficiency Virus of Macaques [PublishAheadOfPrint]

  • Following retrovirus entry, the viral capsid disassembles into its component capsid (CA) proteins. The rate of this uncoating process, which is regulated by CA-CA interactions and by the association of the capsid with host cell factors like cyclophilin A (CypA), can influence the efficiency of reverse transcription. Inspection of the CA sequences of lentiviruses reveals that several species of simian immunodeficiency viruses (SIVs) have lost the glycine-proline motif in the helix 4-5 loop important for CypA binding; instead, the helix 4-5 loop in these SIVs exhibits an increase in the number of glutamine residues. Here we investigate the role of these glutamine residues in SIVmac239 replication. Changes in these residues, particularly glutamine 89 and glutamine 92, resulted in a decreased efficiency of core condensation, decreased stability of the capsids in infected cells, and blocks to reverse transcription. In some cases, coexpression of two different CA mutants produced chimeric virions that exhibited higher infectivity than either parental mutant virus. For this complementation of infectivity, glutamine 89 was apparently required on one of the complementing pair of mutants, and glutamine 92 on the other. Modeling suggests that glutamines 89 and 92 are located on the distal face of hexameric capsid spokes, and thus are well positioned to contribute to inter-hexamer interactions. Requirements to evade host restriction factors like TRIMCyp may drive some SIV lineages to evolve means other than CypA binding to stabilize the capsid. One solution used by several SIV strains consists of glutamine-based bonding.

    IMPORTANCE The retroviral capsid is an assembly of individual capsid proteins that surrounds the viral RNA. After a retrovirus enters a cell, the capsid must disassemble or uncoat at a proper rate. The interactions among capsid proteins contribute to this rate of uncoating. We found that some simian immunodeficiency viruses use arrays of glutamine residues, which can form hydrogen bonds efficiently, to keep their capsids stable. This strategy may allow these viruses to forego the use of capsid-stabilizing factors from the host cell, some of which have antiviral activity.

  • The P4-P2' amino acids surrounding human norovirus polyprotein cleavage sites define the core sequence regulating self-processing order [PublishAheadOfPrint]

  • Noroviruses (NoV) are members of the family Caliciviridae. The human NoV ORF1 encodes a 200 kDa polyprotein which is cleaved by the viral 20 kDa 3C-like protease (Pro, NS6) into 6 non-structural proteins necessary for viral replication. NoV ORF1 polyprotein is processed in a specific order with llsquo;early' sites (NS1/2-3, NS3-4) being cleaved rapidly, and three 'late' sites (NS4-5, NS5-6, NS6-7) processed subsequently and less efficiently. Previously, we demonstrated that NoV polyprotein processing order is directly correlated with enzyme efficiency regulated by the primary amino acid sequence surrounding ORF1 cleavage sites. Using FRET peptides representing the NS2-3 and NS6-7 ORF1 cleavage sites, we now demonstrate that the P4-P2rrsquo; amino acids surrounding each site comprise the core sequence controlling NoV protease enzyme efficiency. Furthermore, NoV polyprotein self-processing order can be altered by interchanging this core sequence between NS2-3 and any of the three llsquo;late' sites in in vitro transcription-translation assays. We also demonstrate that the nature of the side chain at the P3 position for the NS1/2-3 (Nterm/NTPase) site confers significant influence on enzyme catalysis (kcat and kcat/Km), a feature overlooked in previous structural studies. Molecular modeling provides possible explanations for the P3 interactions with NoV protease.

    IMPORTANCE Noroviruses (NoV) are the prevailing cause of nonbacterial acute gastroenteritis worldwide and pose a significant financial burden on healthcare systems. Proteolytic processing of the viral nonstructural polyprotein is required for norovirus replication. Previously, the core sequence of amino acids surrounding the scissile bonds responsible for governing the relative processing order had not been determined. Using both FRET-based peptides and full-length NoV polyprotein, we have successfully demonstrated the core sequences spanning positions P4-P2rrsquo; surrounding the NS2-3, NS4-5, NS5-6, and NS6-7 cleavage sites contain all of the structural information necessary to control processing order. We also provide insight into a previously overlooked role for the NS2-3 P3 residue in enzyme efficiency. This manuscript builds upon our previous studies on NoV protease enzymatic activities and polyprotein processing order. Our work provides significant additional insight into understanding viral polyprotein processing and has important implications for improving the design of inhibitors targeting the NoV protease.

  • Homeostatic Cytokines Induce CD4 Downregulation in African Green Monkeys Independent of Antigen Exposure to Generate SIV-resistant CD8{alpha}{alpha} T Cells [PublishAheadOfPrint]

  • African green monkeys (genus Chlorocebus) are a natural host of simian immunodeficiency virus (SIVagm). As they do not develop simian AIDS, there is great interest in understanding how this species has evolved to avoid immunodeficiency. Adult African green monkeys naturally have low numbers of CD4 T cells and a large population of MHC Class II-restricted CD8aalpha;dim T cells that are generated through CD4 downregulation in CD4+ T cells. Mechanisms that drive this process of CD4 downregulation are unknown. Here we show that juvenile AGMs accelerate CD4 to CD8aalpha;aalpha; conversion upon SIV infection and avoid progression to AIDS. The CD4 downregulation induced by SIV infection is not limited to SIV-specific T cells, and vaccination of an adult AGM who had a negligible number of CD4 T cells demonstrated that CD4 downregulation can occur without antigenic exposure. Finally, we show that the T cell homeostatic cytokines IL-2, IL-7 and IL-15 can induce CD4 downregulation in vitro. These data identify a mechanism that allows AGMs to generate a large, diverse population of T cells that perform CD4 T cell functions but are resistant to SIV infection. A better understanding of this mechanism may allow the development of treatments to induce protective CD4 downregulation in humans.

    Importance Many African primate species are naturally infected with simian immunodeficiency virus (SIV). African green monkeys, one natural host species, avoid simian AIDS by creating a population of T cells that lack CD4, the HIV/SIV receptor, that are therefore resistant to infection. However, these T cells maintain properties of CD4+ T cells even after receptor downregulation, and preserve immune function. Here we show that juvenile AGMs, who have not undergone extensive CD4 downregulation, accelerate this process upon SIV infection. Furthermore, we show that in vivo, CD4 downregulation doesn't occur exclusively in antigen-experienced T cells. Finally, we show that the cytokines IL-2, IL-7 and IL-15, which induce homeostatic T cell proliferation, lead to CD4 downregulation in vitro and therefore can provide signals that lead to antigen-independent CD4 downregulation. These results suggest that if a similar process of CD4 downregulation could be induced in humans, it could theoretically provide a cure for AIDS.

  • The Rhesus Rhadinovirus CD200 Homologue Affects Immune Responses and Viral Loads During in vivo Infection [PublishAheadOfPrint]

  • Rhesus macaque rhadinovirus (RRV) is a gamma-herpesvirus of rhesus macaque (RM) monkeys that is closely related to human herpesvirus 8 (HHV-8)/Kaposi's Sarcoma-associated herpesvirus (KSHV), and is capable of inducing diseases in SIV-infected RM that are similar to those seen in humans co-infected with HIV and HHV-8. Both HHV-8 and RRV encode viral CD200 molecules that are homologues of cellular CD200, a membrane glycoprotein that regulates immune responses and helps maintain immune homeostasis via interactions with CD200 receptor (CD200R). Though the functions of RRV and HHV-8 vCD200 molecules have been examined in vitro, the precise roles that these viral proteins play during in vivo infection remain unknown. Thus, to address the contributions of RRV vCD200 to immune regulation and disease in vivo, we generated a form of RRV lacking expression of vCD200 for use in infection studies in RM. Our data indicate that RRV vCD200 expression limits immune responses against RRV at early times post-infection and also impacts viral loads, but does not appear to have significant effects on disease development. Further, examination of the distribution pattern of CD200R in RM indicates that this receptor is expressed on a majority of cells in PBMC, including B and T cells, suggesting potentially wider regulatory capabilities for both vCD200 and CD200 that are not strictly limited to myeloid lineage cells. In addition, we also demonstrate that RRV infection affects CD200R expression levels in vivo, although vCD200 expression does not play a role in this phenomenon.

    IMPORTANCE Cellular CD200 and its receptor CD200R compose a pathway that is important in regulating immune responses, and is known to play a role in a variety of human diseases. A number of pathogens have been found to modulate the CD200-CD200R pathway during infection, including human herpesvirus 8 (HHV-8), the causative agent of Kaposi's sarcoma and B cell neoplasms in AIDS patients, and a closely related primate virus, rhesus macaque rhadinovirus (RRV), which infects and induces disease in rhesus macaque monkeys. HHV-8 and RRV encode homologues of CD200, termed vCD200, which are thought to play a role in preventing immune responses against these viruses. However, neither molecule has been studied in an in vivo model of infection to address their actual contributions to immunoregulation and disease. Here we report findings from our studies analyzing the properties of a mutant form of RRV lacking vCD200 expression in infected rhesus macaques.

  • Ectopic DNMT3L triggers assembly of a repressive complex for retroviral silencing in somatic cells [PublishAheadOfPrint]

  • Mammalian genomes are replete with retrotransposable elements, including endogenous retroviruses. DNA methyltransferase 3-like (DNMT3L) is an epigenetic regulator expressed in prospermatogonia, growing oocytes, and embryonic stem (ES) cells. Here, we demonstrate that DNMT3L enhances the interaction of repressive epigenetic modifiers, including histone deacetylase 1 (HDAC1), SET domain, bifurcated 1 (SETDB1), DNA methyltransfearse 3A (DNMT3A), and tripartite motif-containing protein 28 (TRIM28; also known as TIF1bbeta; and KAP1) in ES cells and orchestrates retroviral-silencing activity with TRIM28 through mechanisms including, but not limited to, de novo DNA methylation. Ectopic expression of DNMT3L in somatic cells causes methylation-independent retroviral silencing activity by recruitment of the TRIM28/HDAC1/SETDB1/DNMT3A/DNMT3L complex to newly integrated Moloney Murine Leukemia Virus (Mo-MuLV) proviral DNA. Concurrent with this recruitment, we also observed the accumulation of histone H3 lysine 9 trimethylation (H3K9me3) and heterochromatin protein 1 gamma (HP1), as well as reduced H3K9 and H3K27 acetylation at Mo-MuLV proviral sequences. Ectopic expression of DNMT3L in late passage mouse embryonic fibroblasts (MEFs) recruited cytoplasmically localized HDAC1 to the nucleus. The formation of this epigenetic modifying complex requires interaction of DNMT3L with DNMT3A as well as with histone H3. In fetal testes at embryonic day 17.5, endogenous DNMT3L also enhanced the binding among TRIM28, DNMT3A, SETDB1, and HDAC1. We propose that DNMT3L may be involved in initiating a cascade of repressive epigenetic modifications by assisting in the preparation of a chromatin context that further attracts DNMT3A-DNMT3L binding and installs longer-term DNA methylation marks at newly-integrated retroviruses.

    IMPORTANCE Almost half of the mammalian genome is composed of endogenous retroviruses and other retrotransposable elements that threaten genomic integrity. These elements are usually subject to epigenetic silencing. We discovered that two epigenetic regulators that lack enzymatic activity, DNA methyltransferase 3-like (DNMT3L) and tripartite motif-containing protein 28 (TRIM28) collaborate with each other to impose retroviral silencing. In addition to modulating de novo DNA methylation, we found that by interacting with TRIM28, DNMT3L can attract various enzymes to form a DNMT3L-induced repressive complex to remove active marks and add repressive marks to histone proteins. Collectively, these results reveal a novel and pivotal function of DNMT3L in shaping the chromatin modifications necessary for retroviral and retrotransposon silencing.

  • Recent H3N2 influenza virus clinical isolates rapidly acquire hemagglutinin or neuraminidase mutations when propagated for antigenic analyses [PublishAheadOfPrint]

  • Prior to serological testing, influenza viruses are typically propagated in eggs or cell culture. Recent human H3N2 strains bind to cells with low avidity. Here, we isolated 9 primary H3N2 viral isolates from respiratory secretions of children. Upon propagation in vitro, 5 of these isolates acquired hemagglutinin or neuraminidase mutations that increased virus binding to cell surfaces. These mutations can potentially confound serological assays commonly used to identify antigenically novel influenza viruses.

  • Late Endosomal Trafficking of Alternative Serotype Adenovirus Vaccine Vectors Augments Antiviral Innate Immunity [PublishAheadOfPrint]

  • Adenovirus (Ad) vaccine vectors have found widespread use as vaccine platforms against multiple infections and cancers, and multiple serotypes have been shown to differ significantly in their biologic properties and immune phenotypes. Our laboratory and others have previously described differential innate immune stimulation elicited by various Ad serotypes. Here we show that Ad5 traffics rapidly to the nucleus following infection, whereas Ad35 and Ad26 accumulate in late endosomes between 2-8 hours post-infection. Innate immune cytokine elicitation by all Ad serotypes was abrogated by blockade of endosomal acidification, Cathepsin B and Caspase-1, suggesting that virus interactions with acid-dependent sensors such as Toll-like receptor- and cathepsin-dependent inflammasome activation in late endosomes may trigger innate immunity. These data suggest a mechanism by which Ad vectors from various serotypes differentially trigger innate antiviral pathways via distinct intracellular trafficking to late endosomes.

    IMPORTANCE Adenoviruses (Ad) are widely used for applications of vaccination and gene therapy. Importantly, Ad vectors have been shown to differ significantly in their innate immune profiles both in vivo and in vitro. The molecular mechanism which underlies these observed differences has important implications for the development of improved vaccines. In this study, we propose a mechanism in which the degree of late endosomal trafficking of Ad vectors results in differential stimulation of late endosomal pattern recognition receptors.

  • Tobacco etch virus P1 protein traffics to the nucleolus and associates with the host 60S ribosomal subunits during infection [PublishAheadOfPrint]

  • Genus Potyvirus comprises a large group of positive-strand RNA plant viruses whose genome encodes a large polyprotein processed by three viral proteinases. P1 protein, the most amino-terminal product of the polyprotein, is an accessory factor stimulating viral genome amplification whose role during infection is not well understood. We infected plants with Tobacco etch virus (TEV; genus Potyvirus) clones in which P1 was tagged with a fluorescent protein to track its expression and subcellular localization or with an affinity tag to identify host proteins involved in complexes in which P1 also takes part during infection. Our results showed that TEV P1 exclusively accumulates in infected cells at an early stage of infection, and that the protein displays a dynamic subcellular localization, trafficking in and out of the nucleus and nucleolus during infection. Inside the nucleolus, P1 particularly targets the dense granular component. Consistently, we found functional nucleolar localization and nuclear export signals in TEV P1 sequence. Our results also indicated that TEV P1 physically interacts with the host 80S cytoplasmic ribosomes and specifically binds to the 60S ribosomal subunits during infection. In vitro translation assays of reporter proteins suggested that TEV P1 stimulates protein translation, particularly when driven from the TEV internal ribosome entry site. These in vitro assays also suggested that TEV helper-component proteinase (HC-Pro) inhibits protein translation. Based on these findings, we propose that TEV P1 stimulates translation of viral proteins in infected cells.

    IMPORTANCE In this work, we researched the role during infection of tobacco etch virus P1 protease. P1 is the most mysterious protein of potyviruses, a relevant group of RNA viruses infecting plants. Our experiments showed that the viral P1 protein exclusively accumulates in infected cells at an early stage of infection and moves in and out of the nucleus of infected cells, particularly targeting the nucleolus. Our experiments also showed that P1 protein binds host ribosomes during infection. Based on these findings and other in vitro experiments we propose that P1 protein stimulates translation of viral proteins during infection.

  • Modulation of stop codon readthrough efficiency and its effect on the replication of murine leukemia virus [PublishAheadOfPrint]

  • Translational readthrough nndash; suppression of termination at a stop codon nndash; is exploited in the replication cycles of several viruses and represents a potential target for antiviral intervention. In the gammaretroviruses, typified by Moloney murine leukemia virus (MuLV), gag and pol are in the same reading frame, separated by a UAG stop codon, and termination codon readthrough is required for expression of the viral Gag-Pol fusion protein. Here we investigated the effect on MuLV replication of modulating readthrough efficiency. We began by manipulating the readthrough signal in the context of an infectious viral clone to generate a series of MuLV variants in which readthrough was stimulated or reduced. In carefully controlled infectivity assays, it was found that reducing the MuLV readthrough efficiency only four-fold led to a marked defect and that a ten-fold reduction essentially abolished replication. However, up to an ~8.5-fold stimulation of readthrough (up to 60% readthrough) was well tolerated by the virus. These high levels of readthrough were achieved using a two-plasmid system, with Gag and Gag-Pol expressed from separate infectious clones. We also modulated readthrough by silencing expression of release factors 1 and 3 (eRF1 and eRF3) or by introducing aminoglycosides into the cells. The data obtained indicate that gammaretroviruses tolerate a substantial excess of viral Gag-Pol synthesis, but are very sensitive to a reduction in levels of this polyprotein. Thus, as is also the case for ribosomal frameshifting, antiviral therapies targeting readthrough with inhibitory agents are likely to be the most beneficial.

    IMPORTANCE Many pathogenic RNA viruses and retroviruses use ribosomal frameshifting or stop codon readthrough to regulate expression of their replicase enzymes. These translational "recoding" processes are potential targets for antiviral intervention, but we have only a limited understanding of the consequences to virus replication of modulating the efficiency of recoding, particularly for those viruses employing readthrough. In this paper, we describe the first systematic analysis of the effect of increasing or decreasing readthrough efficiency on virus replication using the gammaretrovirus MuLV as a model system. We find unexpectedly that MuLV replication is only slightly inhibited by substantial increases in readthrough frequency, but like other viruses that use recoding strategies, replication is quite sensitive to even modest reductions. These studies provide insights into both the readthrough process and MuLV replication and have implications for the selection of antivirals against gammaretroviruses.

  • Minireview: Unexpected structural features of the hepatitis c virus envelope protein 2 ectodomain. [PublishAheadOfPrint]

  • Hepatitis C virus (HCV), a member of the family Flaviviridae, is a leading cause of chronic liver disease and cancer. Recent advances in HCV therapeutics have resulted in improved cure rates, but an HCV vaccine is not available and is urgently needed to control the global pandemic. Vaccine development has been hampered by the lack of high-resolution structural information for the two HCV envelope glycoproteins E1 and E2. Recently, Kong and co-workers (Science 342, 1090-1094, 2013) and Khan and co-workers (Nature, 509, 381-384. 2014) independently determined the structure of the HCV E2 ectodomain core with some unexpected and informative results. The HCV E2 ecotdomain core features a globular architecture with antiparallel bbeta;-sheets forming a central bbeta; sandwich. The residues comprising the epitopes of several neutralizing and non-neutralizing human monoclonal antibodies were also determined, which is an essential step towards obtaining a fine map of the human humoral response to HCV. Also clarified were the regions of E2 that directly bind CD81, an important HCV cellular receptor. While it has been widely assumed that HCV E2 is a class II viral fusion protein (VFP), the new structure suggests that the HCV E2 ectodomain shares structural and functional similarities only with domain III of class II VFPs. The new structural determinations suggest that the HCV glycoproteins use a different mechanism from class II fusion proteins for cell fusion.

  • The immunodominant Influenza A virus M158-66 CTL epitope exhibits degenerate class I MHC restriction in humans [PublishAheadOfPrint]

  • Cytotoxic T lymphocytes recognizing conserved peptide epitopes are crucial in the protection against influenza A virus (IAV) infection. The CD8 T cell response against the M158-66 (GILGFVFTL) matrix protein epitope is immunodominant when restricted by HLA-A*02, a major histocompatibility complex (MHC) expressed by approximately half of the human population. Herein, we report that the GILGFVFTL peptide is restricted by multiple HLA-C*08 alleles as well. We observed that M158-66 was able to elicit CTL responses in both HLA-A*02 and -C*08 positive individuals; and that GILGFVFTL-specific CTLs in individuals expressing both restriction elements were distinct and not cross-reactive. The crystal structure of GILGFVFTL/HLA-C*08:01 was solved at 1.84AAring; and comparison with the known GILGFVFTL/HLA-A*02:01 structure revealed that the antigen bound both complexes in near-identical conformation, accommodated by binding pockets shaped from shared as well as unique residues. This discovery of degenerate peptide presentation by both HLA-A and HLA-C allelic variants eliciting unique CTL responses to IAV infection contributes fundamental knowledge with important implications for vaccine development strategies.

    Importance The presentation of influenza A virus peptide to elicit immunity is thought to be narrowly restricted, with a single peptide presented by a specific HLA molecule. In this study, we show that the same influenza A peptide can be more broadly presented by both HLA-A and HLA-C molecules. This discovery may help to explain the differences in immunity to influenza A between individuals and populations, and also aid in the design of vaccines.

  • How viruses hijack the ERAD tuning machinery [PublishAheadOfPrint]

  • An essential step during the intracellular life cycle of many positive-strand RNA viruses is the rearrangement of host cell membranes to generate membrane-bound replication platforms. For example Nidovirales and Flaviviridae subvert the membrane of the endoplasmic reticulum (ER) for their replication. However, the absence of conventional ER and secretory pathway markers in virus-induced ER-derived membranes has for long time hampered a thorough understanding of their biogenesis. Recent reports highlight the analogies between mouse hepatitis virus-, equine arteritis virus- and Japanese encephalitis virus-induced replication platforms and ER-associated degradation (ERAD) tuning vesicles (or EDEMosomes) that display non-lipidated LC3 at their cytosolic face and segregate the ERAD factors EDEM1, OS-9 and SEL1L from the ER lumen. In this review, we shortly summarize the current knowledge on ERAD tuning pathways and how they might be hijacked for viral genome replication. As ERAD tuning components such as SEL1L and non-lipidated LC3 appear to contribute to viral infection, these cellular pathways represent novel candidate drug targets to combat positive-strand RNA viruses.

  • SnRK1 Phosphorylation of AL2 Delays Cabbage Leaf Curl Virus Infection in Arabidopsis [PublishAheadOfPrint]

  • Geminivirus AL2/C2 proteins play key roles in establishing infection and causing disease in their plant hosts. They are involved in viral gene expression, counter host defenses by suppressing transcriptional gene silencing, and interfere with host signaling involved in pathogen resistance. We report here that begomovirus and curtovirus AL2/C2 proteins interact strongly with host geminivirus Rep-interacting kinases (GRIKs), which are upstream activating kinases of the protein kinase SnRK1, a global regulator of energy and nutrient levels in plants. We used an in vitro kinase system to show that GRIK-activated SnRK1 phosphorylates recombinant AL2/C2 proteins from several begomoviruses and to map the SnRK1 phosphorylation site to serine-109 in the AL2 proteins of two New World begomoviruses mmdash; Cabbage leaf curl virus (CaLCuV) and Tomato mottle virus. A CaLCuV AL2 S109D phosphomimic mutation did not alter viral DNA levels in protoplast replication assays. In contrast, the phosphomimic mutant was delayed for symptom development and viral DNA accumulation during infection of Arabidopsis thaliana, demonstrating that SnRK1 contributes to host defenses against CaLCuV. Our observation that serine-109 is not conserved in all AL2/C2 proteins that are SnRK1 substrates in vitro suggested that phosphorylation of viral proteins by plant kinases contributes to the evolution of geminivirusmmdash;host interactions.

    IMPORTANCE Geminiviruses are single-stranded DNA viruses that cause serious diseases in many crops. Dicot-infecting geminiviruses encode multi-functional AL2/C2 proteins that are essential for infection. However, it is not clear how AL2/C2 proteins are regulated. Here, we show that the host protein kinase SnRK1, a central regulator of energy balance and nutrient metabolism in plants, phosphorylates serine-109 in AL2 proteins of three subgroups of New World begomoviruses, resulting in a delay in viral DNA accumulation and symptom appearance. Our results support SnRK1's antiviral role and reveal a novel mechanism underlying this function. Phylogenetic analysis suggested that AL2 S109 evolved as begomoviruses migrated from the Old World to the New World, and may have provided a selective advantage as begomoviruses adapted to a different environment and plant hosts. This study provides new insights into the interaction of viral pathogens with their plant hosts at the level of viral protein modification by the host.

  • H7N9 and other pathogenic avian influenza viruses elicit a three-pronged transcriptomic signature that is reminiscent of 1918 influenza and associated with lethal outcome in mice [PublishAheadOfPrint]

  • Modulating the host response is a promising approach to treating influenza, a virus whose pathogenesis is determined in part by the reaction it elicits within the host. Though the pathogenicity of emerging H7N9 influenza virus has been reported in several animal models, these studies have not included a detailed characterization of the host response following infection. To this end, we characterized the transcriptomic response of BALB/c mice infected with H7N9 (A/Anhui/01/2013) virus and compared it to the responses induced by H5N1 (A/Vietnam/1203/2004), H7N7 (A/Netherlands/219/2003) or pandemic 2009 H1N1 (A/Mexico/4482/2009) influenza viruses. We found that responses to the H7 subtype viruses were intermediate to those elicited by H5N1 and pdm09H1N1 early in infection, but that they evolved to resemble the H5N1 response as infection progressed. H5N1, H7N7 and H7N9 viruses were pathogenic in mice, and this pathogenicity correlated with increased transcription of cytokine response genes and decreased transcription of lipid metabolism and coagulation signaling genes. This three-pronged transcriptomic signature was observed in mice infected with pathogenic H1N1 strains such as the 1918 virus, indicating that it may be predictive of pathogenicity across multiple influenza strains. Finally, we used host transcriptomic profiling to computationally predict drugs that reverse the host response to H7N9 infection, and identified six FDA-approved drugs that could potentially be repurposed to treat H7N9 and other pathogenic influenza viruses.

    Importance Emerging avian influenza viruses are of global concern because the human population is immunologically naiiuml;ve to them. Current influenza drugs target viral molecules, but the high mutation rate of influenza viruses eventually leads to the development of antiviral resistance. As the host evolves far more slowly than the virus, and influenza pathogenesis is determined in part by the host response, targeting the host response is a promising approach to treating influenza. Here we characterize the host transcriptomic response to emerging H7N9 influenza virus and compare it with the responses to H7N7, H5N1 and pdm09H1N1. All three avian viruses were pathogenic in mice, and elicited a transcriptomic signature that also occurs in response to the legendary 1918 influenza virus. Our work identifies host responses that could be targeted to treat severe H7N9 influenza and identifies six FDA-approved drugs that could potentially be repurposed as H7N9 influenza therapeutics.

  • The high-resolution structure of a virally encoded DNA translocating conduit and the mechanism of DNA penetration. [PublishAheadOfPrint]

  • Although the X174 DNA pilot protein H is monomeric during procapsid assembly, it forms an oligomeric tube on the surface of host cells. Reminiscent of a double-stranded DNA phage tails in form and function, the H-tube transports the single-stranded X174 genome across the E. coli cell wall. The 2.4 AAring; resolution, H-tube crystal structure suggests functional and energetic mechanisms, which may be common features of DNA transport through virally encoded conduits.

  • Blockade of Type I IFN Production by Retroviral Replicating Vectors and Reduced Tumor Cell Responses to IFN Likely Contribute to Tumor Selectivity [PublishAheadOfPrint]

  • We developed a MLV-based retroviral replicating vector (RRV), Toca 511, which displays tumor specificity in resected brain-tumor material and blood in clinical trials so far. Here, we investigated the interaction between Toca 511 and human host cells and showed that RRV did not induce Type I IFN responses in cultured human tumor cells or cultured human primary cells. However, exogenous Type I IFN inhibited RRV replication in tumor cells and induced IFN-regulated genes, albeit at a lower level than in primary cells. Unexpectedly, RRV did not induce IFNaalpha; production upon incubation in vitro with human plasmacytoid dendritic cells (pDCs), whereas lentiviral vector and heat-treated RRV did. Co-incubation of RRV with heat-treated RRV or with lentiviral vector suppressed IFNaalpha; production in pDCs, suggesting that native RRV has a dominant inhibitory effect on Type I IFN induction and is most sensitive to trypsin treatment. In addition, heat treatment inactivates that activity but exposes an immune stimulatory activity. The immune-stimulating component is sensitive to deglycosidases, trypsin and phospholipase C treatment. Experiments with retroviral non-replicating vectors and virus-like particles demonstrated that the immunosuppressive activity is not associated with the amphotropic envelope or the glyco-gag protein. In summary, our data provide evidence that RRV does not directly trigger Type I IFN responses in IFN-responsive tumor cells. Moreover, RRV appears to carry a heat-labile component that actively suppresses activation of cellular innate immune responses in pDCs. Inhibition of IFN induction by RRV and the reduced response to IFN should facilitate tumor-specific infection in vivo.

    Importance RRV have a convincing preference for replicating in tumor cells in animal models, and we appear to see similar preferences in initial treatment of human GBM patients. This study investigates the basis for the interaction between RRV and human host (tumor vs. non-tumor) cells in vitro. We find that RRV do not trigger IFNaalpha;/bbeta; response in tumor cells, but the cells are capable of responding to Type I interferons and of producing them when stimulated with known agonists. Surprisingly, the data shows that RRV can actively inhibit induction of cellular innate immunity, and that this inhibitory activity is heat labile and trypsin-sensitive, and not attributable to the envelope protein. These data can partially explain the observed in vivo tumor specificity.

  • Verdinexor, a novel Selective Inhibitor of Nuclear Export (SINE), reduces Influenza A virus replication in vitro and in vivo [PublishAheadOfPrint]

  • Influenza is a global health concern causing mortality, morbidity, and economic losses. Chemotherapeutics that target influenza are available; however, rapid emergence of drug resistant strains is common. Therapeutic targeting of host proteins hijacked by influenza virus to facilitate replication is an antiviral strategy to reduce the development of drug resistance. Nuclear export of influenza ribonucleoprotein (vRNP) from infected cells has been shown to be mediated by Exportin 1 (XPO1) interaction with nuclear export signal proteins tethered to vRNP. RNA interference (RNAi) screening has identified XPO1 as a host pro-influenza factor where XPO1 silencing results in reduced influenza replication. The Streptomyces metabolite XPO1 inhibitor, Leptomycin B (LMB), has been shown to limit influenza replication in vitro; however, LMB is toxic in vivo which makes it unsuitable for therapeutic use. In this study, we tested the anti-influenza activity of a new class of orally available small molecule Selective Inhibitors of Nuclear Export (SINE), specifically the XPO1 antagonist, KPT-335 (verdinexor). Verdinexor was shown to potently and selectively inhibit vRNP export, and effectively inhibited replication of various influenza A and B strains in vitro including pandemic H1N1, highly pathogenic H5N1 avian influenza virus, and the recently emerged H7N9 strain. In vivo, prophylactic and therapeutic administration of verdinexor protected mice against disease pathology following challenge with influenza A/California/04/09 or A/Philippines/2/82-X79, as well as reduced lung viral load and pro-inflammatory cytokine expression having minimal toxicity. These studies show that verdinexor acts as a novel anti-influenza therapeutic agent.

    Importance Anti-viral drugs represent important means for influenza virus control. However, substantial drug resistance has developed with currently approved influenza therapeutics. New anti-viral approaches are required to address drug resistance, and reduce the burden of influenza-related disease. This study addressed critical pre-clinical studies for the development of verdinexor (KPT-335) as a novel anti-viral drug. Verdinexor blocks nuclear export of progeny influenza virus genome, thus effectively inhibits virus replication. Verdinexor was found to limit replication of various strains of influenza A and B viruses including a pandemic H1N1 influenza virus, a highly pathogenic H5N1 avian influenza virus, and a recently emerging H7N9 influenza virus strains. Importantly, oral verdinexor treatments, given prophylactically or therapeutically, were efficacious to limit lung virus burden in influenza-infected mice, in addition to limiting lung pro-inflammatory cytokines expression, pathology, and mortality. Thus, this study demonstrated that verdinexor is efficacious against influenza virus infection in vitro and in vivo.

  • Broadly Neutralizing Antibody VRC01 Prevents HIV-1 Transmission from Plasmacytoid Dendritic Cells to CD4 T-Lymphocytes [PublishAheadOfPrint]

  • Plasmacytoid dendritic cells (pDC) poorly replicate HIV-1 but efficiently transfer HIV-1 to adjacent CD4 T-lymphocytes. We found that coculture with T-lymphocytes downregulates SAMHD1 expression, enhances HIV-1 replication, increases pDC maturation and IFN-aalpha; secretion. HIV-1 transfer to T-lymphocytes is inhibited by broadly neutralizing antibody VRC01 with similar efficiency as cell-free infection of T-lymphocytes. Interestingly, prevention of HIV-1 transmission by VRC01 retains IFN-aalpha; secretion. These results emphasize the multiple functions of VRC01 in protection against HIV-1 acquisition.

  • Characterization and Immunogenicity of a Novel Mosaic M HIV-1 gp140 Trimer [PublishAheadOfPrint]

  • The extraordinary diversity of the human immunodeficiency virus type 1 (HIV-1) Envelope (Env) glycoprotein poses a major challenge for the development of an HIV-1 vaccine. One strategy to circumvent this problem utilizes bioinformatically optimized mosaic antigens. However, mosaic Env proteins expressed as trimers have not been previously evaluated for their stability, antigenicity, and immunogenicity. Here we report the production and characterization of a stable HIV-1 mosaic M gp140 Env trimer. The mosaic M trimer bound CD4 as well as multiple broadly neutralizing monoclonal antibodies, and biophysical characterization suggested an intact and stable trimer. The mosaic M trimer elicited higher neutralizing antibody (nAb) titers against clade B viruses than a previously described clade C (C97ZA.012) gp140 trimer in guinea pigs, whereas the clade C trimer elicited higher nAb titers than the mosaic M trimer against clades A and C viruses. A mixture of the clade C and mosaic M trimers elicited nAb responses that were comparable to the better component of the mixture for each virus tested. These data suggest that combinations of relatively small numbers of immunologically complementary Env trimers may improve nAb responses.

    Importance The development of an HIV-1 vaccine remains a formidable challenge due to multiple circulating strains of HIV-1 worldwide. This study describes a candidate HIV-1 Env protein vaccine whose sequence has been designed by computational methods to address HIV-1 diversity. The characteristics and immunogenicity of this Env protein are described, both alone and mixed together with a clade C Env protein vaccine.

  • Structure of the HSV-1 genome: manipulation of nicks and gaps can abrogate infectivity and alter the cellular DNA damage response. [PublishAheadOfPrint]

  • The HSV-1 virion DNA contains nicks and gaps, and in this study a novel assay for estimating the size and number of gaps in virion DNA was developed. Consistent with previous reports, we estimate that there are approximately 15 gaps per genome, and we calculate the average gap length to be approximately 30 bases. Virion DNA was isolated and treated with DNA modifying enzymes in order to fill in the gaps and modify the ends. Interestingly, filling in gaps, blunting the ends, or the addition of random sequences to the 3' ends of DNA producing 3' flaps did not impair the infectivity of treated DNA following transfection of Vero cells. On the other hand, the formation of 5' flaps in the DNA following treatment resulted in a dramatic reduction (95-100%) in infectivity. Virion DNA stimulated DNA-PKcs activity in transfected cells, and DNA with 5' flaps stimulated a higher level of DNA-PKcs activity than that observed in cells transfected with untreated virion DNA. The infectivity of 5'-flapped DNA was restored in cells that do not express DNA-PKcs and in cells co-transfected with the immediate early protein, ICP0, which degrades DNA-PKcs. These results are consistent with previous reports that DNA-PK and the NHEJ repair pathway are intrinsically antiviral and that ICP0 can counteract this effect. We suggest that HSV-1 DNA with 5' flaps may induce an antiviral state due to the induction of a DNA damage response, primarily mediated by NHEJ, that renders the HSV-1 genome less efficient for lytic infection.

    IMPORTANCE For productive lytic infection to occur, HSV-1 must counteract a variety of cellular intrinsic antiviral mechanisms, including the DNA damage response (DDR). DDR pathways have been associated with silencing of gene expression, cell cycle arrest and induction of apoptosis. In addition, the fate of viral genomes is likely to play a role in whether viral genomes adopt a configuration suitable for lytic DNA replication. This study demonstrates that virion DNA activates the cellular DDR kinase, DNA-PK, and that this response is inhibitory to viral infection. Furthermore, we show that HSV-1 ubiquitin ligase, ICP0, plays an important role in counteracting the negative effects of DNA-PK activation. These findings support the notion that DNA-PK is antiviral and suggest that the fate of incoming viral DNA has important consequences for the progression of lytic infection. This study underscores the complex evolutionary relationships between HSV and its host.

  • Fine mapping of murine antibody responses to immunization with a novel soluble form of hepatitis C virus envelope glycoprotein complex [PublishAheadOfPrint]

  • The hepatitis C virus (HCV) envelope glycoprotein E1E2 complex is a candidate vaccine antigen. Previous immunization studies of E1E2 have yielded varying results on its ability to induce virus neutralizing antibodies (NAbs) in animal models and humans. The murine model has become a vital tool for HCV research owing to the development of humanized mice susceptible to HCV infection. In this study, we investigated the antibody responses of mice immunized with E1E2 and a novel soluble form of E1E2 (sE1E2) by a DNA prime and protein boost strategy. The results showed that sE1E2 elicited higher antibody titers and greater breath of reactivity than the wildtype cell-associated E1E2. However, immune sera elicited by either immunogen were only weakly neutralizing. In order to understand the contrasting results in binding and neutralizing serum activities, epitopes targeted by the polyclonal antibody responses were mapped and monoclonal antibodies (MAbs) were generated. The results showed that the majority of serum antibodies were directed to E1 region 211-250 and E2 regions 421-469, 512-539, 568-609, and 638-651, instead of the well-known immunodominant E2 hypervariable region 1 (HVR1). Unexpectedly, in MAb analysis, ~12% of MAbs isolated were specific to the conserved E2 antigenic site 412-423 and 85% of them cross-neutralize multiple HCV isolates. The epitopes recognized by these MAbs are similar but distinct from the previously reported HCV1 and AP33 broadly neutralizing epitopes. In conclusion, E1E2 can prime B cells specific to conserved neutralizing epitopes but the levels of serum NAbs elicited were insufficient for effective virus neutralization. The sE1E2 constructs described in this study can be a useful template for rational antigen engineering.

    Importance Hepatitis C virus infected 2-3% of the world population and is a leading cause of liver failures and the need for liver transplantation. The virus envelope glycoprotein complex E1E2 produced by detergent-extraction of cells over-expressing the protein was evaluated in a Phase I clinical trial but failed to induce neutralizing antibodies in most subjects. In this study, we designed a novel form of E1E2 which is secreted from cells and is soluble, and compared it to wildtype E1E2 by DNA immunization of mice. The results showed that this new E1E2 is more immunogenic than wildtype E1E2. Detailed mapping of the antibody responses revealed that antibodies to the conserved E2 antigenic site 412-423 were elicited but the serum concentrations were too low to neutralize the virus effectively. This soluble E1E2 provides a new reagent for studying HCV and for rational vaccine design.

  • Influenza A virus attenuation by codon deoptimization of the NS gene for vaccine development [PublishAheadOfPrint]

  • Influenza virus infection represents a serious public health problem that causes contagious respiratory disease, which is most effectively prevented through vaccination to reduce transmission and future infection. The non-structural (NS) gene of influenza A virus encodes an mRNA transcript that is alternatively spliced to express two viral proteins, the non-structural protein 1 (NS1) and the nuclear export protein (NEP). The importance of the NS gene of influenza A for viral replication and virulence has been well described and represents an attractive target to generate live attenuated influenza viruses with vaccine potential. Considering that most amino acids can be synthesized from several synonymous codons, this study employs the use of misrepresented mammalian codons (codon deoptimization) for the de novo synthesis of an NS viral RNA segment based on influenza A/Puerto Rico/8/34 (H1N1). We generated three different recombinant influenza PR8 viruses containing codon deoptimized synonymous mutations in coding regions comprising the entire NS gene, or the mRNA corresponding to the individual viral proteins NS1 or NEP, without modifying the respective splicing and packaging signals of the viral segment. The fitness of these synthetic viruses was attenuated in vivo, while retaining immunogenicity, conferring both homologous and heterologous protection against influenza A virus challenges. These results indicate that influenza viruses can be effectively attenuated by synonymous codon deoptimization of the NS gene and open the possibility of their use as a safe vaccine to prevent infections with these important human pathogens.

    IMPORTANCE Vaccination serves as the best therapeutic option to protect humans against influenza viral infections. However, the efficacy of current influenza vaccines is suboptimal and novel approaches are necessary for the prevention of this important human respiratory pathogen. The non-structural (NS) gene of influenza virus encodes both the multifunctional non-structural protein 1 (NS1), essential for innate immune evasion, and the nuclear export protein (NEP), required for the nuclear export of viral ribonucleoproteins and for timing of the virus life cycle. Here, we have generated a recombinant influenza A/Puerto Rico/8/1934 H1N1 (PR8) virus containing a codon deoptimized NS segment that is attenuated in vivo, yet retains immunogenicity and protection efficacy against homologous and heterologous influenza virus challenges. These results open the exciting possibility of using this NS codon deoptimization methodology alone or in combination with other approaches for the future development of vaccine candidates to prevent influenza viral infections.

  • Acute Clearance of Human Metapneumovirus Occurs Independently of Natural Killer Cells [PublishAheadOfPrint]

  • Human metapneumovirus(HMPV) is a major cause of respiratory disease. The role of NK cells in protection against HMPV is unclear. We show that while HMPV-infected C57BL/6 mice had higher numbers of functional lung NK cells than mock-treated mice, comparing NK cell-depleted and control mice did not reveal differences in lung viral titers, histopathology, cytokine levels, or T cell numbers or function. These data indicate that NK cells are not required for host control of HMPV.

  • Unveiling cell surface and type IV secretion proteins responsible for archaeal rudivirus entry [PublishAheadOfPrint]

  • Sulfolobus mutants resistant to archaeal lytic virus SIRV2 were isolated and mutations were identified in two gene clusters, sso3138-3141 and sso2386-2387, encoding cell surface and type IV secretion proteins, respectively. The involvement of the mutations in the resistance was confirmed by genetic complementation. Block of virus entry into the mutants was demonstrated by the lack of early gene transcription strongly supporting a role of the proteins in SIRV2 entry.

  • High-throughput identification of loss-of-function mutations for anti-interferon activity in influenza A virus NS segment [PublishAheadOfPrint]

  • Viral proteins often display several functions which require multiple assays to dissect their genetic basis. Here, we describe a systematic approach to screen for loss-of-function mutations that confer fitness disadvantage in a specified growth condition. Our methodology is achieved by genetically monitoring a mutant library under two growth conditions, with and without interferon, by deep sequencing. We employed a molecular tagging technique to distinguish true mutations from sequencing error. This approach enabled us to identify mutations that were negatively selected against in addition to those that were positively selected for. Using this technique, we identified loss-of-function mutations on the influenza A virus NS segment that were sensitive to type I interferon in a high-throughput fashion. Mechanistic characterization further showed that a single substitution, D92Y, resulted in the inability of NS to inhibit RIG-I ubiquitination. The approach described in this study can be applied under any specified condition for any virus that can be genetically manipulated.

    Importance Traditional genetics focuses on a single genotype-phenotype relationship, whereas high-throughput genetics permits phenotypic characterization of numerous mutants in parallel. High-throughput genetics often involves monitoring of a mutant library with deep sequencing. However, deep sequencing suffers from a high error rate (~0.1-1%), which is usually higher than the occurrence frequency for individual point mutations within a mutant library. Therefore, only mutations that confer fitness advantage can be identified with confidence due to an enrichment in occurrence frequency. In contrast, it is impossible to identify deleterious mutations using most next-generation sequencing technique. In this study, we have applied a molecular tagging technique to distinguish true mutations from sequencing errors. It enabled us to identify mutations that underwent negative selection in addition to mutations that experienced positive selection. This study provides a proof-of-concept by screening for loss-of-function mutations on the influenza A virus NS segment that is involved in its anti-interferon activity.

  • Identification and characterization of two novel spliced genes located in the orf47-orf46-orf45 gene locus of Kaposi's sarcoma-associated herpesvirus [PublishAheadOfPrint]

  • The orf47-orf46-orf45 gene cluster of Kaposi's sarcoma-associated herpesvirus (KSHV) is known to serially encode glycoprotein L (gL), uracial DNA glycosylase, and a viral tegument protein. Here, we identify two novel mRNA variants, orf47/45-A and orf47/45-B, alternatively spliced from a tricistronic orf47+46+45 mRNA that is expressed in the orf47-orf46-orf45 gene locus during the early stages of viral reactivation. The spliced gene products, ORF47/45-A and ORF47/45-B, consist of only a partial region of gL (ORF47), a unique 7-amino acid motif, and the complete tegument protein ORF45. Like ORF45 protein, ORF47/45-A and ORF47/45-B expressed in cells sufficiently activate the phosphorylation of p90 ribosomal S6 kinase (RSK) and extracellular signal-regulated protein kinase (ERK). However, unlike ORF45, both ORF47/45-A and ORF47/45-B contain a signal peptide sequence and are localized at endoplasmic reticulum (ER). Additionally, we found that ORF47/45-A and ORF47/45-B have an extra function that mediates the upregulation of GRP78, a master regulator of ER homeostasis. The important event regarding the GRP78 upregulation can be observed in all tested KSHV-positive cell lines after viral reactivation and knockdown of GRP78 in cells significantly impairs viral lytic-cycle progression, especially at late lytic stages. Compared with some other viral glycoproteins synthesized through ER, our results strongly implicate that ORF47/45s may serve as key effectors for controlling the GRP78 expression and ER homeostasis in cells. Taken together, our findings provide evidence showing the reciprocal association between the modulation of ER homeostasis and the progression of the KSHV lytic cycle.

    Importance Emerging evidence has shown that several viruses appear to use different strategies to control ER homeostasis for supporting their productive infections. This study identifies two aspects regarding the association between the regulation of ER homeostasis and the progression of the KSHV lytic cycle. The first part is the functional characterization of two early lytic cycle proteins, ORF47/45-A and ORF47/45-B, on the activation of a major ER chaperone protein GRP78. In addition to the ability to promote the GRP78 upregulation, ORF47/45s also activate the phosphorylation of RSK and ERK. The second part reveals that upregulation of GRP78 is essential for the progression of the KSHV lytic cycle, especially at late stages. We therefore propose that activation of the GRP78 expression by viral proteins at the early lytic stage may aid to protect host cells from severe ER stress and may directly involve the assembly or release of virions.

  • The ATR signaling pathway is disabled during infection by the parvovirus minute virus of mice [PublishAheadOfPrint]

  • The ATR kinase has essential functions in maintenance of genome integrity in response to replication stress. ATR is recruited to RPA-coated single-stranded DNA at DNA damage sites via its interacting partner ATRIP, which binds to the large subunit of RPA. ATR activation typically leads to activation of the Chk1 kinase among other substrates. We show here that, together with a number of other DNA repair proteins, both ATR and its associated protein ATRIP were recruited to viral nuclear replication compartments (APAR bodies) during replication of the single-stranded parvovirus minute virus of mice (MVM). Chk1, however, was not activated during MVM infection even though viral genomes bearing bound RPA nndash; normally a potent trigger of ATR activation mmdash; accumulate in APAR bodies. Failure to activate Chk1 in response to MVM infection was likely due to our observation that Rad9 failed to associate with chromatin at MVM APAR bodies. Additionally, early in infection, prior to the onset of the virus-induced DNA damage response (DDR), stalling of the replication of MVM genomes with hydroxyurea (HU) resulted in Chk1 phosphorylation in a virus dose-dependent manner. However, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to HU and various other drug treatments. Finally, ATR phosphorylation became undetectable upon MVM infection and, although virus infection induced RPA32 phosphorylation on serine 33, an ATR-associated phosphorylation site, this phosphorylation event could not be prevented by ATR depletion or inhibition. Together our results suggest that MVM infection disables the ATR signaling pathway.

    IMPORTANCE Upon infection, the parvovirus MVM activates a cellular DNA damage response that governs the virus-induced cell cycle arrest and is required for efficient virus replication. ATM and ATR are major cellular kinases that coordinate the DNA damage response to diverse DNA damage stimuli. Although a significant amount has been discovered about ATM activation during parvovirus infection, involvement of the ATR pathway has been less studied. During MVM infection, Chk1, a major downstream target of ATR, is not detectably phosphorylated even though viral genomes bearing the bound cellular single-strand binding protein RPA nndash; normally a potent trigger of ATR activation mmdash; accumulate in viral replication centers. ATR phosphorylation also became undetectable. In addition, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to hydroxyurea and various other drug treatments. Our results suggest that MVM infection disables this important cellular signaling pathway.

  • Comparison of Effects of Inhibitors of Viral and Cellular Protein Kinases on Human Cytomegalovirus Disruption of Nuclear Lamina and Nuclear Egress [PublishAheadOfPrint]

  • The human cytomegalovirus (HCMV) kinase UL97 is required for efficient nuclear lamina disruption during nuclear egress. However, cellular protein kinase C (PKC) has been implicated in this process in other systems. Comparing the effects of UL97 or cellular kinase inhibitors on HCMV nuclear egress confirms a role for UL97 in lamina disruption and nuclear egress. A pan-PKC inhibitor did not affect lamina disruption, but did reduce the number of cytoplasmic capsids more than nuclear capsids.

  • Development of giant bacteriophage FEKZ is independent of the host transcription apparatus [PublishAheadOfPrint]

  • Pseudomonas aeruginosa bacteriophage FEKZ is the type representative of the llsquo;giant' phage genus characterized by unusually large virions and genomes. By unraveling the transcriptional map of the ~280 kb FEKZ genome to single-nucleotide resolution, we combine 369 FEKZ genes into 134 operons. Early transcription is initiated from highly conserved AT-rich promoters distributed across the FEKZ genome and located on the same strand of the genome. Early transcription does not require phage or host protein synthesis. Transcription of middle and late genes is dependent on protein synthesis and mediated by poorly conserved middle and late promoters. Unique to FEKZ is its ability to complete its infection in the absence of bacterial RNA polymerase (RNAP) enzyme activity. We propose that transcription of the FEKZ genome is performed by the consecutive action of two FEKZ-encoded, non-canonical multisubunit RNAPs, one of which is packed within the virion, another being the product of early genes. This unique, rifampicin-resistant transcriptional machinery is conserved within the diverse giant phage genus.

    IMPORTANCE The data presented in this paper offer, for the first time, an insight into the complex transcriptional scheme of giant bacteriophages. We show that Pseudomonas aeruginosa giant phage FEKZ is able to infect and lyse its host cell and produce phage progeny in the absence of functional bacterial transcriptional machinery. This unique property can be attributed to two phage-encoded putative RNAP enzymes, which contain very distant homologues of bacterial bbeta; and bbeta;'-like RNAP subunits.

  • Impaired Nef function is associated with early control of HIV-1 viremia [PublishAheadOfPrint]

  • Host and viral factors influence the HIV-1 infection course. Reduced Nef function has been observed in HIV-1 controllers during the chronic phase, but the kinetics and mechanisms of Nef attenuation in such individuals remain unclear. We examined plasma RNA-derived Nef clones from 10 recently infected individuals who subsequently suppressed viremia to less than 2000 RNA copies/mL within one year post-infection (acute controllers) and 50 recently infected individuals who did not control viremia (acute progressors). Nef clones from acute controllers displayed lower ability to downregulate CD4 and HLA class I from the cell surface and reduced ability to enhance virion infectivity compared to those from acute progressors (all pllt;0.01). HLA class I downregulation activity correlated inversely with days post-infection (Spearman's R= nndash;0.85, p=0.004) and positively with baseline plasma viral load (Spearman's R=0.81, p=0.007) in acute controllers, but not in acute progressors. Nef polymorphisms associated with functional changes over time were identified in follow-up samples from six controllers. For one such individual, mutational analyses indicated that four polymorphisms selected by HLA-A*31 and B*37 acted in combination to reduce Nef steady state protein levels and HLA class I downregulation activity. Our results demonstrate that relative control of initial HIV-1 viremia is associated with Nef clones that display reduced function, which in turn may influence the course of HIV-1 infection. Transmission of impaired Nef sequences likely contribute in part to this observation; however, accumulation of HLA-associated polymorphisms in Nef that impair function also suggests that CD8+ T-cell pressures play a role in this phenomenon.

    Importance Rare individuals can spontaneously control HIV-1 viremia in the absence of antiretroviral treatment. Understanding the host and viral factors that contribute to the controller phenotype may identify new strategies to design effective vaccines or therapeutics. The HIV-1 Nef protein enhances viral pathogenesis through multiple mechanisms. We examined the function of plasma HIV-1 RNA-derived Nef clones isolated from 10 recently infected individuals who subsequently controlled HIV viremia, compared to those from 50 individuals who failed to control viremia. Our results demonstrate that early Nef clones from HIV controllers displayed lower HLA class I and CD4 downregulation activity, as well as a reduced ability to enhance virion infectivity. The accumulation of HLA-associated polymorphisms in Nef during the first year post-infection was associated with impaired protein function in some controllers. This work highlights the potential for host immune responses to modulate HIV pathogenicity and disease outcome by targeting CTL epitopes in Nef.

  • A novel lifecycle modeling system for Ebola virus shows a genome length-dependent role of VP24 on virus infectivity [PublishAheadOfPrint]

  • Work with infectious Ebola viruses is restricted to biosafety level (BSL) 4 laboratories, presenting a significant barrier for studying these viruses. Lifecycle modeling systems, including minigenome systems and transcription and replication-competent virus-like particle (trVLP) systems, allow modeling of the virus lifecycle under BSL2 conditions; however, all current systems model only certain aspects of the virus lifecycle, rely on plasmid-based viral protein expression, and have only been used to model single infectious cycles. We have developed a novel lifecycle modeling system allowing continuous passaging of infectious trVLPs containing a tetracistronic minigenome that encodes a reporter and the viral proteins VP40, VP24, and GP1,2. This system is ideally suited for studying morphogenesis, budding and entry, in addition to genome replication and transcription. Importantly, the specific infectivity of trVLPs in this system was ~500 fold higher than in previous systems. Using this system for functional studies of VP24 we showed that, contrary to previous reports, VP24 only very modestly inhibits genome replication and transcription when expressed in a regulated fashion, which we confirmed using infectious Ebola viruses. Interestingly, we also discovered a genome length-dependent effect of VP24 on particle infectivity, which was previously undetected due to the short length of monocistronic minigenomes, and which is due at least partially to a previously unknown function of VP24 in RNA packaging. Based on our findings we propose a model for the function of VP24 that reconciles all currently available data regarding the role of VP24 in nucleocapsid assembly as well as genome replication and transcription.

    Importance Ebola viruses cause severe hemorrhagic fevers in humans, with no countermeasures currently available, and must be studied in maximum containment laboratories. Only a handful of these laboratories exist worldwide, limiting our ability to study Ebola viruses and develop countermeasures. Here we report the development of a novel reverse genetics-based system that allows the study of Ebola viruses without maximum containment laboratories. We used this system to investigate the Ebola virus protein VP24, showing that contrary to previous reports it only modestly inhibits virus genome replication and transcription but is important for packaging of genomes into virus particles, which constitutes a previously unknown function of VP24 and a potential antiviral target. We further propose a comprehensive model for the function of VP24 in nucleocapsid assembly. Importantly, based on this approach it should be easily possible to develop similar experimental systems for other viruses that are currently restricted to maximum containment laboratories.

  • Virus-induced tubule: a vehicle for rapid spread of virions through basal lamina from midgut epithelium in the insect vector [PublishAheadOfPrint]

  • The plant reoviruses, plant rhabdoviruses, tospoviruses and tenuiviruses are transmitted by insect vectors in a persistent-propagative manner. These viruses induce the formation of viral inclusions to facilitate viral propagation in insect vectors. The intestines of insect vectors are formed by the epithelial cells that lie on the noncellular basal lamina surrounded by visceral muscle tissue. Here, we demonstrate that a recently identified plant reovirus, southern rice black streaked dwarf virus (SRBSDV), exploits virus-containing tubules composed of virus-encoded nonstructural protein P7-1 to directly cross the basal lamina from the initially infected epithelium toward visceral muscle tissues in the intestine of its vector, the white-backed planthopper (Sogatella furcifera). Furthermore, such tubules spread along visceral muscle tissues through a direct interaction of P7-1 and actin. The destruction of tubule assembly by RNA interference with synthesized dsRNA targeting the P7-1 gene inhibits viral spread in insect vector in vitro and in vivo. All these results, for the first time, show that a virus employs virus-induced tubule as a vehicle for viral spread from the initially infected midgut epithelium through the basal lamina, facilitating the rapid dissemination of virus from the intestine of the insect vector.

    Importance: Numerous plant viruses are transmitted in a persistent manner by sap-sucking insects, including thrips, aphids, planthoppers and leafhoppers. These viruses, ingested by the insects, establish their primary infection in the intestinal epithelium of the insect vector. Subsequently, the invading virus manages to transverse the basal lamina, a noncellular layer lining the intestine, a barrier that may theoretically hinder viral spread. The mechanism by which plant viruses cross the basal lamina is unknown. Here, we report that a plant virus has evolved to exploit virus-induced tubules to pass through the basal lamina from the initially infected midgut epithelium of the insect vector, thus revealing the previously undescribed pathway adapted by the virus for rapid dissemination of virions from the intestine of the insect vector.

  • Modification of the RSV F Protein in Virus-like Particles Impacts Generation of B Cell Memory [PublishAheadOfPrint]

  • Immunization with virus-like particles (VLPs) containing the Newcastle disease virus (NDV) core proteins, NP and M, and two chimera proteins (F/F and H/G) containing the respiratory syncytial virus (RSV) F and G protein ectodomains fused to the transmembrane and cytoplasmic domains of NDV F and HN proteins respectively, stimulated durable RSV neutralizing antibodies, F protein specific long lived, bone marrow associated plasma cells (LLPC) and B cell memory, in striking contrast to RSV infection (Schmidt, et al J. Virol. 86: 11654). Here we report the characterization of a VLP with an RSV F protein ectodomain fused to the NDV F HR2, transmembrane, and cytoplasmic domain sequences creating a chimera with two tandem HR2 domains, one from the RSV F protein and the other from the NDV F protein ectodomain (F/HR2F). F/HR2F chimera protein was efficiently assembled into VLPs along with the H/G chimera protein. This VLP (VLP-H/G+F/HR2F) stimulated anti-F protein and anti-G protein IgG, durable RSV neutralizing antibodies, and anti-RSV F protein secreting LLPC. However, the subtypes of anti-F IgG induced were different than those elicited by VLPs containing the F/F chimera (VLP-H/G+F/F). Most importantly, VLP-H/G+F/HR2F did not induce RSV F protein specific B cell memory as shown by adoptive transfer of B cells from immunized animals to immunodeficient animals. The VLP did, however, induce B cell memory specific to the RSV G protein. Thus the form of the F protein has a direct role in inducing anti-F protein B cell memory.

    Importance Development of vaccines for respiratory syncytial virus (RSV) is hampered by a lack of a clear understanding of the requirements for eliciting protective as well as durable human immune responses to virus antigens. The results of this study indicate that the form of the RSV F protein has a direct and significant impact on the type of anti-F IgG antibodies induced and the generation of F protein specific memory. Identification of the conformation of the RSV F protein that most effectively stimulates not only LLPC and but also memory B cells will be important in future development of RSV vaccines.

  • Structure of 2G12 Fab2 in Complex with Soluble and Fully Glycosylated HIV-1 Env by Negative-Stain Single Particle Electron Microscopy [PublishAheadOfPrint]

  • The neutralizing anti-HIV-1 antibody 2G12 is of particular interest due to the sterilizing protection it provides from viral challenge in animals models. 2G12 is a unique, domain-exchanged antibody, which binds exclusively to conserved N-linked glycans that form the high mannose patch on the gp120 outer domain centered on a glycan at position N332. Several glycans in and around the 2G12 epitope have been shown to interact with other potent, broadly neutralizing antibodies; therefore, this region constitutes a supersite of vulnerability on gp120. While crystal structures of 2G12 and 2G12 bound to high mannose glycans have been solved, no structural information that describes the interaction of 2G12 with gp120 or the Env trimer is available. Here we present a negative stain single-particle electron microscopy reconstruction of 2G12 Fab2 in complex with a soluble, trimeric Env at ~17 AAring; resolution that reveals the antibody interaction with its native and fully glycosylated epitope. We also map relevant glycans in this epitope by fitting high-resolution crystal structures and by performing neutralization assays of glycan knock-outs. In addition, a reconstruction at ~26 AAring; of the ternary complex formed by 2G12 Fab2, soluble CD4 and Env indicates that 2G12 may block membrane fusion by induced steric hindrance upon primary receptor binding, thereby abrogating Env interaction with co-receptor(s). These structures provide a basis for understanding 2G12 binding and neutralization and our low-resolution model and glycan assignments provide a basis for higher-resolution studies to determine the molecular nature of the 2G12 epitope.

    Importance HIV-1 is a deadly virus that results in the deaths of millions of people around the world each year. While there are several effective therapeutics available to prolong life, a vaccine is the best long-term solution for curbing this global epidemic. Here we present structural data that reveal the viral binding site of one of the first HIV-1 neutralizing antibodies isolated, 2G12, and provide a rationale for its effectiveness. These structures provide a basis for higher-resolution studies to determine the molecular nature of the 2G12 epitope, which will aid in vaccine design and antibody-based therapies.

  • Impact of Distinct Poxvirus Infections on the Specificities and Functionalities of CD4+ T Cell Responses [PublishAheadOfPrint]

  • The factors that determine CD4+ T cell (TCD4+) specificities, functional capacity, and memory persistence in response to complex pathogens remain unclear. We explored these parameters in the C57Bl/6 mouse through comparison of two highly related (ggt;92% homology) poxviruses: ectromelia virus (ECTV), a natural mouse pathogen, and vaccinia virus (VACV), a heterologous virus that nevertheless elicits potent immune responses. In addition to elucidating several previously unidentified major histocompatibility complex class II (MHCII)-restricted epitopes, we observed many qualitative and quantitative differences between the TCD4+ repertoires, including responses not elicited by VACV despite complete sequence conservation. In addition, we observed functional heterogeneity between ECTV and VACV specific TCD4+ at both a global and individual epitope level, particularly greater expression of the cytolytic marker, CD107a from TCD4+ following ECTV infection. Most striking were differences during the late memory phase where, in contrast to ECTV, VACV infection failed to elicit measurable, epitope specific TCD4+ by intracellular cytokine staining. These findings illustrate the strong influence of epitope-extrinsic factors on TCD4+ responses and memory.

    Importance Much of our understanding concerning host-pathogen relationships in the context of poxvirus infections stems from studies of VACV in mice. However, VACV is not a natural mouse pathogen and, therefore, the relevance of results obtained using this model may be limited. Here, we explored the MHC class II-restricted TCD4+ repertoire induced by mousepox (ECTV) infection and the functional profile of the responding epitope-specific TCD4+, comparing these results to those induced by VACV infection under matched conditions. Despite a high degree of homology between the two viruses, we observed distinct specificity and functional profiles of TCD4+ responses at both acute and memory time-points, with VACV-specific TCD4+ memory being notably compromised. These data offer insight into the impact of "epitope extrinsic" factors on resulting TCD4+ responses.

  • Modulation of CD163 Expression by Metalloprotease ADAM17 Regulates Porcine Reproductive and Respiratory Syndrome Virus Entry [PublishAheadOfPrint]

  • As a consequence of their effects on ectodomain shedding, members of the A disintegrin and metalloprotease (ADAM) family have been implicated in the control of various cellular processes. Although ADAM family members are also involved in cancer, inflammation, and other pathologies, it's unclear whether they affect porcine reproductive and respiratory syndrome virus (PRRSV) infection. Here, we demonstrate for the first time that inhibition of ADAM17 enhances PRRSV entry in Marc-145 and porcine alveolar macrophages (PAMs). We also demonstrate that inhibition of ADAM17 up-regulates membrane CD163 expression, a putative PRRSV receptor that is exogenously expressed in BHK-21 and endogenously expressed in Marc-145 and PAMs. Furthermore, overexpression of ADAM17 induced down-regulation of CD163 expression and a reduction in PRRSV infection, whereas ablation of ADAM17 expression using specific siRNA resulted in up-regulation of CD163 expression with a corresponding increase in PRRSV infection. These ADAM17-mediated effects were confirmed with PRRSV non-permissive BHK-21 cells transfected with CD163 cDNA. Overall, these findings indicate that ADAM17 down-regulates CD163 expression and hinders PRRSV entry. Hence, down-regulation of ADAM17 particular substrates may be an additional component of the anti-infection defenses.

    IMPORTANCE ADAM17 is one of the important membrane-associated metalloproteases that mediate various cellular events as well as inflammation, cancer, and other pathologies. Here, we investigate for the first time the role of the metalloprotease ADAM17 in PRRSV infection. By using inhibitor and genetic modification methods, we demonstrate that ADAM17 negatively regulate PRRSV entry by regulating its substrate(s). More specifically, ADAM 17 mediates the down-regulation of the PRRSV cellular receptor CD163. The reduction in CD163 expression represents another component of the anti-infection response initiated by ADAM17.

  • Murine leukemia virus Gag localizes to the uropod of migrating primary lymphocytes [PublishAheadOfPrint]

  • B and CD4+ T lymphocytes are natural targets of Murine leukemia virus (MLV). Migrating lymphocytes adopt a polarized morphology with a trailing edge designated uropod. Here, we demonstrate that MLV Gag localizes to the uropod in polarized B cells and CD4+ T cells. The uropod localization of MLV Gag was dependent on plasma membrane (PM) association and multimerization of Gag, but independent of the viral glycoprotein Env. Basic residues in MA that are required for MLV Gag recruitment to virological synapses between HEK293 and XC cells, were dispensable for uropod localization in migrating B cells. Ultrastructural studies indicated that both wild-type and basic residue mutant Gag localized to the outer surface of the PM at the uropod. Late-domain mutant virus particles were seen at the uropod in form of budding-arrested intermediates. Finally, uropods mediated contact between MLV-infected B cells and uninfected T cells to form virological synapses. Our results suggest that MLV not unlike HIV accumulate at the uropod of primary lymphocytes to facilitate viral spreading through the formation of uropod mediated cell-cell contacts.

    IMPORTANCE Viruses have evolved mechanisms to coordinate their assembly and budding with cell polarity to facilitate their spreading. In this study, we demonstrate that the viral determinants for MLV Gag to localize to the uropod in polarized B cells are distinct from the requirements to localize to virological synapses in transformed cell lines. Basic residues in MA that are required for the Gag localization to virological synapses between HEK293 and XC cells are dispensable for Gag localization to the uropod in primary B cells. Rather, plasma membrane association and capsid-driven multimerization of Gag are sufficient to drive MLV Gag to the uropod. MLV-laden uropods also mediate contacts between MLV-infected B cells and uninfected T cells to form virological synapses. Our results indicate that MLV accumulates at the uropod of primary lymphocytes to facilitate viral spreading through the formation of uropod-mediated cell-cell contacts.

  • Introductions and evolution of human-origin seasonal influenza A viruses in multinational swine populations [PublishAheadOfPrint]

  • The capacity of influenza A viruses to cross species barriers presents a continual threat to human and animal health. Knowledge of the human-swine interface is particularly important for understanding how viruses with pandemic potential evolve in swine hosts. We sequenced the genomes of 141 influenza viruses collected from North American swine during 2002-2011 and identified a swine virus that possessed all eight genome segments of human seasonal A/H3N2 virus origin. A molecular clock analysis indicates that this virus mmdash; A/sw/Saskatchewan/02903/2009(H3N2) mmdash; has likely circulated undetected in swine for at least seven years. For historical context, we performed a comprehensive phylogenetic analysis of an additional 1,404 whole-genome sequences from swine influenza A viruses collected globally during 1931-2013. Human-to-swine transmission occurred frequently over this time period, with 20 discrete introductions of human seasonal influenza A viruses showing sustained onward transmission in swine for at least one year since 1965. Notably, human-origin hemagglutinin (H1 and H3) and neuraminidase (particularly N2) segments were detected in swine at a much higher rate than the six internal gene segments, suggesting an association between the acquisition of swine-origin internal genes via reassortment and the adaptation of human influenza viruses to new swine hosts. Further understanding of the fitness constraints on the adaptation of human viruses to swine, and vice versa, at a genomic level is central to understanding the complex multi-host ecology of influenza and the disease threats that swine and humans pose to each other.

    Importance The swine origin of the 2009 A/H1N1 pandemic virus underscored the importance of understanding how influenza A virus evolves in these animals hosts. While the importance of reassortment in generating genetically diverse influenza viruses in swine is well documented, the role of human-to-swine transmission has not been as intensively studied. Through a large-scale sequencing effort we identified a novel influenza virus of wholly human origin that has been circulating undetected in swine for at least seven years. In addition, we demonstrate that human-to-swine transmission has occurred frequently at a global scale over the past decades, but that there is little persistence of human virus internal gene segments in swine.

  • Activation of A1-adenosine Receptors Promotes Leukocyte Recruitment to the Lung and Attenuates Acute Lung Injury in Mice Infected with Influenza A/WSN/33 (H1N1) Virus [PublishAheadOfPrint]

  • We have shown that bronchoalveolar epithelial A1-adenosine receptors (A1-AdoR) are activated in influenza A virus-infected mice. Alveolar macrophages and neutrophils also express A1-AdoRs and we hypothesized that activation of A1-AdoRs on these cells will promote macrophage and neutrophil chemotaxis and activation and thereby play a role in the pathogenesis of influenza virus-induced acute lung injury. Wild-type (WT) C57BL/6 mice, congenic A1-AdoR-knockout (A1-KO) mice, and mice that had undergone reciprocal bone marrow transfer were inoculated intranasally with 10,000 PFU/mouse influenza A/WSN/33 (H1N1). Alternatively, WT mice underwent daily treatment with the A1-AdoR antagonist DPCPX from one day prior to inoculation. Infection increased BALF adenosine comparably in WT and A1-KO mice. Infection of WT mice resulted in reduced carotid arterial O2 saturation (hypoxemia), lung pathology, pulmonary edema, reduced lung compliance, increased basal airway resistance, and hyperresponsiveness to methacholine. These effects were absent or significantly attenuated in A1-KO mice. BALF leukocytes, IFN-, and IL-10 were significantly reduced in infected A1-KO mice, but KC, IP-10, and MCP-1 were increased. Reciprocal bone marrow transfer resulted in WT-like lung injury severity, but BALF leukocytes only increased in WT to A1-KO mice. Hypoxemia, pulmonary edema, and BALF alveolar macrophages, neutrophils, IFN-, and IL-10 were reduced in DPCPX-treated WT mice. Viral replication did not differ between mouse strains or treatment groups. These findings indicate that adenosine activation of leukocyte A1-AdoRs plays a significant role in their recruitment to the infected lung and contributes to influenza pathogenesis. A1-AdoR inhibitor therapy may therefore be beneficial in patients with influenza-induced lung injury.

    IMPORTANCE Because antiviral drugs are of limited efficacy in patients hospitalized for influenza virus-induced respiratory failure, there is an urgent need for new therapeutics that can limit progression of lung injury and reduce influenza death rates. We show that influenza A virus infection results in increased production of the nucleoside adenosine in the mouse lung, and that activation of A1-subtype adenosine receptors by adenosine contributes significantly to both recruitment of innate immune cells to the lung and development of acute lung injury following influenza infection. We also show that treatment with an A1-adenosine receptor antagonist reduces the severity of lung injury in influenza-infected mice. Our findings indicate that adenosine plays an important and previously unrecognized role in the innate immune response to influenza infection, and suggest that drugs which can inhibit either adenosine generation or activation of A1-adenosine receptors may be beneficial in treating influenza patients hospitalized for respiratory failure.

  • Long-term Antiretroviral Treatment Initiated in Primary HIV-1 Infection Affects the Size, Composition and Decay Kinetics of the Reservoir of HIV-1 Infected CD4 T Cells [PublishAheadOfPrint]

  • Initiation of antiretroviral therapy during the earliest stages of HIV-1 infection may limit the seeding of a long-lasting viral reservoir, but long-term effects of early antiretroviral treatment initiation remain unknown. Here, we analyzed immunological and virological characteristics of nine patients who started antiretroviral therapy in primary HIV-1 infection and remained on suppressive treatment for ggt;10 years; patients with similar treatment duration but initiation of suppressive therapy in chronic HIV-1 infection served as controls. We observed that independently of the timing of treatment initiation, HIV-1 DNA in CD4 T cells decayed primarily during the initial 3-4 years of treatment. However, in patients who started antiretroviral therapy in early infection, this decay occurred faster and was more pronounced, leading to substantially lower levels of cell-associated HIV-1 DNA after long-term treatment. Despite this smaller size, the viral CD4 T cell reservoir in persons with early treatment initiation consisted more dominantly of the long-lasting central-memory and T memory stem cells. HIV-1-specific T cell responses remained continuously detectable during antiretroviral therapy, independently of the timing of treatment initiation. Together, these data suggest that early HIV-1 treatment initiation, even when continued for ggt;10 years, is unlikely to lead to viral eradication, but the presence of low viral reservoirs and durable HIV-1 T cell responses may make such patients attractive candidates for future interventional studies aiming at HIV-1 eradication and cure.

    Importance Antiretroviral therapy can effectively suppress HIV-1 replication to undetectable levels; however, HIV-1 can persist despite treatment, and viral replication rapidly rebounds when treatment is discontinued. This is mainly due to the presence of latently infected CD4 T cells, which are not susceptible to antiretroviral drugs. Starting treatment in the earliest stages of HIV-1 infection can limit the number of these latently infected cells, raising the possibility that these viral reservoirs are naturally eliminated if suppressive antiretroviral treatment is continued for extremely long periods of time. Here, we analyzed nine patients who started on antiretroviral therapy within the earliest weeks of the disease, and continued treatment for more than 10 years. Our data show that early treatment accelerated the decay of infected CD4 T cells and led to very low residual levels of detectable HIV-1 after long-term therapy nndash; levels that were otherwise detectable in patients who are able to maintain a spontaneous, drug-free control of HIV-1 replication. Thus, long-term antiretroviral treatment started during early infection cannot eliminate HIV-1, but the reduced reservoirs of HIV-1 infected cells in such patients may increase their chances to respond to clinical interventions aiming at inducing a drug-free remission of HIV-1 infection.

  • The Role of Electrostatics in the Assembly Pathway of a Single-Stranded RNA Virus [PublishAheadOfPrint]

  • We have recently discovered (Cadena-Nava, et al. J. Virol. 2012, 86, 3318) that the in vitro packaging of RNA by the capsid protein (CP) of Cowpea Chlorotic Mottle Virus is optimal when there is a significant excess of CP, specifically that complete packaging of all the RNA in solution requires sufficient CP to provide charge matching of the N-terminal positively-charged arginine-rich motifs (ARMS) of the CPs with the negatively-charged phosphate backbone of the RNA. We show here that packaging results from the initial formation of a "charge-matched" protocapsid consisting of RNA decorated by a disordered arrangement of CPs. This protocapsid reorganizes into the final, icosahedrally-symmetric, nucleocapsid by displacing the excess CPs from the RNA to the exterior surface of the emerging capsid, through electrostatic attraction between the ARMs of the excess CP and the negative charge density of the capsid exterior. As a test of this scenario we prepare CP mutants with "extra" and "missing" (relative to wildtype) cationic residues and show that a correspondingly smaller and larger excess of CP is needed, respectively, for complete packaging of RNA.

    IMPORTANCE Cowpea Chlorotic Mottle Virus (CCMV) has long been studied as a model system for the assembly of single-stranded RNA viruses. While much is known about the electrostatic interactions within the CCMV virion, relatively little is known about these interactions during assembly nndash; i.e., within intermediate states preceding the final nucleocapsid structure. Theoretical models and coarse-grained molecular dynamics simulations suggest that viruses like CCMV assemble by the bulk adsorption of CPs onto the RNA nndash; driven by electrostatic attraction nndash; followed by structural reorganization into the final capsid. Such a mechanism facilitates assembly by condensing the RNA for packaging while simultaneously concentrating the local density of CP for capsid nucleation. We provide experimental evidence of such a mechanism by demonstrating that efficient assembly is initiated by the formation of a disordered protocapsid complex whose stoichiometry is governed by electrostatics (charge matching of the anionic RNA and the cationic N-termini of the CP).

  • A novel partitivirus that confers hypovirulence on plant pathogenic fungi [PublishAheadOfPrint]

  • Members of the family Partitiviridae have bisegmented dsRNA genomes and are not generally known to cause obvious symptoms in their natural hosts. An unusual partitivirus, Sclerotinia sclerotiorum partitivirus 1 (SsPV1/WF-1), conferred hypovirulence on its natural plant pathogenic fungal host, Sclerotinia sclerotiorum strain WF-1. Cellular organelles including mitochondria were severely damaged. Hypovirulence and associated traits of strain WF-1 and SsPV1/WF-1 were readily co-transmitted horizontally via hyphal contact to different vegetative compatibility groups of S. sclerotiorum and interspecifically to Sclerotinia nivalis and Sclerotinia minor. S. sclerotiorum strain 1980 transfected with purified SsPV1/WF-1 virions also exhibited hypovirulence and associated traits similar to those of strain WF-1. Moreover, introduction of purified SsPV1/WF-1 virions into strain KY-1 of Botrytis cinerea also resulted in reduction in virulence and mycelial growth, and, unexpectedly, enhanced conidial production. However, virus infection suppressed hyphal growth of most germinating conidia of B. cinerea and was eventually lethal to infected hyphae since very few new colonies could develop following germ tube formation. Taken together, our results support the conclusion that SsPV1/WF-1 causes hypovirulence in Sclerotinia spp. and B. cinerea. CryoEM reconstruction of the SsPV1 particle shows it has a distinct structure with similarity to closely related partitivirus Fusarium poae virus 1 and Penicillium stoloniferum virus F. These findings provide new insights into partitivirus biological activities and clues about molecular interactions between partitiviruses and their hosts.

    IMPORTANCE Members of Partitiviridae are believed to be of common occurrence in their phytopathogenic fungi and plant hosts. However, most partitiviruses examined so far appear to be associated with latent infections. Here we report a partitivirus, SsPV1/WF-1, that was isolated from a hypovirulent strain of Sclerotinia sclerotiorum, and describe its biological and molecular features. We have demonstrated that SsPV1 confers hypovirulence. Furthermore, SsPV1 can infect, and cause hypovirulence in Botrytis cinerea. Our study also suggests that SsPV1 has vigorous ability for proliferation and spread via hyphal contact. SsPV1 can overcome vegetative incompatibility barriers and can be transmitted horizontally among different vegetative compatibility groups of S. sclerotiorum, even transmitted interspecifically. CryoEM reconstruction of SsPV1 shows it has a distinct structure with similarity to closely related partitiviruses. Our studies exploit a novel system, SsPV1 and its hosts, which can provide the means to explore the mechanisms by which partitiviruses interact with their hosts.

  • Host Factors interacting with the Pestivirus N terminal protease, Npro are Components of the Ribonucleoprotein Complex [PublishAheadOfPrint]

  • The viral N-terminal protease Npro of pestiviruses counteracts cellular antiviral defences through inhibition of IRF3. Here we use mass spectrometry to identify a new role for Npro through its interaction with 55 associated proteins, mainly ribosomal proteins and ribonucloproteins, including RNA helicase A (DHX9), Y-box binding protein (YBX1), DDX3, DDX5, eIF3, IGF2BP1, multiple myeloma tumour protein 2, interleukin enhancer binding factor 3 (IEBP3), guanine nucleotide binding protein 3 and polyadenylate-binding protein 1 (PABP-1). These are components of the translation machinery, ribonucleoparticles and stress granules. Significantly, we found that stress granule formation was inhibited in MDBK cells infected with a non-cytopathic BVDV strain Kyle. However, RNPs binding to Npro did not inhibit these proteins from aggregating into stress granules. Npro interacted with YBX1 though its TRASH domain, since the mutant C112R protein with an inactive TRASH domain, no longer redistributed to stress granules. Interestingly, RNA helicase A and La autoantigen relocated from a nuclear location to form cytoplasmic granules with Npro. To address a pro-viral role for Npro in RNP granules, we investigated whether Npro affected RNA interference (RNAi), since interacting proteins have roles in RISC function during RNA silencing. Using GAPDH silencing with siRNAs followed by Northern blot analysis of GAPDH, expression of Npro had no effect on RNAi silencing activity, contrasting with other viral suppressors of interferon. We propose that Npro is involved with virus RNA translation in the cytoplasm for virus particle production, and when translation is inhibited following stress, it redistributes to the replication complex.

    Importance Although the pestivirus N-terminal protease, Npro has shown to have an important role in degrading IRF3 to prevent apoptosis and interferon production during infection, the function of this unique viral protease in the pestivirus life cycle remains to be elucidated. We use proteomic mass spectrometry to identify novel interacting proteins and show Npro is present in ribosomal and ribonucleoprotein particles, indicating a translational role in virus particle production. The virus itself can prevent stress granule assembly from these complexes, but this inhibition is not due to Npro. A pro-viral role to subvert RNA silencing through binding of these host RNP proteins was not identified for this viral suppressor of interferon.

  • Changes in protein domains outside the catalytic site of the bacteriophage Q{beta} replicase reduce the mutagenic effect of 5-azacytidine [PublishAheadOfPrint]

  • The high genetic heterogeneity and great adaptability of RNA viruses is ultimately caused by the low replication fidelity of their polymerases. However, single amino acid substitutions that modify replication fidelity can evolve in response to mutagenic treatments with nucleoside analogues. Here, we investigate how two independent mutants of the bacteriophage Qbbeta; replicase (Thr210Ala and Tyr410His) reduce sensitivity to the nucleoside analogue 5-azacytidine (AZC). Despite being located outside the catalytic site, both mutants reduced the mutation frequency in the presence of the drug. However, they did not modify the type of AZC-induced substitutions, which was mediated mainly by ambiguous base-pairing of the analogue with purines. Furthermore, the Thr210Ala and Tyr410His substitutions had little or no effect on replication fidelity in untreated viruses. Also, both substitutions were costly in the absence of AZC, or when the action of the drug was suppressed by adding an excess of natural pyrimidines, uridine or cytosine. Overall, the phenotypic properties of these two mutants were highly convergent, despite being located in different domains of the Qbbeta; replicase. This suggests that treatment with a given nucleoside analogue tends to select for a unique functional response in the viral replicase.

    IMPORTANCE In the last years, artificial increase of the replication error rate has been proposed as an antiviral therapy. In this study we investigate the mechanisms by which two substitutions in the Qbbeta; replicase confer partial resistance to the mutagenic nucleoside analogue AZC. As opposed to previous work with animal viruses, where different mutations selected sequentially confer nucleoside analogue resistance through different mechanisms, our results suggest that there are few or no alternative AZC resistance phenotypes in Qbbeta;. Also, despite resistance mutations being highly costly in the absence of the drug, there was no sequential fixation of secondary mutations. Bacteriophage Qbbeta; is the virus with the highest reported mutation rate, which should make it particularly sensitive to nucleoside analogue treatments, probably favoring resistance mutations even if they incur in high costs. The results are also relevant for understanding the possible pathways by which fidelity of the replication machinery can be modified.

  • 3D imaging of HIV-1 virological synapses reveals membrane architectures involved in virus transmission [PublishAheadOfPrint]

  • HIV transmission efficiency is greatly increased when viruses are transmitted at virological synapses formed between infected and uninfected cells. We have previously shown that virological synapses formed between HIV-pulsed mature dendritic cells (DCs) and uninfected T cells contain interdigitated membrane surfaces, with T cell filopodia extending toward virions sequestered deep inside invaginations formed on the DC membrane. To explore membrane structural changes relevant to HIV transmission across other types of intercellular conjugates, we used a combination of light and focused ion beam scanning electron microscopy (FIB-SEM) to determine the 3D architectures of contact regions between HIV-1 infected CD4+ T cells and either uninfected human CD4+ T cells or human fetal astrocytes. We present evidence that in each case, membrane extensions that originate from the uninfected cells, either as membrane sheets or filopodial bridges, are present and may be involved in HIV transmission from infected to uninfected cells. We show that individual virions are distributed along the length of astrocyte filopodia, suggesting that virus transfer to the astrocytes is mediated, at least in part, by processes originating from the astrocyte itself. Mechanisms that selectively disrupt the polarization and formation of such membrane extensions could thus represent a possible target for reducing viral spread.

    IMPORTANCE STATEMENT Our findings lead to new insights into unique aspects of HIV transmission in the brain, and at T cell-T cell synapses, which are thought by many to be a predominant mode of rapid HIV transmission early in the infection process.

  • The linker region of NS3 plays a critical role in the replication and infectivity of hepatitis C virus [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) NS3-4A is required for viral replication and assembly. We establish that virus assembly is sensitive to mutations in the linker region between the helicase and protease domains of NS3-4A. However, we find that protease cleavage, RNA binding, and unwinding rates of NS3 are minimally affected in vitro. Thus we conclude that the NS3 linker is critical for mediating protein-protein interactions and dynamic control rather than modulating the enzymatic functions of NS3-4A.

  • Predicting the impact of CD8+ T cell polyfunctionality on HIV disease progression [PublishAheadOfPrint]

  • During the chronic phase of HIV-1 infection, polyfunctional CD8+ T cell responses, which are characterized by a high frequency of cells able to secrete multiple cytokines simultaneously, are associated with lower virus loads and slower disease progression. This relationship could arise for different reasons. Polyfunctional responses could simply be stronger. Alternatively, it could be the increased functional diversity in polyfunctional responses that leads to lower virus loads and slower disease progression. Lastly, polyfunctional responses could contain more CD8+ T cells that mediate a specific key function, which is primarily responsible for viral control. Disentangling the influence of overall strength, functional diversity, and specific function on viral control and disease progression is very relevant for the rational design of vaccines and immunotherapy using cellular immune responses. We developed a mathematical model to study how polyfunctional CD8+ T cell responses mediating lytic and non-lytic effector functions affect the CD4+ T cell count and plasma viral load. We based our model on in vitro data on the efficacy of IFN- and MIP-1bbeta;/RANTES against HIV. We find that the strength of the response is a good predictor of disease progression, while functional diversity has only a minor influence. In addition, our model predicts for realistic levels of cytotoxicity that immune responses dominated by non-lytic effector functions most positively influence disease outcome.

    Importance Why polyfunctional CD8+ T cell responses are associated with better viral control while individual functional correlates of protection have not been identified so far is still an open question in HIV-1 research. Identifying the role of CD8+ T cells in HIV-1 infection has important implications for the potential development of effective T cell based vaccines. Our analysis provides new ways to think about a causative role of CD8+ T cells by studying different hypotheses why polyfunctional CD8+ T cells might be more advantageous. We identify measurements that have to be obtained in order to evaluate the role of CD8+ T cells in HIV-1 infection. In addition, our method shows how individual cell functionality data can be used in population based virus dynamics models.

  • Influenza A virus hemagglutinin and neuraminidase mutually accelerate their apical targeting through clustering of lipid rafts. [PublishAheadOfPrint]

  • In polarized epithelial cells, influenza A virus hemagglutinin (HA) and neuraminidase (NA) are intrinsically associated with lipid rafts and target the apical plasma membrane for viral assembly and budding. Previous studies have indicated that the transmembrane domain (TMD) and cytoplasmic tail (CT) of HA and NA were required for association with lipid rafts, but the raft dependencies of their apical targeting are controversial. Here, we show that coexpression of HA with NA accelerated their apical targeting through accumulation in lipid rafts. HA was targeted to the apical even when expressed alone but the kinetics was much slower than that of HA in infected cells. Coexpression experiments revealed that apical targeting of HA and NA was accelerated by their coexpression. The apical targeting of HA was also accelerated by coexpression with M1 but not M2. The mutations in the outer leaflet of the TMD and the deletion of the CT in HA and NA that reduced their association with lipid rafts abolished the acceleration of their apical transport, indicating that the lipid raft association is essential for efficient apical trafficking of HA and NA. An in situ proximity ligation assay (PLA) revealed that HA and NA were accumulated and clustered in the cytoplasmic compartments, only when both were associated with lipid rafts. Analysis with mutant viruses containing nonraft HA/NA confirmed these findings. We further analyzed lipid raft markers by in situ PLA and suggest a possible mechanism of the accelerated apical transport of HA and NA via clustering of lipid rafts.

    Importance Lipid rafts serve as sites for viral entry, particle assembly, and budding, leading to efficient viral replication. The influenza A virus utilizes lipid rafts for apical plasma membrane targeting and particle budding. The hemagglutinin (HA) and neuraminidase (NA) of influenza virus, key players for particle assembly, contain determinants for apical sorting and lipid raft association. However, it remains to be elucidated how lipid rafts contribute to the apical trafficking and budding. We investigated the relation of lipid raft association of HA and NA to the efficiency of apical trafficking. We show that coexpression of HA and NA induces their accumulation in lipid rafts and accelerates their apical targeting and suggest that the accelerated apical transport likely occurs by clustering of lipid rafts at the TGN. This finding provides the first evidence that two different raft-associated viral proteins induce lipid raft clustering, thereby accelerating apical trafficking of the viral proteins.

  • Uukuniemi phlebovirus assembly and secretion leaves a functional imprint on the virion glycome [PublishAheadOfPrint]

  • Uukuniemi virus (UUKV) is a model system for investigating the Phlebovirus genus of the Bunyaviridae. We report the UUKV glycome, revealing differential processing of the Gn and Gc virion glycoproteins. Both glycoproteins display poly-N-acetyllactosamines, consistent with virion assembly in the medial Golgi apparatus, whereas oligomannose-type glycans required for DC-SIGN-dependent cellular attachment are predominant on Gc. Local virion structure and the route of viral egress from the cell leaves a functional imprint on the phleboviral glycome.

  • Northern-African Strains of HTLV-1 Arose from a Recombination Event [PublishAheadOfPrint]

  • Although recombination is a major source of genetic variability in retroviruses, no recombinant strain had been observed for human T-lymphotropic virus type 1 (HTLV-1), the first isolated pathogenic human retrovirus. Different genotypes exist for HTLV-1: Genotypes b and d-g are restricted to Central Africa, while genotype c is only endemic in Australo-Melanesia. In contrast, the Cosmopolitan genotype a is widely distributed. We applied a combination of phylogenetics, and recombination analysis approaches to a set of new HTLV-1 sequences, which we collected from 19 countries throughout Africa, the continent where the virus has the largest endemic presence. This led us to demonstrate the presence of recombinants in HTLV-1. Indeed, the HTLV-1 strains currently present in North Africa have originated from a recombinant event between strains from Senegal and West Africa. This recombination is estimated to have occurred around 4,000 years ago. This recombination seems to have been generated during reverse transcription. In conclusion, we demonstrate that, albeit rare, recombination can occur in HTLV-1 and may play a role in the evolution of this retrovirus.

    Importance A number of HTLV-1 subtypes have been described in different populations, but none of the genetic differences between these subtypes have been ascribed to recombination events. Here we report an HTLV-1 recombinant virus among infected individuals in North Africa. This demonstrates that, contrary to what was thought, recombination can occur and could play a role in the evolution of HTLV-1.

  • Analysis of Human Rotaviruses from a Single Location Over an 18-year Timespan Suggests Protein Co-adaption Influences Gene Constellations [PublishAheadOfPrint]

  • Rotaviruses (RVs) are eleven-segmented, double-stranded RNA viruses that cause severe gastroenteritis in children. In addition to an error-prone genome replication mechanism, RVs can increase their genetic diversity by reassorting genes during host co-infection. Such exchanges allow RVs to acquire advantageous genes and adapt in the face of selective pressures. Yet, reassortment may also impose fitness costs if it unlinks genes/proteins that have accumulated compensatory, co-adaptive mutations and operate best when kept together. To better understand human RV evolutionary dynamics, we analyzed the genome sequences of 135 strains (genotype G1/G3/G4-P[8]-I1-C1-R1-A1-N1-T1-E1-H1) that were collected at a single location in Washington, DC, during the years of 1974-1991. Intra-genotypic phylogenetic trees were constructed for each viral gene using the nucleotide sequences, thereby defining novel allele-level gene constellations (GCs) and illuminating putative reassortment events. The results showed that RVs with distinct GCs co-circulated during the vast majority of the collection years, and that some of these GCs persisted in the community, unchanged by reassortment. To investigate the influence of protein co-adaptation on GC maintenance, we performed a mutual information-based analysis of the concatenated amino acid sequences and identified an extensive co-variance network. Unexpectedly, amino acid co-variation was highest between VP4 and VP2, which are structural components of the RV virion that are not thought to directly interact. These results suggest that GCs may be influenced by the selective constraints placed on functionally co-adapted, albeit non-interacting, viral proteins. This work raises important questions about the mutation-reassortment interplay and its impact on human RV evolution.

    IMPORTANCE Rotaviruses are devastating human pathogens that cause severe diarrhea and kill ggt;450,000 children each year. The virus can evolve by accumulating mutations and by acquiring new genes from other strains via a process called reassortment. However, little is known about the relationship between mutation accumulation and gene reassortment for rotaviruses and how it impacts viral evolution. In this study, we analyzed the genome sequences of human strains found in clinical fecal specimens that were collected at a single hospital over an 18-year timespan. We found that many rotaviruses did not reassort their genes, but instead maintained them as specific sets (i.e., constellations). By analyzing the encoded proteins, we discovered concurrent amino acid changes among them, which suggests that they are functionally co-adapted to operate best when kept together. This study increases our understanding of how rotaviruses evolve over time in the human population.

  • Endosomal Trafficking of Nanoformulated Antiretroviral Therapy Facilitates Drug Particle Carriage and HIV Clearance [PublishAheadOfPrint]

  • Limitations in antiretroviral therapy (ART) include poor patient adherence, drug toxicities, viral resistance and failures to penetrate viral reservoirs. Recent developments of nanoformulated ART (nanoART) could overcome such limitations. To this end, we now report a novel effect of nanoART that facilitates drug depots within intracellular compartments at sites at or adjacent to the viral replication cycle. Poloxamer 407 coated nanocrystals containing the protease inhibitor atazanavir (ATV) were prepared by high-pressure homogenization. These drug particles readily accumulated in human monocyte-derived macrophages (MDM). NanoATV concentrations were ~1,000 times higher in cells than what could be achieved by native drug. ATV particles in late and recycling endosome compartments were seen following pull down by immunoaffinity chromatography with Rab-specific antibodies conjugated to magnetic beads. Confocal microscopy provided cross validation by immunofluorescent staining of compartments. Mathematical modeling validated drug-endosomal interactions. Measures of reverse transcriptase activity and HIV-1p24 levels in culture media and cells showed that such endosomal drug concentrations enhanced antiviral responses up to 1,000 fold. We conclude that late and recycling endosomes can serve as depots for nanoATV. The co-localization of nanoATV at endosomal sites of viral assembly and its slow release sped antiretroviral activities. Long acting nanoART can serve as a drug carrier in both cells and subcellular compartments and as such facilitate viral clearance.

    IMPORTANCE The need for long acting ART is significant and highlighted by limitations in drug access, toxicity, adherence and reservoir penetrance. We propose that targeting nanoformulated drugs to infected tissues, cells and subcellular sites of viral replication may improve clinical outcomes. Endosomes are sites for human immunodeficiency virus assembly and increasing ART concentration to such sites enhances viral clearance. The current work uncovers a new mechanism for why nanoART can enhance viral clearance over native drug formulations.

  • Activation of NF{kappa}B via Endosomal Toll-like Receptors 7 or 9 Contributes to Limiting Murine Herpes Virus 68 Reactivation [PublishAheadOfPrint]

  • In order to understand and possibly treat B-cell malignancies associated with latent gamma-herpesvirus infection, it is vital to understand the factors that control the balance between the two transcriptional states of gamma-herpesviruses: latency and lytic replication. We used murine gamma-herpesvirus (MHV)-68 as a model system to investigate how engagement of endosomal Toll-like receptors (TLRs) impacts on reactivation from latency in vitro and on establishment of latent infection in vivo. We found that treatment with TLR7 ligand R848 or TLR9 ligand CpG ODN suppresses reactivation of MHV-68 in vitro. These suppressive effects correlated with the ability to activate cellular transcription factor NFB. Downregulation of TLR9 by RNA interference in vitro led to a reduction of nuclear levels of NFB p65 and consequently to an increase of spontaneous reactivation in MHV-68 latently infected cells, indicating that the TLR9 pathway contributes to limiting spontaneous reactivation events. In vivo, sustained stimulation of TLR7 by repeated R848 treatment led to an increased frequency of infected splenocytes compared to mock-treated control. Frequencies of infected splenic B-cells in tlr7-/- or tlr9-/- mice after establishment of latency did not differ from their wild-type counterpart. Nevertheless, MHV-68-infected B-cells from tlr9-/- mice showed a higher frequency of reactivation compared to B-cells from wild-type or tlr7-/- mice in ex vivo reactivation assays. Thus, we show a suppressive effect of TLR7 or TLR9 triggering on MHV-68 reactivation that correlates with NFB activation and that the mere presence of functional TLR9 signaling pathway contributes to dampen lytic gamma-herpesvirus reactivation in infected cells.

    IMPORTANCE A hallmark of gamma-herpesviruses is their establishment of latency in B-cells that is reversible through lytic reactivation. Latency can result in B-cell malignancies. Activation of the innate immune system is thought to contribute to controlling the switch between the transcriptional states of latency and reactivation. Nevertheless, the mechanisms involved are not clear. Here, we show that engagement of Toll-like receptors (TLRs)7 and 9 suppress reactivation of murine gamma-herpesvirus MHV-68 in vitro and that stimulation of TLR7 in vivo increases the frequency of infected cells. TLRs 7 and 9 are innate immunity sensors of nucleic acids localized in endosomes. Additionally, we demonstrate that impairment of TLR9 signaling in latently infected B-cells leads to increased reactivation. Thus, activated endosomal TLR7 and TLR9 pathways play an important role in promoting establishment of latent gamma-herpesvirus infection. Counteracting signaling of these pathways allows for reactivation and could represent treatment targets in gamma-herpesvirus-associated malignancies.

  • All-in-one bacmids: An efficient reverse genetics strategy for influenza A virus vaccines. [PublishAheadOfPrint]

  • Vaccination is the first line of defense against influenza infections, yet influenza vaccine production methods are slow, antiquated, and expensive to effectively reduce the virus' burden during epidemic or pandemic periods. There is a great need for alternative influenza vaccines and vaccination methods with a global scale impact. We demonstrate a strategy to generate influenza A virus in vivo using bacmid DNAs. Compared to the classical reverse genetics system, the "eight-in-one" bacmids (bcmd-RGFlu) showed higher efficiency of virus rescue in various cell types. Using a transfection-based inoculation (TBI) system, intranasal delivery in DBA/2J and Balb/c mice of bcmd-RGFlu plus 293T cells lead to the generation of lethal PR8 virus in vivo. A prime-boost intranasal vaccination strategy using TBI in the context of a bcmd-RGFlu encoding a temperature sensitive H1N1 virus resulted in protection of mice against lethal challenge with the PR8 strain. Taken together, these studies are proof-of-principle to highlight the potential of vaccination against influenza by in vivo reverse genetics.

    IMPORTANCE Vaccination is the first line of defense against influenza infections. A major drawback in the preparation of influenza vaccines is that production rely on a heavily time consuming process of growing the viruses in eggs. We propose a radical change in the way influenza vaccination is approached in which a recombinant bacmid, a shuttle vector that can be propagated in both E. coli and insect cells, carries an influenza infectious clone (bcmd-RGFlu). Using a surrogate cell system, we show that intranasal delivery of the bcmd-RGFlu results in generation of influenza virus in mice. Furthermore, mice vaccinated with this system were protected against lethal influenza challenge. The study serves as a proof-of-principle of a potentially universal vaccine platform against influenza and other pathogens.

  • Intrinsic innate immunity fails to control herpes simplex and vesicular stomatitis virus replication in sensory neurons and fibroblasts. [PublishAheadOfPrint]

  • Herpes simplex virus-1 (HSV-1) establishes lifelong latent infections in the sensory neurons of the trigeminal ganglia (TG) wherein it retains the capacity to reactivate. The interferon (IFN)-driven antiviral response is critical for the control of HSV-1 acute replication. We therefore sought to further investigate this response in TG neurons cultured from adult mice deficient in a variety of IFN signaling components. Parallel experiments were also performed in fibroblasts isolated concurrently. We showed that HSV-1 replication was comparable in wild-type (WT) and IFN signaling-deficient neurons and fibroblasts. Unexpectedly, a similar pattern was observed for the IFN-sensitive vesicular stomatitis virus (VSV). Despite these findings, TG neurons responded to IFNbbeta; pretreatment with STAT1 nuclear localization and restricted replication of both VSV and an HSV-1 strain deficient in 34.5, while wild-type HSV-1 replication was unaffected. This was in contrast to fibroblasts in which all viruses were restricted by the addition of IFNbbeta;. Taken together, these data show that adult TG neurons can mount an effective antiviral response only if provided with an exogenous source of IFNbbeta;, and HSV-1 combats this response through 34.5. These results further our understanding of the antiviral response of neurons and highlight the importance of paracrine IFNbbeta; signaling in establishing an antiviral state.

    IMPORTANCE Herpes simplex virus type 1 (HSV-1) is a ubiquitous virus that establishes a lifelong latent infection in neurons. Reactivation from latency can cause cold sores, blindness, and death from encephalitis. Humans with deficiencies in innate immunity have significant problems controlling HSV infections. In this study we therefore sought to elucidate the role of neuronal innate immunity in the control of viral infection. Using neurons isolated from mice, we found that the intrinsic capacity of neurons to restrict virus replication was unaffected by the presence or absence of innate immunity. In contrast, neurons were able to mount a robust antiviral response when provided with interferon beta, a molecule that strongly stimulates innate immunity, and that HSV-1 can combat this response through the viral gene, 34.5. Our results have important implications for understanding how the nervous system defends itself against virus infections.

  • Enterovirus 71 3C inhibits cytokine expression through cleavage of the TAK1/TAB1/TAB2/TAB3 complex [PublishAheadOfPrint]

  • Enterovirus 71 (EV71) causes hand-foot-and-mouth disease in young children and infants. Severe infection with EV71 can lead to various neurological complications or fatal diseases. However, the mechanism of EV71 pathogenesis is poorly understood. Emerging evidence suggests that EV71 modulates type I IFN and cytokine responses. Here, we show that EV71 disables components of the TAB2 complex through the 3C protein. When expressed in mammalian cells, EV71 3C interacts with TAB2 and TAK1, which inhibits NF-B activation. Furthermore, 3C mediates cleavage of TAB2 and its partners, which requires the protease activity. H40D or C147S substitution in the 3C active sites abolishes its activity whereas R84Q or V154S substitution in the RNA binding domain has no effect. The 3C protein targets TAB2 at Q113-S114, TAK1 at Q360-S361, TAB1 both at Q414-G415 and Q451-S452, and TAB3 at Q173-G174 and Q343-G344. Importantly, overexpression of TAB2 inhibits EV71 replication whereas addition of cleaved fragments has no effect. Thus, an equilibrium between the TAB2 complex and EV71 3C represents a control point of viral infection. These results suggest that TAK1/TAB1/TAB2/TAB3 cleavage-mediated by EV71 may be a mechanism to interfere with inflammatory responses.

    Importance The TAK1 complex plays a critical role in the activation of NF-B and cytokine production. However, little is known about its connection to enterovirus 71 (EV71). We demonstrate that EV71 3C suppresses the cytokines expression via cleavage of the TAK1 complex proteins. EV71 3C interacts with TAB2 and TAK1. Furthermore, overexpression of TAB2 inhibits EV71 replication whereas addition of cleaved fragment has no effect. These results suggest that interplay of EV71 and the TAK1 complex influences the outcome of viral infection.

  • Ebola Virus Modulates Transforming Growth Factor-{beta} Signaling and Cellular Markers of Mesenchymal-Like Transition in Hepatocytes [PublishAheadOfPrint]

  • Ebola virus (EBOV) causes a severe hemorrhagic disease in humans and nonhuman primates with a median case fatality rate of 78.4%. Although EBOV is considered a public-health concern there is a relative paucity of information regarding the modulation of the functional host response during infection. We employed temporal kinome analysis to investigate the relative early, intermediate, and late host kinome responses to EBOV infection in human hepatocytes. Pathway over-representation analysis and functional network analysis of kinome data revealed that transforming growth factor (TGF)-bbeta;-mediated signaling responses were temporally modulated in response to EBOV infection. Up-regulation of TGF-bbeta; signaling in the kinome data sets correlated with the up-regulation of TGF-bbeta; secretion from EBOV infected cells. Kinase inhibitors targeting TGF-bbeta; signaling, or additional cell receptors and downstream signaling pathway intermediates identified from our kinome analysis, also inhibited EBOV replication. Further, the inhibition of select cell signaling intermediates identified from our kinome analysis provided partial protection in a lethal model of EBOV infection. To gain perspective on the cellular consequence of TGF-bbeta; signaling modulation during EBOV infection we assessed cellular markers associated with up-regulation of TGF-bbeta; signaling. We observed up-regulation of matrix metalloproteinase 9, N-cadherin, and fibronectin expression with concomitant reductions in the expression of E-cadherin and claudin-1, responses that are standard characteristics of epithelial to mesenchymal -like transition. Additionally, we identified phosphorylation events downstream of TGF-bbeta; that may contribute to this process. From these observations we propose a model for a broader role of TGF-bbeta;-mediated signaling responses in the pathogenesis of Ebola virus disease.

    Importance: Ebola virus (EBOV), formerly Zaire ebolavirus, causes a severe hemorrhagic disease in humans and nonhuman primates and is the most lethal Ebola virus species with case fatality rates up to 90%. Although EBOV is considered a worldwide concern many questions remain regarding EBOV molecular pathogenesis. As it is appreciated that many cellular processes are regulated through kinase-mediated phosphorylation events we employed temporal kinome analysis to investigate the functional responses of human hepatocytes to EBOV infection. Administration of kinase inhibitors targeting signaling pathway intermediates identified in our kinome analysis inhibited viral replication in vitro and reduced EBOV pathogenesis in vivo. Further analysis of our data also demonstrated that EBOV infection modulated transforming growth factor (TGF)-bbeta;-mediated signaling responses and promoted "mesenchymal-like" phenotypic changes. Taken together, these results demonstrated that EBOV infection specifically modulates TGF-bbeta;-mediated signaling responses in epithelial cells and may have broader implications in EBOV pathogenesis.

  • TALEN Knockout of the PSIP1 Gene in Human cells: Analyses of HIV-1 Replication and Allosteric Integrase Inhibitor Mechanism [PublishAheadOfPrint]

  • HIV-1 utilizes the cellular protein LEDGF/p75 as a chromosome docking and integration cofactor. The LEDGF/p75 gene, PSIP1, is a potential therapeutic target because, like CCR5, depletion of LEDGF/p75 is tolerated well by human CD4+ T cells, and knockout mice have normal immune systems. RNAi has been useful for studying LEDGF/p75, but the potent cofactor activity of small protein residua can be confounding. Here, in human cells with utility for HIV research (293T, Jurkat), we used transcription activator-like effector nucleases (TALENs) to completely eradicate all LEDGF/p75 expression. We performed two kinds of PSIP1 knockouts: whole gene deletion and deletion of the integrase binding domain (IBD)-encoding exons. HIV-1 integration was inhibited and spreading viral replication was severely impaired in PSIP1-/- Jurkat cells infected at high multiplicity. Furthermore, frameshifting the gene in the first coding exon with a single TALEN pair yielded trace LEDGF/p75 levels that were virologically active, affirming the cofactor's potency and the value of definitive gene or IBD exon segment deletion. Some recent studies have suggested LEDGF/p75 may participate in HIV-1 assembly. However, we determined that assembly of infectious viral particles is normal in PSIP1-/- cells. The potency of an allosteric integrase inhibitor, ALLINI-2, for rendering produced virions non-infectious was also unaffected by total eradication of cellular LEDGF/p75. We conclude that HIV-1 particle assembly and the main ALLINI mechanism are LEDGF/p75-independent. The block to HIV-1 propagation in PSIP1-/- human CD4+ T cells raises the possibility of gene targeting PSIP1 combinatorially with CCR5 for HIV-1 cure.

    IMPORTANCE LEDGF/p75 dependence is universally conserved in the retroviral genus Lentivirus. Once inside the nucleus, lentiviral pre-integration complexes are thought to attach to the chromosome when integrase binds to LEDGF/p75. This tethering process is largely responsible for the two-fold preference for integration into active genes, but the cofactor's full role in the lentiviral life cycle is not yet clear. Effective knockdowns are difficult because even trace residua of this tightly chromatin-bound protein can support integration cofactor function. Here, in experimentally useful human cell lines, we used TALENs to definitively eradicate LEDGF/p75 by deleting either all of PSIP1 or the exons that code for the integrase binding domain. HIV-1 replication was severely impaired in these PSIP1 knockout cells. Experiments in these cells also excluded a role for LEDGF/p75 in HIV-1 assembly and showed that the main ALLINI mechanism is LEDGF/p75-independent. Site-specific gene targeting of PSIP1 may have therapeutic potential for HIV-1 disease.

  • Interactions between the Influenza A Virus RNA Polymerase Components and Retinoic Acid-Inducible Gene I [PublishAheadOfPrint]

  • The influenza A virus genome possesses eight negative-strand RNA segments in the form of viral ribonucleoprotein particles (vRNPs) in association with the three viral RNA polymerase subunits (PB2, PB1, and PA) and the nucleoprotein (NP). Through interactions with multiple host factors, the RNP subunits play vital roles in replication, host adaptation, interspecies transmission, and pathogenicity. In order to gain insight into the potential roles of RNP subunits in the modulation of the host's innate immune response, the interactions of each RNP subunit with Retinoic Acid-Inducible Gene I protein (RIG-I) from mammalian and avian species were investigated. Studies using co-immunoprecipitation (Co-IP), bimolecular fluorescence complementation (BiFc), and co-localization using confocal microscopy, provided direct evidence for the RNA-independent binding of PB2, PB1, and PA with RIG-I from various hosts (human, swine, mouse, and duck). In contrast, the binding of NP with RIG-I was found to be RNA-dependent. Expression of the viral NS1 protein, which interacts with RIG-I, did not interfere with the association of RNA polymerase subunits with RIG-I. The association of each individual virus polymerase component with RIG-I failed to significantly affect the IFN induction elicited by RIG-I and 5rrsquo; ppp-RNA in reporter assays, quantitative RT-PCR, and IRF3 phosphorylation tests. Taken together, these findings indicate that viral RNA polymerase components PB2, PB1, and PA directly target RIG-I, but the exact biological significance of these interactions in the replication and pathogenicity of influenza A virus needs to be further clarified.

    IMPORTANCE RIG-I is an important RNA sensor to elicit innate immune response in mammals and some bird species (such as duck) upon influenza A virus infection. Although the 5rrsquo; -triphosphate dsRNA panhandle structure at the end of viral genome RNA is responsible for the binding and subsequent activation of RIG-I, this structure is supposedly wrapped by RNA polymerase complex (PB2, PB1, PA), which may interfere with the induction of RIG-I signaling pathway. In the present study, PB2, PB1, and PA were found to individually interact with RIG-Is from multiple mammalian and avian species in an RNA-independent manner, without significantly affecting the generation of IFN. The data suggest that although RIG-I binding by RNA polymerase complex is conserved in different species, it does not appear to play crucial role in the modulation of IFN in vitro.

  • Inter-continental dispersal of HIV-1 subtype B associated with transmission among men who have sex with men in Japan [PublishAheadOfPrint]

  • Transmission clusters of HIV-1 subtype B uniquely associated with the epidemic among men who having sex with men (MSM) in East Asia have recently been identified. Using the Los Alamos HIV sequence database and UK HIV drug resistance database, we explored possible links between HIV MSM epidemics in East Asia and the rest of the world, using phylogenetic and molecular clock analyses. We found that JP.MSM.B-1, a subtype B MSM variant that accounts for approximately one-third of infections among Japanese MSM, was detected worldwide; in UK (n=13), mainland China (n=3), USA, Germany, Canada and Taiwan (n=1 each). Interestingly, ten UK samples plus two from Germany and the USA formed a distinct monophyletic subgroup within JP.MSM.B-1. The estimated divergence times of JP.MSM.B-1 and the latter subgroup were ~1989 and ~1999, respectively. These dates suggest that JP.MSM.B-1 was circulating for many years in Japan among MSM before disseminating to other countries, most likely through global MSM networks. A significant number of other Asian MSM HIV lineages were also detected in UK database. Our study provides insight into the regional and global dispersal of Asian MSM HIV lineages. Further study of these strains is warranted to elucidate viral migration and the interrelationship of HIV epidemics on a global scale.

    Importance We previously identified several transmission clusters of HIV-1 subtype B uniquely associated with the epidemic among men who have sex with men (MSM) in East Asia. Using the Los Alamos HIV sequence database and UK HIV drug resistance database, we explored the possible interplay of HIV MSM epidemics in the different geographic regions and found previously unrecognized interrelationships among the HIV-1 epidemics in East Asia, UK and the rest of the world. Our study provides insight into the regional and global dispersal of Asian MSM HIV lineages and highlights the importance of strengthening HIV monitoring efforts and the need for implementing effective control measures to reduce HIV transmission on a global scale.

  • HIV-1 and HIV-2 Vif Interact with Human APOBEC3 Proteins Using Completely Different Determinants [PublishAheadOfPrint]

  • Human APOBEC3 (A3) restriction factors provide intrinsic immunity against zoonotic transmission of pathogenic viruses. A3D, A3F, A3G, and A3H-haplotype II (A3H-hapII) can be packaged into virion infectivity factor (Vif)-deficient HIVs to inhibit viral replication. To overcome these restriction factors, Vif binds to the A3 proteins in viral producer cells to target them for ubiquitination and proteasomal degradation, thus preventing their packaging into assembling virions. Therefore, the Vif-A3 interactions are attractive targets for novel drug development. HIV-1 and HIV-2 arose via distinct zoonotic transmission events of simian immunodeficiency viruses from chimpanzees and sooty mangabeys, respectively, and Vifs from these viruses have limited homology. To gain insights into the evolution of virus-host interactions that led to successful cross-species transmission of lentiviruses, we characterized the determinants of interaction between HIV-2 Vif (Vif2) with human A3 proteins and compared them to the previously identified HIV-1 Vif (Vif1) interactions with the A3 proteins. We found that A3G, A3F, and A3H-hapII, but not A3D, were susceptible to Vif2-induced degradation. Alanine scanning mutational analysis of the first 62 amino acids of Vif2 indicates that Vif2 determinants important for degradation of A3G and A3F are completely distinct from these regions in Vif1, as are the determinants in A3G and A3F that are critical for Vif2-induced degradation. These observations suggest that distinct Vif-A3 interactions evolved independently in different SIVs and their non-human primate hosts, and conservation of the A3 determinants targeted by the SIV Vif proteins resulted in successful zoonotic transmission into humans.

    Importance Primate APOBEC3 proteins provide innate immunity against invading pathogens, and Vif proteins of primate lentiviruses have evolved to overcome these host defenses by interacting with them and inducing their proteasomal degradation. HIV-1 and HIV-2 are two human pathogens that induce AIDS, and elucidating interactions between their Vif proteins and human A3 proteins could facilitate development of novel antiviral drugs. Furthermore, understanding Vif-A3 interactions can provide novel insights into cross-species transmission events that led to the HIV-1 and HIV-2 pandemics and evolution of host-virus interactions. We carried out mutational analysis of the N-terminal 62 amino acids of HIV-2 Vif (Vif2) and analyzed A3G/A3F chimeras that retained antiviral activity to identify Vif2 and A3 interacting determinants. Our results show that Vif2-A3 interactions are completely different from the Vif1-A3 interactions, suggesting that these interactions evolved independently and that conservation of the A3 determinants resulted in successful zoonotic transmission into humans.

  • The West Nile-like flavivirus Koutango is highly virulent in mice due to delayed viral clearance and the induction of a poor neutralizing antibody response. [PublishAheadOfPrint]

  • The mosquito-borne West Nile virus (WNV) is responsible for outbreaks of viral encephalitis in humans, horses and birds with particularly virulent strains causing recent outbreaks of disease in Eastern Europe, the Middle East, North America and Australia. Previous studies have phylogenetically separated WNV strains into two main genetic lineages (I and II), containing virulent strains associated with neurological disease. Several WNV-like strains, clustering outside these lineages have been identified and form an additional five proposed lineages. However, little is known about whether these strains have the potential to induce disease. In a comparative analysis with the highly virulent Lineage I American strain (WNVNY99), the low pathogenic Lineage II strain (B956) and a benign Australian strain, Kunjin (WNVKUN); the African WNV-like Koutango virus (WNVKOU) and a WNV-like isolate from Sarawak, Malaysia (WNVSarawak) were assessed for neuroinvasive properties in a murine model and for their replication kinetics in vitro. While WNVNY99 replicated to highest levels in vitro, in vivo mouse challenge revealed that WNVKOU was more virulent with a shorter time to onset of neurological disease and higher morbidity. Histological analysis of WNVKOU- and WNVNY99-infected brain and spinal cords demonstrated a more prominent meningoencephalitis and presence of viral antigen in WNVKOU-infected mice. Enhanced virulence of WNVKOU was also associated with poor viral clearance in the periphery (sera and spleen), a skewed innate immune response and poor neutralizing antibody development. These data demonstrate for the first time potent neuroinvasive and neurovirulent properties of a WNV-like virus outside of Lineage I and II.

    Importance In this study, we characterized the in vitro and in vivo properties of previously uncharacterized West Nile virus strains and West Nile-like viruses. We identified a West Nile-like virus, Koutango (WNVKOU) that was more virulent than a known virulent Lineage 1 virus, (WNVNY99). Enhanced virulence of WNVKOU was associated with poor viral clearance and the induction of a poor neutralization antibody response. These findings provide new insights into the pathogenesis of West Nile virus.

  • Isolation and Characterization of A Novel Alphaherpesvirus in Fruit Bats [PublishAheadOfPrint]

  • Bats are known to harbor emerging RNA viruses. Recent studies have used high-throughput sequencing technology to identify various virus species, including DNA viruses that are harbored by bats; however, little is known about the nature of these potentially novel viruses. Here, we report the characterization of a novel herpesvirus isolated from an Indonesian pteropodid bat. The virus, tentatively named fruit bat alphaherpesvirus 1 (FBAHV1), has a double-stranded DNA genome of 149,459 bp. The phylogenetic analyses suggested that FBAHV1 is phylogenetically grouped with Simplexviruses within the subfamily, Alphaherpesvirinae. Inoculation of FBAHV1 into laboratory mice caused a lethal infection. Virus infection was observed in lung, liver, and brain tissue. Serological and PCR screening revealed that FBAHV1- or its related virus-infected fruit bats were widely distributed in Indonesia. The identification of FBAHV1 makes a considerable contribution to our understanding of simplexviruses associated with bats.

    Importance Bats are known to harbor emerging viruses such as lyssaviruses, henipaviruses, SARS-like coronaviruses, and filoviruses. Although alphaherpesviruses are disseminated in humans and other animals, there is little information about their distribution in bats. Here, we isolated a previously unknown alphaherpesvirus from an Indonesian fruit bat. Genome sequence analysis suggested that the virus is a member of the genus Simplexvirus within the subfamily Alphaherpesvirinae, which also includes common human viruses such as herpes simplex virus type 1 and herpes simplex virus type 2. FBAHV1 is the first bat-derived alphaherpesvirus whose complete genome has been sequenced.

  • Dendritic cells restore CD8+ T cell reactivity to autologous HIV-1 [PublishAheadOfPrint]

  • Recall T cell responses to HIV-1 antigens are used as a surrogate for endogenous cellular immune responses generated during infection. Current methods of identifying antigen-specific T cell reactivity in HIV-1 infection use bulk PBMC, yet ignore professional antigen presenting cells (APC) that could reveal otherwise hidden responses. In the present study, peptides representing autologous variants of MHC class I - restricted epitopes from HIV-1 Gag and Env were used as antigen in IFN ELISpot and polyfunctional cytokine assays. Here we show that DC enhance T cell reactivity at all stages of disease progression, but specifically restored T cell reactivity after combination antiretroviral therapy (cART) to early infection levels. Type-1 cytokine secretion was also enhanced by DC and was most apparent late post-cART. We additionally show that DC reveal polyfunctional T cell responses after many years of treatment, when potential immunotherapies would be implemented. These data underscore the potential efficacy of a DC immunotherapy that aims to awaken a dormant, autologous HIV-1-specific CD8+ T cell response.

    Importance Assessment of endogenous HIV-1-specific T cell responses is critical for generating immunotherapies for subjects on cART. Current assays ignore the ability of dendritic cells to reveal these responses and may therefore underestimate the breadth and magnitude of T cell reactivity. As DC do not prime new responses in these assays, it can be assumed that the observed responses are not detected without appropriate stimulation. This is important because dogma states that HIV-1 mutates to evade host recognition, and that CD8+ cytotoxic T lymphocyte (CTL) failure is due to the inability of T cells to recognize the autologous virus. The results presented here indicate that responses to autologous virus are generated during infection but may need additional stimulation to be effective. Detecting the breadth and magnitude of HIV-1-specific T cell reactivity generated in vivo is of the utmost importance for generating effective DC immunotherapies.

  • Regulation of Tissue-Dependent Differences in CD8+ T Cell Apoptosis During Viral Infection [PublishAheadOfPrint]

  • Virus-specific CD8+ T cells in the lymphoid organs contract at the resolution of virus infections by apoptosis or by dissemination into peripheral tissues, and those residing in non-lymphoid organs, including the peritoneal cavity and fat pads, are more resistant to apoptosis than those in the spleen and lymph nodes. This stability of memory T cells in the non-lymphoid tissues may enhance protection to secondary challenges. Here we show that LCMV-specific CD8+ T cells in non-lymphoid tissues were enriched for memory precursors (IL7Rhi KLRG1lo) and had higher expression of CD27, CXCR3 and T cell factor-1, each marker of which individually correlated with decreased apoptosis. CD8+ T cells in the peritoneal cavity of TCF-1-deficient mice had decreased survival, suggesting a role for TCF-1 in promoting survival in the non-lymphoid tissues. CXCR3+ CD8+ T cells resisted apoptosis and accumulated in the lymph nodes of mice treated with FTY720, which blocks the export of lymph node cells into peripheral tissue. The peritoneal exudate cells (PECs) expressed increased amounts of CXCR3 ligands, CXCL9 and CXCL10, which may normally recruit these non-apoptotic cells from the lymph nodes. In addition, adoptive transfer of splenic CD8+ T cells into PEC or spleen environments showed that the peritoneal environment promoted survival of CD8+ T cells. Thus, intrinsic stability of T cells which are present in the non-lymphoid tissues along with preferential migration of apoptosis-resistant CD8+ T cells into peripheral sites and the availability of tissue-specific factors that enhance memory cell survival may collectively account for the tissue-dependent apoptotic differences.

    IMPORTANCE: Most infections are initiated at non-lymphoid tissue sites, and the presence of memory T cells in non-lymphoid tissues is critical for protective immunity in various viral infection models. Virus-specific CD8+T cells in the non-lymphoid tissues are more resistant to apoptosis than those in lymphoid organs during the resolution and memory phase of the immune response to acute LCMV infection. Here we investigated the mechanisms promoting stability of T cells in the non-lymphoid tissues. This increased resistance to apoptosis of virus-specific CD8+T cells in non-lymphoid tissues was due to several factors. Non-lymphoid tissues were enriched in memory-phenotype CD8+T cells, which were intrinsically resistant to apoptosis irrespective of the tissue environment. Furthermore, apoptosis-resistant CD8+T cells preferentially migrated into the non-lymphoid tissues where the availability of tissue-specific factors may enhance memory cell survival. Our findings are relevant for the generation of long lasting vaccines providing protection at peripheral infection sites.

  • Triggering TLR2, 3, 4, 5 and 8 reinforces the restrictive nature of M1- and M2-polarized macrophages to HIV [PublishAheadOfPrint]

  • Macrophages must react to a large number of pathogens and their effects. In chronic HIV infection, the microenvironment changes with an influx of microbial products that trigger TLRs. That dynamic nature can be replicated ex vivo by the pro-inflammatory (M1-) and alternatively activated (M2-) macrophages. Thus, we determined how polarized macrophages primed by various TLR agonists support HIV replication. Triggering TLR2, 3, 4, 5 and 8 reinforced the low level of permissiveness in polarized macrophages. HIV was inhibited even more in M1-polarized macrophages than in macrophages activated only by TLR agonists. HIV was inhibited before its integration into the host chromosome. Polarization and triggering by various TLR agonists resulted in distinct cytokine profiles, endocytic activity, and distinct upregulation of restriction factors of HIV. Thus, different mechanisms likely contribute to the HIV inhibitory effects. In chronic HIV infection, macrophages might become less permissive to HIV due to changes in the microenvironment. The high level of reactivity of polarized macrophages to TLR triggering may be exploited for immunotherapeutic strategies.

    Importance Macrophages are a major target of HIV-1 infection. Different cell types in this very heterogeneous cell population respond differently to stimuli. In vitro, the heterogeneity is mimicked by their polarization into pro-inflammatory and alternatively activated macrophages. Here we explored the extent to which agonists triggering the TLR family affect HIV replication in polarized macrophages. We found that a number of TLR agonists blocked HIV replication substantially when given before infection. We also report the mechanisms of how TLR agonists exert their inhibitory action. Our findings may advance our understanding of which and how TLR agonists block HIV infection in polarized macrophages and may facilitate the design of novel immunotherapeutic approaches.

  • A novel role of the potyviral helper component proteinase contributes to enhance the yield of viral particles [PublishAheadOfPrint]

  • The helper component proteinase (HCPro) is an indispensable, multifunctional protein from members of the genus Potyvirus and other viruses of the family Potyviridae. This viral factor is directly involved in diverse steps of the viral infection, such as aphid transmission, polyprotein processing, and suppression of host antiviral RNA silencing. In this paper, we show that although a chimeric virus based on the potyvirus Plum pox virus lacking HCPro, which was replaced by an heterologous silencing suppressor, caused an efficient infection in Nicotiana benthamiana plants, its viral progeny had a very reduced infectivity. Making use of different approaches, here we provide direct evidences of a previously unknown function of HCPro, by which this viral factor enhances the stability of its cognate capsid protein (CP), positively affecting the yield of virions, and consequently improving the infectivity of the viral progeny. Site-directed mutagenesis revealed that the ability of HCPro to stabilize CP and enhance the yield of infectious viral particles is unlinked to any of its previously known activities, and helped us to delimit the region of HCPro involved in this function in the central region of the protein. Moreover, this function is highly specific and cannot be fulfilled by the HCPro of a heterologous potyvirus. The importance of this novel requirement in regulating the sorting of the viral genome to be subjected to replication, translation and encapsidation, thus contributing to the synchronization of these viral processes, is discussed.

    IMPORTANCE Potyviruses form one of the most numerous groups of plant viruses, being a major cause of crop loss worldwide. It is well known that these pathogens make use of virus-derived multitasking proteins, as well as dedicated host factors, to successfully infect their hosts. Here, we describe a novel requirement for the proper yield and infectivity of a potyviral progeny. In this case, such a function is accomplished by the extensively studied viral factor HCPro, which seems to use an unknown mechanism that is not linked to its previously described activities. To our knowledge, it is the first time that a factor different from CP is directly involved in the yield of potyviral particles. With the data presented here, we hypothesize that this capacity of HCPro might be involved in the coordination of mutually exclusive activities of the viral genome by controlling correct assembly of CP in stable virions.

  • Interaction pattern between Potato virus Y and eIF4E-mediated recessive resistance in the Solanaceae [PublishAheadOfPrint]

  • The structural pattern of infectivity matrices, where a set of parasites is confronted to a set of hosts, is a key parameter for our understanding of biological interactions and their evolution. This pattern determines evolution of parasite pathogenicity and host resistance, the spatio-temporal distribution of host and parasite genotypes and the efficiency of disease control strategies. Two major patterns have been proposed for plant-virus genotypes infectivity matrices. In the gene-for-gene model, infectivity matrices show a nested pattern, where the host range of specialist virus genotypes is a subset of the host range of less-specialized viruses. In contrast, in the matching-allele (MA) model, each virus genotype is specialized to infect one (or a small set of) host genotype(s). The corresponding infectivity matrix shows a modular pattern, where infection is frequent for plants and viruses belonging to the same module but rare for those belonging to different modules. We analyzed the structure of infectivity matrices between Potato virus Y (PVY) and plant genotypes in the family Solanaceae carrying different eukaryotic initiation factor 4E (eIF4E)-coding alleles conferring recessive resistance. Whereas this system corresponds mechanistically to a MA model, the expected modular pattern was rejected for our experimental data. This was mostly because PVY mutations involved in adaptation to a particular plant genotype displayed frequent pleiotropic effects, conferring simultaneously an adaptation to additional plant genotypes carrying different eIF4E alleles. Such effects should be taken into account for the design of strategies of sustainable control of PVY through plant varietal mixtures or rotations.

    Importance The interaction pattern between host and virus genotypes has important consequences on their respective evolution and on applied issues regarding disease control. We found that the structure of the interaction between Potato virus Y (PVY) variants and host plants in the family Solanaceae departs significantly from the current model of interaction considered for these organisms, because of frequent pleiotropic effects of virus mutations. These mutational effects allow the virus to expand rapidly its range of host plant genotypes, make it very difficult to predict the effects of mutations in PVY infectivity factor and raise concerns about strategies of sustainable management of plant genetic resistance to viruses.

  • A Comparative Analysis of the Capacity of Elite Suppressor CD4+ and CD8+ T cells to Inhibit HIV-1 Replication in Monocyte-Derived Macrophages [PublishAheadOfPrint]

  • Elite controllers or suppressors (ES) are HIV-1 infected individuals who are able to maintain viral loads below the limit of detection of clinical assays without antiretroviral therapy. The mechanisms of virologic control are not fully understood, but ES have been shown to have a more effective CD8+ T cell response to infected CD4+ T cells than chronic progressors (CP). While macrophages are another cell type productively infected by HIV-1, few studies have examined the ability of primary effector T-cells to suppress HIV-1 replication in these target cells. Here, we compared the ability of unstimulated primary CD4+ and CD8+ effector T cells to suppress viral replication in monocyte-derived macrophages (MDMs) in ES and CP. While CD4+ effector T cells were capable of inhibiting viral replication in MDMs, the magnitude of this response was not significantly different between ES and CP. In contrast, the CD8+ T cells from ES were significantly more effective than those from CP at inhibiting viral replication in MDMs. The CD4+ T cell response was partially mediated by soluble factors while the CD8+ T cell response required cell to cell interaction. Our results suggest that the individual contributions of various effector cells should be considered in rational vaccine design and in ongoing eradication efforts.

    IMPORTANCE Elite suppressors are individuals capable of maintaining low-level viremia in HIV-1 infection without antiretroviral drugs. Their T cell responses have been implicated in eliminating infected CD4+ T cells, and as such elite suppressors may represent a model of a functional cure of HIV-1 infection. Here, we sought to determine whether the suppressive T cell responses against infected CD4+ T cells also apply to infected macrophages by comparing responses seen with elite suppressors and HIV-1+ individuals on HAART. Our results show that CD8+ but not CD4+ T cells of elite suppressors have a superior response against infected macrophages than those of patients on HAART. Our results suggest that the induction of a CD8+ T cell response effective against infected macrophages is an outcome to consider in rational vaccine design.

  • Immune activation is associated with CD8 T-cell IL-21 production in HIV-1-infected individuals [PublishAheadOfPrint]

  • Interleukin-21 (IL-21) can be produced by CD8 T-cells from HIV-1-infected individuals and those with autoimmune disease, but the mechanism remains poorly understood. Here we demonstrate that IL-21-producing CD8 T-cells are not associated with CD4 depletion and are absent in patients with idiopathic CD4 lymphocytopenia. Instead, IL-21 production by CD8 T-cells was associated with high levels of activation suggesting that these cells emerge as a consequence of excessive chronic immune activation, rather than CD4 lymphopenia.

  • Herpes Simplex Virus Type 2 Glycoprotein H Interacts with Integrin {alpha}v{beta}3 to Facilitate Viral Entry and Calcium Signaling in Human Genital Tract Epithelial Cells [PublishAheadOfPrint]

  • HSV entry requires multiple interactions at the cell surface and activation of a complex calcium-signaling cascade. Previous studies demonstrated that integrins participate in this process, but the precise role has not been determined. These studies were designed to test the hypothesis that integrin aalpha;vbbeta;3 signaling promotes the release of intracellular calcium (Ca2+) stores and contributes to viral entry and cell-to-cell spread. Transfection of cells with small interfering RNA (siRNA) targeting integrin aalpha;vbbeta;3, but not other integrin subunits, or treatment with cilengitide, an RGD mimetic, impaired HSV-induced Ca2+ release, viral entry, plaque formation and cell-to-cell spread of HSV-1 and HSV-2 in human cervical and primary genital tract epithelial cells. Co-immunoprecipitation studies and proximity ligation assays indicated that integrin aalpha;vbbeta;3 interacts with glycoprotein H (gH). An HSV-2 gH-null virus was engineered to further assess the role of gH in the viral induced signaling cascade. The gH-2 null virus bound to cells and activated Akt to induce a small Ca2+ response at the plasma membrane, but failed to trigger the release of cytoplasmic Ca2+ stores and was impaired for entry and cell-to-cell spread. Silencing of integrin aalpha;vbbeta;3 and deletion of gH prevented phosphorylation of focal adhesion kinase (FAK) and the transport of viral capsids to the nuclear pore. Together these findings demonstrate that integrin signaling is activated downstream of viral-induced Akt signaling and facilitates viral entry through interactions with gH by activating the release of intracellular Ca2+ and FAK phosphorylation. These findings suggest a new target for HSV treatment and suppression.

    Importance Herpes simplex viruses (HSV) are the leading cause of genital disease worldwide, the most common infection associated with neonatal encephalitis, and a major co-factor for HIV acquisition and transmission. There is no effective vaccine. These epidemiological findings underscore the urgency to develop novel HSV treatment or prevention strategies. These studies address this gap by further defining the signaling pathways the virus usurps to enter human genital tract epithelial cells. Specifically the studies define the role played by integrins and by the viral envelope glycoprotein H in entry and cell-to-cell spread. This knowledge will facilitate the identification of new targets for the development of treatment and prevention.

  • Murine cytomegalovirus virion-associated protein M45 mediates rapid NF-{kappa}B activation after infection [PublishAheadOfPrint]

  • Murine cytomegalovirus (MCMV) rapidly induces activation of nuclear factor B (NF-B) upon infection of host cells. After a transient phase of activation, the MCMV M45 protein blocks all canonical NF-B activating pathways by inducing degradation of the NF-B essential modulator (NEMO), the gamma subunit of the inhibitor of B kinase complex (IKK). Here we show that the viral M45 protein also mediates the rapid NF-B activation immediately after infection. MCMV mutants lacking M45 or expressing C-terminally truncated M45 proteins induced neither NF-B activation nor transcription of NF-B-dependent genes within the first three hours of infection. The rapid NF-B activation was absent in MCMV-infected NEMO-deficient fibroblasts, indicating that activation occurs at or upstream of the IKK complex. NF-B activation was strongly reduced in murine fibroblasts lacking receptor-interacting protein 1 (RIP1), a known M45-interacting protein, but restored upon complementation with murine RIP1. However, the ability of M45 to interact with RIP1 and NEMO was not sufficient to induce NF-B activation upon infection. In addition, incorporation of the M45 protein into virions was required. This was dependent on a C-terminal region of M45, which is not required for interaction with RIP1 and NEMO. We propose a model in which M45 delivered by viral particles activates NF-B, presumably involving an interaction with RIP1 and NEMO. Later in infection, expression of M45 induces the degradation of NEMO and shutdown of canonical NF-B activation.

    Importance Transcription factor NF-B is an important regulator of innate and adaptive immunity. Its activation can be beneficial or detrimental for viral pathogens. Therefore, many viruses interfere with NF-B signaling by stimulating or inhibiting the activation of this transcription factor. Cytomegaloviruses, opportunistic pathogens that cause lifelong infections in their respective hosts, activate NF-B rapidly and transiently upon infection, but block NF-B signaling soon thereafter. Here we report the surprising finding that the murine cytomegalovirus protein M45, a component of viral particles, plays a dual role in NF-B signaling. It not only blocks NF-B signaling later on in infection but also triggers the rapid activation of NF-B immediately following virus entry into host cells. Both, activation and inhibition, involve M45 interaction with the cellular signaling mediators RIP1 and NEMO. Similar dual functions in NF-B signaling are likely to be found in other viral proteins.

  • CD4 ligation on human blood monocytes triggers macrophage differentiation and enhances HIV infection [PublishAheadOfPrint]

  • A unique aspect of human monocytes, compared to monocytes from many other species, is that they express the CD4 molecule. However, the role of the CD4 molecule in human monocyte development and function is not known. We determined that the activation of CD4 via interaction with major histocompatibility complex class II (MHC II) triggers cytokine expression and the differentiation of human monocytes into functional mature macrophages. Importantly, we identified that CD4 activation induces intracellular signaling in monocytes and inhibition of the MAPK and Src-family kinase pathways blocked the ability of CD4 ligation to trigger macrophage differentiation. We observed that ligation of CD4 by MHC II on activated endothelial cells induced CD4-mediated macrophage differentiation of blood monocytes. Finally, CD4 ligation by MHC II increases the susceptibility of blood-derived monocytes to HIV binding and subsequent infection. In all, our studies have identified a novel function for the CD4 molecule on peripheral monocytes and suggest that a unique set of events that lead to innate immune activation differ between humans and mice. Further these events can have effects on HIV infection and persistence in the macrophage compartment.

    Importance The CD4 molecule, as the primary receptor for HIV, plays an important role in HIV pathogenesis. There are many cell types that express CD4 other than the primary HIV target, the CD4+ T cell. Other than allowing HIV infection, the role of the CD4 molecule on human monocytes or macrophages is not known. We were interested in determining the role of CD4 in human monocyte/macrophage development and function and the potential effects of this on HIV infection. We identified a role for the CD4 molecule in triggering the activation and development of a monocyte into a macrophage following its ligation. Activation of the monocyte through the CD4 molecule in this manner increases the ability of monocytes to bind to and become infected with HIV. Our studies have identified a novel function for the CD4 molecule on peripheral monocytes in triggering macrophage development that has direct consequences on HIV infection.

  • Interactions between E6, FAK, and GIT1 at Paxillin LD4 Are Necessary for Transformation by Bovine Papillomavirus Type 1 E6 [PublishAheadOfPrint]

  • Bovine Papillomavirus Type 1 E6 interacts with two similar proteins that regulate cell attachment and cell migration called paxillin (PXN) and HIC-5 (also known as HIC5, ARA55, HIC-5, TSC-5, and TGFB1I1). Despite the similarity between HIC-5 and paxillin, paxillin is required for E6 to transform mouse embryo fibroblasts while HIC-5 is not. Using mutants of paxillin we find that dynamic competitive interactions between E6, Focal Adhesion Kinase and the GIT1 ARF-GAP protein for binding to paxillin are required but not sufficient for transformation by E6. Using mutants of paxillin and chimeric proteins between HIC-5 and Paxillin, we demonstrate that a critical difference between HIC-5 and paxillin is within the LIM domains of paxillin that do not directly interact with E6. Mutational analysis indicates that at least six distinct domains of paxillin are required for E6 transformation.

    Importance Papillomaviruses cause epitheliomas in vertebrates through the action of virally encoded oncoproteins. Despite the immense diversity of papillomavirus types, our understanding of the mechanisms by which the virally encoded E6 oncoproteins contribute to cell transformation is restricted to human papillomavirus types that are associated with cancer. Bovine papillomavirus type 1 (BPV-1) E6 has served as a model system for studies of E6 structure and function. This study examines the mechanisms by which BPV-1 E6 association with the cellular focal adhesion adapter protein paxillin contributes to cell transformation and extends our knowledge of the diverse mechanisms by which papillomaviruses transform host cells.

  • STAT4-deficiency Fails to Induce Th2 or Th17 Immunity with Primary or Secondary Respiratory Syncytial Virus Challenge but Enhances the RSV-specific Lung CD8+ T cell Immune Response to Secondary Challenge. [PublishAheadOfPrint]

  • Immune-mediated lung injury is a hallmark of RSV lower respiratory tract illness. STAT4 plays a critical role in CD4+ Th1 lineage differentiation and IFN- protein expression by CD4+ T cells. As CD4+ Th1 differentiation is associated with negative regulation of CD4+ Th2 and Th17 differentiation, we hypothesized that RSV infection of STAT4-/- mice would result in enhanced lung Th2 and Th17 inflammation and impaired lung Th1 inflammation compared to WT mice. We performed primary and secondary RSV challenge in WT and STAT4-/- mice, and used STAT1-/- mice as a primary challenge positive control for the development of RSV-specific lung Th2 and Th17 inflammation. Primary RSV challenge of STAT4-/- mice resulted in decreased T-bet and IFN- expression in CD4+ T cells compared to WT mice. Lung Th2 and Th17 inflammation did not develop in primary RSV-challenged STAT4-/- mice. Decreased IFN- expression by NK cells, CD4+ T cells, and CD8+ T cells was associated with attenuated weight loss and enhanced viral clearance with primary challenge in STAT4-/- mice compared to WT mice. Following secondary challenge, WT and STAT4-/- mice also did not develop lung Th2 or Th17 inflammation. In contrast to primary challenge, secondary RSV challenge of STAT4-/- mice resulted in enhanced weight loss, increased lung IFN- expression, and an increased lung RSV-specific CD8+ T cell response compared to WT mice. These data demonstrate that STAT4 regulates the RSV-specific CD8+ T cell response to secondary infection, but does not independently regulate lung Th2 or Th17 immune responses to RSV challenge.

    Importance STAT4 is a protein critical for both innate and adaptive immune responses to viral infection. Our results show that STAT4 regulates the immune response to primary and secondary challenge with RSV, but does not restrain RSV-induced lung Th2 or Th17 immune responses. These findings suggest that STAT4 expression may influence lung immunity and severity of illness following primary and secondary RSV infection.

  • Newcastle Disease Virus Vector Producing Human Norovirus-like Particles Induces Serum, Cellular, and Mucosal Immune Responses in Mice [PublishAheadOfPrint]

  • Human norovirus infection is the most common cause of viral gastroenteritis worldwide. Development of an effective vaccine is required for reducing norovirus outbreaks. Inability to grow human norovirus in cell culture has hindered development of live attenuated vaccines. To overcome this obstacle, we generated a recombinant Newcastle disease virus (rNDV) vectored experimental norovirus vaccine by expressing the capsid protein (VP1) of norovirus strain VA387. We compared two different NDV vectors, a conventional rNDV vector and a modified rNDV vector for their efficiency to express VP1 protein. Our results showed that the modified vector replicated to higher titers and expressed higher levels of VP1 protein in DF1 cells and in allantoic fluid of embryonated chicken eggs than the conventional vector. We further demonstrated that the VP1 protein produced by rNDVs was able to self-assemble into virus-like particles (VLPs), which are morphologically similar to baculovirs-expressed VLPs. Evaluation of their immunogenicity in mice showed that the modified rNDV vector induced higher level of IgG response than that induced by the conventional vector and by the baculovirus-expressed VLPs. The rNDV vectors predominantly induced subclass IgG2a antibody for Th1 response, and specifically, high levels of IFN-, TNF-aalpha;, and IL-2 were detected in the splenocytes. In addition, the modified rNDV vector induced higher level of fecal IgA response in mice than baculovirus-expressed VLPs. Our findings suggest that rNDV vector is an efficient system to produce cost-effective VLPs in embryonated chicken eggs as well as has the potential to be used as a live attenuated vaccine in humans.

    IMPORTANCE Noroviruses are the major cause of viral gastroenteritis worldwide. Currently, effective vaccines against norovirus infection are not available. In this study, we have evaluated Newcastle disease virus (NDV) as a vaccine vector for norovirus. Our results suggest that NDV can not only be used as a cost-effective method for large-scale production of norovirus-like particle vaccines but also can be used as a live-attenuated vectored vaccine.

  • The tegument protein pp65 of human cytomegalovirus acts as an optional scaffold protein that optimizes protein uploading into viral particles [PublishAheadOfPrint]

  • The mechanisms that lead to the tegumentation of herpesviral particles are only poorly defined. The phosphoprotein 65 (pp65) is the most abundant constituent of the virion tegument of human cytomegalovirus (HCMV). It is, however, non-essential for virion formation. This seeming discrepancy has not met with a satisfactory explanation regarding the role of pp65 in HCMV particle morphogenesis. Here we addressed the question how the overall tegument composition of the HCMV virion depended on pp65 and how the lack of pp65 influenced the packaging of particular tegument proteins. To investigate this, we analyzed the proteome of pp65pos and pp65neg virions by label-free quantitative mass spectrometry and determined the relative abundance of tegument proteins. Surprisingly, only pUL35 was elevated in pp65neg virions. As the abundance of pUL35 in the HCMV tegument is low, it unlikely replaced pp65 as a structural component in pp65neg virions. A subset of proteins, including the third most abundant tegument protein pUL25 as well as pUL43, pUL45, pUL71 were reduced in pp65neg or pp65low virions, indicating that the packaging of these proteins was related to pp65. The levels of tegument components, like pp28 and the capsid associated tegument proteins pp150, pUL48 and pUL47, were unaffected by the lack of pp65. Our analyses demonstrate that deletion of pp65 is not compensated by other viral proteins in the process of virion tegumentation. The results are concordant with a model of pp65 serving as an optional scaffold protein that facilitates protein upload into the outer tegument of HCMV particles.

    IMPORTANCE The assembly of the tegument of herpesviruses is only poorly understood. Particular proteins, like the HCMV pp65 are abundant tegument constituents. The pp65 is thus considered to play a major role in tegument assembly in the process of virion morphogenesis. We show here that deletion of the pp65 gene leads to reduced packaging of a subset of viral proteins indicating that pp65 acts as an optional scaffold protein mediating protein upload into the tegument.

  • The HSV-1 virion host shutoff protein enhances translation of viral late mRNAs by preventing mRNA overload. [PublishAheadOfPrint]

  • We recently demonstrated that the virion host shutoff (vhs) protein, an mRNA specific endonuclease, is required for efficient HSV-1 replication and translation of viral true late mRNAs, but not other viral and cellular mRNAs, in many cell types. Here, we evaluated whether the structure of true late mRNAs or the timing of their trancription is responsible for the poor translation efficiency in the absence of vhs. To test whether the highly structured 5'UTR of the true late gC mRNA is the primary obstacle for translation initiation we replaced it with the less structured 5' UTR of the -actin mRNA. However, this mutation did not restore translation in the context of a vhs-deficient virus. We then examined whether the timing of transcription affects translation efficiency at late times. To this end, we engineered a vhs-deficient virus mutant that transcribes the true late gene US11 with IE kinetics (IEUS11Sma). Interestingly, IEUS11Sma showed increased translational activity on the Us11 transcript at late times postinfection and US11 protein levels were restored to wild-type levels. These results suggest that mRNAs can maintain translational activity throughout the late stage of infection if they are present before translation factors and/or ribosomes become limiting. Taken together these results provide evidence that in the absence of the mRNA destabilizing function of vhs, accumulation of viral mRNAs overwhelms the capacity of the host translational machinery, leading to functional exclusion of the last mRNAs that are made during infection.

    IMPORTANCE The process of mRNA translation accounts for a significant portion of a cell's energy consumption. To ensure efficient use of cellular resources, transcription, translation and mRNA decay are tightly linked and highly regulated. However, during virus infection the overall amount of mRNA may increase drastically, possibly overloading the capacity of the translation apparatus. Our results suggest that the HSV-1 vhs protein, an mRNA-specific endoribonuclease, prevents mRNA overload during infection, thereby allowing translation of late viral mRNAs. The requirement for vhs varies between cell types. Further studies of the basis for this difference will likely offer insights into how cells regulate overall mRNA levels and access to the translational apparatus.

  • Locking and Blocking the Viral Landscape of an Alphavirus with Neutralizing Antibodies [PublishAheadOfPrint]

  • Alphaviruses can be serious, sometimes lethal human pathogens that belong to the family Togaviridae. Structures of human Venezuelan equine encephalitis virus (VEEV), an alphavirus, in complex with two strongly neutralizing antibody Fab fragments (F5 and 3B4C-4) have been determined using a combination of cryo-electron microscopy (cryo-EM) and homology modeling. Here we characterize these monoclonal antibody Fab fragments known to abrogate VEEV infectivity by binding to the E2 (envelope) surface glycoprotein. Both these antibody Fab fragments cross-link the surface E2 glycoproteins and, therefore, probably inhibit infectivity by blocking the conformational changes that are required for making the virus fusogenic. The F5 Fab fragment cross-links E2 proteins within one trimeric spike, whereas the 3B4C-4 Fab fragment cross-links E2 proteins from neighboring spikes. Furthermore, F5 probably blocks the receptor-binding site, whereas 3B4C-4 sterically hinders the exposure of the fusion loop at the end of the E2 B-domain.

    IMPORTANCE Alphaviral infections are transmitted mainly by mosquitoes. Venezuelan equine encephalitis virus (VEEV) is an alphavirus with a wide distribution across the globe. No effective vaccines exist for alphaviral infections. Therefore, a better understanding of VEEV and its associated neutralizing antibodies will help with the development of effective drugs and vaccines.

  • Intranasal P particle vaccine provided partial cross-variant protection against human GII.4 norovirus diarrhea in gnotobiotic pigs [PublishAheadOfPrint]

  • Noroviruses (NoVs) are the leading cause of nonbacterial acute gastroenteritis worldwide in people of all ages. The P particle is a novel vaccine candidate derived from the protruding (P) domain of the NoV VP1 capsid protein. This study utilized the neonatal gnotobiotic pig model to evaluate the protective efficacies of primary infection, P particles, and VLPs against NoV infection and disease and the T cell responses to these treatments. Pigs were intranasally vaccinated with GII.4/1997 NoV (VA387)-derived P particles or virus-like particles (VLPs) or orally inoculated with a GII.4/2006b NoV variant. At post-inoculation day (PID) 28, pigs were euthanized or challenged with the GII.4/2006b variant and monitored for diarrhea and virus shedding for 7 days. The T cell responses in intestinal and systemic lymphoid tissues were examined. Primary NoV infection provided 83% homologous protection against diarrhea and 49% against virus shedding, while P particle and VLP vaccines provided cross-variant protection (47% and 60%, respectively) against diarrhea. The protection rates against diarrhea are significantly inversely correlated with T cell expansion in duodenum and positively correlated with T cell expansion in ileum and spleen. The P particle vaccine primed for stronger immune responses than VLPs, including significantly higher activated CD4+ T cells in all tissues, IFN-+CD8+ T cells in duodenum, regulatory T cells (Tregs) in blood, and TGF-bbeta; producing CD4+CD25-FoxP3+ Tregs in spleen post-challenge, indicating P particles are more immunogenic than VLPs at the same dose. In conclusion, the P particle vaccine is a promising vaccine candidate worthy of further development.

    Importance The norovirus (NoV) P particle is a vaccine candidate derived from protruding (P) domain of NoV VP1 capsid protein. P particles can be easily produced in E. coli at high yield, thus may be more economically viable than the virus-like particle (VLP) vaccine. This study demonstrated for the first time the cross-variant protection (46.7%) of the intranasal P particle vaccine against human NoV diarrhea and revealed in detail the intestinal and systemic T cell responses using the gnotobiotic pig model. The cross-variant protective efficacy of the P particle vaccine was comparable to that of the virus-like particle (VLP) vaccine in pigs (60%) and the homologous protective efficacy in humans (47%). NoV is now the leading cause of pediatric dehydrating diarrhea, responsible for approximately 1 million hospital visits for U.S. children and 218,000 deaths in developing countries. The P particle vaccine has promise to reduce the disease burden and mortality.

  • Genomic Flexibility of Human Endogenous Retrovirus Type K [PublishAheadOfPrint]

  • Human endogenous retrovirus type K (HERV-K) proviruses are scattered throughout the human genome, but as no infectious HERV-K virus has been detected to date, the mechanism by which these viruses replicated, and thus populated the genome, remains unresolved. Here, we provide evidence that, in addition to the RNA genomes that canonical retroviruses package, modern HERV-K viruses can contain reverse transcribed DNA (RT-DNA) genomes. Indeed, reverse transcription of genomic HERV-K RNA into the DNA form is able to occur in three distinct times and locations: 1) in the virus-producing cell prior to viral release, yielding a DNA-containing extracellular virus particle similar to the Spumaviruses; 2) within the extracellular virus particle itself, transitioning from an RNA-containing particle to a DNA-containing particle; and 3) after entry of the RNA-containing virus into the target cell, similar to canonical retroviruses such as MLV and HIV. Moreover, using a resuscitated HERV-K virus construct, we show that both viruses with RNA genomes and viruses with DNA genomes are capable of infecting target cells. This high level of genomic flexibility historically could have permitted these viruses to replicate in variable host cell environments, potentially assisting in their many integration events and resulting in their high prevalence in the human genome. Moreover, the ability of modern HERV-K viruses to proceed through reverse transcription and package RT-DNA genomes suggests a higher level of replication competency than was previously understood, and may be relevant in HERV-K associated human diseases.

    Importance Retroviral elements comprise at least 8% of the human genome. Of all the endogenous retroviruses, HERV-K are the most intact and biologically active. While a modern infectious HERV-K has yet to be found, HERV-K activation has been associated with cancers, autoimmune diseases, and HIV-1 infection. Thus, determining how this virus family became such a prevalent member of our genome, and what it is capable of in its current form, is of the utmost importance. Here, we provide evidence that HERV-K viruses currently found in the human genome are able to proceed through reverse transcription, and historically utilized a life cycle with a surprising degree of genomic flexibility in which both RNA and DNA containing viruses were capable of mediating infection.

  • Impaired immune response to vaccination against infection with human respiratory syncytial virus at advanced age [PublishAheadOfPrint]

  • Elderly humans are prone to severe infection with human respiratory syncytial virus (HRSV). Aging of today's human population warrants the development of protective vaccination strategies aimed specifically at the elderly. This may require special approaches due to deteriorating immune function. To design and test vaccination strategies tailored to the elderly population, we need to understand the host response to HRSV vaccination and infection at old age. Moreover, preclinical need of testing candidate vaccines requires translational models resembling susceptibility to the (unadapted) human pathogen.

    Here, we explored effects of aging on immunity and protection induced by a model human HRSV vaccine candidate in a translational aging model in cotton rats (Sigmodon hispidus), and examined possibilities to optimize vaccination concepts for the elderly. We immunized young and aged cotton rats with a live-attenuated recombinant HRSV vaccine candidate, and analyzed the induced immune response and protection to challenge with HRSV.

    In old cotton rats HRSV-infection persisted longer, and vaccination induced less protection against infection. Aged animals developed lower levels of vaccine-induced IgG, virus-neutralizing serum antibodies and IgA in lungs. Moreover, booster responses to HRSV-challenge virus were impaired in animals vaccinated at older age. However, increased dose and reduced attenuation of vaccine could improve protection even in old animals.

    This study shows cotton rats provide a model for studying effects of aging on the immune response to the human respiratory pathogen HRSV, and possibilities to optimize vaccine concepts for the elderly.

    IMPORTANCE HRSV infection poses a risk for severe disease in the elderly. Aging of the population urges to increase efforts to prevent disease at old age, whereas HRSV vaccines are only in the developmental phase. Preclinical need of testing candidate human vaccines requires translational models resembling susceptibility to the natural human virus. Moreover, we need to gain insight in the waning immunity at old age, as this is a special concern in vaccine development. In this study we explored the effect of age on protection and immunity against an experimental HRSV vaccine in aged cotton rats (Sigmodon hispidus), a rodent species that provides a model representing natural susceptibility to human viruses. Older animals generate less antibodies upon vaccination, and require a higher vaccine dose for protection. Notably, during the early secondary immune response to subsequent HRSV infection older animals show less protection and a slower rise of the virus-neutralizing antibody titer.

  • Influenza virus infectivity and virulence following ocular-only aerosol inoculation of ferrets. [PublishAheadOfPrint]

  • The study of respiratory pathogens has traditionally been performed by examining virus exposure to and infection of respiratory tract tissues. However, these studies typically overlook the role of ocular surfaces, which represent both a potential site of virus replication and a portal of entry for the establishment of a respiratory infection. To model transocular virus entry in a mammalian species, we established a novel inoculation method that delivers an aerosol inoculum exclusively to the ferret ocular surface. Using influenza as a representative respiratory pathogen, we found that both human and avian viruses mounted a productive respiratory infection in ferrets following ocular-only aerosol inoculation, and demonstrated that H5N1 exposure can result in a fatal infection at viral doses below 10 PFU or for as little as 2 minutes of virus exposure. Ferrets inoculated by the ocular aerosol route with avian (H7N7, H7N9) or human (H1N1, H3N2v) viruses were capable of transmitting virus to naiiuml;ve animals in direct contact or respiratory droplet models, respectively. Our results reveal that ocular-only exposure to virus-containing aerosols constitutes a valid exposure route for a potentially fatal respiratory infection, even among viruses which do not demonstrate an ocular tropism, underscoring the public health implications of ocular exposure in clinical or occupational health settings.

    Importance. In the absence of eye protection, the human ocular surface remains vulnerable to infection with aerosolized respiratory viruses. In this study, we present a way to inoculate laboratory mammals that excludes respiratory exposure, infecting ferrets only by ocular exposure to influenza virus-containing aerosols. This study demonstrates that the use of respiratory protection alone does not fully protect against influenza virus exposure, infection, and severe disease.

  • Mapping of functional domains of the lipid kinase phosphatidylinositol 4-kinase type III alpha involved in enzymatic activity and hepatitis c virus replication [PublishAheadOfPrint]

  • The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIaalpha;) is an ER-resident enzyme that synthesizes phosphatidylinositol 4-phosphate (PI4P). PI4KIIIaalpha; is an essential host factor for hepatitis C virus (HCV) replication. Interaction with HCV non-structural protein 5A (NS5A) leads to kinase activation and accumulation of PI4P at intracellular membranes. In this study, we investigated structural requirements of PI4KIIIaalpha; involved in HCV replication and enzymatic activity. Therefore, we analyzed PI4KIIIaalpha; mutants for subcellular localization, reconstitution of HCV replication in PI4KIIIaalpha; knockdown cell lines, PI4P induction in HCV-positive cells and lipid kinase activity in vitro. All mutants still interacted with NS5A and localized similarly as the full-length enzyme, suggesting multiple regions of PI4KIIIaalpha; involved in NS5A interaction and subcellular localization. Interestingly, the N-terminal 1152 amino acids were dispensable for HCV replication, PI4P induction and enzymatic function, whereas further N-terminal or C-terminal deletions were deleterious, thereby defining the minimal PI4KIIIaalpha; core enzyme to a size of ca. 108 kDa. Additional deletion of predicted functional motifs within the C-terminal half of PI4KIIIaalpha; were also detrimental for enzymatic activity and for the ability of PI4KIIIaalpha; to rescue HCV replication, with the exception of a proposed nuclear localization signal, suggesting that the entire C-terminal half of PI4KIIIaalpha; is involved in the formation of a minimal enzymatic core. This view was supported by structural modeling of the PI4KIIIaalpha; C-terminus suggesting a catalytic center formed by an N- and C-terminal lobe and an armadillo-fold motif, which is preceded by three distinct alpha-helical domains probably involved in regulation of enzymatic activity.

    Importance The lipid kinase phosphatidylinositol 4-phosphate kinase type IIIaalpha; (PI4KIIIaalpha;, PIK4CA, PI4KA) is of central importance for the cellular phosphatidylinositol metabolism and a key host cell factor of hepatitis C virus replication. However, little is known so far about the structure of this 240 kDa protein and the functional importance of specific subdomains regarding lipid kinase activity and viral replication. This work focuses on the phenotypic analysis of distinct PI4KIIIaalpha; mutants in different biochemical and cell-based assays and develops a structural model of the C-terminal enzymatic core. The results shed light on the structural and functional requirements of enzymatic activity and the determinants required for HCV replication.

  • Polyinosinic-polycytidylic acid treatment leads to the interferon-dependent clearance of hepatitis B virus in a hydrodynamic injection mouse model [PublishAheadOfPrint]

  • We have previously shown that polyinosinic-polycytidylic acid (poly(I:C)) activates murine hepatic cells to produce interferon (IFN) and suppresses HBV replication in vitro. Therefore, we addressed whether poly(I:C) is able to induce the clearance of HBV in vivo. The chronic HBV replication mouse model was established by the hydrodynamic injection (HI) of pAAV-HBV1.2 into the tail veins of wild-type, IFN-aalpha;/bbeta;R-, IFN--, and CXCR3-deficient C57BL/6 mice. Fourteen days post-HI of pAAV-HBV1.2, mice were administered poly(I:C) by intraperitoneal injection, intramuscular injection or HI. Only treatment of poly(I:C) by HI led to HBV clearance in wild-type C57BL/6 mice. Serum HBsAg disappeared within 40 dpi in mice that received poly(I:C) by HI, and this was accompanied by the appearance of anti-HBs antibodies. HBV-specific T-cell and antibody responses were significantly enhanced by HI of poly(I:C). HBV replication intermediates and HBcAg-positive hepatocytes were eliminated in the liver. HI of poly(I:C) induced the production of IFNs in mice and enhanced the levels of cytokines, IFN-stimulated genes, and T-cell markers in the liver. Importantly, poly(I:C)-induced HBV clearance was impaired in IFN-aalpha;/bbeta;R-, IFN--, and CXCR3-deficient mice, indicating that the induction of type I IFN as well as the stimulation and recruitment of T-cells into the liver are essential for HBV clearance in this model. Taken together, the application of poly(I:C) by HI into the liver enhances innate and adaptive immune responses and leads to HBV clearance in an HBV mouse model, implicating the potential of intrahepatic TLR3 activation for the treatment of chronic chronic hepatitis B patients.

    Importance: It became well-accepted that immunomodulation is a potentially useful approach to treat chronic viral infection. Recently, combinations of antiviral treatment and therapeutic vaccinations were evaluated for therapies of chronic hepatitis B virus (HBV) infection. Activation of the innate immune branch may also be important for viral control and contributes to HBV clearance. Our present study demonstrated that hepatic TLR3 activation led to clearance of hepatitis B virus (HBV) in an HBV mouse model (Fig.I1). For the first time, we showed that HBV clearance in this model is not only dependent on type I interferon (IFN) but also on type II IFN, indicating a coordinated action of innate and adaptive immune responses. T cell recruitment appeared to be critical for the success of TLR3-mediated antiviral action. These findings implicate the potential of intrahepatic TLR3 activation for the treatment of chronic HBV infection.

  • CD40L-adjuvanted DNA/MVA SIV239 vaccine enhances SIV-specific humoral and cellular immunity, and improves protection against a heterologous SIVE660 mucosal challenge [PublishAheadOfPrint]

  • It remains a challenge to develop a successful HIV vaccine that is capable of preventing infection. Here, we utilized the benefits of CD40L, a co-stimulatory molecule that can stimulate both dendritic cells (DCs) and B cells, as an adjuvant for our simian immunodeficiency virus (SIV) DNA vaccine in rhesus macaques. We co-expressed the CD40L with our DNA/SIV vaccine such that the CD40L is anchored on the membrane of SIV virus-like particle (VLP). These CD40L containing SIV VLPs showed enhanced activation of DCs in vitro. We then tested the potential of DNA/SIV-CD40L vaccine to adjuvant the DNA prime of a DNA/modified vaccinia Ankara (MVA) vaccine in rhesus macaques. Our results demonstrated that the CD40L adjuvant enhanced the functional quality of anti-Env antibody response and breadth of anti-SIV CD8 and CD4 T cell responses, significantly delayed the acquisition of heterologous mucosal SIV infection and improved viral control. Notably, the CD40L adjuvant enhanced the control of viral replication in the gut at the site of challenge that was associated with lower mucosal CD8 immune activation, one of the strong predictors of disease progression. Collectively, our results highlight the benefits of CD40L adjuvant for enhancing anti-viral humoral and cellular immunity leading to enhanced protection against a pathogenic SIV. A single adjuvant that enhances both humoral and cellular immunity is rare and thus underlines the importance and practicality of CD40L as an adjuvant for vaccines against infectious diseases including HIV-1.

    Importance Despite many advances in the field of AIDS research, an effective AIDS vaccine that can prevent infection remains elusive. CD40L is a key stimulator of dendritic cells and B cells, and can therefore enhance T cell and antibody responses but its overly potent nature can lead to adverse effects, unless if used in small doses. In order to modulate local expression of CD40L at relatively lower levels, we expressed CD40L in a membrane-bound form along with SIV antigens in a nucleic acid (DNA) vector and an attenuated poxvirus (rMVA) vector. We tested immunogenicity and efficacy of the CD40L-adjuvanted vaccine in macaques using a heterologous mucosal SIV infection. The CD40L-adjuvanted vaccine enhanced the functional quality of anti-Env antibody response and breadth of anti-SIV T cell responses, and improved protection. These results demonstrate that VLP-membrane-bound CD40L serves as a novel adjuvant for a HIV vaccine.

  • The Effect of HIV-1 Integrase Resistance Mutations when Introduced into SIVmac239 on Susceptibility to Integrase Strand Transfer Inhibitors [PublishAheadOfPrint]

  • Studies on the in vitro susceptibility of SIV to integrase strand transfer inhibitors (INSTIs) have been rare. In order to determine the susceptibility of SIVmac239 to INSTIs and characterize the genetic pathways that might lead to drug resistance, we inserted various integrase (IN) mutations that had been selected with HIV under drug pressure with raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG) into the IN gene of SIV. We evaluated the effects of these mutations on SIV susceptibility to INSTIs and on viral infectivity.

    Sequence alignments of SIVmac239 IN with various HIV-1 isolates showed a high degree of homology and conservation of each of the catalytic triad and the key residues involved in drug resistance. Each of the G118R, Y143R, Q148R, R263K, and G140S/Q148R mutations, when introduced into SIV, impaired infectiousness and replication fitness compared with wild-type (WT) virus. Using TZM-bl cells, we demonstrated that the Q148R and N155H mutational pathways conferred resistance to EVG (36- and 62-fold, respectively) whereas R263K also displayed moderate resistance to EVG (12-fold). In contrast, Y143R, Q148R and N155H all yielded low levels of resistance to RAL. The combination of G140S/Q148R conferred high-level resistance to both RAL and EVG (ggt;300 and 286-fold). DTG remained fully effective against all site-directed mutants except G118R and R263K.

    Thus, HIV INSTI-mutations when inserted into SIV result in a similar phenotype. These findings suggest that SIV and HIV may share similar resistance pathways profiles and that SIVmac239 could be a useful non-human primate model for studies of HIV resistance to INSTIs.

    Importance of the study: The goal of our project was to establish whether drug resistance against integrase inhibitors in SIV are likely to be the same as those responsible for drug resistance in HIV. Our data answer this question in the affirmative and show that SIV can probably serve as a good animal model for studies of INSTIs and as an early indicator for possible emergent mutations that may cause treatment failure. A SIV-primate model remains an invaluable tool to investigate questions related to the potential role of INSTIs in HIV therapy, transmission, and pathogenesis, and the present study will facilitate each of the above.

  • Molecular mechanism of the airborne transmissibility of H9N2 avian influenza A viruses in chickens [PublishAheadOfPrint]

  • H9N2 avian influenza virus has been prevalent in poultry in many parts of the world since the 1990s and occasionally crosses the host barrier, transmitting to mammals, including humans. In recent years, these viruses have contributed genes to H5N1 and H7N9 influenza viruses, threatening the public health. To explore the molecular mechanism for the airborne transmission of H9N2 virus, we compared two genetically close strains isolated from chickens in 2001, A/chicken/Shanghai/7/2001(SH7) and A/chicken/Shanghai/14/2001(SH14). SH7 is airborne transmissible between chickens, whereas SH14 is not. We used reverse genetics and gene swapping to derive rSH7, rSH14 and a panel of reassortant viruses. Among the reassortant viruses, we identified segments HA and PA as governing the airborne transmission among chickens. In addition, the NP and NS genes also contributed to a lesser extent. Furthermore, the mutational analyses showed the transmissibility phenotype predominantly mapped to the HA and PA genes with HA-K363 and PA-L672 being important for airborne transmissibility among chickens. In addition, the viral infectivity and acid stability are related to the airborne transmissibility. Importantly, airborne transmission studies of 18 arbitrarily chosen H9N2 viruses from our collections confirmed the importance of both 363K in HA and 672L in PA in determining their transmissibilities. Our finding elucidates the genetic contributions to H9N2 transmissibility in chickens and highlights their importance in the prevalence in poultry.

    Importance Our study investigates the airborne transmissibility of H9N2 viruses in chickens and the subsequent epidemic. H9N2 virus is the donor for several prevalent reassortant influenza viruses, such as H7N9/2013 and the H5N1 viruses. Poultry as the reservoir hosts of influenza is closely related to associated with human society. Airborne transmission is an efficient pathway for influenza transmission among flocks and individuals. Exploring the mechanism of the airborne transmission of the H9N2 virus in chickens could provide essential data regarding prevention and control of influenza endemics and pandemics.

  • Rewiring of Cellular Membrane Homeostasis by Picornaviruses [PublishAheadOfPrint]

  • Viruses are obligatory intracellular parasites and utilize host elements to support key viral processes including the penetration of the plasma membrane, initiation of infection, replication, and suppression of the host's anti-viral defenses. In this review we focus on picornaviruses, a family of positive strand RNA viruses and discuss the mechanisms by which these viruses hijack the cellular machinery to form and operate membranous replication complexes. Studies aimed at revealing factors required for the establishment of viral replication structures identified several cellular membrane remodeling proteins and led to the development of models in which the virus used a pre-existing cellular membrane-shaping pathway "as is" for generating its replication organelles. However, as more data accumulate, this view is being increasingly questioned, and it is becoming clearer that viruses may utilize cellular factors in ways that are distinct from the normal functions of these proteins in uninfected cells. In addition, the protein-centric view is being supplemented by important new studies showing a previously unappreciated deep remodeling of lipid homeostasis, including extreme changes to phospholipid biosynthesis and cholesterol trafficking. The data on viral modifications of lipid biosynthetic pathways are still rudimentary, but it appears that once again, the viruses may rewire existing pathways to generate novel functions. Despite remarkable progress, our understanding of how a handful of viral proteins can completely overrun the multi-layered complex mechanisms that control membrane organization of a eukaryotic cell remains very limited.

  • Unfixed endogenous retroviral insertions in the human population [PublishAheadOfPrint]

  • One lineage of human endogenous retroviruses, HERV-K(HML2), is upregulated in many cancers, some autoimmune/inflammatory diseases, and in HIV-infected cells. Despite three decades of research it is not known if these viruses play a causal role in disease, and there has been recent interest in whether they can be used as immunotherapy targets. Resolution of both these questions will be helped by an ability to distinguish between the effects of different integrated copies of the virus (loci). Research so far has concentrated on the 20 or so recently integrated loci that, with one exception, are in the human reference genome sequence. However, this viral lineage has been copying in the human population within the last million years, so some loci will inevitably be present in the human population but absent from the reference sequence. We therefore performed the first detailed search for such loci by mining whole genome sequences generated by Next Generation Sequencing. We found a total of 17 loci: ranging from being present in only two of the 358 individuals examined to being present in over 95% of them. On average, each individual had six loci that are not in the human reference genome sequence. Comparing the number of loci we found to an expectation derived from a neutral population genetic model suggests that the lineage was copying till at least ~250,000 years ago.

    IMPORTANCE. About 5% of our genome sequence is composed of the remains of retroviruses that over millions of years have integrated into the chromosomes of egg and/or sperm precursor cells. There are indications that protein expression of these viruses is higher in some diseases, and we need to know (a) whether these viruses have a role in causing disease and (b) whether they can be used as immunotherapy targets in some of them. Answering both questions requires a better understanding of how individuals differ in the viruses they carry. We therefore carried out the first careful search for new viruses in some of the many human genome sequences that are now available thanks to advances in sequencing technology. We also compare the number we find to a theoretical expectation to see if it is likely that these viruses are still replicating in the human population today.

  • Hepatitis A virus 3C protease cleaves NEMO to impair induction of beta interferon [PublishAheadOfPrint]

  • NEMO (NF-B essential modulator) is a bridging adaptor indispensable for viral activation of interferon (IFN) antiviral response. Herein, we show that hepatitis A virus (HAV) 3C protease (3Cpro) cleaves NEMO at Q304 residue, negating its signaling adaptor function and abrogating viral induction of IFN-bbeta; synthesis via RIG-I/MDA5 and TLR3 pathways. NEMO cleavage and IFN antagonism, however, were lost upon ablating the catalytic activity of 3Cpro. These data describe a novel immune evasion mechanism of HAV.

  • Intranasal adenovirus vectored vaccine for induction of long-lasting humoral immunity-mediated broad protection against influenza in mice [PublishAheadOfPrint]

  • Influenza vaccines aimed at inducing antibody (Ab) responses against viral surface hemagglutinin (HA) and neuraminidase (NA) provide sterile immunity to infection with the same subtypes. Vaccines targeting viral conserved determinants shared by the influenza A viruses (IAV) offer heterosubtypic immunity (HSI), a broad protection against different subtypes. We proposed that vaccines targeting both HA and the conserved ectodomain of matrix protein 2 (M2e) would provide protection against infection with the same subtype and also HSI against other subtypes. Here we report that single intranasal (i.n.) immunization with a recombinant adenovirus (rAd) vector encoding both HA of H5 virus and M2e (rAdH5/M2e) induced significant HA and M2e specific Ab responses along with protection against heterosubtypic challenge in mice. The protection is superior as compared to that induced by rAd vector encoding either HA (rAdH5), or M2e (rAdM2e). While protection against homotypic H5 virus is primarily mediated by virus neutralizing (VN) antibodies (Abs), the cross-protection is associated with Abs directed to conserved stalk HA and M2e that seem to have additive effect. Consistently, adoptive transfer of antisera induced by rAdH5/M2e provided the best protection against heterosubtypic challenge as compared to that provided by antisera derived from mice immunized with rAdH5 or rAdM2e. These results support the development of rAd vectored vaccines encoding both H5 and M2e as universal vaccines against different IAV subtypes.

    IMPORTANCE: Current licensed influenza vaccines provide protection limited to the infection with same virus strains, therefore, the composition of influenza vaccines has to be revised every year. We have developed a new universal influenza vaccine that is highly efficient in induction of long-lasting cross-protection against different influenza virus strains. The cross-protection is associated with high level of vaccine-induced antibodies against conserved stalk domain of influenza virus hemagglutinin and ectodomain of matrix protein. The vaccine could be used to stimulate cross-protective antibodies for prevention and treatment of influenza with immediate effect for individuals who fail to respond to or receive the vaccine in due time. The vaccine offers a new tool to control influenza outbreaks including pandemics.

  • A Novel Mode of Poxvirus Superinfection Exclusion that Prevents Fusion of the Lipid Bilayers of Viral and Cellular Membranes [PublishAheadOfPrint]

  • Superinfection exclusion is a widespread phenomenon that prevents secondary infections by closely related viruses. Previous studies showed that the vaccinia virus A56 and K2 proteins in the cell membrane prevent superinfection by interacting with the entry-fusion complex of subsequent viruses. Here we described another form of exclusion that is established earlier in infection and does not require the A56 or K2 proteins. Cells infected with one or more infectious virions excluded hundreds of superinfecting vaccinia virus particles. A related orthopoxvirus, but neither a flavivirus nor a rhabdovirus, was also excluded indicating selectivity. Although superinfecting vaccinia virus bound to cells, infection was inhibited at the membrane fusion step, thereby preventing core entry into the cytoplasm and early gene expression. In contrast, A56/K2 protein-mediated exclusion occurred subsequent to membrane fusion. Induction of resistance to superinfection depended on viral RNA and protein synthesis by the primary virus but did not require DNA replication. Although superinfection-resistance correlated with virus-induced changes in the cytoskeleton, studies with mutant vaccinia viruses indicated that the cytoskeletal changes were not necessary for resistance to superinfection. Interferon-inducible transmembrane proteins, which can inhibit membrane fusion in other viral systems, did not prevent vaccinia virus membrane fusion, suggesting that these interferon-inducible proteins are not involved in superinfection exclusion. While the mechanism remains to be determined, the early establishment of superinfection exclusion may provide a "winner-take-all" reward to the first poxvirus particles that successfully initiates infection and prevent the entry and genome reproduction of defective or less fit particles.

    IMPORTANCE The replication of a virus usually follows a defined sequence of events: attachment, entry into the cytoplasm or nucleus, gene expression, genome replication, assembly of infectious particles and spread to other cells. Although multiple virus particles may enter a cell at the same time, mechanisms exist to prevent infection by subsequent viruses. The latter phenomenon, known as superinfection exclusion, can occur by a variety of mechanisms that are not well understood. We showed that superinfection by vaccinia virus was prevented at the membrane fusion step, which closely followed virion attachment. Thus, neither gene expression nor genome replication of the superinfecting virus occurred. Expression of early proteins by the primary virus was necessary and sufficient to induce the superinfection-resistant state. Superinfection exclusion may be beneficial to vaccinia virus by selecting particles that can infect cells rapidly, excluding defective particles and synchronizing the replication cycle.

  • A comprehensive analysis of contributions from protein conformational stability and MHCII-peptide binding affinity to CD4+ epitope immunogenicity in HIV-1 envelope glycoprotein [PublishAheadOfPrint]

  • Helper T-cell epitope dominance in HIV-1 envelope glycoprotein gp120 is not adequately explained by peptide-binding to MHC proteins. Antigen processing potentially influences epitope dominance, but few, if any, studies have attempted to reconcile the influences of antigen processing and MHC protein binding for all helper T-cell epitopes of an antigen. Epitopes of gp120 identified in both humans and mice occur on the C-terminal flanks of flexible segments that are likely to be proteolytic cleavage sites. In this study, the influence of gp120 conformation on the dominance pattern in gp120 from HIV strain 89.6 was examined in CBA mice, whose class II MHC protein has one of the most well-defined peptide-binding preferences. Only one of six dominant epitopes contained the most conserved element of the I-Ak binding motif, an aspartic acid. Destabilization of the gp120 conformation by deletion of single disulfide bonds preferentially enhanced responses to the cryptic I-Ak motif-containing sequences, as reported by T-cell proliferation or cytokine secretion. Conversely, inclusion of CpG in the adjuvant with gp120 enhanced responses to the dominant CD4+ T-cell epitopes. The gp120 destabilization affected secretion of some cytokines more than others, suggesting that antigen conformation could modulate T-cell functions through mechanisms of antigen processing.

    Importance CD4+ helper T-cells play an essential role in protection against HIV and other pathogens. Thus, the sites of helper T-cell recognition, the dominant epitopes, are targets for vaccine design; and the corresponding T cells may provide markers for monitoring infection and immunity. However, T-cell epitopes are difficult to identify and predict. It is also unclear whether CD4+ T cells specific for one epitope are more protective than T cells specific for other epitopes. This work shows that the 3D structure of an HIV protein partially determines which epitopes are dominant, most likely by controlling the breakdown of HIV into peptides. Moreover, some types of signals from CD4+ T cells are affected by the HIV protein 3D structure; and thus the protectiveness of a particular peptide vaccine could be related to its location in the 3D structure.

  • Stabilizing the native trimer of HIV-1 Env by destabilizing the heterodimeric interface of the gp41 post-fusion six helix bundle [PublishAheadOfPrint]

  • HIV-1 envelope glycoprotein (Env) is a trimer of gp120-gp41 heterodimers and is essential for viral entry. The gp41 subunit in native, pre-fusion trimeric Env exists in a metastable conformation and attains a stable six helix bundle (6HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers, that drives fusion of viral and cellular membranes. We attempt to stabilize native Env trimers by incorporation of mutations at the NHR:CHR interface that disrupt the post-fusion 6HB of gp41. The mutations V570D and I573D stabilize native JRFL Env and occlude non-neutralizing epitopes to a greater extent than the previously identified I559P mutation that is at the interface of the NHR trimers in the 6HB. The mutations prevent sCD4 induced gp120 shedding and 6HB formation. In the context of cell surface expressed JRFL Env, introduction of a previously reported additional disulfide between residues A501 and T605 perturbs the native conformation, though this effect is partially alleviated by furin co-expression. The data suggest that positions 570 and 573 are surface proximal in native Env and that the NHR homotrimeric coiled coil in native Env terminates before or close to residue 573. Aspartic acid substitutions at these positions stabilize native trimers through destabilization of the post fusion 6HB conformation. These mutations can be used to stabilize Env in a DNA vaccine format.

    Importance: The major protein on the surface of HIV-1 is the envelope (Env) glycoprotein. Env is a trimer of gp120-gp41 heterodimers. gp120 is involved in receptor/co-receptor binding and gp41 in fusion of viral and cellular membranes. Like many other viral fusion proteins, the gp41 subunit in native, trimeric Env exists in a metastable conformation. gp41 readily forms a stable six helix bundle (6HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers, that drives fusion of viral and cellular membranes. While it is expected that native Env would be a good immunogen, its metastability results in exposure of immunodominant, non-neutralizing epitopes. In the present study, we stabilize native Env trimers by incorporation of a number of different mutations at the NHR:CHR interface that disrupt the post-fusion 6HB of gp41. The stabilized constructs described here can be incorporated in DNA vaccine candidates.

  • Defining the chemokine basis for leukocyte recruitment during viral encephalitis. [PublishAheadOfPrint]

  • The encephalitic response to viral infection requires local chemokine production and the ensuing recruitment of immune and inflammatory leukocytes. Accordingly, chemokine receptors present themselves as plausible therapeutic targets for drugs aimed at limiting encephalitic responses. However, it remains unclear which chemokines are central to this process and whether leukocyte recruitment is important for limiting viral proliferation and survival in the brain or whether it is predominantly a driver of coincident inflammatory pathogenesis. Here we examine chemokine expression and leukocyte recruitment in the context of avirulent, and virulent, Semliki Forest Virus, as well as West Nile Virus, infection and demonstrate rapid and robust expression of a variety of inflammatory CC and CXC-chemokines in all models. On this basis we define a chemokine axis involved in leukocyte recruitment to the encephalitic brain during SFV infection. CXCR3 is most active, CCR2 is also active but less so and CCR5 plays only a modest role in leukocyte recruitment. Importantly, inhibition of each of these receptors individually, and the resulting suppression of leukocyte recruitment to the infected brain, has no effect on viral titre or survival following infection with a virulent SFV strain. In contrast, simultaneous blockade of CXCR3 and CCR2 results in significantly reduced mortality in response to virulent SFV infection. In summary, therefore, our data provide an unprecedented level of insight into chemokine orchestration of leukocyte recruitment in viral encephalitis. Our data also highlight CXCR3 and CCR2 as possible therapeutic targets for limiting inflammatory damage in response to viral infection of the brain.

    Importance Brain inflammation (encephalitis) in response to viral infection can lead to severe illness and even death. This therefore represents an important clinical problem and one that requires the development of new therapeutic approaches. Central to the pathogenesis of encephalitis is the recruitment of inflammatory leukocytes to the infected brain, a process driven by members of the chemokine family. Here we provide an in-depth analysis of the chemokines involved in leukocyte recruitment to the virally infected brain and demonstrate that simultaneous blockade of two of these receptors, namely CXCR3 and CCR2, does not alter viral titres within the brain but markedly reduces inflammatory leukocyte recruitment and enhances survival in a murine model of lethal viral encephalitis. Our results therefore highlight chemokine receptors as plausible therapeutic targets in treating viral encephalitis.

  • Transcriptional and Post-transcriptional Regulation of Cytokine Gene Expression in HIV-1 Antigen-specific CD8+ T Cells that Mediate Virus Inhibition [PublishAheadOfPrint]

  • The ability of CD8+ T cells to effectively limit HIV-1 replication and block HIV-1 acquisition is determined by the capacity to rapidly respond to HIV-1 antigens. Understanding both the functional properties and regulation of an effective CD8+ response would enable better evaluation of T cell directed vaccine strategies and may inform the design of new therapies. We assessed the antigen-specificity, cytokine signature, and mechanisms that regulate antiviral gene expression in CD8+ T cells from a cohort of HIV-1 virus controllers (VCs; llt;2000 HIV-1 RNA copies/ml and CD4+ lymphocyte counts ggt;400 cells/mmu;l) capable of soluble HIV-1 inhibition. Gag p24 and Nef CD8+ T cell specific soluble inhibition was common among the VCs and correlated with substantial increases in the abundance of mRNAs encoding the antiviral cytokines MIP-1aalpha;, MIP-1aalpha;P (CCL3L1), MIP-1bbeta;, GMCSF, XCL1, TNFRSF9 and IFN-. The induction of several of these mRNAs was driven through a coordinated response of both increased transcription and stabilization of mRNA which, together, accounted for the observed increase in mRNA abundance. This coordinated response allows for a rapid and robust induction of mRNA messages that can enhance the CD8+ T cells' ability to inhibit virus upon antigen encounter.

    Importance We show that mRNA stability, in addition to transcription, is key in regulating the direct HIV-1 antiviral function of antigen specific memory CD8+ T cells. Regulation at the level of RNA helps enable rapid recall of memory CD8+ T cells' effector functions for HIV-1 inhibition. By uncovering and understanding the mechanisms employed by CD8+ T cell subsets with antigen specific anti-HIV-1 activity, we can identify new strategies for comprehensive identification of other important antiviral genes. This will, in turn, enhance our ability to inhibit virus replication by informing both cure strategies and HIV-1 vaccine designs that aim to reduce transmission and can aid in blocking HIV-1 acquisition.

  • Down-regulation of IL-18 mediated cell signalling and IFN-{gamma} expression by the hepatitis B virus e antigen. [PublishAheadOfPrint]

  • The mechanisms by which hepatitis B virus (HBV) establishes and maintains chronic hepatitis B infection (CHB) are poorly defined. Innate immune responses play a critical role in reducing HBV replication and pathogenesis. HBV has developed numerous mechanisms to escape these responses including the production of the secreted hepatitis B e antigen (HBeAg), which has been shown to regulate the antiviral toll like receptor (TLR) and interleukin 1 (IL-1) signalling. IL-18 is a related cytokine that inhibits HBV replication in hepatoma cell lines and in the liver through the induction of interferon gamma (IFN-) by NK cells and T cells. We hypothesized that HBV, or HBV proteins inhibits IFN- expression by NK cells as an accessory immunomodulatory function. We show that HBeAg protein inhibits the NFB pathway and thereby down-regulates NK cell IFN- expression. Additionally IFN- expression was significantly inhibited by exposure to serum from individuals with HBeAg-positive, but not HBeAg-negative chronic HBV infection. Further, we show that the HBeAg protein suppresses IL-18 mediated NF-kB signalling in NK and hepatoma cells, via modulation of the NF-kB pathway. Together these findings show that the HBeAg inhibits IL-18 signalling and IFN- expression, which may play an important role in the establishment and/or maintenance of persistent HBV infection.

    Importance It is becoming increasingly apparent that NK cells play a role in the establishment and/or maintenance of chronic hepatitis B infection. The secreted hepatitis B e antigen (HBeAg) is an important regulator of innate and adaptive immune responses. We now show that the HBeAg down-regulates NK cell mediated IFN- production and IL-18 signalling which may contribute to the establishment of infection and/or viral persistence. Our findings build on previous studies showing that the HBeAg also suppresses the toll like receptor (TLR) and interleukin 1 (IL-1) signaling pathways, suggesting that this viral protein is a key regulator of antiviral innate immune responses.

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