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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]

  • Population Bottlenecks and Pathogen Extinction: "Make This Everyone's Mission to Mars, Including Yours" [Commentary]

  • Kapusinszky et al. (J Virol 89:8152nndash;8161, 2015, report that host population bottlenecks may result in pathogen extinction, which provides a compelling argument for an alternative approach to vaccination for the control of virus spread. By comparing the prevalence levels of three viral pathogens in two populations of African green monkeys (AGMs) (Chlorocebus sabaeus) from Africa and two Caribbean Islands, they convincingly show that a major host bottleneck resulted in the eradication of select pathogens from a given host.

  • Dark Matter of the Biosphere: the Amazing World of Bacteriophage Diversity [Gems]

  • Bacteriophages are the most abundant biological entities in the biosphere, and this dynamic and old population is, not surprisingly, highly diverse genetically. Relative to bacterial genomics, phage genomics has advanced slowly, and a higher-resolution picture of the phagosphere is only just emerging. This view reveals substantial diversity even among phages known to infect a common host strain, but the relationships are complex, with mosaic genomic architectures generated by illegitimate recombination over a long period of evolutionary history.

  • Innovations in Undergraduate Science Education: Going Viral [Gems]

  • Bacteriophage discovery and genomics provides a powerful and effective platform for integrating missions in research and education. Implementation of the Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program facilitates a broad impact by including a diverse array of schools, faculty, and students. The program generates new insights into the diversity and evolution of the bacteriophage population and presents a model for introducing first-year undergraduate students to discovery-based research experiences.

  • Marine Snails and Slugs: a Great Place To Look for Antiviral Drugs [Minireviews]

  • Molluscs, comprising one of the most successful phyla, lack clear evidence of adaptive immunity and yet thrive in the oceans, which are rich in viruses. There are thought to be nearly 120,000 species of Mollusca, most living in marine habitats. Despite the extraordinary abundance of viruses in oceans, molluscs often have very long life spans (10 to 100 years). Thus, their innate immunity must be highly effective at countering viral infections. Antiviral compounds are a crucial component of molluscan defenses against viruses and have diverse mechanisms of action against a wide variety of viruses, including many that are human pathogens. Antiviral compounds found in abalone, oyster, mussels, and other cultured molluscs are available in large supply, providing good opportunities for future research and development. However, most members of the phylum Mollusca have not been examined for the presence of antiviral compounds. The enormous diversity and adaptations of molluscs imply a potential source of novel antiviral compounds for future drug discovery.

  • A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication [Genome Replication and Regulation of Viral Gene Expression]

  • The process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting the in vitro data. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis.

    IMPORTANCE Reverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting the in vitro data. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity.

  • Characterization and Implementation of a Diverse Simian Immunodeficiency Virus SIVsm Envelope Panel in the Assessment of Neutralizing Antibody Breadth Elicited in Rhesus Macaques by Multimodal Vaccines Expressing the SIVmac239 Envelope [Vaccines and Antiviral Agents]

  • Antibodies that can neutralize diverse viral strains are likely to be an important component of a protective human immunodeficiency virus type 1 (HIV-1) vaccine. To this end, preclinical simian immunodeficiency virus (SIV)-based nonhuman primate immunization regimens have been designed to evaluate and enhance antibody-mediated protection. However, these trials often rely on a limited selection of SIV strains with extreme neutralization phenotypes to assess vaccine-elicited antibody activity. To mirror the viral panels used to assess HIV-1 antibody breadth, we created and characterized a novel panel of 14 genetically and phenotypically diverse SIVsm envelope (Env) glycoproteins. To assess the utility of this panel, we characterized the neutralizing activity elicited by four SIVmac239 envelope-expressing DNA/modified vaccinia virus Ankara vector- and protein-based vaccination regimens that included the immunomodulatory adjuvants granulocyte-macrophage colony-stimulating factor, Toll-like receptor (TLR) ligands, and CD40 ligand. The SIVsm Env panel exhibited a spectrum of neutralization sensitivity to SIV-infected plasma pools and monoclonal antibodies, allowing categorization into three tiers. Pooled sera from 91 rhesus macaques immunized in the four trials consistently neutralized only the highly sensitive tier 1a SIVsm Envs, regardless of the immunization regimen. The inability of vaccine-mediated antibodies to neutralize the moderately resistant tier 1b and tier 2 SIVsm Envs defined here suggests that those antibodies were directed toward epitopes that are not accessible on most SIVsm Envs. To achieve a broader and more effective neutralization profile in preclinical vaccine studies that is relevant to known features of HIV-1 neutralization, more emphasis should be placed on optimizing the Env immunogen, as the neutralization profile achieved by the addition of adjuvants does not appear to supersede the neutralizing antibody profile determined by the immunogen.

    IMPORTANCE Many in the HIV/AIDS vaccine field believe that the ability to elicit broadly neutralizing antibodies capable of blocking genetically diverse HIV-1 variants is a critical component of a protective vaccine. Various SIV-based nonhuman primate vaccine studies have investigated ways to improve antibody-mediated protection against a heterologous SIV challenge, including administering adjuvants that might stimulate a greater neutralization breadth. Using a novel SIV neutralization panel and samples from four rhesus macaque vaccine trials designed for cross comparison, we show that different regimens expressing the same SIV envelope immunogen consistently elicit antibodies that neutralize only the very sensitive tier 1a SIV variants. The results argue that the neutralizing antibody profile elicited by a vaccine is primarily determined by the envelope immunogen and is not substantially broadened by including adjuvants, resulting in the conclusion that the envelope immunogen itself should be the primary consideration in efforts to elicit antibodies with greater neutralization breadth.

  • Local Virus Extinctions following a Host Population Bottleneck [Genetic Diversity and Evolution]

  • A small number of African green monkeys (AGMs) were introduced into the Caribbean from West Africa in the 1600s. To determine the impact of this population bottleneck on the AGM virome, we used metagenomics to compare the viral nucleic acids in the plasma of 43 wild AGMs from West Africa (Gambia) to those in 44 AGMs from the Caribbean (St. Kitts and Nevis). Three viruses were detected in the blood of Gambian primates: simian immunodeficiency virus (SIVagm; in 42% of animals), a novel simian pegivirus (SPgVagm; in 7% of animals), and numerous novel simian anelloviruses (in 100% of animals). Only anelloviruses were detected in the Caribbean AGMs with a prevalence and levels of viral genetic diversity similar to those in the Gambian animals. A host population bottleneck therefore resulted in the exclusion of adult-acquired SIV and pegivirus from the Caribbean AGMs. The successful importation of AGM anelloviruses into the Caribbean may be the result of their early transmission to infants, very high prevalence in African AGMs, and frequent coinfections with as many as 11 distinct variants.

    IMPORTANCE The extent to which viruses can persist in small isolated populations depends on multiple host, viral, and environmental factors. The absence of prior infections may put an immunologically naive population at risk for disease outbreaks. Isolated populations originating from a small number of founder individuals are therefore considered at increased risk following contact with populations with a greater variety of viruses. Here, we compared the plasma virome of West African green monkeys to that in their descendants after importation of a small number of animals to the Caribbean. A lentivirus and a pegivirus were found in the West African population but not in the Caribbean population. Highly diverse anelloviruses were found in both populations. A small founder population, limited to infants and young juvenile monkeys, may have eliminated the sexually transmitted viruses from the Caribbean AGMs, while anelloviruses, acquired at an earlier age, persisted through the host population bottleneck.

  • Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells [Virus-Cell Interactions]

  • During HIV-1 assembly, the Gag viral proteins are targeted and assemble at the inner leaflet of the cell plasma membrane. This process could modulate the cortical actin cytoskeleton, located underneath the plasma membrane, since actin dynamics are able to promote localized membrane reorganization. In addition, activated small Rho GTPases are known for regulating actin dynamics and membrane remodeling. Therefore, the modulation of such Rho GTPase activity and of F-actin by the Gag protein during virus particle formation was considered. Here, we studied the implication of the main Rac1, Cdc42, and RhoA small GTPases, and some of their effectors, in this process. The effect of small interfering RNA (siRNA)-mediated Rho GTPases and silencing of their effectors on Gag localization, Gag membrane attachment, and virus-like particle production was analyzed by immunofluorescence coupled to confocal microscopy, membrane flotation assays, and immunoblot assays, respectively. In parallel, the effect of Gag expression on the Rac1 activation level was monitored by G-LISA, and the intracellular F-actin content in T cells was monitored by flow cytometry and fluorescence microscopy. Our results revealed the involvement of activated Rac1 and of the IRSp53-Wave2-Arp2/3 signaling pathway in HIV-1 Gag membrane localization and particle release in T cells as well as a role for actin branching and polymerization, and this was solely dependent on the Gag viral protein. In conclusion, our results highlight a new role for the Rac1-IRSp53-Wave2-Arp2/3 signaling pathway in the late steps of HIV-1 replication in CD4 T lymphocytes.

    IMPORTANCE During HIV-1 assembly, the Gag proteins are targeted and assembled at the inner leaflet of the host cell plasma membrane. Gag interacts with specific membrane phospholipids that can also modulate the regulation of cortical actin cytoskeleton dynamics. Actin dynamics can promote localized membrane reorganization and thus can be involved in facilitating Gag assembly and particle formation. Activated small Rho GTPases and effectors are regulators of actin dynamics and membrane remodeling. We thus studied the effects of the Rac1, Cdc42, and RhoA GTPases and their specific effectors on HIV-1 Gag membrane localization and viral particle release in T cells. Our results show that activated Rac1 and the IRSp53-Wave2-Arp2/3 signaling pathway are involved in Gag plasma membrane localization and viral particle production. This work uncovers a role for cortical actin through the activation of Rac1 and the IRSp53/Wave2 signaling pathway in HIV-1 particle formation in CD4 T lymphocytes.

  • The Baculovirus Antiapoptotic p35 Protein Functions as an Inhibitor of the Host RNA Interference Antiviral Response [Cellular Response to Infection]

  • RNA interference (RNAi) is considered an ancient antiviral defense in diverse organisms, including insects. Virus infections generate double-strand RNAs (dsRNAs) that trigger the RNAi machinery to process dsRNAs into virus-derived short interfering RNAs (vsiRNAs), which target virus genomes, mRNAs, or replication intermediates. Viruses, in turn, have evolved viral suppressors of RNAi (VSRs) to counter host antiviral RNAi. Following recent discoveries that insects mount an RNAi response against DNA viruses, in this study, we found that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection similarly induces an RNAi response in Spodoptera frugiperda cells by generating a large number of vsiRNAs postinfection. Interestingly, we found that AcMNPV expresses a potent VSR to counter RNAi. The viral p35 gene, which is well known as an inhibitor of apoptosis, was found to be responsible for the suppression of RNAi in diverse insect and mammalian cells. The VSR activity of p35 was further confirmed by a p35-null AcMNPV that did not suppress the response. In addition, our results showed that the VSR activity is not due to inhibition of dsRNA cleavage by Dicer-2 but acts downstream in the RNAi pathway. Furthermore, we found that the VSR activity is not linked to the antiapoptotic activity of the protein. Overall, our results provide evidence for the existence of VSR activity in a double-stranded DNA virus and identify the responsible gene, which is involved in the inhibition of RNAi as well as apoptosis.

    IMPORTANCE Our findings demonstrate the occurrence of an insect RNAi response against a baculovirus (AcMNPV) that is highly utilized in microbial control, biological and biomedical research, and protein expression. Moreover, our investigations led to the identification of a viral suppressor of RNAi activity and the gene responsible for the activity. Notably, this gene is also a potent inhibitor of apoptosis. The outcomes signify the dual role of a virus-encoded protein in nullifying two key antiviral responses, apoptosis and RNAi.

  • Nonglycosylated G-Protein Vaccine Protects against Homologous and Heterologous Respiratory Syncytial Virus (RSV) Challenge, while Glycosylated G Enhances RSV Lung Pathology and Cytokine Levels [Vaccines and Antiviral Agents]

  • New efforts are under way to develop a vaccine against respiratory syncytial virus (RSV) that will provide protective immunity without the potential for vaccine-associated disease enhancement such as that observed in infants following vaccination with formalin-inactivated RSV vaccine. In addition to the F fusion protein, the G attachment surface protein is a target for neutralizing antibodies and thus represents an important vaccine candidate. However, glycosylated G protein expressed in mammalian cells has been shown to induce pulmonary eosinophilia upon RSV infection in a mouse model. In the current study, we evaluated in parallel the safety and protective efficacy of the RSV A2 recombinant unglycosylated G protein ectodomain (amino acids 67 to 298) expressed in Escherichia coli (REG) and those of glycosylated G produced in mammalian cells (RMG) in a mouse RSV challenge model. Vaccination with REG generated neutralizing antibodies against RSV A2 in 7/11 BALB/c mice, while RMG did not elicit neutralizing antibodies. Total serum binding antibodies against the recombinant proteins (both REG and RMG) were measured by surface plasmon resonance (SPR) and were found to be ggt;10-fold higher for REG- than for RMG-vaccinated animals. Reduction of lung viral loads to undetectable levels after homologous (RSV-A2) and heterologous (RSV-B1) viral challenge was observed in 7/8 animals vaccinated with REG but not in RMG-vaccinated animals. Furthermore, enhanced lung pathology and elevated Th2 cytokines/chemokines were observed exclusively in animals vaccinated with RMG (but not in those vaccinated with REG or phosphate-buffered saline [PBS]) after homologous or heterologous RSV challenge. This study suggests that bacterially produced unglycosylated G protein could be developed alone or as a component of a protective vaccine against RSV disease.

    IMPORTANCE New efforts are under way to develop vaccines against RSV that will provide protective immunity without the potential for disease enhancement. The G attachment protein represents an important candidate for inclusion in an effective RSV vaccine. In the current study, we evaluated the safety and protective efficacy of the RSV A2 recombinant unglycosylated G protein ectodomain produced in E. coli (REG) and those of glycosylated G produced in mammalian cells (RMG) in a mouse RSV challenge model (strains A2 and B1). The unglycosylated G generated high protective immunity and no lung pathology, even in animals that lacked anti-RSV neutralizing antibodies prior to RSV challenge. Control of viral loads correlated with antibody binding to the G protein. In contrast, the glycosylated G protein provided poor protection and enhanced lung pathology after RSV challenge. Therefore, bacterially produced unglycosylated G protein holds promise as an economical approach to a protective vaccine against RSV.

  • Global Diversity within and between Human Herpesvirus 1 and 2 Glycoproteins [Genetic Diversity and Evolution]

  • Human herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) are large-genome DNA viruses that establish a persistent infection in sensory neurons and commonly manifest with recurring oral or genital erosions that transmit virus. HSV encodes 12 predicted glycoproteins that serve various functions, including cellular attachment, entry, and egress. Glycoprotein G is currently the target of an antibody test to differentiate HSV-1 from HSV-2; however, this test has shown reduced capacity to differentiate HSV strains in East Africa. Until the recent availability of 26 full-length HSV-1 and 36 full-length HSV-2 sequences, minimal comparative information was available for these viruses. In this study, we use a variety of sequence analysis methods to compare all available sequence data for HSV-1 and HSV-2 glycoproteins, using viruses isolated in Europe, Asia, North America, the Republic of South Africa, and East Africa. We found numerous differences in diversity, nonsynonymous/synonymous substitution rates, and recombination rates between HSV-1 glycoproteins and their HSV-2 counterparts. Phylogenetic analysis revealed that while most global HSV-2 glycoprotein G sequences did not form clusters within or between continents, one clade (supported at 60.5%) contained 37% of the African sequences analyzed. Accordingly, sequences from this African subset contained unique amino acid signatures, not only in glycoprotein G, but also in glycoproteins I and E, which may account for the failure of sensitive antibody tests to distinguish HSV-1 from HSV-2 in some African individuals. Consensus sequences generated in the study can be used to improve diagnostic assays that differentiate HSV-1 from HSV-2 in global populations.

    IMPORTANCE Human herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) are large DNA viruses associated with recurring oral or genital erosions that transmit virus. Up to 12 HSV-1 and HSV-2 glycoproteins are involved in HSV cell entry or are required for viral spread in animals, albeit some are dispensable for replication in vitro. The recent availability of comparable numbers of full-length HSV-1 and HSV-2 sequences enabled comparative analysis of gene diversity of glycoproteins within and between HSV types. Overall, we found less glycoprotein sequence diversity within HSV-2 than within the HSV-1 strains studied, while at the same time, several HSV-2 glycoproteins were evolving under less selective pressure. Because HSV glycoproteins are the focus of antibody tests to detect and differentiate between infections with the two strains and are constituents of vaccines in clinical-stage development, these findings will aid in refining the targets for diagnostic tests and vaccines.

  • Genome Sequencing and Analysis of Geographically Diverse Clinical Isolates of Herpes Simplex Virus 2 [Genetic Diversity and Evolution]

  • Herpes simplex virus 2 (HSV-2), the principal causative agent of recurrent genital herpes, is a highly prevalent viral infection worldwide. Limited information is available on the amount of genomic DNA variation between HSV-2 strains because only two genomes have been determined, the HG52 laboratory strain and the newly sequenced SD90e low-passage-number clinical isolate strain, each from a different geographical area. In this study, we report the nearly complete genome sequences of 34 HSV-2 low-passage-number and laboratory strains, 14 of which were collected in Uganda, 1 in South Africa, 11 in the United States, and 8 in Japan. Our analyses of these genomes demonstrated remarkable sequence conservation, regardless of geographic origin, with the maximum nucleotide divergence between strains being 0.4% across the genome. In contrast, prior studies indicated that HSV-1 genomes exhibit more sequence diversity, as well as geographical clustering. Additionally, unlike HSV-1, little viral recombination between HSV-2 strains could be substantiated. These results are interpreted in light of HSV-2 evolution, epidemiology, and pathogenesis. Finally, the newly generated sequences more closely resemble the low-passage-number SD90e than HG52, supporting the use of the former as the new reference genome of HSV-2.

    IMPORTANCE Herpes simplex virus 2 (HSV-2) is a causative agent of genital and neonatal herpes. Therefore, knowledge of its DNA genome and genetic variability is central to preventing and treating genital herpes. However, only two full-length HSV-2 genomes have been reported. In this study, we sequenced 34 additional HSV-2 low-passage-number and laboratory viral genomes and initiated analysis of the genetic diversity of HSV-2 strains from around the world. The analysis of these genomes will facilitate research aimed at vaccine development, diagnosis, and the evaluation of clinical manifestations and transmission of HSV-2. This information will also contribute to our understanding of HSV evolution.

  • Discovery of Dengue Virus NS4B Inhibitors [Spotlight]

  • The four serotypes of dengue virus (DENV-1 to -4) represent the most prevalent mosquito-borne viral pathogens in humans. No clinically approved vaccine or antiviral is currently available for DENV. Here we report a spiropyrazolopyridone compound that potently inhibits DENV both in vitro and in vivo. The inhibitor was identified through screening of a 1.8-million-compound library by using a DENV-2 replicon assay. The compound selectively inhibits DENV-2 and -3 (50% effective concentration [EC50], 10 to 80 nM) but not DENV-1 and -4 (EC50, ggt;20 mmu;M). Resistance analysis showed that a mutation at amino acid 63 of DENV-2 NS4B (a nonenzymatic transmembrane protein and a component of the viral replication complex) could confer resistance to compound inhibition. Genetic studies demonstrate that variations at amino acid 63 of viral NS4B are responsible for the selective inhibition of DENV-2 and -3. Medicinal chemistry improved the physicochemical properties of the initial "hit" (compound 1), leading to compound 14a, which has good in vivo pharmacokinetics. Treatment of DENV-2-infected AG129 mice with compound 14a suppressed viremia, even when the treatment started after viral infection. The results have proven the concept that inhibitors of NS4B could potentially be developed for clinical treatment of DENV infection. Compound 14a represents a potential preclinical candidate for treatment of DENV-2- and -3-infected patients.

    IMPORTANCE Dengue virus (DENV) threatens up to 2.5 billion people and is now spreading in many regions in the world where it was not previously endemic. While there are several promising vaccine candidates in clinical trials, approved vaccines or antivirals are not yet available. Here we describe the identification and characterization of a spiropyrazolopyridone as a novel inhibitor of DENV by targeting the viral NS4B protein. The compound potently inhibits two of the four serotypes of DENV (DENV-2 and -3) both in vitro and in vivo. Our results validate, for the first time, that NS4B inhibitors could potentially be developed for antiviral therapy for treatment of DENV infection in humans.

  • Comparative Analysis of the Glycosylation Profiles of Membrane-Anchored HIV-1 Envelope Glycoprotein Trimers and Soluble gp140 [Vaccines and Antiviral Agents]

  • The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer, which consists of the gp120 and gp41 subunits, is the focus of multiple strategies for vaccine development. Extensive Env glycosylation provides HIV-1 with protection from the immune system, yet the glycans are also essential components of binding epitopes for numerous broadly neutralizing antibodies. Recent studies have shown that when Env is isolated from virions, its glycosylation profile differs significantly from that of soluble forms of Env (gp120 or gp140) predominantly used in vaccine discovery research. Here we show that exogenous membrane-anchored Envs, which can be produced in large quantities in mammalian cells, also display a virion-like glycan profile, where the glycoprotein is extensively decorated with high-mannose glycans. Additionally, because we characterized the glycosylation with a high-fidelity profiling method, glycopeptide analysis, an unprecedented level of molecular detail regarding membrane Env glycosylation and its heterogeneity is presented. Each glycosylation site was characterized individually, with about 500 glycoforms characterized per Env protein. While many of the sites contain exclusively high-mannose glycans, others retain complex glycans, resulting in a glycan profile that cannot currently be mimicked on soluble gp120 or gp140 preparations. These site-level studies are important for understanding antibody-glycan interactions on native Env trimers. Additionally, we report a newly observed O-linked glycosylation site, T606, and we show that the full O-linked glycosylation profile of membrane-associated Env is similar to that of soluble gp140. These findings provide new insight into Env glycosylation and clarify key molecular-level differences between membrane-anchored Env and soluble gp140.

    IMPORTANCE A vaccine that protects against human immunodeficiency virus type 1 (HIV-1) infection should elicit antibodies that bind to the surface envelope glycoproteins on the membrane of the virus. The envelope glycoproteins have an extensive coat of carbohydrates (glycans), some of which are recognized by virus-neutralizing antibodies and some of which protect the virus from neutralizing antibodies. We found that the HIV-1 membrane envelope glycoproteins have a unique pattern of carbohydrates, with many high-mannose glycans and also, in some places, complex glycans. This pattern was very different from the carbohydrate profile seen for a more easily produced soluble version of the envelope glycoprotein. Our results provide a detailed characterization of the glycans on the natural membrane envelope glycoproteins of HIV-1, a carbohydrate profile that would be desirable to mimic with a vaccine.

  • Functional Analysis of the 60-Nucleotide Duplication in the Respiratory Syncytial Virus Buenos Aires Strain Attachment Glycoprotein [Virus-Cell Interactions]

  • There are two subgroups of respiratory syncytial virus (RSV), A and B, and within each subgroup, isolates are further divided into clades. Several years ago, multiple subgroup B isolates which contained a duplication of 60 nucleotides in the glycoprotein (G) gene were described. These isolates were given a new clade designation of BA based on the site of isolation, Buenos Aires, Argentina. BA RSV strains have since become the predominant circulating clade of RSV B viruses. We hypothesized that the duplicated region in G serves to enhance the function of G in the virus life cycle. We generated recombinant viruses that express a consensus BA G gene or a consensus BA G gene lacking the duplication (Gdup). We determined that the duplicated region functions during virus attachment to cells. Additionally, we showed that in vitro, the virus containing the duplication has a fitness advantage compared to the virus without the duplication. Our data demonstrate that the duplicated region in the BA strain G protein augments virus attachment and fitness.

    IMPORTANCE Respiratory syncytial virus (RSV) is an important pathogen for infants for which there is no vaccine. Different strains of RSV circulate from year to year, and the predominating strains change over time. Subgroup B RSV strains with a duplication in the attachment glycoprotein (G) emerged and then became the dominant B genotype. We found that a recombinant virus harboring the duplication bound more efficiently to cells and was more fit than a recombinant strain lacking the duplication. Our work advances a mechanism for an important natural RSV mutation.

  • Robust Protection against Highly Virulent Foot-and-Mouth Disease Virus in Swine by Combination Treatment with Recombinant Adenoviruses Expressing Porcine Alpha and Gamma Interferons and Multiple Small Interfering RNAs [Vaccines and Antiviral Agents]

  • Because the currently available vaccines against foot-and-mouth disease (FMD) provide no protection until 4 to 7 days postvaccination, the only alternative method to halt the spread of the FMD virus (FMDV) during outbreaks is the application of antiviral agents. Combination treatment strategies have been used to enhance the efficacy of antiviral agents, and such strategies may be advantageous in overcoming viral mechanisms of resistance to antiviral treatments. We have developed recombinant adenoviruses (Ads) for the simultaneous expression of porcine alpha and gamma interferons (Ad-porcine IFN-aalpha;) as well as 3 small interfering RNAs (Ad-3siRNA) targeting FMDV mRNAs encoding nonstructural proteins. The antiviral effects of Ad-porcine IFN-aalpha; and Ad-3siRNA expression were tested in combination in porcine cells, suckling mice, and swine. We observed enhanced antiviral effects in porcine cells and mice as well as robust protection against the highly pathogenic strain O/Andong/SKR/2010 and increased expression of cytokines in swine following combination treatment. In addition, we showed that combination treatment was effective against all serotypes of FMDV. Therefore, we suggest that the combined treatment with Ad-porcine IFN-aalpha; and Ad-3siRNA may offer fast-acting antiviral protection and be used with a vaccine during the period that the vaccine does not provide protection against FMD.

    IMPORTANCE The use of current foot-and-mouth disease (FMD) vaccines to induce rapid protection provides limited effectiveness because the protection does not become effective until a minimum of 4 days after vaccination. Therefore, during outbreaks antiviral agents remain the only available treatment to confer rapid protection and reduce the spread of foot-and-mouth disease virus (FMDV) in livestock until vaccine-induced protective immunity can become effective. Interferons (IFNs) and small interfering RNAs (siRNAs) have been reported to be effective antiviral agents against FMDV, although the virus has associated mechanisms of resistance to type I interferons and siRNAs. We have developed recombinant adenoviruses for the simultaneous expression of porcine alpha and gamma interferons (Ad-porcine IFN-aalpha;) as well as 3 small interfering RNAs (Ad-3siRNA) to enhance the inhibitory effects of these antiviral agents observed in previous studies. Here, we show enhanced antiviral effects against FMDV by combination treatment with Ad-porcine IFN-aalpha; and Ad-3siRNA to overcome the mechanisms of resistance of FMDV in swine.

  • Distinct Immune Responses in Resistant and Susceptible Strains of Mice during Neurovirulent Alphavirus Encephalomyelitis [Pathogenesis and Immunity]

  • Susceptibility to alphavirus encephalomyelitis is dependent on a variety of factors, including the genetic background of the host. Neuroadapted Sindbis virus (NSV) causes uniformly fatal disease in adult C57BL/6 (B6) mice, but adult BALB/c (Bc) mice recover from infection. In B6 mice, fatal encephalomyelitis is immune mediated rather than a direct result of virus infection. To identify the immunological determinants of host susceptibility to fatal NSV-induced encephalomyelitis, we compared virus titers and immune responses in adult B6 and Bc mice infected intranasally with NSV. B6 mice had higher levels of virus replication, higher levels of type I interferon (IFN), and slower virus clearance than did Bc mice. B6 mice had more neuronal apoptosis, more severe neurologic disease, and higher mortality than Bc mice. B6 mice had more infiltration of inflammatory cells and higher levels of IL1b, IL-6, TNFa, Csf2, and CCL2 mRNAs and interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-aalpha;), IFN-, and C-C motif ligand 2 (CCL2) protein in brains than Bc mice. However, Bc mice had more brain antibody at day 7 and a higher percentage of CD4+ T cells. CD4+ T cells in the brains of Bc mice included fewer Th17 cells and more regulatory T cells (Tregs) producing IL-10 than B6 mice, accompanied by higher levels of Il2 and Cxcl10 mRNAs. In the absence of IL-10, resistant Bc mice became susceptible to fatal encephalomyelitis after NSV infection. These studies demonstrate the importance of the immune response and its regulation in determining host survival during alphavirus encephalomyelitis.

    IMPORTANCE Mosquito-borne alphavirus infections are an important cause of encephalomyelitis in humans. The severity of disease is dependent both on the strain of the virus and on the age and genetic background of the host. A neurovirulent strain of Sindbis virus causes immune-mediated fatal encephalomyelitis in adult C57BL/6 mice but not in BALB/c mice. To determine the host-dependent immunological mechanisms underlying the differences in susceptibility between these two strains of mice, we compared their immune responses to infection. Resistance to fatal disease in BALB/c mice was associated with better antibody responses, more-rapid virus clearance, fewer Th17 cells, and more-potent regulatory T cell responses than occurred in susceptible C57BL/6 mice. In the absence of interleukin-10, a component of the regulatory immune response, resistant mice became susceptible to lethal disease. This study demonstrates the importance of the immune response and its regulation for host survival during alphavirus encephalomyelitis.

  • mRNA Capping by Venezuelan Equine Encephalitis Virus nsP1: Functional Characterization and Implications for Antiviral Research [Genome Replication and Regulation of Viral Gene Expression]

  • Alphaviruses are known to possess a unique viral mRNA capping mechanism involving the viral nonstructural protein nsP1. This enzyme harbors methyltransferase (MTase) and nsP1 guanylylation (GT) activities catalyzing the transfer of the methyl group from S-adenosylmethionine (AdoMet) to the N7 position of a GTP molecule followed by the formation of an m7GMP-nsP1 adduct. Subsequent transfer of m7GMP onto the 5' end of the viral mRNA has not been demonstrated in vitro yet. Here we report the biochemical characterization of Venezuelan equine encephalitis virus (VEEV) nsP1. We have developed enzymatic assays uncoupling the different reactions steps catalyzed by nsP1. The MTase and GT reaction activities were followed using a nonhydrolyzable GTP (GIDP) substrate and an original Western blot assay using anti-m3G/m7G-cap monoclonal antibody, respectively. The GT reaction is stimulated by S-adenosyl-l-homocysteine (Ado-Hcy), the product of the preceding MTase reaction, and metallic ions. The covalent linking between nsP1 and m7GMP involves a phosphamide bond between the nucleotide and a histidine residue. Final guanylyltransfer onto RNA was observed for the first time with an alphavirus nsP1 using a 5'-diphosphate RNA oligonucleotide whose sequence corresponds to the 5' end of the viral genome. Alanine scanning mutagenesis of residues H37, H45, D63, E118, Y285, D354, R365, N369, and N375 revealed their respective roles in MT and GT reactions. Finally, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA), and ribavirin triphosphate on MTase and capping reactions were investigated, providing possible avenues for antiviral research.

    IMPORTANCE Emergence or reemergence of alphaviruses represents a serious health concern, and the elucidation of their replication mechanisms is a prerequisite for the development of specific inhibitors targeting viral enzymes. In particular, alphaviruses are able, through an original reaction sequence, to add to their mRNA a cap required for their protection against cellular nucleases and initiation of viral proteins translation. In this study, the capping of a 5' diphosphate synthetic RNA mimicking the 5' end of an alphavirus mRNA was observed in vitro for the first time. The different steps for this capping are performed by the nonstructural protein 1 (nsP1). Reference compounds known to target the viral capping inhibited nsP1 enzymatic functions, highlighting the value of this enzyme in antiviral development.

  • Frequency, Private Specificity, and Cross-Reactivity of Preexisting Hepatitis C Virus (HCV)-Specific CD8+ T Cells in HCV-Seronegative Individuals: Implications for Vaccine Responses [Pathogenesis and Immunity]

  • T cell responses play a critical role in controlling or clearing viruses. Therefore, strategies to prevent or treat infections include boosting T cell responses. T cells specific for various pathogens have been reported in unexposed individuals and an influence of such cells on the response toward vaccines is conceivable. However, little is known about their frequency, repertoire, and impact on vaccination. We performed a detailed characterization of CD8+ T cells specific to a hepatitis C virus (HCV) epitope (NS3-1073) in 121 HCV-seronegative individuals. We show that in vitro HCV NS3-1073-specific CD8+ T cell responses were rather abundantly detectable in one-third of HCV-seronegative individuals irrespective of risk factors for HCV exposure. Ex vivo, these NS3-1073-specific CD8+ T cells were found to be both naive and memory cells. Importantly, recognition of various peptides derived from unrelated viruses by NS3-1073-specific CD8+ T cells showed a considerable degree of T cell cross-reactivity, suggesting that they might in part originate from previous heterologous infections. Finally, we further provide evidence that preexisting NS3-1073-specific CD8+ T cells can impact the T cell response toward peptide vaccination. Healthy, vaccinated individuals who showed an in vitro response toward NS3-1073 already before vaccination displayed a more vigorous and earlier response toward the vaccine.

    IMPORTANCE Preventive and therapeutic vaccines are being developed for many viral infections and often aim on inducing T cell responses. Despite effective antiviral drugs against HCV, there is still a need for a preventive vaccine. However, the responses to vaccines can be highly variable among different individuals. Preexisting T cells in unexposed individuals could be one reason that helps to explain the variable T cell responses to vaccines. Based on our findings, we suggest that HCV CD8+ T cells are abundant in HCV-seronegative individuals but that their repertoire is highly diverse due to the involvement of both naive precursors and cross-reactive memory cells of different specificities, which can influence the response to vaccines. The data may emphasize the need to personalize immune-based therapies based on the individual's T cell repertoire that is present before the immune intervention.

  • A Kinome-Wide Small Interfering RNA Screen Identifies Proviral and Antiviral Host Factors in Severe Acute Respiratory Syndrome Coronavirus Replication, Including Double-Stranded RNA-Activated Protein Kinase and Early Secretory Pathway Proteins [Virus-Cell Interactions]

  • To identify host factors relevant for severe acute respiratory syndrome-coronavirus (SARS-CoV) replication, we performed a small interfering RNA (siRNA) library screen targeting the human kinome. Protein kinases are key regulators of many cellular functions, and the systematic knockdown of their expression should provide a broad perspective on factors and pathways promoting or antagonizing coronavirus replication. In addition to 40 proteins that promote SARS-CoV replication, our study identified 90 factors exhibiting an antiviral effect. Pathway analysis grouped subsets of these factors in specific cellular processes, including the innate immune response and the metabolism of complex lipids, which appear to play a role in SARS-CoV infection. Several factors were selected for in-depth validation in follow-up experiments. In cells depleted for the bbeta;2 subunit of the coatomer protein complex (COPB2), the strongest proviral hit, we observed reduced SARS-CoV protein expression and a ggt;2-log reduction in virus yield. Knockdown of the COPB2-related proteins COPB1 and Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) also suggested that COPI-coated vesicles and/or the early secretory pathway are important for SARS-CoV replication. Depletion of the antiviral double-stranded RNA-activated protein kinase (PKR) enhanced virus replication in the primary screen, and validation experiments confirmed increased SARS-CoV protein expression and virus production upon PKR depletion. In addition, cyclin-dependent kinase 6 (CDK6) was identified as a novel antiviral host factor in SARS-CoV replication. The inventory of pro- and antiviral host factors and pathways described here substantiates and expands our understanding of SARS-CoV replication and may contribute to the identification of novel targets for antiviral therapy.

    IMPORTANCE Replication of all viruses, including SARS-CoV, depends on and is influenced by cellular pathways. Although substantial progress has been made in dissecting the coronavirus replicative cycle, our understanding of the host factors that stimulate (proviral factors) or restrict (antiviral factors) infection remains far from complete. To study the role of host proteins in SARS-CoV infection, we set out to systematically identify kinase-regulated processes that influence virus replication. Protein kinases are key regulators in signal transduction, controlling a wide variety of cellular processes, and many of them are targets of approved drugs and other compounds. Our screen identified a variety of hits and will form the basis for more detailed follow-up studies that should contribute to a better understanding of SARS-CoV replication and coronavirus-host interactions in general. The identified factors could be interesting targets for the development of host-directed antiviral therapy to treat infections with SARS-CoV or other pathogenic coronaviruses.

  • Broadly Neutralizing Human Immunodeficiency Virus Type 1 Antibody Gene Transfer Protects Nonhuman Primates from Mucosal Simian-Human Immunodeficiency Virus Infection [Spotlight]

  • Broadly neutralizing antibodies (bnAbs) can prevent lentiviral infection in nonhuman primates and may slow the spread of human immunodeficiency virus type 1 (HIV-1). Although protection by passive transfer of human bnAbs has been demonstrated in monkeys, durable expression is essential for its broader use in humans. Gene-based expression of bnAbs provides a potential solution to this problem, although immune responses to the viral vector or to the antibody may limit its durability and efficacy. Here, we delivered an adeno-associated viral vector encoding a simianized form of a CD4bs bnAb, VRC07, and evaluated its immunogenicity and protective efficacy. The expressed antibody circulated in macaques for 16 weeks at levels up to 66 mmu;g/ml, although immune suppression with cyclosporine (CsA) was needed to sustain expression. Gene-delivered simian VRC07 protected against simian-human immunodeficiency virus (SHIV) infection in monkeys 5.5 weeks after treatment. Gene transfer of an anti-HIV antibody can therefore protect against infection by viruses that cause AIDS in primates when the host immune responses are controlled.

    IMPORTANCE Sustained interventions that can prevent HIV-1 infection are needed to halt the spread of the HIV-1 pandemic. The protective capacity of anti-HIV antibody gene therapy has been established in mouse models of HIV-1 infection but has not been established for primates. We show here a proof-of-concept that gene transfer of anti-HIV antibody genes can protect against infection by viruses that cause AIDS in primates when host immune responses are controlled.

  • New Insights into the Understanding of Hepatitis C Virus Entry and Cell-to-Cell Transmission by Using the Ionophore Monensin A [Virus-Cell Interactions]

  • In our study, we characterized the effect of monensin, an ionophore that is known to raise the intracellular pH, on the hepatitis C virus (HCV) life cycle. We showed that monensin inhibits HCV entry in a pangenotypic and dose-dependent manner. Monensin induces an alkalization of intracellular organelles, leading to an inhibition of the fusion step between viral and cellular membranes. Interestingly, we demonstrated that HCV cell-to-cell transmission is dependent on the vesicular pH. Using the selective pressure of monensin, we selected a monensin-resistant virus which has evolved to use a new entry route that is partially pH and clathrin independent. Characterization of this mutant led to the identification of two mutations in envelope proteins, the Y297H mutation in E1 and the I399T mutation in hypervariable region 1 (HVR1) of E2, which confer resistance to monensin and thus allow HCV to use a pH-independent entry route. Interestingly, the I399T mutation introduces an N-glycosylation site within HVR1 and increases the density of virions and their sensitivity to neutralization with anti-apolipoprotein E (anti-ApoE) antibodies, suggesting that this mutation likely induces conformational changes in HVR1 that in turn modulate the association with ApoE. Strikingly, the I399T mutation dramatically reduces HCV cell-to-cell spread. In summary, we identified a mutation in HVR1 that overcomes the vesicular pH dependence, modifies the biophysical properties of particles, and drastically reduces cell-to-cell transmission, indicating that the regulation by HVR1 of particle association with ApoE might control the pH dependence of cell-free and cell-to-cell transmission. Thus, HVR1 and ApoE are critical regulators of HCV propagation.

    IMPORTANCE Although several cell surface proteins have been identified as entry factors for hepatitis C virus (HCV), the precise mechanisms regulating its transmission to hepatic cells are still unclear. In our study, we used monensin A, an ionophore that is known to raise the intracellular pH, and demonstrated that cell-free and cell-to-cell transmission pathways are both pH-dependent processes. We generated monensin-resistant viruses that displayed different entry routes and biophysical properties. Thanks to these mutants, we highlighted the importance of hypervariable region 1 (HVR1) of the E2 envelope protein for the association of particles with apolipoprotein E, which in turn might control the pH dependency of cell-free and cell-to-cell transmission.

  • Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22 [Virus-Cell Interactions]

  • Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm.

    IMPORTANCE Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex executes endosomal membrane fission and cargo sorting to the Rab11-positive and Rab22-positive recycling pathway, resulting in membrane fusion and virus core uncoating in the cytoplasm.

  • Herpes Simplex Virus 1 Reactivates from Autonomic Ciliary Ganglia Independently from Sensory Trigeminal Ganglia To Cause Recurrent Ocular Disease [Spotlight]

  • Herpes simplex virus 1 (HSV-1) and HSV-2 establish latency in sensory and autonomic neurons after ocular or genital infection, but their recurrence patterns differ. HSV-1 reactivates from latency to cause recurrent orofacial disease, and while HSV-1 also causes genital lesions, HSV-2 recurs more efficiently in the genital region and rarely causes ocular disease. The mechanisms regulating these anatomical preferences are unclear. To determine whether differences in latent infection and reactivation in autonomic ganglia contribute to differences in HSV-1 and HSV-2 anatomical preferences for recurrent disease, we compared HSV-1 and HSV-2 clinical disease, acute and latent viral loads, and viral gene expression in sensory trigeminal and autonomic superior cervical and ciliary ganglia in a guinea pig ocular infection model. HSV-2 produced more severe acute disease, correlating with higher viral DNA loads in sensory and autonomic ganglia, as well as higher levels of thymidine kinase expression, a marker of productive infection, in autonomic ganglia. HSV-1 reactivated in ciliary ganglia, independently from trigeminal ganglia, to cause more frequent recurrent symptoms, while HSV-2 replicated simultaneously in autonomic and sensory ganglia to cause more persistent disease. While both HSV-1 and HSV-2 expressed the latency-associated transcript (LAT) in the trigeminal and superior cervical ganglia, only HSV-1 expressed LAT in ciliary ganglia, suggesting that HSV-2 is not reactivation competent or does not fully establish latency in ciliary ganglia. Thus, differences in replication and viral gene expression in autonomic ganglia may contribute to differences in HSV-1 and HSV-2 acute and recurrent clinical disease.

    IMPORTANCE Herpes simplex virus 1 (HSV-1) and HSV-2 establish latent infections, from which the viruses reactivate to cause recurrent disease throughout the life of the host. However, the viruses exhibit different manifestations and frequencies of recurrent disease. HSV-1 and HSV-2 establish latency in both sensory and autonomic ganglia. Autonomic ganglia are more responsive than sensory ganglia to stimuli associated with recurrent disease in humans, such as stress and hormone fluctuations, suggesting that autonomic ganglia may play an important role in recurrent disease. We show that HSV-1 can reactivate from autonomic ganglia, independently from sensory ganglia, to cause recurrent ocular disease. We found no evidence that HSV-2 could reactivate from autonomic ganglia independently from sensory ganglia after ocular infection, but HSV-2 did replicate in both ganglia simultaneously to cause persistent disease. Thus, viral replication and reactivation in autonomic ganglia contribute to different clinical disease manifestations of HSV-1 and HSV-2 after ocular infection.

  • H3K27 Demethylation at the Proviral Promoter Sensitizes Latent HIV to the Effects of Vorinostat in Ex Vivo Cultures of Resting CD4+ T Cells [Virus-Cell Interactions]

  • Histone methyltransferase inhibitors (HMTis) and histone deacetylase inhibitors (HDACis) are reported to synergistically induce the expression of latent human immunodeficiency virus type 1 (HIV-1), but studies have largely been performed with cell lines. As specific and potent HMTis directed at EZH1 (enhancer of zeste 2 Polycomb repressive complex 2 subunit 1)/EZH2 are now in human testing, we wished to rigorously test such an inhibitor in a primary resting T-cell model of HIV latency. We found that GSK343, a potent and selective EZH2/EZH1 inhibitor, reduced trimethylation of histone 3 at lysine 27 (H3K27) of the HIV provirus in resting cells. Remarkably, this epigenetic change was not associated with increased proviral expression in latently infected resting cells. However, following the reduction in H3K27 at the HIV long terminal repeat (LTR), subsequent exposure to the HDACi suberoylanilide hydroxamic acid or vorinostat (VOR) resulted in increases in HIV gag RNA and HIV p24 antigen production that were up to 2.5-fold greater than those induced by VOR alone. Therefore, in primary resting CD4+ T cells, true mechanistic synergy in the reversal of HIV latency may be achieved by the combination of HMTis and HDACis. Although other cellular effects of EZH2 inhibition may contribute to the sensitization of the HIV LTR to subsequent exposure to VOR, and to increase viral antigen production, this synergistic effect is directly associated with H3K27 demethylation at nucleosome 1 (Nuc-1). Based upon our findings, the combination of HMTis and HDACis should be considered for testing in animal models or clinical trials.

    IMPORTANCE Demethylation of H3K27 mediated by the histone methyltransferase inhibitor GSK343 in primary resting T cells is slow, occurring over 96 h, but by itself does not result in a significant upregulation of cell-associated HIV RNA expression or viral antigen production. However, following H3K27 demethylation, latent viral expression within infected primary resting CD4+ T cells is synergistically increased upon exposure to the histone deacetylase inhibitor vorinostat. Demethylation at H3K27 sensitizes the HIV promoter to the effects of an HDACi and provides a proof-of-concept for the testing of combination epigenetic approaches to disrupt latent HIV infection, a necessary step toward the eradication of HIV infection.

  • Poxvirus Protein MC132 from Molluscum Contagiosum Virus Inhibits NF-{kappa}B Activation by Targeting p65 for Degradation [Spotlight]

  • Molluscum contagiosum virus (MCV) is unique in being the only known extant, human-adapted poxvirus, yet to date, it is very poorly characterized in terms of host-pathogen interactions. MCV causes persistent skin lesions filled with live virus, but these are generally immunologically silent, suggesting the presence of potent inhibitors of human antiviral immunity and inflammation. Fewer than five MCV immunomodulatory genes have been characterized in detail, but it is likely that many more remain to be discovered given the density of such sequences in all well-characterized poxviruses. Following virus infection, NF-B activation occurs in response to both pattern recognition receptor (PRR) signaling and cellular activation by virus-elicited proinflammatory cytokines, such as tumor necrosis factor (TNF). As such, NF-B activation is required for virus detection, antiviral signaling, inflammation, and clearance of viral infection. Hence, we screened a library of MCV genes for effects on TNF-stimulated NF-B activation. This revealed MC132, a unique protein with no orthologs in other poxviral genomes, as a novel inhibitor of NF-B. Interestingly, MC132 also inhibited PRR- and virus-activated NF-B, since MC132 interacted with the NF-B subunit p65 and caused p65 degradation. Unbiased affinity purification to identify host targets of MC132 revealed that MC132 acted by targeting NF-B p65 for ubiquitin-dependent proteasomal degradation by recruiting p65 to a host Cullin-5/Elongin B/Elongin C complex. These data reveal a novel mechanism for poxviral inhibition of human innate immunity and further clarify how the human-adapted poxvirus MCV can so effectively evade antiviral immunity to persist in skin lesions.

    IMPORTANCE How human cells detect and respond to viruses is incompletely understood, but great leaps in our understanding have been made by studying both the early innate immune response and the ways that viruses evade it. Poxviruses adapt to specific hosts over time by evolving elegantly precise inhibitors targeting the rate-limiting steps of immunity. These inhibitors reveal new features of the antiviral response while also offering potent new tools for approaching therapeutic intervention in autoimmunity. Molluscum contagiosum virus (MCV) is the only known extant poxvirus specifically adapted to human infection, yet it remains poorly characterized. In this study, we report the identification of the MCV protein MC132 as a potent inhibitor of NF-B, an essential regulatory crux of innate immunity. Furthermore, identification of the mechanism of inhibition of NF-B by MC132 reveals an elegant example of convergent evolution with human herpesviruses. This discovery greatly expands our understanding of how MCV so effectively evades human immunity.

  • Coronavirus nsp10/nsp16 Methyltransferase Can Be Targeted by nsp10-Derived Peptide In Vitro and In Vivo To Reduce Replication and Pathogenesis [Vaccines and Antiviral Agents]

  • The 5' cap structures of eukaryotic mRNAs are important for RNA stability and protein translation. Many viruses that replicate in the cytoplasm of eukaryotes have evolved 2'-O-methyltransferases (2'-O-MTase) to autonomously modify their mRNAs and carry a cap-1 structure (m7GpppNm) at the 5' end, thereby facilitating viral replication and escaping innate immune recognition in host cells. Previous studies showed that the 2'-O-MTase activity of severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural protein 16 (nsp16) needs to be activated by nsp10, whereas nsp16 of feline coronavirus (FCoV) alone possesses 2'-O-MTase activity (E. Decroly et al., J Virol 82:8071nndash;8084, 2008,; M. Bouvet et al., PLoS Pathog 6:e1000863, 2010,; E. Decroly et al., PLoS Pathog 7:e1002059, 2011,; Y. Chen et al., PLoS Pathog 7:e1002294, 2011, . In this study, we demonstrate that stimulation of nsp16 2'-O-MTase activity by nsp10 is a universal and conserved mechanism in coronaviruses, including FCoV, and that nsp10 is functionally interchangeable in the stimulation of nsp16 of different coronaviruses. Based on our current and previous studies, we designed a peptide (TP29) from the sequence of the interaction interface of mouse hepatitis virus (MHV) nsp10 and demonstrated that the peptide inhibits the 2'-O-MTase activity of different coronaviruses in biochemical assays and the viral replication in MHV infection and SARS-CoV replicon models. Interestingly, the peptide TP29 exerted robust inhibitory effects in vivo in MHV-infected mice by impairing MHV virulence and pathogenesis through suppressing virus replication and enhancing type I interferon production at an early stage of infection. Therefore, as a proof of principle, the current results indicate that coronavirus 2'-O-MTase activity can be targeted in vitro and in vivo.

    IMPORTANCE Coronaviruses are important pathogens of animals and human with high zoonotic potential. SARS-CoV encodes the 2'-O-MTase that is composed of the catalytic subunit nsp16 and the stimulatory subunit nsp10 and plays an important role in virus genome replication and evasion from innate immunity. Our current results demonstrate that stimulation of nsp16 2'-O-MTase activity by nsp10 is a common mechanism for coronaviruses, and nsp10 is functionally interchangeable in the stimulation of nsp16 among different coronaviruses, which underlies the rationale for developing inhibitory peptides. We demonstrate that a peptide derived from the nsp16-interacting domain of MHV nsp10 could inhibit 2'-O-MTase activity of different coronaviruses in vitro and viral replication of MHV and SARS-CoV replicon in cell culture, and it could strongly inhibit virus replication and pathogenesis in MHV-infected mice. This work makes it possible to develop broad-spectrum peptide inhibitors by targeting the nsp16/nsp10 2'-O-MTase of coronaviruses.

  • Novel Arenavirus Entry Inhibitors Discovered by Using a Minigenome Rescue System for High-Throughput Drug Screening [Vaccines and Antiviral Agents]

  • Certain members of the Arenaviridae family are category A agents capable of causing severe hemorrhagic fevers in humans. Specific antiviral treatments do not exist, and the only commonly used drug, ribavirin, has limited efficacy and can cause severe side effects. The discovery and development of new antivirals are inhibited by the biohazardous nature of the viruses, making them a relatively poorly understood group of human pathogens. We therefore adapted a reverse-genetics minigenome (MG) rescue system based on Junin virus, the causative agent of Argentine hemorrhagic fever, for high-throughput screening (HTS). The MG rescue system recapitulates all stages of the virus life cycle and enables screening of small-molecule libraries under biosafety containment level 2 (BSL2) conditions. The HTS resulted in the identification of four candidate compounds with potent activity against a broad panel of arenaviruses, three of which were completely novel. The target for all 4 compounds was the stage of viral entry, which positions the compounds as potentially important leads for future development.

    IMPORTANCE The arenavirus family includes several members that are highly pathogenic, causing acute viral hemorrhagic fevers with high mortality rates. No specific effective treatments exist, and although a vaccine is available for Junin virus, the causative agent of Argentine hemorrhagic fever, it is licensed for use only in areas where Argentine hemorrhagic fever is endemic. For these reasons, it is important to identify specific compounds that could be developed as antivirals against these deadly viruses.

  • The Torsin Activator LULL1 Is Required for Efficient Growth of Herpes Simplex Virus 1 [Cellular Response to Infection]

  • TorsinA is a membrane-tethered AAA+ ATPase implicated in nuclear envelope dynamics as well as the nuclear egress of herpes simplex virus 1 (HSV-1). The activity of TorsinA and the related ATPase TorsinB strictly depends on LAP1 and LULL1, type II transmembrane proteins that are integral parts of the Torsin/cofactor AAA ring, forming a composite, membrane-spanning assembly. Here, we use CRISPR/Cas9-mediated genome engineering to create single- and double knockout (KO) cell lines of TorA and TorB as well as their activators, LAP1 and LULL1, to investigate the effect on HSV-1 production. Consistent with LULL1 being the more potent Torsin activator, a LULL1 KO reduces HSV-1 growth by one order of magnitude, while the deletion of other components of the Torsin system in combination causes subtle defects. Notably, LULL1 deficiency leads to a 10-fold decrease in the number of viral genomes per host cell without affecting viral protein production, allowing us to tentatively assign LULL1 to an unexpected role that precedes HSV-1 nuclear egress.

    IMPORTANCE In this study, we conduct the first comprehensive genetic and phenotypic analysis of the Torsin/cofactor system in the context of HSV-1 infection, establishing LULL1 as the most important component of the Torsin system with respect to viral production.

  • Influenza A Virus Coinfection through Transmission Can Support High Levels of Reassortment [Genetic Diversity and Evolution]

  • The reassortment of gene segments between influenza viruses increases genomic diversity and plays an important role in viral evolution. We have shown previously that this process is highly efficient within a coinfected cell and, given synchronous coinfection at moderate or high doses, can give rise to ~60 to 70% of progeny shed from an animal host. Conversely, reassortment in vivo can be rendered undetectable by lowering viral doses or extending the time between infections. One might also predict that seeding of transmitted viruses into different sites within the target tissue could limit subsequent reassortment. Given the potential for stochastic factors to restrict reassortment during natural infection, we sought to determine its efficiency in a host coinfected through transmission. Two scenarios were tested in a guinea pig model, using influenza A/Panama/2007/99 (H3N2) virus (wt) and a silently mutated variant (var) thereof as parental virus strains. In the first, coinfection was achieved by exposing a naive guinea pig to two cagemates, one infected with wt and the other with var virus. When such exposure led to coinfection, robust reassortment was typically seen, with 50 to 100% of isolates carrying reassortant genomes at one or more time points. In the second scenario, naive guinea pigs were exposed to a cagemate that had been coinoculated with wt and var viruses. Here, reassortment occurred in the coinoculated donor host, multiple variants were transmitted, and reassortants were prevalent in the recipient host. Together, these results demonstrate the immense potential for reassortment to generate viral diversity in nature.

    IMPORTANCE Influenza viruses evolve rapidly under selection due to the generation of viral diversity through two mechanisms. The first is the introduction of random errors into the genome by the viral polymerase, which occurs with a frequency of approximately 10nndash;5 errors/nucleotide replicated. The second is reassortment, or the exchange of gene segments between viruses. Reassortment is known to occur readily under well-controlled laboratory conditions, but its frequency in nature is not clear. Here, we tested the hypothesis that reassortment efficiency following coinfection through transmission would be reduced compared to that seen with coinoculation. Contrary to this hypothesis, our results indicate that coinfection achieved through transmission supports high levels of reassortment. These results suggest that reassortment is not exquisitely sensitive to stochastic effects associated with transmission and likely occurs in nature whenever a host is infected productively with more than one influenza A virus.

  • Early Steps of Jaagsiekte Sheep Retrovirus-Mediated Cell Transformation Involve the Interaction between Env and the RALBP1 Cellular Protein [Transformation and Oncogenesis]

  • Ovine pulmonary adenocarcinoma is a naturally occurring lung cancer in sheep induced by the Jaagsiekte sheep retrovirus (JSRV). Its envelope glycoprotein (Env) carries oncogenic properties, and its expression is sufficient to induce in vitro cell transformation and in vivo lung adenocarcinoma. The identification of cellular partners of the JSRV envelope remains crucial for deciphering mechanisms leading to cell transformation. We initially identified RALBP1 (RalA binding protein 1; also known as RLIP76 or RIP), a cellular protein implicated in the ras pathway, as a partner of JSRV Env by yeast two-hybrid screening and confirmed formation of RALBP1/Env complexes in mammalian cells. Expression of the RALBP1 protein was repressed in tumoral lungs and in tumor-derived alveolar type II cells. Through its inhibition using specific small interfering RNA (siRNA), we showed that RALBP1 was involved in envelope-induced cell transformation and in modulation of the mTOR (mammalian target of rapamycin)/p70S6K pathway by the retroviral envelope.

    IMPORTANCE JSRV-induced lung adenocarcinoma is of importance for the sheep industry. While the envelope has been reported as the oncogenic determinant of the virus, the cellular proteins directly interacting with Env are still not known. Our report on the formation of RALBP/Env complexes and the role of this interaction in cell transformation opens up a new hypothesis for the dysregulation observed upon virus infection in sheep.

  • Inhibition of O-Linked N-Acetylglucosamine Transferase Reduces Replication of Herpes Simplex Virus and Human Cytomegalovirus [Virus-Cell Interactions]

  • O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential cellular enzyme that posttranslationally modifies nuclear and cytoplasmic proteins via O-linked addition of a single N-acetylglucosamine (GlcNAc) moiety. Among the many targets of OGT is host cell factor 1 (HCF-1), a transcriptional regulator that is required for transactivation of the immediate-early genes of herpes simplex virus (HSV). HCF-1 is synthesized as a large precursor that is proteolytically cleaved by OGT, which may regulate its biological function. In this study, we tested whether inhibition of the enzymatic activity of OGT with a small molecule inhibitor, OSMI-1, affects initiation of HSV immediate-early gene expression and viral replication. We found that inhibiting OGT's enzymatic activity significantly decreased HSV replication. The major effect of the inhibitor occurred late in the viral replication cycle, when it reduced the levels of late proteins and inhibited capsid formation. However, depleting OGT levels with small interfering RNA (siRNA) reduced the expression of HSV immediate-early genes, in addition to reducing viral yields. In this study, we identified OGT as a novel cellular factor involved in HSV replication. Our results obtained using a small molecule inhibitor and siRNA depletion suggest that OGT's glycosylation and scaffolding functions play distinct roles in the replication cycle of HSV.

    IMPORTANCE Antiviral agents can target viral or host gene products essential for viral replication. O-GlcNAc transferase (OGT) is an important cellular enzyme that catalyzes the posttranslational addition of GlcNAc sugar residues to hundreds of nuclear and cytoplasmic proteins, and this modification regulates their activity and function. Some of the known OGT targets are cellular proteins that are critical for the expression of herpes simplex virus (HSV) genes, suggesting a role for OGT in the replication cycle of HSV. In this study, we found that OGT is required for efficient expression of viral genes and for assembly of new virions. Thus, we identify OGT as a novel host factor involved in the replication of HSV and a potential target for antiviral therapy.

  • Tracking the Emergence of Host-Specific Simian Immunodeficiency Virus env and nef Populations Reveals nef Early Adaptation and Convergent Evolution in Brain of Naturally Progressing Rhesus Macaques [Genetic Diversity and Evolution]

  • While a clear understanding of the events leading to successful establishment of host-specific viral populations and productive infection in the central nervous system (CNS) has not yet been reached, the simian immunodeficiency virus (SIV)-infected rhesus macaque provides a powerful model for the study of human immunodeficiency virus (HIV) intrahost evolution and neuropathogenesis. The evolution of the gp120 and nef genes, which encode two key proteins required for the establishment and maintenance of infection, was assessed in macaques that were intravenously inoculated with the same viral swarm and allowed to naturally progress to simian AIDS and potential SIV-associated encephalitis (SIVE). Longitudinal plasma samples and immune markers were monitored until terminal illness. Single-genome sequencing was employed to amplify full-length env through nef transcripts from plasma over time and from brain tissues at necropsy. nef sequences diverged from the founder virus faster than gp120 diverged. Host-specific sequence populations were detected in nef (~92 days) before they were detected in gp120 (~182 days). At necropsy, similar brain nef sequences were found in different macaques, indicating convergent evolution, while gp120 brain sequences remained largely host specific. Molecular clock and selection analyses showed weaker clock-like behavior and stronger selection pressure in nef than in gp120, with the strongest nef selection in the macaque with SIVE. Rapid nef diversification, occurring prior to gp120 diversification, indicates that early adaptation of nef in the new host is essential for successful infection. Moreover, the convergent evolution of nef sequences in the CNS suggests a significant role for nef in establishing neurotropic strains.

    IMPORTANCE The SIV-infected rhesus macaque model closely resembles HIV-1 immunopathogenesis, neuropathogenesis, and disease progression in humans. Macaques were intravenously infected with identical viral swarms to investigate evolutionary patterns in the gp120 and nef genes leading to the emergence of host-specific viral populations and potentially linked to disease progression. Although each macaque exhibited unique immune profiles, macaque-specific nef sequences evolving under selection were consistently detected in plasma samples at 3 months postinfection, significantly earlier than in gp120 macaque-specific sequences. On the other hand, nef sequences in brain tissues, collected at necropsy of two animals with detectable infection in the central nervous system (CNS), revealed convergent evolution. The results not only indicate that early adaptation of nef in the new host may be essential for successful infection, but also suggest that specific nef variants may be required for SIV to efficiently invade CNS macrophages and/or enhance macrophage migration, resulting in HIV neuropathology.

  • A Dual-Modality Herpes Simplex Virus 2 Vaccine for Preventing Genital Herpes by Using Glycoprotein C and D Subunit Antigens To Induce Potent Antibody Responses and Adenovirus Vectors Containing Capsid and Tegument Proteins as T Cell Immunogens [Vaccines and Antiviral Agents]

  • We evaluated a genital herpes prophylactic vaccine containing herpes simplex virus 2 (HSV-2) glycoproteins C (gC2) and D (gD2) to stimulate humoral immunity and UL19 (capsid protein VP5) and UL47 (tegument protein VP13/14) as T cell immunogens. The HSV-2 gC2 and gD2 proteins were expressed in baculovirus, while the UL19 and UL47 genes were expressed from replication-defective adenovirus vectors. Adenovirus vectors containing UL19 and UL47 stimulated human and murine CD4+ and CD8+ T cell responses. Guinea pigs were either (i) mock immunized; (ii) immunized with gC2/gD2, with CpG and alum as adjuvants; (iii) immunized with the UL19/UL47 adenovirus vectors; or (iv) immunized with the combination of gC2/gD2-CpG/alum and the UL19/UL47 adenovirus vectors. Immunization with gC2/gD2 produced potent neutralizing antibodies, while UL19 and UL47 also stimulated antibody responses. After intravaginal HSV-2 challenge, the mock and UL19/UL47 adenovirus groups developed severe acute disease, while 2/8 animals in the gC2/gD2-only group and none in the combined group developed acute disease. No animals in the gC2/gD2 or combined group developed recurrent disease; however, 5/8 animals in each group had subclinical shedding of HSV-2 DNA, on 15/168 days for the gC2/gD2 group and 13/168 days for the combined group. Lumbosacral dorsal root ganglia were positive for HSV-2 DNA and latency-associated transcripts for 5/8 animals in the gC2/gD2 group and 2/8 animals in the combined group. None of the differences comparing the gC2/gD2-only group and the combined group were statistically significant. Therefore, adding the T cell immunogens UL19 and UL47 to the gC2/gD2 vaccine did not significantly reduce genital disease and vaginal HSV-2 DNA shedding compared with the excellent protection provided by gC2/gD2 in the guinea pig model.

    IMPORTANCE HSV-2 infection is a common cause of genital ulcer disease and a significant public health concern. Genital herpes increases the risk of transmission and acquisition of HIV-1 infection 3- to 4-fold. A herpes vaccine that prevents genital lesions and asymptomatic genital shedding will have a substantial impact on two epidemics, i.e., both the HSV-2 and HIV-1 epidemics. We previously reported that a vaccine containing HSV-2 glycoprotein C (gC2) and glycoprotein D (gD2) reduced genital lesions and asymptomatic HSV-2 genital shedding in guinea pigs, yet the protection was not complete. We evaluated whether adding the T cell immunogens UL19 (capsid protein VP5) and UL47 (tegument protein VP13/14) would enhance the protection provided by the gC2/gD2 vaccine, which produces potent antibody responses. Here we report the efficacy of a combination vaccine containing gC2/gD2 and UL19/UL47 for prevention of genital disease, vaginal shedding of HSV-2 DNA, and latent infection of dorsal root ganglia in guinea pigs.

  • Thioredoxin 2 Is a Novel E2-Interacting Protein That Inhibits the Replication of Classical Swine Fever Virus [Virus-Cell Interactions]

  • The E2 protein of classical swine fever virus (CSFV) is an envelope glycoprotein that is involved in virus attachment and entry. To date, the E2-interacting cellular proteins and their involvement in viral replication have been poorly documented. In this study, thioredoxin 2 (Trx2) was identified to be a novel E2-interacting partner using yeast two-hybrid screening from a porcine macrophage cDNA library. Trx2 is a mitochondrion-associated protein that participates in diverse cellular events. The Trx2-E2 interaction was further confirmed by glutathione S-transferase (GST) pulldown, in situ proximity ligation, and laser confocal assays. The thioredoxin domain of Trx2 and the asparagine at position 37 (N37) in the E2 protein were shown to be critical for the interaction. Silencing of the Trx2 expression in PK-15 cells by small interfering RNAs significantly promotes CSFV replication, and conversely, overexpression of Trx2 markedly inhibits viral replication of the wild-type (wt) CSFV and to a greater extent that of the CSFV N37D mutant, which is defective in binding Trx2. The wt CSFV but not the CSFV N37D mutant was shown to reduce the Trx2 protein expression in PK-15 cells. Furthermore, we demonstrated that Trx2 increases nuclear factor kappa B (NF-B) promoter activity by promoting the nuclear translocation of the p65 subunit of NF-B. Notably, activation of the NF-B signaling pathway induced by tumor necrosis factor alpha (TNF-aalpha;) significantly inhibits CSFV replication in PK-15 cells, whereas blocking the NF-B activation in Trx2-overexpressing cells no longer suppresses CSFV replication. Taken together, our findings reveal that Trx2 inhibits CSFV replication via the NF-B signaling pathway.

    IMPORTANCE Thioredoxin 2 (Trx2) is a mitochondrion-associated protein that participates in diverse cellular events, such as antioxidative and antiapoptotic processes and the modulation of transcription factors. However, little is known about the involvement of Trx2 in viral replication. Here, we investigated, for the first time, the role of Trx2 in the replication of classical swine fever virus (CSFV), a devastating pestivirus of pigs. By knockdown and overexpression, we showed that Trx2 negatively regulates CSFV replication. Notably, we demonstrated that Trx2 inhibits CSFV replication by promoting the nuclear translocation of the p65 subunit of NF-B, a key regulator of the host's innate immunity and inflammatory response. Our findings reveal a novel role of Trx2 in the host's antiviral response and provide new insights into the complex mechanisms by which CSFV interacts with the host cell.

  • Head-to-Head Comparison of Poxvirus NYVAC and ALVAC Vectors Expressing Identical HIV-1 Clade C Immunogens in Prime-Boost Combination with Env Protein in Nonhuman Primates [Vaccines and Antiviral Agents]

  • We compared the HIV-1-specific cellular and humoral immune responses elicited in rhesus macaques immunized with two poxvirus vectors (NYVAC and ALVAC) expressing the same HIV-1 antigens from clade C, Env gp140 as a trimeric cell-released protein and a Gag-Pol-Nef polyprotein as Gag-induced virus-like particles (VLPs) (referred to as NYVAC-C and ALVAC-C). The immunization protocol consisted of two doses of the corresponding poxvirus vector plus two doses of a combination of the poxvirus vector and a purified HIV-1 gp120 protein from clade C. This immunogenicity profile was also compared to that elicited by vaccine regimens consisting of two doses of the ALVAC vector expressing HIV-1 antigens from clades B/E (ALVAC-vCP1521) plus two doses of a combination of ALVAC-vCP1521 and HIV-1 gp120 protein from clades B/E (similar to the RV144 trial regimen) or clade C. The results showed that immunization of macaques with NYVAC-C stimulated at different times more potent HIV-1-specific CD4+ T-cell responses and induced a trend toward higher-magnitude HIV-1-specific CD8+ T-cell immune responses than did ALVAC-C. Furthermore, NYVAC-C induced a trend toward higher levels of binding IgG antibodies against clade C HIV-1 gp140, gp120, or murine leukemia virus (MuLV) gp70-scaffolded V1/V2 and toward best cross-clade-binding IgG responses against HIV-1 gp140 from clades A, B, and group M consensus, than did ALVAC-C. Of the linear binding IgG responses, most were directed against the V3 loop in all immunization groups. Additionally, NYVAC-C and ALVAC-C also induced similar levels of HIV-1-neutralizing antibodies and antibody-dependent cellular cytotoxicity (ADCC) responses. Interestingly, binding IgA antibody levels against HIV-1 gp120 or MuLV gp70-scaffolded V1/V2 were absent or very low in all immunization groups. Overall, these results provide a comprehensive survey of the immunogenicity of NYVAC versus ALVAC expressing HIV-1 antigens in nonhuman primates and indicate that NYVAC may represent an alternative candidate to ALVAC in the development of a future HIV-1 vaccine.

    IMPORTANCE The finding of a safe and effective HIV/AIDS vaccine immunogen is one of the main research priorities. Here, we generated two poxvirus-based HIV vaccine candidates (NYVAC and ALVAC vectors) expressing the same clade C HIV-1 antigens in separate vectors, and we analyzed in nonhuman primates their immunogenicity profiles. The results showed that immunization with NYVAC-C induced a trend toward higher HIV-1-specific cellular and humoral immune responses than did ALVAC-C, indicating that this new NYVAC vector could be a novel optimized HIV/AIDS vaccine candidate for human clinical trials.

  • Primer ID Validates Template Sampling Depth and Greatly Reduces the Error Rate of Next-Generation Sequencing of HIV-1 Genomic RNA Populations [Genetic Diversity and Evolution]

  • Validating the sampling depth and reducing sequencing errors are critical for studies of viral populations using next-generation sequencing (NGS). We previously described the use of Primer ID to tag each viral RNA template with a block of degenerate nucleotides in the cDNA primer. We now show that low-abundance Primer IDs (offspring Primer IDs) are generated due to PCR/sequencing errors. These artifactual Primer IDs can be removed using a cutoff model for the number of reads required to make a template consensus sequence. We have modeled the fraction of sequences lost due to Primer ID resampling. For a typical sequencing run, less than 10% of the raw reads are lost to offspring Primer ID filtering and resampling. The remaining raw reads are used to correct for PCR resampling and sequencing errors. We also demonstrate that Primer ID reveals bias intrinsic to PCR, especially at low template input or utilization. cDNA synthesis and PCR convert ca. 20% of RNA templates into recoverable sequences, and 30-fold sequence coverage recovers most of these template sequences. We have directly measured the residual error rate to be around 1 in 10,000 nucleotides. We use this error rate and the Poisson distribution to define the cutoff to identify preexisting drug resistance mutations at low abundance in an HIV-infected subject. Collectively, these studies show that ggt;90% of the raw sequence reads can be used to validate template sampling depth and to dramatically reduce the error rate in assessing a genetically diverse viral population using NGS.

    IMPORTANCE Although next-generation sequencing (NGS) has revolutionized sequencing strategies, it suffers from serious limitations in defining sequence heterogeneity in a genetically diverse population, such as HIV-1 due to PCR resampling and PCR/sequencing errors. The Primer ID approach reveals the true sampling depth and greatly reduces errors. Knowing the sampling depth allows the construction of a model of how to maximize the recovery of sequences from input templates and to reduce resampling of the Primer ID so that appropriate multiplexing can be included in the experimental design. With the defined sampling depth and measured error rate, we are able to assign cutoffs for the accurate detection of minority variants in viral populations. This approach allows the power of NGS to be realized without having to guess about sampling depth or to ignore the problem of PCR resampling, while also being able to correct most of the errors in the data set.

  • African Swine Fever Virus Georgia 2007 with a Deletion of Virulence-Associated Gene 9GL (B119L), when Administered at Low Doses, Leads to Virus Attenuation in Swine and Induces an Effective Protection against Homologous Challenge [Vaccines and Antiviral Agents]

  • African swine fever virus (ASFV) is the etiological agent of an often lethal disease of domestic pigs. Disease control strategies have been hampered by the unavailability of vaccines against ASFV. Since its introduction in the Republic of Georgia, a highly virulent virus, ASFV Georgia 2007 (ASFV-G), has caused an epizootic that spread rapidly into Eastern European countries. Currently no vaccines are available or under development to control ASFV-G. In the past, genetically modified ASFVs harboring deletions of virulence-associated genes have proven attenuated in swine, inducing protective immunity against challenge with homologous parental viruses. Deletion of the gene 9GL (open reading frame [ORF] B119L) in highly virulent ASFV Malawi-Lil-20/1 produced an attenuated phenotype even when administered to pigs at 106 50% hemadsorption doses (HAD50). Here we report the construction of a genetically modified ASFV-G strain (ASFV-G-9GLv) harboring a deletion of the 9GL (B119L) gene. Like Malawi-Lil-20/1-9GL, ASFV-G-9GL showed limited replication in primary swine macrophages. However, intramuscular inoculation of swine with 104 HAD50 of ASFV-G-9GL produced a virulent phenotype that, unlike Malawi-Lil-20/1-9GL, induced a lethal disease in swine like parental ASFV-G. Interestingly, lower doses (102 to 103 HAD50) of ASFV-G-9GL did not induce a virulent phenotype in swine and when challenged protected pigs against disease. A dose of 102 HAD50 of ASFV-G-9GLv conferred partial protection when pigs were challenged at either 21 or 28 days postinfection (dpi). An ASFV-G-9GL HAD50 of 103 conferred partial and complete protection at 21 and 28 dpi, respectively. The information provided here adds to our recent report on the first attempts toward experimental vaccines against ASFV-G.

    IMPORTANCE The main problem for controlling ASF is the lack of vaccines. Studies on ASFV virulence lead to the production of genetically modified attenuated viruses that induce protection in pigs but only against homologous virus challenges. Here we produced a recombinant ASFV lacking virulence-associated gene 9GL in an attempt to produce a vaccine against virulent ASFV-G, a highly virulent virus isolate detected in the Caucasus region in 2007 and now spreading though the Caucasus region and Eastern Europe. Deletion of 9GL, unlike with other ASFV isolates, did not attenuate completely ASFV-G. However, when delivered once at low dosages, recombinant ASFV-G-9GL induces protection in swine against parental ASFV-G. The protection against ASFV-G is highly effective after 28 days postvaccination, whereas at 21 days postvaccination, animals survived the lethal challenge but showed signs of ASF. Here we report the design and development of an experimental vaccine that induces protection against virulent ASFV-G.

  • Characterization of a Novel Megabirnavirus from Sclerotinia sclerotiorum Reveals Horizontal Gene Transfer from Single-Stranded RNA Virus to Double-Stranded RNA Virus [Genetic Diversity and Evolution]

  • Mycoviruses have been detected in all major groups of filamentous fungi, and their study represents an important branch of virology. Here, we characterized a novel double-stranded RNA (dsRNA) mycovirus, Sclerotinia sclerotiorum megabirnavirus 1 (SsMBV1), in an apparently hypovirulent strain (SX466) of Sclerotinia sclerotiorum. Two similarly sized dsRNA segments (L1- and L2-dsRNA), the genome of SsMBV1, are packaged in rigid spherical particles purified from strain SX466. The full-length cDNA sequence of L1-dsRNA/SsMBV1 comprises two large open reading frames (ORF1 and ORF2), which encode a putative coat protein and an RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis of the RdRp domain clearly indicates that SsMBV1 is related to Rosellinia necatrix megabirnavirus 1 (RnMBV1). L2-dsRNA/SsMBV1 comprises two nonoverlapping ORFs (ORFA and ORFB) encoding two hypothetical proteins with unknown functions. The 5'-terminal regions of L1- and L2-dsRNA/SsMBV1 share strictly conserved sequences and form stable stem-loop structures. Although L2-dsRNA/SsMBV1 is dispensable for replication, genome packaging, and pathogenicity of SsMBV1, it enhances transcript accumulation of L1-dsRNA/SsMBV1 and stability of virus-like particles (VLPs). Interestingly, a conserved papain-like protease domain similar to a multifunctional protein (p29) of Cryphonectria hypovirus 1 was detected in the ORFA-encoded protein of L2-dsRNA/SsMBV1. Phylogenetic analysis based on the protease domain suggests that horizontal gene transfer may have occurred from a single-stranded RNA (ssRNA) virus (hypovirus) to a dsRNA virus, SsMBV1. Our results reveal that SsMBV1 has a slight impact on the fundamental biological characteristics of its host regardless of the presence or absence of L2-dsRNA/SsMBV1.

    IMPORTANCE Mycoviruses are widespread in all major fungal groups, and they possess diverse genomes of mostly ssRNA and dsRNA and, recently, circular ssDNA. Here, we have characterized a novel dsRNA virus (Sclerotinia sclerotiorum megabirnavirus 1 [SsMBV1]) that was isolated from an apparently hypovirulent strain, SX466, of Sclerotinia sclerotiorum. Although SsMBV1 is phylogenetically related to RnMBV1, SsMBV1 is markedly distinct from other reported megabirnaviruses with two features of VLPs and conserved domains. Our results convincingly showed that SsMBV1 is viable in the absence of L2-dsRNA/SsMBV1 (a potential large satellite-like RNA or genuine genomic virus component). More interestingly, we detected a conserved papain-like protease domain that commonly exists in ssRNA viruses, including members of the families Potyviridae and Hypoviridae. Phylogenetic analysis based on the protease domain suggests that horizontal gene transfer might have occurred from an ssRNA virus to a dsRNA virus, which may provide new insights into the evolutionary history of dsRNA and ssRNA viruses.

  • Characterization of Ribosomal Frameshifting in Theiler's Murine Encephalomyelitis Virus [Spotlight]

  • Theiler's murine encephalomyelitis virus (TMEV) is a member of the genus Cardiovirus in the Picornaviridae, a family of positive-sense single-stranded RNA viruses. Previously, we demonstrated that in the related cardiovirus, Encephalomyocarditis virus, a programmed nndash;1 ribosomal frameshift (nndash;1 PRF) occurs at a conserved G_GUU_UUU sequence within the 2B-encoding region of the polyprotein open reading frame (ORF). Here we show that nndash;1 PRF occurs at a similar site during translation of the TMEV genome. In addition, we demonstrate that a predicted 3' RNA stem-loop structure at a noncanonical spacing downstream of the shift site is required for efficient frameshifting in TMEV and that frameshifting also requires virus infection. Mutating the G_GUU_UUU shift site to inhibit frameshifting results in an attenuated virus with reduced growth kinetics and a small-plaque phenotype. Frameshifting in the virus context was found to be extremely efficient at 74 to 82%, which, to our knowledge, is the highest frameshifting efficiency recorded to date for any virus. We propose that highly efficient nndash;1 PRF in TMEV provides a mechanism to escape the confines of equimolar expression normally inherent in the single-polyprotein expression strategy of picornaviruses.

    IMPORTANCE Many viruses utilize programmed nndash;1 ribosomal frameshifting (nndash;1 PRF) to produce different protein products at a defined ratio, or to translate overlapping ORFs to increase coding capacity. With few exceptions, nndash;1 PRF occurs on specific "slippery" heptanucleotide sequences and is stimulated by RNA structure beginning 5 to 9 nucleotides (nt) downstream of the slippery site. Here we describe an unusual case of nndash;1 PRF in Theiler's murine encephalomyelitis virus (TMEV) that is extraordinarily efficient (74 to 82% of ribosomes shift into the alternative reading frame) and, in stark contrast to other examples of nndash;1 PRF, is dependent upon a stem-loop structure beginning 14 nt downstream of the slippery site. Furthermore, in TMEV-based reporter constructs in transfected cells, efficient frameshifting is critically dependent upon virus infection. We suggest that TMEV evolved frameshifting as a novel mechanism for removing ribosomes from the message (a "ribosome sink") to downregulate synthesis of the 3'-encoded replication proteins.

  • The Canonical Immediate Early 3 Gene Product pIE611 of Mouse Cytomegalovirus Is Dispensable for Viral Replication but Mediates Transcriptional and Posttranscriptional Regulation of Viral Gene Products [Genome Replication and Regulation of Viral Gene Expression]

  • Transcription of mouse cytomegalovirus (MCMV) immediate early ie1 and ie3 is controlled by the major immediate early promoter/enhancer (MIEP) and requires differential splicing. Based on complete loss of genome replication of an MCMV mutant carrying a deletion of the ie3-specific exon 5, the multifunctional IE3 protein (611 amino acids; pIE611) is considered essential for viral replication. Our analysis of ie3 transcription resulted in the identification of novel ie3 isoforms derived from alternatively spliced ie3 transcripts. Construction of an IE3-hemagglutinin (IE3-HA) virus by insertion of an in-frame HA epitope sequence allowed detection of the IE3 isoforms in infected cells, verifying that the newly identified transcripts code for proteins. This prompted the construction of an MCMV mutant lacking ie611 but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using ie611 MCMV, we demonstrated the dispensability of the canonical ie3 gene product pIE611 for viral replication. To determine the role of pIE611 for viral gene expression during MCMV infection in an unbiased global approach, we used label-free quantitative mass spectrometry to delineate pIE611-dependent changes of the MCMV proteome. Interestingly, further analysis revealed transcriptional as well as posttranscriptional regulation of MCMV gene products by pIE611.

    IMPORTANCE Cytomegaloviruses are pathogenic betaherpesviruses persisting in a lifelong latency from which reactivation can occur under conditions of immunosuppression, immunoimmaturity, or inflammation. The switch from latency to reactivation requires expression of immediate early genes. Therefore, understanding of immediate early gene regulation might add insights into viral pathogenesis. The mouse cytomegalovirus (MCMV) immediate early 3 protein (611 amino acids; pIE611) is considered essential for viral replication. The identification of novel protein isoforms derived from alternatively spliced ie3 transcripts prompted the construction of an MCMV mutant lacking ie611 but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using ie611 MCMV, we demonstrated the dispensability of the canonical ie3 gene product pIE611 for viral replication and delineated pIE611-dependent changes of the MCMV proteome. Our findings have fundamental implications for the interpretation of earlier studies on pIE3 functions and highlight the complex orchestration of MCMV gene regulation.

  • Restriction of HIV-1 Requires the N-Terminal Region of MxB as a Capsid-Binding Motif but Not as a Nuclear Localization Signal [Virus-Cell Interactions]

  • The interferon alpha (IFN-aalpha;)-inducible restriction factor MxB blocks HIV-1 infection after reverse transcription but prior to integration. Fate-of-capsid experiments have correlated the ability of MxB to block HIV-1 infection with stabilization of viral cores during infection. We previously demonstrated that HIV-1 restriction by MxB requires capsid binding and oligomerization. Deletion and gain-of-function experiments have mapped the HIV-1 restriction ability of MxB to its N-terminal 25 amino acids. This report reveals that the N-terminal 25 amino acids of MxB exhibit two separate functions: (i) the ability of MxB to bind to HIV-1 capsid and (ii) the nuclear localization signal of MxB, which is important for the ability of MxB to shuttle into the nucleus. To understand whether MxB restriction of HIV-1 requires capsid binding and/or nuclear localization, we genetically separated these two functions and evaluated their contributions to restriction. Our experiments demonstrated that the 11RRR13 motif is important for the ability of MxB to bind capsid and to restrict HIV-1 infection. These experiments suggested that capsid binding is necessary for the ability of MxB to block HIV-1 infection. Separately from the capsid binding function of MxB, we found that residues 20KY21 regulate the ability of the N-terminal 25 amino acids of MxB to function as a nuclear localization signal; however, the ability of the N-terminal 25 amino acids to function as a nuclear localization signal was not required for restriction.

    IMPORTANCE MxB/Mx2 blocks HIV-1 infection in cells from the immune system. MxB blocks infection by preventing the uncoating process of HIV-1. The ability of MxB to block HIV-1 infection requires that MxB binds to the HIV-1 core by using its N-terminal domain. The present study shows that MxB uses residues 11RRR13 to bind to the HIV-1 core during infection and that these residues are required for the ability of MxB to block HIV-1 infection. We also found that residues 20KY21 constitute a nuclear localization signal that is not required for the ability of MxB to block HIV-1 infection.

  • Identification of Owl Monkey CD4 Receptors Broadly Compatible with Early-Stage HIV-1 Isolates [Genetic Diversity and Evolution]

  • Most HIV-1 variants isolated from early-stage human infections do not use nonhuman primate versions of the CD4 receptor for cellular entry, or they do so poorly. We and others have previously shown that CD4 has experienced strong natural selection over the course of primate speciation, but it is unclear whether this selection has influenced the functional characteristics of CD4 as an HIV-1 receptor. Surprisingly, we find that selection on CD4 has been most intense in the New World monkeys, animals that have never been found to harbor lentiviruses related to HIV-1. Based on this, we sampled CD4 genetic diversity within populations of individuals from seven different species, including five species of New World monkeys. We found that some, but not all, CD4 alleles found in Spix's owl monkeys (Aotus vociferans) encode functional receptors for early-stage human HIV-1 isolates representing all of the major group M clades (A, B, C, and D). However, only some isolates of HIV-1 subtype C can use the CD4 receptor encoded by permissive Spix's owl monkey alleles. We characterized the prevalence of functional CD4 alleles in a colony of captive Spix's owl monkeys and found that 88% of surveyed individuals are homozygous for permissive CD4 alleles, which encode an asparagine at position 39 of the receptor. We found that the CD4 receptors encoded by two other species of owl monkeys (Aotus azarae and Aotus nancymaae) also serve as functional entry receptors for early-stage isolates of HIV-1.

    IMPORTANCE Nonhuman primates, particularly macaques, are used for preclinical evaluation of HIV-1 vaccine candidates. However, a significant limitation of the macaque model is the fact that most circulating HIV-1 variants cannot use the macaque CD4 receptor to enter cells and have to be adapted to these species. This is particularly true for viral variants from early stages of infection, which represent the most relevant vaccine targets. In this study, we found that some individuals from captive owl monkey populations harbor CD4 alleles that are compatible with a broad collection of HIV-1 isolates, including those isolated from early in infection in highly affected populations and representing diverse subtypes.

  • SIRT1 Suppresses Human T-Cell Leukemia Virus Type 1 Transcription [Genome Replication and Regulation of Viral Gene Expression]

  • Human T-cell leukemia virus type 1 (HTLV-1)-associated diseases are poorly treatable, and HTLV-1 vaccines are not available. High proviral load is one major risk factor for disease development. HTLV-1 encodes Tax oncoprotein, which activates transcription from viral long terminal repeats (LTR) and various types of cellular promoters. Counteracting Tax function might have prophylactic and therapeutic benefits. In this work, we report on the suppression of Tax activation of HTLV-1 LTR by SIRT1 deacetylase. The transcriptional activity of Tax on the LTR was largely ablated when SIRT1 was overexpressed, but Tax activation of NF-B was unaffected. On the contrary, the activation of the LTR by Tax was boosted when SIRT1 was depleted. Treatment of cells with resveratrol shunted Tax activity in a SIRT1-dependent manner. The activation of SIRT1 in HTLV-1-transformed T cells by resveratrol potently inhibited HTLV-1 proviral transcription and Tax expression, whereas compromising SIRT1 by specific inhibitors augmented HTLV-1 mRNA expression. The administration of resveratrol also decreased the production of cell-free HTLV-1 virions from MT2 cells and the transmission of HTLV-1 from MT2 cells to uninfected Jurkat cells in coculture. SIRT1 associated with Tax in HTLV-1-transformed T cells. Treatment with resveratrol prevented the interaction of Tax with CREB and the recruitment of CREB, CRTC1, and p300 to Tax-responsive elements in the LTR. Our work demonstrates the negative regulatory function of SIRT1 in Tax activation of HTLV-1 transcription. Small-molecule activators of SIRT1 such as resveratrol might be considered new prophylactic and therapeutic agents in HTLV-1-associated diseases.

    IMPORTANCE Human T-cell leukemia virus type 1 (HTLV-1) causes a highly lethal blood cancer or a chronic debilitating disease of the spinal cord. Treatments are unsatisfactory, and vaccines are not available. Disease progression is associated with robust expression of HTLV-1 genes. Suppressing HTLV-1 gene expression might have preventive and therapeutic benefits. It is therefore critical that host factors controlling HTLV-1 gene expression be identified and characterized. This work reveals a new host factor that suppresses HTLV-1 gene expression and a natural compound that activates this suppression. Our findings not only provide new knowledge of the host control of HTLV-1 gene expression but also suggest a new strategy of using natural compounds for prevention and treatment of HTLV-1-associated diseases.

  • Context-Dependent Cleavage of the Capsid Protein by the West Nile Virus Protease Modulates the Efficiency of Virus Assembly [Structure and Assembly]

  • The molecular mechanisms that define the specificity of flavivirus RNA encapsulation are poorly understood. Virions composed of the structural proteins of one flavivirus and the genomic RNA of a heterologous strain can be assembled and have been developed as live attenuated vaccine candidates for several flaviviruses. In this study, we discovered that not all combinations of flavivirus components are possible. While a West Nile virus (WNV) subgenomic RNA could readily be packaged by structural proteins of the DENV2 strain 16681, production of infectious virions with DENV2 strain New Guinea C (NGC) structural proteins was not possible, despite the very high amino acid identity between these viruses. Mutagenesis studies identified a single residue (position 101) of the DENV capsid (C) protein as the determinant for heterologous virus production. C101 is located at the P1' position of the NS2B/3 protease cleavage site at the carboxy terminus of the C protein. WNV NS2B/3 cleavage of the DENV structural polyprotein was possible when a threonine (Thr101 in strain 16681) but not a serine (Ser101 in strain NGC) occupied the P1' position, a finding not predicted by in vitro protease specificity studies. Critically, both serine and threonine were tolerated at the P1' position of WNV capsid. More extensive mutagenesis revealed the importance of flanking residues within the polyprotein in defining the cleavage specificity of the WNV protease. A more detailed understanding of the context dependence of viral protease specificity may aid the development of new protease inhibitors and provide insight into associated patterns of drug resistance.

    IMPORTANCE West Nile virus (WNV) and dengue virus (DENV) are mosquito-borne flaviviruses that cause considerable morbidity and mortality in humans. No specific antiflavivirus therapeutics are available for treatment of infection. Proteolytic processing of the flavivirus polyprotein is an essential step in the replication cycle and is an attractive target for antiviral development. The design of protease inhibitors has been informed by insights into the molecular details of the interactions of proteases and their substrates. In this article, studies of the processing of WNV and DENV capsid proteins by the WNV protease identified an unexpected contribution of the sequence surrounding critical residues within the cleavage site on protease specificity. This demonstration of context-dependent protease cleavage has implications for the design of chimeric flaviviruses, new therapeutics, and the interpretation of flavivirus protease substrate specificity studies.

  • Vaccine-Induced Linear Epitope-Specific Antibodies to Simian Immunodeficiency Virus SIVmac239 Envelope Are Distinct from Those Induced to the Human Immunodeficiency Virus Type 1 Envelope in Nonhuman Primates [Vaccines and Antiviral Agents]

  • To evaluate antibody specificities induced by simian immunodeficiency virus (SIV) versus human immunodeficiency virus type 1 (HIV-1) envelope antigens in nonhuman primate (NHP), we profiled binding antibody responses to linear epitopes in NHP studies with HIV-1 or SIV immunogens. We found that, overall, HIV-1 Env IgG responses were dominated by V3, with the notable exception of the responses to the vaccine strain A244 Env that were dominated by V2, whereas the anti-SIVmac239 Env responses were dominated by V2 regardless of the vaccine regimen.

  • Protective Efficacy of Recombinant Modified Vaccinia Virus Ankara Delivering Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein [Vaccines and Antiviral Agents]

  • Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans. We tested a recombinant modified vaccinia virus Ankara (MVA) vaccine expressing full-length MERS-CoV spike (S) glycoprotein by immunizing BALB/c mice with either intramuscular or subcutaneous regimens. In all cases, MVA-MERS-S induced MERS-CoV-specific CD8+ T cells and virus-neutralizing antibodies. Vaccinated mice were protected against MERS-CoV challenge infection after transduction with the human dipeptidyl peptidase 4 receptor. This MERS-CoV infection model demonstrates the safety and efficacy of the candidate vaccine.

  • Arenavirus Coinfections Are Common in Snakes with Boid Inclusion Body Disease [Genetic Diversity and Evolution]

  • Recently, novel arenaviruses were found in snakes with boid inclusion body disease (BIBD); these form the new genus Reptarenavirus within the family Arenaviridae. We used next-generation sequencing and de novo sequence assembly to investigate reptarenavirus isolates from our previous study. Four of the six isolates and all of the samples from snakes with BIBD contained at least two reptarenavirus species. The viruses sequenced comprise four novel reptarenavirus species and a representative of a new arenavirus genus.

  • Mapping of a Region of the PA-X Protein of Influenza A Virus That Is Important for Its Shutoff Activity [Genome Replication and Regulation of Viral Gene Expression]

  • Influenza A virus PA-X comprises an N-terminal PA endonuclease domain and a C-terminal PA-X-specific domain. PA-X reduces host and viral mRNA accumulation via its endonuclease function. Here, we found that the N-terminal 15 amino acids, particularly six basic amino acids, in the C-terminal PA-X-specific region are important for PA-X shutoff activity. These six basic amino acids enabled a PA deletion mutant to suppress protein expression at a level comparable to that of wild-type PA-X.

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    JVI Accepts: Articles Published Ahead of Print

  • Fitness-balanced escape determines resolution of dynamic founder virus escape processes in HIV-1 infection [PublishAheadOfPrint]

  • To understand the interplay between host cytotoxic T-lymphocyte (CTL) responses and the mechanisms by which HIV-1 evades them, we studied viral evolutionary patterns associated with host CTL responses in six linked transmission pairs. HIV-1 sequences corresponding to full-length p17 and p24 gag were generated by 454 pyrosequencing for all pairs near the time of transmission, and seroconverting partners were followed for a median of 847 days post infection. T-cell responses were screened by IFN/IL-2 FluoroSpot using autologous peptide sets reflecting any Gag variant present in at least 5% of sequence reads in the individual's viral population. While we found little evidence for the occurrence of CTL reversions, CTL escape processes were found to be highly dynamic, with multiple epitope variants emerging simultaneously. We found a correlation between epitope entropy and the number of epitope variants per response (r=0.43 p=0.05). In cases in which multiple escape mutations developed within a targeted epitope, a variant with no fitness cost became fixed in the viral population. When multiple mutations within an epitope achieved fitness-balanced escape, these escape mutants were each maintained in the viral population. Additional mutations found to confer escape but undetected in viral populations incurred high fitness costs, suggesting that functional constraints limit the available sites tolerable to escape mutations. These results further our understanding of the impact of CTL escape and reversion from the founder virus in HIV infection and contribute to the identification of immunogenic Gag regions most vulnerable to a targeted T-cell attack.

    Importance Rapid diversification of the viral population is a hallmark of HIV-1 infection, and understanding the selective forces driving the emergence of viral variants can provide critical insight into the interplay between host immune responses and viral evolution. We used deep sequencing to comprehensively follow viral evolution over time in six linked HIV transmission pairs. We then mapped T-cell responses to explore if mutations arose due to adaption to the host, and found that escape processes were often highly dynamic, with multiple mutations arising within targeted epitopes. When we explored the impact of these mutations on replicative capacity, we found that dynamic escape processes only resolve with the selection of mutations that conferred escape with no fitness cost to the virus. These results provide further understanding of the complicated viral-host interactions that occur during early HIV-1 infection and may help inform the design of future vaccine immunogens.

  • R5 Macrophage-Tropic HIV-1 in the Male Genital Tract [PublishAheadOfPrint]

  • The entry tropism of HIV-1 Env proteins from virus isolated from the blood and genital tract of five men with compartmentalized lineages was determined. The Env proteins isolated from the genital tract of subject C018 were macrophage-tropic, while the remaining cloned env genes were R5 T cell-tropic. The detection of a macrophage-tropic lineage of HIV-1 within the male genital tract strongly suggests that evolution of macrophage-tropic viruses can occur in anatomically isolated sites outside of the central nervous system.

  • Inhibition of HIV expression and integration in macrophages by Methylglyoxal-bis-guanylhydrazone [PublishAheadOfPrint]

  • Macrophages are a target for infection with HIV and represent one of the viral reservoirs that are relatively resistant to current anti-retroviral drugs. Here we demonstrate that methylglyoxal-bis-guanylhydrazone (MGBG), a polyamine analog and potent S-adenosylmethionine decarboxylase inhibitor, decreases HIV expression in monocytes and macrophages. MGBG is selectively concentrated by these cells through a mechanism consistent with active transport by the polyamine transporter. Using a macrophage-tropic reporter virus tagged with the enhanced green fluorescent protein, we demonstrate that MGBG decreases the frequency of HIV infected cells. The effect is dose dependent and correlates with the production of HIV p24 in culture supernatants. This anti-HIV effect was further confirmed using three macrophage tropic primary HIV isolates. Viral life cycle mapping studies show that MGBG inhibits HIV DNA integration into the cellular DNA in both monocytes and macrophages.

    IMPORTANCE Our work demonstrates for the first time the selective concentration of MGBG by monocytes/macrophages, leading to the inhibition of HIV-1 expression and a reduction in proviral load within macrophage cultures. These results suggest that MGBG may be useful in adjunctive macrophage targeted therapy for HIV infection.

  • RIG-I mediates an antiviral response to Crimean-Congo hemorrhagic fever virus [PublishAheadOfPrint]

  • In the cytoplasm, the retinoic acid-inducible gene I (RIG-I) senses the RNA genomes of several RNA viruses. RIG-I binds to viral RNA, eliciting an antiviral response via the cellular adaptor MAVS. Crimean-Congo hemorrhagic fever virus (CCHFV), a negative sense RNA virus with a 5rrsquo; -monophosphorylated genome, is a highly pathogenic zoonotic agent with significant public health and clinical implications. We found that, during CCHFV infection, RIG-I mediated a type-I interferon (IFN) response via MAVS. Interfering with RIG-I signaling reduced IFN production and IFN-stimulated gene expression, and increased viral replication. Immunostimulatory RNA was isolated from CCHFV-infected cells and from virion preparations, and RIG-I co-immunoprecipitation of infected cell lysates isolated immunostimulatory CCHFV RNA. This report serves as the first description of a pattern recognition receptor for CCHFV and highlights a critical signaling pathway in the antiviral response to CCHFV.

    IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus with significant public health and clinical impact. In order for cells to respond to virus infection, they must recognize the virus as foreign and initiate antiviral signaling. To date, the receptors involved in recognition of CCHFV are not known. Herein we investigate and identify retinoic acid-inducible gene I (RIG-I) as a receptor involved in initiating an antiviral response to CCHFV. This receptor was initially not expected to play a role in CCHFV recognition because of characteristics of the viral genome. These findings are important in understanding the antiviral response to CCHFV and support continued investigation into the spectrum of potential viruses recognized by RIG-I.

  • Bub1 in complex with LANA recruits PCNA to regulate KSHV latent replication and DNA translesion synthesis [PublishAheadOfPrint]

  • Latent DNA replication of Kaposi's sarcoma-associated herpesvirus (KSHV) initiates at the terminal repeat (TR) element and requires trans-acting elements, both viral and cellular, such as ORCs, MCMs and LANA. However, how cellular proteins are recruited to the viral genome is not very clear. Here we demonstrated that the host cellular protein, Bub1, is involved in KSHV latent DNA replication. We show that Bub1 constitutively interacts with PCNA via a highly conserved PIP-box motif within the Kinase domain. Furthermore, we demonstrated that Bub1 can form a complex with LANA and PCNA in KSHV positive cells. This strongly indicated that Bub1 may serve as a scaffold or molecular bridge between LANA and PCNA. LANA recruited PCNA to the KSHV genome via Bub1 to initiate viral replication in S-phase and interacted with PCNA to promote its mono-ubiquitination in response to UV-induced damage for translesion DNA synthesis. This resulted in increased survival of KSHV infected cells.

    IMPORTANCE In KSHV latency infected cells, the viral episomal DNA replicates once each cell cycle. KSHV does not express DNA replication proteins during latency. Instead, KSHV latency-associated nuclear antigen (LANA) recruits the host cell DNA replication machinery to the replication origin. However, the mechanism by which LANA mediates replication is uncertain. Here, we show that LANA is able to form a complex with proliferating cell nuclear antigen (PCNA), a critical protein for viral DNA replication. Furthermore our findings suggest that Bub1, a spindle checkpoint protein, may serve as a scaffold or molecular bridge between LANA and PCNA. Our data further support a role for Bub1 and LANA in PCNA-mediated cellular DNA replication processes as well as mono-ubiquitination of PCNA in response to UV damage. These data reveal a therapeutic target for inhibition of KSHV persistence in malignant cells.

  • Human dendritic cell response signatures distinguish 1918, pandemic and seasonal H1N1 influenza viruses [PublishAheadOfPrint]

  • Influenza viruses continue to present global threats to human health. Antigenic drift and shift, genetic reassortment, and cross-species transmission generate new strains with differences in epidemiology and clinical severity. We compared the temporal transcriptional responses of human dendritic cells (DC) to infection with two pandemic (A/Brevig Mission/1/1918, A/California/4/09) and two seasonal (A/New Caledonia/20/99, A/Texas/36/1991) H1N1 influenza viruses. Strain-specific response differences included stronger activation of NFB following infection with A/New Caledonia/20/99 and a unique cluster of genes expressed following infection with A/Brevig Mission/1/1918. A common anti-viral program showing strain-specific timing was identified in the early DC response and found to correspond with reported transcript changes in blood during symptomatic human influenza infection. Comparison of the global response to the seasonal and pandemic strains showed that a dramatic divergence occurred after 4 h, with only the seasonal strains inducing widespread mRNA loss.

    Importance Continuously evolving influenza viruses present a global threat to human health however, these host responses display strain-dependent differences that are incompletely understood. Thus we conducted a detailed comparative study comparing the immune response of human DC to infection with two pandemic and two seasonal H1N1 influenza strains. We identified in the immune response to viral infection both common, but also strain specific, features. Among the stain specific elements were a time shift of the ISG response, selective induction of NFB signaling by one of the seasonal strains and massive RNA degradation as early as 4 hours post-infection by the seasonal, but not the pandemic, viruses. These findings illuminate new aspects that characterize the distinct differences in the immune response to pandemic or seasonal influenza viruses.

  • The structure of immature-like Rous sarcoma virus Gag particles reveals a structural role for the p10 domain in assembly [PublishAheadOfPrint]

  • The polyprotein Gag is the primary structural component of retroviruses. Gag consists of independently folded domains connected by flexible linkers. Interactions between the conserved CA domains of Gag mediate formation of hexameric protein lattices that drive assembly of immature virus particles. Proteolytic cleavage of Gag by the viral protease (PR) is required for maturation of retroviruses from an immature form into an infectious form. Within the assembled Gag lattices of HIV-1 and Mason-Pfizer monkey virus (M-PMV), the C-terminal domain of CA adopts similar quaternary arrangements, while the N-terminal domain of CA is packed in very different manners. Here we have employed cryo-electron tomography and subtomogram averaging to study in vitro assembled, immature virus-like Rous sarcoma virus (RSV) Gag particles, and have determined the structure of CA and the surrounding regions to a resolution of ~ 8 AAring;. We found that the C-terminal domain of RSV CA is arranged similarly to HIV-1 and M-PMV, while the N-terminal domain of CA adopts a novel arrangement in which the upstream p10 domain folds back into the CA lattice. In this position the cleavage site between CA and p10 appears inaccessible to PR. Below CA, an extended density is consistent with the presence of a six-helix bundle formed by the spacer-peptide region. We have also assessed the affect of lattice assembly on proteolytic processing by exogenous PR. The cleavage between p10 and CA is indeed inhibited in the assembled lattice, consistent with structural regulation of proteolytic maturation.

    IMPORTANCE Retroviruses first assemble into immature virus particles, requiring interactions between Gag proteins that form a protein layer under the viral membrane. Subsequently, Gag is cleaved by the viral protease enzyme into separate domains, leading to rearrangement of the virus into its infectious form. It is important to understand how Gag is arranged within immature retroviruses, in order to understand how virus assembly occurs, and how maturation takes place. Here we have used techniques called cryo-electron tomography and subtomogram averaging to obtain a detailed structural picture of the CA domains in immature assembled Rous sarcoma virus Gag particles. We find that part of Gag next to CA, called p10, folds back and interacts with CA when Gag assembles. This arrangement is different from that seen in HIV-1 and Mason-Pfizer monkey virus, illustrating further structural diversity of retroviral structures. The structure provides new information on how the virus assembles and undergoes maturation.

  • Innate immune activity correlates with CD4 T cell-associated HIV-1 DNA decline during latency-reversing treatment with panobinostat [PublishAheadOfPrint]

  • Pharmaceutical reactivation of dormant HIV-1 proviruses by histone deacetylase inhibitors (HDACi) represents a possible strategy to reduce the reservoir of HIV-1 infected cells in individuals treated with suppressive antiretroviral combination therapy (cART). However, effects of such latency-reversing agents on the viral reservoir size are likely to be influenced by host immune responses. Here, we analyzed immune factors associated with changes in proviral HIV-1 DNA levels during treatment with the potent HDACi panobinostat in a human clinical trial involving 15 cART-treated HIV-1 patients. We observed that the magnitude, breadth, and cytokine-secretion profile of HIV-1-specific CD8 T cell responses were unrelated to HIV-1 DNA changes in CD4 T cells during panobinostat treatment. In contrast, proportions of CD3- CD56+ total NK cells and CD16+ CD56dim NK cells were inversely correlated with HIV-1 DNA throughout the study, and changes of HIV-1 DNA during panobinostat treatment were negatively associated with corresponding changes in CD69+ NK cells. Decreasing levels of HIV-1 DNA during latency-reversing treatment were also related to proportions of plasmacytoid dendritic cells, to distinct gene expression patterns of interferon-stimulated genes and to expression of the IL28B "CC" genotype. Together, these data suggest that innate immune activity can critically modulate effects of latency-reversing agents on the viral reservoir and may represent a target for future immunotherapeutic interventions in HIV-1 eradication studies.

    IMPORTANCE Currently available antiretroviral drugs are highly effective in suppressing HIV-1 replication, but the virus persists despite treatment in a latent form that does not actively express HIV-1 gene products. One approach to eliminate these cells, colloquially termed as the "shock and kill" strategy, focuses on the use of latency-reversing agents that induce active viral gene expression in latently infected cells, followed by immune-mediated killing. Panobinostat, a histone deacetylase inhibitor, demonstrated potent activities in reversing HIV-1 latency in a recent pilot clinical trial, and reduce HIV-1 DNA in a subset of patients. Interestingly, we found that innate immune factors, such as natural killer cells, plasmacytoid dendritic cells and expression patterns of Interferon-stimulated genes, were most closely linked to a decline of HIV-1 DNA during treatment with panobinostat. These data suggest that innate immune activity may play an important role in reducing the residual reservoir of HIV-1 infected cells.

  • Pathogenesis and transmission of novel HPAI H5N2 and H5N8 avian influenza viruses in ferrets and mice [PublishAheadOfPrint]

  • A novel highly pathogenic avian influenza (HPAI) H5N8 virus, first detected in January 2014 in poultry and wild birds in South Korea, has spread throughout Asia and Europe, and caused outbreaks in Canada and the United States by the end of the year. The spread of H5N8 and the novel reassortant viruses, H5N2 and H5N1 (H5Nx), in domestic poultry across multiple states in the U.S. pose a potential public health risk. To evaluate the potential of cross-species infection, we determined the pathogenesis and transmissibility of two Asian-origin H5Nx viruses in mammalian animal models. The newly isolated H5N2 and H5N8 viruses were able to cause severe disease in mice only at high doses. Both viruses replicated efficiently in the upper and lower respiratory tracts of ferrets; however clinical symptoms were generally mild and there was no evidence of systemic dissemination of virus to multiple organs. Moreover, these influenza H5Nx viruses lacked the ability to transmit between ferrets in a direct contact setting. We further assessed viral replication kinetics of the novel H5Nx viruses in a human bronchial epithelium cell line, Calu-3. Both H5Nx viruses replicated to a level comparable to a human seasonal H1N1 virus, but significantly lower than a virulent Asian-lineage H5N1 HPAI virus. Although the recently isolated H5N2 and H5N8 viruses displayed moderate pathogenicity in mammalian models, their ability to rapidly spread among avian species, reassort, and generate novel strains underscores the need for continued risk assessment in mammals.

    IMPORTANCE In 2015, highly pathogenic avian influenza (HPAI) H5 viruses have caused outbreaks in domestic poultry in multiple U.S. states. The economic losses incurred with H5N8 and H5N2 subtype virus infection have raised serious concerns for the poultry industry and the general public due to the potential risk of human infection. This recent outbreak underscores the need to better understand the pathogenesis and transmission of these viruses in mammals, which is an essential component of pandemic risk assessment. This study demonstrates that the newly isolated H5N2 and H5N8 viruses lacked the ability to transmit between ferrets and exhibited low to moderate virulence in mammals. In human bronchial epithelial (Calu-3) cells, both H5N8 and H5N2 viruses replicated to a level comparable to a human seasonal virus, but significantly lower than a virulent Asian-lineage H5N1 (A/Thailand/16/2004) virus. The results of this study are important for the evaluation of public health risk.

  • Attenuated Human Parainfluenza Virus Type 1 (HPIV1) Expressing the Fusion F Glycoprotein of Human Respiratory Syncytial Virus (RSV) as a Bivalent HPIV1/RSV Vaccine [PublishAheadOfPrint]

  • Live attenuated recombinant human parainfluenza virus type 1 (rHPIV1) was investigated as a vector to express the respiratory syncytial virus (RSV) fusion (F) glycoprotein, to provide a bivalent vaccine against RSV and HPIV1. The RSV F gene was engineered to include HPIV1 transcription signals and inserted individually into three gene locations in each of the two attenuated rHPIV1 backbones. Each backbone contained a single previously-described attenuating mutation that was stabilized against de-attenuation: specifically, a non-temperature-sensitivity deletion mutation involving six nucleotides in the overlapping P/C ORFs (C170), or a temperature-sensitivity missense mutation in the L ORF (LY942A). The insertion sites in the genome were pre-N (F1), N-P (F2), or P-M (F3) and were identical for both backbones. In vitro, the presence of the F insert reduced the rate of virus replication, but the final titers were the same as wt HPIV1. High levels of RSV F expression in cultured cells were observed with rHPIV1-C170-F1, -F2, and -F3, and rHPIV1-LY942A-F1. In hamsters, the rHPIV1-C170-F1, -F2, and -F3 vectors were moderately restricted in the nasal turbinates and highly restricted in lungs, and were genetically stable in vivo. Among the C170 vectors, the F1 virus was the most immunogenic and protective against wt RSV challenge. The rHPIV1-LY942A vectors were highly restricted in vivo and were not detectably immunogenic or protective, indicative of over-attenuation. The C170-F1 construct appears to be suitably attenuated and immunogenic for further development as a bivalent intranasal pediatric vaccine.

    IMPORTANCE There are no vaccines for the pediatric respiratory pathogens RSV and HPIVs. We are developing live attenuated RSV and HPIV vaccines for use in virus-naiiuml;ve infants. Live attenuated RSV strains in particular are difficult to develop due to its poor growth and physical instability, but these obstacles could be avoided by the use of a vaccine vector. We describe the development and pre-clinical evaluation of live attenuated rHPIV1 vectors expressing the RSV F protein. Two different attenuated rHPIV1 backbones were engineered to express RSV F from three different gene positions each. The rHPIV1-C170-F1 vector, bearing an attenuating deletion mutation (C170) in the P/C gene and expressing RSV F from the pre-N position, was attenuated, stable, and immunogenic in the hamster model against RSV F protein and HPIV1 and provided substantial protection against RSV challenge. This study provided a candidate rHPIV1-RSV-F vaccine virus suitable for continued development as a bivalent vaccine against two major childhood pathogens.

  • Identification and Functional Analysis of Novel Non-structural Proteins of Human Bocavirus 1 [PublishAheadOfPrint]

  • Human bocavirus 1 (HBoV1) is a single-stranded DNA parvovirus that causes lower respiratory tract infections in young children worldwide. In this study, we identified novel splice acceptor and donor sites, namely A1rrsquo; and D1rrsquo;, in the large non-structural protein (NS1)-encoding region of the HBoV1 precursor mRNA. The novel small NS proteins (NS2, NS3, and NS4) were confirmed to be expressed following transfection of an HBoV1 infectious proviral plasmid and viral infection of polarized human airway epithelium cultured at an air-liquid interface (HAE-ALI). We constructed mutant pIHBoV1 infectious plasmids which harbor silent mutations smA1rrsquo; and smD1rrsquo; at the A1rrsquo; and D1rrsquo; splice sites, respectively. The mutant infectious plasmids maintained production of HBoV1 progeny virions at a level less than five times lower than that of the wild-type plasmid. Importantly, the smA1rrsquo; mutant virus that does not express NS3 and NS4 replicated in HAE-ALI as effectively as the wild-type virus; however, the smD1rrsquo; mutant virus that does not express NS2 and NS4 underwent an abortive infection in HAE-ALI. Thus, our study identified three novel NS proteins, NS2, NS3, and NS4, and suggests an important function of the NS2 in HBoV1 replication in HAE-ALI.

    IMPORTANCE Human bocavirus 1 infection causes respiratory diseases, including acute wheezing in infants, of which life-threatening cases have been reported. In vitro, human bocavirus 1 infects polarized human bronchial airway epithelium cultured at an air-liquid interface, which mimics the environment of human lower respiratory airways. Viral non-structural proteins are often important for virus replication and pathogenesis in infected tissues or cells. In this report, we identified three new non-structural proteins of human bocavirus 1 during infection of polarized human bronchial airway epithelium. Among them, we proved that one non-structural protein is critical to the virus replication in polarized human bronchial airway epithelium. The creation of non-replicating infectious HBoV1 mutant may have particular utility in vaccine development for this virus.

  • Vaccinia B1 kinase is required for post-replicative stages of the viral lifecycle in a BAF-independent manner in U2OS cells [PublishAheadOfPrint]

  • The vaccinia B1R gene encodes a highly conserved protein kinase that is essential for the poxviral lifecycle. As demonstrated in many cell types, B1 plays a critical role during viral DNA replication when it inactivates the cellular host defense effector BAF (barrier to autointegration factor or BANF1). To better understand the role of B1 during infection, we have characterized the growth of a B1-deficient temperature sensitive mutant virus (Cts2) in U2OS osteosarcoma cells. In contrast to all other cell lines tested to date, we found that in U2OS cells Cts2 viral DNA replication is unimpaired at non-permissive temperature. However, Cts2 viral yield in these cells is reduced more than 10 fold, thus indicating that B1 is required at another stage of the vaccinia viral lifecycle. Our results further suggest that the host defense function of endogenous BAF may be absent in U2OS cells, but can be recovered either through overexpression of BAF or fusion of U2OS cells with mouse cells in which the antiviral function of BAF is active. Interestingly, examination of late viral proteins during Cts2 infection demonstrates that B1 is required for optimal processing of the L4 protein. Finally, execution point analyses as well as electron microscopy studies uncover a role for B1 during maturation of poxviral virions. Overall, this work demonstrates that U2OS cells are a novel model system for studying the cell-type specific regulation of BAF and reveal a role for B1 beyond DNA replication during the late stages of the viral life cycle.

    IMPORTANCE The most well characterized role for the vaccinia B1 kinase is to facilitate viral DNA replication by phosphorylating and inactivating BAF, a cellular host defense responsive to foreign DNA. Additional roles for B1 later in the viral lifecycle have been postulated for decades, but are difficult to examine directly due to the importance of B1 during DNA replication. Herein, we demonstrate that in U2OS cells, a B1-mutant virus escapes the block in DNA replication observed in other cell types and instead this mutant virus exhibits impaired late protein accumulation and incomplete maturation of new virions. These data provide the clearest evidence to date that B1 is needed for multiple critical junctures in the poxviral lifecycle, both dependent and independent of BAF.

  • Elite control, gut CD4 T cell sparing, and enhanced mucosal T cell responses in Macaca nemestrina infected by an SIV lacking a gp41 trafficking motif [PublishAheadOfPrint]

  • Deletion of Gly-720 and Tyr-721 from a highly conserved GYxxOOslash; trafficking signal in the SIVmac239 envelope glycoprotein cytoplasmic domain, producing a virus termed GY, leads to a striking perturbation in pathogenesis in rhesus macaques (M. mulatta). Infected macaques develop immune activation and progress to AIDS, but with only limited and transient infection of intestinal CD4+ T cells and an absence of microbial translocation. Here we evaluated GY in pig-tailed macaques (M. nemestrina), a species in which SIVmac239 infection typically leads to increased immune activation and more rapid progression to AIDS than in rhesus macaques. In pig-tailed macaques GY also replicated acutely to high peak plasma RNA levels identical to SIVmac239, and caused only transient infection of CD4+ T cells in the gut lamina propria and no microbial translocation. However, in marked contrast to rhesus macaques, 19 of 21 pig-tailed macaques controlled GY replication with plasma viral loads of llt;15-50 RNA copies/ml. CD4+ T cells were preserved in blood and gut for up to 100 weeks with no immune activation or disease progression. Robust, anti-viral CD4+ T cell responses were seen, particularly in the gut. Anti-CD8 antibody depletion demonstrated CD8+ cellular control of viral replication. Two pig-tailed macaques progressed to disease with persisting viremia and possible compensatory mutations in the cytoplasmic tail. These studies demonstrate a marked perturbation in pathogenesis caused by GY's ablation of the GYxxOOslash; trafficking motif and reveal, paradoxically, that viral control is enhanced in a macaque species typically predisposed to more pathogenic manifestations of SIV infection.

    IMPORTANCE Pathogenesis of human (HIV) and simian (SIV) immunodeficiency viruses reflects a balance between viral replication host innate and adaptive anti-viral immune responses, and sustained immune activation that in humans and Asian macaques is associated with persistent viremia, immune escape and AIDS. Among nonhuman primates, pig-tailed macaques following SIV infection are predisposed to more rapid disease progression than are rhesus macaques. Here, we show that disruption of a conserved tyrosine-based cellular trafficking motif in the viral transmembrane envelope glycoprotein cytoplasmic tail leads in pig-tailed macaques to a unique phenotype in which high levels of acute viral replication are followed by elite control, robust cellular responses in mucosal tissues, and no disease. Paradoxically, control of this virus in rhesus macaques is only partial, and progression to AIDS occurs. This novel model should provide a powerful tool to help identify host-specific determinants for viral control with potential relevance for vaccine development.

  • Adenovirus E4-ORF3 targets PIAS3 and together with E1B-55K remodels SUMO interactions in the nucleus and at virus genome replication domains [PublishAheadOfPrint]

  • Adenovirus E4-ORF3 and E1B-55K converge in subverting critical overlapping cellular pathways to facilitate virus replication. Here we show that E1B-55K and E4-ORF3 induce sumoylation and the assembly of SUMO2/3 viral genome replication domains. Using a conjugation deficient SUMO2 construct, we demonstrate that SUMO2/3 is recruited to E2A viral genome replication domains through non-covalent interactions. E1B-55K and E4-ORF3 have critical functions in inactivating MRN and ATM to facilitate viral genome replication. We show that ATM kinase inhibitors rescue E1B-55K/E4-ORF3 viral genome replication and that the assembly of E2A domains recruits SUMO2/3 independently of E1B-55K and E4-ORF3. However, the morphology and organization of SUMO2/3 associated E2A domains is strikingly different to that in wild type Ad5 infected cells. These data reveal that E1B-55K and E4-ORF3 specify the nuclear compartmentalization and structure of SUMO2/3 associated E2A domains, which could have important functions in viral replication. We show that E4-ORF3 specifically targets and sequesters the cellular E3 SUMO ligase PIAS3 but not PIAS1, 2, or 4. The assembly of E4-ORF3 into a multivalent nuclear matrix is required to target PIAS3. In contrast to MRN, PIAS3 is targeted by E4-ORF3 proteins from disparate adenovirus subgroups. Our studies reveal that PIAS3 is a novel and evolutionary conserved target of E4-ORF3 in human adenovirus infections. Furthermore, we reveal that viral proteins not only disrupt but also usurp SUMO2/3 to transform the nucleus and assemble novel genomic domains that could facilitate pathological viral replication.

    IMPORTANCE SUMO is a key post-translational modification that modulates the function, localization, and assembly of protein complexes. In the ever-escalating host-pathogen arms race, viruses have evolved strategies to subvert sumoylation. Adenovirus is a small DNA tumor virus that is a global human pathogen and key biomedical agent in basic research and therapy. We show that adenovirus infection induces global changes in SUMO localization and conjugation. Using virus and SUMO mutants, we demonstrate that E1B-55K and E4-ORF3 disrupt and usurp SUMO2/3 interactions to transform the nucleus and assemble highly structured and compartmentalized viral genome domains. We reveal that the cellular E3 SUMO ligase PIAS3 is a novel and conserved target of E4-ORF3 proteins from disparate adenovirus subgroups. The induction of sumoylation and SUMO2/3 viral replication domains by early viral proteins could play an important role in determining the outcome of viral infection.

  • An Antagonistic Relationship between Human Cytomegalovirus pUL27 and pUL97 Activities during Infection [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) is a member of the beta-herpesvirus family. During infection, an array of viral proteins manipulates the host cell cycle. We have previously shown that expression of HCMV pUL27 results in increased levels of the cyclin-dependent kinase (CDK) inhibitor p21Cip1. In addition, pUL27 is necessary for the full antiviral activity of the pUL97 kinase inhibitor maribavir (MBV). The purpose of this study was to define the relationship between pUL27 and pUL97, and its role in MBV antiviral activity. We observed that expression of wild-type but not kinase-inactive pUL97 disrupted pUL27-dependent induction of p21Cip1. Furthermore, pUL97 associated with and promoted the phosphorylation of pUL27. During infection, inhibiting the kinase resulted in elevated levels of p21Cip1 in wild-type but not a pUL27-deficient virus. We manipulated p21Cip1 levels to evaluate the functional consequence to MBV. Overexpression of p21Cip1 restored MBV activity against a pUL27-deficient virus while disruption reduced activity against wild-type virus. We provide evidence that the functional target of p21Cip1 in the context of MBV activity is CDK1. One CDK-like activity of pUL97 is to phosphorylate nuclear lamin A/C resulting in altered nuclear morphology and increased viral egress. In the presence of MBV, we observed that infection using a pUL27-deficient virus still altered nuclear morphology. This was prevented by the addition of a CDK inhibitor. Overall, our results demonstrate an antagonistic relationship between pUL27 and pUL97 activities centering on p21Cip1 and support the idea that CDKs can complement some activities of pUL97.

    IMPORTANCE HCMV infection results in severe disease upon immunosuppression and is a leading cause of congenital birth defects. Effective antiviral compounds exist yet exhibit high levels of toxicity, are not approved for use during pregnancy and can result in antiviral resistance. Our studies have uncovered new information regarding the antiviral efficacy of the HCMV pUL97 kinase inhibitor MBV as it relates to the complex interplay between pUL97 and a second HCMV protein, pUL27. We demonstrate that pUL97 functions antagonistically against pUL27 by phosphorylation-dependent inactivation of pUL27-mediated induction of p21Cip1. In contrast, we provide evidence that p21Cip1 functions to antagonize overlapping activities between pUL97 and cellular CDKs. In addition, these studies further support the notion that CDK inhibitors or p21Cip1 activators might be useful in combination with MBV to effectively inhibit HCMV infections.

  • A14U Substitution in 3' Non-Coding Region of M vRNA Supports Replication of NS1-Deleted Influenza Virus by Modulating Alternative Splicing of M mRNAs [PublishAheadOfPrint]

  • The NS1 protein of influenza virus has multiple functions and is a determinant of virulence. NS1-deleted (DelNS1) influenza viruses are a useful tool for studying virus replication and can serve as effective live attenuated vaccines, but deletion of NS1 severely diminishes virus replication, hampering functional studies and vaccine production. We found that WSN-DelNS1 viruses passaged in cells consistently adapted to gain an A14U substitution in the 3rrsquo; non-coding region of the M vRNA segment which restored replicative ability. DelNS1-M-A14U viruses cannot inhibit interferon expression in virus infected-cells, providing an essential model for studying virus replication in the absence of the NS1 protein. Characterization of DelNS1-M-A14U virus found that lack of NS1 has no apparent effect on expression of other viral proteins, with the exception of M mRNAs. Expression of the M transcripts, M1, M2, mRNA3 and mRNA4, is regulated by alternative splicing. The A14U substitution changes the splicing donor site consensus sequence of mRNA3, altering expression of M transcripts, with M2 expression significantly increased and mRNA3 markedly suppressed in DelNS1-M-A14U, but not DelNS1-M-WT, virus infected cells. Further analysis revealed that the A14U substitution also affects promoter function during replication of the viral genome. The M-A14U mutation increases M vRNA synthesis in DelNS1 virus infection and enhances alternative splicing of M2 mRNA in the absence of other viral proteins. The findings demonstrate that NS1 is directly involved in influenza virus replication through modulation of alternative splicing of M transcripts, and provide strategic information important to construction of NS1-deleted vaccine strains.

    IMPORTANCE The non-structural protein (NS1) of influenza virus has multiple functions. Besides its role in antagonizing host antiviral activity, NS1 is also believed to be involved in regulating virus replication, but mechanistic details are not clear. The NS1 protein is a virulence determinant which inhibits both innate and adaptive immunity and NS1 deleted live attenuated viruses show promise as effective vaccines. However, deletion of NS1 causes severe attenuation of virus replication during infection, impeding functional studies and vaccine development. We characterized a replication competent DelNS1 virus which carries an A14U substitution in the 3rrsquo; non-coding region of the M vRNA segment. We found that M-A14U mutation supports virus replication through modulation of alternative splicing of mRNAs transcribed from the M segment. Our findings give insight into the role of NS1 in influenza virus replication and provide an approach for constructing replication-competent NS1-deleted strains for use in functional and vaccine studies.

  • Going Viral with Fluorescent Proteins [PublishAheadOfPrint]

  • Many longstanding questions about dynamics of virus-cell interactions can be answered by combining fluorescence imaging techniques with fluorescent protein (FP) tagging strategies. Successfully creating a FP fusion with a cellular or viral protein of interest first requires selecting the appropriate FP. However, while viral architecture and cellular localization will often dictate the suitability of a FP, a FP's chemical and physical properties must also be considered. Here, we discuss the challenges of and offer suggestions for identifying the optimal FPs for studying the cell biology of viruses.

  • Pim Kinase Interacts with Nonstructural 5A Protein and Regulates Hepatitis C Virus Entry [PublishAheadOfPrint]

  • The life cycle of hepatitis C virus (HCV) is highly dependent on host cellular proteins for its propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assay using the HCV NS5A protein as a probe. Of ~9,000 human cellular proteins immobilized in a microarray, approximately 90 cellular proteins were identified as NS5A interactors. Of these candidates, Pim1, a member of serine/threonine kinase family composed of three different isoforms (Pim1, Pim2 and Pim3), was selected for further study. Pim kinases share a common consensus sequence which overlaps with kinase activity. Pim kinase activity has been implicated in tumorigenesis. In the present study, we verified the physical interaction between NS5A and Pim1 by both in vitro pulldown and coimmunoprecipitation assays. Pim1 interacted with NS5A through amino acid residues 141 to 180 of Pim1. We demonstrated that protein stability of Pim1 was increased by NS5A protein and this increase was mediated by protein interplay. siRNA-mediated knockdown or pharmacological inhibition of Pim kinase abrogated HCV propagation. By employing HCV pseudoparticle entry and single cycle HCV infection assays, we further demonstrated that Pim kinase was involved in HCV entry at a post-binding step. These data suggest that Pim kinase may represent a new host factor for HCV entry.

    IMPORTANCE Pim1 is an oncogenic serine/threonine kinase. HCV NS5A protein physically interacts with Pim1 and contributes to Pim1 protein stability. Since Pim1 protein expression level is upregulated in many cancers, NS5A-mediated protein stability may be associated with HCV pathogenesis. Either gene silencing or chemical inhibition of Pim kinase abrogated HCV propagation in HCV-infected cells. We further showed that Pim kinase was specifically required at an early entry step of the HCV life cycle. Thus we have not only identified Pim kinase as an HCV cell entry factor but also a new anti-HCV therapeutic target.

  • The nuclear protein Sam68 interacts with Enterovirus 71 internal ribosome entry site and positively regulates viral protein translation [PublishAheadOfPrint]

  • Enterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism, but also has the potential for the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68), acts as an internal ribosome entry site (IRES) trans-acting factor (ITAF) that binds specifically to the EV71 5rrsquo; untranslated region (UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the KH domain of Sam68 protein were further mapped using electrophoretic mobility shift assay (EMSA) and biotin RNA pull-down assay. More importantly, dual-firefly luciferase reporter analysis suggested that over-expression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells when compared with the negative control ShRNA-treated cells. However, down-regulation of Sam68 did not have a significant inhibitory effect on the accumulation of EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factor, such as PCBP2 and PABP, during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation.

    IMPORTANCE The nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection and could potentially enhance the translation of virus protein. To our knowledge, this is the first report that describes Sam68 actively participating in the life cycle of EV71 in molecular details. These studies will not only improve our understanding of the replication of EV71, but also have the potential for developing a therapeutic strategy against EV71 infection.

  • Determinants of human CD134 essential for entry of human herpesvirus 6B [PublishAheadOfPrint]

  • We identified two key amino acid residues within human CD134 (hCD134) that are required for its interaction with HHV-6B and for HHV-6B entry into cells. One of the residues (K79) allows access of HHV-6B ligand to hCD134. Murine CD134 (mCD134) functioned as a HHV-6B receptor when these two amino acid residues were substituted with homologous human residues. This study identifies both the HHV-6B receptor-ligand interaction and the species-specific determinants of hCD134 essential for HHV-6B entry.

  • Epstein-Barr virus BDLF4 Gene Is Required for Efficient Expression of Viral Late Lytic Genes [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) is a gamma-herpesvirus, associated with infectious mononucleosis and various types of malignancy. We here focused on the BDLF4 of EBV and identified it as a lytic gene, expressed with early kinetics. Viral late gene expression of the BDLF4 knockout strain was severely restricted; this could be restored by an exogenous supply of BDLF4. These results indicate that BDLF4 is important for the EBV lytic replication cycle, especially late gene expression.

    IMPORTANCE EBV is a causative agent of infectious mononucleosis and various types of proliferative disorders. To combat such diseases, determining the molecular mechanism(s) of the virus replication is of great significance. However, among the ggt; 80 genes encoded by EBV, there remain genes that have not yet been identified or characterized. Focusing on one of these genes, BDLF4, we show that it encodes a ~22 kDa protein and that it affects late gene expression by preparing knockout viruses. Our results provide basic empirical data for understanding regulation of non-canonical TATA-mediated late gene transcription, which is conserved in beta/gamma herpesviruses.

  • Association of HIV-1 Envelope-Specific Breast Milk IgA Responses with Reduced Risk of Postnatal Mother-to-Child Transmission of HIV-1 [PublishAheadOfPrint]

  • Infants born to HIV-1 infected mothers in resource-limited areas where replacement feeding is unsafe and impractical are repeatedly exposed to HIV-1 throughout breastfeeding. Despite this, the majority of infants do not contract HIV-1 postnatally, even in absence of maternal antiretroviral therapy. This suggests that immune factors in breast milk of HIV-1-infected mothers help to limit vertical transmission. We compared the HIV-1 envelope-specific breast milk and plasma antibody responses of clade C HIV-1-infected postnatally transmitting and nontransmitting mothers in the control arm of the Malawi-based Breastfeeding Antiretrovirals and Nutrition study using multivariable logistic regression modeling. We found no association between milk or plasma neutralization activity, antibody-dependent cell-mediated cytotoxicity, or HIV-1 envelope-specific IgG responses and postnatal transmission risk. While the envelope-specific breast milk and plasma IgA responses also did not reach significance in predicting postnatal transmission risk in the primary model after correction for multiple comparisons, subsequent exploratory analysis using two distinct assay methodologies demonstrated that the magnitudes of breast milk total and secretory IgA responses against a consensus HIV-1 envelope gp140 (B.con env03) were associated with reduced postnatal transmission risk. These results suggest a protective role for mucosal HIV-1 envelope-specific IgA responses in the context of postnatal virus transmission. This finding supports further investigations into the mechanisms by which mucosal IgA reduces risk of HIV-1 transmission via breast milk, and into immune interventions aimed at enhancing this response.

    IMPORTANCE Infants born to HIV-1 infected mothers are repeatedly exposed to virus in breast milk. Remarkably, the transmission rate is low, suggesting that immune factors in breast milk of HIV-1-infected mothers help to limit transmission. We compared the antibody responses in plasma and breast milk of HIV-1 transmitting and nontransmitting mothers to identify responses that correlated with reduced risk of postnatal HIV-1 transmission. We found that neither plasma nor breast milk IgG antibody responses were associated with risk of HIV-1 transmission. In contrast, the magnitude of the breast milk IgA and secretory IgA response against HIV-1 envelope proteins was associated with reduced risk of postnatal HIV-1 transmission. The results of this study support further investigations of the mechanisms by which mucosal IgA may reduce the risk of HIV-1 transmission via breastfeeding, and development of strategies to enhance milk envelope-specific IgA responses to reduce mother-to-child HIV transmission and promote an HIV-free generation.

  • A higher frequency of NKG2A+ than NKG2A- NK cells respond to autologous HIV-infected CD4 cells irrespective of whether they co-express KIR3DL1 [PublishAheadOfPrint]

  • Epidemiological and functional studies implicate NK cells in HIV control. However, there is little information available on what NK cell populations, as defined by inhibitory NK cell receptors (iNKRs) they express, respond to autologous HIV-infected CD4+ T cells (iCD4). NK cells acquire anti-viral functions through education, which requires signals received from iNKRs, such as NKG2A and KIR3DL1 (3DL1), engaging their ligands. NKG2A interacts with HLA-E and 3DL1 interacts with HLA-A/B antigens expressing the Bw4 epitope. HIV infected cells downregulate HLA-A/B, which should interrupt negative signaling through 3DL1, leading to NK cell activation, provided there is sufficient engagement of activating NKRs. We examined the functionality of NK cells expressing or not NKG2A and 3DL1 stimulated by HLA null and autologous iCD4 cells. Flow cytometry was used to gate on each NKG2A+/-3DL1+/- population and to measure the frequency of all possible combinations of CD107a expression and IFN-, and CCL4 secretion. The highest frequency of functional NK cells responding to HLA-null cell stimulation was the NKG2A+3DL1+ NK cell population. The highest frequencies of functional NK cells responding to autologous iCD4 were those expressing NKG2A; co-expression of 3DL1 did not further modulate responsiveness. This was the case for the functional subsets characterized by the sum of all functions tested (total responsiveness), as well as, by the tri-functional, CD107a+IFN-+, total CD107a+ and total IFN-+ functional subsets. These results indicate the NKG2A receptor has a role in NK cell mediated anti-HIV responses.

    Importance HIV-infected CD4 cells (iCD4) activate NK cells, which then control HIV replication. However, little is known regarding which NK cell populations iCD4 stimulate to develop anti-viral activity. Here, we examine the frequency of NK cell populations, defined by the presence/absence of the NK cell receptors (NKR), NKG2A and 3DL1, that respond to iCD4. NKG2A and 3DL1 are involved in priming NK cells for anti-viral functions upon encountering virus-infected cells. A higher frequency of NKG2A+ compared with NKG2A- NK cells responded to iCD4 by developing anti-viral functions such as CD107a expression, which correlates with NK cell killing, and secretion of Interferon- and CCL4. Co-expression of 3DL1 on the NKG2A+ and NKG2A- NK cells did not modulate responses to iCD4. Understanding the mechanisms underlying the interaction of NK cells with iCD4, that lead to HIV control, may contribute to developing strategies that harness NK cells for preventing or controlling HIV infection.

  • The cellular factor NXP2/MORC3 is a positive regulator for influenza virus multiplication [PublishAheadOfPrint]

  • Transcription and replication of the influenza A virus are carried out in the nucleus of the infected cells in the context of viral ribonucleoproteins (RNPs). The viral polymerase responsible for these processes is a protein complex composed by the PB1, PB2 and PA proteins. We previously identified a set of polymerase-associated cellular proteins by proteomic analysis of polymerase-containing intracellular complexes expressed and purified from human cells. Here we characterize the role of NXP2/MORC3 in the infection cycle. NXP2/MORC3 is a member of the Microrchidia (MORC) family that is associated to the nuclear matrix and has RNA-binding activity. Influenza virus infection led to a slight increase in NXP2/MORC3 expression and partial relocalization to the cytoplasm. Co-immunoprecipitation and immunofluorescence experiments indicated an association of NXP2/MORC3 with viral polymerase and RNPs during infection. Down-regulation of NXP2/MORC3 with two independent shRNAs reduced virus titers in low-multiplicity infections. Consistent with these findings, analysis of virus-specific RNA in high-multiplicity infections indicated a reduction of vRNA and mRNA after NXP2/MORC3 down-regulation. Silencing of NXP2/MORC3 in a recombinant mini-replicon system, where virus transcription and replication are uncoupled, showed a reduction in cat mRNA and CAT protein accumulation, but no alterations in cat vRNA levels, suggesting that NXP2/MORC3 is important for influenza virus transcription.

    IMPORTANCE Influenza infections appear as yearly epidemics and occasional pandemics of respiratory disease with large morbidity and occasional mortality. Influenza viruses are intracellular parasites that replicate and transcribe their genomic ribonucleoproteins in the nucleus of infected cells, in a complex interplay with host cell factors. Here we characterized the role of human NXP2/MORC3 protein, a member of the Microrchidia family that is associated to the nuclear matrix, during virus infection. NXP2/MORC3 associates to the viral ribonucleoproteins in infected cells. Down-regulation of NXP2/MORC3 reduced virus titers and the accumulations of viral genomic and mRNAs. Silencing of NXP2/MORC3 in an influenza CAT mini-replicon system diminished CAT protein and cat mRNA but not genomic RNA. We propose that NXP2/MORC3 plays a role in influenza virus transcription.


  • Coronaviruses (CoVs) have shown neuroinvasive properties in human and animal secondary to replication in peripheral organ but the mechanism of neuroinvasion is unknown. The major aim of our work was to evaluate the ability of CoVs to enter the central nervous system (CNS) through the blood brain barrier (BBB). Using the high hepatotropic mouse hepatitis virus (MHV) type 3, its attenuated variant 51.6-MHV3, showing low tropism for endothelial cells, and the low hepatotropic MHV-A59 strains from murine coronavirus group, we investigated the viral-induced dysfunctions of BBB in vivo and in brain microvascular endothelial cells (BMECs) in vitro. We report here a MHV strain-specific ability to cross the BBB during acute infection according to their virulence for liver. Brain invasion was only observed in MHV3-infected mice and correlated with enhanced BBB permeability associated with decreased expression of ZO-1, VE-cadherin and occludin but not claudin-5 in the brain or in cultured BMECs. BBB breakdown in MHV3 infection was not related to production of barrier-dysregulating inflammatory cytokines or chemokines by infected BMECs but rather to a downregulation of barrier protective IFN-bbeta; production. Our findings highlight the importance of IFN-bbeta; production by infected BMECs in preserving BBB function and preventing access of blood-borne infectious viruses to the brain.

    IMPORTANCE Coronaviruses (CoVs) infect several mammals including humans and are associated with respiratory, gastrointestinal and/or neurological diseases. Some evidences suggest that human respiratory CoVs may show neuroinvasive properties. Indeed, the SARS-CoV, causing severe acute respiratory syndrome, and the CoVs OC43 and 229E were found in the brains of SARS and multiple sclerosis patients respectively. These finding suggest that hematogenous spread CoVs may gain access to CNS at the BBB level. Herein we report for the first time that CoVs exhibit ability to cross the BBB according to strain virulence. BBB invasion by CoVs correlates with viral-induced disruption of tight junctions on BMECs, leading to BBB dysfunction and enhanced permeability. We provide evidence that production of IFN-bbeta; by BMECs during CoV infection may prevent BBB breakdown and brain viral invasion.


  • Previous studies have demonstrated an interaction between sorting nexin 17 and the L2 capsid proteins from a variety of papillomavirus types. This interaction is required for late endosomal trafficking of the L2 protein and entry of the L2/DNA complex into the nucleus during infection. Here we show an interaction between papillomavirus L2 proteins and the related PX-Ferm family member, sorting nexin 27, which is mediated in part by a novel interaction between the PDZ domain of SNX27 and sequences in a central portion of L2. The interaction is direct, and unlike that with SNX17, is variable in strength depending on the papillomavirus type. We show that siRNA-mediated knockdown of SNX27 alone leads to a marginal reduction in the efficiency of viral infection, but double knockdown of both sorting nexins results in a striking reduction in infection, greater than that observed for knockdown of either sorting nexin alone. These results suggest that the HPV L2 proteins can interact through distinct mechanisms with multiple components of the cellular cargo-sorting machinery.

    IMPORTANCE The trafficking of papillomaviruses to the host cell nucleus during their natural infectious life-cycle is a process that is incompletely understood. Studies have suggested that the virus L2 minor capsid protein can interact with the endosomal recycling pathway, in part, by association with sorting nexin 17, to ensure that virus DNA bound to L2 is recycled through the trans-golgi network rather than back to the plasma membrane. In this study we characterize the interaction between L2 and a second sorting nexin, SNX27, which is also part of the retromer complex. The study furthers our understanding of papillomavirus infection dynamics as well as providing potential tools to further dissect endosomal structure and function.

  • Alteration of Mature Nucleocapsid and Enhancement of Covalently Closed Circular DNA Formation by Hepatitis B Virus Core Mutants Defective in Complete Virion Formation [PublishAheadOfPrint]

  • Assembly of hepatitis B virus (HBV) begins with packaging of the pregenomic RNA (pgRNA) into immature nucleocapsids (NC), which are converted to mature NCs containing the genomic relaxed circular (RC) DNA as a result of reverse transcription. Mature NCs have two alternative fates, envelopment by viral envelope proteins leading to secretion extracellularly as virions or disassembly (uncoating) to deliver their RC DNA content into the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, the template for viral transcription. How these two alternative fates are regulated remains to be better understood. The NC shell is composed of multiple copies of a single viral protein, the HBV core (HBc) protein. HBc mutations located on the surface of NC have been identified that allow NC maturation but block its envelopment. The potential effects of some of these mutations on NC uncoating and CCC DNA formation have been analyzed by transfecting HBV replication constructs into hepatoma cells. All envelopment-defective HBc mutations tested were competent for CCC DNA formation, indicating that core functions in envelopment and uncoating/nuclear delivery of RC DNA were genetically separable. Some of the envelopment-defective HBc mutations were found to alter specifically the integrity of mature, but not immature, NCs such that RC DNA became susceptible to nuclease digestion. Furthermore, CCC DNA formation could be enhanced by NC surface mutations that did or didn't significantly affect mature NC integrity, indicating that the NC surface residues may be closely involved in NC uncoating and/or nuclear delivery of RC DNA.

    IMPORTANCE Hepatitis B virus (HBV) infection is a major health issue worldwide. HBV assembly begins with the packaging into immature nucleocapsids (NCs) of a viral RNA pregenome, which is converted to the DNA genome in mature NCs. Mature NCs are then selected for envelopment and secretion as complete virion particles, or alternatively, can deliver their DNA to host cell nucleus to maintain the viral genome as nuclear episomes, which are the basis for virus persistence. Previous studies have identified mutations on the capsid surface that selectively block NC envelopment without affecting NC maturation. We have now discovered that some of these same mutations result in preferential alteration of mature NCs and increased viral nuclear episomes. These findings provide important new insights into the regulation of the two alternative fates of mature NCs and suggest new ways to perturb viral persistence by manipulating levels of viral nuclear episomes.

  • Polyphenols Inhibit Hepatitis C Virus Entry by a New Mechanism of Action [PublishAheadOfPrint]

  • Despite the validation of direct acting antivirals for hepatitis C treatment, discovery of new compounds with different modes of action may still be of importance for the treatment of special patient populations. We recently identified a natural molecule, epigallocatechin-3-gallate (EGCG) as an inhibitor of hepatitis C virus (HCV) targeting the viral particle. The aim of this work was to discover new natural compounds with higher anti-HCV activity than EGCG and determine their mode of action. Eight natural molecules with structure similarity to EGCG were selected. HCV JFH1 in cell culture and HCV pseudoparticle systems were used to determine antiviral activity and mechanism of action of the compounds. We identified delphinidin, a polyphenol belonging to the anthocyanidin family, as a new inhibitor of HCV entry. Delphinidin inhibits HCV entry, in a pan-genotypic manner, by acting directly on the viral particle and impairing its attachment to the cell surface. Importantly, it is also active against HCV in primary human hepatocytes with no apparent cytotoxicity and in combination with interferon and boceprevir in cell culture. Different approaches showed that neither aggregation nor destruction of the particle occurred. Cryo-transmission electron microscopy observations of HCV pseudoparticles treated with delphinidin or EGCG showed a bulge on particles that was not observed in control conditions. In conclusion, EGCG and delphinidin inhibit HCV entry by a new mechanism, i.e. alteration of the viral particle structure that impairs its attachment to the cell surface.

    IMPORTANCE In this article, we identify a new inhibitor of hepatitis C virus (HCV) infection, delphinidin, which prevents HCV entry. This natural compound, a plant pigment responsible for the blue-purple color of flowers and berries, belongs to the flavonoid family, like the catechin EGCG, the major component present in green tea extract, which is also an inhibitor of HCV entry. We studied the mode of action of these two compounds against HCV and demonstrated that they both act directly on the virus, inducing a bulging of the viral envelope. This deformation might be responsible for the inhibition of the virus attachment to the cell surface. The discovery of such anti-HCV inhibitors with an unusual mode of action is important to better characterize the mechanism of HCV entry into hepatocyte and to help developing a new class of anti-HCV entry inhibitors.

  • Type I IFN released by Myeloid Dendritic Cells reversibly impairs Cytomegalovirus replication by inhibiting immediate early gene expression [PublishAheadOfPrint]

  • Cytomegalovirus (CMV) is a ubiquitous beta-herpes virus, whose reactivation from latency is a major cause of morbidity and mortality in immunocompromised hosts. Mouse CMV (MCMV) is a well-established model virus to study virus-host interactions.

    We show in this study that the CD8-independent antiviral function of myeloid dendritic cells (mDC) is biologically relevant for the inhibition of MCMV replication in vivo and in vitro. In vivo ablation of CD11c+ DC resulted in higher viral titers and increased susceptibility to MCMV infection in the first three days post infection. We developed in vitro co-culture systems, where we co-cultivated MCMV infected endothelial or fibroblasts cells with T-cell subsets and/or dendritic cells. While CD8 T-cells failed to control MCMV replication, bone marrow-derived mDC reduced viral titers by a factor of up to 10,000. Contact of mDC with the infected endothelial cells was crucial for their antiviral activity. Soluble factors secreted by the mDC blocked MCMV replication at the level of immediate-early gene expression, yet the viral lytic cycle reinitiated once the mDC were removed from the cells. On the other hand the mDC did not impair MCMV replication in cells deficient for the interferon-aalpha;/bbeta; receptor (IFNAR), arguing that type I interferons (IFN) were critical for viral control by mDC. In light of our recent observation that type I IFN is sufficient for the induction of latency immediately upon infection, our results imply that IFN secreted by mDC may play an important role in the establishment of CMV latency.

    IMPORTANCE Numerous studies have focused on the infection of DC with Cytomegaloviruses, and on the establishment of latency within them. However, almost all of these studies have relied on the infection of DC mono-cultures in vitro, whereas DC are just one among many cell types present in an infection site in vivo. To mimic this aspect of the in vivo situation, we co-cultured DC with infected endothelial cells or fibroblasts. Our data suggest that direct contact with virus-infected endothelial cells activates CD11c+ DC, which leads to reversible suppression of MCMV replication at the level of IE gene expression by a mechanism that depends on type I IFN. The effect matches the formal definition of viral latency. Therefore, our data argue that the interplay of dendritic cells and infected neighboring cells might play an important role in the establishment of viral latency.

  • Transmission of H7N9 influenza viruses with polymorphism at PB2 residue 627 in chickens and ferrets [PublishAheadOfPrint]

  • Poultry exposure is a major risk factor for human H7N9 zoonotic infections of which the mode of transmission remains unclear. We studied the transmission of genetically related poultry and human H7N9 influenza viruses differed by four amino acids including the host determinant PB2 residue 627. A/Silkie Chicken/HK/1772/2014 (SCk1772) and A/HK/3263/14 (HK3263) replicated to comparable titers in chickens with superior oropharyngeal over cloacal shedding; both viruses transmitted efficiently among chickens via direct contact but inefficiently via airborne route. Interspecies transmission via the airborne route was observed for ferrets exposed to the SCk1772 or HK3263 infected chickens while low numbers of copies of influenza viral genome were detected in the air, predominantly at particle sizes larger than 4mmu;m. In ferrets, the human isolate HK3263 replicated to higher titers and transmitted more efficiently via direct contact than SCk1772. We monitored "intra-host" and "inter-host" adaptive changes at PB2 residue 627 during infection and transmission of the SCK1772 that carried E627 and HK3263 that carried V/K/E polymorphism at 60%, 20%, and 20%, respectively. For SCk1772, positive selection for K627 over E627 was observed in ferrets during the chicken-to-ferret or ferret-to-ferret transmission. For HK3263 that contained V/K/E polymorphism, mixed V627 and E627 genotypes were transmitted among chickens while either V627 or K627 was transmitted from chicken-to-ferret or ferret-to-ferret with a narrow transmission bottleneck. Overall, our results suggest direct contact as the main mode for H7N9 transmission and identify the PB2-V627 genotype with uncompromised fitness and transmissibility in both avian and mammalian species.

    IMPORTANCE We studied the modes of H7N9 transmission, as this information is crucial for developing effective control measures for prevention. Using chicken (SCk1772) and human (HK3263) H7N9 isolates that differed by four amino acids, including the host determinant PB2 residue 627, we observed both viruses transmitted efficiently among chickens via direct contact but inefficiently via airborne route. Chicken-to-ferret transmission via airborne route was observed along with the detection of viral genome in the air at low copies. In ferrets, HK3263 transmitted more efficiently than SCk1772 via direct contact. During the transmission of SCk1772 that contained E and HK3263 that contained V/K/E polymorphism at PB2 residue 627, positive selections of E627 and K627 were observed in chickens and ferrets, respectively. In addition, PB2-V627 was transmitted and stably maintained in both avian and mammalian species. Our results support applying intervention strategies that minimize direct and indirect contact at the poultry markets during epidemics.

  • Inhibition of Ebola and Marburg viral entry by G protein-coupled receptor antagonists [PublishAheadOfPrint]

  • Filoviruses, consisting of Ebola virus (EBOV) and Marburg virus (MARV), are among the most lethal infectious threats to mankind. Infections of these viruses can cause severe hemorrhagic fevers in humans and nonhuman primates with high mortality rates. Since there is currently no vaccine or antiviral therapy approved for humans, there is an urgent need to developing prophylactic and therapeutic options for use during filoviral outbreaks and bioterrorist attacks. One of the ideal targets against filoviral infection and diseases is at the entry step, which is mediated by the filoviral glycoprotein (GP). In this report, we screened a chemical library of small molecules and identified numerous inhibitors which are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs including histamine receptors, 5-HT (serotonin) receptors, muscarinic acetylcholine receptor, and adrenergic receptor. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. The time-of-addition experiment and microscopic studies suggest that GPCR antagonists block filoviral entry at a step post the initial attachment but prior to viral/cell membrane fusion. These results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy.

    IMPORTANCE Infection of Ebola virus and Marburg virus can cause severe illness in humans with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The 2013-2015 epedemic in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we have identified numerous inhibitors which are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. Our results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy.

  • Intermolecular Complementation between Two Varicella-Zoster Virus pORF30 Terminase Domains Essential for DNA Encapsidation [PublishAheadOfPrint]

  • The herpesviral terminase complex is part of the intricate machinery that delivers a single viral genome into empty preformed capsids (encapsidation). The Varicella-zoster virus (VZV) terminase components (pORF25, pORF30, and pORF45/42) have not been as extensively studied as those of herpes simplex virus type 1 and human cytomegalovirus. In this report, VZV BACs were generated with small (30S), medium (30M) or large (30L) ORF30 internal deletions. In addition, recombinant viruses were isolated with specific alanine substitutions in the putative zinc finger motif (30-ZF3A) or in a conserved region (region IX) with predicted structural similarity to the human topoisomerase I core subdomains I and II (30-IXAla, 30-620A, 30-622A). Recombinant viruses replicated in an ORF30 complementing cell line (ARPE30) but failed to replicate in non-complementing ARPE19 and MeWo cells. Transmission electron microscopy of 30-IXAla, 30-620A, and 30-622A infected ARPE19 cells revealed only empty VZV capsids. Southern analysis showed that cells infected with parental VZV (VZVLUC) and a repaired virus (30R) contained DNA termini, whereas cells infected with 30L, 30-IXAla, 30-620A, or 30-622A contained little to no processed viral DNA. These results demonstrated that pORF30, and more specifically, amino acids 619-624 (region IX) were required for DNA encapsidation. A luciferase based assay was employed to assess potential intermolecular complementation between the zinc finger domain and conserved region IX. Complementation between 30-ZF3A and 30-IXAla provided evidence that distinct pORF30 domains can function independently. The results suggest that pORF30 may exist as a multimer or participate in higher-order assemblies during viral DNA encapsidation.

    IMPORTANCE Antivirals with novel mechanisms of action are sought as additional therapeutic options to treat human herpesvirus infections. Proteins involved in the viral DNA encapsidation process have become promising antiviral targets. For example, Letermovir is a small molecule drug targeting HCMV terminase that is currently in phase III clinical trials. It is important to define the structural and functional characteristics of proteins that make up viral terminase complexes to identify or design additional terminase specific compounds. The VZV ORF30 mutants described in this study represent the first VZV terminase mutants reported to date. Targeted mutations confirmed the importance of a conserved zinc finger domain found in all herpesvirus ORF30 terminase homologs but also identified a novel, highly conserved region (IX) essential for terminase function. Homology modeling suggested that the structure of region IX is present in all human herpesviruses and thus represents a potential, structurally conserved antiviral target.

  • Protection from severe influenza infections in mice carrying the Mx1 influenza resistance gene strongly depends on genetic background [PublishAheadOfPrint]

  • Influenza infections represent a serious threat to human health. Both extrinsic and intrinsic factors determine the severity of influenza disease. The MX dynamin-like GTPase 1 (Mx1) gene has been shown to confer strong resistance to influenza A virus infections in mice. Most laboratory mouse strains, including C57BL/6J, carry nonsense or deletion mutations in Mx1 and thus a non-functional allele, whereas wild-derived mouse strains carry a wild type Mx1 allele. Congenic C57BL/6J (B6-Mx1r/r) mice expressing a wild type allele from the A2G mouse strain are highly resistant to influenza A infections, to both mono- and poly-basic subtypes. Furthermore, in genetic mapping studies, Mx1 was identified as the major resistance locus to influenza infections. Here, we investigated whether the Mx1 protective function is influenced by the genetic background. For this, we generated a congenic mouse strain carrying the A2G wild type Mx1 resistance allele on a DBA/2J background (D2-Mx1r/r). Most remarkably, congenic D2-Mx1r/r mice expressing a functional Mx1 wild type allele are still highly susceptible to H1N1 virus. However, pre-treatment of D2-Mx1r/r mice with interferon aalpha; protected them from lethal infections. Our results showed, for the first time, that the presence of an Mx1 wild type allele from A2G as such does not fully protect mice from lethal influenza A virus infections. These observations are also highly relevant for susceptibility to influenza infections in humans.

    IMPORTANCE Influenza A virus represents a major health threat to humans. Seasonal influenza epidemics cause high economic loss, morbidity and deaths each year. Genetic factors of the host strongly influence susceptibility and resistance to virus infections. The Mx1 (MX dynamin-like GTPase 1) gene has been described as a major resistance gene in mice and human. Most inbred laboratory mouse strains are deficient in Mx1 but congenic B6-Mx1r/r mice that carry the wild type Mx1 gene from the A2G mouse strain are highly resistant. Here, we show that, very unexpectedly, congenic D2-Mx1r/r mice carrying the wild type Mx1 gene from the A2G strain are not fully protected against lethal influenza infections. These observations demonstrate that the genetic background is very important for the protective function of the Mx1 resistance gene. Our results are also highly relevant for understanding genetic susceptibility to influenza infections in humans.

  • Modulation of glycosaminoglycans affects PrPSc metabolism but does not block PrPSc uptake [PublishAheadOfPrint]

  • Mammalian prions are unconventional infectious agents composed primarily of misfolded aggregated host prion protein PrP, termed PrPSc. Prions propagate by the recruitment and conformational conversion of cellular prion protein into abnormal prion aggregates on the cell surface or along the endocytic pathway. Cellular glycosaminoglycans have been implicated as first attachment sites for prions and cofactors for cellular prion replication. Glycosaminoglycan mimetics and obstruction of glycosaminoglycan sulfation affect prion replication, but the inhibitory effect on different strains and different stages of the cell infection has not been thoroughly addressed. We have examined the effect of a glycosaminoglycan mimetic and undersulfation on cellular prion protein metabolism, prion uptake and the establishment of productive infections in L929 cells by two mouse-adapted prion strains. Surprisingly, both treatments reduced endogenous sulfated glycosaminoglycans but had divergent effects on cellular PrP levels. Chemical or genetic manipulation of glycosaminoglycans did not prevent PrPSc uptake, arguing against their role as essential prion attachment sites. However, both treatments effectively antagonized de novo prion infection independent of the prion strain and reduced PrPSc formation in chronically infected cells. Our results demonstrate that sulfated glycosaminoglycans are dispensable for prion internalization but play a pivotal role in persistently maintained PrPSc formation independent of the prion strain.

    IMPORTANCE Recently, GAGs came into the focus of neurodegenerative disease research as general attachment sites for cell invasion by pathogenic protein aggregates. GAGs influence amyloid formation in vitro. GAGs are also found in intra- and extracellular amyloid deposits. In light of the essential role GAGs play in proteinopathies, understanding the effect of GAGs on protein aggregation and aggregate dissemination is crucial for therapeutic intervention. Here we show that GAGs are dispensable for prion uptake but play essential roles in downstream infection processes. GAG mimetics also affect cellular GAG levels and localization and might thus affect prion propagation by depleting intracellular cofactor pools.

  • Genetic and pathological follow-up study in goats experimentally and naturally exposed to an sheep scrapie isolate. [PublishAheadOfPrint]

  • Thirty-seven goats carrying different prion-protein genotypes (PRNP) were orally infected with a classical scrapie brain homogenate from wildtype (ARQ/ARQ) sheep, and then mated to obtain 2 additional generations of offspring, which were kept in the same environment and allowed to be naturally exposed to scrapie.

    Occurrence of clinical or subclinical scrapie was observed in the experimentally infected goats (F0) and only in one (F1b) of the naturally exposed offspring groups. In both groups (F0 and F1b) goats carrying the R154H, H154H, R211Q and P168Q-P240P dimorphisms died of scrapie after a longer incubation period compared to that of wildtype, G37V, Q168Q-P240P, and S240P. Differently, D145D and Q222K goats were resistant to infection. The immunobiochimical signature of the scrapie isolate and its pathological aspects observed in the sheep donors were substantially maintained over 2 goat generations i.e. after experimental and natural transmission. This demonstrates that the prion protein gene sequence, which is shared by sheep and goats, is more powerful than any possible but unknown species-related factors in determining scrapie phenotypes.

    With regards to genetics, our study confirms that the K222 mutation protects goats even against ovine scrapie isolates and, for the first time, a possible association of D145 mutation with scrapie resistance is shown. In addition, it is possible that the sole diverse frequencies of these genetic variants might, at least in part, shape the prevalence of scrapie among the naturally exposed progenies in the affected herds.

    IMPORTANCE This study was aimed at investigating the genetic and pathological features characterizing sheep to goat interspecies transmission. We show that in goats with different prion protein gene mutations the K222 genetic variant is associated with scrapie resistance after natural and experimental exposure to ovine prion infectivity. In addition, we observed for the first time a protective effect of the D145 goat variant against scrapie. Importantly, our results demonstrate that the phenotypic characteristic of the wildtype sheep scrapie isolate is substantially preserved in goats carrying different susceptible PRNP gene variants, thus indicating that the prion protein gene sequence, which is shared by sheep and goats, plays a fundamental role in determining scrapie phenotypes.

  • Experimental evolution identifies VACV mutations in A24R and A35R that antagonize the PKR pathway and accompany collapse of an extragenic gene amplification [PublishAheadOfPrint]

  • Most new human infectious diseases emerge from cross-species pathogen transmissions; however, it's not clear how viruses adapt to productively infect new hosts. Host restriction factors represent one species-specific barrier that viruses may initially have little ability to inhibit in new hosts. For example, viral antagonists of protein kinase R (PKR) vary in their ability to block PKR-mediated inhibition of viral replication, in part due to PKR allelic variation between species. We previously reported that amplification of a weak PKR antagonist encoded by rhesus cytomegalovirus, rhtrs1, improved replication of a recombinant poxvirus (VVEK+RhTRS1) in several resistant primate cells. To test whether amplification increases the opportunity for mutations to evolve that improve virus replication with only a single copy of rhtrs1, we passaged rhtrs1-amplified viruses in semi-permissive primate cells. After passage, we isolated two viruses that contained only a single copy of rhtrs1 yet replicated as well as the amplified virus. Surprisingly, rhtrs1 was not mutated in these viruses; instead, we identified mutations in two VACV genes, A24R and A35R, either of which was sufficient to improve VVEK+RhTRS1 replication. Neither of these genes has previously been implicated in PKR antagonism. Furthermore, the mutation in A24R, but not A35R, increased resistance to the anti-poxviral drug isatin-bbeta;-thiosemicarbazone, suggesting that these mutations employ different mechanisms to evade PKR. This study supports our hypothesis that gene amplification may provide a "molecular foothold", broadly improving replication to facilitate rapid adaptation while subsequent mutations maintain this efficient replication in the new host without requiring gene amplification.

    IMPORTANCE Understanding how viruses adapt to a new host may help identify viruses poised to cross species barriers before an outbreak occurs. Amplification of rhtrs1, a weak viral antagonist of the host antiviral protein PKR, enabled a recombinant vaccinia virus to replicate in resistant cells from humans and other primates. After serial passage of rhtrs1-amplified viruses, mutations arose in two vaccinia virus genes that improved viral replication without requiring rhtrs1 amplification. Neither of these genes has previously been associated with inhibition of the PKR pathway. These data suggest that gene amplification can improve viral replication in a resistant host species and facilitate the emergence of novel adaptations that maintain the foothold needed for continued replication and spread in the new host.

  • Puzzling origins of the Ebola outbreak in the Democratic Republic of the Congo, 2014 [PublishAheadOfPrint]

  • Maganga et al. (1) and Naccache et al. (2) recently reported the genome sequences of the Zaire Ebolavirus (ZEBOV) that caused the 2014 outbreak in the Democratic Republic of the Congo (COD)....

  • Temporal Characterization of Marburg Angola Infection Following Aerosol Challenge in Rhesus Macaques [PublishAheadOfPrint]

  • Marburg virus (MARV) infection is a lethal hemorrhagic fever for which no licensed vaccines or therapeutics are available. Development of appropriate medical countermeasures requires a thorough understanding of the interaction between the host and the pathogen and the resulting disease course. In this study, fifteen rhesus macaques were sequentially sacrificed following aerosol exposure to MARV variant Angola, with longitudinal changes in physiology, immunology, and histopathology used to assess disease progression. Immunohistochemical evidence of infection and resulting histopathological changes were identified as early as day 3 PE. The appearance of fever in infected animals coincided with the detection of serum viremia and plasma viral genomes on day 4 postexposure (PE). High (ggt;107 PFU/mL) viral loads were detected in all major organs (lung, liver, spleen, kidney, brain, etc.) beginning day 6 PE. Clinical pathology findings included coagulopathy, leukocytosis, and profound liver destruction as indicated by elevated liver transaminases, azotemia, and hypoalbuminemia. Altered cytokine expression in response to infection included early increases in Th2 cytokines such as IL-10 and IL-5 and late stage increases in Th1 cytokines such as IL-2, IL-15, and GM-CSF. This study provides a longitudinal examination of clinical disease of aerosol MARV Angola infection in the rhesus macaque model.

    IMPORTANCE In this study we carefully analyzed the timeline of Marburg virus infection in nonhuman primates in order to provide a well-characterized model of disease progression following an aerosol exposure.

  • Transcriptional Profiling of the Immune Response to Marburg Virus Infection [PublishAheadOfPrint]

  • Marburg virus is a genetically simple RNA virus that causes a severe hemorrhagic fever in humans and nonhuman primates. The mechanism of pathogenesis of the infection is not well understood, but it is well accepted that pathogenesis is appreciably driven by a hyperactive immune response. To better understand the overall response to Marburg virus challenge, we undertook a transcriptomic analysis of immune cells circulating in the blood following aerosol exposure of rhesus macaques to a lethal dose of Marburg virus. Using two-color microarrays, we analyzed the transcriptome of peripheral blood mononuclear cells that were collected throughout the course of infection from 1 to 9 days postexposure, representing the full course of the infection. The response followed a 3-stage induction (early,1nndash;3 days postexposure; mid-infection, 5 days postexposure; late infection, 7nndash;9 days postexposure) that was led by a robust innate immune response. The host response to aerosolized Marburg was evident at 1 day postexposure. Analysis of cytokine transcripts that were overexpressed during infection indicated that previously unanalyzed cytokines are likely induced in response to exposure to Marburg virus, and further suggested that the early immune response is skewed towards a Th2 response that would hamper the development of an effective antiviral immune response early in disease. Late infection events included the up-regulation of coagulation associated factors. These findings demonstrate very early host responses to Marburg virus infection and provide a rich dataset of factors expressed throughout the course of infection that can be investigated as markers of infection and targets for therapy.

    IMPORTANCE Marburg virus causes a severe infection that is associated with high mortality and hemorrhage. The disease is associated with an immune response that contributes to the lethality of the disease. In this study we have investigated how the immune cells circulating in the blood of infected primates respond following exposure to Marburg virus. Our results show that there are three discernable stages of response to infection that correlate with pre-symptomatic, early, and late symptomatic stages of infection, a response format similar to that seen following challenge with other hemorrhagic fever viruses. In contrast to the ability of the virus to block innate immune signaling in vitro, the earliest and most sustained response is an interferon-like response. Our analysis also identifies a number of cytokines that are transcriptionally upregulated during late stages of infection and suggest that there is a Th2-skewed response to infection. When correlated with companion data describing the animal model from which our samples were collected our results suggest that the innate immune response may be contributing to overall pathogenesis.

  • Redundant Function of Plasmacytoid and Conventional Dendritic Cells is Required to Survive a Natural Viral Infection. [PublishAheadOfPrint]

  • Viruses that spread systemically from a peripheral site of infection cause morbidity and mortality in the human population. Innate myeloid cells, including monocytes, macrophages, monocyte-derived dendritic cells (mo-DC), and dendritic cells (DC), respond early during viral infection to control viral replication, reducing virus spread from the peripheral site. Ectromelia virus (ECTV), an orthopoxvirus that naturally infects the mouse, spreads systemically from the peripheral site of infection and results in death of susceptible mice. While phagocytic cells have a requisite role in the response to ECTV, the requirement of individual myeloid cell populations during acute immune responses to peripheral viral infection is unclear. In this study, a variety of myeloid-specific depletion methods were used to dissect the role of individual myeloid cell subsets in the survival of ECTV infection. We showed that DC are the primary producers of Type I interferon (T1-IFN), a requisite cytokine for survival, following ECTV infection. DC, but not macrophages, monocytes or granulocytes were required for control of the virus and survival of mice following ECTV infection. Depletion of either plasmacytoid DC (pDC) alone or the lymphoid-resident DC subset (CD8aalpha;+ DC) alone did not confer lethal susceptibility to ECTV. However, the function of at least one of the pDC or CD8aalpha;+ DC subsets is required for survival of ECTV infection, as mice depleted of both populations were susceptible to ECTV challenge. The presence of at least one of these DC subsets is sufficient for cytokine production that reduces ECTV replication and virus spread, facilitating survival following infection.

    IMPORTANCE Prior to the eradication of variola virus, the orthopoxvirus that causes smallpox, one-third of infected people succumbed to the disease. Following successful eradication of smallpox, vaccination rates with the smallpox vaccine have significantly dropped. There is now an increasing incidence of zoonotic orthopoxvirus infections, for which there are no effective treatments. Moreover, the safety of the smallpox vaccine is of great concern as complications may arise resulting in morbidity. Like many viruses that cause significant human diseases, orthopoxviruses spread from a peripheral site of infection to become systemic. This study elucidates the early requirement of innate immune cells in controlling a peripheral infection with ECTV, the causative agent of mousepox. We report that there is redundancy in the function of two innate immune cell subsets in controlling the virus spread early during infection. The viral control mediated by these cell subsets presents a potential target for therapies and rational vaccine design.

  • Molecular Epidemiology and Evolution of Influenza Viruses Circulating within European Swine between 2009 and 2013 [PublishAheadOfPrint]

  • The emergence in humans of the A(H1N1)pdm09 influenza virus, a complex reassortant virus of swine origin, highlighted the importance of worldwide influenza virus surveillance in swine. To date, large-scale surveillance studies have been reported for southern China and North America, but such data has not yet been described for Europe. We report the first large-scale genomic characterization of 290 swine influenza viruses collected from 14 European countries between 2009 and 2013. 23 distinct genotypes were identified, with the seven most common comprising 82% of the incidence. Contrasting epidemiological dynamics were observed for two of these genotypes, H1huN2 and H3N2, with the former showing multiple long-lived geographically-isolated lineages, whilst the latter had short-lived geographically-diffuse lineages. At least 32 human-swine transmission events have resulted in A(H1N1)pdm09 becoming established at a mean frequency of 8% across European countries. Notably, swine in the UK have largely had a replacement of the endemic Eurasian llsquo;avian-like' genotypes with A(H1N1)pdm09-derived genotypes. The high number of reassortant genotypes observed in European swine, combined with the identification of a genotype similar to the A(H3N2)v in North America, underlines the importance of continued swine surveillance in Europe for the purposes of public health. This study further reveals that the emergence and drivers of virus evolution in swine differ at a global level.

    IMPORTANCE The influenza A(H1N1)pdm09 virus contains a reassortant genome with segments derived from separate virus lineages that evolved in different regions of world. In particular its neuraminidase and matrix segments were derived from the Eurasian llsquo;avian-like' lineage that emerged in European swine in the 1970s. However, while large-scale genomic characterization of swine has been reported for southern China and North America, no equivalent study has yet been reported for Europe. Surveillance of swine herds across Europe between 2009 and 2013 revealed that the A(H1N1)pdm09 virus is established in European swine, increasing the number of circulating lineages in the region and increasing the possibility of the emergence of a genotype with human pandemic-potential. It also has implications for veterinary health, making prevention through vaccination more challenging. The identification of a genotype similar to the A(H3N2)v, causing zoonoses at North American agricultural fairs, underlines the importance of continued genomic characterization in European swine.

  • Reduced SIV replication in macrophages of sooty mangabeys is associated with increased expression of host restriction factors. [PublishAheadOfPrint]

  • Macrophages are target cells of HIV/SIV infection that may play a role in AIDS pathogenesis and contribute to the long-lived reservoir of latently infected cells during antiretroviral therapy (ART). In previous work, we and others have shown that during pathogenic SIV infection of rhesus macaques (RMs), rapid disease progression is associated with high levels of in vivo macrophage infection. In contrast, during nonpathogenic SIV infection of sooty mangabeys (SMs), neither spontaneous nor experimental CD4+ T cell depletion results in substantial levels of in vivo macrophage infection. To test the hypothesis that SM macrophages are intrinsically more resistant to SIV infection than RM macrophages, we undertook an in vitro comparative assessment of monocyte-derived macrophages (MDMs) from both nonhuman primate species. Using the primary isolate SIVM949, which replicates well in lymphocytes from both RMs and SMs, we found that infection of RM macrophages resulted in persistent SIV-RNA production while SIV-RNA levels in SM macrophage cultures decreased 10- to 100-fold over a similar temporal course of in vitro infection. To explore potential mechanisms responsible for the lower levels of SIV replication and/or production in macrophages from SMs we comparatively assessed, in the two studied species, the expression of the SIV co-receptor as well as the expression of a number of host restriction factors. While previous studies showed that SM monocytes express lower levels of CCR5 (but not CD4) than RM monocytes, the level of CCR5 expression in MDMs was similar in the two species. Interestingly, we found that SM macrophages exhibited a significantly greater increase in the expression of tetherin (P=0.003) and TRIM22 (P=0.0006) in response to interferon (IFN)-aalpha; stimulation and increased expression of multiple host restriction factors in response to lipopolysaccharide (LPS) stimulation and exposure to SIV. Overall these findings confirm, in an in vitro infection system, that SM macrophages are relatively more resistant to SIV infection compared to RM macrophages, and suggest that a combination of entry and post-entry restriction mechanisms may protect these cells from productive SIV infection.

    IMPORTANCE This manuscript represents the first in vivo comparative analysis of monocyte-derived macrophages (MDMs) between rhesus macaques, i.e., experimental SIV hosts in which the infection is pathogenic and macrophages can be infected, and sooty mangabeys, i.e., natural SIV hosts in which the infection is non-pathogenic and macrophages are virtually never infected in vivo. This study demonstrates that mangabey-derived MDMs are more resistant to SIV infection in vitro as compared to macaque-derived MDMs, and provides a potential explanation for this observation by showing increased expression of specific retrovirus restriction factors in mangabey-derived macrophages. Overall, this study is important as it contributes to our understanding of why SIV infection is non-pathogenic in sooty mangabeys while it is pathogenic in macaques, and is consistent with a pathogenic role for in vivo macrophage infection during pathogenic lentiviral infection.

  • Apolipoprotein E, but not Apolipoprotein B, is essential for efficient HCV cell-to-cell transmission [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) infects hepatocytes through two different routes: cell-free particle diffusion followed by engagement with specific cellular receptors, and cell-to-cell direct transmission mediated by mechanisms not well defined yet. HCV exits host cells in association with very low-density lipoproteins (VLDL). VLDL particles contain apolipoprotein B (ApoB) and E (ApoE), which are required for viral assembly and/or infectivity. Based on these precedents, we decided to study whether these VLDL components participate in HCV cell-to-cell transmission in vitro. We observed that cell-to-cell viral spread was compromised after ApoE interference in donor but not in acceptor cells. In contrast, ApoB knockdown in either donor or acceptor cells did not impair cell-to-cell viral transmission. Interestingly, ApoB participated in the assembly of cell-free infective virions, suggesting a differential regulation of cell-to-cell and cell-free HCV infection. This study identifies host-specific factors involved in these distinct routes of infection that may unveil new therapeutic targets and advance our understanding of HCV pathogenesis.

    IMPORTANCE This work demonstrates that cell-to-cell transmission of HCV depends on ApoE but not ApoB. The data also indicate that ApoB is required for the assembly of cell-free infective particles, strongly suggesting the existence of mechanisms involving VLDL components that differentially regulate cell-free and cell-to-cell HCV transmission. These data clarify some of the questions regarding the role of VLDL in HCV pathogenesis and the transmission of the virus cell to cell as a possible mechanism of immune evasion and opens the door to therapeutic intervention.

  • Cellular protein WDR11 interacts with specific herpes simplex virus proteins at the trans-Golgi network to promote virus replication [PublishAheadOfPrint]

  • It has recently been proposed that the herpes simplex virus (HSV) protein ICP0 has cytoplasmic roles in blocking antiviral signaling and in promoting viral replication in addition to its well-known proteasome-dependent functions in the nucleus. However, the mechanisms through which it produces these effects remain unclear. While investigating this further, we identified a novel cytoplasmic interaction between ICP0 and the poorly characterized cellular protein WDR11. During an HSV infection, WDR11 undergoes a dramatic change in localization at late times in the viral replication cycle, moving from defined perinuclear structures to a dispersed cytoplasmic distribution. While this relocation was not observed during infection with viruses other than HSV-1 and correlated with efficient HSV-1 replication, the redistribution was found to occur independently of ICP0 expression, instead requiring viral late gene expression. We demonstrate for the first time that WDR11 is localized to the trans-Golgi network (TGN), where it interacts specifically with some, but not all, HSV virion components, in addition to ICP0. Knockdown of WDR11 in cultured human cells resulted in a modest but consistent decrease in yields of both wildtype and ICP0-null viruses, in the supernatant and cell-associated fractions, without affecting viral gene expression. Although further study is required, we propose that WDR11 participates in viral assembly and/or secondary envelopment.

    IMPORTANCE While the TGN has been proposed to be the major site of secondary envelopment, this process is incompletely understood, and in particular, the role of cellular TGN components in this pathway is unknown. Additionally, little is known about the cellular functions of WDR11, although the disruption of this protein has been implicated in multiple human diseases. Therefore, our finding that WDR11 is a TGN-resident protein that interacts with specific viral proteins to enhance viral yields improves both our understanding of basic cellular biology as well as how this is co-opted by HSV.

  • HIV Vpu interferes with NF-{kappa}B activity but not with Interferon Regulatory Factor 3 [PublishAheadOfPrint]

  • The accessory HIV protein Vpu inhibits a number of cellular pathways that trigger host innate restriction mechanisms. HIV Vpu mediated degradation of Tetherin allows efficient particle release and hampers the activation of the NF-B pathway thereby limiting the expression of proinflammatory genes. In addition, Vpu reduces cell surface expression of several cellular molecules such as newly synthesized CD4. However the role of HIV Vpu in regulating the type 1 interferon response to viral infection by degradation of the Interferon Regulatory Factor 3 (IRF3) has been subject of conflicting reports. We therefore systematically investigated the expression of IRF3 in primary CD4+ T cells and macrophages infected with HIV at different time points. In addition, we also tested the ability of Vpu to interfere with innate immune signaling pathways such as the NF-B and the IRF3 pathways. Here we report that HIV Vpu failed to degrade IRF3 in infected primary cells. Moreover, we observed that HIV NL4.3 Vpu had no effect on IRF3 dependent gene expression in reporter assays. On the other hand, HIV NL4.3 Vpu down-modulated NF-B dependent transcription. Mutation of two serines (position 52 and 56) involved in the binding of NL4.3 Vpu to the bbeta;TrCP ubiquitin ligase abolishes its ability to inhibit NF-B activity. Taken together, these results suggest that HIV Vpu regulates antiviral innate response in primary human cells by acting specifically on the NF-B pathway.

    IMPORTANCE HIV Vpu plays a pivotal role in enhancing HIV infection by counteraction of Tetherin. However, Vpu also regulates host response to HIV infection by hampering the type 1 interferon response. The molecular mechanism by which Vpu inhibits the interferon response is still controversial. Here we report that Vpu affects interferon expression by inhibiting NF-B activity without affecting IRF3 levels or activity. These data suggest that Vpu facilitates HIV infection by regulating NF-B transcription to levels sufficient for viral transcription while limiting cellular responses to infection.

  • Differential ability of primary HIV-1 Nef isolates to down-regulate HIV-1 entry receptors [PublishAheadOfPrint]

  • HIV-1 Nef down-regulates the viral entry receptor CD4 as well as the co-receptors CCR5 and CXCR4 from the surface of HIV-infected cells, leading to promotion of viral replication through super-infection resistance and other mechanisms. Nef sequence motifs that modulate these functions have been identified via in vitro mutagenesis of laboratory HIV-1 strains. However, it remains unclear whether the same motifs contribute to Nef activity in patient-derived sequences, and whether these motifs may differ in Nef sequences isolated at different infection stages and/or from patients with different disease phenotypes. Here, nef clones from 45 elite controllers (EC), 46 chronic progressors (CP), and 43 acute progressors (AP) were examined for their CD4, CCR5, and CXCR4 down-regulation functions. Nef clones from EC exhibited statistically significantly impaired CD4 and CCR5 down-regulation ability, and modestly impaired CXCR4 down-regulation activity, compared to those from CP and AP. Nef's ability to down-regulate CD4 and CCR5 correlated positively in all cohorts, suggesting that they are functionally linked in vivo. Moreover, impairments in Nef's receptor down-regulation functions increased the susceptibility of Nef-expressing cells to HIV-1 infection. Mutagenesis studies on three functionally-impaired EC Nef clones revealed that multiple residues, including those at novel sites, were involved in the alteration of Nef functions and steady-state protein levels. Specifically, polymorphisms at highly conserved tryptophan residues (e.g., Trp-57 and Trp-183) and immune-escape associated sites were responsible for reduced Nef functions in these clones. Our results suggest that the functional modulation of primary Nef sequences is mediated by complex polymorphism networks.

    IMPORTANCE HIV-1 Nef, a key factor for viral pathogenesis, down-regulates functionally important molecules from the surface of infected cells, including the viral entry receptor CD4 and co-receptors CCR5 and CXCR4. This activity enhances viral replication by protecting infected cells from cytotoxicity associated with superinfection, and may also serve as an immune evasion strategy. However, how these activities are maintained under selective pressure in vivo remains elusive. We addressed this question by analyzing functions of primary Nef clones isolated from patients at various infection stages and with different disease phenotypes including elite controllers, who spontaneously control HIV-1 viremia to undetectable levels. The results indicate that down-regulation of HIV-1 entry receptors, particularly CCR5, was impaired in Nef clones from elite controllers. These functional impairments were driven by rare Nef polymorphisms and adaptations associated with cellular immune responses, underscoring the complex molecular pathways responsible for maintaining and attenuating viral protein function in vivo.

  • Distribution and Redistribution of HIV-1 Nucleocapsid Protein in Immature, Mature, and Integrase-inhibited Virions: A Role for Integrase in Maturation [PublishAheadOfPrint]

  • During virion maturation, HIV-1 capsid protein assembles into a conical core containing the viral ribonucleoprotein (vRNP) complex, thought to be composed mainly of the viral RNA and nucleocapsid protein (NC). After infection, the viral RNA is reverse-transcribed into double-stranded DNA, which is then incorporated into host chromosomes by integrase (IN) catalysis. Certain IN mutations (class II) and antiviral drugs (allosteric IN inhibitors, ALLINIs) adversely affect maturation, resulting in virions that contain "eccentric condensates", electron-dense aggregates located outside seemingly empty capsids. Here we demonstrate that in addition to this mislocalization of electron density, a class II IN mutation and ALLINIs each increase the fraction of virions with malformed capsids (from ~12% to ~53%). Eccentric condensates have a high NC content as demonstrated by "tomo-bubblegram" imaging, a novel labeling technique that exploits NC's susceptibility to radiation damage. Tomo-bubblegrams also localized NC inside wild-type cores and lining the spherical Gag shell in immature virions. We conclude that eccentric condensates represent non-packaged vRNPs and that either genetic or pharmacological inhibition of IN can impair vRNP incorporation into mature cores. Supplying IN in trans as part of a Vpr-IN fusion protein partially restored the formation of conical cores with internal electron density and the infectivity of a class II IN deletion mutant virus. Moreover the ability of ALLINIs to induce eccentric condensate formation required both IN and viral RNA. Based on these observations, we propose a role for IN in initiating core morphogenesis and vRNP incorporation into the mature core during HIV-1 maturation.

    IMPORTANCE Maturation, a process essential for HIV-1 infectivity, involves core assembly, whereby the viral ribonucleoprotein (vRNP, composed by vRNA and nucleocapsid protein (NC)) is packaged into a conical capsid. Allosteric integrase inhibitors (ALLINIs) affect multiple viral processes. We have characterized ALLINIs and integrase mutants that have the same phenotype. First, comparing the effect of ALLINIs on several steps of the viral cycle, we show that inhibition of maturation accounts for compound potency. Second, using cryo-electron tomography we find that ALLINIs impair conical capsid assembly. Third, developing tomo-bubblegram imaging nndash; which specifically labels NC protein nndash; we find that ALLINIs block vRNP packaging; instead, vRNPs form "eccentric condensates" outside the core. Fourth, malformed cores, typical of integrase-deleted virus, are partially replaced by conical cores when integrase is supplied in trans. Fifth, vRNA is necessary for ALLINI-induced eccentric condensate formation. These observations suggest that integrase is involved in capsid morphogenesis and vRNP packaging.

  • Reactivation kinetics of HIV-1 and susceptibility of reactivated latently infected CD4+ T cells to HIV-1-specific CD8+ T cells [PublishAheadOfPrint]

  • The "shock and kill" model of HIV-1 eradication involves the induction of transcription of HIV-1 genes in latently infected CD4+ T cells followed by the elimination of these infected CD4+ T cells by CD8+ T cells or other effector cells. CD8+ T cells may also be needed to control the spread of new infection if residual infected cells are present at the time cART is discontinued. In order to determine the time frame needed for CD8+ T cells to effectively prevent the spread of HIV-1 infection, we examined the kinetics of HIV transcription and virus release in latently infected cells reactivated ex vivo. Isolated resting, primary CD4+ T cells from HIV+ subjects on suppressive cART regimens were found to upregulate cell-associated HIV-1 mRNA within 1 hour of stimulation and produce extracellular virus as early as 6 hours post-stimulation. In spite of the rapid kinetics of virus production, we show that CD8+ T cells from 2 out of 4 viremic controllers were capable of effectively eliminating reactivated autologous CD4+ cells that upregulate cell associated HIV-1 mRNA. The results have implications for devising strategies to prevent rebound viremia due to reactivation of rare latently infected cells that persist after potentially curative therapy.

    IMPORTANCE A prominent HIV-1 cure strategy termed "shock and kill" involves the induction of HIV-1 transcription in latently infected CD4+ T cells with the goal of elimination of these cells by either the cytotoxic T lymphocyte response or other immune cell subsets. However, the cytotoxic T cell response may also be required post-curative treatment if residual latently infected cells remain. The kinetics of HIV-1 reactivation indicate rapid upregulation of cell-associated HIV-1 mRNA and a 5-hour window between transcription and virus release. Thus, HIV-specific CD8+ T cell responses likely have a very short time frame to eliminate residual latently infected CD4+ T cell that become reactivated after discontinuation of antiretroviral therapy following potentially curative treatment strategies.

  • Structures of adenovirus incomplete particles clarify capsid architecture and show maturation changes of packaging protein L1 52/55k [PublishAheadOfPrint]

  • Adenovirus is one of the most complex icosahedral, non-enveloped viruses. Even after its structure was solved at near atomic resolution by both cryo-electron microscopy and X-ray crystallography, the location of minor coat proteins is still a subject of debate. The elaborated capsid architecture is the product of a correspondingly complex assembly process, of which many aspects remain unknown. Genome encapsidation involves the concerted action of five virus proteins, and proteolytic processing by the virus protease is needed to prime the virion for sequential uncoating. Protein L1 52/55k is required for packaging, and multiple cleavages by the maturation protease facilitate its release from the nascent virion. Light density particles are routinely produced in adenovirus infections, and thought to represent assembly intermediates. Here we present the molecular and structural characterization of two different types of human adenovirus light particles produced by a mutant with delayed packaging. We show that these particles lack core polypeptide V but do not lack the density corresponding to this protein in the X-ray structure, therefore adding support to the adenovirus cryo-electron microscopy model. The two types of light particles present different degrees of proteolytic processing. Their structure provides the first glimpse on the organization of L1 52/55k protein inside the capsid shell, and on how this organization changes upon partial maturation. Immature, full length L1 52/55k is poised beneath the vertices to engage the virus genome. Upon proteolytic processing, L1 52/55k disengages from the capsid shell, facilitating genome release during uncoating.

    IMPORTANCE Adenoviruses have been extensively characterized as experimental systems in molecular biology, human pathogens and therapeutic vectors. However, a clear picture of many aspects in their basic biology is still lacking. Two of these aspects are the location of minor coat proteins in the capsid, and the molecular details of capsid assembly. Here we provide evidence supporting one of the two current models for capsid architecture. We also show for the first time the location of the packaging protein L1 52/55k in particles lacking the virus genome, and how this location changes during maturation. Our results contribute to clarify standing questions in adenovirus capsid architecture and provide new details on the role of L1 52/55k protein in assembly.

  • Vaccination with a Live Attenuated Cytomegalovirus Devoid of a Protein Kinase R Inhibitory Gene Results in Reduced Maternal Viremia and Improved Pregnancy Outcome in a Guinea Pig Congenital Infection Model [PublishAheadOfPrint]

  • Background. Development of a vaccine to prevent congenital cytomegalovirus infection is a major public health priority. Live vaccines attenuated through mutations targeting viral mechanisms responsible for evasion of host defense may be both safe and efficacious.

    Methods. Safety and vaccine efficacy was evaluated using a guinea pig cytomegalovirus (GPCMV) model. Recombinant GPCMV 145 was generated with a targeted deletion of gp145, a viral protein kinase R (PKR) inhibitor. Attenuation was evaluated following inoculation of 107 pfu of 145 or parental virus into guinea pigs immunosuppressed with cyclophosphamide. Efficacy was evaluated by immunizing GPCMV-naive guinea pigs twice with either 105 or 106 pfu of 145, establishing pregnancy, and challenging with salivary gland-adapted GPCMV. Immune response, maternal viral load, pup mortality, and congenital infection rates were compared in vaccine and control groups.

    Results. 145 was substantially attenuated for replication in immunocompromised guinea pigs. Vaccination with 145 induced ELISA and neutralizing antibody levels comparable to natural infection. In the higher- and lower-dose vaccine groups, pup mortality was reduced to 1/24 (4%) and 4/29 (14%), respectively, compared to 26/31 (81%) in unvaccinated controls (both groups pllt;0.0001 vs. control). Congenital infection occurred in 20/31 (65%) of control pups compared to 8/24 (33%) pups in the 106 vaccination group (pllt;0.05). Significant reductions in the magnitude of maternal DNAemia and pup viral load were noted in the vaccine groups, compared to controls.

    Conclusion. Deletion of a GPCMV-encoded PKR inhibitor results in a highly attenuated virus that is immunogenic and protective as a vaccine against transplacental infection.

    IMPORTANCE Previous attempts to develop a successful immunization against cytomegalovirus have largely centered on subunit vaccination against virion proteins, but have yielded disappointing results. The advent of BAC technologies has enabled engineering of recombinant CMVs, deleted of virally encoded immune modulation genes, toward the goal of developing a safe and potentially more efficacious live attenuated vaccine. Here we report studies of such a vaccine against congenital CMV infection, based on a virus with a targeted deletion in gp145, a virally-encoded inhibitor of protein kinase R, using the guinea pig model of vertical CMV transmission. The deletion virus was attenuated for dissemination in immunocompromised guinea pigs, but elicited ELISA and neutralizing responses. The vaccine conferred protection against maternal DNAemia and congenital transmission, and resulted in reduced viral load in newborn guinea pigs. These results provide support for future studies of attenuated CMV vaccines.

  • pUL69 of human cytomegalovirus recruits the cellular protein arginine methyltransferase 6 via a domain that is crucial for mRNA export and efficient viral replication [PublishAheadOfPrint]

  • The regulatory protein pUL69 of human cytomegalovirus acts as a viral mRNA export factor facilitating the cytoplasmic accumulation of unspliced RNA via interaction with the cellular mRNA export factor UAP56. Here we provide evidence for a post-translational modification of pUL69 via arginine methylation within the functionally important N-terminus. Firstly, we demonstrate a specific immunoprecipitation of full-length pUL69 as well as pUL69aa1-146 by a mono/dimethylarginine-specific antibody. Second, we observed a specific electrophoretic mobility shift upon overexpression of catalytically active protein arginine methyltransferase 6 (PRMT6). Third, a direct interaction of pUL69 and PRMT6 could be confirmed by yeast two-hybrid and coimmunoprecipitation analyses. We mapped the PRMT6 interaction motif to the pUL69 N-terminus and identified critical amino acids within the arginine-rich R1 box of pUL69 that were crucial for PRMT6- and/or UAP56-recruitment. In order to test the impact of putative methylation substrates on the functions of pUL69 we constructed various pUL69-derivatives harboring arginine to alanine substitutions and tested them for RNA export activity. Thus, we were able to discriminate between arginines within the R1 box of pUL69 that were crucial for UAP56/PRMT6-interaction and/or mRNA export activity. Remarkably, NMR-analyses revealed the same aalpha;-helical structures for pUL69-peptides encoding either the wild type R1/R2 boxes or a UAP56/PRMT6-binding deficient derivative, thereby excluding that R/A amino acid substitutions within R1 affected the secondary structure of pUL69. We therefore conclude that the pUL69 N-terminus is methylated by PRMT6 and this critically affects the functions of pUL69 for efficient mRNA export and replication of human cytomegalovirus.

    IMPORTANCE The UL69 protein of human cytomegalovirus is a multifunctional regulatory protein that acts as a viral RNA export factor with a critical role for efficient replication. Here, we demonstrate that pUL69 is posttranslationally modified via arginine methylation and that the protein methyltransferase PRMT6 mediates this modification. Furthermore, arginine residues with a crucial function for RNA export and for binding of the cellular RNA export factor UAP56 as well as PRMT6 were mapped within the arginine-rich R1 motif of pUL69. Importantly, we demonstrated that mutation of those arginines did not alter the secondary structure of R1, altogether suggesting that they may serve as critical methylation substrates. In summary, our study reveals a novel posttranslational modification of pUL69 which has significant impact on the function of this important viral regulatory protein. Since PRMTs appear to be amenable to selective inhibition by small molecules this may constitute a novel target for antiviral therapy.

  • Efficacy of HSV-2 Glycoprotein D/AS04 vaccine against genital HSV-2 and HSV-1 infection and disease in the cotton rat Sigmodon hispidus model [PublishAheadOfPrint]

  • Subunit vaccines based on the HSV-2 glycoprotein D (gD-2) have been the major focus of HSV-2 vaccine development for the past two decades. Based on the promising data generated in the guinea pig model, a formulation containing truncated gD-2, aluminium salt, and MPL (gD/AS04) advanced to clinical trials. The results of these trials, however, were unexpected, as the vaccine protected against HSV-1 infection, but not against HSV-2. To address this discrepancy, we developed a Depot medroxyprogesterone (DMPA) treated cotton rat S. hispidus model of HSV-2 and HSV-1 genital infection. Severity of HSV-1 genital herpes was less compared to HSV-2 in cotton rats, yet the model allowed for comparative evaluation of gD/AS04 immunogenicity and efficacy. Cotton rats were intramuscularly vaccinated using a prime boost strategy with gD/AS04 (Simplirixttrade; vaccine) or control vaccine formulation (hepatitis B vaccine FENDrixttrade;) and subsequently challenged intravaginally with HSV-2 or HSV-1. gD/AS04 vaccine was immunogenic in cotton rats, induced serum IgG directed against gD-2 and serum HSV-2 neutralizing antibodies, but failed to efficiently protect against HSV-2 disease or to decrease HSV-2 viral load. However, gD/AS04 significantly reduced vaginal titers of HSV-1 and better protected animals against HSV-1 compared to HSV-2 genital disease. The latter finding is generally consistent with the clinical outcome of Herpevac trial of Simplirixttrade;. Passive transfer of serum from gD/AS04-immunized cotton rats conferred stronger protection against HSV-1 genital disease. These findings suggest the need for alternative vaccine strategies and the identification of new correlates of protection.

    IMPORTANCE In spite of the high health burden of genital herpes, there is still no effective intervention against the disease. The significant gap in knowledge on genital herpes pathogenesis has been further highlighted by the recent failure of GSK HSV-2 vaccine Simplirixttrade; (gD/AS04) to protect humans against HSV-2 and the surprising finding that the vaccine protected against HSV-1 genital herpes instead. In this paper we report that gD/AS04 has higher efficacy against HSV-1 compared to HSV-2 genital herpes in the novel DMPA-synchronized cotton rat model of HSV-1 and HSV-2 infection. The findings help explain the results of the Simplirixttrade; trial.

  • Simian Varicella Virus is Present in Macrophages, Dendritic Cells and T Cells in Lymph Nodes of Rhesus Macaques after Experimental Reactivation [PublishAheadOfPrint]

  • Like varicella zoster virus (VZV), simian varicella virus (SVV) reactivates to produce zoster. Herein, 5 rhesus macaques were inoculated intrabronchially with SVV and 5 months later, 4 monkeys were immunosuppressed; one monkey was not immunosuppressed, but was subjected to the stress of transportation. In 4 monkeys, zoster rash developed 7-12 weeks after immunosuppression, and rash also developed in the monkey that was not immunosuppressed. Analysis at 24-48 h after zoster revealed SVV antigen in the lung alveolar wall, in ganglionic neurons and non-neuronal cells, in skin and in lymph nodes. In skin, SVV was found primarily in sweat glands. In lymph nodes, SVV antigen co-localized mostly with macrophages, dendritic cells and, to a lesser extent, with T cells. The presence of SVV in lymph nodes, as verified by qPCR detection of SVV DNA, might reflect sequestration of virus by macrophages and dendritic cells in lymph nodes or presentation of viral antigens to T cells to initiate an immune response against SVV or both.

    IMPORTANCE VZV causes varicella (chickenpox), becomes latent in ganglia, and reactivates to produce zoster and multiple other serious neurological disorders. SVV in non-human primates has proved to be a useful model that parallels VZV pathogenesis in humans. Here, we show that SVV antigens are present in sweat glands in skin and in macrophages and dendritic cells in lymph nodes after SVV reactivation in monkeys, raising the possibility that macrophages and dendritic cells in lymph nodes serve as antigen-presenting cells to activate T cell responses against SVV after reactivation.

  • Critical Role for the Adenosine Pathway in Controlling SIV-Related Immune Activation and Inflammation in Gut Mucosal Tissues [PublishAheadOfPrint]

  • The role of adenosine (ADO) pathway in HIV-1/SIV infection remains unclear. We compared SIVsab-induced changes of markers related to ADO production (CD39 and CD73) and breakdown (CD26 and adenosine deaminase) on T cells from blood, lymph nodes, and intestine collected from pigtailed macaques (PTMs) and African green monkeys (AGMs) that experience different SIVsab infection outcomes. We also measured ADO and inosine (INO) levels in tissues by mass spectrometry. Finally, we assessed the suppressive effect of ADO on proinflammatory cytokine production after T cell receptor stimulation. The baseline level of both CD39 and CD73 coexpression on regulatory T cells and ADO levels were higher in AGMs than PTMs. Conversely, high INO levels associated with dramatic increases in CD26 expression and adenosine deaminase activity were observed in PTMs during chronic SIV infection. Immune activation and inflammation markers in the gut and periphery inversely correlated with ADO and directly correlated with INO. Ex vivo administration of ADO significantly suppressed proinflammatory cytokine production by T cells in both species. In conclusion, the opposite dynamics of ADO pathway-related markers and contrasting ADO/INO levels in species with divergent proinflammatory responses to SIV infection support a key role of ADO in controlling immune activation/inflammation in nonprogressive SIV infections. Changes in ADO levels predominately occurred in the gut, suggesting that the ADO pathway may be involved in sparing nonhuman primates from developing SIV-related gut dysfunction and warrant focus of studies of ADO pathway to mucosal sites of viral replication.

    IMPORTANCE The mechanisms responsible for the severe gut dysfunction characteristic to progressive HIV and SIV infection in humans and macaques are not completely elucidated. We report that ADO may play a key role in controlling immune activation/inflammation in nonprogressive SIV infections by limiting SIV-related gut inflammation. Conversely, in progressive SIV infection, significant degradation of ADO occur possibly due to an early increase of ADO deaminase complexing protein 2 (CD26) and adenosine deaminase. Our study supports therapeutic interventions to offset alterations of this pathway during progressive HIV/SIV infections. These potential approaches to control chronic immune activation and inflammation during pathogenic SIV infection may prevent HIV disease progression.

  • Latency expression of the Epstein-Barr virus-encoded MHC class I TAP inhibitor, BNLF2a in EBV-positive gastric carcinomas. [PublishAheadOfPrint]

  • The EBV BNLF2a gene product provides immune evasion properties to infected cells through inhibition of TAP-mediated transport of antigen peptides. Although BNLF2a is considered to be a lytic gene, we demonstrate that it is expressed in nearly half of the EBV-associated gastric carcinomas analyzed. Further, we show that BNLF2a expression is dissociated from lytic gene expression. BNLF2a is therefore expressed in this latency setting, potentially helping protect the infected tumor cells from immuno-surveillance.

  • Similarities between HIV-1 and HCV genetic and phenotypic protease quasispecies diversity [PublishAheadOfPrint]

  • Human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) are two highly variable RNA viruses that cause chronic infections in humans. Although HCV likely preceded the AIDS epidemic by some decades, the global spread of both viruses is a relatively recent event. Nevertheless, HCV global diversity is higher than that of HIV-1. To identify differences in mutant diversity, we compared the HIV-1 protease and HCV NS3 protease quasispecies. Three protease gene quasispecies samples per virus, isolated from a total of six infected patients, were genetically and phenotypically analyzed at high resolution (HIV-1, 308 individual clones; HCV, 299). Single nucleotide variant frequency did not differ between quasispecies from the two viruses (HIV-1, 2.4 x 10-3 pplusmn; 0.4 vs HCV 2.1 x 10-3 pplusmn; 0.5; p = 0.1680). The proportion of synonymous substitutions to potential synonymous sites was similar (3.667 pplusmn; 0.6667 and 2.183 pplusmn; 0.9048, respectively; p = 0.2573), and Shannon's entropy values did not differ between HIV-1 and HCV (0.84 pplusmn; 0.02 and 0.83 pplusmn; 0.12, respectively; p = 0.9408). Of note, 65% (HIV-1) and 67% (HCV) of the analyzed enzymes displayed detectable protease activity, suggesting that both proteases have a similar mutational robustness. In both viruses, there was a rugged protease enzymatic activity landscape characterized by a sharp peak, representing the master sequence, surrounded by a collection of diverse variants present at lower frequencies. These results indicate that nucleotide quasispecies diversification during chronic infection may not be responsible for the higher worldwide genetic diversity observed in HCV.

    IMPORTANCE HCV global diversity is higher than that of HIV-1. We asked whether HCV genetic diversification during infection might be responsible of the higher worldwide genetic diversity observed in HCV. To this end, we analyzed and compared the genotype and enzymatic activities of HIV-1 and HCV protease quasispecies existing in infected individuals. Our results indicate that HIV-1 and HCV protease quasispecies have very similar genetic diversity and comparable rugged enzymatic activity landscapes. Therapy for HCV has expanded with the new therapeutic agents such as the direct acting antivirals (DAAs). DAAs, which target HCV NS3 protease and other virus proteins, have improved cure rates. However, major questions remain to be elucidated regarding the virologic correlates of HCV eradication. The findings shown here may help our understanding of the different therapeutic responses observed during chronic HCV infection.

  • 2' -5' -Oligoadenylate Synthetase-Like Inhibits Respiratory Syncytial Virus Replication and is Targeted by the Viral Nonstructural Protein 1 [PublishAheadOfPrint]

  • 2rrsquo; -5rrsquo; -oligoadenylate synthetase-like (OASL) is an interferon inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, the nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that the RSV NS1 protein suppresses this function to evade cellular innate immunity and allow virus growth.

  • The Telomerase Inhibitor MST-312 Interferes with Multiple Steps in the Herpes Simplex Virus Life Cycle [PublishAheadOfPrint]

  • The life-cycle of HSV has the potential to be further manipulated to yield novel, more effective therapeutic treatments. Recent research has demonstrated that HSV-1 can increase telomerase activity and that expression of the catalytic component of telomerase, TERT, alters sensitivity to HSV-dependent apoptosis. Telomerase is a cellular enzyme that synthesizes nucleotide repeats at the ends of chromosomes (telomeres), which prevents shortening of the 3rrsquo; ends of DNA with each cell division. Once telomeres reach a critical length, cells undergo senescence and apoptosis. Here, we used a cell permeable, reversible inhibitor of the telomerase enzyme, MST-312, to investigate telomerase activity during HSV infection. Human mammary epithelial cells immortalized through TERT expression and the human carcinoma HEp-2 cells were infected with the KOS1.1 strain of HSV-1 in the presence of MST-312. MST-312 treatment reduced the number of cells displaying cytopathic effect and accumulation of immediate early and late viral proteins. Moreover, the presence of 20mmu;M - 100 mmu;M MST-312 during infection led to a 2.5 nndash; 5.5 log10 decrease in viral titer. MST-312 also inhibited the replication of HSV-2 and a recent clinical isolate of HSV-1. Additionally, we determined that MST-312 has the largest impact on viral events that take place prior to 5 hpi. Furthermore, MST-312 treatment inhibited virus replication as measured by adsorption assays and quantification of genome replication. Together these findings demonstrate that MST-312 interferes with the HSV life cycle. Further investigation into the mechanism for MST-312 is warranted and may provide novel targets for HSV therapies.

    Importance Herpes simplex virus (HSV) infections can lead to cold sores, blindness, and brain damage. Identifying host factors that are important for the virus life cycle may provide novel targets for HSV antivirals. One such factor, telomerase, is the cellular enzyme which synthesizes DNA repeats at the ends of chromosomes during replication to prevent DNA shortening. In this study, we investigate role of telomerase on HSV infection. The data demonstrate that the telomerase inhibitor, MST-312, suppressed HSV replication at multiple steps of the viral infection.

  • Expansion of SIV-specific CD8 T cell lines from SIV-naive Mauritian cynomolgus macaques for adoptive transfer [PublishAheadOfPrint]

  • CD8 T cells play a crucial role in the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). However, the specific qualities and characteristics of an effective CD8 T cell response remain unclear. Although targeting breadth, cross-reactivity, polyfunctionality, avidity, and specificity are correlated with HIV control, further investigation is needed to determine the precise contributions of these various attributes to CD8 T cell efficacy. We developed protocols for isolating and expanding SIV-specific CD8 T cells from SIV-naiiuml;ve Mauritian cynomolgus macaques (MCM). These cells exhibited an effector memory phenotype, produced cytokines in response to cognate antigen, and suppressed viral replication in vitro. We further cultured cell lines specific for four SIV-derived epitopes: Nef 103-111 RM9, Gag 389-394 GW9, Env 338-346 RF9, and Nef 254-262 LT9. These lines were up to 94.4% pure as determined by MHC-tetramer analysis. After autologous transfer into two MCM recipients, expanded CD8 T cells persisted in peripheral blood and lung tissue for at least 24 weeks, and trafficked to multiple extra-lymphoid tissues. However, these cells did not impact acute phase SIV load after challenge compared to historic controls. The expansion and autologous transfer of SIV-specific T cells into naiiuml;ve animals provides a unique model for exploring cellular immunity and the control of SIV infection, and facilitates a systematic evaluation of therapeutic adoptive transfer strategies for eradication of the latent reservoir.

    IMPORTANCE CD8 T cells play a crucial role in the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). Autologous adoptive transfer studies followed by SIV challenge may help define the critical elements of an effective T cell response to HIV and SIV infection. We developed protocols for isolating and expanding SIV-specific CD8 T cells from SIV-naiiuml;ve Mauritian cynomolgus macaques. This is an important first step toward the development of autologous transfer strategies to explore cellular immunity and potential therapeutic applications in the SIV model.

  • Circulative non-propagative aphid-transmission of nanoviruses: an oversimplified view. [PublishAheadOfPrint]

  • Plant virus species of the family Nanoviridae have segmented genomes with the highest known number of segments encapsidated individually. They thus likely represent the most extreme case of the so-called multipartite or multicomponent viruses. All species of this family are believed to be transmitted in a circulative non-propagative manner by aphid vectors, meaning that the virus simply crosses cellular barriers within the aphid body, from the gut to the salivary glands, without replicating or even expressing any of its genes. However, this assumption is largely based on analogy with the transmission of other plant viruses such as geminiviruses or luteoviruses, and the details of the molecular and cellular interactions between aphids and nanoviruses is poorly investigated. When comparing the relative frequencies of the eight genome segments in populations of the species Faba bean necrotic stunt virus (FBNSV, genus Nanovirus) within host plants and within aphid vectors fed on these plants, we unexpectedly evidenced reproducible changes of the frequency of some specific segments. We further show that these changes occur within the gut during early stages of the virus cycle in the aphid, and not later when the virus is translocated into the salivary glands. This peculiar observation, which is similarly confirmed in three aphid vector species, Acyrthosiphon pisum, Aphis craccivora and Myzus persicae, calls for a revisiting of the mechanisms of nanovirus transmission. It reveals an unexpected intimate interaction that may not fit the canonical circulative non-propagative transmission.

    IMPORTANCE A specific mode of interaction between viruses and arthropod vectors has been extensively described in plant viruses, in the three families Luteoviridae, Geminiviridae and Nanoviridae, but never in arboviruses of animals. This so-called "circulative non-propagative transmission" contrasts with the classical biological transmission of animal arboviruses in that the corresponding viruses are thought to cross the vector cellular barriers, from the gut lumen to the hemolymph and to the salivary glands, without expressing any of their genes and without replicating. By monitoring the genetic composition of viral populations during the life cycle of Faba bean necrotic stunt virus (FBNSV, Nanovirus), we demonstrate reproducible genetic changes during the transit of the virus within the body of the aphid vector. These changes do not fit the view where viruses simply traverse the body of their arthropod vectors, and suggest more intimate interactions questioning the current understanding of the circulative non-propagative transmission.

  • Replication of Tomato yellow leaf curl virus in its whitefly vector Bemisia tabaci [PublishAheadOfPrint]

  • Tomato yellow leaf curl virus (TYLCV) is a begomovirus exclusively transmitted by the whitefly Bemisia tabaci in a persistent, circulative manner. Replication of TYLCV in its vector remains controversial and thus far, the virus has been considered to be non-propagative. Following 8 h acquisition on TYLCV-infected tomato plants or purified virions and then transfer to non-TYLCV-host cotton plants, the amounts of virus inside whitefly adults significantly increased (more than twofold) during the first few days and then continuously decreased, as measured by amounts of genes on both virus DNA strands. Reported alterations in insect immune and defense responses upon virus retention led us to hypothesize a role for the immune response in suppressing virus replication. After virus acquisition, stress conditions were imposed on the whitefly, and the levels of three viral gene sequences were measured over time. When whiteflies were exposed to TYLCV acquisition and treatment with two different pesticides, the virus levels continuously increased. Upon exposure to heat stress, the virus levels gradually decreased, without any initial accumulation. Switching whiteflies between pesticide, heat-stress and control treatments caused fluctuating increases and decreases in virus levels. Fluorescence in situ hybridization analysis confirmed these results and showed virus signals inside midgut epithelial cell nuclei. Combining the pesticide and heat treatments with virus acquisition had significant effects on fecundity. Altogether, our results demonstrate for the first time that a single-stranded DNA plant virus can replicate in its hemipteran vector.

    IMPORTANCE Plant viruses are of great concern in agricultural crops worldwide. Many of them are transmitted from infected to healthy plants by insects. Persistently transmitted viruses often have a complex association with their vectors; however, most are believed not to replicate within those vectors. Such replication is important as it contributes to the virus's spread and can impact vector biology. Tomato yellow leaf curl virus (TYLCV) is a devastating begomovirus that infects tomatoes. It is persistently transmitted by the whitefly Bemisia tabaci, but is believed not to replicate in the insect. To demonstrate that TYLCV is, in fact, propagative (i.e., it replicates in its insect host), we hypothesized that insect defenses play a role in suppressing virus replication. We thus exposed the whitefly to pesticide and heat-stress conditions to manipulate its physiology, and we showed that under such conditions, the virus is able to replicate and significantly influence the insect's fecundity.

  • RNA and nucleocapsid are dispensable for mature HIV-1 capsid core assembly [PublishAheadOfPrint]

  • Human immunodeficiency virus type 1 (HIV-1) is released from infected cells in an immature, non-infectious form in which the structural polyprotein Gag is arranged in a hexameric lattice, forming an incomplete spherical shell. Maturation to the infectious form is mediated by the viral protease that cleaves Gag at five sites, releasing the CA (capsid) protein which forms a conical capsid encasing the condensed RNA genome. The pathway of this structural rearrangement is currently not understood and it is unclear how cone assembly is initiated. RNA represents an integral structural component of retroviruses, and the viral nucleoprotein core has previously been proposed to nucleate mature capsid assembly. We addressed this hypothesis by replacing the RNA-binding NC (nucleocapsid) domain of HIV-1 Gag by a leucine zipper protein-protein interaction domain (LZ) in the viral context. We found that GagLZ-carrying virus was efficiently released and viral polyproteins were proteolytically processed, though with reduced efficiency. Cryo-electron tomography revealed that the particles lacked a condensed nucleoprotein, and contained an increased proportion of aberrant core morphologies, caused by either the absence of RNA or by altered Gag processing. Nevertheless, a significant proportion of HIV(LZ) particles contained mature capsids with wild-type morphology. These results clearly demonstrate that the nucleoprotein complex is dispensable as a nucleator for mature HIV-1 capsid assembly in the viral context.

    IMPORTANCE Formation of a closed conical capsid encasing the viral RNA genome is essential for HIV-1 infectivity. It is currently unclear what viral components initiate and regulate the formation of the capsid during virus morphogenesis, but it has been proposed that the ribonucleoprotein complex plays a role. To test this, we prepared virus-like particles lacking the viral nucleocapsid protein and RNA, and analysed their three-dimensional structure by cryo-electron tomography. While most virions displayed abnormal morphology under these conditions, some particles showed a normal mature morphology with closed conical capsids. These data demonstrate that the presence of RNA and the nucleocapsid protein are not required for formation of a mature, cone-shaped HIV-1 capsid.

  • The multiplicity of cellular infection changes depending on the route of cell infection in a plant virus [PublishAheadOfPrint]

  • The multiplicity of cellular infection (MOI) is the number of virus genomes of a given virus species that infect individual cells. This parameter chiefly impacts the severity of within-host population bottlenecks as well as the intensity of genetic exchange, competition and complementation among viral genotypes. Only a handful of formal estimations of the MOI is currently available, and most theoretical reports have considered this parameter as constant within the infected host. Nevertheless, the colonization of a multicellular host is a complex process during which the MOI may dramatically change in different organs and at different stages of the infection. We have used both qualitative and quantitative approaches to analyze the MOI during the colonization of turnip plants by the Turnip mosaic virus. Remarkably, different MOI values were observed at two phases of the systemic infection of a leaf. The MOI was very low in primary infections from virus circulating within the vasculature, generally leading to primary foci founded by a single genome. Then, each lineage moved from cell to cell at a very high MOI. Despite this elevated MOI during cell-to-cell progression, co-infection of cells by lineages originating in different primary foci is severely limited by the rapid onset of a mechanism inhibiting secondary infection. Our results thus unveil an intriguing colonization pattern where individual viral genomes initiate distinct lineages within a leaf. Therein, kin genomes massively co-infect cells but co-infection by two distinct lineages is strictly limited.

    IMPORTANCE The multiplicity of cellular infection (MOI) is the size of the viral population colonizing cells and defines major phenomena in virus evolution like the intensity of genetic exchange and the size of within-host population bottlenecks. However, few studies have quantified the MOI and most consider this parameter as constant during infection. Our results reveal that the MOI can largely depend on the route of cell infection in a systemically infected leaf. The MOI is usually one genome per cell when cells are infected from virus particles moving long distance in the vasculature, whereas it is much higher during subsequent cell-to-cell movement in mesophyl. However, a fast-acting superinfection exclusion prevents cell coinfection by merging populations originating in different primary foci within a leaf. This complex colonization pattern results in a situation where within-cell interactions are almost exclusively occuring among kin, and explains the common but uncharacterized phenomenom of genotype spatial segregation in infected plants.

  • Rapid development of gp120-focused, neutralizing B cell responses during acute simian immunodeficiency virus infection of African green monkeys [PublishAheadOfPrint]

  • The initial phases of acute human immunodeficiency virus (HIV-1) infection may be critical for development of effective envelope (Env)-specific antibodies capable of impeding the establishment of the latent pool of HIV-1-infected CD4+ T cells, preventing virally-induced immune hyperactivation to limit disease progression, and blocking vertical virus transmission. However, the initial systemic HIV-1 Env-specific antibody response targets gp41 epitopes and fails to control acute-phase viremia. African-origin, natural simian immunodeficiency virus (SIV) hosts do not typically progress to AIDS and rarely postnatally transmit virus to their infants, despite high milk viral loads. Conversely, SIV-infected rhesus macaques (RMs), Asian-origin non-natural SIV hosts, sustain pathogenic SIV infections and exhibit higher rates of postnatal virus transmission. In this study of acute SIV infection, we compared the initial systemic Env-specific B cell responses of AGMs and RMs in order to probe potential factors influencing the lack of disease progression observed in AGMs. AGMs developed higher magnitude plasma gp120-specific IgA and IgG responses than RMs, whereas RMs developed more robust gp140-directed IgG responses. These gp120-focused antibody responses were accompanied by rapid autologous neutralizing responses during acute SIV infection in AGMs compared to RMs. Moreover, acute SIV infection elicited a higher number of circulating Env-specific memory B cells in peripheral blood of AGMs compared to RMs. These findings indicate that AGMs have distinct initial systemic Env-specific B cell responses to SIV infection compared to a non-natural SIV host, resulting in more functional SIV-specific humoral responses, which may be involved in impairing pathogenic disease progression and minimizing postnatal transmission.

    IMPORTANCE Due to the worldwide prevalence of HIV-1 infections, development of a vaccine to prevent infection or limit the viral reservoir remains an important goal. HIV-1 infected humans, as well as SIV infected non-natural SIV hosts, develop pathogenic infections and readily transmit the virus to their infants. Conversely, natural SIV hosts do not develop pathogenic infections and rarely transmit the virus to their infants. The immunologic factors contributing to these favorable outcomes in natural SIV hosts could prove invaluable for directing HIV-1 vaccine and therapy design. This study identified distinctions in the specificity and function of the initial systemic SIV envelope-specific B cell response that developed during acute SIV infection in natural and non-natural SIV host species. Identification of distinct acute B cell responses in natural SIV hosts may inform vaccine strategies seeking to elicit similar responses prior to or during the initial phases of acute HIV-1 infection.

  • The gut-associated lymphoid tissues in the small intestine, not the large intestine, play a major role in oral prion disease pathogenesis [PublishAheadOfPrint]

  • Prion diseases are infectious neurodegenerative disorders characterised by accumulations of abnormally folded cellular prion protein in affected tissues. Many natural prion diseases are acquired orally and following exposure the early replication of some prion isolates upon follicular dendritic cells (FDC) within gut-associated lymphoid tissues (GALT) is important for the efficient spread of disease to the brain (neuroinvasion). Prion detection within large intestinal GALT biopsies has been used to estimate human and animal disease prevalence. However, the relative contributions of the small and large intestinal GALT to oral prion pathogenesis were unknown. To address this issue we created mice that specifically lacked FDC-containing GALT only in the small intestine. Our data show that oral prion disease susceptibility was dramatically reduced in mice lacking small intestinal GALT. Although these mice had FDC-containing GALT throughout their large intestines, these tissues were not early sites of prion accumulation or neuroinvasion. We also determined whether pathology specifically within the large intestine might influence prion pathogenesis. Congruent infection with the nematode parasite Trichuris muris in the large intestine around the time of oral prion exposure did not affect disease pathogenesis. Together, these data demonstrate that the small intestinal GALT are the major early sites of prion accumulation and neuroinvasion after oral exposure. This has important implications for our understanding of the factors that influence the risk to infection and the pre-clinical diagnosis of disease.

    IMPORTANCE Many natural prion diseases are acquired orally. After exposure the accumulation of some prion diseases in the gut-associated lymphoid tissues (GALT) is important for efficient spread of disease to the brain. However, the relative contributions of GALT in the small and large intestines to oral prion pathogenesis were unknown. We show that the small intestinal GALT are the essential early sites of prion accumulation. Furthermore, congruent infection with a large intestinal helminth (worm) around the time of oral prion exposure did not affect disease pathogenesis. This is important for our understanding of the factors that influence the risk to prion infection and the pre-clinical diagnosis of disease. The detection of prions within large intestinal GALT biopsies has been used to estimate human and animal disease prevalence. However, our data suggest that these biopsies may miss individuals in the early stages of oral prion infection and significantly underestimate the disease prevalence.

  • Insights into CWD and BSE species barriers using real-time conversion [PublishAheadOfPrint]

  • The propensity for trans-species prion transmission is related to the structural characteristics of the enciphering and new host PrP, although the exact mechanism remains incompletely understood. The effects of variability in prion protein on cross-species prion transmission have been studied with animal bioassays, but the influence of prion protein structure vs. host co-factors (e.g. cellular constituents, trafficking, and innate immune interactions) remains difficult to dissect. To isolate the effects of protein:protein interactions on trans-species conversion, we used recombinant PrPC and real-time quaking-induced conversion (RT-QuIC) and compared chronic wasting disease (CWD) and classical bovine spongiform encephalopathy (cBSE) prions. To assess the impact of transmission to a new species, we studied feline CWD (fCWD) and feline BSE (i.e. feline spongiform encephalopathy, FSE). We cross-seeded fCWD and FSE into each species' full-length, recombinant PrPC and measured the time required for conversion to the amyloid (PrPRes) form, which we describe here as the rate of amyloid conversion. These studies revealed that: (1) CWD and BSE seeded their homologous species' PrP best; (2) fCWD was a more efficient seed for feline rPrP than for white-tailed deer rPrP; (3) conversely, FSE more efficiently converted bovine than feline rPrP; (4) and CWD, fCWD, BSE, and FSE all converted human rPrP, although not as efficiently as homologous sCJD prions. These results suggest that: (1) at the level of protein: protein interactions, CWD adapts to a new species more readily than does BSE, and (2) the barrier preventing transmission of CWD to humans may be less robust than estimated.

    IMPORTANCE We demonstrate that bovine spongiform encephalopathy prions maintain their trans-species conversion characteristics upon passage to cats, but that chronic wasting disease prions adapt to the cat and are distinguishable from the original prion. Additionally, we showed that chronic wasting disease prions are effective at seeding the conversion of normal human prion protein to an amyloid conformation, perhaps the first step in crossing the species barrier.

  • Structural Basis of Human Parechovirus Neutralization by Human Monoclonal Antibodies [PublishAheadOfPrint]

  • Since it was first recognized in 2004 that human parechoviruses (HPeV) are a significant cause of central nervous system and neonatal sepsis, their clinical importance, primarily in children, has started to emerge. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases and has given moderate success. Direct inhibition of parechovirus infection using monoclonal antibodies is a potential treatment. We have developed two neutralising monoclonal antibodies against HPeV1 and HPeV2, namely AM18 and AM28 that also cross-neutralise other viruses. Here we present the mapping of their epitopes using peptide scanning, surface plasmon resonance, fluorescence-based thermal shift assays, electron cryo-microscopy and image reconstruction. We determined by peptide scanning and surface plasmon resonance that AM18 recognizes a linear epitope motif including the llsquo;arginine-glycine-aspartic acid' on the C-terminus of capsid protein VP1. This epitope is normally used by the virus to attach to host cell-surface integrins during entry and is found in 3 other viruses that AM18 neutralises. Therefore, AM18 is likely to cause virus neutralization by aggregation and by blocking integrin binding to the capsid. Further, we show by electron cryo-microscopy, three-dimensional reconstruction and pseudo-atomic model fitting that ordered RNA interacts with HPeV1 VP1 and VP3. AM28 recognizes quaternary epitopes on the capsid composed of VP0 and VP3 loops from neighbouring pentamers, thereby increasing the RNA accessibility temperature for the virus-AM28 complex compared to the virus alone. Thus, inhibition of RNA uncoating probably contributes to neutralisation by AM28.

    IMPORTANCE Human parechoviruses can cause mild infections to severe diseases in young children such as neonatal sepsis, encephalitis and cardiomyopathy. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases. In order to develop more targeted treatment we have searched for human monoclonal antibodies that would neutralize human parechovirus 1 and 2, associated with mild infections such as gastroenteritis and severe infections of the central nervous system and thus to allow safe treatment. In the current study we show how two such promising antibodies interact with the virus, modelling the atomic interactions between the virus and the antibody to propose how neutralization occurs. Both antibodies can cause aggregation, in addition, one antibody interferes with the virus recognising its target cell, the other, recognising only the whole virus, inhibits the genome uncoating and replicating in the cell.

  • Microplitis demolitor Bracovirus Proviral Loci and Clustered Replication Genes Exhibit Distinct DNA Amplification Patterns During Replication [PublishAheadOfPrint]

  • Polydnaviruses are large, double-stranded DNA viruses that are beneficial symbionts of parasitoid wasps. Polydnaviruses in the genus Bracovirus (BVs) persist in wasps as proviruses and their genomes consist of two functional components referred to as proviral segments and nudivirus-like genes. Prior studies establish that the DNA domains where proviral segments reside are amplified during replication and that segments within amplified loci are circularized before packaging into nucleocapsids. One DNA domain where nudivirus-like genes are located is also amplified but never packaged into virions. We recently sequenced the genome of the braconid Microplitis demolitor, which carries M. demolitor bracovirus (MdBV). Here, we took advantage of this resource to characterize the DNAs that are amplified during MdBV replication using a combination of Illumina and PacBio sequencing approaches. Results showed that specific nucleotide sites identify the boundaries of amplification for proviral loci. Yet surprisingly, amplification of loci 3, 4, 6 and 8 produced head-to-tail concatemeric intermediates, loci 1, 2 and 5 produced head-to-head/tail-to-tail concatemers, and locus 7 yielded no identified concatemers. Sequence differences at amplification junctions correlated with the type of amplification intermediates loci produced, while concatemer processing gave rise to the circularized DNAs that are packaged into nucleocapsids. The MdBV nudivirus-like gene cluster was also amplified, albeit more weakly than most proviral loci and with non-discrete boundaries. Overall, the MdBV genome exhibited three patterns of DNA amplification during replication. Our data also suggest that PacBio sequencing could be useful in studying the replication intermediates produced by other DNA viruses.

    IMPORTANCE Polydnaviruses are of fundamental interest because they provide a novel example of viruses evolving into beneficial symbionts. All polydnaviruses are associated with insects called parasitoid wasps, which are of additional applied interest because many are biological control agents of pest insects. Polydnaviruses in the genus Bracovirus (BV) evolved ~100 million years ago from an ancestor related to the baculovirus-nudivirus lineage, but have also established many novelties due to their symbiotic lifestyle. These include that BVs are only transmitted vertically as proviruses and produce replication-defective virions that package only a portion of the viral genome. Here we studied Microplitis demolitor bracovirus (MdBV) and report its genome exhibits three distinct patterns of DNA amplification during replication. We also identify several previously unknown features of BV genomes that correlate with these different amplification patterns.

  • Enhanced Neutralizing Antibody Response Induced by Respiratory Syncytial Virus Pre-fusion F Protein Expressed by a Vaccine Candidate [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are the first and second leading viral agents of severe respiratory tract disease in infants and young children worldwide. Vaccines are not available, and an RSV vaccine is particularly needed. A live attenuated chimeric bovine/human PIV3 (rB/HPIV3) vector expressing the RSV fusion (F) glycoprotein from an added gene has been under development as a bivalent vaccine against RSV and HPIV3. Previous clinical evaluation of this vaccine candidate suggested that increased genetic stability and immunogenicity of the RSV F insert were needed. This was investigated in the present study. RSV F expression was enhanced 5-fold by codon-optimization and by modifying the amino acid sequence to be identical to that of an early passage of the original clinical isolate. This conferred a hypo-fusogenic phenotype that presumably reflects the original isolate. We then compared vectors expressing stabilized pre- and post-fusion versions of RSV F. In a hamster model, pre-fusion F induced increased quantity and quality of RSV-neutralizing serum antibodies and increased protection against wt RSV challenge. In contrast, vector expressing the post-fusion F was less immunogenic and protective. The genetic stability of the RSV F insert was high and was not affected by enhanced expression or the pre- or post-fusion conformation of RSV F. These studies provide an improved version of the previously-well-tolerated rB/HPIV3-RSV F vaccine candidate that induces a superior RSV-neutralizing serum antibody response.

    Importance Respiratory syncytial virus (RSV) and parainfluenza virus type 3 (HPIV3) are two major causes of pediatric pneumonia and bronchiolitis. The rB/HPIV3 vector expressing RSV F protein is a candidate bivalent live vaccine against HPIV3 and RSV. Previous clinical evaluation indicated the need to increase the immunogenicity and genetic stability of the RSV F insert. Here, we increased RSV F expression by codon optimization and by modifying the RSV F amino acid sequence to conform to that of an early passage of the original isolate. This resulted in a hypo-fusogenic phenotype, which likely represents the original phenotype before adaptation to cell culture. We also included stabilized versions of pre- and post-fusion RSV F protein. Pre-fusion RSV F induced a higher quantity and quality of RSV-neutralizing serum antibodies and was highly protective. This provides an improved candidate for further clinical evaluation.

  • The Vaginal Acquisition and Dissemination of HIV-1 Infection in a Novel Transgenic Mouse Model is Facilitated by Coinfection with HSV-2 and is Inhibited by Microbicide Treatment [PublishAheadOfPrint]

  • Epidemiological studies have demonstrated that HSV-2 infection significantly increases the risk of HIV-1 acquisition, thereby contributing to the expanding HIV-1 epidemic. To investigate whether HSV-2 infection directly facilitates mucosal HIV-1 acquisition, we used our transgenic hu-CD4/R5/cT1 mouse model which circumvents major entry and transcription blocks preventing murine HIV-1 infection by targeting transgenic expression of human CD4, CCR5 and cyclinT1 genes to CD4+ T cells and myeloid-committed cells. Productive infection of mucosal leukocytes, predominantly CD4+ T cells, was detected in all hu-CD4/R5/cT1 mice intravaginally challenged with an HIV-1 infectious molecular clone, HIV-Du151.2env-NLuc, which expresses an env gene (C.Du151.2) cloned from an acute heterosexually infected woman and a NanoLuc luciferase reporter gene. Lower genital HIV-1 infection after HIV-Du151.2env-NLuc intravaginal challenge was increased ~4-fold in hu-CD4/R5/cT1 mice co-infected with HSV-2. Furthermore, HIV-1 dissemination to draining lymph nodes was detected only in HSV-2 co-infected mice. HSV-2 infection stimulated local infiltration and activation of CD4+ T cells and dendritic cells, likely contributing to the enhanced HIV-1 infection and dissemination in HSV-2 co-infected mice. We then used this model to demonstrate that a novel gel containing tenofovir disproxil fumerate (TDF), the more potent prodrug of tenofovir (TFV), but not the TFV microbicide gel utilized in the recent CAPRISA 004, VOICE and FACTS 001 clinical trials, was effective as pre-exposure prophylaxis (PrEP) to prevent vaginal HIV-1 infection in almost half of HSV-2-coinfected mice. These results also support utilization of huCD4/R5/cT1 mice as a highly reproducible immunocompetent preclinical model to evaluate HIV-1 acquisition across the female genital tract.

    IMPORTANCE Multiple epidemiological studies reported that genital Herpes simplex virus-2 (HSV-2) infection increases the risk of HIV-1 sexual acquisition by several-fold. Understanding the underlying mechanisms by which HSV-2 facilitates HIV-1 infection and optimizing the efficacy of therapies to inhibit HIV-1 infection during HSV-2 co-infection should contribute to reducing HIV-1 transmission. Using our novel transgenic hu-CD4/R5/cT1 mouse model infectable with HIV-1, we demonstrated that HSV-2 infection enhances vaginal transmission and dissemination of HIV-1 infection while stimulating recruitment and activation of CD4+ T cells and dendritic cells in the lower genital tract. HIV acquisition by hu-CD4/R5/cT1 mice vaginally coinfected with HSV-2 could be prevented in almost half the mice by pre-exposure prophylaxis (PrEP) with a novel gel containing tenofovir disproxil fumerate (TDF), the tenofovir prodrug, but not with the tenofovir microbicide gel utilized in CAPRISA-004, VOICE and FACTS-001 clinical trials. huCD4/R5/cT1 mice represent a new preclinical mouse model to evaluate vaginal HIV-1 acquisition.

  • A Dynein light chain 1 binding motif in RABV polymerase L protein plays a role in microtubule re-organization and viral primary transcription [PublishAheadOfPrint]

  • Rabies virus (RABV) polymerase L together with phosphoprotein P forms the PL polymerase complex that is essential for replication and transcription. However, its exact mechanism of action, interactions with cellular factors and its intracellular distribution are yet to be understood. Here by imaging a fluorescently tagged polymerase (mCherry-RABV-L) we show that L accumulates at acetylated and reorganized microtubules (MT). In silico analysis revealed a dynein light chain 1 (DLC1) binding motif in L that could mediate MT binding through dynein motors. As DLC1 binding by polymerase cofactor P is known, we compared the impact of the DLC1-binding motifs in P and L. Viruses with mutations in the respective motifs revealed that both motifs are required for efficient primary transcription, indicating that DLC1 acts as a transcription enhancer by binding to both, P and L. Notably, also the levels of cellular DLC1 protein were regulated by both motifs, suggesting regulation of the DLC1-gene expression by both, P and L. Finally, disruption of the motif in L resulted in a cell type specific loss of MT localization, demonstrating that DLC1 is involved in L-mediated cytoskeleton reorganization. Overall, we conclude that DLC1 acts as a transcription factor that stimulates primary RABV transcription by binding to both, P and L. We further conclude that L influences MT organization and posttranslational modification, suggesting a model in which MT manipulation by L contributes to efficient intracellular transport of virus components and thus may serve as an important step virus replication.

    Importance Regulation of rabies virus polymerase complex by viral and cellular factors thus far has not been fully understood. Although cellular dynein light chain 1 (DLC1) has been reported to increase primary transcription by binding to polymerase cofactor phosphoprotein P, the detailed mechanism is unknown and it is also not known whether the large enzymatic polymerase subunit L is involved.

    By fluorescence microscopy analysis of fluorescence tagged rabies virus L, in silico identification of a potential DLC1 binding site in L, and characterization of recombinant rabies virus mutants, we show that a DLC1 binding motif in L is involved in cytoskeleton localization and re-organisation, in primary transcription regulation by DLC1 and in regulation of cellular DLC1 gene expression. By providing evidence for a direct contribution of a DLC1 binding motif in L our data significantly increase the understanding of rabies virus polymerase regulation and host manipulation by the virus as well.

  • Comparative phylodynamics of rabbit haemorrhagic disease virus (RHDV) in Australia and New Zealand [PublishAheadOfPrint]

  • The introduction of Rabbit Haemorrhagic Disease Virus (RHDV) into Australia and New Zealand during the 1990s as a means of controlling feral rabbits is an important case study in viral emergence. Both epidemics are exceptional in that the founder viruses share a common origin and the timing of their releases are known, providing a unique opportunity to compare the evolution of a single virus in distinct naiiuml;ve populations. We examined the evolution and spread of RHDV in Australia and New Zealand through a genome-wide evolutionary analysis, including data from 28 newly sequenced RHDV field isolates. Following the release of the Australian inoculum strain into New Zealand no subsequent mixing of the populations occurred, with viruses from both countries forming distinct groups. Strikingly, the rate of evolution in the capsid gene was higher in the Australian viruses than those from New Zealand, most likely due to the presence of transient deleterious mutations in the former. However, estimates of both substitution rates and population dynamics were strongly sample-dependent, such that small changes in sample composition had an important impact on evolutionary parameters. Phylogeographic analysis revealed clear spatial structure in the Australian RHDV strains, with a major division between those viruses from western and eastern states. Importantly, RHDV sequences from the state where the virus was first released, South Australia, had the greatest diversity and were diffuse throughout both geographic lineages, such that this region was likely a source population for the subsequent spread of the virus across the country.

    IMPORTANCE Most studies of viral emergence lack detailed knowledge on which strains were founders for the outbreak or when these events occurred. Hence, the human-mediated introduction of Rabbit Haemorrhagic Disease Virus (RHDV) into Australia and New Zealand from known starting stocks provides a unique opportunity to understand viral evolution and emergence. Within Australia we revealed a major phylogenetic division between viruses sampled from the east and west of the country, both regions likely seeded by viruses from South Australia. Despite their common origins, marked differences in evolutionary rates were observed between the Australian and New Zealand RHDV, and which led to conflicting conclusions about population growth rates. An analysis of mutational patterns suggested that evolutionary rates have been elevated in the Australian viruses, at least in part due to the presence of low fitness (deleterious) variants that have yet to be selectively purged.

  • Systemic foot-and-mouth disease vaccination in cattle promotes specific antibody secreting cells at the respiratory tract and triggers local anamnestic-compatible responses upon aerosol infection [PublishAheadOfPrint]

  • Foot-and-mouth disease (FMD) is a highly contagious viral disease affecting biungulate species. Commercial vaccines, formulated with inactivated FMD virus (FMDV), are regularly used worldwide to control the disease. Here, we studied the generation of antibody responses in local lymphoid tissues along the respiratory system in vaccinated and further aerosol-infected cattle. Animals immunized with a high payload monovalent FMD vaccine developed high titers of neutralizing antibodies at 7 days post-vaccination (dpv), reaching a plateau until 29 dpv. FMDV-specific antibody-secreting cells (ASC), predominantly IgM, were evident at 7 dpv in the prescapular lymph node (LN) draining the vaccination site and in distal LN draining the respiratory mucosa, though in lower numbers. Twenty-nine dpv a significant switch to IgG1 was clear in prescapular LN, while FMDV-specific ASC were detected in all lymphoid tissues draining the respiratory tract, mostly as IgM-secreting cells. None of the animals (n=10) exhibited FMD symptoms after oronasal challenge at 30 dpv. Three days post-infection, a large increase in ASC numbers and rapid isotype switches to IgG1 were observed particularly in LN draining virus replication sites already described. These results indicate for the first time that systemic FMD vaccination in cattle effectively promotes the presence of anti-FMDV ASC in lymphoid tissues associated with the respiratory system. Oronasal infection triggered an immune reaction compatible with a local anamnestic response upon contact with the replicating FMDV, suggesting that FMD vaccination might induce circulation of virus-specific B-lymphocytes, including memory B-cells that differentiate into ASC soon after contact with the infective virus.

    Importance Over the last decades, world animal health organizations as well as national sanitary authorities have supported the use of vaccination as an essential component of the official FMD control programs in both endemic and disease-free settings. Very few works studied the local immunity induced by FMD-vaccines at the respiratory mucosa and local responses induced in vaccinated animals after aerosol infection have not been described yet. In this work, we demonstrate for the first time that systemic FMD vaccination: (i) induced the early presence of active antigen-specific ASC along the respiratory tract, and (ii) prompted a rapid local antibody response in the respiratory mucosa, triggered upon oronasal challenge and congruent with a memory B-cell response. This information may help to understand novel aspects of protective responses induced by current FMD vaccines, as well as to provide alternative parameters to establish protection efficiency for new vaccine developments.

  • Human and murine IFIT1 do not restrict infection of negative sense RNA viruses of the orthomyxoviridae, bunyaviridae, and filoviridae families [PublishAheadOfPrint]

  • Interferon (IFN)-induced protein with tetratricopeptide repeats 1 (IFIT1) is a host gene with reported cell-intrinsic antiviral activity against several RNA viruses. The proposed basis for the activity against negative sense RNA viruses is the binding to exposed 5rrsquo; -triphosphate (5rrsquo; -ppp) on the genome of viral RNA. However, recent studies reported relatively low binding affinities of IFIT1 for 5rrsquo; -ppp-RNA, suggesting that IFIT1 may not interact efficiently with this moiety under physiological conditions. To evaluate the ability of IFIT1 to impact negative sense RNA viruses, we infected Ifit1-/- and control wild-type mice and primary cells with four negative sense RNA viruses (influenza A virus (IAV), La Crosse virus (LACV), Oropouche virus (OROV), and Ebola virus) corresponding to three distinct families. Unexpectedly, a lack of Ifit1 gene expression did not result in increased infection of any of these viruses in cell culture. Analogously, morbidity, mortality, and viral burden in tissues were identical between Ifit1-/- and control mice after infection with IAV, LACV, or OROV. Finally, deletion of human IFIT1 protein in A549 cells did not affect IAV replication or infection and reciprocally, ectopic expression of IFIT1 in HEK293T cells did not inhibit IAV infection. To explain the lack of antiviral activity against IAV, we measured the binding affinity of IFIT1 for RNA oligonucleotides resembling the 5rrsquo; -end of IAV gene-segments. The affinity for 5rrsquo; -ppp-RNA was approximately 10-fold lower compared to that of non-2rrsquo; -O-methylated cap 0-RNA oligonucleotides. Based on this analysis, we conclude that IFIT1 is not a dominant restriction factor against negative sense RNA viruses.

    IMPORTANCE Negative sense RNA viruses including Influenza and Ebola are responsible for some of the most deadly outbreaks in recent history. The host interferon response and induction of antiviral genes contributes to the control of infection by these viruses. IFIT1 is highly induced after virus infection and reportedly has antiviral activity against several RNA and DNA viruses. However, its role in restricting infection by negative sense RNA viruses remains unclear. Here, we evaluated the ability of IFIT1 to inhibit negative sense RNA virus replication and pathogenesis both in vitro and in vivo. Detailed cell culture and animal studies demonstrate that that IFIT1 is not a dominant restriction factor against three different families of negative sense RNA viruses.

  • Acute Hepatitis C Virus Infection Induces Consistent Changes in Circulating miRNAs that are Associated with Non-Lytic Hepatocyte Release [PublishAheadOfPrint]

  • Plasma microRNAs (miRNAs) change in abundance in response to disease, and have been associated with liver fibrosis severity in chronic hepatitis C virus (HCV) infection. However, the early dynamics of miRNA release during acute HCV infection are poorly understood. In addition, circulating miRNA signatures have been difficult to reproduce among separate populations. We studied plasma miRNA abundance during acute HCV infection to identify a miRNA signature of early infection. We measured 754 plasma miRNAs by quantitative PCR array in a discovery cohort of 22 individuals before and during acute HCV infection and after spontaneous resolution (n=11) or persistence (n=11) to identify a plasma miRNA signature. The discovery cohort derived from Baltimore Before and After Acute Study of Hepatitis. During acute HCV infection, increases in miR-122 (Pllt;.01) and miR-885-5p (Pcorrectedllt;.05) and a decrease in miR-494 (Pcorrectedllt;.05) were observed at the earliest time-points after virus detection. Changes in miR-122 and miR-885-5p were sustained in persistent (Pllt;.001) but not resolved HCV infection. The circulating miRNA signature of acute HCV infection was confirmed in a separate validation cohort that derived from the San Francisco-based UFO Study (n=28). As further confirmation, cellular changes of signature miRNAs were examined in a tissue-culture model of HCV in hepatoma cells: HCV infection induced extracellular release of miR-122 and miR-885-5p despite unperturbed intracellular levels. In contrast, miR-494 accumulated intracellularly (Pllt;.05). Collectively, these data are inconsistent with necrolytic release of hepatocyte miRNAs into the plasma during acute HCV infection of humans.

    IMPORTANCE MicroRNAs (miRNAs) are small non-coding RNA molecules that emerging research shows can transmit regulatory signals between cells in health and disease. Hepatitis C virus (HCV) infects 2% of humans worldwide, and chronic HCV infection is a major cause of severe liver disease. We profiled plasma miRNAs in injection drug users before, during, and (in the people with resolution) after HCV infection. We discovered miRNA signatures of acute and persistent viremia, and confirmed these findings two ways: i) in a separate cohort of people with newly acquired HCV infection and ii) in an HCV cell culture system. Our results demonstrate that acute HCV infection induces early changes in the abundance of specific plasma miRNAs that may affect the host response to HCV infection.

  • Selective Involvement of the Checkpoint Regulator VISTA in Suppression of B-cell, but not T-cell, Responsiveness by Monocytic Myeloid Derived Suppressor Cells from Mice Infected by an Immunodeficiency-causing Retrovirus. [PublishAheadOfPrint]

  • Inhibition by myeloid-derived suppressor cells (MDSCs) against T-cell responses in tumor microenvironments is widely accepted. We demonstrated augmentation of monocytic MDSCs whose suppression of not only T-, but also B-cell, responsiveness paralleled the immunodeficiency during LP-BM5 retrovirus infection. MDSCs inhibited T-cells by iNOS/NO, but uniquely, inhibition of B-cells was ~50% dependent each on iNOS/NO and MDSC-expressed, negative-checkpoint regulator, VISTA. Combining iNOS/NO and VISTA blockade caused additive or synergistic abrogation of MDSC-mediated suppression of B-cell responsiveness.

  • Measles Virus Infection Inactivates Cellular Protein Phosphatase 5 with Consequent Suppression of Sp1 and c-Myc Activities [PublishAheadOfPrint]

  • Measles virus (MeV) causes several unique syndromes including transient immunosuppression. To clarify the cellular responses to MeV infection, we previously analyzed a MeV-infected epithelial cell line and a lymphoid cell line by microarray, and showed that the expression of numerous genes was up- or downregulated in the epithelial cells. In particular, there was a characteristic comprehensive downregulation of housekeeping genes during late stage infection. To identify the mechanism underlying this phenomenon, we examined the phosphorylation status of transcription factors and kinase/phosphatase activities in epithelial cells after infection. MeV infection inactivated cellular protein phosphatase 5 (PP5) that consequently inactivated DNA-dependent protein kinase (DNA-PK), which reduced Sp1 phosphorylation levels, and c-Myc degradation, both of which downregulated the expression of many housekeeping genes. In addition, intracellular accumulation of viral nucleocapsid inactivated PP5 and subsequent downstream responses. These findings demonstrate a novel strategy of MeV during infection, which causes the collapse of host cellular functions.

    IMPORTANCE MeV is one of the most important pathogens in humans. We previously showed that MeV infection induces the comprehensive downregulation of housekeeping genes in epithelial cells. By examining this phenomenon, we clarified the molecular mechanism underlying the constitutive expression of housekeeping genes in cells, which is maintained by PP5 and DNA-PK. We also demonstrated that MeV targets PP5 for downregulation in epithelial cells. This is the first report to show how MeV infection triggers a reduction in overall cellular functions of infected host cells. Our findings will help uncover unique pathogenicities caused by MeV.

  • Generation of a Recombinant Akabane Virus Expressing Enhanced Green Fluorescent Protein [PublishAheadOfPrint]

  • We generated a recombinant Akabane virus (AKAV) expressing enhanced green fluorescence protein (eGFP-AKAV) by using reverse genetics. We artificially constructed an ambisense AKAV S genome encoding N/NSs on the negative-sense strand, and eGFP on the positive-sense strand, with an intergenic region (IGR) derived from the Rift Valley fever virus (RVFV) S genome. The recombinant virus exhibited eGFP fluorescence and had a cytopathic effect in cell cultures, even after several passages. These results indicate that the gene encoding eGFP in the ambisense RNA could be stably maintained. Transcription of N/NSs and eGFP mRNAs of eGFP-AKAV was terminated within the IGR. The mechanism responsible for this appears to be different for that in RVFV, where the termination sites for N and NSs are determined by a defined signal sequence. We inoculated suckling mice intraperitoneally with eGFP-AKAV, which resulted in neurological signs and lethality equivalent to that seen for the parent AKAV. Fluorescence from eGFP in frozen brain slices from the eGFP-AKAV-infected mice was localized to the cerebellum, pons, and medulla oblongata. Our approach to producing a fluorescent virus, using an ambisense genome, helped obtain eGFP-AKAV, a fluorescent bunyavirus whose viral genes are intact and which can be easily visualized.

    IMPORTANCE AKAV is the etiological agent of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic loss to the livestock industry. We successfully generated a recombinant enhanced green fluorescent protein-tagged AKAV containing an artificial ambisense S genome. This virus could become a useful tool for analyzing AKAV pathogenesis in host animals. In addition, our approach of using an ambisense genome to generate an orthobunyavirus stably expressing a foreign gene could contribute to establishing alternative vaccine strategies, such as bivalent vaccine virus constructs, for veterinary use against infectious diseases.

  • Down-regulation of poly (ADP-ribose) polymerase-1 by a viral processivity factor facilitates gammaherpesvirus lytic replication [PublishAheadOfPrint]

  • In Kaposi's sarcoma-associated herpesvirus (KSHV), poly (ADP-ribose) polymerase-1 (PARP-1) acts as an inhibitor of lytic replication. Here, we demonstrate that KSHV down-regulated PARP-1 upon reactivation. The viral processivity factor of KSHV (PF-8) interacted with PARP-1 and was sufficient to degrade PARP-1 in a proteasome-dependent manner; this effect was conserved in murine gammaherpesvirus 68. PF-8 knockdown in KSHV-infected cells resulted in reduced lytic replication upon reactivation with increased levels of PARP-1, when compared to control cells. PF-8 overexpression reduced the levels of PARylated RTA and further enhanced RTA-mediated transactivation. These results suggest a novel viral mechanism for overcoming the inhibitory effect of a host factor, PARP-1, thereby promoting gammaherpesvirus lytic replication.

    IMPORTANCE Gammaherpesviruses are important human pathogens, as they are associated with various kinds of tumors and establish latency mainly in host B lymphocytes. Replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is a central molecular switch for lytic replication and its expression is tightly regulated by many host and viral factors. In this study, we investigated a viral strategy to overcome the inhibitory effect of poly (ADP-ribose) polymerase-1 (PARP-1) on the RTA activity. PARP-1, an abundant multifunctional nuclear protein, was down-regulated during KSHV reactivation. The viral processivity factor of KSHV (PF-8) directly interacted with PARP-1 and was sufficient and necessary to degrade PARP-1 protein in a proteasome-dependent manner. PF-8 reduced the levels of PARylated RTA and further promoted RTA-mediated transactivation. As this was also conserved in another gammaherpesvirus, murine gammaherpesvirus 68, our results suggest a conserved viral modulation of a host inhibitory factor to facilitate its lytic replication.

  • The Impact of Nucleoporin Mediated Chromatin localization and Nuclear Architecture on HIV Integration Site Selection [PublishAheadOfPrint]

  • It has been known for a number of years that integration sites of human immunodeficiency virus 1 (HIV-1) DNA show a preference for actively expressed chromosomal locations. A number of viral and cellular proteins are implicated in this process, but the underlying mechanism is not clear. Two recent breakthrough publications advance our understanding of HIV integration site selection by focusing on the localization of the preferred target genes of integration. These studies reveal that knockdown of certain nucleoporins and components of nucleocytoplasmic trafficking alter integration site preference; not by altering the trafficking of the viral genome, but by altering the chromatin subtype localization relative to the structure of the nucleus. Here we describe the link between the nuclear basket nucleoporins (Tpr and Nup153) and chromatin organization and how altering the host environment by manipulating nuclear structure could have important implications for the preferential integration of HIV into actively transcribed genes facilitating efficient viral replication.

  • Blockage of CD59 function restores activities of neutralizing and non-neutralizing antibodies in triggering antibody-dependent complement-mediated lysis of HIV-1 virions and provirus-activated latently infected cells [PublishAheadOfPrint]

  • Both HIV-1 virions and infected cells use their surface regulators of complement activation (RCA) to resist antibody-dependent complement-mediated lysis (ADCML). Blockage of the biological function of RCA members, particularly CD59 (a key RCA member that controls formation of the membrane attack complex at the terminal stage of the complement activation cascades via all three activation pathways), has rendered both HIV-1 virions and infected cells sensitive to ADCML mediated by anti-Env antibodies (Abs) or sera/plasma from patients at different stages of viral infection. In the current study, we used the well-characterized anti-HIV-1 neutralizing Abs (nAbs) including 2G12, 2F5, and 4E10 and non-nAbs including 2.2C, A32, N5-i5, and N12-i15 to investigate whether the enhancement of ADCML by blockage of CD59 function is mediated by nAbs, non-nAbs, or both. We found that all nAbs and two non-nAbs (N5-i5 and A32) strongly reacted to three HIV-1 laboratory strains (R5, X4, and R5/X4), six primary isolates, and provirus-activated ACH-2 cells examined. In contrast, two non-nAbs 2.2C and N12-i15 reacted weakly and did not react to these targets, respectively. After blockage of CD59 function, the reactive Abs, regardless of their neutralizing activities, significantly enhanced specific ADCML of HIV-1 virions (both laboratory strains and primary isolates) and provirus-activated latently infected cells. The ADMCL efficacy positively correlated with the ELISA-reactive intensity of those Abs with their targets. Thus, blockage of RCA function represents a novel approach to restore activities of both nAbs and non-nAbs in triggering ADCML of HIV-1 virions and provirus-activated latently infected cells.

    Importance There is a renewed interest in the potential role of non-nAbs in the control of HIV-1 infection. Our data, for the first time, demonstrated that blockage of the biological function of RCA members rendered both HIV-1 virions and infected cells sensitive to ADCML mediated by not only nAbs, but also non-nAbs. Our results are significant in developing novel immune-based approaches to restore the functions of nAbs and non-nAbs in the circulation of HIV-1-infected individuals to specifically target and clear HIV-1 virions and infected cells. Our data also provide new insights into the mechanisms by which HIV-1 virions and infected cells escape Ab-mediated immunity and could aid in the design and/or development of therapeutic HIV-1 vaccines. In addition, a combination of antiretroviral therapy (ART) with RCA blockage, provirus activators, and therapeutic vaccines may represent a novel approach to eliminate HIV-1 reservoirs, i.e. the infected cells harboring replication-competent proviruses and residual viremia.

  • The intracellular domain of the coxsackievirus and adenovirus receptor differentially influences adenovirus entry [PublishAheadOfPrint]

  • The coxsackievirus and adenovirus receptor (CAR) is a cell adhesion molecule used as a docking molecule by some adenoviruses (AdVs) and group B coxsackieviruses. We previously proposed that the preferential transduction of neurons by canine adenovirus type 2 (CAV-2) is due to CAR-mediated internalization. Our proposed pathway of CAV-2 entry is in contrast to that of human AdV type 5 (HAdV-C5) in non-neuronal cells, where internalization is mediated by auxiliary receptors such as integrins. We therefore asked if in fibroblast-like cells the intracellular domain (ICD) of CAR plays a role in the internalization of the CAV-2 fiber knob (FKCAV), CAV-2, or HAdV-C5 when the capsid cannot engage integrins. Here, we show that in fibroblast-like cells, the CAR ICD is needed for FKCAV entry and efficient CAV-2 transduction, but dispensable for HAdV-C5 and a HAdV-C5 capsid lacking the RGD sequence (an integrin-interacting motif) in the penton. Moreover, the deletion of CAR ICD further impacts CAV-2 intracellular trafficking, highlighting the crucial role of CAR in CAV-2 intracellular dynamics. These data demonstrate that CAR ICD contains sequences important for the recruitment of the endocytic machinery that differentially influences AdV cell entry.

    Importance Understanding how viruses interact with the host cell surface and reach the intracellular space is of crucial importance for applied and fundamental virology. Here, we compare the role of a cell adhesion molecule (CAR), in the internalization of adenoviruses that naturally infect humans and Canidae. We show that the intracellular domain of CAR differentially regulate AdV entry and trafficking. Our study highlights the mechanistic differences that a receptor can have for two viruses from the same family.

  • Role of nectin-1 and HVEM as cellular receptors for Herpes Simplex Virus Type 1 on primary murine dermal fibroblasts [PublishAheadOfPrint]

  • The cellular proteins nectin-1 and HVEM can both mediate the entry of herpes simplex virus type 1 (HSV-1). We have recently shown how these receptors contribute to infection of skin by investigating HSV-1 entry into murine epidermis. Ex vivo infection studies reveal nectin-1 as the primary receptor in epidermis whereas HVEM has a more limited role. While the epidermis represents the outermost layer of skin, the contribution of nectin-1 and HVEM in the underlying dermis is still open. Here we analyzed the role of each receptor during HSV-1 entry in murine dermal fibroblasts that were deficient in expression of either nectin-1 or HVEM or both receptors. Because infection was not prevented by the absence of either nectin-1 or HVEM, we conclude that they can act as alternative receptors. Although HVEM was found to be highly expressed on fibroblasts, entry was delayed in nectin-1-deficient cells suggesting that nectin-1 acts as the more efficient receptor. In the absence of both receptors, entry was strongly delayed leading to a much reduced viral spread and virus production. These results suggest an unidentified cellular component that acts as alternate but inefficient receptor for HSV-1 on dermal fibroblasts. Characterization of the cellular entry mechanism sugeests that HSV-1 can enter dermal fibroblasts both by direct fusion with the plasma membrane and via endocytic vesicles and that this is not dependent on the presence or absence of nectin-1. Entry was also shown to require dynamin and cholesterol, suggesting comparable entry pathways in keratinocytes and dermal fibroblasts.

    Importance Herpes simplex virus (HSV) is a human pathogen which infects its host via mucosal surfaces or abraded skin. To understand how HSV-1 overcomes the protective barrier of mucosa or skin and reaches its receptors in tissue, it is essential to know which receptors contribute to the entry into individual skin cells. Previously, we have explored the contribution of nectin-1 and HVEM as receptors for HSV-1 entry into murine epidermis where keratinocytes form the major cell type. Since the underlying dermis consists primarily of fibroblasts, we have now extended our study of HSV-1 entry to dermal fibroblasts isolated from nectin-1- or HVEM-deficient mice or from mice deficient in both receptors. Our results demonstrate a role for both nectin-1 and HVEM as receptors and suggest a further receptor which appears much less efficient.

  • Double-strand RNA is detected by immunofluorescence analysis in RNA and DNA virus infections including those by negative-strand RNA viruses [PublishAheadOfPrint]

  • Early biochemical studies of viral replication suggested that most viruses produce dsRNA that is essential for the induction of the host immune response. However, it was reported in 2006 that dsRNA could be detected by immunofluorescence antibody staining in dsDNA and positive-strand RNA but not in negative-strand RNA virus infections. Other reports in the literature seemed to support these observations. This suggested that negative-strand RNA viruses produce little, if any dsRNA, or that more efficient viral countermeasures to mask dsRNA are mounted. Because of our interest in use of dsRNA antibodies for virus discovery, particularly in pathologic specimens, we wanted to determine how universal immunostaining for dsRNA might be in animal virus infections. We have detected in situ formation of dsRNA in cells infected with vesicular stomatitis virus, measles virus, influenza A virus and Nyamanini virus infections that represent different negative-strand RNA virus families. dsRNA was also detected in cells infected with lymphocytic choriomeningitis virus, an ambisense RNA virus, and minute virus of mice, a ssDNA parvovirus, but not hepatitis B virus. Although dsRNA staining was primarily observed in the cytoplasm it was also seen in the nucleus of cells infected with influenza A virus Nyamanini virus and minute virus of mice (MVM). Thus, it is likely that most animal virus infections produce dsRNA species that can be detected by immunofluorescence staining. Apoptosis induced in several uninfected cell lines failed to up-regulate dsRNA formation.

    IMPORTANCE An effective antiviral host immune response depends on recognition of viral invasion and an intact innate immune system as a first line of defense. Double-stranded RNA (dsRNA) is an essential viral product in the induction of innate immunity, leading to the production of type I interferons (IFN) and activation of hundreds of IFN-stimulated genes. The present study demonstrates that infections, including those by ssDNA and positive- and negative-strand RNA viruses, produce dsRNAs detectable by standard immunofluorescence staining. While dsRNA staining was primarily observed in the cytoplasm, nuclear staining was also present in some RNA and DNA virus infections. The nucleus is unlikely to have pathogen associated molecular pattern (PAMP) receptors for dsRNA because of the presence of host dsRNA molecules. Thus, it is likely that most animal virus infections produce dsRNA species detectable by immunofluorescence staining that may prove useful in viral discovery as well.

  • A Coronavirus E Protein is Present in Two Distinct Pools with Different Effects on Assembly and the Secretory Pathway [PublishAheadOfPrint]

  • Coronaviruses (CoVs) assemble by budding into the lumen of the early Golgi prior to exocytosis. The small CoV envelope (E) protein plays roles in assembly, virion release, and pathogenesis. CoV E has a single hydrophobic domain (HD), is targeted to Golgi membranes, and has cation channel activity in vitro. However, the precise functions of the CoV E protein during infection are still enigmatic. Structural data for the severe acute respiratory syndrome (SARS)-CoV E protein suggests that it assembles into a homo-pentamer. Specific residues in the HD regulate the ion-conducting pore formed by SARS-CoV E in artificial bilayers and the pathogenicity of the virus during infection. The E protein from the avian infectious bronchitis virus (IBV) has dramatic effects on the secretory system, which requires residues in the HD. Here, we use the known structural data from SARS-CoV E to infer residues important for ion channel activity and oligomerization of IBV E. We present biochemical data for the formation of two distinct oligomeric pools of IBV E in transfected and infected cells, and residues required for their formation. A high-order oligomer of IBV E is required for the production of virus-like particles (VLPs), implicating this form of the protein in virion assembly. Additionally, disruption of the secretory pathway by IBV E correlates with a form that is likely monomeric, suggesting that the effects on the secretory pathway are independent of E ion channel activity.

    IMPORTANCE CoVs are important human pathogens with significant zoonotic potential as demonstrated by the emergence of SARS-CoV and Middle East respiratory syndrome (MERS)-CoV. Progress has been made toward identifying potential vaccine candidates in mouse models of CoV infection, including use of attenuated viruses that lack the CoV E protein or express E mutants. However, no approved vaccines and anti-viral therapeutics exist. We previously reported that the hydrophobic domain of the IBV E protein, a putative viroporin, causes disruption of the mammalian secretory pathway when exogenously expressed in cells. Understanding the mechanism of this disruption could lead to the identification of novel anti-viral therapeutics. Here, we present biochemical evidence for two distinct oligomeric forms of IBV E, one essential for assembly and the other with a role in disruption of the secretory pathway. Discovery of two forms of CoV E protein will provide additional targets for anti-viral therapeutics.

  • Myd88 initiates early innate responses and promotes CD4 T cells during coronavirus encephalomyelitis [PublishAheadOfPrint]

  • Myd88 signaling is critical to control numerous central nervous system (CNS) infections by promoting both innate and adaptive immune responses. Nevertheless, the extent to which Myd88 regulates type I IFN versus proinflammatory factors and T cell function, as well as anatomical site of action varies extensively with the pathogen. CNS infection by neurotropic coronavirus with confined replication in brain and spinal cord induces protective IFNaalpha;/bbeta; via Myd88 independent activation of melanoma differentiation-associated gene 5 (MDA5). However, a contribution of Myd88 dependent signals to CNS pathogenesis has not been assessed. Infected Myd88-/- mice failed to control virus, exhibited enhanced clinical disease coincident with increased demyelination, and succumbed to infection within three weeks. Induction of IFNaalpha;/bbeta;, as well as proinflammatory cytokines and chemokines was impaired early during infection. However, defects in both IFNaalpha;/bbeta; and select proinflammatory factors were rapidly overcome prior to T cell recruitment. Myd88 deficiency also specifically blunted myeloid and CD4 T cell recruitment into the CNS without affecting CD8 T cells. Moreover, CD4 T cells, but not CD8 T cells, were impaired in IFN production. Ineffective virus control indeed correlated most prominently with reduced anti-viral IFN in the CNS of Myd88-/- mice. The results demonstrate a crucial role for Myd88 both in early induction of innate responses during coronavirus induced encephalomyelitis and in specifically promoting protective CD4 T cell activation. In the absence of these responses, functional CD8 T cells are insufficient to control viral spread within the CNS resulting in severe demyelination.

    Importance During central nervous system (CNS) infections signaling through the adaptor protein Myd88 promotes both innate and adaptive immune responses. The extent to which Myd88 regulates antiviral type I IFN, pro inflammatory factors, adaptive immunity and pathology is pathogen dependent. These results reveal that Myd88 protects from lethal neurotropic coronavirus induced encephalomyelitis by accelerating, but not enhancing, IFNaalpha;/bbeta; as well as promoting peripheral activation and CNS accumulation of virus specific CD4 T cells secreting IFN. By controlling both early innate responses and CD4 T cell mediated antiviral IFN, Myd88 signaling limits initial viral dissemination and is vital for T cell mediated control of viral load. Uncontrolled viral replication in the absence of Myd88 leads to severe demyelination and pathology despite overall reduced inflammatory responses. These data support a vital role of Myd88 signaling in protective antimicrobial function in the CNS by promoting proinflammatory mediators and T cell mediated IFN production.

  • P2X1 receptor antagonists inhibit HIV-1 fusion by blocking virus-coreceptor interactions [PublishAheadOfPrint]

  • HIV-1 Env glycoprotein-mediated fusion is initiated upon sequential binding of Env to CD4 and coreceptors, CXCR4 or CCR5. Whereas these interactions are thought to be necessary and sufficient to promote HIV-1 fusion, other host factors can modulate this process. Previous studies have reported potent inhibition of HIV-1 fusion by selective P2X1 receptor antagonists, including NF279, and suggested that these receptors play a role in HIV-1 entry. Here we investigated the mechanism of anti-viral activity of NF279 and found that this compound does not inhibit HIV-1 fusion by preventing the activation of P2X1 channels, but effectively blocks the virus binding to CXCR4 or CCR5. The notion of an off-target effect of NF279 on HIV-1 fusion is supported by the lack of detectable expression of P2X1 receptors in cells used in fusion experiments and by the fact that addition of ATP or enzymatic depletion of ATP in a culture medium does not modulate viral fusion. Importantly, NF279 fails to inhibit HIV-1 fusion with cell lines and primary macrophages when added at an intermediate stage downstream of Env-CD4-coreceptor engagement. Conversely, in the presence of NF279, HIV-1 fusion is arrested downstream of CD4 binding but prior to coreceptor engagement. NF279 also antagonizes the signaling function of CCR5, CXCR4 and another chemokine receptor, as evidenced by the suppression of calcium responses elicited by specific ligands and by recombinant gp120. Collectively, our results demonstrate that NF279 is a dual HIV-1 coreceptor inhibitor that interferes with the functional engagement of CCR5 and CXCR4 by Env.

    Importance The well-documented inhibition of HIV-1 fusion and replication by purinergic receptor antagonists implicated these proteins in viral entry/fusion and suggested new targets for anti-viral therapy. Mechanistic studies of the role of P2X1 receptor in HIV-1 fusion performed in this study strongly imply that this channel is not expressed in target cells or involved in viral fusion. Instead, we found that inhibition of HIV-1 fusion by a specific P2X1 receptor antagonist NF279 is due to blocking the virus interactions with both CXCR4 and CCR5 coreceptors. The ability of NF279 to abrogate cellular calcium signaling induced by respective chemokines showed that this compound acts as a dual-coreceptor antagonist. P2X1 receptor antagonists could thus represent a new class of dual-coreceptor inhibitors with a structure and mechanism of action that are distinct from known HIV-1 coreceptor antagonists.

  • Salmon gill poxvirus, the deepest representative of the Chordopoxvirinae [PublishAheadOfPrint]

  • Poxviruses are large DNA viruses of vertebrates and insects causing disease in many animal species including reptiles, birds and mammals. Although poxvirus-like particles were detected in diseased farmed koi carp, ayu and Atlantic salmon, their genetic relationships to poxviruses were not established. Here, we provide the first genome sequence of a fish poxvirus, which was isolated from farmed Atlantic salmon, and use qPCR and immunohistochemistry to describe aspects of salmon gill poxvirus disease. The gill was the main target organ where immature and mature poxvirus particles were detected in detaching, apoptotic respiratory epithelial cells, preceding clinical disease in the form of lethargy, respiratory distress and mortality. In moribund salmon blocking of gas exchange would likely be caused by adherence of respiratory lamellae and epithelial proliferation obstructing respiratory surfaces. The virus was not found in healthy salmon or in controls of gill disease without apoptotic cells, although transmission remains to be demonstrated. PCR on archival tissue confirmed virus infection in 14 cases with gill apoptosis in Norway starting from 1995. Phylogenomic analyses showed that the fish poxvirus is the deepest available branch of chordopoxviruses. The virus encompasses most key chordopoxvirus genes that are required for genome replication and expression although the gene order is substantially different. Nevertheless, many highly conserved chordopoxvirus genes involved in viral membranes biogenesis or virus-host interactions are missing. Instead, the salmon poxvirus carries numerous genes encoding unknown proteins many of which have low sequence complexity and contain simple repeats suggestive of intrinsic disorder or distinct protein structures.

    IMPORTANCE Aquaculture is an increasingly important global source of high quality food. To sustain this growth, disease control in fish farming is essential. Moreover, the spread of disease from farmed fish to wild life is a concern. Serious poxviral diseases are emerging in aquaculture, but very little is known about the viruses and the diseases they cause. There is a possibility that viruses may spread to new species with enhanced virulence as has occurred with the myxoma poxvirus in rabbits. Provision of the first fish poxvirus genome sequence and specific diagnostics for the salmon gill poxvirus in Atlantic salmon may help curb this disease and provide comparative knowledge. Furthermore, because salmon gill poxvirus represents the deepest branch of chordopoxvirus so far discovered, the genome analysis provided substantial insight into the evolution of different functional modules in this important group of viruses.

  • Exploring the balance between DNA pressure and capsid stability in Herpes and phage. [PublishAheadOfPrint]

  • We have recently shown in both herpesviruses and phages that packaged viral DNA creates a pressure of tens of atmospheres pushing against the interior capsid wall. For the first time, using differential scanning microcalorimetry, we directly measure the energy powering the release of pressurized DNA from the capsid. Furthermore, using a new calorimetric assay to accurately determine the temperature inducing DNA release, we found a direct influence of internal DNA pressure on the stability of the viral particle. We show that the balance of forces between the DNA pressure and capsid strength, required for DNA retention between rounds of infection, is conserved between evolutionarily diverse bacterial viruses (phage and P22), as well as a eukaryotic virus, human Herpes Simplex 1 (HSV-1). Our data also suggest that the portal vertex in these viruses is the weakest point in the overall capsid structure and presents the "Achilles' heel" of virus's stability. Comparison between these viral systems shows that viruses with higher DNA packing density (resulting in higher capsid pressure) have inherently stronger capsid structures preventing spontaneous genome release prior to infection. This force balance is of key importance for viral survival and replication. Investigating the ways to disrupt this balance can lead to development of new mutation resistant anti-virals.

    IMPORTANCE A virus can generally be described as a nucleic acid genome contained within a protective protein shell, called the capsid. For many double-stranded DNA viruses, confinement of the large DNA molecule within the small protein capsid results in an energetically stressed DNA state exerting tens of atmospheres of pressures of on the inner capsid wall. We show that stability of viral particles (which directly relates to infectivity) is strongly influenced by the state of the packaged genome. Using scanning calorimetry on a bacterial virus (phage ), as an experimental model system, we investigate the thermodynamics of genome release associated with destabilizing the viral particle. Furthermore, we compare the influence of tight genome confinement on the relative stability for diverse bacterial and eukaryotic viruses. These comparisons reveal an evolutionarily conserved force balance between the capsid stability and the density of the packaged genome.

  • Longitudinal Detection of Prion Shedding in Saliva and Urine by CWD-Infected Deer by RT-QuIC [PublishAheadOfPrint]

  • Chronic Wasting Disease (CWD) is an emergent, rapidly spreading prion disease of cervids. Shedding of infectious prions in saliva and urine is thought to be an important factor in CWD transmission. To help elucidate this issue, we applied an in vitro amplification assay to determine the onset, duration, and magnitude of prion shedding in longitudinally collected saliva and urine samples from CWD-exposed white-tailed deer. We detected prion shedding as early as 3 months after CWD exposure and sustained shedding throughout the disease course. We estimated that a 50% lethal dose (LD50) for cervidized transgenic mice would be contained in 1 ml of infected deer saliva or 10 ml or urine. Given the average course of infection and daily production of these body fluids, an infected deer would shed thousands of prion infectious dosesover the course of CWD infection. The direct and indirect environmental impact of this magnitude of prion shedding for cervid and non-cervid species is surely significant.

    Importance: Chronic wasting disease (CWD) is an emerging and uniformly fatal prion disease affecting free ranging deer and elk and now recognized in 22 United States and 2 C anadian Provinces. It is unique among prion diseases in that it is transmitted naturally though wild populations. A major hypothesis for CWD's florid spread is that prions are shed in excreta and transmitted via direct or indirect environmental contact. Here we use a rapid in vitro assay to show that infectious doses of CWD prions are in fact shed throughout the multi-year disease course in deer. This finding is an important advance in assessing the risks posed by shed CWD prions to animals as well as humans.

  • Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of the Ebola Virus [PublishAheadOfPrint]

  • Lipid enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. The Ebola virus, which buds from the plasma membrane of the host cell causes viral hemorrhagic fever and has a high fatality rate. To date little is known about how budding and egress of the Ebola virus is mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of the Ebola virus matrix protein VP40. VP40 binds PS containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress; whereas PS is typically only on the inner leaflet. Together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus like particles.

    IMPORTANCE The lipid-enveloped Ebola virus causes severe infection, has a high mortality rate, and currently lacks FDA approved therapeutics or vaccines. The Ebola virus harbors just seven genes in its genome and during the replication process there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell it infects. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This study, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry.

  • Characterization of T-antigens including Middle T and ALTO expressed by the human polyomavirus associated with trichodysplasia spinulosa [PublishAheadOfPrint]

  • The polyomavirus tumor (T) antigens play crucial roles in viral replication, transcription and cellular transformation. They are encoded by partially overlapping open reading frames located in the early region through alternative mRNA splicing. The T expression pattern of the Trichodysplasia Spinulosa-associated Polyomavirus (TSPyV) has not been established yet, hampering further study of its pathogenic mechanisms and taxonomic relationship.

    Here we characterized TSPyV T-expression in human cell lines transfected with the TSPyV early region. Sequencing of T-encoded RT-PCR products revealed three splice donor and acceptor sites creating six mRNA splice products that potentially encode Small T (ST), Middle T (MT), Large T (LT), TinyT, 21kT and alternative T (ALTO). Except for 21kT, these splice products were also detected in skin of TSPyV-infected patients. At least three splice products were confirmed by Northern blotting likely encoding LT, MT, ST, 21kT and ALTO. Protein expression was demonstrated for LT, ALTO and possibly MT, of which LT was detected in the nucleus and ALTO in the cytoplasm of transfected cells. Splice site and start codon mutations indicated that ALTO is encoded by the same splice product that encodes LT and uses internal start codons for initiation. Genuineness of ALTO was indicated by the identification of acetylated N-terminal ALTO peptides by mass spectrometry.

    Summarizing, TSPyV exhibits an expression pattern characterized by both MT and ALTO expression, combining features of rodent and human polyomaviruses. This unique expression pattern provides important leads to further study of polyomavirus-related disease and to understand polyomavirus evolution.

    IMPORTANCE The human Trichodysplasia Spinulosa-associated Polyomavirus (TSPyV) is distinguished among polyomaviruses for combining productive infection with cell-transforming properties. In the research presented here, we further substantiate this unique position by indicating expression of both Middle (MT) and alternative T-(ALTO) antigens in TSPyV. So far, none of the human polyomaviruses was shown to express MT, which is considered the most important viral oncoprotein of rodent polyomaviruses. Co-expression of ALTO and MT, which involves a conserved, recently recognized overlapping ORF subject to positive selection, has not been observed before for any polyomavirus. Altogether, our study provides valuable new insights in polyomavirus T gene use and expression. Obviously these insights will be instrumental in further study and understanding of TSPyV pathogenicity. More importantly, however, they provide important leads with regard to interrelationship, functionality and evolution of polyomaviruses as a whole, pushing TSPyV forward as a suitable model virus to further study these entities.

  • Evolution of influenza B virus in Kuala Lumpur, Malaysia between 1995 and 2008 [PublishAheadOfPrint]

  • Influenza B virus causes significant disease but remains understudied in tropical regions. We sequenced 72 influenza B viruses collected in Kuala Lumpur, Malaysia from 1995nndash;2008. The predominant circulating lineage (Victoria or Yamagata) changed every 1nndash;3 years, and these shifts were associated with increased incidence of influenza B. We also found poor lineage matches with recommended influenza vaccine strains. While most influenza B lineages in Malaysia were short-lived, one circulated for 3nndash;4 years.

  • Phylodynamics of Enterovirus A71-Associated Hand, Foot and Mouth Disease in Viet Nam [PublishAheadOfPrint]

  • Enterovirus A71 (EV-A71) is a major cause of hand, foot and mouth disease (HFMD) and is particularly prevalent in parts of Southeast Asia, affecting thousands of children and infants each year. Revealing the evolutionary and epidemiological dynamics of EV-A71 through time and space is central to understanding its outbreak potential. We generated the full genome sequences of 200 EV-A71 strains sampled from various locations in Viet Nam between 2011-2013, and used these sequence data to determine the evolutionary history and phylodynamics of EV-A71 in Viet Nam, providing estimates of the effective reproduction number (Re) of the infection through time. In addition, we described the phylogeography of EV-A71 throughout Southeast Asia, documenting patterns of viral gene flow. Accordingly, our analysis reveals that a rapid genogroup switch from C4 to B5 likely took place during 2012 in Viet Nam. We show that the Re of subgenogroup C4 decreased during the time-frame of sampling, while that of B5 increased and remained ggt;1 at the end of 2013, corresponding to a rise in B5 prevalence. Our study reveals that the subgenogroup B5 virus that emerged into Viet Nam is closely related to variants that were responsible for large epidemics in Malaysia and Taiwan and therefore extends our knowledge regarding its associated endemic area. Subgenogroup B5 evidently has the potential to cause more widespread outbreaks across Southeast Asia.

    Importance EV-A71 is one of many viruses that cause HFMD, a common syndrome that largely affects infants and children. HFMD usually causes only mild illness with no long-term consequences. Occasionally, however, severe infection may arise, especially in very young children, causing neurological complications and even death. EV-A71 is highly contagious and is associated with the most severe HFMD cases, with large and frequent epidemics of the virus recorded worldwide. Although major advances have been made in the development of a potential EV-A71 vaccine, there is no current prevention and little is known about the patterns and dynamics of EV-A71 spread. In this study we utilize full-length genome sequence data obtained from HFMD patients in Viet Nam, a geographical region where the disease has been endemic since 2003, to characterize the phylodynamics of this important emerging virus.

  • HIV-1 cell-free and cell-to-cell infection are differentially regulated by distinct determinants in the Env gp41 cytoplasmic tail [PublishAheadOfPrint]

  • The HIV-1 envelope (Env) glycoprotein mediates viral entry during both cell-free and cell-to-cell infection of CD4+ T cells. The highly conserved long cytoplasmic tail (CT) of Env is required in a cell type-dependent manner for optimal infectivity of cell-free virus. To probe the role of the CT in cell-to-cell infection, we tested a panel of mutants in the CT region that maintain or attenuate cell-free infection to ask whether the functions of the CT are conserved during cell-free and cell-to-cell infection. The mutants tested included truncations of structural motifs in the gp41 CT, and two point mutants in lentiviral lytic peptide 3 (LLP-3) previously described as disrupting the infectivity of cell-free virus. We found that small truncations of 28 to 43 amino acids (aa) or two LLP-3 point mutations YW_SL and LL_RQ severely impaired single round cell-free infectivity by 10-fold or more relative to wild type full-length CT. These mutants showed a modest 2-fold reduction in cell-to-cell infection assays. Conversely, large truncations of 93 to 124 aa severely impaired cell-to-cell infectivity by 20-fold or more, while displaying a 50% increase in infectivity of cell-free viral particles when produced in 293T cells. Intermediate truncations of 46 to 90 aa showed profound impairment of both modes of infection. Our results show that the ability of Env to support cell-free and cell-to-cell infection are genetically distinct. These differences are cell-type dependent for large CT truncation mutants. Additionally, point mutants in LLP-3 can maintain multi-round propagation from cell-to-cell in primary CD4+ T cells.

    Importance The functions of HIV Env gp41 CT remain poorly understood despite being widely studied in the context of cell-free infection. We have identified domains of the gp41 CT responsible for striking selective deficiencies in either cell-free or cell-to-cell infectivity. These differences may reflect a different intrinsic regulatory influence of the CT on cell-associated versus particle-associated Env, or differential interaction with host or viral proteins. Our findings provide novel insight into the key regulatory potential of the gp41 CT in cell-free and cell-to-cell HIV-1 infection, particularly for short truncation mutants lle;43 amino acids, or point mutations in the LLP-3 helical domain of the CT which are able to propagate via cell-to-cell in the absence of infectious cell-free virus production. These mutants may also serve as tools to further define the contributions of cell-free and cell-to-cell infection in vitro and in vivo.

  • An aptamer against the matrix binding domain on the hepatitis B virus capsid impairs virion formation [PublishAheadOfPrint]

  • The hepatitis B virus (HBV) particle is an icosahedral nucleocapsid surrounded by a lipid envelope containing viral surface proteins. A small domain (matrix domain, MD) in the large surface protein L and a narrow region (matrix binding domain, MBD) including isoleucine 126 on the capsid surface have been mapped where point mutations like core-I126A specifically blocked nucleocapsid envelopment. Possibly, both domains interact with each other during virion morphogenesis. By the SELEX method we evolved DNA aptamers from an oligonucleotide library binding to purified recombinant capsids but not binding to the corresponding I126A mutant capsids. Aptamers bound to capsids were separated from unbound molecules by filtration. After 13 rounds of selections and amplifications 16 different aptamers were found among 73 clones. The four most frequent aptamers represented more than 50 % of the clones. The main aptamer AO-01 (13 clones, 18 %) showed the lowest dissociation constant (Kd) of 180 +/- 82 nM for capsid binding among the four molecules. Its Kd value for I126A capsids was 1306 +/- 503 nM. Cotransfection of Huh7 cells with AO-01 and an HBV genomic construct resulted in 47 % inhibition of virion production 3 days post transfection but showed no inhibition by cotransfection of an aptamer with random sequence. The half-life of AO-01 in cells was 2 hours which might explain the incomplete inhibition. The results support the importance of the MBD for nucleocapsid envelopment. Inhibiting the MD-MBD interaction by a low molecular weight substance might represent a new approach for an antiviral therapy.

    Importance Approximately 240 million people are persistently infected with HBV. To date, antiviral therapies depend on a single target, the viral reverse transcriptase. Future additional targets could be viral protein-protein interactions. We selected a 55 base long single stranded DNA molecule (aptamer) which binds with relatively high affinity to a region on the HBV capsid interacting with viral envelope proteins during budding. This aptamer inhibits virion formation in cell culture. The result substantiates the current model for HBV morphogenesis and shows that the capsid envelope interaction is a potential antiviral target.

  • The great (nuclear) escape: new insights into the role of the nuclear egress complex of herpesviruses [PublishAheadOfPrint]

  • Herpesviruses are unusual among enveloped viruses because they bud twice yet acquire a single envelope. They are also the only known viruses that bud into the nuclear envelope. We discovered that the herpesvirus nuclear egress complex (NEC) could bud membranes without the help of other proteins by forming a coat-like hexagonal scaffold inside the budding membrane. This finding raises a possibility that a phenotypically similar nuclear export of large RNAs is cargo-driven.

  • Impaired EBV-specific neutralizing antibody response during acute infectious mononucleosis is coincident with global B-cell dysfunction [PublishAheadOfPrint]

  • Here we present evidence for previously unappreciated B-cell immune dysregulation during acute EBV-associated infectious mononucleosis (IM). Longitudinal analyses revealed that patients with acute IM have undetectable EBV-specific neutralizing antibodies and gp350-specific B-cell responses, which was associated with a significant reduction in memory B-cells and no evidence of circulating antibody-secreting cells. These observations correlate with dysregulation of tumour necrosis factor (TNF)-family members BAFF and APRIL and increased expression of FAS on circulating B-cells.

  • Functional and structural characterization of human V3-specific monoclonal antibody 2424 with neutralizing activity against HIV-1 JRFL [PublishAheadOfPrint]

  • The V3 region of HIV-1 gp120 is important for virus-coreceptor interaction and highly immunogenic. Although most anti-V3 antibodies neutralize only the sensitive Tier 1 viruses, anti-V3 antibodies effective against the more resistant viruses exist, and a better understanding about these antibodies and their epitopes would be beneficial for the development of novel vaccine immunogens against HIV. The HIV-1 isolate JRFL with its cryptic V3 is resistant to most V3-specific monoclonal antibodies (mAbs). However, the V3 mAb 2424 achieves 100% neutralization against JRFL. 2424 is encoded by IgH V3-53 and IgL V2-28 genes, a pairing rarely used by the other V3 mAbs.. 2424 also has distinct binding and neutralization profiles. Studies of 2424-mediated neutralization of JRFL produced with a mannosidase inhibitor further revealed that its neutralizing activity is unaffected by the glycan composition of the virus envelope. To understand the distinct activity of 2424, we determined the crystal structure of 2424 Fab in complex with a JRFL V3 peptide, and showed that the 2424 epitope is located at the tip of the V3 crown (307IHIGPGRAFY318), dominated by interactions with HisP308, ProP313, and ArgP315. The binding mode of 2424 is similar to that of the well-characterized 447-52D, although 2424 is more side-chain dependent. The 2424 epitope is focused on the very apex of V3, away from nearby glycans, facilitating antibody access. This feature distinguishes the 2424 epitope from the other V3 crown epitopes and indicates that the tip of V3 is a potential site to target and incorporate into HIV vaccine immunogens.

    IMPORTANCE HIV/AIDS vaccines are crucial for controlling the HIV epidemics that continue to afflict millions of people worldwide. However, HIV vaccine development has been hampered by significant scientific challenges, one of which is the inability of HIV vaccine candidates evaluated thus far to elicit production of potent and broadly neutralizing antibodies. The V3 loop is one of the few immunogenic targets on the virus envelope glycoprotein that can induce neutralizing antibodies, but in many viruses, parts of V3 are inaccessible for antibody recognition. This study examined a V3-specific monoclonal antibody that can completely neutralize HIV-1 JRFL, a virus isolate resistant to most V3 antibodies. Our data reveal that this antibody recognizes the most distal tip of V3 that is not as occluded as other parts of V3. Hence, the epitope of 2424 is in one of the vulnerable sites on the virus that may be exploited in designing HIV vaccine immunogens.

  • Dual-Tropic CXCR6/CCR5 Simian Immunodeficiency Virus Infection of Sooty Mangabey Primary Lymphocytes: Distinct Coreceptor Use in SIV Natural versus Pathogenic Hosts [PublishAheadOfPrint]

  • Natural host sooty mangabeys (SM) infected with SIV exhibit high viral loads but do not develop disease, whereas infection of rhesus macaques (RM) causes CD4+ T cell loss and AIDS. Several mechanisms have been proposed to explain these divergent outcomes, including differences in cell targeting, which has been linked to low expression of the canonical SIV entry receptor CCR5 on CD4+ T cells of SM and other natural hosts. We previously showed that infection and high-level viremia occur even in a subset of SM that genetically lack functional CCR5, which indicates that alternative entry coreceptors are used by in SM in vivo in these animals. We also showed that SM CXCR6 is a robust coreceptor for SIVsmm in vitro. Here we identify CXCR6 as a principal entry pathway for SIV in SM primary lymphocytes. We show that ex vivo SIVinfection of lymphocytes from CCR5 wild-type SM is mediated by both CXCR6 and CCR5. In contrast, infection of RM lymphocytes is fully dependent on CCR5. These data raise the possibility that CXCR6-directed tropism in CCR5-low natural hosts may alter CD4+ T cell subset targeting compared with non-natural hosts, enabling SIV to maintain high level replication without leading to widespread CD4+ T cell loss.

    IMPORTANCE Natural hosts of SIV such as sooty mangabeys sustain high viral load but do not develop disease, while non-natural hosts like rhesus macaques develop AIDS. Understanding this difference may help elucidate mechanisms of pathogenesis. Natural hosts have very low levels of the SIV entry coreceptor CCR5 suggesting that restricted entry may limit infection of certain target cells, although it is unclear how the virus replicates so robustly. Here we show that, in sooty mangabey lymphocytes, infection is mediated by the alternative entry coreceptor CXCR6, as well as CCR5. In rhesus macaque lymphocytes, however, infection occurs entirely through CCR5. The use of CXCR6 for entry, combined with very low CCR5 levels, may re-direct the virus to different cell targets in natural hosts. It is possible that differential targeting may favor infection of non-essential cells and limit infection of critical cells in natural hosts, and thus contribute to benign outcome of infection.

  • Patient-specific neutralizing antibody responses to herpes simplex virus are attributed to epitopes on either gD, gB, or both and can be type-specific [PublishAheadOfPrint]

  • Herpes simplex virus (HSV) types 1 and 2 infect many humans and establish a latent infection in sensory ganglia. While some infected people suffer periodic recurrences, others do not. Infected people mount both cell-mediated and humoral responses, including the production of virus-neutralizing antibodies (Abs) directed at viral entry glycoproteins. Previously, we examined IgGs from 10 HSV-seropositive individuals; all neutralized virus and were directed primarily against gD or gD+gB. Here, we expand our studies and examine 32 additional sera from HSV-infected individuals, 23 of whom had no recurrent disease. Using an Octet RED96rreg; system, we screened all 32 serum samples directly for both glycoprotein binding and competition with known neutralizing anti-gD and -gB monoclonal Abs (MAbs). On average, the recurrent cohort exhibited higher binding to gD and gB and had higher neutralization titers. There were similar trends in the blocking of MAbs to critical gD and gB epitopes. When we depleted 6 sera of Abs to specific glycoproteins, we found different types of responses, but always directed primarily at gD and/or gB. Interestingly, in one dual-infected person, the neutralizing response to HSV-2 was due to gD2 and gB2 while HSV-1 neutralization was due to gD1 and gB1. In another case, virus neutralization was HSV-1 specific with the Ab response directed entirely at gB1, despite this serum blocking type-common anti-gD and -gB neutralizing MAbs. These data are pertinent in the design of future HSV vaccines as they demonstrate the importance of both serotypes of gD and gB as immunogens.

    IMPORTANCE We previously showed that people infected with HSV produce neutralizing Abs directed against gD or a combination of gD+gB (and in one case, gD+gB+gC, which was type-1 specific). In this more extensive study, we again found that gD or gD+gB can account for the virus neutralizing response and critical epitopes of one or both of these proteins are represented in sera of naturally-infected humans. However, we also found that some individuals produced a strong response against gB alone. In addition, we identified type-specific contributions to HSV neutralization from both gD and gB. Contributions from the other entry glycoproteins, gC and gH/gL, were minimal and limited to HSV-1 neutralization. Knowing the variations in how humans see and mount a response to HSV will be important to vaccine development.

  • A dual role for the non-receptor tyrosine kinase Pyk2 during the intracellular trafficking of human papillomavirus 16 [PublishAheadOfPrint]

  • The infectious process of human papillomaviruses (HPV) has been studied considerably and many cellular components required for viral entry and trafficking continue to be revealed. In this study we investigated the role of the non-receptor tyrosine kinase Pyk2 during HPV16 pseudovirion infection of human keratinocytes. We found that Pyk2 is necessary for infection and appears to be involved in the intracellular trafficking of the virus. siRNA-mediated reduction of Pyk2 resulted in a significant decrease in infection, but did not prevent viral entry at the plasma membrane. Pyk2 depletion resulted in altered endo-lysosomal trafficking of HPV16 and accelerated unfolding of the viral capsid. Furthermore, we observed retention of the HPV16 pseudogenome in the trans-Golgi network (TGN) in Pyk2-depleted cells, suggesting that the kinase could be required for the viral DNA to exit the TGN. While Pyk2 has previously been shown to function during entry of enveloped viruses at the plasma membrane, the kinase has not yet been implicated during the intracellular trafficking of a non-enveloped virus, such as HPV. Additionally, these data enrich the current literature on Pyk2's function in human keratinocytes.

    IMPORTANCE In this study we investigate the role of the non-receptor tyrosine kinase, Pyk2, during human papillomavirus (HPV) infection of human skin cells. Infections with high-risk types of HPV, such as HPV16, are the leading cause of cervical cancer and major cause of genital and oropharyngeal cancers. As a non-enveloped virus, HPV enters cells by interacting with cellular receptors and established cellular trafficking routes to ensure that the viral DNA reaches the nucleus for productive infection. This study identifies Pyk2 as a required cellular component for the intracellular trafficking of HPV16 during infection. Understanding the infectious pathways of HPVs is critical for developing additional preventative therapies. Furthermore, this study advances knowledge on intracellular trafficking processes in keratinocytes.

  • Screening of human kinome identifies MSK1/2-CREB1 as an essential pathway mediating KSHV lytic replication during primary infection [PublishAheadOfPrint]

  • Viruses often hijack cellular pathways to facilitate infection and replication. Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus etiologically associated with Kaposi's sarcoma, a vascular tumor of endothelial cells. Despite intensive studies, cellular pathways mediating KSHV infection and replication are still not well defined. Using an antibody array approach, we examined cellular proteins phosphorylated during KSHV primary infection of primary human umbilical vein endothelial cells. Enrichment analysis identified integrin/MAPK, insulin/EGFR and JAK/STAT as the activated networks during KSHV primary infection. Transcriptional factor CREB1 (cAMP responsive element-binding protein 1) had the strongest increase in phosphorylation. While knock down of CREB1 had no effect on KSHV entry and trafficking, it drastically reduced the expression of lytic transcripts and proteins, and production of infectious virions. Chemical activation of CREB1 significantly enhanced viral lytic replication. In contrast, CREB1 neither influenced the expression of latent gene LANA nor affected KSHV infectivity. Mechanistically, CREB1 was neither activated through the classic cAMP/PKA pathway, nor via the AKT, MK2 and RSK pathways. Rather, CREB1 was activated by the mitogen- and stress-activated protein kinases-1 and -2 (MSK1/2). Consequently, chemical inhibition or knock down of MSKs significantly inhibited KSHV lytic replication program; however, it had minimal effect on LANA expression and KSHV infectivity. Together, these results identify MSK1/2-CREB1 as novel essential effectors for KSHV lytic replication during primary infection. The differential effect of the MSK1/2-CREB1 pathway on the expression of viral latent and lytic genes might control the robustness of viral lytic replication, and therefore the KSHV replication program during primary infection.

    Importance Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus associated with several cancers. Through genome-wide kinase screening, we have found that KSHV activates the mitogen- and stress-activated protein kinases-1 and -2 (MSK1/2) and cAMP responsive element-binding protein 1 (CREB1) pathway during primary infection, and depends on this pathway for viral lytic replication. Inhibition of this pathway blocks KSHV lytic replication. These results illustrate a mechanism by which KSHV hijacks a cellular pathway for its replication and identify a potential therapeutic target.

  • A Screen for ERK Primed GSK-3 Substrates Identifies the p53 Inhibitor iASPP [PublishAheadOfPrint]

  • The Kaposi sarcoma associated herpesvirus (KSHV) LANA protein is essential for the replication and maintenance of virus genomes in latently KSHV infected cells. LANA also drives dysregulated cell growth through a multiplicity of mechanisms that include altering the activity of the cellular kinases ERK (extracellular signal-regulated kinase) and GSK-3 (glycogen synthase kinase -3). To investigate the potential impact of these changes in enzyme activity, we used protein microarrays to identify cell proteins that were phosphorylated by the combination of ERK and GSK-3. The assays identified 58 potential ERK primed GSK-3 substrates of which 23 had evidence for in vivo phosphorylation in mass spectrometry databases. Two of these, SMAD4 and iASPP, were selected for further analysis and were confirmed as ERK primed GSK-3 substrates. Co-transfection experiments revealed that iASPP, but not SMAD4, was targeted for degradation in the presence of GSK-3. iASPP interferes with apoptosis induced by p53 family members. To determine the importance of iASPP to KSHV infected cell growth, PEL cells were treated with an iASPP inhibitor, in the presence or absence of the MDM2 inhibitor Nutlin-3. Drug inhibition of iASPP activity induced apoptosis in BC3 and BCBL1 PEL cells but did not induce PARP cleavage in virus negative BJAB cells. The effect of iASPP inhibition was additive with Nutlin-3. Interfering with iASPP function is therefore another mechanism that can sensitize KSHV positive PEL cells to cell death.

    IMPORTANCE: KSHV is associated with several malignancies including primary effusion lymphoma (PEL). The KSHV encoded LANA protein is multi-functional and promotes both cell growth and resistance to cell death. LANA is known to activate the ERK kinase and limit the activity of another kinase, GSK -3. To discover ways in which LANA manipulation of these two kinases might impact on the PEL cell survival, we screened a human protein microarray for ERK primed, GSK-3 substrates. One of the proteins identified, iASPP, showed reduced levels in the presence of GSK-3. Further, blocking iASPP activity increased cell death, particularly in p53 wild-type BC3 PEL cells.

  • HIV-1 Reservoir Dynamics after Vaccination and Antiretroviral Therapy Interruption Are Associated with Dendritic Cell-Vaccine Induced T-Cell Responses [PublishAheadOfPrint]

  • HIV-1 specific immune responses induced by a dendritic cells (DCs) therapeutic vaccine might have some effect on viral reservoir. We measured total and integrated HIV-1 DNA in isolated CD4 T cells in patients on cART randomized to receive DC pulsed with autologous HIV-1 (n=24) (DC-HIV-1) or with non-pulsed DCs (n=12) (DC-control) at 6 time-points: before any cART, before STOP1 (first cART interruption 56 weeks before the first immunization to isolate virus for pulsing DCs), before and after vaccinations (VAC1 and VAC2) and at weeks 12 and 48 after second cART interruption. Vaccinations did not influence HIV-1 DNA levels in vaccinated subjects. After cART interruption post-vaccination (week 12), while total HIV-1 DNA significantly increased in both arms, integrated HIV-1 DNA did not change in vaccinees (1.8 to 1.9, p=0.22) and increased in controls (1.8 to 2.1, p=0.02) (p=0.03 for the difference between groups). However, this lack of increase of integrated HIV-1 DNA observed in DC-HIV-1 group was transient and at week 48 after cART interruption no differences were observed between groups. HIV-1 specific T cells responses at VAC2 time-point were inversely correlated with total and integrated HIV-1 after cART interruption in vaccinees (r=-0.69, p=0.002 and r=-0.82, pllt;0.0001, respectively). No correlations were found in controls. HIV-1-specific T-cell immune responses elicited by DC therapeutic vaccines drive changes in HIV-1 DNA after vaccination and cART interruption.

    IMPORTANCE There is an intense interest in developing strategies to target HIV-1 reservoirs that create barriers to cure. The development of therapeutic vaccines aimed at enhancing immune mediated clearance of virus producing cells is of high priority. Few therapeutic vaccine clinical trials have investigate the role of therapeutic vaccines as a strategy to safely eliminate or control viral reservoirs. We recently reported that a dendritic cell based therapeutic vaccine was able to decrease significantly viral set-point in vaccinated patients with a concomitant increase in HIV-1--specific T cell responses. HIV-1 specific T cell immune responses elicited by this therapeutic dendritic cell vaccine drove changes of viral reservoir after vaccinations and significantly delayed the replenishment of integrated HIV-1 DNA after cART interruption. These data help to understand how an immunization could shift the virus/host balance and are instrumental to better design strategies to reach the functional cure of HIV-1 infection.

  • Superoxide-generating Nox5{alpha} is functionally required for HTLV-1-induced cell transformation phenotype [PublishAheadOfPrint]

  • Human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and transforms T-cells in vitro. To our knowledge, the functional role of reactive oxygen species (ROS)-generating NADPH oxidase (Nox) 5 in HTLV-1 transformation remains undefined. Here, we found that the Nox5aalpha; expression was upregulated in 88% of 17 ATL patient samples but not in normal peripheral blood T-cells. Upregulation of Nox5aalpha; variant was transcriptionally sustained by the constitutive Jak-STAT5 signaling pathway in IL-2-independent HTLV-1-transformed cell lines including MT1 and MT2, whereas it was transiently induced by the IL-2-triggered Jak-STAT5 axis in uninfected T-cells. A Nox inhibitor, diphenylene iodonium and anti-oxidants such as N-acetyl cysteine blocked proliferation of MT1 and MT2 cells. Ablation of Nox5aalpha; by small interfering RNAs abrogated ROS production, inhibited cellular activities including proliferation, migration, and survival, and suppressed tumorigenicity in immunodeficient NOG mice. The findings suggest that Nox5aalpha; is a key molecule for redox-signal-mediated maintenance of HTLV-1 transformation phenotype and could be a potential molecular target for therapeutic intervention of cancer development.

    IMPORTANCE HTLV-1 is the first human oncogenic retrovirus associated with ATL. Despite the extensive study over the years, the mechanism underlying HTLV-1-induced cell transformation is not fully understood. In this study, we addressed the expression and function of ROS-generating Nox family genes in HTLV-1-transformed cells. Our study provides the first evidence that the upregulated expression of Nox5aalpha; is associated with the pathological state of ATL peripheral blood mononuclear cells, and that Nox5aalpha; is an integral component of the Jak-STAT5 signaling pathway in HTLV-1-transformed T-cells. Nox5aalpha;-derived ROS are critically involved in the regulation of cellular activities in HTLV-1-transformed cells including proliferation, migration, survival, and tumorigenicity. These results indicate that Nox5aalpha;-derived ROS are functionally required for maintenance of HTLV-1 transformation phenotype. The finding provides new insight into the redox-dependent mechanism of HTLV-1 transformation and raises an intriguing possibility that Nox5aalpha; serves as a potential molecular target to treat HTLV-1-related leukemia.

  • Interferon-inducible Protein Tetherin Inhibits Hepatitis B Virus Virion Secretion [PublishAheadOfPrint]

  • Interferon alpha (IFN-aalpha;) is an approved medication for chronic hepatitis B therapy. Besides acting as an immunomodulator, IFN-aalpha; elicits a pleiotropic antiviral state in HBV infected hepatocytes, but whether or not IFN-aalpha; impedes the late steps of HBV life cycle, such as HBV secretion, remains elusive. Herein we report that IFN-aalpha; treatment of HepAD38 cells with established HBV replication selectively reduced HBV virion release without altering the intracellular viral replication or the secretion of HBV subviral particles and nonenveloped capsids. In search of the interferon stimulated gene(s) that is responsible for HBV virion reduction, we found that tetherin, a broad spectrum antiviral transmembrane protein that inhibits the egress of a variety of enveloped viruses, was highly induced by IFN-aalpha; in HepAD38 cells and in primary human hepatocytes. We further demonstrated that the expression of full-length tetherin, but not the C-terminal glycosylphosphatidylinositol (GPI) anchor-truncated form, inhibited HBV virion egress from HepAD38 cells. In addition, the GPI anchor-truncated tetherin exhibited a dominant negative effect and was incorporated into the liberated virions. We also found the colocalization of tetherin and HBV L protein at intracellular multivesicular body, where the budding of HBV virion takes place. In line with this, electron microscopy demonstrated the HBV virion was tethered in the lumen of the cisterna membrane under tetherin expression. Finally, knock down of tetherin or overexpression of the dominant-negative tetherin attenuated IFN-aalpha;-mediated reduction of HBV virion release. Taken together, our study suggests that IFN-aalpha; inhibits HBV virion egress from hepatocytes through the induction of tetherin.

    IMPORTANCE Tetherin is a host restriction factor that blocks the egress of a variety of enveloped viruses through tethering the budding virions on the cell surface by its membrane anchor domains. Herein we report that interferon directly and selectively inhibits the secretion of HBV virion, but not subviral particles or nonenveloped capsids, through the induction of tetherin in hepatocyte-derived cells. The antiviral function of tetherin requires the carboxyl-terminal GPI anchor, while the GPI anchor deletion mutant exhibits dominant negative activity and attaches to the liberated HBV virion. Consistent with the fact that HBV is an intracellular budding virus, microscopy analyses demonstrated that the tethering of HBV virion occurs in the intracellular cisterna, and that tetherin colocalizes with HBV virion on multivesicular body, where is the HBV virion budding site. Our study not only expands the antiviral spectrum of tetherin, but also sheds light on the mechanisms of interferon-elicited anti-HBV responses.

  • The expected effect of deleterious mutations on within-host adaptation of pathogens [PublishAheadOfPrint]

  • Adaptation is a common theme in both pathogen emergence, for example in zoonotic cross-species transmission, and pathogen control, where adaptation might limit the effect of the immune response and antiviral treatment. When such evolution requires deleterious intermediate mutations, fitness ridges and valleys arise in the pathogen's fitness landscape. The effect of deleterious intermediate mutations on within-host pathogen adaptation is examined with deterministic calculations, appropriate for pathogens replicating in large populations with high error rates. The effect of deleterious intermediates on pathogen adaptation is smaller than their name might suggest: when two mutations are required, and each individual single mutation is fully deleterious, the pathogen can jump across the fitness valley by obtaining two mutations at once, leading to a proportion of adapted mutant that is 20-fold lower than for the situation where all mutants are neutral. The negative effects of deleterious intermediates are typically substantially smaller, and outweighed, by fitness advantages of the adapted mutant. Moreover, requiring a specific mutation order has a substantially smaller effect on pathogen adaptation than the effect of all intermediates being deleterious. These results can be rationalized when calculating the number of routes of mutation available to the pathogen, providing a simple approach to estimate the effect of deleterious mutations. The calculations discussed here are applicable when assessing the effect of deleterious mutations on the within-host adaptation of pathogens, for example in the context of zoonotic emergence, antigenic escape, and drug resistance.

    IMPORTANCE Adaptation is critical for pathogens after zoonotic transmission into a new host species, or to achieve antigenic immune escape and drug resistance. Using a deterministic approach, the effects of deleterious intermediate mutations on pathogen adaptation are calculated whilst avoiding commonly made simplifications that do not apply to large pathogen populations replicating with high mutations rates. Perhaps unexpectedly, pathogen adaptation does not halt when the intermediate mutations are fully deleterious. Negative effects of deleterious mutations are substantially outweighed by fitness gains of adaptation. To gain an understanding of the effect of deleterious mutations on pathogen adaptation, a simple approach is introduced that counts the number of routes available to the pathogen with and without deleterious intermediate mutations. This methodology enables a straightforward calculation of the proportion of the pathogen population that will cross a fitness valley or traverse a fitness ridge, without reverting to more complicated mathematical models.

  • Serotonin Receptor Agonist 5-nonyloxytryptamine Alters the Kinetics of Reovirus Cell Entry [PublishAheadOfPrint]

  • Mammalian orthoreoviruses (reoviruses) are nonenveloped double-stranded RNA viruses that infect most mammalian species including humans. Reovirus binds to cell-surface glycans, junctional adhesion molecule-A (JAM-A), and the Nogo-1 receptor (depending on the cell type) and enters cells by receptor-mediated endocytosis. Within the endocytic compartment, reovirus undergoes stepwise disassembly, which is followed by release of the transcriptionally active viral core into the cytoplasm. In a small-molecule screen to identify host mediators of reovirus infection, we found that treatment of cells with 5-nonyloxytryptamine (5-NT), a prototype serotonin receptor agonist, diminished reovirus cytotoxicity. 5-NT also blocked reovirus infection. In contrast, treatment of cells with methiothepin mesylate, a serotonin antagonist, enhanced infection by reovirus. 5-NT did not alter cell-surface expression of JAM-A or attachment of reovirus to cells. However, 5-NT altered the distribution of early endosomes with a concomitant impairment of reovirus transit to late endosomes and a delay in reovirus disassembly. Consistent with an inhibition of viral disassembly, 5-NT treatment did not alter infection by in vitro-generated infectious subvirion particles, which bind to JAM-A but bypass a requirement for proteolytic uncoating in endosomes to infect cells. We also found that treatment of cells with 5-NT decreased infectivity of alphavirus chikungunya virus and coronavirus mouse hepatitis virus. These data suggest that serotonin receptor signaling influences cellular activities that regulate entry of diverse virus families and provide a new, potentially broad-spectrum target for antiviral drug development.

    IMPORTANCE Identification of well-characterized small molecules that modulate viral infection can accelerate development of antiviral therapeutics while also providing new tools to increase our understanding of the cellular processes that underlie virus-mediated cell injury. We conducted a small-molecule screen to identify compounds capable of inhibiting cytotoxicity caused by reovirus, a prototype double-stranded RNA virus. We found that 5-NT impairs reovirus infection by altering viral transport during cell entry. Remarkably, 5-NT also inhibits infection by an alphavirus and a coronavirus. The antiviral properties of 5-NT suggest that serotonin receptor signaling is an important regulator of infection by diverse virus families and illuminate a potential new drug target.

  • Tetherin sensitivity of influenza A viruses is strain specific: Role of hemagglutinin and neuraminidase [PublishAheadOfPrint]

  • The expression of the antiviral host cell factor tetherin is induced by interferon and can inhibit the release of enveloped viruses from infected cells. The Vpu protein of HIV-1 antagonizes the antiviral activity of tetherin and tetherin antagonists with Vpu-like activity have been identified in other viruses. In contrast, it is incompletely understood whether tetherin inhibits influenza A virus (FLUAV) release and whether FLUAV encodes tetherin antagonists. Here, we show that release of several laboratory-adapted and a seasonal FLUAV strain is inhibited by tetherin while pandemic FLUAV A/Hamburg/4/2009 is resistant. Studies with a virus-like particle system and analysis of reassortant viruses provided evidence that the viral hemagglutinin (HA) is an important determinant of tetherin antagonism but requires the presence of its cognate neuraminidase (NA) to inhibit tetherin. Finally, tetherin antagonism by FLUAV was dependent on the virion context, since retrovirus release from tetherin-positive cells was not rescued, and correlated with a HA, NA-dependent reduction in tetherin expression. In sum, our study identifies HA and NA proteins of certain pandemic FLUAV as tetherin antagonists, which has important implications for understanding FLUAV pathogenesis.

    IMPORTANCE Influenza A virus (FLUAV) infection is responsible for substantial global morbidity and mortality and understanding how the virus evades immune defenses of the host may uncover novel targets for antiviral intervention. Tetherin is an antiviral effector molecule of the innate immune system which can contribute to control of viral invasion. However, it has been unclear whether FLUAV are inhibited by tetherin and whether these viruses encode tetherin antagonizing proteins. Our observation that several pandemic FLUAV can counteract tetherin via their HA and NA proteins identifies these proteins as novel tetherin antagonists and indicates that HA/NA-dependent inactivation of innate defenses may contribute to the efficient spread of pandemic FLUAV.

  • Alfalfa leaf curl virus: An aphid-transmitted geminivirus [PublishAheadOfPrint]

  • The family Geminiviridae comprises seven genera differentiated by genome organisation, sequence similarity and insect vector. Capulavirus, an eighth genus, has been proposed so as to accommodate two newly discovered highly divergent geminiviruses that presently have no known vector. Alfalfa leaf curl virus, identified here as a third capulavirus is shown to be transmitted by Aphis craccivora: This is the first report of an aphid-transmitted geminivirus.

  • HIV-1 Resistance to the Capsid-Targeting Inhibitor PF74 Results in Altered Dependence on Host Factors Required for Virus Nuclear Entry [PublishAheadOfPrint]

  • During HIV-1 infection of cells, the viral capsid plays critical roles in reverse transcription and nuclear entry of the virus. The capsid-targeting small molecule PF74 inhibits HIV-1 at early stages of infection. HIV-1 resistance to PF74 is complex, requiring multiple amino acid substitutions in the viral CA protein. Here we report the identification and analysis of a novel PF74-resistant mutant encoding amino acid changes in both domains of CA, three of which are near the pocket where PF74 binds. Interestingly, the mutant virus retained partial PF74 binding, and its replication was stimulated by the compound. The mutant capsid structure was not significantly perturbed by binding of PF74; rather, the mutations inhibited capsid interactions with CPSF6 and Nup153 and altered HIV-1 dependence on these host factors and on TNPO3. Moreover, the replication of the mutant virus was markedly impaired in activated primary CD4+ T cells and macrophages. Our results suggest that HIV-1 escapes a capsid-targeting small molecule inhibitor by altering the virus's dependence on host factors normally required for entry into the nucleus. They further imply that clinical resistance to inhibitors targeting the PF74 binding pocket is likely to be strongly limited by functional constraints on HIV-1 evolution.

    IMPORTANCE The HIV-1 capsid plays critical roles in early steps of infection and is an attractive target for therapy. Here we show that selection for resistance to a capsid-targeting small molecule inhibitor can result in viral dependence on the compound. The mutant virus was debilitated in primary T cells and macrophages---cellular targets of infection in vivo. The mutations also altered the virus's dependence on cellular factors that are normally required for HIV-1 entry into the nucleus. This work provides new information regarding mechanisms of HIV-1 resistance that should be useful in efforts to develop clinically useful drugs targeting the HIV-1 capsid.

  • Cataloguing of Potential HIV Susceptibility Factors during the Menstrual Cycle of Pigtail Macaques Using a Systems Biology Approach [PublishAheadOfPrint]

  • Our earlier studies in pig-tailed macaques demonstrated varying SHIV susceptibility during the menstrual cycle, likely caused by cyclic variations in immune responses in the female genital tract. There is concern that high-dose, long-lasting, injectable progestin-based contraception could mimic the high-progesterone luteal phase and predispose women to HIV-1 acquisition and transmission. In this study, we adopted a systems biology approach employing proteomics (tandem mass spectrometry), transcriptomics (RNA microarray hybridization), and other specific protein assays (enzyme-linked immunosorbent assays and multiplex chemokine-cytokine measurements) to characterize the effects of hormonal changes on the expression of innate factors and secreted proteins in the macaque vagina. Several antiviral factors and pathways (including acute phase response signaling and complement system) were overexpressed in the follicular phase. Conversely, during the luteal phase there were factors overexpressed (including moesins, syndecans, integrins, among others) that could play direct or indirect roles in enhancing HIV-1 infection. Thus, our study showed that specific pathways and proteins/genes might be working in tandem to regulate innate immunity, thus fostering further investigation and future design of approaches to help counter HIV-1 acquisition in the female genital tract.

    IMPORTANCE HIV infection in women is poorly understood. High levels of the hormone progesterone may make women more vulnerable to infection. This could be the case during the menstrual cycle, when using hormone-based birth control, or during pregnancy. The biological basis for increased HIV vulnerability is not known. We used an animal model with high risk for infection during periods of high progesterone. Genital secretions and tissues were studied during the menstrual cycle. Our goal was to identify biological factors upregulated at high progesterone levels, and we indeed show an upregulation of genes and proteins which enhance the ability of HIV to infect when progesterone is high. In contrast, during low progesterone periods, we find more HIV inhibitory factors. This basic research study contributes to our understanding of mechanisms that may regulate HIV infection in females under hormonal influences. Such knowledge is needed for the development of novel prevention strategies.

  • Rotavirus NSP3 is a translational surrogate of the PABP-poly(A) complex [PublishAheadOfPrint]

  • Through its interaction with the 5rrsquo; translation initiation factor eIF4G, poly(A) binding protein (PABP) facilitates the translation of 5rrsquo; -capped and 3rrsquo; -poly(A)-tailed mRNAs. Rotavirus mRNAs are capped but not polyadenylated, instead terminating in a 3rrsquo; GACC motif that is recognized by the viral protein NSP3, which competes with PABP for eIF4G binding. Upon rotavirus infection, viral, GACC-tailed mRNAs are efficiently translated while host poly(A)-tailed mRNA translation is, in contrast, severely impaired. To explore the roles of NSP3 in these two opposing events, the translational capabilities of three capped mRNAs, distinguished by either a GACC, a poly(A) or a non-GACC and non-poly(A) 3rrsquo; end, have been monitored after electroporation of cells expressing all rotavirus proteins (infected cells) or only NSP3 (stably or transiently transfected cells). In infected cells, we found that the magnitudes of translation induction (GACC-tailed mRNA) and translation reduction (poly(A)-tailed mRNA) both depended on the rotavirus strain used but were not genetically linked to NSP3. In transfected cells, even a small amount of NSP3 was sufficient to dramatically enhance GACC-tailed mRNA translation and, surprisingly, to slightly favor the translation of both poly(A)- and non-poly(A)-tailed mRNAs, likely through by stabilizing the eIF4E-eIF4G interaction. These data suggest that NSP3 is a translational surrogate of the PABP-poly(A) complex and therefore cannot by itself be responsible for inhibiting the translation of host poly(A)-tailed mRNAs upon rotavirus infection.

    IMPORTANCE To control host cell physiology and to circumvent innate immunity, many viruses have evolved powerful mechanisms aimed at inhibiting host mRNA translation while stimulating translation of their own mRNAs. How rotavirus tackles this challenge is still a matter of debate. Using rotavirus-infected cells, we show that the magnitude of cellular poly(A) mRNA translation differs with respect to rotavirus strains but is not genetically linked to NSP3. Using cells expressing rotavirus NSP3, we show that NSP3 alone not only dramatically enhances rotavirus-like mRNA translation but also enhances poly(A) mRNA translation rather than inhibiting it, likely by stabilizing the eIF4E-eIF4G complex. Thus, the inhibition of cellular polyadenylated mRNA translation during rotavirus infection cannot be solely attributed to NSP3 and is more likely the result of global competition between viral and host mRNAs for the cellular translation machinery.

  • Identification of the Receptor Binding Domain of the Spike Glycoprotein of Human Betacoronavirus HKU1. [PublishAheadOfPrint]

  • Coronavirus spike (S) glycoproteins mediate receptor binding, membrane fusion, and virus entry and determine host range. Murine betacoronavirus (bbeta;-CoV) in group A uses the N-terminal domain (NTD) of S protein to bind to its receptor, whereas bbeta;-CoVs SARS-CoV in group B and MERS-CoV in group C, respectively, and several aalpha;-CoVs use the downstream C-domain in their S proteins to recognize their receptor proteins. To identify the receptor-binding domain in the spike of human bbeta;-CoV HKU1 in group A, we generated and mapped a panel of monoclonal antibodies (mAbs) to the ectodomain of HKU1 spike. They did not cross-react with S proteins of any other CoV tested. Most of the HKU1 spike mAbs recognized epitopes in the C-domain, between amino acids 535 to 673, indicating that this region is immunodominant. Two of the mAbs blocked HKU1 virus infection of primary human tracheal-bronchial epithelial (HTBE) cells. Pre-incubation of HTBE cells with a truncated HKU1 S protein that includes the C-domain blocked infection with HKU1 virus, but pre-incubation of cells with truncated S protein containing only the NTD did not block infection. These data suggest that the receptor-binding domain (RBD) of HKU1 spike protein is located in the C-domain, where the spike proteins of aalpha;-CoVs and bbeta;-CoVs in groups B and C bind to their specific receptor proteins. Thus, two bbeta;-CoVs in group A, HKU1 and murine CoV, have evolved to use different regions of their spike glycoproteins to recognize their respective receptor proteins.

    IMPORTANCE Mouse hepatitis virus, a bbeta;-CoV in group A, uses the galectin-like NTD in its spike protein to bind its receptor protein, while HCoV-OC43, another bbeta;-CoV in group A, uses the NTD to bind to its sialic acid containing receptor. In marked contrast, the NTD of the spike glycoprotein of human respiratory bbeta;-CoV HKU1, which is also in group A, does not bind sugar. In this study, we showed that for the spike protein of HKU1, the purified C-domain, downstream of the NTD, could block HKU1 virus infection of human respiratory epithelial cells, and that several monoclonal antibodies that mapped to the C-domain neutralized virus infectivity. Thus the receptor-binding domain of HKU1 spike glycoprotein is located in the C-domain. Surprisingly, two bbeta;-CoVs in group A, MHV and HKU1, have evolved to use different regions of their spike glycoproteins to recognize their respective receptors.

  • The human adenovirus type 5 E4orf4 protein targets two phosphatase regulators of the Hippo signaling pathway [PublishAheadOfPrint]

  • When expressed alone at high levels, the human adenovirus E4orf4 protein exhibits tumor cell-specific p53-independent toxicity. A major E4orf4 target is the B55 class of PP2A regulatory subunits and we have shown recently that binding of E4orf4 inhibits PP2AB55 phosphatase activity in a dose-dependent fashion by preventing access of substrates. While interaction with B55 subunits is essential for toxicity, E4orf4 mutants exist that, despite binding B55 at high levels, are defective in cell killing, suggesting that other essential targets may exist. In an attempt to identify additional targets we undertook a proteomics approach to characterize E4orf4-interacting proteins. Our findings indicated that, in addition to PP2AB55 subunits, ASPP-PP1 complex subunits were found among the major E4orf4-binding species. Both the PP2A and ASPP-PP1 phosphatases are known to positively regulate effectors of the Hippo signaling pathway that controls expression of cell growth/survival genes by dephosphorylating the YAP transcriptional co-activator. We find here that expression of E4orf4 results in hyperphosphorylation of YAP, suggesting that Hippo signaling may be affected by E4orf4 interactions with PP2AB55 and/or ASPP-PP1 phosphatases. Furthermore, knockdown YAP1 expression was seen to enhance E4orf4 killing, again consistent with a link between E4orf4 toxicity and inhibition of the Hippo pathway. This effect may in fact contribute to the cancer cell specificity of E4orf4 toxicity as many human cancer cells rely heavily on the Hippo pathway for their enhanced proliferation.

    Importance The human adenovirus E4orf4 protein has been known for some time to induce tumor cell-specific death when expressed at high levels, and thus knowledge of its mode of action could be of importance for development of new cancer therapies. Although the B55 form of the phosphatase PP2A has long been known as an essential E4orf4 target, genetic analyses indicated that others must exist. To identify additional E4orf4 targets we performed, for the first time, a large scale affinity purification/mass spectrometry analysis of E4orf4 binding partners. Several additional candidates were detected, including key regulators of the Hippo signaling pathway that enhances cell viability in many cancers, and results of preliminary studies suggested a possible link between inhibition of Hippo signaling and E4orf4 toxicity.

  • The Nucleocapsid Protein of Coronaviruses Acts as a Viral Suppressor of RNA Silencing in Mammalian Cells [PublishAheadOfPrint]

  • RNA interference (RNAi) is a process of eukaryotic posttranscriptional gene silencing that functions in antiviral immunity in plants, nematodes, and insects. However, recent studies provided strong supports that RNAi also plays a role in antiviral mechanism in mammalian cells. To combat RNAi-mediated antiviral responses, many viruses encode viral suppressors of RNA silencing (VSR) to facilitate their replication. VSRs have been widely studied for plant and insect viruses but only few have been defined for mammalian viruses currently. Here, we identified a novel VSR from coronaviruses, a group of medically important mammalian viruses including Severe acute respiratory syndrome coronavirus (SARS-CoV), and showed that the nucleocapsid protein (N protein) of coronaviruses suppresses RNAi triggered by either short hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs) in mammalian cells. Mouse hepatitis virus (MHV) is closely related to SARS-CoV in the family Coronaviridae and was used as a coronavirus replication model. The replication of MHV increased when the N proteins were expressed in trans, while knockdown of Dicer1 or Ago2 transcripts facilitated the MHV replication in mammalian cells. These results support the hypothesis that RNAi is a part of the antiviral immunity responses in mammalian cells.

    Importance RNAi has been well known to play important antiviral roles from plants to invertebrates. However, recent studies provided strong supports that RNAi is also involved in antiviral response in mammalian cells. An important indication for RNAi-mediated antiviral activity in mammals is the fact that a number of mammalian viruses encode potent suppressor of RNA silencing (VSR). Our current results demonstrate that coronaviruses N protein could function as a VSR through its dsRNA binding activity. Mutational analysis of N protein allowed us to find out the critical residues for the VSR activity. Using the MHV-A59 as the coronavirus replication model, we showed that ectopic expression of SARS-CoV N protein could promote MHV replication in RNAi-active cells but did not in RNAi depleted cells. These results indicate that coronaviruses encode a VSR that functions in the replication cycle, and provide further evidences to support that RNAi-mediated antiviral response exists in mammalian cells.

  • Human cytomegalovirus gH/gL/gO promotes the fusion step of entry into all cell types whereas gH/gL/UL128-131 broadens virus tropism through a distinct mechanism. [PublishAheadOfPrint]

  • Interaction between gH/gL and the fusion protein gB is likely a conserved feature of the entry mechanism for all herpesviruses. Human cytomegalovirus (HCMV) gH/gL can be bound by gO, or by the set of proteins UL128, UL130, and UL131, forming gH/gL/gO and gH/gL/UL128-131. The mechanisms by which these complexes facilitate entry are poorly understood. Mutants lacking UL128-131 replicate well on fibroblasts, but fail to enter epithelial/endothelial cells, and this has lead to the general assumption that gH/gL/UL128-131 promotes gB-mediated fusion on epithelial/endothelial cells whereas gH/gL/gO provides this function on fibroblasts. This was challenged by observations that gO-null mutants were defective on all of these cell types, suggesting that entry into epithelial/endothelial cells requires both of the gH/gL complexes, but the severe replication defect of the gO mutants precluded detailed analysis. We previously reported that the ratio of gH/gL/gO and gH/gL/UL128-131 in the virion envelope varied dramatically among HCMV strains. Here, we show that stains not only differ in the ratio, but also vary in the total amount of gH/gL in the virion. Cell-type specific particle-to-PFU ratios of HCMV strains that contained different amounts of gH/gL/gO and gH/gL/UL128-131 were determined. Infection of both fibroblasts and epithelial cells was generally correlated with the abundance of gH/gL/gO, but not with gH/gL/UL128-131. The low infectivity of virions rich in gH/gL/UL128-131, but low in gH/gL/gO could be overcome by treatment with the chemical fusogen PEG, strongly arguing that gH/gL/gO provides the conserved herpesvirus "gH/gL entry function" of promoting gB-mediated fusion for entry into all cell types, whereas gH/gL/UL128-131 acts through a distinct mechanism to allow infection of select cell types.

    IMPORTANCE The functions of HCMV gH/gL complexes in entry are unclear. Unlike the well-studied Epstein-Barr virus (EBV), where gH/gL and gH/gL/gp42 complexes both seem capable of promoting gB fusion during entry into different cell types, our studies here suggest that for HCMV, gH/gL/gO promotes gB fusion on all cell types, whereas gH/gL/UL128-131 broadens virus tropism through a distinct, as yet unknown mechanism. To our knowledge, this is the first suggestion of a herpesvirus gH/gL that does not act by promoting gB fusion, which might make HCMV a useful model to study the fundamental mechanisms by which herpesvirus gH/gL regulates gB fusion. Moreover, gH/gL/UL128-131 is a candidate vaccine target. Our findings help to explain the cell type-dependent virus neutralization exhibited by anti-gH/gL/UL128-131 antibodies, and underscore the importance of gH/gL/gO as another important part of vaccine or therapeutic strategies.

  • Structural Determinants for the Selective Anti-HIV-1 Activity of the All-{beta} Alternative Conformer of XCL1 [PublishAheadOfPrint]

  • HIV-1 replication is regulated in vivo by a complex network of cytokines and chemokines. XCL1/lymphotactin, a unique metamorphic chemokine, was recently identified as a broad-spectrum endogenous HIV-1 inhibitor that blocks viral entry via direct interaction with the gp120 envelope glycoprotein. HIV-1 inhibition by XCL1 requires access to the alternative all-bbeta; conformation, which interacts with glycosaminoglycans (GAG) but not with the specific XCL1 receptor, XCR1. To investigate the structural determinants of the HIV-inhibitory function of XCL1, we performed a detailed structure-function analysis of a stabilized all-bbeta; variant, XCL1 W55D. Individual alanine substitutions of two basic residues within the 40s' loop, K42 and R43, abrogated the ability of XCL1 to bind to the viral envelope and block HIV-1 infection; moreover, a loss of HIV-inhibitory function, albeit less marked, was seen upon individual mutation of three additional basic residues, R18, R35 and K46. In contrast, mutation of K42 to arginine did not cause any loss of function, suggesting that the interaction with gp120 is primarily electrostatic in nature. Strikingly, four of these five residues cluster to form a large (~350AAring;2) positively-charged surface in the all-bbeta; XCL1 conformation, while they are dissociated in the classic chemokine fold, which is inactive against HIV-1, providing a structural basis for the selective antiviral activity of the alternatively-folded XCL1. Furthermore, we observed that changes to the N-terminal domain, which is proximal to the cluster of putative HIV-1 gp120-interacting residues, also affect the antiviral activity of XCL1. Interestingly, the complement of residues involved in HIV-1 blockade is partially overlapping, but distinct from those involved in the GAG-binding function of XCL1. These data identify key structural determinants of anti-HIV activity in XCL1, providing new templates for the development of HIV-1 entry inhibitors.

    Importance The host immune system controls HIV-1 infection through a wide array of inhibitory responses, including the induction of cytotoxic effector cells and the secretion of non-cytolytic soluble antiviral factors such as cytokines and chemokines. We recently identified XCL1/lymphotactin, a chemokine primarily produced by CD8+ T cells, as a novel endogenous factor with broad anti-HIV activity. Strikingly, only one of the two conformations that XCL1 can adopt in solution, the alternative all-bbeta; fold, mediates antiviral activity. At variance with the classic HIV-inhibitory chemokines such as CCL5/RANTES, XCL1 acts via direct interaction with the external viral envelope glycoprotein, gp120. Here, we identify the interactive surface of XCL1 that is implicated in binding to the HIV-1 envelope and HIV-1 inhibition, providing a structural basis to explain why only the all-bbeta; XCL1 conformer is effective against HIV-1. Our findings may be useful in guiding the rational design of new inhibitors of HIV-1 entry.

  • Hepatitis B Virus Covalently Closed Circular DNA Formation in Immortalized Mouse Hepatocytes Associated with Nucleocapsid Destabilization [PublishAheadOfPrint]

  • Hepatitis B virus (HBV) infects hundreds of millions of people worldwide and causes acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. HBV is an enveloped virus with a relaxed circular (RC) DNA genome. In the nuclei of infected human hepatocytes, conversion of RC DNA from the incoming virion or cytoplasmic mature nucleocapsid (NC) to the covalently closed circular (CCC) DNA, which serves as the template for producing all viral transcripts, is essential to establish and sustain viral replication. For reasons yet to be understood, HBV is apparently unable to make CCC DNA in normal mouse hepatocytes in the liver. We report here that HBV CCC DNA was formed efficiently in an immortalized mouse hepatocyte cell line, AML12HBV10, and this is associated with destabilization of mature NCs in these cells. These results suggest that destabilization of mature HBV NCs in AML12HBV10 cells facilitates efficient NC uncoating and subsequent CCC DNA formation. They further implicate NC uncoating as an important step in CCC DNA formation that is subject to host regulation and potentially a critical determinant of host range and/or cell tropism of HBV.

    Importance Persistent infection by hepatitis B virus (HBV), afflicting hundreds of millions worldwide, is sustained by the episomal viral covalently closed circular (CCC) DNA in the nuclei of infected hepatocytes. CCC DNA is converted from the viral genomic (precursor) DNA contained in cytoplasmic viral nucleocapsids. The conversion process remains ill-defined but host cell factors are thought to play an essential role. In particular, HBV fails to make CCC DNA in normal mouse hepatocytes despite the presence of large amounts of nucleocapsids containing the precursor viral DNA. We have found that in an immortalized mouse hepatocyte cell line, HBV is able to make abundant amounts of CCC DNA. This ability correlates with increased instability of viral nucleocapsids in these cells, which likely facilitates nucleocapsid disassembly (uncoating) to release the genomic DNA for conversion to CCC DNA. Our studies have thus revealed a novel mechanism of controlling viral persistence via regulating nucleocapsid disassembly.

  • Host Range Restriction of Epstein-Barr Virus and Related Lymphocryptoviruses [PublishAheadOfPrint]

  • Epstein-Barr related herpesviruses, or lymphocryptoviruses (LCV) naturally infect humans and nonhuman primates (NHP), but their host range is not well characterized. Using LCV and B cells from multiple species of Hominidae and Cercopithicidae, we show LCV can immortalize B cells from some non-native species, but growth transformation is restricted to B cells from their own family of hominoids or Old World NHP suggesting a high degree of LCV adaptation to their natural primate host.

  • Co-crystal structures of antibody N60-i3 and antibody JR4 in complex with gp120 define more Cluster A epitopes involved in effective antibody-dependent effector function against HIV-1 [PublishAheadOfPrint]

  • Accumulating evidence indicates a role for Fc receptor (FcR)-mediated effector functions of antibodies, including antibody-dependent cell-mediated cytotoxicity (ADCC), in prevention of HIV-1 acquisition and in post-infection control of viremia. Consequently, an understanding of the molecular basis for Env epitopes that constitute effective ADCC targets is of fundamental interest for humoral anti-HIV-1 immunity and for HIV-1 vaccine design. A substantial portion of FcR-effector function of potentially protective anti-HIV-1 antibodies is directed toward non-neutralizing, transitional, CD4-induceable (CD4i) epitopes associated with the gp41 reactive region of gp120 (Cluster A epitopes). Our previous studies defined the A32-like epitope within the Cluster A region and mapped it to the highly conserved and mobile layers 1 and 2 of the gp120 inner domain within C1-C2 regions of gp120. Here we elucidate additional Cluster A epitope structures, including an A32-like epitope, recognized by human mAb N60-i3 and a hybrid A32-C11-like epitope, recognized by rhesus macaque mAb JR4. These studies define for the first time a hybrid A32-C11-like epitope and map it to elements of both the A32-like sub-region and the 7 layered bbeta;-sheet of the gp41-interactive region of gp120. These studies provide additional evidence that effective antibody-dependent effector function to the Cluster A region depends on precise epitope targeting nndash; a combination of epitope footprint and mode of antibody attachment. All together these findings help in understanding how Cluster A epitopes are targeted by humoral responses

    Importance HIV/AIDS has claimed the lives of over 30 million people. Although antiretroviral drugs can control viral replication no vaccine has yet been developed to prevent the spread of the disease. Studies of natural HIV-1 infection, SIV or SHIV infected non-human primates (NHPs) and HIV-1-infected humanized mice models, passive transfer studies in infants born to HIV-infected mothers and the RV144 clinical trial have linked FcR-mediated effector functions of anti-HIV-1 antibodies with post-infection control of viremia and/or blocking viral acquisition. With this report we provide additional definition of the molecular determinants for Env antigen engagement which lead to effective antibody-dependent effector function directed to the non-neutralizing CD4-dependent epitopes in the gp41 reactive region of gp120. These findings have important implications for the development of an effective HIV-1 vaccine.

  • Role of Host Cell p32 in Herpes Simplex Virus 1 De-envelopment During Viral Nuclear Egress [PublishAheadOfPrint]

  • To clarify the function(s) of the herpes simplex virus 1 (HSV-1) major virion structural protein UL47 (also designated VP13/14), we screened cells overexpressing UL47 for UL47-binding cellular proteins. Tandem affinity purification of transiently expressed UL47 coupled with mass spectrometry-based proteomics technology and subsequent analyses showed that UL47 interacted with cell protein p32 in HSV-1-infected cells. Unlike in mock-infected cells, p32 accumulated at the nuclear rim in HSV-1-infected cells and this p32 recruitment to the nuclear rim required UL47. p32 formed a complex(es) with HSV-1 proteins UL31, UL34, Us3, UL47 and/or ICP22 in HSV-1-infected cells. All these HSV-1 proteins were previously reported to be important for HSV-1 nuclear egress, in which nucleocapsids bud through the inner nuclear membrane (primary envelopment) and the enveloped nucleocapsids then fuse with the outer nuclear membrane (de-envelopment). Like viral proteins UL31, UL34, Us3 and UL47, p32 was detected in primary enveloped virions. p32 knock-down reduced viral replication and induced membranous invaginations adjacent to the nuclear rim containing primary enveloped virions and aberrant localization of UL31 and UL34 in punctate structures at the nuclear rim. These effects of p32 knock-down were reduced in the absence of UL47. Therefore, the effects of p32 knock-down in HSV-1 nuclear egress were similar to those of the previously reported mutation(s) in HSV-1 regulatory proteins for HSV-1 de-envelopment during viral nuclear egress. Collectively, these results suggested that p32 regulated HSV-1 de-envelopment and replication, in a UL47-dependent manner.

    IMPORTANCE In this study, we have presented data suggesting that: (i) the HSV-1 major virion structural protein UL47 interacted with host cell protein p32 and mediated the recruitment of p32 to the nuclear rim in HSV-1-infected cells; (ii) p32 was a component of the HSV-1 nuclear egress complex (NEC), whose core components were UL31 and UL34; and (iii) p32 regulated HSV-1 de-envelopment during viral nuclear egress. It has been reported that p32 was a component of human cytomegalovirus NEC and was required for efficient disintegration of nuclear lamina, which has been thought to facilitate HSV-1 primary envelopment during viral nuclear egress. Thus, p32 appeared to be a core component of herpesvirus NECs, like UL31 and UL34 homologues in other herpesviruses, and to play multiple roles in herpesvirus nuclear egress.

  • Immunoglobulin with High-Titer In-vitro Cross Neutralizing Hepatitis C Virus Antibody Passively Protects Chimpanzee from Homologous, but not Heterologous Challenge [PublishAheadOfPrint]

  • The importance of neutralizing antibodies (NtAb) in protection against hepatitis C virus (HCV) remains controversial. We infused a chimpanzee with H06-immunoglobulin from a genotype 1a HCV-infected patient, and challenged with genotype strains efficiently neutralized by H06 in vitro. 1a-NtAb afforded no protection against 4a or 5a. Protection against homologous 1a lasted 18 weeks, but infection emerged when NtAb-titers waned. However, 6a-infection was prevented. The differential in vivo neutralization patterns have implications for HCV vaccine development.

  • Cellular and protein-directed glycosylation of native cleaved HIV-1 envelope [PublishAheadOfPrint]

  • The gp120/gp41 HIV-1 envelope glycoprotein (Env) is highly glycosylated with up to 50% of its mass consisting of N-linked glycans. This dense carbohydrate coat has emerged as a promising vaccine target, with its glycans acting as epitopes for a number of potent and broadly neutralizing antibodies (bnAbs). Characterizing the glycan structures present on native HIV-1 Env is thus a critical goal for the design of Env immunogens. Here, we use a complementary, multistep approach involving ion-mobility mass spectrometry and high performance liquid chromatography to comprehensively characterize the glycan structures present on HIV-1 gp120 produced in peripheral blood mononuclear cells (PBMCs). The capacity of different expression systems, including pseudoviral particles and recombinant cell surface trimers, to reproduce native-like glycosylation was then assessed. A population of oligomannose glycans on gp120 was reproduced across all expression systems, supporting this as an intrinsic property of Env that can be targeted for vaccine design. In contrast, Env produced in HEK 293T cells failed to accurately reproduce the highly processed complex-type glycan structures observed on PBMC-derived gp120, and in particular the precise linkage of sialic acid residues that cap these glycans. Finally we show that, unlike gp120, the glycans decorating gp41 are mostly complex-type sugars, consistent with the glycan specificity of bnAbs that target this region. These findings provide insights into the glycosylation of native and recombinant HIV-1 Env and can be used to inform strategies for immunogen design and preparation.

    Importance: Development of an HIV vaccine is desperately needed to control new infections and elicitation of HIV bnAbs will likely be an important component of an effective vaccine. Increasingly, HIV bnAbs are being identified that bind to the N-linked glycans coating the HIV envelope glycoproteins gp120 and gp41, highlighting them as important targets for vaccine design. It is therefore important to characterize the glycan structures present on native, virion-associated gp120 and gp41 for development of vaccines that accurately mimic native-Env glycosylation. Here we use a number of analytical techniques to precisely study the structures of both the oligomannose and complex-type glycans present on native Env to provide a reference for determining the ability of potential HIV immunogens to accurately replicate the glycosylation pattern on these native structures.

  • The ORF61 protein encoded by Simian Varicella Virus and Varicella Zoster Virus inhibits NF{kappa}B signaling by interfering with I{kappa}B{alpha} degradation [PublishAheadOfPrint]

  • Varicella Zoster Virus (VZV) causes chickenpox upon primary infection and establishes latency in ganglia. Reactivation from latency causes herpes zoster, which may be complicated by post-herpetic neuralgia. Innate immunity mediated by interferon and pro-inflammatory cytokines represent the first line of immune defense upon infection and reactivation. VZV is known to interfere with multiple innate immune signaling pathways including the central transcription factor NFB. However the role of these inhibitory mechanisms in vivo is unknown. Simian varicella virus (SVV)-infection of rhesus macaques recapitulates key aspects of VZV pathogenesis and this model thus permits examining the role of immune evasion mechanisms in vivo. Here we compare SVV and VZV with respect to interference of NFB activation. We demonstrate that both viruses prevent ubiquitination of the NFB inhibitor IĸBaalpha;, whereas SVV additionally prevents IĸBaalpha; phosphorylation. We show that the ORF61 proteins of VZV and SVV are sufficient to prevent IĸBaalpha; ubiquitination upon ectopic expression. We further demonstrate that SVV ORF61 interacts with bbeta;-TrCP, a subunit of the SCF ubiquitin ligase complex that mediates the degradation of IĸBaalpha;. This interaction seems to inactivate SCF-mediated protein degradation in general since the unrelated bbeta;-TrCP-target Snail is also stabilized by ORF61. In addition to ORF61, SVV seems to encode additional inhibitors of the NFB pathway since ORF61-deleted SVV still prevented IĸBaalpha; phosphorylation and degradation. Taken together, our data demonstrate that SVV interferes with TNFaalpha;-induced NFB activation at multiple levels which is consistent with the importance of these counter mechanisms for Varicella Virus infection.

    Importance The role of innate immunity during the establishment of primary infection, latency and reactivation by Varicella Zoster Virus (VZV) is incompletely understood. Since infection of rhesus macaques by Simian Varicella Virus (SVV) is being used as an animal model of VZV infection we characterized the molecular mechanism by which SVV interferes with innate immune activation. Specifically, we studied how SVV prevents activation of the transcription factor NFB, a central factor in eliciting pro-inflammatory responses. The identification of molecular mechanisms that counteract innate immunity might ultimately lead to better vaccines and treatments of VZV since overcoming these mechanisms either by small molecule inhibition of by genetic modification of vaccine strains is expected to reduce the pathogenic potential of VZV. Moreover, using SVV-infection of rhesus macaques it will be possible to study increasing the vulnerability of varicella viruses to innate immunity will impact viral pathogenesis.

  • Assembly of Epstein-Barr virus capsid in promyelocytic leukemia nuclear bodies [PublishAheadOfPrint]

  • The Epstein-Barr virus (EBV) capsid contains a major capsid protein, VCA; two minor capsid proteins, BDLF1 and BORF1; and a small capsid protein, BFRF3. During the lytic cycle, these capsid proteins are synthesized and imported into the host nucleus for capsid assembly. This study finds that EBV capsid proteins colocalize with promyelocytic leukemia nuclear bodies (PML-NBs) in P3HR1 cells during the viral lytic cycle, appearing as nuclear speckles under a confocal laser-scanning microscope. In a GST-pulldown study, we show that BORF1 interacts with PML-NBs in vitro. BORF1 also colocalizes with PML-NBs in EBV-negative Akata cells after transfection, and is responsible for bringing VCA and the VCA-BFRF3 complex from the cytoplasm to PML-NBs in the nucleus. Furthermore, BDLF1 is dispersed throughout the cell when expressed alone, but colocalizes with PML-NBs when BORF1 is also present in the cell. In addition, this study finds that knockdown of PML expression by shRNA does not influence intracellular levels of capsid proteins, but reduces the number of viral particles produced by P3HR1 cells. Together, these results demonstrate that BORF1 plays a critical role in bringing capsid proteins to PML-NBs, which may likely be the assembly sites of EBV capsids. The mechanisms elucidated in this study are critical to understanding the process of EBV capsid assembly.

    IMPORTANCE Capsid assembly is an important event during the Epstein-Barr virus (EBV) lytic cycle, as this process is required for the production of virions. In this study, confocal microscopy revealed that the EBV capsid protein BORF1 interacts with promyelocytic leukemia nuclear bodies (PML-NBs) in the host nucleus, and is responsible for transporting the other EBV capsid proteins, including VCA, BDLF1, and BFRF3, to these subnuclear locations prior to initiation of capsid assembly. This study also found that knockdown of PML expression by shRNA significantly reduces EBV capsid assembly capabilities. This enhanced understanding of capsid assembly offers potential for the development of novel antiviral strategies and therapies that can prevent the propagation and spread of EBV.

  • Preliminary Evaluation of a Bunyavirus Vector for Cancer Immunotherapy [PublishAheadOfPrint]

  • Replicon particles of Rift Valley fever virus, referred to as nonspreading RVFV (NSR), are intrinsically safe and highly immunogenic. Here, we demonstrate that NSR-infected human dendritic cells can activate CD8+ T-cells in vitro, and that prophylactic and therapeutic vaccination of mice with NSR encoding a tumor associated CD8 peptide can control outgrowth of lymphoma cells in vivo. These results suggest that the NSR system holds promise for cancer immunotherapy.

  • The partial attenuation of Small Hydrophobic (SH) gene deleted RSV is associated with elevated IL-1{beta} responses. [PublishAheadOfPrint]

  • The small hydrophobic (SH) gene of respiratory syncytial virus (RSV), a major cause of infant hospitalisation, encodes a viroporin of unknown function. SH gene knockout virus (RSV SH) is partially attenuated in vivo but not in vitro, suggesting that the SH protein may have an immunomodulatory role. RSV SH has been tested as a live attenuated vaccine in humans and cattle and here we demonstrate that it protected against viral re-challenge in mice. We compared the immune response to infection with wild type and SH RSV, in vivo using BALB/c mice and in vitro using epithelial cells, neutrophils and macrophages. Strikingly, the IL-1bbeta; response to RSV SH infection was greater than wild type RSV, in spite of decreased viral load and when IL-1bbeta; was blocked in vivo, viral load returned to wild type levels. A significantly higher IL-1bbeta; response to RSV SH was also detected in vitro, with greater magnitude responses in neutrophils and macrophages than epithelial cells. Depleting macrophages (with clodronate liposome) and neutrophils (with anti-Ly6G/1A8) demonstrated the contribution of these cells to the IL-1bbeta; response in vivo, the first demonstration of neutrophilic IL-1bbeta; production in response to viral lung infection. In this study we describe an increased IL-1bbeta; response to RSV SH, which may explain the attenuation in vivo and supports targeting the SH gene in live attenuated vaccines.

    Importance There is a pressing need for a vaccine for Respiratory Syncytial Virus (RSV). A number of live attenuated RSV vaccine strains have been developed in which the small hydrophobic (SH) gene has been deleted, even though the function of the SH protein is unknown. The structure of the SH protein has recently been solved showing it is a pore forming protein (viroporin). Here we demonstrate that the IL-1bbeta; response to RSV SH is greater in spite of lower viral load, which contributes to the attenuation in vivo. This potentially suggests a novel method by which viruses can evade the host response. As all Pneumovirinae and some Paramyxovirinae encode similar SH genes this new understanding may also enable the development of live attenuated vaccines for both RSV and other members of the Paramyxoviridae.

  • Genomic and Proteomic Analyses Indicate that Banchine and Campoplegine Polydnaviruses Have Similar, if not Identical, Viral Ancestors [PublishAheadOfPrint]

  • Polydnaviruses form a group of unconventional dsDNA viruses transmitted by endoparasitic wasps during egg laying into caterpillar hosts, where viral gene expression is essential to immature wasp survival. A copy of the viral genome is present in wasp chromosomes, thus ensuring vertical transmission. Polydnaviruses comprise two taxa, Bracovirus and Ichnovirus, shown to have distinct viral ancestors whose genomes were "captured" by ancestral wasps. While evidence indicates bracoviruses derive from a nudivirus ancestor, the identity of the ichnovirus progenitor remains unknown. In addition, ichnoviruses are found in two ichneumonid wasp subfamilies, Campopleginae and Banchinae, where they constitute morphologically and genomically different virus types. To address the question of whether these two ichnovirus subgroups have distinct ancestors, we used genomic, proteomic and transcriptomic analyses to characterize particle proteins of the banchine Glypta fumiferanae ichnovirus and the genes encoding them. Several proteins were found to be homologous to those identified earlier for campoplegine ichnoviruses while the corresponding genes were located in clusters of the wasp genome similar of those observed previously in a campoplegine wasp. However, for the first time in a polydnavirus system, these clusters also revealed sequences encoding enzymes presumed to form the replicative machinery of the progenitor virus and observed to be overexpressed in the virogenic tissue. Homology searches pointed to nucleocytoplasmic large DNA viruses as the likely source of these genes. These data, along with an analysis of the chromosomal form of five viral genome segments, provide clear evidence for the relatedness of the banchine and campoplegine ichnovirus ancestors.

    Importance Recent work indicates that the two recognized polydnavirus taxa, bracovirus and ichnovirus, are derived from distinct viruses whose genomes integrated into the genomes of ancestral wasps. However, the identity of the ichnovirus ancestor is unknown and questions remain regarding the possibility that the two described ichnovirus subgroups, banchine and campoplegine ichnoviruses, have distinct origins. Our study provides unequivocal evidence that these two ichnovirus types are derived from related viral progenitors. This suggests that morphological and genomic differences observed between the ichnovirus lineages, including features unique to banchine ichnovirus genome segments, result from evolutionary divergence either before or after their endogenization. Strikingly, analysis of selected wasp genomic regions revealed genes presumed to be part of the replicative machinery of the progenitor virus, shedding new light on the likely identity of this virus. Finally, these genes could well play a role in ichnovirus replication as they were overexpressed in the virogenic tissue.

  • The ERdj5 reductase cooperates with PDI to promote SV40 ER membrane translocation [PublishAheadOfPrint]

  • The non-enveloped polyomavirus (PyV) SV40 traffics from the cell surface to the endoplasmic reticulum (ER) where it penetrates the ER membrane to reach the cytosol before mobilizing into the nucleus to cause infection. Prior to ER membrane penetration, ER lumenal factors impart structural rearrangements to the virus, generating a translocation-competent virion capable of crossing the ER membrane. Here we identify ERdj5 as an ER enzyme that reduces SV40's disulfide bonds, a reaction important for its ER membrane transport and infection. ERdj5 also mediates human BK PyV infection. This enzyme cooperates with protein disulfide isomerase (PDI), a redox chaperone previously implicated in unfolding SV40, to fully stimulate membrane penetration. Negative stain electron microscopy of ER-localized SV40 suggests that ERdj5/PDI impart structural rearrangements to the virus. These conformational changes enable SV40 to engage BAP31, an ER membrane protein essential for supporting membrane penetration of the virus. Uncoupling SV40 from BAP31 traps the virus in an ER subdomain called foci that likely serve as depots from where SV40 gains access to the cytosol. Our study thus pinpoints two ER lumenal factors that coordinately prime SV40 for ER membrane translocation, and establishes a functional connection between lumenal and membrane events driving this process.

    Importance PyVs are established etiologic agents of many debilitating human diseases, especially in immunocompromised individuals. To infect cells at the cellular level, this virus family must penetrate the host ER membrane to reach the cytosol, a critical entry step. In this report, we identify two ER lumenal factors that prepare the virus for ER membrane translocation, and connect these lumenal events with events on the ER membrane. Pinpointing cellular components necessary for supporting PyV infection should lead to rational therapeutic strategies in preventing and treating PyV-related diseases.

  • Lentiviral protein transfer vectors are an efficient vaccine-platform inducing strong antigen-specific cytotoxic T cell response [PublishAheadOfPrint]

  • To induce and trigger innate and adaptive immune responses, antigen presenting cells (APCs) take up and process antigens. Retroviral particles are capable of transferring not only genetic information, but also foreign cargo proteins when genetically fused to viral structural proteins. Here, we demonstrate the capacity of lentiviral protein transfer vectors (PTV) for targeted antigen transfer directly into APCs and thereby induction of cytotoxic T cell responses. Targeting of lentiviral PTVs to APCs can be achieved analogously to gene transfer vectors by pseudotyping the particles with truncated wild-type measles virus (MV) glycoproteins (GPs), which use human SLAM as main entry receptor. SLAM is expressed on stimulated lymphocytes and APCs including dendritic cells. SLAM-targeted PTVs transferred the reporter proteins GFP or Cre recombinase with strict receptor specificity into SLAM-expressing CHO and B cell lines, in contrast to broadly transducing VSV-G pseudotyped PTVs. Primary myeloid dendritic cells (mDCs) incubated with targeted or non-targeted, ovalbumin (Ova) transferring PTVs stimulated Ova-specific T lymphocytes, especially CD8+ T cells. Administration of Ova-PTVs into SLAM-transgenic and control mice confirmed the observed predominant induction of antigen-specific CD8+ T cells and demonstrated capacity of protein transfer vectors as suitable vaccines for the induction of antigen-specific immune responses.

    Importance This study demonstrates specificity and efficacy of antigen transfer by SLAM-targeted and non-targeted lentiviral protein transfer vectors into antigen presenting cells to trigger antigen-specific immune responses in vitro and in vivo. The observed predominant activation of antigen-specific CD8+ T cells indicates the suitability of SLAM-targeted, but also non-targeted PTVs as vaccine for the induction of cytotoxic immune responses. Since cytotoxic CD8+ T lymphocytes are a mainstay of anti-tumoral immune responses, PTVs could be engineered for the transfer of specific tumor antigens provoking tailored anti-tumoral immunity. Therefore, PTVs can be used as safe and efficient alternative to gene transfer vectors or life-attenuated replicating vector platforms avoiding genotoxicity or general toxicity in highly immunocompromised patients, respectively. Thereby, the potential for easy envelope exchange allows to circumvent neutralizing antibodies e.g. during repeated boost immunizations.

  • Possible HPV38 contamination of endometrial cancer RNA-Seq samples in The Cancer Genome Atlas database [PublishAheadOfPrint]

  • Viruses are causally associated with a number of human malignancies. In this study, we sought to identify new viral-cancer associations by searching RNA-Sequencing datasets from ggt;2000 patients, encompassing 21 cancers from The Cancer Genome Atlas (TCGA), for the presence of viral sequences. In agreement with previous studies, we found human papillomavirus type 16 (HPV16) and HPV18 in oropharyngeal cancer and hepatitis B and C viruses in liver cancer. Unexpectedly, however, we found HPV38, a cutaneous form of HPV associated with skin cancer, in 32 of 168 samples with endometrial cancer. In 12 of the HPV38+ samples, we observed at least one paired read that mapped to both human and HPV38 genomes, indicative of viral integration into host DNA, something not previously demonstrated for HPV38. The expression levels of HPV38 transcripts were relatively low, and all 32 HPV38+ samples belonged to the same experimental batch of 40 samples, whereas none of the other 128 endometrial carcinoma samples were HPV38+, raising doubts about the significance of the HPV38 association. Moreover, the HPV38+ samples contained the same 10 novel single nucleotide variations (SNVs), leading us to hypothesize that one patient was infected with this new isolate of HPV38, which was integrated into his/her genome and may have cross-contaminated other TCGA samples within batch #228. Based on our analysis, we propose guidelines to examine batch effect, virus expression level, and SNVs as part of NGS data analysis for evaluating the significance of viral/pathogen sequences in clinical samples.

    Importance High-throughput RNA-Sequencing followed by computational analysis has vastly accelerated the identification of viral and other pathogenic sequences in clinical samples, but cross-contamination during the processing of the samples remain a major problem that can lead to erroneous conclusions. We found HPV38 sequences specifically present in RNA-Seq samples of endometrial cancer patients from TCGA, a virus not previously associated with this type of cancer. However, multiple lines of evidence suggest possible cross-contamination in these samples, which were processed together in the same batch. Despite this potential cross-contamination, our data indicate that we have detected a new isolate of HPV38 that appears to be integrated into the human genome. We also herein provide a general guideline for computational detection and interpretation of pathogen-disease associations.

  • Molecular signatures of immune activation and epithelial barrier remodeling are enhanced during the luteal phase of the menstrual cycle: implications for HIV susceptibility [PublishAheadOfPrint]

  • Variable infectivity and transmissibility of HIV/SHIV has been recently associated with the menstrual cycle, with particular susceptibility observed during the luteal phase in non-human primate models and ex vivo human explant cultures, but the mechanism is poorly understood. Here, we performed an unbiased, mass spectrometry-based proteomic analysis to better understand the mucosal immunological processes underpinning this observed susceptibility to HIV infection. Cervicovaginal lavage samples (n=19) were collected, characterized as follicular or luteal phase using days since last menstrual period, and analyzed by tandem-mass spectrometry. Biological insights from these data were gained using a spectrum of computational methods including hierarchical clustering, pathway analysis, gene set enrichment analysis, and partial least-squares discriminant analysis with LASSO feature selection. Of the 384 proteins identified, 43 were differentially abundant between phases (pllt;0.05, gge;2 fold change). Cell-cell adhesion proteins and antiproteases were reduced, and leukocyte recruitment (IL-8 pathway, p=1.41E-5) and extravasation proteins (p=5.62E-4) were elevated during the luteal phase. LASSO/PLSDA identified a minimal profile of 18 proteins that best distinguished the luteal phase. This profile included cytoskeletal elements and proteases known to be involved in cellular movement. Gene set enrichment analysis associated CD4+ T cell and neutrophil gene set signatures with the luteal phase (pllt;0.05). Taken all together, our findings indicate a strong association between proteins involved in tissue remodeling and leukocyte infiltration with the luteal phase, which may represent potential hormone-associated mechanisms of increased susceptibility to HIV.

    Importance Recent studies have discovered an enhanced susceptibility to HIV infection during the progesterone-dominant luteal phase of the menstrual cycle. Yet, the mechanism responsible for this enhanced susceptibility has yet to be determined. Understanding the source of this vulnerability will be important for designing efficacious HIV prevention technologies for women. Furthermore, these findings may also be extrapolated to better understand the impact of exogenous hormone application, such as the use of hormonal contraceptives, on HIV acquisition risk. Hormonal contraceptives are the most widely used contraceptive method in sub-Saharan Africa, the most HIV burdened area of the world. For this reason, research conducted to better understand how hormones impact host immunity and susceptibility factors important for HIV infection is a global health priority.

  • Newly-emergent highly pathogenic H5N9 subtype avian influenza A virus [PublishAheadOfPrint]

  • The Novel H7N9 avian influenza virus (AIV), was demonstrated to cause severe human respiratory infections in China. Here, we examined poultry specimens from live bird markets linked to human H7N9 infection in Hangzhou, China. Metagenomic sequencing revealed mixed subtypes (H5, H7, H9, N1, N2 and N9). Subsequently AIV subtypes H5N9, H7N9 and H9N2 were isolated. Evolutionary analysis showed that the hemagglutination and neuraminidase genes of the novel H5N9 virus originated from A/Muscovy duck/Vietnam/LBM227/2012 (H5N1) belonging to Clade and human-infective A/Hangzhou/1/2013 (H7N9), six internal genes were similar to those of the H5N1, H7N9 and H9N2 viruses. The virus harbored the PQRERRRKR/GL motif characteristic of highly pathogenic AIVs at the HA cleavage site. Receptor-binding experiments demonstrated that the virus binds aalpha;-2,3 sialic acid, but not aalpha;-2,6 sialic acid. Identically, pathogenicity experiment also showed that the virus caused low mortality rates in mice. This newly isolated H5N9 virus is a highly pathogenic reassortant virus originating from H5N1, H7N9 and H9N2 subtypes. Live bird markets represent a potential transmission risk to public health and the poultry industry.

    IMPORTANCE This investigation confirm that the novel H5N9 subtype avian influenza A virus is a reassortant strain originating from H5N1, H7N9 and H9N2 subtypes, which is totally different from those H5N9 viruses reported before. The novel H5N9 virus got a highly pathogenic H5 gene and an N9 gene from human-infecting H7N9, but caused low mortality rates in mice. Whether this novel H5N9 virus will cause human infections from its avian host and become a pandemic subtype, is not known yet. So it is interesting to assess the risk of the emergence of novel reassortant virus with potential transmissibility to public health.

  • An enhanced synthetic multi-clade DNA prime induces improved cross-clade reactive functional antibodies when combined with an adjuvanted protein boost in non-human primates [PublishAheadOfPrint]

  • The search for an efficacious human immunodeficiency virus (HIV-1) vaccine remains a pressing need. The moderate success of the RV144 Thai clinical vaccine trial suggested that vaccine-induced HIV-1 specific antibodies can reduce the risk of HIV-1 infection. We have made several improvements to the DNA platform and have previously shown that improved DNA vaccines alone are capable of inducing both binding and neutralizing antibodies in small animal models. In this study, we explored how an improved DNA prime and recombinant protein boost would impact HIV-specific vaccine immunogenicity in rhesus macaques (RhM). After DNA immunization with either a single HIV Env consensus sequence or multiple constructs expressing HIV subtype-specific Env consensus sequences, we detected both CD4+ and CD8+ T-cell responses to all vaccine immunogens. These T-cell responses were further increased after protein boosting to levels exceeding those of DNA only or protein only immunization. In addition, we observed antibodies that exhibited robust cross-clade binding, neutralizing and ADCC activity after immunization with the DNA prime-protein boost regimen with the multiple Env formulation inducing a more robust and broad response compared to the single Env formulation. The magnitude and functionality of these responses emphasize the strong priming effect improved DNA immunogens can induce which are further expanded upon protein boost. These results support further study of an improved synthetic DNA prime together with a protein boost for enhancing anti-HIV immune responses.

    Importance Even with effective anti-retroviral drugs, HIV remains an enormous global health burden. Vaccine development has been problematic in part due to the high degree of diversity and poor immunogenicity of the HIV Env protein. Studies suggest that a relevant HIV vaccine will likely need to induce broad cellular and humoral responses from a simple vaccine regimen due to the resource limited setting in which the HIV pandemic is most rampant. DNA vaccination lends itself well to increasing the amount of diversity included in a vaccine due to the ease of manufacturing multiple plasmids and formulating them as a single immunization. By increasing the number of Envs within a formulation, we were able to show increased breadth of responses as well as improved functionality induced in a non-human primate model. This increased breadth could be built upon, leading to better coverage against circulating strains with broader vaccine-induced protection.

  • Large tailed spindle viruses of Archaea: A new way of doing viral business [PublishAheadOfPrint]

  • Viruses of Archaea continue to surprise us. Archaeal viruses have revealed new morphologies, protein folds, and gene content. This is especially true for large spindle viruses, which only infect Archaea. We present a comparison of particle morphology, major coat protein structure, and gene content among the five characterized large spindle viruses to elucidate defining characteristics. Structural similarities and a core set of genes support the grouping of the large spindle viruses into a new superfamily.

  • Structural analysis of the roles of influenza A virus membrane-associated proteins in assembly and morphology. [PublishAheadOfPrint]

  • The assembly of influenza A virus at the plasma membrane of infected cells leads to release of enveloped virions that are typically round in tissue-culture adapted strains, but filamentous in strains isolated from patients. The viral proteins hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1) and the M2 ion channel all contribute to virus assembly. When expressed individually or in combination in the cells, they can all, under certain conditions, mediate release of membrane-enveloped particles, but their relative roles in virus assembly, release and morphology remain unclear. To investigate this, we have produced membrane-enveloped particles by plasmid-derived expression of combinations of HA, NA and M proteins (M1 and M2), or by infection with influenza A virus. We have monitored particle release, particle morphology, and plasma membrane morphology using biochemical methods, electron microscopy, electron tomography and cryo-electron tomography. Our data suggest that HA, NA or HANA expression leads to particle release through non-specific induction of membrane curvature. In contrast, co-expression with the M proteins clusters the glycoproteins into filamentous membrane protrusions, which by forming a constricted neck at the base, can be released as particles. HA and NA preferentially distribute to differently curved membranes within these particles. Both the budding intermediates and the released particles are morphologically similar to those produced during infection with influenza A virus. Together, our data provide new insights into influenza virus assembly and show that the M segment together with either of the glycoproteins is the minimal requirement to assemble and release membrane-enveloped particles that are truly virus-like.

    IMPORTANCE Influenza A virus is a major respiratory pathogen. It assembles membrane-enveloped virus particles whose shapes vary from spherical to filamentous. Here we have studied the roles of individual viral proteins in mediating virus assembly and in determining virus shape. To do this, we used a range of electron microscopy techniques to obtain and compare 2D and 3D images of virus particles and virus-like particles during and after assembly. The virus-like particles were produced using different combinations of viral proteins. Among our results, we found that co-expression of one or both of the viral surface proteins (hemagglutinin and neuraminidase), together with the viral membrane associated proteins encoded in the M segment, results in assembly and release of filamentous virus-like particles in a manner very similar to the budding and release of influenza virions. These data provide novel insights into the roles played by individual viral proteins in influenza A virus assembly.

  • Targeting importin-{alpha}7 as a therapeutic approach against pandemic influenza viruses [PublishAheadOfPrint]

  • Viral drug resistance is believed to occur less likely if compounds are directed against cellular rather than viral proteins. In this study, we analyzed the feasibility of a crucial viral replication factor, namely importin-aalpha;7, as a potential cellular drug target to combat pandemic influenza. Surprisingly, only five viral lung-to-lung passages were required to achieve 100% lethality in importin-aalpha;7-/- mice that are otherwise resistant. Viral escape from importin-aalpha;7 requirement was mediated by five mutations in the viral ribonucleoprotein complex and the surface glycoproteins. Moreover, the importin-aalpha;7-/- mouse-adapted strain became even more virulent for wild-type mice compared to the parental strain. These studies show that targeting host proteins may still result in viral escape by alternative pathways eventually giving rise to even more virulent virus strains. Thus, therapeutic intervention strategies should consider a multi-target approach to reduce viral drug resistance.

    Importance Here, we investigated the longstanding hypothesis based on in vitro studies that viral drug resistance occurs less likely if compounds are directed against cellular rather than viral proteins. Here, we challenged this hypothesis by analyzing in an in vivo animal model the feasibility of targeting the cellular factor importin-aalpha;7 - that is crucial for human influenza virus replication and pathogenesis - as an efficient antiviral strategy against pandemic influenza viruses. In summary, our studies suggest that resistance against cellular factors is possible in vivo and the emergence of even more virulent viral escape variants calls for particular caution. Thus, therapeutic intervention strategies should consider a multi-target approach using compounds against viral as well as cellular factors to reduce the risk of viral drug resistance and potentially increased virulence.

  • Structure of C-Terminal Domain of the Multifunctional ICP27 Protein from Herpes Simplex Virus-1 [PublishAheadOfPrint]

  • Herpes viruses are nuclear-replicating viruses that have successfully evolved to evade the immune system of humans, establishing life-long infections. ICP27 from herpes simplex virus (HSV) is a multifunctional regulatory protein that is functionally conserved in all known human herpes viruses. It has the potential to interact with an array of cellular proteins as well as intronless viral RNAs. ICP27 plays an essential role in viral transcription, nuclear export of intronless RNAs, translation of viral transcripts and virion host shut-off function. It has also been implicated in several signaling pathways and prevention of apoptosis. Although much is known about its central role in viral replication and infection, very little is known about the structure and mechanistic properties of ICP27 and its homologs. We present the first crystal structure of ICP27 C-terminal domain at 2.0 AAring; resolution. The structure reveals the C-terminal half of ICP27 to have a novel fold consisting of aalpha;-helices and long loops, along with a unique CHCC-type of zinc-binding motif. The two termini of this domain extend out from the central core and hint to possibilities of making interactions. ICP27 essential domain is capable of forming self-dimers as seen in the structure, which is confirmed by analytical ultracentrifugation study. Preliminary in vitro phosphorylation assays reveal that this domain may be regulated by cellular kinases.

    IMPORTANCE ICP27 is a key regulatory protein of the Herpes Simplex Virus and has functional homologs in all known human herpes viruses. Understanding the structure of this protein is a step ahead in deciphering the mechanism by which the virus thrives. In this study, we present the first structure of the C-terminal domain of ICP27 and describe its novel features. We critically analyze the structure and compare our results to the information available form earlier studies. This structure can act as a guide in future experimental designs, and can add to a better understanding of mechanism of ICP27 as well as that of its homologs.


  • Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family that includes Dengue, West Nile, Yellow Fever and Japanese encephalitis viruses, causing a mosquito-borne disease transmitted by the Aedes genus, with recent outbreaks in the South Pacific. Here, we determine the importance of the human skin in the entry of ZIKV and its contribution to the induction of anti-viral immune responses. We show that human dermal fibroblasts, epidermal keratinocytes and immature dendritic cells are permissive to the most recent ZIKV isolate, responsible for the epidemic in French Polynesia. Several entry and/or adhesion factors, among which DC-SIGN, AXL, TYRO3, and to a lesser extent, TIM-1, permitted ZIKV entry with a major role for the TAM receptor AXL. ZIKV permissiveness of human skin fibroblasts was confirmed by the use of a neutralizing Ab and specific RNA silencing. ZIKV induced the transcription of TLR-3, RIG-I and MDA5, as well as several interferon-stimulated genes, including OAS2, ISG15 and MX1, characterized by a strongly enhanced interferon-bbeta; gene expression. ZIKV was found to be sensitive to the antiviral effect of both type I and type II interferons. Finally, infection of skin fibroblasts resulted in the formation of autophagosomes whose presence was associated with enhanced viral replication, as shown by the use of Torin 1, a chemical inducer of autophagy or the specific autophagy inhibitor 3-Methyladenine. The results presented herein permit to gain better insight in the biology of ZIKV and to devise strategies aiming to interfere with the pathology caused by this emerging Flavivirus.

    IMPORTANCE Zika virus (ZIKV) is an arbovirus belonging to Flaviviridae family. Vector-mediated transmission of ZIKV is initiated when a blood-feeding female Aedes mosquito injects the virus into the skin of its mammalian host, followed by infection of permissive cells via specific receptors. Indeed, skin immune cells, including dermal fibroblasts, epidermal keratinocytes and immature dendritic cells, were all found to be permissive to ZIKV infection. The results also show a major role for the phosphatidylserine receptor AXL as a ZIKV entry receptor, and cellular autophagy in enhancing ZIKV replication in permissive cells. ZIKV replication leads to activation of an antiviral innate immune response and the production of type I interferons in infected cells. Taken together, these results provide for the first time a general insight into the interaction between ZIKV and its mammalian host.

  • Mutation of a Single Envelope N-linked Glycosylation Site Enhances the Pathogenicity of Bovine Leukemia Virus [PublishAheadOfPrint]

  • Viruses have co-evolved with their host to ensure efficient replication and transmission without inducing excessive pathogenicity that would indirectly impair their persistence. This is exemplified by the bovine leukemia virus (BLV) system in which lymphoproliferative disorders develop in ruminants after latency periods of several years. In principle, the equilibrium reached between the virus and its host could be disrupted by emergence of more pathogenic strains. Intriguingly but fortunately, such a hyperpathogenic BLV strain was never observed in the field nor designed in vitro. In this study, we aimed at understanding the role of envelope N-linked glycosylation with the hypothesis that this posttranslational modification could either favor BLV infection by allowing viral entry or allow immune escape by using glycans as a shield. Using reverse genetics of an infectious molecular provirus, we have identified a N-linked envelope glycosylation site (N230) that limits viral replication and pathogenicity. Indeed, mutation N230E unexpectedly leads to enhanced fusogenicity and protein stability.

    IMPORTANCE Infection by retroviruses requires the interaction of the viral envelope protein (SU) with a membrane-associated receptor allowing fusion and release of the viral genomic RNA into the cell. Here, we have shown that N-linked glycosylation of the Bovine Leukemia Virus (BLV) SU protein is, as expected, essential for cell infection in vitro. Consistently, mutation of all glycosylation sites of a BLV provirus destroys infectivity in vivo. However, single mutations do not significantly modify replication in vivo. Instead, a particular mutation at SU codon 230 increases replication and accelerates pathogenesis. This unexpected observation has important consequences in terms of disease control and managing.

  • Characterization of Two Human Monoclonal Antibodies Neutralizing Influenza A H7N9 Viruses [PublishAheadOfPrint]

  • H7N9 caused a significant global health concern, due to its severe infection and approximately 35% mortality in humans. By screening a Fab antibody phage library derived from patients who recovered from H7N9 infections, we characterized two human monoclonal antibodies (HuMAbs), HNIgGD5 and HNIgGH8. The epitope of these two antibodies was dependent on two residues in the receptor binding site at positions V186 and L226 of the hemagglutinin glycoprotein. Both antibodies possessed high neutralizing activity.

  • Two mutations were critical for bat-to-human transmission of MERS coronavirus [PublishAheadOfPrint]

  • To understand how MERS coronavirus (MERS-CoV) transmitted from bats to humans, we compared the virus-surface spikes of MERS-CoV and a related bat coronavirus HKU4. Although HKU4 spike cannot mediate viral entry into human cells, two mutations enabled it to do so by allowing it to be activated by human proteases. These mutations are present in MERS-CoV spike, explaining why MERS-CoV infects human cells. These mutations therefore played critical roles in the bat-to-human transmission of MERS-CoV.

  • VP2 exchange and NS3/NS3a deletion in African horsesickness virus (AHSV) towards AHS Disabled Infectious Single Animal (DISA) vaccine candidates [PublishAheadOfPrint]


    African horsesickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae. There are nine serotypes of AHSV showing different levels of cross neutralization. AHSV is transmitted by species of Culicoides biting midges and causes African Horsesickness (AHS) in equids with a mortality rate of up to 95% in naiiuml;ve horses. AHS has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climate conditions appear to be competent vectors for the related bluetongue virus (BTV). To control AHS, live-attenuated vaccines (LAVs) are used in Africa. We used reverse genetics to generate llsquo;synthetic' reassortants of AHSV for all nine serotypes by exchange of genome segment 2 (Seg-2). This segment encodes VP2 which is the serotype determining protein and the dominant target for neutralizing antibodies. Single Seg-2 AHSV reassortants showed similar cytopathogenic effect in mammalian cells, but displayed different growth kinetics. Reverse genetics for AHSV was also used to study Seg-10 expressing NS3/NS3a proteins. We demonstrated that NS3/NS3a proteins are not essential for AHSV replication in vitro. NS3/NS3a of AHSV is however involved in cytopathogenic effect in mammalian cells, and is very important for virus release from cultured insect cells in particular. Similar to the concept of BT Disabled Infectious Single Animal (DISA) vaccine platform, an AHS DISA vaccine platform lacking NS3/NS3a expression was developed. Using Seg-2[lsqb]VP2[rsqb] exchange we will be able to develop AHS DISA vaccine candidates for all current AHSV serotypes.

    IMPORTANCE African horsesickness virus is transmitted by species of Culicoides biting midges and causes African Horsesickness in equids with a mortality rate of up to 95% in naiiuml;ve horses. African Horsesickness has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climate conditions are supposed to be competent vectors. Using reverse genetics, viruses for all nine serotypes were constructed by exchange of Seg-2 expressing serotype determining VP2 protein. Further, we demonstrated that non-structural protein NS3/NS3a is not essential for virus replication in vitro. However, potential spread of virus by biting midges is supposed to be blocked, since in vitro release of virus is strongly reduced due to this deletion. VP2 exchange and NS3/NS3a deletion in African horsesickness virus were combined in the concept of Disabled Infectious Single Animal vaccine for all nine serotypes.

  • The epidemiology, evolution and recent outbreaks of avian influenza viruses in China: A Review [PublishAheadOfPrint]

  • Novel reassortants of H7N9, H10N8, and H5N6 avian influenza viruses (AIVs) are currently circulating in China's poultry flocks, occasionally infecting humans and other mammals. Combined with the sometimes enzootic H5N1 and H9N2 strains, this cauldron of genetically diverse AIVs pose significant risks to public health. Here, we review the epidemiology, evolution and recent outbreaks of AIVs in China, discuss reasons behind the recent increase in the emergence of novel AIVs, and identify warning signs which may point to the emergence of a potentially virulent and highly transmissible AIV to humans. This review will be useful to authorities who consider options for the detection and control of AIV transmission in animals and humans, with the goal of preventing future epidemics and pandemics.

  • Infection patterns induced in naive adult woodchucks by virions of woodchuck hepatitis virus collected during either acute or chronic phases of infection. [PublishAheadOfPrint]

  • The infectivity of hepadnavirus virions produced during either acute or chronic stages of infection was compared by testing the ability of the virions of woodchuck hepatitis virus (WHV) to induce productive acute infection in naiiuml;ve adult woodchucks. Serum WHV collected during acute infection was compared to virions harvested from WHV-infected woodchucks during either (i) early chronic infection, when WHV-induced hepatocelluar carcinoma (HCC) was not yet developed; or (ii) late chronic infection, when established HCC was terminal. All tested types of WHV inoculum were related, because they were collected from woodchucks that originally were infected with standardized WHV7 inoculum. Despite the individual differences between animals, the kinetics of accumulation of serum relaxed circular DNA of WHV demonstrated that the virions produced during early or late chronic infection are fully capable of inducing productive acute infection with long lasting high viremia. These findings were further supported by the analysis of such intrahepatic markers of WHV infection, as replicative intermediate DNA, covalently-closed circular DNA, pre-genomic RNA and percentage of WHV core antigen-positive hepatocytes measured at several time points over the course of seventeen and a half weeks after the inoculation. In addition, the observed relationship between the production of antibodies against WHV surface antigens and parameters of WHV infection appears to be complex. Taken together, the generated data suggest that in vivo hepadnavirus virions produced during different phases of chronic infection did not demonstrate any considerable deficiencies in infectivity, when compared to that of virions generated during the acute phase of infection.

    IMPORTANCE The generated data suggest that infectivity of virions produced during the early or late stages of chronic hepadnavirus infection is not compromised. Our novel results provided several lines of further evidence supporting the idea that during the state of chronic infection in vivo, the limitations of hepadnavirus cell-to-cell spread/superinfection (observed recently in woodchuck model) are not due to diminished infectivity of the virions circulating in the blood; and are likely (i) related to the properties of hepatocytes (i.e., their capacity to support hepadnavirus infection/replication) and (ii) influenced by the immune system. The obtained results further extend the understanding of the mechanisms regulating the persistence of hepadnavirus infection. The follow-up studies that will further investigate hepadnavirus cell-to-cell spread as a potential regulator of the chronic state of the infection are warranted.

  • Differential impact of in vivo CD8+ T-lymphocyte depletion in controller versus progressor SIV-infected macaques. [PublishAheadOfPrint]

  • Numerous studies have demonstrated that CD8+ T-lymphocytes suppress virus replication during HIV/SIV infection. However, the mechanisms underlying this activity of T-cells remain incompletely understood. Here we conducted CD8+ T-lymphocyte depletion in 15 rhesus macaques (RMs) infected i.v. with SIVmac239. At day 70 post-infection the animals (10 "progressors" with high viremia and 5 "controllers" with low viremia) were CD8-depleted by s.c. administration of the antibody M-T807R1. As expected, CD8 depletion resulted in increased virus replication, more prominently in controllers as compared to progressors, which correlated inversely with pre-depletion viremia. Of note, the feature of CD8+ T-lymphocytes pre-depletion that correlated best with the increase in viremia post-depletion was the level of CD8+T-bet+ lymphocytes. We next found that CD8 depletion resulted in a homogenous increase of SIV-RNA in superficial and mesenteric lymph nodes, spleen, and the gastro-intestinal tract of both controllers and progressors. Interestingly, the level of SIV-DNA increased post-depletion in both central-memory (TCM) and effector-memory (TEM) CD4+ T-lymphocytes in progressor RMs, but decreased in the CD4+ TCM of 4 out of 5 controllers. Finally, we found that CD8 depletion is associated with a greater increase in CD4+ T-lymphocyte activation (measured by Ki-67 expression) in controllers as compared to progressors. Overall, these data reveal a differential impact of CD8+ T-lymphocyte depletion between controller and progressor SIV-infected RMs, thus emphasizing the complexity of the in vivo antiviral role of CD8+ T-lymphocytes.

    Importance In this study we further dissect the impact of CD8+ T-lymphocytes on HIV/SIV replication during SIV infection. CD8+ T-lymphocyte depletion leads to a relatively homogenous increase in viral replication in peripheral blood and tissues. CD8+ T-lymphocytes depletion resulted in a more prominent increase in viral loads and CD4+ T-lymphocyte activation in controllers relative to progressors. Interestingly, we found T-bet expression on CD8+ T-lymphocytes to be the best predictor of viral load increase following depletion. The levels of SIV-DNA increase post-depletion in both central-memory (TCM) and effector-memory (TEM) CD4+ T-lymphocytes in progressors RMs, but decrease in the CD4+ TCM of controllers. The findings described in this study provide key insights into the differential function of CD8+ T-lymphocytes in controller and progressor RMs.

  • Lower Respiratory Tract Infection of the Ferret by 2009 H1N1 Pandemic Influenza A Virus Triggers Biphasic, Systemic and Local Neutrophil Recruitment [PublishAheadOfPrint]

  • Infection of the lower respiratory tract by influenza A viruses results in an increase in inflammation and immune cell infiltration in the lung. The dynamic relationships among the lung microenvironments, the lung and systemic host responses during infection remain poorly understood. Herein, we used an extensive systematic histologic analysis coupled with live imaging to gain access to these relationships in ferrets infected with the pandemic A(H1N1)2009 virus [H1N1pdm]. Neutrophil levels rose in lungs of H1N1pdm-infected ferrets 6 hours post-infection and became concentrated at areas of H1N1pdm-infected bronchiolar epithelium by 1 dpi (days post-infection). In addition, neutrophils were increased throughout the alveolar spaces during the first 3 dpi, and returned to baseline density by 6 dpi. Histochemical staining revealed neutrophil infiltration in the lungs occurred in two waves, at 1 and 3 dpi, and gene expression within microenvironments suggested two types of neutrophils. Specifically, CCL3, but not CXCL8/IL-8, levels were greater within discrete lung microenvironments, and coincided with increased infiltration of neutrophils in the lung. We used live imaging of ferrets to monitor host responses within the lung over time with 18fluorodeoxyglucose (FDG). Sites within the H1N1pdm-infected ferret lung with high FDG had high levels of proliferative epithelium. In summary, neutrophils invaded the H1N1pdm-infected ferret lung, globally, and focally, at sites of infection. The microenvironments with increased neutrophils, did not correlate with FDG, and hence, FDG-uptake may reflect prior infection and inflammation that has experienced damage as reflected by bronchial regeneration of tissues in the lungs at sites of high FDG.

    IMPORTANCE Severe influenza disease is characterized by an acute infection of the lower airways that may rapidly progress to organ failure and death. Well-developed animal models that mimic human disease are essential to understanding the complex relationships of the microenvironment, organ and system in controlling virus replication, inflammation, and disease progression. Employing the ferret model of H1N1pdm infection, we used live imaging and comprehensive histological analyses to address specific hypothesis regarding spatial and temporal relationships that occur over the progression of infection and inflammation. We show the general invasion of neutrophils at the organ level (lung), but a distinct pattern of localized accumulation within the local microenvironment at the site of infection. Moreover, we show that these responses were biphasic within the lung. Finally, live imaging revealed an early and sustained host metabolic response at sites of infection that may reflect damage and repair of tissues in the lungs.

  • Novel receptor specificity of avian gammacoronaviruses causing enteritis [PublishAheadOfPrint]


    Viruses exploit molecules on target membrane as receptors for attachment and entry into the host cells. Thus, receptor expression patterns can define viral tissue tropism and might to some extent predict the susceptibility of a host for a particular virus. Previously, others and we have shown that respiratory pathogens of the genus gammacoronavirus, including chicken infectious bronchitis virus (IBV), require specific aalpha;2-3-linked sialylated glycans for attachment and entry. Here, we studied determinants for binding of enterotropic avian gammacoronaviruses, including those of turkey (TCoV-US), guineafowl (GfCoV) and quail (QCoV), which are evolutionary distant from respiratory avian coronaviruses based on the viral attachment protein spike (S1). We profiled the binding of recombinantly expressed S1 proteins of TCoV, GfCoV and QCoV to tissues of their respective hosts. Protein histochemistry showed that the tissue binding specificity of S1 of turkey, quail, and guineafowl CoVs was limited to intestinal tissues of each particular host, in accordance with the reported pathogenicity of these viruses in vivo. Glycan array analyses revealed that, in contrast to IBV, S1 of enteric gammacoronaviruses recognize a unique set of non-sialylated type 2 poly-N-acetyl lactosamines. Lectin histochemistry as well as tissue binding patterns of TCoV-S1 further indicated that these complex N-glycans are prominently expressed on the intestinal tract of various avian species. In conclusion, our data demonstrates not only that enteric gammacoronaviruses recognize a novel glycan receptor, but also that enterotropism may be correlated with the high specificity of spike proteins for such glycans expressed in the intestines of the avian host.

    IMPORTANCE Avian coronaviruses are economically important viruses for the poultry industry. While infectious bronchitis virus (IBV), a respiratory pathogen of chickens, is rather well known, other viruses of the genus gammacoronavirus, including those causing enteric disease, are hardly studied. In turkey, guineafowl and quail, coronaviruses have been reported to be the major causative agent of enteric diseases. Specifically, turkey coronavirus outbreaks have been reported in North America, Europe and Australia for several decades. Recently a gammacoronavirus was isolated from guineafowl with fulminating disease. To date, it is not clear why these avian coronaviruses are enteropathogenic, whereas other closely related avian coronaviruses like IBV cause respiratory disease. A comprehensive understanding of the virus' tropism and pathogenicity explained by their receptor specificity, and the receptor expression on tissues was therefore needed. Here we identify a novel glycan receptor for enteric avian coronaviruses, which will further support the development of vaccines.

  • Human Papillomavirus Infectious Entry and Trafficking is a Rapid Process [PublishAheadOfPrint]


    Previous studies have indicated that Human Papillomavirus (HPV) infectious entry is slow requiring many hours after initial infection for the virus to gain entry into the nucleus. However intracellular transport pathways are typically very rapid and in the context of a natural HPV infection in a wounded epithelium, such slow intracellular transport would seem at odds with a normal viral infection. Using synchronised cell populations we show that HPV trafficking can however be a rapid process. In cells that are infected in the late S-early G2/M phase of the cell cycle, HPV16 pseudovirion (PsV) reporter DNA gene expression is detectable by 8hrs post-infection. Likewise reporter DNA can be visualised within the nucleus in conjunction with PML nuclear bodies 1-2hrs post-infection in cells that are infected with PsVs just prior to mitotic entry. This demonstrates that endosomal trafficking of HPV is rapid, with mitosis being the main restriction on nuclear entry.

    IMPORTANCE HPV infectious entry appears to be slow and requires mitosis to occur before the incoming viral DNA can access the nucleus. In this study we show that HPV trafficking in the cell is actually very rapid. This demonstrates that in the context of a normal virus infection, the cell cycle state will have a major influence on the time it takes for an incoming virus to enter the nucleus and initiate viral gene expression.

  • Lung CD8+ T cell impairment occurs during human metapneumovirus infection despite virus-like particle (VLP) induction of functional CD8+ T cells [PublishAheadOfPrint]

  • Human metapneumovirus (HMPV) is a major cause of respiratory disease in infants, the elderly, and immunocompromised individuals worldwide. There is currently no licensed HMPV vaccine. Virus-like particles (VLPs) are an attractive vaccine candidate because they are non-infectious and elicit a neutralizing antibody response. However, studies show that serum neutralizing antibodies are insufficient for complete protection against reinfection, and that adaptive T cell immunity is important for viral clearance. HMPV and other respiratory viruses induce lung CD8+ T cell (TCD8) impairment, mediated by programmed death-1 (PD-1). In this study, we generated HMPV VLPs by expressing the fusion and matrix proteins in mammalian cells and tested whether VLP immunization induces functional HMPV-specific TCD8 responses in mice. C57BL/6 mice vaccinated twice with VLPs and subsequently challenged with HMPV were protected from lung viral replication for at least 20 weeks post-immunization. A single VLP dose elicited F- and M-specific lung TCD8 with higher function and lower expression of PD-1 and other inhibitory receptors compared to TCD8 from HMPV-infected mice. However, after HMPV challenge, lung TCD8 from VLP-vaccinated mice exhibited inhibitory receptor expression and functional impairment similar to those of mice experiencing secondary infection. HMPV challenge of VLP-immunized mmu;MT mice also elicited a large percentage of impaired lung TCD8, similar to mice experiencing secondary infection. Together, these results indicate that VLPs are a promising vaccine candidate but do not prevent lung TCD8 impairment upon HMPV challenge.

    IMPORTANCE Human metapneumovirus (HMPV) is a leading cause of acute respiratory disease for which there is no licensed vaccine. Virus-like particles (VLPs) are an attractive vaccine candidate and induce antibodies, but T cell responses are less defined. Moreover, HMPV and other respiratory viruses induce lung CD8+ T cell (TCD8) impairment mediated by programmed death-1 (PD-1). In this study, HMPV VLPs containing viral fusion and matrix proteins elicited epitope-specific TCD8 that were functional and low in PD-1 expression. Two VLP doses conferred sterilizing immunity in C57BL/6 mice and facilitated HMPV clearance in antibody-deficient mmu;MT mice without enhancing lung pathology. However, regardless of whether responding lung TCD8 had previously encountered HMPV antigens in the context of VLPs or virus, a similar proportion were impaired and expressed comparable levels of PD-1 upon viral challenge. These results suggest that VLPs are a promising vaccine candidate but do not prevent lung TCD8 impairment upon HMPV challenge.

  • Careers in virology: Science writing and journalism [PublishAheadOfPrint]

  • This article condenses some highlights from a presentation I've now given at several universities about the bench-to-newsroom career path. For readers who simply want a short explanation of how to parlay their hard-earned critical thinking skills from graduate school into a lucrative job in a growing industry: go to law school.

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