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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]

  • Viral Sensing of the Subcellular Environment Regulates the Assembly of New Viral Replicase Complexes during the Course of Infection [Gems]

  • Replication of plus-stranded RNA [(+)RNA] viruses depends on the availability of coopted host proteins and lipids. But, how could viruses sense the accessibility of cellular resources? An emerging concept based on tombusviruses, small plant viruses, is that viruses might regulate viral replication at several steps depending on what cellular factors are available at a given time point. I discuss the role of phospholipids, sterols, and cellular WW domain proteins and eukaryotic elongation factor 1A (eEF1A) in control of activation of the viral RNA-dependent RNA polymerase (RdRp) and regulation of the assembly of viral replicase complexes (VRCs). These regulatory mechanisms might explain how tombusviruses could adjust the efficiency of RNA replication and new VRC assembly to the limiting resources of the host cells during infections.

  • Exosomal Communication Goes Viral [Gems]

  • Exosomes are small vesicles secreted from cells that participate in intercellular communication events. Accumulating evidence demonstrates that host exosome pathways are hijacked by viruses and that virally modified exosomes contribute to virus spread and immune evasion. In the case of tumor viruses, recent findings suggest that alterations in normal exosome biology may promote the development and progression of cancer. These studies will be discussed in the context of our current knowledge of Epstein-Barr virus (EBV)-modified exosomes.

  • The Road Less Traveled: HIV's Use of Alternative Routes through Cellular Pathways [Minireviews]

  • Pathogens such as HIV-1, with their minimalist genomes, must navigate cellular networks and rely on hijacking and manipulating the host machinery for successful replication. Limited overlap of host factors identified as vital for pathogen replication may be explained by considering that pathogens target, rather than specific cellular factors, crucial cellular pathways by targeting different, functionally equivalent, protein-protein interactions within that pathway. The ability to utilize alternative routes through cellular pathways may be essential for pathogen survival when restricted and provide flexibility depending on the viral replication stage and the environment in the infected host. In this minireview, we evaluate evidence supporting this notion, discuss specific HIV-1 examples, and consider the molecular mechanisms which allow pathogens to flexibly exploit different routes.

  • Peptide-Dependent Recognition of HLA-B*57:01 by KIR3DS1 [Pathogenesis and Immunity]

  • Killer cell immunoglobulin-like receptors (KIRs) play an important role in the activation of natural killer (NK) cells, which in turn contribute to the effective immune control of many viral infections. In the context of HIV infection, the closely related KIR3DL1 and KIR3DS1 molecules, in particular, have been associated with disease outcome. Inhibitory signals via KIR3DL1 are disrupted by downregulation of HLA class I ligands on the infected cell surface and can also be impacted by changes in the presented peptide repertoire. In contrast, the activatory ligands for KIR3DS1 remain obscure. We used a structure-driven approach to define the characteristics of HLA class I-restricted peptides that interact with KIR3DL1 and KIR3DS1. In the case of HLA-B*57:01, we used this knowledge to identify bona fide HIV-derived peptide epitopes with similar properties. Two such peptides facilitated productive interactions between HLA-B*57:01 and KIR3DS1. These data reveal the presence of KIR3DS1 ligands within the HIV-specific peptide repertoire presented by a protective HLA class I allotype, thereby enhancing our mechanistic understanding of the processes that enable NK cells to impact disease outcome.

    IMPORTANCE Natural killer (NK) cells are implicated as determinants of immune control in many viral infections, but the precise molecular mechanisms that initiate and control these responses are unclear. The activating receptor KIR3DS1 in combination with HLA-Bw4 has been associated with better outcomes in HIV infection. However, evidence of a direct interaction between these molecules is lacking. In this study, we demonstrate that KIR3DS1 recognition of HLA-Bw4 is peptide dependent. We also identify HIV-derived peptide epitopes presented by the protective HLA-B*57:01 allotype that facilitate productive interactions with KIR3DS1. Collectively, these findings suggest a mechanism whereby changes in the peptide repertoire associated with viral infection provide a trigger for KIR3DS1 engagement and NK cell activation.

  • Genome Diversity of Epstein-Barr Virus from Multiple Tumor Types and Normal Infection [Genetic Diversity and Evolution]

  • Epstein-Barr virus (EBV) infects most of the world's population and is causally associated with several human cancers, but little is known about how EBV genetic variation might influence infection or EBV-associated disease. There are currently no published wild-type EBV genome sequences from a healthy individual and very few genomes from EBV-associated diseases. We have sequenced 71 geographically distinct EBV strains from cell lines, multiple types of primary tumor, and blood samples and the first EBV genome from the saliva of a healthy carrier. We show that the established genome map of EBV accurately represents all strains sequenced, but novel deletions are present in a few isolates. We have increased the number of type 2 EBV genomes sequenced from one to 12 and establish that the type 1/type 2 classification is a major feature of EBV genome variation, defined almost exclusively by variation of EBNA2 and EBNA3 genes, but geographic variation is also present. Single nucleotide polymorphism (SNP) density varies substantially across all known open reading frames and is highest in latency-associated genes. Some T-cell epitope sequences in EBNA3 genes show extensive variation across strains, and we identify codons under positive selection, both important considerations for the development of vaccines and T-cell therapy. We also provide new evidence for recombination between strains, which provides a further mechanism for the generation of diversity. Our results provide the first global view of EBV sequence variation and demonstrate an effective method for sequencing large numbers of genomes to further understand the genetics of EBV infection.

    IMPORTANCE Most people in the world are infected by Epstein-Barr virus (EBV), and it causes several human diseases, which occur at very different rates in different parts of the world and are linked to host immune system variation. Natural variation in EBV DNA sequence may be important for normal infection and for causing disease. Here we used rapid, cost-effective sequencing to determine 71 new EBV sequences from different sample types and locations worldwide. We showed geographic variation in EBV genomes and identified the most variable parts of the genome. We identified protein sequences that seem to have been selected by the host immune system and detected variability in known immune epitopes. This gives the first overview of EBV genome variation, important for designing vaccines and immune therapy for EBV, and provides techniques to investigate relationships between viral sequence variation and EBV-associated diseases.

  • Multiple Genome Segments Determine Virulence of Bluetongue Virus Serotype 8 [Pathogenesis and Immunity]

  • Bluetongue virus (BTV) causes bluetongue, a major hemorrhagic disease of ruminants. In order to investigate the molecular determinants of BTV virulence, we used a BTV8 strain minimally passaged in tissue culture (termed BTV8L in this study) and a derivative strain passaged extensively in tissue culture (BTV8H) in in vitro and in vivo studies. BTV8L was pathogenic in both IFNARnndash;/nndash; mice and in sheep, while BTV8H was attenuated in both species. To identify genetic changes which led to BTV8H attenuation, we generated 34 reassortants between BTV8L and BTV8H. We found that partial attenuation of BTV8L in IFNARnndash;/nndash; mice was achieved by simply replacing genomic segment 2 (Seg2, encoding VP2) or Seg10 (encoding NS3) with the BTV8H homologous segments. Fully attenuated viruses required at least two genome segments from BTV8H, including Seg2 with either Seg1 (encoding VP1), Seg6 (encoding VP6 and NS4), or Seg10 (encoding NS3). Conversely, full reversion of virulence of BTV8H required at least five genomic segments of BTV8L. We also demonstrated that BTV8H acquired an increased affinity for glycosaminoglycan receptors during passaging in cell culture due to mutations in its VP2 protein. Replication of BTV8H was relatively poor in interferon (IFN)-competent primary ovine endothelial cells compared to replication of BTV8L, and this phenotype was determined by several viral genomic segments, including Seg4 and Seg9. This study demonstrated that multiple viral proteins contribute to BTV8 virulence. VP2 and NS3 are primary determinants of BTV pathogenesis, but VP1, VP5, VP4, VP6, and VP7 also contribute to virulence.

    IMPORTANCE Bluetongue is one of the major infectious diseases of ruminants, and it is listed as a notifiable disease by the World Organization for Animal Health (OIE). The clinical outcome of BTV infection varies considerably and depends on environmental and host- and virus-specific factors. Over the years, BTV serotypes/strains with various degrees of virulence (including nonpathogenic strains) have been described in different geographical locations. However, no data are available to correlate the BTV genotype to virulence. This study shows that BTV virulence is determined by different viral genomic segments. The data obtained will help to characterize thoroughly the pathogenesis of bluetongue. The possibility to determine the pathogenicity of virus isolates on the basis of their genome sequences will help in the design of control strategies that fit the risk posed by new emerging BTV strains.

  • Changes in Susceptibility to Oncolytic Vesicular Stomatitis Virus during Progression of Prostate Cancer [Cellular Response to Infection]

  • A major challenge to oncolytic virus therapy is that individual cancers vary in their sensitivity to oncolytic viruses, even when these cancers arise from the same tissue type. Variability in response may arise due to differences in the initial genetic lesions leading to cancer development. Alternatively, susceptibility to viral oncolysis may change during cancer progression. These hypotheses were tested using cells from a transgenic mouse model of prostate cancer infected with vesicular stomatitis virus (VSV). Primary cultures from murine cancers derived from prostate-specific Pten deletion contained a mixture of cells that were susceptible and resistant to VSV. Castration-resistant cancers contained a higher percentage of susceptible cells than cancers from noncastrated mice. These results indicate both susceptible and resistant cells can evolve within the same tumor. The role of Pten deletion was further investigated using clonal populations of murine prostate epithelial (MPE) progenitor cells and tumor-derived Ptennndash;/nndash; cells. Deletion of Pten in MPE progenitor cells using a lentivirus vector resulted in cells that responded poorly to interferon and were susceptible to VSV infection. In contrast, tumor-derived Ptennndash;/nndash; cells expressed higher levels of the antiviral transcription factor STAT1, activated STAT1 in response to VSV, and were resistant to VSV infection. These results suggest that early in tumor development following Pten deletion, cells are primarily sensitive to VSV, but subsequent evolution in tumors leads to development of cells that are resistant to VSV infection. Further evolution in castration-resistant tumors leads to tumors in which cells are primarily sensitive to VSV.

    IMPORTANCE There has been a great deal of progress in the development of replication-competent viruses that kill cancer cells (oncolytic viruses). However, a major problem is that individual cancers vary in their sensitivity to oncolytic viruses, even when these cancers arise from the same tissue type. The experiments presented here were to determine whether both sensitive and resistant cells are present in prostate cancers originating from a single genetic lesion in transgenic mice, prostate-specific deletion of the gene for the tumor suppressor Pten. The results indicate that murine prostate cancers are composed of both cells that are sensitive and cells that are resistant to oncolytic vesicular stomatitis virus (VSV). Furthermore, androgen deprivation led to castration-resistant prostate cancers that were composed primarily of cells that were sensitive to VSV. These results are encouraging for the use of VSV for the treatment of prostate cancers that are resistant to androgen deprivation therapy.

  • Anti-V3/Glycan and Anti-MPER Neutralizing Antibodies, but Not Anti-V2/Glycan Site Antibodies, Are Strongly Associated with Greater Anti-HIV-1 Neutralization Breadth and Potency [Vaccines and Antiviral Agents]

  • The membrane-proximal external region (MPER), the V2/glycan site (initially defined by PG9 and PG16 antibodies), and the V3/glycans (initially defined by PGT121nndash;128 antibodies) are targets of broadly neutralizing antibodies and potential targets for anti-HIV-1 antibody-based vaccines. Recent evidence shows that antibodies with moderate neutralization breadth are frequently attainable, with 50% of sera from chronically infected individuals neutralizing gge;50% of a large, diverse set of viruses. Nonetheless, there is little systematic information addressing which specificities are preferentially targeted among such commonly found, moderately broadly neutralizing sera. We explored associations between neutralization breadth and potency and the presence of neutralizing antibodies targeting the MPER, V2/glycan site, and V3/glycans in sera from 177 antiretroviral-naive HIV-1-infected (ggt;1 year) individuals. Recognition of both MPER and V3/glycans was associated with increased breadth and potency. MPER-recognizing sera neutralized 4.62 more panel viruses than MPER-negative sera (95% prediction interval [95% PI], 4.41 to 5.20), and V3/glycan-recognizing sera neutralized 3.24 more panel viruses than V3/glycan-negative sera (95% PI, 3.15 to 3.52). In contrast, V2/glycan site-recognizing sera neutralized only 0.38 more panel viruses (95% PI, 0.20 to 0.45) than V2/glycan site-negative sera and no association between V2/glycan site recognition and breadth or potency was observed. Despite autoreactivity of many neutralizing antibodies recognizing MPER and V3/glycans, antibodies to these sites are major contributors to neutralization breadth and potency in this cohort. It may therefore be appropriate to focus on developing immunogens based upon the MPER and V3/glycans.

    IMPORTANCE Previous candidate HIV vaccines have failed either to induce wide-coverage neutralizing antibodies or to substantially protect vaccinees. Therefore, current efforts focus on novel approaches never before successfully used in vaccine design, including modeling epitopes. Candidate immunogen models identified by broadly neutralizing antibodies include the membrane-proximal external region (MPER), V3/glycans, and the V2/glycan site. Autoreactivity and polyreactivity of anti-MPER and anti-V3/glycan antibodies are thought to pose both direct and indirect barriers to achieving neutralization breadth. We found that antibodies to the MPER and the V3/glycans contribute substantially to neutralization breadth and potency. In contrast, antibodies to the V2/glycan site were not associated with neutralization breadth/potency. This suggests that the autoreactivity effect is not critical and that the MPER and the V3/glycans should remain high-priority vaccine candidates. The V2/glycan site result is surprising because broadly neutralizing antibodies to this site have been repeatedly observed. Vaccine design priorities should shift toward the MPER and V3/glycans.

  • Human Papillomavirus 16 Oncoprotein Expression Is Controlled by the Cellular Splicing Factor SRSF2 (SC35) [Transformation and Oncogenesis]

  • High-risk human papillomaviruses (HR-HPV) cause anogenital cancers, including cervical cancer, and head and neck cancers. Human papillomavirus 16 (HPV16) is the most prevalent HR-HPV. HPV oncogenesis is driven by two viral oncoproteins, E6 and E7, which are expressed through alternative splicing of a polycistronic RNA to yield four major splice isoforms (E6 full length, E6*I, E6*II, E6*X). The production of multiple mRNA isoforms from a single gene is controlled by serine/arginine-rich splicing factors (SRSFs), and HPV16 infection induces overexpression of a subset of these, SRSFs 1, 2, and 3. In this study, we examined whether these proteins could control HPV16 oncoprotein expression. Small interfering RNA (siRNA) depletion experiments revealed that SRSF1 did not affect oncoprotein RNA levels. While SRSF3 knockdown caused some reduction in E6E7 expression, depletion of SRSF2 resulted in a significant loss of E6E7 RNAs, resulting in reduced levels of the E6-regulated p53 proteins and E7 oncoprotein itself. SRSF2 contributed to the tumor phenotype of HPV16-positive cervical cancer cells, as its depletion resulted in decreased cell proliferation, reduced colony formation, and increased apoptosis. SRSF2 did not affect transcription from the P97 promoter that controls viral oncoprotein expression. Rather, RNA decay experiments showed that SRSF2 is required to maintain stability of E6E7 mRNAs. These data show that SRSF2 is a key regulator of HPV16 oncoprotein expression and cervical tumor maintenance.

    IMPORTANCE Expression of the HPV16 oncoproteins E7 and E6 drives HPV-associated tumor formation. Although increased transcription may yield increased levels of E6E7 mRNAs, it is known that the RNAs can have increased stability upon integration into the host genome. SR splicing factors (SRSFs) control splicing but can also control other events in the RNA life cycle, including RNA stability. Previously, we demonstrated increased levels of SRSFs 1, 2, and 3 during cervical tumor progression. Now we show that SRSF2 is required for expression of E6E7 mRNAs in cervical tumor but not nontumor cells and may act by inhibiting their decay. SRSF2 depletion in W12 tumor cells resulted in increased apoptosis, decreased proliferation, and decreased colony formation, suggesting that SRSF2 has oncogenic functions in cervical tumor progression. SRSF function can be targeted by known drugs that inhibit SRSF phosphorylation, suggesting a possible new avenue in abrogating HPV oncoprotein activity.

  • Gammaherpesvirus Tegument Protein ORF33 Is Associated with Intranuclear Capsids at an Early Stage of the Tegumentation Process [Structure and Assembly]

  • Herpesvirus nascent capsids, after assembly in the nucleus, must acquire a variety of tegument proteins during maturation. However, little is known about the identity of the tegument proteins that are associated with capsids in the nucleus or the molecular mechanisms involved in the nuclear egress of capsids into the cytoplasm, especially for the two human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), due to a lack of efficient lytic replication systems. Murine gammaherpesvirus 68 (MHV-68) is genetically related to human gammaherpesviruses and serves as an excellent model to study the de novo lytic replication of gammaherpesviruses. We have previously shown that open reading frame 33 (ORF33) of MHV-68 is a tegument protein of mature virions and is essential for virion assembly and egress. However, it remains unclear how ORF33 is incorporated into virions. In this study, we first show that the endogenous ORF33 protein colocalizes with capsid proteins at discrete areas in the nucleus during viral infection. Cosedimentation analysis as well as an immunoprecipitation assay demonstrated that ORF33 is associated with both nuclear and cytoplasmic capsids. An immunogold labeling experiment using an anti-ORF33 monoclonal antibody revealed that ORF33-rich areas in the nucleus are surrounded by immature capsids. Moreover, ORF33 is associated with nucleocapsids prior to primary envelopment as well as with mature virions in the cytoplasm. Finally, we show that ORF33 interacts with two capsid proteins, suggesting that nucleocapsids may interact with ORF33 in a direct manner. In summary, we identified ORF33 to be a tegument protein that is associated with intranuclear capsids prior to primary envelopment, likely through interacting with capsid proteins in a direct manner.

    IMPORTANCE Morphogenesis is an essential step in virus propagation that leads to the generation of progeny virions. For herpesviruses, this is a complicated process that starts in the nucleus. Although the process of capsid assembly and genome packaging is relatively well understood, how capsids acquire tegument (the layer between the capsid and the envelope in a herpesvirus virion) and whether the initial tegumentation process takes place in the nucleus remain unclear. We previously showed that ORF33 of MHV-68 is a tegument protein and functions in both the nuclear egress of capsids and final virion maturation in the cytoplasm. In the present study, we show that ORF33 is associated with intranuclear capsids prior to primary envelopment and identify novel interactions between ORF33 and two capsid proteins. Our work provides new insights into the association between tegument proteins and nucleocapsids at an early stage of the virion maturation process for herpesviruses.

  • Viral Bcl-2 Encoded by the Kaposi's Sarcoma-Associated Herpesvirus Is Vital for Virus Reactivation [Genome Replication and Regulation of Viral Gene Expression]

  • The Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 16 (orf16) encodes a viral Bcl-2 (vBcl-2) protein which shares sequence and functional homology with the Bcl-2 family. Like its cellular homologs, vBcl-2 protects various cell types from apoptosis and can also negatively regulate autophagy. vBcl-2 is transcribed during lytic infection; however, its exact function has not been determined to date. By using bacterial artificial chromosome 16 (BAC16) clone carrying the full-length KSHV genome, we have generated recombinant KSHV mutants that fail to express vBcl-2 or express mCherry-tagged vBcl-2. We show that the vBcl-2 protein is expressed at relatively low levels during lytic induction and that a lack of vBcl-2 largely reduces the efficiency of KSHV reactivation in terms of lytic gene expression, viral DNA replication, and production of infectious particles. In contrast, the establishment of latency was not affected by the absence of vBcl-2. Our findings suggest an important role for vBcl-2 during initial phases of lytic reactivation and/or during subsequent viral propagation. Given the known functions of vBcl-2 in regulating apoptosis and autophagy, which involve its direct interaction with cellular proteins and thus require high levels of protein expression, it appears that vBcl-2 may have additional regulatory functions that do not depend on high levels of protein expression.

    IMPORTANCE The present study shows for the first time the expression of endogenous vBcl-2 protein in KSHV-infected cell lines and demonstrates the importance of vBcl-2 during the initial phases of lytic reactivation and/or during its subsequent propagation. It is suggested that vBcl-2 has additional regulatory functions beyond apoptosis and autophagy repression that do not depend on high levels of protein expression.

  • Identification of the Essential Role of Viral Bcl-2 for Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication [Virus-Cell Interactions]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) evades host defenses through tight suppression of autophagy by targeting each step of its signal transduction: by viral Bcl-2 (vBcl-2) in vesicle nucleation, by viral FLIP (vFLIP) in vesicle elongation, and by K7 in vesicle maturation. By exploring the roles of KSHV autophagy-modulating genes, we found, surprisingly, that vBcl-2 is essential for KSHV lytic replication, whereas vFLIP and K7 are dispensable. Knocking out vBcl-2 from the KSHV genome resulted in decreased lytic gene expression at the mRNA and protein levels, a lower viral DNA copy number, and, consequently, a dramatic reduction in the amount of progeny infectious viruses, as also described in the accompanying article (A. Gelgor, I. Kalt, S. Bergson, K. F. Brulois, J. U. Jung, and R. Sarid, J Virol 89:5298nndash;5307, 2015). More importantly, the antiapoptotic and antiautophagic functions of vBcl-2 were not required for KSHV lytic replication. Using a comprehensive mutagenesis analysis, we identified that glutamic acid 14 (E14) of vBcl-2 is critical for KSHV lytic replication. Mutating E14 to alanine totally blocked KSHV lytic replication but showed little or no effect on the antiapoptotic and antiautophagic functions of vBcl-2. Our study indicates that vBcl-2 harbors at least three important and genetically separable functions to modulate both cellular signaling and the virus life cycle.

    IMPORTANCE The present study shows for the first time that vBcl-2 is essential for KSHV lytic replication. Removal of the vBcl-2 gene results in a lower level of KSHV lytic gene expression, impaired viral DNA replication, and consequently, a dramatic reduction in the level of progeny production. More importantly, the role of vBcl-2 in KSHV lytic replication is genetically separated from its antiapoptotic and antiautophagic functions, suggesting that the KSHV Bcl-2 carries a novel function in viral lytic replication.

  • Single-Chain Soluble BG505.SOSIP gp140 Trimers as Structural and Antigenic Mimics of Mature Closed HIV-1 Env [Vaccines and Antiviral Agents]

  • Similar to other type I fusion machines, the HIV-1 envelope glycoprotein (Env) requires proteolytic activation; specifically, cleavage of a gp160 precursor into gp120 and gp41 subunits creates an N-terminal gp41 fusion peptide and permits folding from an immature uncleaved state to a mature closed state. While the atomic-level consequences of cleavage for HIV-1 Env are still being determined, the uncleaved state is antigenically distinct from the mature closed state, and cleavage has been reported to be essential for mimicry of the mature viral spike by soluble versions of Env. Here we report the redesign of a current state-of-the-art soluble Env mimic, BG505.SOSIP, to make it cleavage independent. Specifically, we replaced the furin cleavage site between gp120 and gp41 with Gly-Ser linkers of various lengths. The resultant linked gp120-gp41 constructs, termed single-chain gp140 (sc-gp140), exhibited different levels of structural and antigenic mimicry of the parent cleaved BG505.SOSIP. When constructs were subjected to negative selection to remove subspecies recognized by poorly neutralizing antibodies, trimers of high antigenic mimicry of BG505.SOSIP could be obtained; negative-stain electron microscopy indicated these to resemble the mature closed state. Higher proportions of BG505.SOSIP-trimer mimicry were observed in sc-gp140s with linkers of 6 or more residues, with a linker length of 15 residues exhibiting especially promising traits. Overall, flexible linkages between gp120 and gp41 in BG505.SOSIP can thus substitute for cleavage, and sc-gp140s that closely mimicked the vaccine-preferred mature closed state of Env could be obtained.

    IMPORTANCE The trimeric HIV-1 envelope glycoprotein (Env) is the sole target of virus-directed neutralizing antibody responses and a primary focus of vaccine design. Soluble mimics of Env have proven challenging to obtain and have been thought to require proteolytic cleavage into two-component subunits, gp120 and gp41, to achieve structural and antigenic mimicry of mature Env spikes on virions. Here we show that replacement of the cleavage site between gp120 and gp41 in a lead soluble gp140 construct, BG505.SOSIP, with flexible linkers can result in molecules that do not require cleavage to fold efficiently into the mature closed state. Our results provide insights into the impact of cleavage on HIV-1 Env folding. In some contexts such as genetic immunization, optimized cleavage-independent soluble gp140 constructs may have utility over the parental BG505.SOSIP, as they would not require furin cleavage to achieve mimicry of mature Env spikes on virions.

  • Clinical Control of HIV-1 by Cytotoxic T Cells Specific for Multiple Conserved Epitopes [Pathogenesis and Immunity]

  • Identification and characterization of CD8+ T cells effectively controlling HIV-1 variants are necessary for the development of AIDS vaccines and for studies of AIDS pathogenesis, although such CD8+ T cells have been only partially identified. In this study, we sought to identify CD8+ T cells controlling HIV-1 variants in 401 Japanese individuals chronically infected with HIV-1 subtype B, in which protective alleles HLA-B*57 and HLA-B*27 are very rare, by using comprehensive and exhaustive methods. We identified 13 epitope-specific CD8+ T cells controlling HIV-1 in Japanese individuals, though 9 of these epitopes were not previously reported. The breadths of the T cell responses to the 13 epitopes were inversely associated with plasma viral load (P = 2.2 x 10nndash;11) and positively associated with CD4 count (P = 1.2 x 10nndash;11), indicating strong synergistic effects of these T cells on HIV-1 control in vivo. Nine of these epitopes were conserved among HIV-1 subtype B-infected individuals, whereas three out of four nonconserved epitopes were cross-recognized by the specific T cells. These findings indicate that these 12 epitopes are strong candidates for antigens for an AIDS vaccine. The present study highlighted a strategy to identify CD8+ T cells controlling HIV-1 and demonstrated effective control of HIV-1 by those specific for 12 conserved or cross-reactive epitopes.

    IMPORTANCE HLA-B*27-restricted and HLA-B*57-restricted cytotoxic T lymphocytes (CTLs) play a key role in controlling HIV-1 in Caucasians and Africans, whereas it is unclear which CTLs control HIV-1 in Asian countries, where HLA-B*57 and HLA-B*27 are very rare. A recent study showed that HLA-B*67:01 and HLA-B*52:01-C*12:02 haplotypes were protective alleles in Japanese individuals, but it is unknown whether CTLs restricted by these alleles control HIV-1. In this study, we identified 13 CTLs controlling HIV-1 in Japan by using comprehensive and exhaustive methods. They included 5 HLA-B*52:01-restricted and 3 HLA-B*67:01-restricted CTLs, suggesting that these CTLs play a predominant role in HIV-1 control. The 13 CTLs showed synergistic effects on HIV-1 control. Twelve out of these 13 epitopes were recognized as conserved or cross-recognized ones. These findings strongly suggest that these 12 epitopes are candidates for antigens for AIDS vaccines.

  • A Metagenomics and Case-Control Study To Identify Viruses Associated with Bovine Respiratory Disease [Genetic Diversity and Evolution]

  • Bovine respiratory disease (BRD) is a common health problem for both dairy and beef cattle, resulting in significant economic loses. In order to identify viruses associated with BRD, we used a metagenomics approach to enrich and sequence viral nucleic acids in the nasal swabs of 50 young dairy cattle with symptoms of BRD. Following deep sequencing, de novo assembly, and translated protein sequence similarity searches, numerous known and previously uncharacterized viruses were identified. Bovine adenovirus 3, bovine adeno-associated virus, bovine influenza D virus, bovine parvovirus 2, bovine herpesvirus 6, bovine rhinitis A virus, and multiple genotypes of bovine rhinitis B virus were identified. The genomes of a previously uncharacterized astrovirus and picobirnaviruses were also partially or fully sequenced. Using real-time PCR, the rates of detection of the eight viruses that generated the most reads were compared for the nasal secretions of 50 animals with BRD versus 50 location-matched healthy control animals. Viruses were detected in 68% of BRD-affected animals versus 16% of healthy control animals. Thirty-eight percent of sick animals versus 8% of controls were infected with multiple respiratory viruses. Significantly associated with BRD were bovine adenovirus 3 (P llt; 0.0001), bovine rhinitis A virus (P = 0.005), and the recently described bovine influenza D virus (P = 0.006), which were detected either alone or in combination in 62% of animals with BRD. A metagenomics and real-time PCR detection approach in carefully matched cases and controls can provide a rapid means to identify viruses associated with a complex disease, paving the way for further confirmatory tests and ultimately to effective intervention strategies.

    IMPORTANCE Bovine respiratory disease is the most economically important disease affecting the cattle industry, whose complex root causes include environmental, genetics, and infectious factors. Using an unbiased metagenomics approach, we characterized the viruses in respiratory secretions from BRD cases and identified known and previously uncharacterized viruses belonging to seven viral families. Using a case-control format with location-matched animals, we compared the rates of viral detection and identified 3 viruses associated with severe BRD signs. Combining a metagenomics and case-control format can provide candidate pathogens associated with complex infectious diseases and inform further studies aimed at reducing their impact.

  • Complementary Assays Reveal a Low Level of CA Associated with Viral Complexes in the Nuclei of HIV-1-Infected Cells [Virus-Cell Interactions]

  • During uncoating, the conical capsid of HIV disassembles by dissociation of the p24 capsid protein (CA). Uncoating is known to be required for HIV replication, but the mechanism is poorly defined. Here, we examined the timing and effect of two capsid binding drugs (PF74 and BI2) on infectivity and capsid integrity in HIV-1-infected cells. The virus remained susceptible to the action of PF74 and BI2 for hours after uncoating as defined in parallel drug addition and cyclosporine (CsA) washout assays to detect the kinetics of drug susceptibility and uncoating, respectively. Resistance mutations in CA decreased the potency of these compounds, demonstrating that CA is the target of drug action. However, neither drug altered capsid integrity in a fluorescence microscopy-based assay. These data suggest that PF74 and BI2 do not alter HIV-1 uncoating but rather affect a later step in viral replication. Because both drugs bind CA, we hypothesized that a residual amount of CA associates with the viral complex after the loss of the conical capsid to serve as a target for these drugs. Superresolution structured illumination microscopy (SIM) revealed that CA localized to viral complexes in the nuclei of infected cells. Using image quantification, we determined that viral complexes localized in the nucleus displayed a smaller amount of CA than complexes at the nuclear membrane, in the cytoplasm, or in controls. Collectively, these data suggest that a subset of CA remains associated with the viral complex after uncoating and that this residual CA is the target of PF74 and BI2.

    IMPORTANCE The HIV-1 capsid is a target of interest for new antiviral therapies. This conical capsid is composed of monomers of the viral CA protein. During HIV-1 replication, the capsid must disassemble by a poorly defined process called uncoating. CA has also been implicated in later steps of replication, including nuclear import and integration. In this study, we used cell-based assays to examine the effect of two CA binding drugs (PF74 and BI2) on viral replication in infected cells. HIV-1 was susceptible to both drugs for hours after uncoating, suggesting that these drugs affect later steps of viral replication. High-resolution structured illumination microscopy (SIM) revealed that a subset of CA localized to viral complexes in the nuclei of cells. Collectively, these data suggest that a subset of CA remains associated with the viral complex after uncoating, which may facilitate later steps of viral replication and serve as a drug target.

  • Protease Inhibitors Block Multiple Functions of the NS3/4A Protease-Helicase during the Hepatitis C Virus Life Cycle [Vaccines and Antiviral Agents]

  • Hepatitis C virus (HCV) NS3 is a multifunctional protein composed of a protease domain and a helicase domain linked by a flexible linker. Protease activity is required to generate viral nonstructural (NS) proteins involved in RNA replication. Helicase activity is required for RNA replication, and genetic evidence implicates the helicase domain in virus assembly. Binding of protease inhibitors (PIs) to the protease active site blocks NS3-dependent polyprotein processing but might impact other steps of the virus life cycle. Kinetic analyses of antiviral suppression of cell culture-infectious genotype 1a strain H77S.3 were performed using assays that measure different readouts of the viral life cycle. In addition to the active-site PI telaprevir, we examined an allosteric protease-helicase inhibitor (APHI) that binds a site in the interdomain interface. By measuring nucleotide incorporation into HCV genomes, we found that telaprevir inhibits RNA synthesis as early as 12 h at high but clinically relevant concentrations. Immunoblot analyses showed that NS5B abundance was not reduced until after 12 h, suggesting that telaprevir exerts a direct effect on RNA synthesis. In contrast, the APHI could partially inhibit RNA synthesis, suggesting that the allosteric site is not always available during RNA synthesis. The APHI and active-site PI were both able to block virus assembly soon (llt;12 h) after drug treatment, suggesting that they rapidly engage with and block a pool of NS3 involved in assembly. In conclusion, PIs and APHIs can block NS3 functions in RNA synthesis and virus assembly, in addition to inhibiting polyprotein processing.

    IMPORTANCE The NS3/4A protease of hepatitis C virus (HCV) is an important antiviral target. Currently, three PIs have been approved for therapy of chronic hepatitis C, and several others are in development. NS3-dependent cleavage of the HCV polyprotein is required to generate the mature nonstructural proteins that form the viral replicase. Inhibition of protease activity can block RNA replication by preventing expression of mature replicase components. Like many viral proteins, NS3 is multifunctional, but how PIs affect stages of the HCV life cycle beyond polyprotein processing has not been well studied. Using cell-based assays, we show here that PIs can directly inhibit viral RNA synthesis and also block a late stage in virus assembly/maturation at clinically relevant concentrations.

  • Spread and Persistence of Influenza A Viruses in Waterfowl Hosts in the North American Mississippi Migratory Flyway [Genetic Diversity and Evolution]

  • While geographic distance often restricts the spread of pathogens via hosts, this barrier may be compromised when host species are mobile. Migratory waterfowl in the order Anseriformes are important reservoir hosts for diverse populations of avian-origin influenza A viruses (AIVs) and are assumed to spread AIVs during their annual continental-scale migrations. However, support for this hypothesis is limited, and it is rarely tested using data from comprehensive surveillance efforts incorporating both the temporal and spatial aspects of host migratory patterns. We conducted intensive AIV surveillance of waterfowl using the North American Mississippi Migratory Flyway (MMF) over three autumn migratory seasons. Viral isolates (n = 297) from multiple host species were sequenced and analyzed for patterns of gene dispersal between northern staging and southern wintering locations. Using a phylogenetic and nucleotide identity framework, we observed a larger amount of gene dispersal within this flyway rather than between the other three longitudinally identified North American flyways. Across seasons, we observed patterns of regional persistence of diversity for each genomic segment, along with limited survival of dispersed AIV gene lineages. Reassortment increased with both time and distance, resulting in transient AIV constellations. This study shows that within the MMF, AIV gene flow favors spread along the migratory corridor within a season, and also that intensive surveillance during bird migration is important for identifying virus dispersal on time scales relevant to pandemic responsiveness. In addition, this study indicates that comprehensive monitoring programs to capture AIV diversity are critical for providing insight into AIV evolution and ecology in a major natural reservoir.

    IMPORTANCE Migratory birds are a reservoir for antigenic and genetic diversity of influenza A viruses (AIVs) and are implicated in the spread of virus diversity that has contributed to previous pandemic events. Evidence for dispersal of avian-origin AIVs by migratory birds is rarely examined on temporal scales relevant to pandemic or panzootic threats. Therefore, characterizing AIV movement by hosts within a migratory season is important for implementing effective surveillance strategies. We conducted surveillance following birds along a major North American migratory route and observed that within a migratory season, AIVs rapidly reassorted and gene lineages were dispersed primarily within the migratory corridor. Patterns of regional persistence were observed across seasons for each gene segment. We show that dispersal of AIV gene lineages by migratory birds occurs quickly along migratory routes and that surveillance for AIVs threatening human and animal health should focus attention on these routes.

  • Mutational Analysis of Vaccinia Virus E3 Protein: the Biological Functions Do Not Correlate with Its Biochemical Capacity To Bind Double-Stranded RNA [Virus-Cell Interactions]

  • Vaccinia E3 protein has the biochemical capacity of binding to double-stranded RNA (dsRNA). The best characterized biological functions of the E3 protein include its host range function, suppression of cytokine expression, and inhibition of interferon (IFN)-induced antiviral activity. Currently, the role of the dsRNA binding capacity in the biological functions of the E3 protein is not clear. To further understand the mechanism of the E3 protein biological functions, we performed alanine scanning of the entire dsRNA binding domain of the E3 protein to examine the link between its biochemical capacity of dsRNA binding and biological functions. Of the 115 mutants examined, 20 were defective in dsRNA binding. Although the majority of the mutants defective in dsRNA binding also showed defective replication in HeLa cells, nine mutants (I105A, Y125A, E138A, F148A, F159A, K171A, L182A, L183A, and I187/188A) retained the host range function to various degrees. Further examination of a set of representative E3L mutants showed that residues essential for dsRNA binding are not essential for the biological functions of E3 protein, such as inhibition of protein kinase R (PKR) activation, suppression of cytokine expression, and apoptosis. Thus, data described in this communication strongly indicate the E3 protein performs its biological functions via a novel mechanism which does not correlate with its dsRNA binding activity.

    IMPORTANCE dsRNAs produced during virus replication are important pathogen-associated molecular patterns (PAMPs) for inducing antiviral immune responses. One of the strategies used by many viruses to counteract such antiviral immune responses is achieved by producing dsRNA binding proteins, such as poxvirus E3 family proteins, influenza virus NS1, and Ebola virus V35 proteins. The most widely accepted model for the biological functions of this class of viral dsRNA binding proteins is that they bind to and sequester viral dsRNA PAMPs; thus, they suppress the related antiviral immune responses. However, no direct experimental data confirm such a model. In this study of vaccinia E3 protein, we found that the biological functions of the E3 protein are not necessarily linked to its biochemical capacity of dsRNA binding. Thus, our data strongly point to a new concept of virus modulation of cellular antiviral responses triggered by dsRNA PAMPs.

  • Alterations in Hemagglutinin Receptor-Binding Specificity Accompany the Emergence of Highly Pathogenic Avian Influenza Viruses [Virus-Cell Interactions]

  • Highly pathogenic avian influenza viruses (HPAIVs) of hemagglutinin H5 and H7 subtypes emerge after introduction of low-pathogenic avian influenza viruses (LPAIVs) from wild birds into poultry flocks, followed by subsequent circulation and evolution. The acquisition of multiple basic amino acids at the endoproteolytical cleavage site of the hemagglutinin (HA) is a molecular indicator for high pathogenicity, at least for infections of gallinaceous poultry. Apart from the well-studied significance of the multibasic HA cleavage site, there is only limited knowledge on other alterations in the HA and neuraminidase (NA) molecules associated with changes in tropism during the emergence of HPAIVs from LPAIVs. We hypothesized that changes in tropism may require alterations of the sialyloligosaccharide specificities of HA and NA. To test this hypothesis, we compared a number of LPAIVs and HPAIVs for their HA-mediated binding and NA-mediated desialylation of a set of synthetic receptor analogs, namely, aalpha;2-3-sialylated oligosaccharides. NA substrate specificity correlated with structural groups of NAs and did not correlate with pathogenic potential of the virus. In contrast, all HPAIVs differed from LPAIVs by a higher HA receptor-binding affinity toward the trisaccharides Neu5Acaalpha;2-3Galbbeta;1-4GlcNAcbbeta; (3'SLN) and Neu5Acaalpha;2-3Galbbeta;1-3GlcNAcbbeta; (SiaLec) and by the ability to discriminate between the nonfucosylated and fucosylated sialyloligosaccharides 3'SLN and Neu5Acaalpha;2-3Galbbeta;1-4(Fucaalpha;1-3)GlcNAcbbeta; (SiaLex), respectively. These results suggest that alteration of the receptor-binding specificity accompanies emergence of the HPAIVs from their low-pathogenic precursors.

    IMPORTANCE Here, we have found for the first time correlations of receptor-binding properties of the HA with a highly pathogenic phenotype of poultry viruses. Our study suggests that enhanced receptor-binding affinity of HPAIVs for a typical "poultry-like" receptor, 3'SLN, is provided by substitutions in the receptor-binding site of HA which appeared in HA of LPAIVs in the course of transmission of LPAIVs from wild waterfowl into poultry flocks, with subsequent adaptation in poultry. The identification of LPAIVs with receptor characteristics of HPAIVs argues that the sialic acid-binding specificity of the HA may be used as a novel phenotypic marker of HPAIVs.

  • Origins and Evolutionary Dynamics of H3N2 Canine Influenza Virus [Genetic Diversity and Evolution]

  • Influenza A viruses (IAVs) are maintained mainly in wild birds, and despite frequent spillover infections of avian IAVs into mammals, only a small number of viruses have become established in mammalian hosts. A new H3N2 canine influenza virus (CIV) of avian origin emerged in Asia in the mid-2000s and is now circulating in dog populations of China and South Korea, and possibly in Thailand. The emergence of CIV provides new opportunities for zoonotic infections and interspecies transmission. We examined 14,764 complete IAV genomes together with all CIV genomes publicly available since its first isolation until 2013. We show that CIV may have originated as early as 1999 as a result of segment reassortment among Eurasian and North American avian IAV lineages. We also identified amino acid changes that might have played a role in CIV emergence, some of which have not been previously identified in other cross-species jumps. CIV evolves at a lower rate than H3N2 human influenza viruses do, and viral phylogenies exhibit geographical structure compatible with high levels of local transmission. We detected multiple intrasubtypic and heterosubtypic reassortment events, including the acquisition of the NS segment of an H5N1 avian influenza virus that had previously been overlooked. In sum, our results provide insight into the adaptive changes required by avian viruses to establish themselves in mammals and also highlight the potential role of dogs to act as intermediate hosts in which viruses with zoonotic and/or pandemic potential could originate, particularly with an estimated dog population of ~700 million.

    IMPORTANCE Influenza A viruses circulate in humans and animals. This multihost ecology has important implications, as past pandemics were caused by IAVs carrying gene segments of both human and animal origin. Adaptive evolution is central to cross-species jumps, and this is why understanding the evolutionary processes that shape influenza A virus genomes is key to elucidating the mechanisms underpinning viral emergence. An avian-origin canine influenza virus (CIV) has recently emerged in dogs and is spreading in Asia. We reconstructed the evolutionary history of CIV and show that it originated from both Eurasian and North American avian lineages. We also identified the mutations that might have been responsible for the cross-species jump. Finally, we provide evidence of multiple reassortment events between CIV and other influenza viruses (including an H5N1 avian virus). This is a cause for concern, as there is a large global dog population to which humans are highly exposed.

  • Neuraminidase Mutations Conferring Resistance to Oseltamivir in Influenza A(H7N9) Viruses [Vaccines and Antiviral Agents]

  • Human infections by avian influenza A(H7N9) virus entail substantial morbidity and mortality. Treatment of infected patients with the neuraminidase (NA) inhibitor oseltamivir was associated with emergence of viruses carrying NA substitutions. In the NA inhibition (NI) assay, R292K conferred highly reduced inhibition by oseltamivir, while E119V and I222K each caused reduced inhibition. To facilitate establishment of laboratory correlates of clinically relevant resistance, experiments were conducted in ferrets infected with virus carrying wild-type or variant NA genes recovered from the A/Taiwan/1/2013 isolate. Oseltamivir treatment (5 or 25 mg/kg of body weight/dose) was given 4 h postinfection, followed by twice-daily treatment for 5 days. Treatment of ferrets infected with wild-type virus resulted in a modest dose-dependent reduction (0.7 to 1.5 log10 50% tissue culture infectious dose [TCID50]) in nasal wash viral titers and inflammation response. Conversely, treatment failed to significantly inhibit the replication of R292K or E119V virus. A small reduction of viral titers was detected on day 5 in ferrets infected with the I222K virus. The propensity for oseltamivir resistance emergence was assessed in oseltamivir-treated animals infected with wild-type virus; emergence of R292K virus was detected in 3 of 6 ferrets within 5 to 7 days postinfection. Collectively, we demonstrate that R292K, E119V, and I222K reduced the inhibitory activity of oseltamivir, not only in the NI assay, but also in infected ferrets, judged particularly by viral loads in nasal washes, and may signal the need for alternative therapeutics. Thus, these clinical outcomes measured in the ferret model may correlate with clinically relevant oseltamivir resistance in humans.

    IMPORTANCE This report provides more evidence for using the ferret model to assess the susceptibility of influenza A(H7N9) viruses to oseltamivir, the most prescribed anti-influenza virus drug. The information gained can be used to assist in the establishment of laboratory correlates of human disease and drug therapy. The rapid emergence of viruses with R292K in treated ferrets correlates well with the multiple reports on this NA variant in treated human patients. Our findings highlight the importance of the discovery and characterization of new antiviral drugs with different mechanisms of action and the use of combination treatment strategies against emerging viruses with pandemic potential, such as avian H7N9 virus, particularly against those carrying drug resistance markers.

  • Diversifying Selection Analysis Predicts Antigenic Evolution of 2009 Pandemic H1N1 Influenza A Virus in Humans [Genetic Diversity and Evolution]

  • Although a large number of immune epitopes have been identified in the influenza A virus (IAV) hemagglutinin (HA) protein using various experimental systems, it is unclear which are involved in protective immunity to natural infection in humans. We developed a data mining approach analyzing natural H1N1 human isolates to identify HA protein regions that may be targeted by the human immune system and can predict the evolution of IAV. We identified 16 amino acid sites experiencing diversifying selection during the evolution of prepandemic seasonal H1N1 strains and found that 11 sites were located in experimentally determined B-cell/antibody (Ab) epitopes, including three distinct neutralizing Caton epitopes: Sa, Sb, and Ca2 [A. J. Caton, G. G. Brownlee, J. W. Yewdell, and W. Gerhard, Cell 31:417nndash;427, 1982, http://dx.doi.org/10.1016/0092-8674(82)90135-0]. We predicted that these diversified epitope regions would be the targets of mutation as the 2009 H1N1 pandemic (pH1N1) lineage evolves in response to the development of population-level protective immunity in humans. Using a chi-squared goodness-of-fit test, we identified 10 amino acid sites that significantly differed between the pH1N1 isolates and isolates from the recent 2012-2013 and 2013-2014 influenza seasons. Three of these sites were located in the same diversified B-cell/Ab epitope regions as identified in the analysis of prepandemic sequences, including Sa and Sb. As predicted, hemagglutination inhibition (HI) assays using human sera from subjects vaccinated with the initial pH1N1 isolate demonstrated reduced reactivity against 2013-2014 isolates. Taken together, these results suggest that diversifying selection analysis can identify key immune epitopes responsible for protective immunity to influenza virus in humans and thereby predict virus evolution.

    IMPORTANCE The WHO estimates that approximately 5 to 10% of adults and 20 to 30% of children in the world are infected by influenza virus each year. While an adaptive immune response helps eliminate the virus following acute infection, the virus rapidly evolves to evade the established protective memory immune response, thus allowing for the regular seasonal cycles of influenza virus infection. The analytical approach described here, which combines an analysis of diversifying selection with an integration of immune epitope data, has allowed us to identify antigenic regions that contribute to protective immunity and are therefore the key targets of immune evasion by the virus. This information can be used to determine when sequence variations in seasonal influenza virus strains have affected regions responsible for protective immunity in order to decide when new vaccine formulations are warranted.

  • Role of EXT1 and Glycosaminoglycans in the Early Stage of Filovirus Entry [Virus-Cell Interactions]

  • Filoviruses, including both Ebola virus (EBOV) and Marburg virus (MARV), can infect humans and other animals, causing hemorrhagic fever with a high mortality rate. Entry of these viruses into the host is mediated by a single filoviral glycoprotein (GP). GP is composed of two subunits: GP1, which is responsible for attachment and binding to receptor(s) on susceptible cells, and GP2, which mediates viral and cell membrane fusion. Although numerous host factors have been implicated in the entry process, the initial attachment receptor(s) has not been well defined. In this report, we demonstrate that exostosin 1 (EXT1), which is involved in biosynthesis of heparan sulfate (HS), plays a role in filovirus entry. Expression knockdown of EXT1 by small interfering RNAs (siRNAs) impairs GP-mediated pseudoviral entry and that of infectious EBOV and MARV in tissue cultured cells. Furthermore, HS, heparin, and other related glycosaminoglycans (GAGs), to different extents, can bind to and block GP-mediated viral entry and that of infectious filoviruses. These results strongly suggest that HS and other related GAGs are attachment receptors that are utilized by filoviruses for entry and infection. These GAGs may have therapeutic potential in treating EBOV- and MARV-infected patients.

    IMPORTANCE Infection by 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 ongoing 2014 outbreak 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 provide several pieces of evidence that demonstrate that heparan sulfate and other closely related glycosaminoglycans are the molecules that are used by filoviruses for initial attachment. Furthermore, we demonstrate that these glycosaminoglycans can block entry of and infection by filoviruses. Thus, this work provides mechanistic insights on the early step of filoviral infection and suggests a possible therapeutic option for diseases caused by filovirus infection.

  • Retroviral Cyclin Controls Cyclin-Dependent Kinase 8-Mediated Transcription Elongation and Reinitiation [Transformation and Oncogenesis]

  • Walleye dermal sarcoma virus (WDSV) infection is associated with the seasonal development and regression of walleye dermal sarcoma. Previous work showed that the retroviral cyclin (RV-cyclin), encoded by WDSV, has separable cyclin box and transcription activation domains. It binds to cyclin-dependent kinase 8 (CDK8) and enhances its kinase activity. CDK8 is evolutionarily conserved and is frequently overexpressed in human cancers. It is normally activated by cyclin C and is required for transcription elongation of the serum response genes (immediate early genes [IEGs]) FOS, EGR1, and cJUN. The IEGs drive cell proliferation, and their expression is brief and highly regulated. Here we show that constitutive expression of RV-cyclin in the HCT116 colon cancer cell line significantly increases the level of IEG expression in response to serum stimulation. Quantitative reverse transcription-PCR (RT-PCR) and nuclear run-on assays provide evidence that RV-cyclin does not alter the initiation of IEG transcription but does enhance the overall rate of transcription elongation and maintains transcription reinitiation. RV-cyclin does not increase activating phosphorylation events in the mitogen-activated protein kinase pathway and does not inhibit decay of IEG mRNAs. At the EGR1 gene locus, RV-cyclin increases and maintains RNA polymerase II (Pol II) occupancy after serum stimulation, in conjunction with increased and extended EGR1 gene expression. The RV-cyclin increases CDK8 occupancy at the EGR1 gene locus before and after serum stimulation. Both of RV-cyclin's functional domains, i.e., the cyclin box and the activation domain, are necessary for the overall enhancement of IEG expression. RV-cyclin presents a novel and ancient mechanism of retrovirus-induced oncogenesis.

    IMPORTANCE The data reported here are important to both virology and cancer biology. The novel mechanism pinpoints CDK8 in the development of walleye dermal sarcoma and sheds light on CDK8's role in many human cancers. CDK8 controls expression from highly regulated genes, including the interferon-stimulated genes. Its function is likely the target of many viral interferon-resistance mechanisms. CDK8 also controls cellular responses to metabolic stimuli, stress, and hypoxia, in addition to the serum response. The retroviral cyclin (RV-cyclin) represents a highly selected probe of CDK8 function. RV-cyclin does not control CDK8 specificity but instead enhances CDK8's effects on regulated genes, an important distinction for its use to delineate natural CDK8 targets. The outcomes of this research are applicable to investigations of normal and abnormal CDK8 functions. The mechanisms defined here will contribute directly to the dermal sarcoma model in fish and clarify an important path for oncogenesis and innate resistance to viruses.

  • Dynamic Interaction of Stress Granules, DDX3X, and IKK-{alpha} Mediates Multiple Functions in Hepatitis C Virus Infection [Virus-Cell Interactions]

  • The ubiquitous ATP-dependent RNA helicase DDX3X is involved in many cellular functions, including innate immunity, and is a pivotal host factor for hepatitis C virus (HCV) infection. Recently, we showed that DDX3X specifically recognizes the HCV 3' untranslated region (UTR), leading to the activation of IKK-aalpha; and a cascade of lipogenic signaling to facilitate lipid droplet biogenesis and viral assembly (Q. Li, V. Pene, S. Krishnamurthy, H. Cha, and T. J. Liang, Nat Med 19:722nndash;729, 2013, http://dx.doi.org/10.1038/nm.3190). The interaction of DDX3X with HCV core protein seems to be dispensable for its proviral role. In this study, through systematic imaging and biochemical and virologic approaches, we identified a dynamic association between DDX3X and various cellular compartments and viral elements mediating multiple functions of DDX3X in productive HCV infection. Upon HCV infection, the HCV 3'UTR interacts with DDX3X and IKK-aalpha;, which redistribute to speckle-like cytoplasmic structures shown to be stress granules (SGs). As viral proteins accumulate in infected cells, DDX3X granules together with SG-associated proteins redistribute and colocalize with HCV core protein around lipid droplets (LDs). IKK-aalpha;, however, does not relocate to the LD but translocates to the nucleus. In HCV-infected cells, various HCV nonstructural proteins also interact or colocalize with DDX3X in close proximity to SGs and LDs, consistent with the tight juxtaposition of the replication complex and the assembly site at the surface of LDs. Short interfering RNA (siRNA)-mediated silencing of DDX3X and multiple SG components markedly inhibits HCV infection. Our data suggest that DDX3X initiates a multifaceted cellular program involving dynamic associations with HCV RNA and proteins, IKK-aalpha;, SG, and LD surfaces for its crucial role in the HCV life cycle.

    IMPORTANCE DDX3X is a proviral host factor for HCV infection. Recently, we showed that DDX3X binds to the HCV 3'UTR, activating IKK-aalpha; and cellular lipogenesis to facilitate viral assembly (Q. Li et al., Nat Med 19:722nndash;729, 2013, http://dx.doi.org/10.1038/nm.3190). Here, we report associations of DDX3X with various cellular compartments and viral elements that mediate its multiple functions in the HCV life cycle. Upon infection, the HCV 3'UTR redistributes DDX3X and IKK-aalpha; to speckle-like cytoplasmic structures shown to be SGs. Subsequently, interactions between DDX3X, SG, and HCV proteins facilitate the translocation of DDX3X-SG complexes to the LD surface. HCV nonstructural proteins are shown to colocalize with DDX3X in close proximity to SGs and LDs, consistent with the tight juxtaposition of the HCV replication complex and assembly site at the LD surface. Our data demonstrate that DDX3X initiates a multifaceted cellular program involving dynamic associations with HCV elements, IKK-aalpha;, SGs, and LDs for its critical role in HCV infection.

  • Transmitted/Founder Viruses Rapidly Escape from CD8+ T Cell Responses in Acute Hepatitis C Virus Infection [Cellular Response to Infection]

  • The interaction between hepatitis C virus (HCV) and cellular immune responses during very early infection is critical for disease outcome. To date, the impact of antigen-specific cellular immune responses on the evolution of the viral population establishing infection and on potential escape has not been studied. Understanding these early host-virus dynamics is important for the development of a preventative vaccine. Three subjects who were followed longitudinally from the detection of viremia preseroconversion until disease outcome were analyzed. The evolution of transmitted/founder (T/F) viruses was undertaken using deep sequencing. CD8+ T cell responses were measured via enzyme-linked immunosorbent spot (ELISpot) assay using HLA class I-restricted T/F epitopes. T/F viruses were rapidly extinguished in all subjects associated with either viral clearance (n = 1) or replacement with viral variants leading to establishment of chronic infection (n = 2). CD8+ T cell responses against 11 T/F epitopes were detectable by 33 to 44 days postinfection, and 5 of these epitopes had not previously been reported. These responses declined rapidly in those who became chronically infected and were maintained in the subject who cleared infection. Higher-magnitude CD8+ T cell responses were associated with rapid development of immune escape variants at a rate of up to 0.1 per day. Rapid escape from CD8+ T cell responses has been quantified for the first time in the early phase of primary HCV infection. These rapid escape dynamics were associated with higher-magnitude CD8+ T cell responses. These findings raise questions regarding optimal selection of immunogens for HCV vaccine development and suggest that detailed analysis of individual epitopes may be required.

    IMPORTANCE A major limitation in our detailed understanding of the role of immune response in HCV clearance has been the lack of data on very early primary infection when the transmitted viral variants successfully establish the acute infection. This study was made possible through the availability of specimens from a unique cohort of asymptomatic primary infection cases in whom the first available viremic samples were collected approximately 3 weeks postinfection and at regular intervals thereafter. The study included detailed examination of both the evolution of the viral population and the host cellular immune responses against the T/F viruses. The findings here provide the first evidence of host cellular responses targeting T/F variants and imposing a strong selective force toward viral escape. The results of this study provide useful insight on how virus escapes the host response and consequently on future analysis of vaccine-induced immunity.

  • Characterization of Two Novel Linear B-Cell Epitopes in the Capsid Protein of Avian Hepatitis E Virus (HEV) That Are Common to Avian, Swine, and Human HEVs [Structure and Assembly]

  • Antisera raised against the avian hepatitis E virus (HEV) capsid protein are cross-reactive with human and swine HEV capsid proteins. In this study, two monoclonal antibodies (MAbs) against the avian HEV capsid protein, namely, 3E8 and 1B5, were shown to cross-react with the swine HEV capsid protein. The motifs involved in binding both MAbs were identified and characterized using phage display biopanning, peptide synthesis, and truncated or mutated protein expression, along with indirect enzyme-linked immunosorbent assay (ELISA) and Western blotting. The results showed that the I/VPHD motif is a necessary core sequence and that P and H are two key amino acids for recognition by MAb 3E8. The VKLYM/TS motif is the minimal amino acid sequence necessary for recognition by MAb 1B5. Cross-reactivity between the two epitopes and antibodies against avian, swine, and human HEVs in sera showed that both epitopes are common to avian, swine, and human HEVs. In addition, amino acid sequence alignment of the capsid proteins revealed that the key motifs of both novel epitopes are the same in HEVs from different animal species, predicting that they may be common to HEV isolates from boars, rabbits, rats, ferrets, mongooses, deer, and camels as well. Protein modeling analysis showed that both epitopes are at least partially exposed on the surface of the HEV capsid protein. Protective capacity analysis demonstrated that the two epitopes are nonprotective against avian HEV infection in chickens. Collectively, these studies characterize two novel linear B-cell epitopes common to avian, swine, and human HEVs, which furthers the understanding of HEV capsid protein antigenic structure.

    IMPORTANCE More and more evidence indicates that the host range diversity of hepatitis E virus (HEV) is a global public health concern. A better understanding of the antigenic structure of the HEV capsid protein may improve disease diagnosis and prevention. In this study, binding site mapping and localization as well as the antigenic biology of two novel linear B-cell epitopes common to several different species of HEV were characterized. These findings partially reveal the antigenic structure of the HEV capsid protein and provide potential applications for the development of diagnostics and interventions for HEV infection.

  • Increased Levels of Macrophage Inflammatory Proteins Result in Resistance to R5-Tropic HIV-1 in a Subset of Elite Controllers [Virus-Cell Interactions]

  • Elite controllers (ECs) are a rare group of HIV seropositive individuals who are able to control viral replication without antiretroviral therapy. The mechanisms responsible for this phenotype, however, have not been fully elucidated. In this study, we examined CD4+ T cell resistance to HIV in a cohort of elite controllers and explored transcriptional signatures associated with cellular resistance. We demonstrate that a subgroup of elite controllers possess CD4+ T cells that are specifically resistant to R5-tropic HIV while remaining fully susceptible to X4-tropic and vesicular stomatitis virus G (VSV-G)-pseudotyped viruses. Transcriptome analysis revealed 17 genes that were differentially regulated in resistant elite controllers relative to healthy controls. Notably, the genes encoding macrophage inflammatory protein 1aalpha; (MIP-1aalpha;), CCL3 and CCL3L1, were found to be upregulated. The MIP-1aalpha;, MIP-1bbeta;, and RANTES chemokines are natural ligands of CCR5 and are known to interfere with HIV replication. For three elite controllers, we observed increased production of MIP-1aalpha; and/or MIP-1bbeta; at the protein level. The supernatant from resistant EC cells contained MIP-1aalpha; and MIP-1bbeta; and was sufficient to confer R5-tropic resistance to susceptible CD4+ T cells. Additionally, this effect was reversed by using inhibitory anti-MIP antibodies. These results suggest that the T cells of these particular elite controllers may be naturally resistant to HIV infection by blocking R5-tropic viral entry.

    IMPORTANCE HIV is a pandemic health problem, and the majority of seropositive individuals will eventually progress to AIDS unless antiretroviral therapy (ART) is administered. However, rare patients, termed elite controllers, have a natural ability to control HIV infection in the absence of ART, but the mechanisms by which they achieve this phenotype have not been fully explored. This paper identifies one mechanism that may contribute to this natural resistance: some elite controllers have CD4+ T cells that produce high levels of MIP chemokines, which block R5-tropic HIV entry. This mechanism could potentially be exploited to achieve a therapeutic effect in other HIV-seropositive individuals.

  • B Virus (Macacine herpesvirus 1) Glycoprotein D Is Functional but Dispensable for Virus Entry into Macaque and Human Skin Cells [Virus-Cell Interactions]

  • Glycoprotein D (gD) plays an essential role in cell entry of many simplexviruses. B virus (Macacine herpesvirus 1) is closely related to herpes simplex virus 1 (HSV-1) and encodes gD, which shares more than 70% amino acid similarity with HSV-1 gD. Previously, we have demonstrated that B virus gD polyclonal antibodies were unable to neutralize B virus infectivity on epithelial cell lines, suggesting gD is not required for B virus entry into these cells. In the present study, we confirmed this finding by producing a B virus mutant, BV-gDZ, in which the gD gene was replaced with a lacZ expression cassette. Recombinant plaques were selected on complementing VD60 cells expressing HSV-1 gD. Virions lacking gD were produced in Vero cells infected with BV-gDZ. In contrast to HSV-1, B virus lacking gD was able to infect and form plaques on noncomplementing cell lines, including Vero, HEp-2, LLC-MK2, primary human and macaque dermal fibroblasts, and U373 human glioblastoma cells. The gD-negative BV-gDZ also failed to enter entry-resistant murine B78H1 cells bearing a single gD receptor, human nectin-1, but gained the ability to enter when phenotypically supplemented with HSV-1 gD. Cell attachment and penetration rates, as well as the replication characteristics of BV-gDZ in Vero cells, were almost identical to those of wild-type (wt) B virus. These observations indicate that B virus can utilize gD-independent cell entry and transmission mechanisms, in addition to generally used gD-dependent mechanisms.

    IMPORTANCE B virus is the only known simplexvirus that causes zoonotic infection, resulting in approximately 80% mortality in untreated humans or in lifelong persistence with the constant threat of reactivation in survivors. Here, we report that B virus lacking the gD envelope glycoprotein infects both human and monkey cells as efficiently as wild-type B virus. These data provide evidence for a novel mechanism(s) utilized by B virus to gain access to target cells. This mechanism is different from those used by its close relatives, HSV-1 and -2, where gD is a pivotal protein in the virus entry process. The possibility remains that unidentified receptors, specific for B virus, permit virus entry into target cells through gD-independent pathways. Understanding the molecular mechanisms of B virus entry may help in developing rational therapeutic strategies for the prevention and treatment of B virus infection in both macaques and humans.

  • Flexibility of Bunyavirus Genomes: Creation of an Orthobunyavirus with an Ambisense S Segment [Genome Replication and Regulation of Viral Gene Expression]

  • The Bunyamwera (BUNV) orthobunyavirus NSs protein has proven a challenge to study in the context of viral infection. NSs is encoded in a reading frame that overlaps that of the viral nucleocapsid (N) protein thus limiting options for mutagenesis. In addition, NSs is poorly immunogenic, and antibodies only work in certain techniques while the protein itself is subject to proteasomal degradation. In order to generate a virus that expresses NSs independently of N, an ambisense S RNA segment was designed by mutating the 5'- and 3'-terminal nucleotide sequences. These mutations were previously shown to alter promoter activity so that both replication and transcription were promoted from both the genome and the antigenome RNAs (J. N. Barr et al., J. Virol. 79:12602nndash;12607, 2005). As proof of principle, a recombinant BUNV was created that expressed green fluorescent protein (GFP) in the ambisense orientation. GFP expression was detected throughout at least 10 passages. Recombinant BUNV encoding epitope-tagged versions of NSs in the ambisense orientation expressed NSs via a subgenomic mRNA, and two viruses grew to titers only modestly lower than parental rBUNdelNSs2 virus. The ambisense viruses were temperature sensitive, and NSs was shown to localize to both the nucleus and the cytoplasm during infection. These viruses will be useful in further studies on structure-function relationships of the orthobunyavirus NSs protein.

    IMPORTANCE Bunyamwera virus (BUNV) is the type species and model system for both the family Bunyaviridae and the genus Orthobunyavirus, a group that includes many significant human and animal pathogens. Studying the basic molecular biology of these viruses is of great importance to underpin research into vaccines and antivirals. We demonstrate here the plasticity of the BUNV genome by generating recombinant viruses where the normal negative-sense S segment has been converted into an ambisense segment, allowing independent expression of either a foreign gene (green fluorescent protein) or the viral nonstructural NSs protein. These new reagents will allow detailed investigation of NSs, the orthobunyavirus interferon antagonist.

  • Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Inhibits Major Histocompatibility Complex Class II Expression by Disrupting Enhanceosome Assembly through Binding with the Regulatory Factor X Complex [Virus-Cell Interactions]

  • Major histocompatibility complex class II (MHC-II) molecules play a central role in adaptive antiviral immunity by presenting viral peptides to CD4+ T cells. Due to their key role in adaptive immunity, many viruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), have evolved multiple strategies to inhibit the MHC-II antigen presentation pathway. The expression of MHC-II, which is controlled mainly at the level of transcription, is strictly dependent upon the binding of the class II transactivator (CIITA) to the highly conserved promoters of all MHC-II genes. The recruitment of CIITA to MHC-II promoters requires its direct interactions with a preassembled MHC-II enhanceosome consisting of cyclic AMP response element-binding protein (CREB) and nuclear factor Y (NF-Y) complex and regulatory factor X (RFX) complex proteins. Here, we show that KSHV-encoded latency-associated nuclear antigen (LANA) disrupts the association of CIITA with the MHC-II enhanceosome by binding to the components of the RFX complex. Our data show that LANA is capable of binding to all three components of the RFX complex, RFX-associated protein (RFXAP), RFX5, and RFX-associated ankyrin-containing protein (RFXANK), in vivo but binds more strongly with the RFXAP component in in vitro binding assays. Levels of MHC-II proteins were significantly reduced in KSHV-infected as well as LANA-expressing B cells. Additionally, the expression of LANA in a luciferase promoter reporter assay showed reduced HLA-DRA promoter activity in a dose-dependent manner. Chromatin immunoprecipitation assays showed that LANA binds to the MHC-II promoter along with RFX proteins and that the overexpression of LANA disrupts the association of CIITA with the MHC-II promoter. These assays led to the conclusion that the interaction of LANA with RFX proteins interferes with the recruitment of CIITA to MHC-II promoters, resulting in an inhibition of MHC-II gene expression. Thus, the data presented here identify a novel mechanism used by KSHV to downregulate the expressions of MHC-II genes.

    IMPORTANCE Kaposi's sarcoma-associated herpesvirus is the causative agent of multiple human malignancies. It establishes a lifelong latent infection and persists in infected cells without being detected by the host's immune surveillance system. Only a limited number of viral proteins are expressed during latency, and these proteins play a significant role in suppressing both the innate and adaptive immunities of the host. Latency-associated nuclear antigen (LANA) is one of the major proteins expressed during latent infection. Here, we show that LANA blocks MHC-II gene expression to subvert the host immune system by disrupting the MHC-II enhanceosome through binding with RFX transcription factors. Therefore, this study identifies a novel mechanism utilized by KSHV LANA to deregulate MHC-II gene expression, which is critical for CD4+ T cell responses in order to escape host immune surveillance.

  • Role of Hydrogen Sulfide in Paramyxovirus Infections [Virus-Cell Interactions]

  • Hydrogen sulfide (H2S) is an endogenous gaseous mediator that has gained increasing recognition as an important player in modulating acute and chronic inflammatory diseases. However, its role in virus-induced lung inflammation is currently unknown. Respiratory syncytial virus (RSV) is a major cause of upper and lower respiratory tract infections in children for which no vaccine or effective treatment is available. Using the slow-releasing H2S donor GYY4137 and propargylglycin (PAG), an inhibitor of cystathionine--lyase (CSE), a key enzyme that produces intracellular H2S, we found that RSV infection led to a reduced ability to generate and maintain intracellular H2S levels in airway epithelial cells (AECs). Inhibition of CSE with PAG resulted in increased viral replication and chemokine secretion. On the other hand, treatment of AECs with the H2S donor GYY4137 reduced proinflammatory mediator production and significantly reduced viral replication, even when administered several hours after viral absorption. GYY4137 also significantly reduced replication and inflammatory chemokine production induced by human metapneumovirus (hMPV) and Nipah virus (NiV), suggesting a broad inhibitory effect of H2S on paramyxovirus infections. GYY4137 treatment had no effect on RSV genome replication or viral mRNA and protein synthesis, but it inhibited syncytium formation and virus assembly/release. GYY4137 inhibition of proinflammatory gene expression occurred by modulation of the activation of the key transcription factors nuclear factor B (NF-B) and interferon regulatory factor 3 (IRF-3) at a step subsequent to their nuclear translocation. H2S antiviral and immunoregulatory properties could represent a novel treatment strategy for paramyxovirus infections.

    IMPORTANCE RSV is a global health concern, causing significant morbidity and economic losses as well as mortality in developing countries. After decades of intensive research, no vaccine or effective treatment, with the exception of immunoprophylaxis, is available for this infection as well as for other important respiratory mucosal viruses. This study identifies hydrogen sulfide as a novel cellular mediator that can modulate viral replication and proinflammatory gene expression, both important determinants of lung injury in respiratory viral infections, with potential for rapid translation of such findings into novel therapeutic approaches for viral bronchiolitis and pneumonia.

  • Characterization of the Influence of Semen-Derived Enhancer of Virus Infection on the Interaction of HIV-1 with Female Reproductive Tract Tissues [Virus-Cell Interactions]

  • The majority of human immunodeficiency virus type 1 (HIV-1) transmission events occur in women when semen harboring infectious virus is deposited onto the mucosal barriers of the vaginal, ectocervical, and endocervical epithelia. Seminal factors such as semen-derived enhancer of virus infection (SEVI) fibrils were previously shown to greatly enhance the infectivity of HIV-1 in cell culture systems. However, when SEVI is intravaginally applied to living animals, there is no effect on vaginal transmission. To define how SEVI might function in the context of sexual transmission, we applied HIV-1 and SEVI to intact human and rhesus macaque reproductive tract tissues to determine how it influences virus interactions with these barriers. We show that SEVI binds HIV-1 and sequesters most virions to the luminal surface of the stratified squamous epithelium, significantly reducing the number of virions that penetrated the tissue. In the simple columnar epithelium, SEVI was no longer fibrillar in structure and was detached from virions but allowed significantly deeper epithelial virus penetration. These observations reveal that the action of SEVI in intact tissues is very different in the anatomical context of sexual transmission and begin to explain the lack of stimulation of infection observed in the highly relevant mucosal transmission model.

    IMPORTANCE The most common mode of HIV-1 transmission in women occurs via genital exposure to the semen of HIV-infected men. A productive infection requires the virus to penetrate female reproductive tract epithelial barriers to infect underlying target cells. Certain factors identified within semen, termed semen-derived enhancers of virus infection (SEVI), have been shown to significantly enhance HIV-1 infectivity in cell culture. However, when applied to the genital tracts of living female macaques, SEVI did not enhance virus transmission. Here we show that SEVI functions very differently in the context of intact mucosal tissues. SEVI decreases HIV-1 penetration of squamous epithelial barriers in humans and macaques. At the mucus-coated columnar epithelial barrier, the HIV-1/SEVI interaction is disrupted. These observations suggest that SEVI may not play a significant stimulatory role in the efficiency of male-to-female sexual transmission of HIV.

  • Profiling of Virus-Encoded MicroRNAs in Epstein-Barr Virus-Associated Gastric Carcinoma and Their Roles in Gastric Carcinogenesis [Transformation and Oncogenesis]

  • Epstein-Barr virus (EBV) is one of the major oncogenic viruses and is found in nearly 10% of gastric carcinomas. EBV is known to encode its own microRNAs (miRNAs); however, their roles have not been fully investigated. The present report is the largest series to comprehensively profile the expression of 44 known EBV miRNAs in tissue samples from patients with EBV-associated gastric carcinoma. Several miRNAs were highly expressed in EBV-associated gastric carcinoma, and in silico analysis revealed that the target genes of these EBV miRNAs had functions associated with cancer-related pathways, especially the regulation of apoptosis. Apoptosis was reduced in EBV-associated gastric carcinoma tissue samples, and gastric carcinoma cell lines infected with EBV exhibited downregulation of the proapoptotic protein Bid (the BH3-interacting domain death agonist), a member of the Bcl-2 family. The luciferase activity of the reporter vector containing the 3' untranslated region of BID was inhibited by an ebv-miR-BART4-5p mimic in gastric cancer cell lines. Transfection of an ebv-miR-BART4-5p mimic reduced Bid expression in EBV-negative cell lines, leading to reduced apoptosis under serum deprivation. The inhibition of ebv-miR-BART4-5p expression was associated with partial recovery of Bid levels in EBV-positive cell lines. The results demonstrated the antiapoptotic role of EBV miRNA via regulation of Bid expression in EBV-associated gastric carcinoma. These findings provide novel insights in the roles of EBV miRNAs in gastric carcinogenesis, which would be a potential therapeutic target.

    IMPORTANCE This report is the largest series to comprehensively profile the expression of 44 known EBV miRNAs in clinical samples from EBV-associated gastric carcinoma patients. Of the EBV miRNAs, ebv-miR-BART4-5p plays an important role in gastric carcinogenesis via regulation of apoptosis.

  • Dengue Virus Evolution under a Host-Targeted Antiviral [Genetic Diversity and Evolution]

  • The host-targeted antiviral drug UV-4B reduces viral replication and promotes survival in a mouse model of experimental dengue virus (DENV) infection. UV-4B is an iminosugar that inhibits the aalpha;-glucosidase family of enzymes and subsequently the folding of glycosylated proteins, both viral and host. Here, we utilized next-generation sequencing to investigate evolution of a flavivirus under selective pressure by a host-targeted antiviral in vivo. In viral populations recovered from UV-4B-treated mice, there was a significant increase in the number of single-nucleotide polymorphisms (SNPs) and the ratio of nonsynonymous to synonymous SNPs compared to findings in viral populations from vehicle-treated mice. The strongest evidence of positive selection was in the glycosylated membrane protein, thereby providing in vivo validation of the mechanism of action of an iminosugar. In addition, mutations in glycosylated proteins were present only in drug-treated mice after a single passage. However, the bulk of the other mutations were present in both populations, indicating nonspecific selective pressure. Together with the continued control of viremia by UV-4B, these findings are consistent with the previously predicted high genetic barrier to escape mutations in host-targeted antivirals.

    IMPORTANCE Although hundreds of millions of people are infected with DENV every year, there is currently no approved vaccine or antiviral therapy. UV-4B has demonstrated antiviral activity against DENV and is expected to enter clinical trials soon. Therefore, it is important to understand the mechanisms of DENV resistance to UV-4B. Host-targeted antivirals are thought to have a higher genetic barrier to escape mutants than directly acting antivirals, yet there are very few published studies of viral evolution under host-targeted antivirals. No study to date has described flavivirus evolution in vivo under selective pressure by a host-based antiviral drug. We present the first in vivo study of the sequential progression of viral evolution under selective pressure by a host-targeted antiviral compound. This study bolsters support for the clinical development of UV-4B as an antiviral drug against DENV, and it provides a framework to compare how treatment with other host-targeted antiflaviviral drugs in humans and different animal models influence viral genetic diversity.

  • Viral Infection of the Central Nervous System and Neuroinflammation Precede Blood-Brain Barrier Disruption during Japanese Encephalitis Virus Infection [Pathogenesis and Immunity]

  • Japanese encephalitis is an acute zoonotic, mosquito-borne disease caused by Japanese encephalitis virus (JEV). Japanese encephalitis is characterized by extensive inflammation in the central nervous system (CNS) and disruption of the blood-brain barrier (BBB). However, the pathogenic mechanisms contributing to the BBB disruption are not known. Here, using a mouse model of intravenous JEV infection, we show that virus titers increased exponentially in the brain from 2 to 5 days postinfection. This was accompanied by an early, dramatic increase in the level of inflammatory cytokines and chemokines in the brain. Enhancement of BBB permeability, however, was not observed until day 4, suggesting that viral entry and the onset of inflammation in the CNS occurred prior to BBB damage. In vitro studies revealed that direct infection with JEV could not induce changes in the permeability of brain microvascular endothelial cell monolayers. However, brain extracts derived from symptomatic JEV-infected mice, but not from mock-infected mice, induced significant permeability of the endothelial monolayer. Consistent with a role for inflammatory mediators in BBB disruption, the administration of gamma interferon-neutralizing antibody ameliorated the enhancement of BBB permeability in JEV-infected mice. Taken together, our data suggest that JEV enters the CNS, propagates in neurons, and induces the production of inflammatory cytokines and chemokines, which result in the disruption of the BBB.

    IMPORTANCE Japanese encephalitis (JE) is the leading cause of viral encephalitis in Asia, resulting in 70,000 cases each year, in which approximately 20 to 30% of cases are fatal, and a high proportion of patients survive with serious neurological and psychiatric sequelae. Pathologically, JEV infection causes an acute encephalopathy accompanied by BBB dysfunction; however, the mechanism is not clear. Thus, understanding the mechanisms of BBB disruption in JEV infection is important. Our data demonstrate that JEV gains entry into the CNS prior to BBB disruption. Furthermore, it is not JEV infection per se, but the inflammatory cytokines/chemokines induced by JEV infection that inhibit the expression of TJ proteins and ultimately result in the enhancement of BBB permeability. Neutralization of gamma interferon (IFN-) ameliorated the enhancement of BBB permeability in JEV-infected mice, suggesting that IFN- could be a potential therapeutic target. This study would lead to identification of potential therapeutic avenues for the treatment of JEV infection.

  • Dynamics of Human Cytomegalovirus Infection in CD34+ Hematopoietic Cells and Derived Langerhans-Type Dendritic Cells [Virus-Cell Interactions]

  • Acquisition of human cytomegalovirus (CMV) usually occurs by contact between contaminated bodily fluids, such as urine and saliva, and host mucosal cells. Langerhans-type dendritic cells (LC) are the only type of immune cells found in the outermost layers of the oral mucosae, where they not only provide a first line of defense against CMV but can easily be targeted by orally administered vaccines, while their bone marrow resident progenitors are important sites of virus latency. In this work, we tracked the progress of infection in CD34+ progenitor cells, immature LC (iLC), and mature LC (mLC) exposed to the clinical-like strain TB40-BAC4 or to the vaccine strain AD169varATCC, prior to their long-term maintenance under either immature or mature conditions. We show that the genomes of both strains are efficiently maintained in CD34+ cells during their differentiation into iLC, although this requires the presence of larger amounts of input AD169varATCC DNA. Lipopolysaccharide- and CD40 ligand-induced maturation of iLC derived from latently infected progenitors was not associated with robust viral genome replication and progeny production, while maturation of directly infected iLC increased and prolonged expression of the viral immediate early proteins. While effective replication of viral genomes from both strains occurred only in mLC, both iLC and mLC produced viral progeny, suggesting that both types of LC may contribute to CMV horizontal transmission in vivo.

    IMPORTANCE Human CMV is usually acquired via the oral and nasal mucosae. Langerhans-type dendritic cells (LC) are the only type of immune cells found in the outermost layers of these tissues. Understanding how CMV interacts with LC and their hematopoietic progenitors is thus essential to develop innovative means of defense against this virus. Here we show that the genomes of a virulent and an attenuated strain of CMV are maintained in hematopoietic progenitor cells during their differentiation into immature LC and that maturation of these cells by exposure to lipopolysaccharide and CD40 ligand is not sufficient to trigger virus reactivation. While the extents of viral protein expression and genome replication were broadest in directly infected mature LC populations, similar amounts of viral progeny were detected in the supernatants of immature and mature LC, suggesting that these immune cells of the oral mucosa are likely to be important for CMV transmission within the human population.

  • The pH Stability of Foot-and-Mouth Disease Virus Particles Is Modulated by Residues Located at the Pentameric Interface and in the N Terminus of VP1 [Virus-Cell Interactions]

  • The picornavirus foot-and-mouth disease virus (FMDV) is the etiological agent of a highly contagious disease that affects important livestock species. The FMDV capsid is highly acid labile, and viral particles lose infectivity due to their disassembly at pH values slightly below neutrality. This acid sensitivity is related to the mechanism of viral uncoating and genome penetration from endosomes. In this study, we have analyzed the molecular basis of FMDV acid-induced disassembly by isolating and characterizing a panel of novel FMDV mutants differing in acid sensitivity. Amino acid replacements altering virion stability were preferentially distributed in two different regions of the capsid: the N terminus of VP1 and the pentameric interface. Even more, the acid labile phenotype induced by a mutation located at the pentameric interface in VP3 could be compensated by introduction of an amino acid substitution in the N terminus of VP1. These results indicate that the acid sensitivity of FMDV can be considered a multifactorial trait and that virion stability is the fine-tuned product of the interaction between residues from different capsid proteins, in particular those located within the N terminus of VP1 or close to the pentameric interface.

    IMPORTANCE The viral capsid protects the viral genome from environmental factors and contributes to virus dissemination and infection. Thus, understanding of the molecular mechanisms that modulate capsid stability is of interest for the basic knowledge of the biology of viruses and as a tool to improve the stability of conventional vaccines based on inactivated virions or empty capsids. Using foot-and-mouth disease virus (FMDV), which displays a capsid with extreme acid sensitivity, we have performed a genetic study to identify the molecular determinants involved in capsid stability. A panel of FMDV mutants with differential sensitivity to acidic pH was generated and characterized, and the results showed that two different regions of FMDV capsid contribute to modulating viral particle stability. These results provide new insights into the molecular mechanisms of acid-mediated FMDV uncoating.

  • Tristetraprolin Recruits the Herpes Simplex Virion Host Shutoff RNase to AU-Rich Elements in Stress Response mRNAs To Enable Their Cleavage [Virus-Cell Interactions]

  • Herpes simplex viruses (HSV) package and bring into cells an RNase designated virion host shutoff (VHS) RNase. In infected cells, the VHS RNase targets primarily stress response mRNAs characterized by the presence of AU-rich elements in their 3' untranslated regions (UTRs). In uninfected cells, these RNAs are sequestered in exosomes or P bodies by host proteins that bind to the AU-rich elements. In infected cells, the AU-rich RNAs are deadenylated and cleaved close to the AU-rich elements, leading to long-term persistence of nontranslatable RNAs consisting of the 5' portions of the cleavage products. The host proteins that bind to the AU-rich elements are either resident in cells (e.g., TIA-1) or induced (e.g., tristetraprolin). Earlier, this laboratory reported that tristetraprolin binds VHS RNase. To test the hypothesis that tristetraprolin directs VHS RNase to the AU-rich elements, we mapped the domains of VHS and tristetraprolin required for their interactions. We report that VHS binds to the domain of tristetraprolin that enables its interaction with RNA. A single amino acid substitution in that domain abolished the interaction with RNA but did not block the binding to VHS RNase. In transfected cells, the mutant but not the wild-type tristetraprolin precluded the degradation of the AU-rich RNAs by VHS RNase. We conclude that TTP mediates the cleavage of the 3' UTRs of stress response mRNAs by recruiting the VHS RNase to the AU-rich elements.

    IMPORTANCE The primary host response to HSV infection is the synthesis of stress response mRNAs characterized by the presence of AU-rich elements in their 3' UTRs. These mRNAs are the targets of the virion host shutoff (VHS) RNase. The VHS RNase binds both to mRNA cap structure and to tristetraprolin, an inducible host protein that sequesters AU-rich mRNAs in exosomes or P bodies. Here we show that tristetraprolin recruits VHS RNase to the AU-rich elements and enables the degradation of the stress response mRNAs.

  • Swine Influenza Virus PA and Neuraminidase Gene Reassortment into Human H1N1 Influenza Virus Is Associated with an Altered Pathogenic Phenotype Linked to Increased MIP-2 Expression [Pathogenesis and Immunity]

  • Swine are susceptible to infection by both avian and human influenza viruses, and this feature is thought to contribute to novel reassortant influenza viruses. In this study, the influenza virus reassortment rate in swine and human cells was determined. Coinfection of swine cells with 2009 pandemic H1N1 virus (huH1N1) and an endemic swine H1N2 (A/swine/Illinois/02860/09) virus (swH1N2) resulted in a 23% reassortment rate that was independent of aalpha;2,3- or aalpha;2,6-sialic acid distribution on the cells. The reassortants had altered pathogenic phenotypes linked to introduction of the swine virus PA and neuraminidase (NA) into huH1N1. In mice, the huH1N1 PA and NA mediated increased MIP-2 expression early postinfection, resulting in substantial pulmonary neutrophilia with enhanced lung pathology and disease. The findings support the notion that swine are a mixing vessel for influenza virus reassortants independent of sialic acid distribution. These results show the potential for continued reassortment of the 2009 pandemic H1N1 virus with endemic swine viruses and for reassortants to have increased pathogenicity linked to the swine virus NA and PA genes which are associated with increased pulmonary neutrophil trafficking that is related to MIP-2 expression.

    IMPORTANCE Influenza A viruses can change rapidly via reassortment to create a novel virus, and reassortment can result in possible pandemics. Reassortments among subtypes from avian and human viruses led to the 1957 (H2N2 subtype) and 1968 (H3N2 subtype) human influenza pandemics. Recent analyses of circulating isolates have shown that multiple genes can be recombined from human, avian, and swine influenza viruses, leading to triple reassortants. Understanding the factors that can affect influenza A virus reassortment is needed for the establishment of disease intervention strategies that may reduce or preclude pandemics. The findings from this study show that swine cells provide a mixing vessel for influenza virus reassortment independent of differential sialic acid distribution. The findings also establish that circulating neuraminidase (NA) and PA genes could alter the pathogenic phenotype of the pandemic H1N1 virus, resulting in enhanced disease. The identification of such factors provides a framework for pandemic modeling and surveillance.

  • Structure-Based Mutational Analysis of Several Sites in the E Protein: Implications for Understanding the Entry Mechanism of Japanese Encephalitis Virus [Virus-Cell Interactions]

  • Japanese encephalitis virus (JEV), which causes viral encephalitis in humans, is a serious risk to global public health. The JEV envelope protein mediates the viral entry pathway, including receptor-binding and low-pH-triggered membrane fusion. Utilizing mutagenesis of a JEV infectious cDNA clone, mutations were introduced into the potential receptor-binding motif or into residues critical for membrane fusion in the envelope protein to systematically investigate the JEV entry mechanism. We conducted experiments evaluating infectious particle, recombinant viral particle, and virus-like particle production and found that most mutations impaired virus production. Subcellular fractionation confirmed that five mutationsmmdash;in I0, ij, BC, and FG and the R9A substitutionmmdash;impaired virus assembly, and the assembled virus particles of another five mutationsmmdash;in kl and the E373A, F407A, L221S, and W217A substitutionsmmdash;were not released into the secretory pathway. Next, we examined the entry activity of six mutations yielding infectious virus. The results showed N154 and the DE loop are not the only or major receptor-binding motifs for JEV entry into BHK-21 cells; four residues, H144, H319, T410, and Q258, participating in the domain I (DI)-DIII interaction or zippering reaction are important to maintain the efficiency of viral membrane fusion. By continuous passaging of mutants, adaptive mutations from negatively charged amino acids to positively charged or neutral amino acids, such as E138K and D389G, were selected and could restore the viral entry activity.

    IMPORTANCE Recently, there has been much interest in the entry mechanism of flaviviruses into host cells, including the viral entry pathway and membrane fusion mechanism. Our study provides strong evidence for the critical role of several residues in the envelope protein in the assembly, release, and entry of JEV, which also contributes to our understanding of the flaviviral entry mechanism. Furthermore, we demonstrate that the H144A, H319A, T410A, and Q258A mutants exhibit attenuated fusion competence, which may be used to develop novel vaccine candidates for flaviviruses.

  • HIV-1 Nef and Vpu Interfere with L-Selectin (CD62L) Cell Surface Expression To Inhibit Adhesion and Signaling in Infected CD4+ T Lymphocytes [Virus-Cell Interactions]

  • Leukocyte recirculation between blood and lymphoid tissues is required for the generation and maintenance of immune responses against pathogens and is crucially controlled by the L-selectin (CD62L) leukocyte homing receptor. CD62L has adhesion and signaling functions and initiates the capture and rolling on the vascular endothelium of cells entering peripheral lymph nodes. This study reveals that CD62L is strongly downregulated on primary CD4+ T lymphocytes upon infection with human immunodeficiency virus type 1 (HIV-1). Reduced cell surface CD62L expression was attributable to the Nef and Vpu viral proteins and not due to increased shedding via matrix metalloproteases. Both Nef and Vpu associated with and sequestered CD62L in perinuclear compartments, thereby impeding CD62L transport to the plasma membrane. In addition, Nef decreased total CD62L protein levels. Importantly, infection with wild-type, but not Nef- and Vpu-deficient, HIV-1 inhibited the capacity of primary CD4+ T lymphocytes to adhere to immobilized fibronectin in response to CD62L ligation. Moreover, HIV-1 infection impaired the signaling pathways and costimulatory signals triggered in primary CD4+ T cells by CD62L ligation. We propose that HIV-1 dysregulates CD62L expression to interfere with the trafficking and activation of infected T cells. Altogether, this novel HIV-1 function could contribute to virus dissemination and evasion of host immune responses.

    IMPORTANCE L-selectin (CD62L) is an adhesion molecule that mediates the first steps of leukocyte homing to peripheral lymph nodes, thus crucially controlling the initiation and maintenance of immune responses to pathogens. Here, we report that CD62L is downmodulated on the surfaces of HIV-1-infected T cells through the activities of two viral proteins, Nef and Vpu, that prevent newly synthesized CD62L molecules from reaching the plasma membrane. We provide evidence that CD62L downregulation on HIV-1-infected primary T cells results in impaired adhesion and signaling functions upon CD62L triggering. Removal of cell surface CD62L may predictably keep HIV-1-infected cells away from lymph nodes, the privileged sites of both viral replication and immune response activation, with important consequences, such as systemic viral spread and evasion of host immune surveillance. Altogether, we propose that Nef- and Vpu-mediated subversion of CD62L function could represent a novel determinant of HIV-1 pathogenesis.

  • Degradation of SAMHD1 by Vpx Is Independent of Uncoating [Virus-Cell Interactions]

  • Sterile alpha motif domain and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in myeloid and resting T cells. Lentiviruses such as HIV-2 and some simian immunodeficiency viruses (SIVs) counteract the restriction by encoding Vpx or Vpr, accessory proteins that are packaged in virions and which, upon entry of the virus into the cytoplasm, induce the proteasomal degradation of SAMHD1. As a tool to study these mechanisms, we generated HeLa cell lines that express a fusion protein termed NLS.GFP.SAM595 in which the Vpx binding domain of SAMHD1 is fused to the carboxy terminus of green fluorescent protein (GFP) and a nuclear localization signal is fused to the amino terminus of GFP. Upon incubation of Vpx-containing virions with the cells, the NLS.GFP.SAM595 fusion protein was degraded over several hours and the levels remained low over 5 days as the result of continued targeting of the CRL4 E3 ubiquitin ligase. Degradation of the fusion protein required that it contain a nuclear localization sequence. Fusion to the cytoplasmic protein muNS rendered the protein resistant to Vpx-mediated degradation, confirming that SAMHD1 is targeted in the nucleus. Virions treated with protease inhibitors failed to release Vpx, indicating that Gag processing was required for Vpx release from the virion. Mutations in the capsid protein that altered the kinetics of virus uncoating and the Gag binding drug PF74 had no effect on the Vpx-mediated degradation. These results suggest that Vpx is released from virions without a need for uncoating of the capsid, allowing Vpx to transit to the nucleus rapidly upon entry into the cytoplasm.

    IMPORTANCE SAMHD1 restricts lentiviral replication in myeloid cells and resting T cells. Its importance is highlighted by the fact that viruses such as HIV-2 encode an accessory protein that is packaged in the virion and is dedicated to inducing SAMHD1 degradation. Vpx needs to act rapidly upon infection to allow reverse transcription to proceed. The limited number of Vpx molecules in a virion also needs to clear the cell of SAMHD1 over a prolonged period of time. Using an engineered HeLa cell line that expresses a green fluorescent protein (GFP)-SAMHD1 fusion protein, we showed that the Vpx-dependent degradation occurs without a need for viral capsid uncoating. In addition, the fusion protein was degraded only when it was localized to the nucleus, confirming that SAMHD1 is targeted in the nucleus and thus explaining why Vpx also localizes to the nucleus.

  • Activation of Tomato Bushy Stunt Virus RNA-Dependent RNA Polymerase by Cellular Heat Shock Protein 70 Is Enhanced by Phospholipids In Vitro [Genome Replication and Regulation of Viral Gene Expression]

  • Similar to other positive-strand RNA viruses, tombusviruses are replicated by the membrane-bound viral replicase complex (VRC). The VRC consists of the p92 virus-coded RNA-dependent RNA polymerase (RdRp), the viral p33 RNA chaperone, and several co-opted host proteins. In order to become a functional RdRp after its translation, the p92 replication protein should be incorporated into the VRC, followed by its activation. We have previously shown in a cell-free yeast extract-based assay that the activation of the Tomato bushy stunt virus (TBSV) RdRp requires a soluble host factor(s). In this article, we identify the cellular heat shock protein 70 (Hsp70) as the co-opted host factor required for the activation of an N-terminally truncated recombinant TBSV RdRp. In addition, small-molecule-based blocking of Hsp70 function inhibits RNA synthesis by the tombusvirus RdRp in vitro. Furthermore, we show that neutral phospholipids, namely, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), enhance RdRp activation in vitro. In contrast, phosphatidylglycerol (PG) shows a strong and dominant inhibitory effect on in vitro RdRp activation. We also demonstrate that PE and PC stimulate RdRp-viral plus-strand RNA [(+)RNA] interaction, while PG inhibits the binding of the viral RNA to the RdRp. Based on the stimulatory versus inhibitory roles of various phospholipids in tombusvirus RdRp activation, we propose that the lipid composition of targeted subcellular membranes might be utilized by tombusviruses to regulate new VRC assembly during the course of infection.

    IMPORTANCE The virus-coded RNA-dependent RNA polymerase (RdRp), which is responsible for synthesizing the viral RNA progeny in infected cells of several positive-strand RNA viruses, is initially inactive. This strategy is likely to avoid viral RNA synthesis in the cytosol that would rapidly lead to induction of RNA-triggered cellular antiviral responses. During the assembly of the membrane-bound replicase complex, the viral RdRp becomes activated through an incompletely understood process that makes the RdRp capable of RNA synthesis. By using TBSV RdRp, we show that the co-opted cellular Hsp70 chaperone and neutral phospholipids facilitate RdRp activation in vitro. In contrast, phosphatidylglycerol (PG) has a dominant inhibitory effect on in vitro RdRp activation and RdRp-viral RNA interaction, suggesting that the membranous microdomain surrounding the RdRp greatly affects its ability for RNA synthesis. Thus, the activation of the viral RdRp likely depends on multiple host components in infected cells.

  • SLAM- and Nectin-4-Independent Noncytolytic Spread of Canine Distemper Virus in Astrocytes [Virus-Cell Interactions]

  • Measles and canine distemper viruses (MeV and CDV, respectively) first replicate in lymphatic and epithelial tissues by using SLAM and nectin-4 as entry receptors, respectively. The viruses may also invade the brain to establish persistent infections, triggering fatal complications, such as subacute sclerosis pan-encephalitis (SSPE) in MeV infection or chronic, multiple sclerosis-like, multifocal demyelinating lesions in the case of CDV infection. In both diseases, persistence is mediated by viral nucleocapsids that do not require packaging into particles for infectivity but are directly transmitted from cell to cell (neurons in SSPE or astrocytes in distemper encephalitis), presumably by relying on restricted microfusion events. Indeed, although morphological evidence of fusion remained undetectable, viral fusion machineries and, thus, a putative cellular receptor, were shown to contribute to persistent infections. Here, we first showed that nectin-4-dependent cell-cell fusion in Vero cells, triggered by a demyelinating CDV strain, remained extremely limited, thereby supporting a potential role of nectin-4 in mediating persistent infections in astrocytes. However, nectin-4 could not be detected in either primary cultured astrocytes or the white matter of tissue sections. In addition, a bioengineered "nectin-4-blind" recombinant CDV retained full cell-to-cell transmission efficacy in primary astrocytes. Combined with our previous report demonstrating the absence of SLAM expression in astrocytes, these findings are suggestive for the existence of a hitherto unrecognized third CDV receptor expressed by glial cells that contributes to the induction of noncytolytic cell-to-cell viral transmission in astrocytes.

    IMPORTANCE While persistent measles virus (MeV) infection induces SSPE in humans, persistent canine distemper virus (CDV) infection causes chronic progressive or relapsing demyelination in carnivores. Common to both central nervous system (CNS) infections is that persistence is based on noncytolytic cell-to-cell spread, which, in the case of CDV, was demonstrated to rely on functional membrane fusion machinery complexes. This inferred a mechanism where nucleocapsids are transmitted through macroscopically invisible microfusion events between infected and target cells. Here, we provide evidence that CDV induces such microfusions in a SLAM- and nectin-4-independent manner, thereby strongly suggesting the existence of a third receptor expressed in glial cells (referred to as GliaR). We propose that GliaR governs intercellular transfer of nucleocapsids and hence contributes to viral persistence in the brain and ensuing demyelinating lesions.

  • Efficient Interaction between Arenavirus Nucleoprotein (NP) and RNA-Dependent RNA Polymerase (L) Is Mediated by the Virus Nucleocapsid (NP-RNA) Template [Structure and Assembly]

  • In this study, we document that efficient interaction between arenavirus nucleoprotein (NP) and RNA-dependent RNA polymerase (L protein), the two trans-acting viral factors required for both virus RNA replication and gene transcription, requires the presence of virus-specific RNA sequences located within the untranslated 5' and 3' termini of the viral genome.

  • BH3 Profiling Reveals Selectivity by Herpesviruses for Specific Bcl-2 Proteins To Mediate Survival of Latently Infected Cells [Virus-Cell Interactions]

  • Herpesviruses, including human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and Kaposirrsquo;s sarcoma-associated herpesvirus, establish latency by modulating or mimicking antiapoptotic Bcl-2 proteins to promote survival of carrier cells. BH3 profiling, which assesses the contribution of Bcl-2 proteins towards cellular survival, was able to globally determine the level of dependence on individual cellular and viral Bcl-2 proteins within latently infected cells. Moreover, BH3 profiling predicted the sensitivity of infected cells to small-molecule inhibitors of Bcl-2 proteins.

  • Latent Herpes Simplex Virus 1 Infection Does Not Induce Apoptosis in Human Trigeminal Ganglia [Virus-Cell Interactions]

  • Herpes simplex virus 1 (HSV-1) can establish lifelong latency in human trigeminal ganglia. Latently infected ganglia contain CD8+ T cells, which secrete granzyme B and are thus capable of inducing neuronal apoptosis. Using immunohistochemistry and single-cell reverse transcription-quantitative PCR (RT-qPCR), higher frequency and transcript levels of caspase-3 were found in HSV-1-negative compared to HSV-1-positive ganglia and neurons, respectively. No terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay-positive neurons were detected. The infiltrating T cells do not induce apoptosis in latently infected neurons.

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    JVI Accepts: Articles Published Ahead of Print

  • Multifunctionality of a picornavirus polymerase domain: nuclear localization signal and nucleotide recognition [PublishAheadOfPrint]

  • The N-terminal region of the FMDV 3D polymerase contains the sequence MRKTKLAPT (residues 16 to 24) that acts as a nuclear localization signal. A previous study showed that substitutions K18E and K20E diminished the transport to the nucleus of 3D and 3CD, and severely impaired virus infectivity. These residues have also been implicated in template binding as seen in the crystal structures of different 3D-RNA elongation complexes. Here we report the biochemical and structural characterization of different mutant polymerases harboring substitutions at residues 18 and 20, in particular K18E, K18A, K20E, K20A and the double mutant KAKA. All mutant enzymes exhibit low RNA binding activity, low processivity and alterations in nucleotide recognition, including increased incorporation of ribavirin monophosphate (RMP) relative to the incorporation of cognate nucleotides, as compared with the wild type enzyme. The structural analysis shows an unprecedented flexibility of the 3D mutant polymerases, including both, global rearrangements of the closed hand architecture and local conformational changes at loop bbeta;9-aalpha;11 (within the polymerase motif B) and at the template-binding channel. Specifically, in 3D bound to RNA both K18E and K20E induced the opening of new pockets in the template channel where the downstream templating nucleotide at position +2 binds. The comparisons of free and RNA bound enzymes suggest that the structural rearrangements may occur in a concerted mode to regulate both RNA replication, processivity and fidelity. Thus, the N-terminal region of FMDV 3D that acts as a nuclear localization signal and in template binding, is also involved in nucleotide recognition, and can affect the incorporation of nucleotide analogues.

    IMPORTANCE The study documents multifunctionality of a nuclear localization signal (NLS) located at the N-terminal region of the foot-and-mouth disease viral polymerase (3D). Amino acid substitutions at this polymerase region can impair the transport of 3D to the nucleus, reduce 3D binding to RNA, and alter the relative incorporation of standard nucleoside-monophosphate versus ribavirin-monophosphate. Structural data reveal that the conformational changes in this region, forming part of the template channel entry, would be involved in nucleotide discrimination. The results have implications for the understanding of viral polymerase function, and for lethal mutagenesis mechanisms.

  • Move over bacteria! Viruses make their mark as mutualistic microbial symbionts [PublishAheadOfPrint]

  • Viruses are being redefined as more than just pathogens. They are also critical symbiotic partners in the health of their hosts. In some cases viruses have fused with their hosts in symbiogenenic relationships. Mutualistic interactions are found in plant, insect and mammalian viruses, as well as with eukaryotic and prokaryotic microbes, and some interactions involve multiple players of the holobiont. With increased virus discovery more mutualistic interactions are being described, and more will undoubtedly be discovered.

  • FACS-based analysis reveals an asymmetric induction of interferon stimulated genes in response to seasonal influenza a virus [PublishAheadOfPrint]

  • Influenza A virus (IAV) infection provokes an antiviral response involving the expression of type I and III interferons (IFN) and IFN-stimulated genes (ISGs) in infected cell cultures. However, the spatio-temporal dynamics of the IFN reaction are incompletely understood as previous studies investigated mainly the population responses of virus infected cultures, although substantial cell-to-cell variability has been documented. We devised a fluorescence activated cell sorting-based assay to simultaneously quantify expression of viral antigen and ISGs such as ISG15, MxA and IFIT1 in IAV-infected cell cultures on the level of single cells. This approach revealed that seasonal IAV triggers an unexpected asymmetric response as the major cell populations expressed either viral antigen or ISG, but rarely both. Further investigations identified a role of the viral NS1 protein in blocking ISG expression in infected cells, which surprisingly did not reduce paracrine IFN signaling to non-infected cells. Interestingly, viral ISG control was impaired in cultures infected with avian-origin IAV including H7N9 virus from Eastern China. This phenotype was traced back to polymorphic NS1 amino acids known to be important for stable binding of the polyadenylation factor CPSF30 and concomitant suppression of host cell gene expression. Most significantly, mutation of two amino acids within the CPSF30 attachment site of NS1 from seasonal IAV diminished the strict control of ISG expression in infected cells and substantially attenuated virus replication. In conclusion, our approach revealed an asymmetric, NS1-dependent ISG induction in cultures infected with seasonal IAV, which appears to be essential for efficient virus propagation.

    IMPORTANCE Interferons are expressed by infected cells in response to IAV infection and play important roles in the antiviral immune response by inducing hundreds of interferon-stimulated genes (ISGs). Unlike many previous studies, we investigated the ISG response on the level of single cells enabling novel insights into this virus-host interaction. Hence, cell cultures infected with seasonal IAV displayed an asymmetric ISG induction that was confined almost exclusively to non-infected cells. In comparison, ISG expression was observed in larger cell populations infected with avian-origin IAV suggesting a more resolute antiviral response towards these strains. Strict control of ISG expression by seasonal IAV was explained by the binding of the viral NS1 protein to the polyadenylation factor CPSF30, which reduces host cell gene expression. Mutational disruption of CPSF30 binding within NS1 concomitantly attenuated ISG control and replication of seasonal IAV illustrating the importance of maintaining an asymmetric ISG response for efficient virus propagation.

  • Non-neutralizing antibodies induced by HIV-1 gp41 NHR domain gain neutralizing activity in the presence of HIV fusion inhibitor enfuvirtide: a potential therapeutic vaccine strategy [PublishAheadOfPrint]

  • A key barrier against developing preventive and therapeutic HIV vaccines is the inability of viral envelope glycoproteins to elicit broad and potent neutralizing antibodies. However, in the presence of fusion inhibitor enfuvirtide, we show that the non-neutralizing antibodies induced by HIV-1 gp41 NHR domain (N63) exhibit potent and broad neutralizing activity against laboratory-adapted HIV-1 strains, including the drug-resistant variants, and primary HIV-1 isolates with different subtypes, suggesting the potential of developing gp41-targeted HIV therapeutic vaccines.

  • Accumulation of cytotoxic CD16+ NK Cells in SIV-infected lymph nodes associated with in situ differentiation and functional anergy [PublishAheadOfPrint]

  • Recent evidence suggests that even in treated infections, HIV and SIV replication may continue in lymph nodes (LN), serving as a potential virus reservoir. Here we investigated the effects of lentivirus infection on NK cell frequencies, phenotypes, and functions in naiiuml;ve and acutely or chronically SIVmac239-infected rhesus macaques. Compared to naiiuml;ve animals, we observed a three-fold greater frequency of cytotoxic CD16+CD56- NK cells in LN of chronically infected macaques. However, NK cells did not appear to be trafficking to LN, as homing markers CD62L and CCR7 did not increase on circulating NK cells during infection. LN NK cells demonstrated enhanced cytotoxicity in acute and chronic infection with two-fold increases in perforin expression and three-fold increases in CD107a expression following mitogen stimulation. Lysis of K562 cells by LN NK cells from acutely infected animals was greater than lysis by pre-infection samples from the same animals. LN NK cells from chronically infected animals lysed K562 cells more efficiently than LN NK cells from uninfected animals, but importantly surrogate markers of cytotoxicity in infected macaques were disproportionately greater than ex vivo killing. Furthermore, Tim-3, an indicator of activation and/or exhaustion, was upregulated three-fold on LN NK cells in infected animals. Collectively, these data suggest that LN NK cells are skewed toward a cytotoxic phenotype during SIV infection, but may become dysfunctional and exhausted in chronic disease.

    Importance The accumulation of CD16+CD56- NK cells in the SIV-infected lymph node without changes in NK homing to the LN could suggest that these cells are differentiating in situ. Surprisingly, this increase in frequency of the cytotoxic subset of NK cells is not accompanied by an increase of similar magnitude in the cytolytic function of LN lymphocytes. This functional modulation, together with the higher Tim-3 expression observed on LN NK cells isolated from chronically infected animals compared to those from naiiuml;ve macaques is indicative of an exhausted phenotype. This exhaustion could contribute to the robust replication of HIV and SIV in the LN during acute and chronic stages of infection allowing the survival of infected cells and maintenance of a viral reservoir.

  • Particle to plaque-forming unit ratio of Ebola virus influences disease course and survival in cynomolgus macaques [PublishAheadOfPrint]

  • This study addresses the role of Ebola virus (EBOV) specific infectivity in virulence. Filoviruses are highly lethal, enveloped, single-stranded negative-sense RNA viruses that can cause hemorrhagic fever. No approved vaccines or therapies exist for filovirus infections and infectious virus must be handled in maximum containment. Efficacy testing of counter measures, in addition to investigations of pathogenicity and immune response, often require a well-characterized animal model. For EBOV, an obstacle in performing accurate disease modeling is a poor understanding of what constitutes an infectious dose in animal models. One well recognized consequence of viral passage in cell culture is a change in specific infectivity, often measured as a particle to plaque forming unit (PFU) ratio. Here we report that serial passages of EBOV in cell culture resulted in a decrease in particle to PFU ratio. Notably, this correlated with decreased potency in a lethal cynomolgus macaque (Macaca fascicularis) model of infection; animals were infected with the same viral-dose as determined by plaque assay, but animals that received more virus particles exhibited increased disease. This suggests that some particles are unable to form a plaque in a cell culture assay but are able to result in lethal disease in vivo. These results have a significant impact on how future studies are designed to model EBOV disease and test counter measures.

    Importance Ebola virus (EBOV) can cause severe hemorrhagic disease with a high case-fatality-rate and there are no approved vaccines or therapies. Specific-infectivity can be considered the total number of viral particles per plaque forming unit (PFU) and its impact on disease is poorly understood. In stocks of most mammalian viruses there are particles that are unable to complete an infectious cycle or unable to cause cell pathology in cultured cells. We asked if these particles cause disease in nonhuman primates by infecting monkeys with an equal infectious-dose of genetically-identical stocks possessing either high or low specific-infectivities. Interestingly, some particles that did not yield plaques in cell culture assays were able to result in lethal disease in vivo. Furthermore, the number of PFU needed to induce lethal disease in animals was very low. Our results have significant impact on how future studies are designed to model EBOV disease and test counter measures.

  • Identification of cholesterol-25-hydroxylase as a novel host restriction factor as a part of primary innate immune responses against hepatitis C virus infection [PublishAheadOfPrint]

  • Hepatitis C virus (HCV), a single-stranded positive-sense RNA virus of the Flaviviridae family, causes chronic liver diseases including hepatitis, cirrhosis and cancer. HCV infection is critically dependent on host lipid metabolism which contributes to all stages of the viral life cycle, including virus entry, replication, assembly and release. 25-hydroxycholesterol (25HC) plays a critical role in regulating lipid metabolism, modulating immune responses and suppressing viral pathogens. In this study, we showed that 25HC and its synthesizing enzyme cholesterol 25-hydroxylase (CH25H) efficiently inhibit HCV infection at a post-entry stage. CH25H inhibits HCV infection by suppressing the maturation of SREBPs, critical transcription factors for host lipid biosynthesis. Interestingly, CH25H is up-regulated upon poly(I:C) treatment or HCV infection in hepatocytes which triggers type I and III interferon responses, suggesting the CH25H induction constitutes a part of host innate immune response. To our surprise, in contrast to studies in mice, CH25H is not induced by interferons in human cells and knockdown of STAT-1 has no effect on the induction of CH25H, suggesting CH25H is not an interferon-stimulated gene in human, but rather represents a primary and direct host response to viral infection. Finally, knockdown of CH25H in human hepatocytes significantly increases HCV infection. In summary, our results demonstrate that CH25H constitutes a primary innate response against HCV infection through regulating host lipid metabolism. Manipulation of CH25H expression and function should provide a new strategy for anti-HCV therapeutics.

    IMPORTANCE Recent studies have expanded the critical roles of oxysterols in regulating immune response and antagonizing viral pathogens. Here we showed that one of the oxysterols, 25HC and its synthesizing enzyme CH25H efficiently inhibit HCV infection at a post-entry stage via suppressing the maturation of transcription factor SREBPs that regulate lipid biosynthesis. Furthermore, we found that CH25H expression is up-regulated upon poly(I:C) stimulation or HCV infection, suggesting CH25H induction constitutes a part of host innate immune response. Interestingly, in contrast to studies in mice that ch25h is an interferon-stimulated gene, CH25H cannot be induced by interferons in human cells, but rather represents a primary and direct host response to viral infection. Our studies demonstrate that the induction of CH25H represents an important host innate response against virus infection and highlight the role of lipid effectors in host antiviral strategy.

  • Deciphering Poxvirus Gene Expression by RNA Sequencing and Ribosome Profiling [PublishAheadOfPrint]

  • The more than 200 closely spaced annotated open reading frames, extensive transcriptional read-through and numerous unpredicted RNA start sites have made the analysis of vaccinia virus gene expression challenging. Genome-wide ribosome profiling provided an unprecedented assessment of poxvirus gene expression. By 4 h after infection, approximately 80% of the ribosome-associated mRNA was viral. Ribosome-associated mRNAs were detected for most annotated early genes at 2 h and most intermediate and late genes at 4 and 8 h. Cluster analysis identified a subset of early mRNAs that continued to be translated at the later times. At 2 h, there was excellent correlation between abundance of individual mRNAs and numbers of associated ribosomes, indicating that expression was primarily transcriptionally regulated. However, extensive transcriptional read through invalidated similar correlations at later times. The mRNAs with the highest density of ribosomes had host response, DNA replication and transcription roles at early times and were virion components at late times. Translation inhibitors were used to map initiation sites at single nucleotide resolution at the start of most annotated open reading frames, although in some cases a downstream methionine was used instead. Additional putative translational initiation sites with AUG or alternative codons occurred mostly within open reading frames and fewer in untranslated leader sequences, antisense strands and intergenic regions. However, most open reading frames associated with these additional translation initiation sites were short, raising questions regarding their biological roles. The data were used to construct a high-resolution genome-wide map of the vaccinia virus translatome.

    Importance This report contains the first genome-wide, high-resolution analysis of poxvirus gene expression at both transcriptional and translational levels. The study was made possible by recent methodological advances allowing examination of the translated regions of mRNAs including start sites at single nucleotide resolution. Vaccinia virus ribosome-associated mRNA sequences were detected for most annotated early genes at 2 h and most intermediate and late genes at 4 and 8 h after infection. The ribosome profiling approach was particularly valuable for poxviruses because of the close spacing of approximately 200 open reading frames and extensive transcriptional read-through resulting in overlapping mRNAs. The expression of intermediate and late genes, in particular, was visualized with unprecedented clarity and quantitation. We also identified novel putative translation initiation sites that were mostly associated with short protein coding sequences. The results provide a framework for further studies of poxvirus gene expression.

  • Equine and canine influenza H3N8 viruses show minimal biological differences despite phylogenetic divergence [PublishAheadOfPrint]

  • The A/H3N8 canine influenza virus (CIV) emerged from A/H3N8 equine influenza virus (EIV) around the year 2000 through the transfer of a single virus from horses to dogs. We defined and compared the biological properties of EIV and CIV by examining their genetic variation, infection and growth in different cell cultures, receptor specificity, hemagglutinin (HA) cleavage, and infection and growth in horse and dog tracheal explant cultures. Comparison of sequences of viruses from horses and dogs revealed mutations that may be linked to host adaptation and tropism. We prepared infectious clones of representative EIV and CIV strains that were similar to the consensus sequences of viruses from each host. The rescued viruses, including HA and NA double reassortants, exhibited similar long-term growth in MDCK cells. Different host cells showed varying levels of susceptibility to infection, but no differences in infectivity were seen when comparing viruses. All viruses preferred aalpha;2-3 over aalpha;2-6 linked sialic acids for infections, and glycan microarray analysis showed EIV and CIV HA-Fc fusion proteins bound only to aalpha;2-3 linked sialic acids. Cleavage assays showed EIV and CIV HA proteins required trypsin for efficient cleavage, and no differences in cleavage efficiency were seen. Inoculation of the viruses into tracheal explants revealed similar levels of infection and replication by each virus in dog trachea, although EIV was more infectious in horse trachea than CIV.

    IMPORTANCE Influenza A viruses can cross species barriers and cause severe disease in their new hosts. Infections with highly pathogenic avian H5N1 virus, and more recently avian H7N9 virus, have resulted in high rates of lethality in humans. Unfortunately, our current understanding of how influenza viruses jump species barriers is limited. Our aim was to provide an overview and biological characterization of H3N8 equine and canine influenza viruses using various experimental approaches, since the canine virus emerged from horses approximately 15 years ago. We showed that although there were numerous genetic differences between the equine and canine viruses, this variation did not result in dramatic biological differences between the viruses from the two hosts, and they appeared phenotypically equivalent in most assays conducted here. These findings suggest the cross species transmission and adaptation of influenza viruses may be mediated by subtle changes in virus biology.

  • Studies on the Contribution of Human Cytomegalovirus UL21a and UL97 to Viral Growth and Inactivation of the Anaphase Promoting Complex/Cyclosome (APC/C) E3 Ubiquitin Ligase Reveal a Unique Cellular Mechanism for Down-modulation of the APC/C Subunits APC1, APC4, and APC5. [PublishAheadOfPrint]

  • Human Cytomegalovirus (HCMV) deregulates the cell cycle by several means, including inactivation of the Anaphase Promoting Complex/Cyclosome (APC/C) E3 ubiquitin ligase. Viral proteins UL97 and UL21a, respectively, affect the APC/C by phosphorylation of APC/C co-activator Cdh1 and by inducing degradation of subunits APC4 and APC5, which with APC1 form the APC/C platform subcomplex. The aim of this study was to further characterize the mechanism of APC/C inactivation and define the relative contributions of UL21a and UL97 to APC/C substrate accumulation and to viral growth. We show that in uninfected cells UL21a, but not UL97, can disrupt APC/C function, leading to accumulation of substrates. We find that UL21a is necessary and sufficient to induce degradation of APC1, in addition to the previously reported APC4 and APC5. We also demonstrate that there is a previously unreported cellular mechanism for a specific decrease in the levels of all three platform subunits, APC1, APC4, and APC5, upon depletion of any one of these subunits or subunit APC8. Finally, we show that at low MOI either UL97 or UL21a can partially complement a growth-defective mutant virus lacking both UL21a and UL97, with significantly greater benefit afforded by expression of both proteins. This double mutant also can be partially rescued by inactivation of the APC/C using siRNA against specific subunits. These results further our understanding of HCMV's interaction with the cell cycle machinery and reveal a new cellular pattern of APC/C subunit down-modulation.

    IMPORTANCE HCMV lytic infection subverts the host cell cycle machinery in multiple ways. A major effect is inactivation of the APC/C, which plays a central role in control of cell cycle progression. This study provides further insight into the mechanism of inactivation. We discovered that the APC1 subunit, which along with APC4 and APC5 form the platform subcomplex of the APC/C, is an additional target of the degradation induced by HCMV protein UL21a. This study also shows for the first time that there is a unique cellular process in uninfected cells whereby depletion of APC1, APC4, APC5, or APC8 recapitulates the pattern of HCMV-mediated APC/C subunit degradation.

  • Immunocompetent Adults from Human Norovirus Challenge Studies do not Exhibit Norovirus Viremia [PublishAheadOfPrint]

  • Noroviruses (NoV) are among the leading causes of acute gastroenteritis worldwide (1)....

  • Murine Immune Responses to Virus-like Particle Associated Pre- and Post-Fusion Forms of the Respiratory Syncytial Virus F Protein [PublishAheadOfPrint]

  • Virus-like particles (VLPs) built on the Newcastle disease virus (NDV) core proteins, NP and M, and containing two chimera proteins, F/F and H/G, composed of respiratory syncytial virus (RSV) fusion protein (F) and glycoprotein (G) ectodomains fused to the transmembrane and cytoplasmic domains of the NDV F and HN proteins, respectively, stimulate durable, protective RSV neutralizing antibodies in mice. Here we report the properties of VLPs constructed to contain mutant RSV F protein ectodomains stabilized in pre-fusion (Pre-F/F) or post-fusion (Post-F/F) configurations. The structures of the chimera proteins assembled into VLPs were verified immunologically by their reactivities with a conformationally restricted anti-F protein monoclonal antibody. Following immunization of mice, without adjuvant, Pre-F/F VLPs induced significantly higher neutralizing antibody titers than the Post-F/F VLPs or the wild type F/F containing VLPs after a single immunization but not after prime and boost immunization. The specificities of anti-F IgG induced by the two mutant VLPs were assessed by ELISA using soluble forms of the pre-fusion and post-fusion forms of the F protein as targets. While both VLPs stimulated similar levels of IgG specific for the soluble post-fusion F protein, titers of IgG specific for pre-fusion F induced by the Pre-F/F VLPs were higher than those induced by Post-F/F VLPs. Thus VLPs containing a stabilized pre-fusion form of the RSV F protein represent a promising RSV vaccine candidate.

    Importance The development of vaccines for respiratory syncytial virus has been hampered by a lack of understanding of the requirements for eliciting high titers of neutralizing antibodies. The results of this study suggest that particle associated RSV F protein containing mutations that stabilize the structure in a pre-fusion conformation may stimulate higher titers of protective antibodies than particles containing F protein in a wild type or post fusion conformation. These findings indicate that the pre-fusion F protein assembled into VLPs has the potential to produce a successful RSV vaccine candidate.

  • Human Cytomegalovirus Infection Dysregulates the Localization and Stability of NICD1 and Jag1 in Neural Progenitor Cells [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) infection of the developing fetus frequently results in major neural developmental damage. In previous studies HCMV was shown to down-regulate neural progenitor/stem cell (NPC) markers and induce abnormal differentiation. As Notch signaling plays a vital role in the maintenance of stem cell status and is a switch governing NPC differentiation, the effect of HCMV infection on the Notch signaling pathway in NPCs was investigated. HCMV down-regulated mRNA levels of Notch1 and its ligand Jag1, and reduced protein levels and altered the intracellular localization of Jag1 and the intracellular effector form of Notch1, NICD1. These effects required HCMV gene expression and appeared to be mediated through enhanced proteasomal degradation. Transient expression of the viral tegument proteins of pp71 and UL26 reduced NICD1 and Jag1 protein levels endogenously and exogenously. Given the critical role of Notch signaling in NPC cell growth and differentiation, these findings reveal important mechanisms by which HCMV disturbs neural cell development in vitro. Similar events in vivo may be associated with HCMV-mediated neuropathogenesis during congenital infection in the fetal brain.

    IMPORTANCE Congenital human cytomegalovirus (HCMV) infection is the leading cause of birth defects that primarily manifest as neurological disabilities. Neural progenitor cells (NPCs), key players in fetal brain development, are the most susceptible cell type for HCMV infection in the fetal brain. Studies have shown that NPCs are fully permissive for HCMV infection which causes neural cell loss and premature differentiation, thereby perturbing NPC fate. Elucidating virus-host interactions that govern NPC proliferation and differentiation is critical to understanding neuropathogenesis. The Notch signaling pathway is critical for maintaining stem cell status and functions as a switch for differentiation of NPCs. Our investigation into the impact of HCMV infection on this pathway revealed that HCMV dysregulates Notch signaling by altering expression of the Notch ligand, Jag1, Notch1 and its active effector in NPCs. These results suggest a mechanism for the neuropathogenesis induced by HCMV infection that includes altered NPC differentiation and proliferation.

  • Plasmacytoid dendritic cell infection and sensing capacity during pathogenic and non-pathogenic SIV infection [PublishAheadOfPrint]

  • HIV in humans and SIV in macaques (MAC) lead to chronic inflammation and AIDS. Natural hosts, such as African green monkeys (AGM) and sooty mangabeys (SM), are protected against SIV-induced chronic inflammation and AIDS. Here, we report that AGM plasmacytoid dendritic cells (pDC) express extremely low levels of CD4, unlike MAC and human pDC. Despite this, AGM pDC efficiently sensed SIVagm, but not heterologous HIV/SIV isolates, indicating a virus-host adaptation. Moreover, both AGM and SM pDC were found to be, in contrast to MAC pDC, predominantly negative for CCR5. Despite such limited CD4 and CCR5 expression, lymphoid tissue pDC were infected to a similar degree as CD4+ T cells, both in MAC and AGM. Altogether, our finding of efficient pDC infection by SIV in vivo identifies pDC as a potential viral reservoir in lymphoid tissues. We discovered low expression of CD4 on AGM pDC, which did not preclude efficient sensing of host-adapted viruses. Therefore, pDC infection and efficient sensing are not prerequisites for chronic inflammation. The high level of pDC infection by SIVagm suggests that if CCR5 paucity on immune cells is important for non-pathogenesis of natural hosts, it is possibly not due to its role as a co-receptor.

    Importance The ability of certain key immune cell subsets to resist infection might contribute to the asymptomatic nature of simian immunodeficiency virus (SIV) infection in its natural hosts, such as African green monkeys (AGM) and sooty mangabeys (SM). This relative resistance to infection has been correlated with reduced expression of CD4 and/or CCR5. We show that plasmacytoid dendritic cells (pDC) of natural hosts display a reduced CD4 and/or CCR5 expression, unlike macaque pDC. Surprisingly, this did not protect AGM pDC, as infection levels were similar to those found in MAC pDC. Furthermore, we show that AGM pDC did not consistently produce IFN-I upon heterologous SIVmac/HIV-1 encounter, while they sensed autologous SIVagm isolates. Pseudotyping SIVmac/HIV-1 overcame this deficiency, suggesting that reduced uptake of heterologous viral strains underlay this lack of sensing. The distinct IFN-I responses depending on host species and HIV/SIV isolates reveal host/virus species-specificity of pDC sensing.

  • A single dose of an avian H3N8 vaccine is highly immunogenic and efficacious against a recently emerged seal influenza virus in mice and ferrets [PublishAheadOfPrint]

  • H3N8 influenza viruses are a commonly found subtype in wild birds, usually causing mild or no disease in infected birds. However, they have crossed the species barrier and have been associated with outbreaks in dogs, pigs, donkeys and seals and therefore pose a threat to humans. A live attenuated cold-adapted (ca) H3N8 vaccine virus was generated by reverse genetics using the wild-type (wt) hemagglutinin (HA) and neuraminidase (NA) genes from the A/blue-winged teal/Texas/Sg-00079/2007 [H3N8] (tl/TX/079/07) wt virus, and the six internal protein gene segments from the cold-adapted (ca) influenza A virus vaccine donor strain, A/Ann Arbor/6/60 ca (H2N2), the backbone of the licensed seasonal live attenuated influenza vaccine. One dose of the tl/TX/079/07 ca vaccine induced a robust neutralizing antibody response against the homologous (tl/TX/079/07) and two heterologous influenza viruses including the recently emerged A/harbor seal/New Hampshire/179629/2011 [H3N8] and A/northern pintail/Alaska/44228-129/2006 [H3N8] and conferred robust protection against the homologous and heterologous influenza viruses. We also analyzed human sera against the tl/TX/079/07 H3N8 avian influenza virus and observed low but detectable antibody reactivity in elderly subjects suggesting that older H3N2 influenza viruses confer some cross reactive antibody. The latter observation was confirmed in a ferret study. The safety, immunogenicity and efficacy of the tl/TX/079/07 ca vaccine in mice and ferrets support further evaluation of this vaccine in humans for use in the event of transmission of an H3N8 avian influenza virus to humans. The human and ferret serology data suggest single dose of the vaccine may be sufficient in older subjects.

    IMPORTANCE Although natural infection of humans with an avian H3N8 influenza virus has not yet been reported, this influenza virus subtype has already crossed the species barrier and productively infected mammals. Pandemic preparedness is an important public health priority. Therefore, we generated a live attenuated avian H3N8 vaccine candidate and demonstrated that a single dose of the vaccine was highly immunogenic and protected mice and ferrets against homologous and heterologous H3N8 avian viruses.

  • Lentivector knock-down of CCR5 in hematopoietic stem cells confers functional and persistent HIV-1 resistance in humanized mice [PublishAheadOfPrint]

  • Gene-engineered CD34+ hematopoietic stem and progenitor cells (HSPCs) can be used to generate an HIV-1-resistant immune system. However, a certain threshold of transduced HSPCs might be required for transplantation into mice for creating an HIV-resistant immune system. Here, we combined CCR5 knock-down by a highly efficient miRNA lentivector with pre-transplantation selection of transduced HSPCs to obtain a rather pure population of gene engineered CD34+ cells. Low-level transduction of HSPCs and subsequent sorting by flow cytometry yielded ggt;70% transduced cells. Mice transplanted with these cells showed functional and persistent resistance to a CCR5-tropic HIV strain: viral load was significantly decreased over months, and human CD4+ T-cells were preserved. In one mouse, viral mutations, resulting presumably in a CXCR4-tropic strain, overcame HIV resistance. Our results suggest that HSPC-based CCR5 knock-down may lead to efficient control of HIV in vivo. We overcome a major limitation of previous HIV gene therapy in humanized mice studies in which only a proportion of the cells in chimeric mice in vivo are anti-HIV engineered. Our strategy underlines the promising future of gene engineering HIV-resistant CD34+ cells that produce a constant supply of HIV resistant progeny.

    Importance Major issues in experimental long-term in vivo HIV gene therapy have been (i) low efficacy of cell transduction at the time of implantation and (ii) transduction resulting in multiple copies of heterologous DNA in target cells. In this study, we demonstrated the efficacy of a transplantation approach with a selection step for transduced cells that allows transplantation of an enriched population of HSPCs expressing a single (low) copy of a CCR5 miRNA. Efficient maintenance of CD4+ T-cells and a low viral titer resulted only when at least 70% of the HIV target cells were genetically modified. These findings imply that clinical protocols of HIV gene therapy require a selective enrichment of genetically targeted cells because positive selection of modified cells is likely to be insufficient below this threshold. This selection approach may not only be beneficial for HIV patients, but also other patients requiring transplantation of genetically modified cells.

  • ORF45-mediated prolonged c-Fos accumulation accelerates viral transcription during the late stage of lytic replication of Kaposi's sarcoma-associated herpesvirus [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) encodes multiple viral proteins that activate ERK-MAPK cascades. One of these viral proteins, ORF45, mediates sustained ERK-RSK activation during KSHV lytic replication and facilitates viral translation through the phosphorylation of an eukaryotic translation initiation factor, eIF4B. The importance of ERK-RSK activation for KSHV viral transcription has been shown; however, which transcription factor senses the sustained MAPK signaling and leads to viral transcription remains poorly understood. Here, we show that the presence of ORF45 leads to the prolonged accumulation of c-Fos during the late stage of KSHV lytic replication through ERK-RSK-dependent phosphorylation and stabilization and that the depletion of c-Fos disrupts viral lytic transcription. Genome-wide screening revealed that c-Fos directly binds to multiple viral gene promoters and enhances viral transcription. Mutation of the ERK-RSK phosphorylation sites of c-Fos restrains KSHV lytic gene expression and virion production. These results indicate that the prolonged accumulation of c-Fos promotes the progression of viral transcription from early to late stage and accelerates viral lytic replication upon sustained ORF45-ERK-RSK activation during the KSHV lytic life cycle.

    Importance During KSHV lytic replication, transient and sustained activation of ERK-RSK induce viral immediate early (IE) transcription and late transcription, respectively. Studies have revealed that ERK-RSK activates several transcription factors involved in IE gene expression, including Ets, AP-1, CREB and C/EBP, which lead to the transient ERK-RSK activation-dependent IE transcription. Whereas c-Fos acts as a sensor of sustained ERK-RSK activation, ORF45-ERK-RSK signaling mediates c-Fos phosphorylation and accumulation during late KSHV lytic replication, consequently promoting viral transcription through the direct binding of c-Fos to multiple KSHV promoters. This finding indicates that c-Fos mediates distinct viral transcriptional progression following sustained ERK-RSK signaling during the KSHV lytic life cycle.

  • The Serum Very-Low-Density Lipoprotein Serves as a Restriction Factor of Hepatitis C Virus Infection [PublishAheadOfPrint]

  • Recent studies demonstrated that transgenic mice expressing key human HCV receptors are susceptible to HCV infection albeit at very low efficiency. Robust mouse models of HCV infection and replication are needed to determine the importance of host factors in HCV replication, pathogenesis, and carcinogenesis as well as to facilitate the development of antiviral agents and vaccines. The low efficiency of HCV replication in the humanized mouse models is likely due to either the lack of essential host factors or presence of restriction factors for HCV infection and/or replication in mouse hepatocytes. To determine whether HCV infection is affected by restriction factors present in serum, we examined the effect of mouse and human sera on HCV infectivity. Strikingly, we found that mouse and human sera potently inhibited HCV infection. Mechanistic studies demonstrated that mouse serum blocked HCV cell attachment without significant effect on HCV replication. Fractionation analysis of mouse serum in conjunction with targeted mass spectrometric analysis suggested that serum very-low-density lipoprotein (VLDL) was responsible for the blockade of HCV cell attachment, as VLDL-depleted mouse serum lost HCV-inhibitory activity. Both purified mouse and human VLDL could efficiently inhibit HCV infection. Collectively, these findings suggest that serum VLDL serves as a major restriction factor of HCV infection in vivo. It also implies that reduction or elimination of VLDL production will likely enhance HCV infection in the humanized mouse model of HCV infection and replication.

    IMPORTANCE HCV is a major cause of liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Recently, several studies suggested that humanized mouse or transgenic mouse expressing key HCV human receptors became susceptible to HCV infection. However, HCV infection and replication in the humanized animals were very inefficient, suggesting either the lack of cellular genes important for HCV replication or the presence of restriction factors inhibiting HCV infection and replication in mouse. In this study, we found that both mouse and human sera effectively inhibited HCV infection. Mechanistic studies demonstrated that VLDL is the major restriction factor that blocks HCV infection. These findings suggest that VLDL is beneficial to patients by restricting HCV infection. More importantly, our findings suggest that elimination of VLDL will lead to the development of more robust mouse models for the study of HCV pathogenesis, host response to HCV infection, and evaluation of HCV vaccines.

  • Impact of the MRN complex on Adeno-Associated Virus integration and replication during co-infection with Herpes Simplex Virus type 1. [PublishAheadOfPrint]

  • Adeno-Associated Virus (AAV) is a helper dependent parvovirus that requires co-infection with adenovirus (AdV) or herpes simplex virus type 1 (HSV-1) to replicate. In the absence of the helper virus, AAV can persist in an episomal or integrated form. Previous studies have analyzed the DNA damage response (DDR) induced upon AAV replication to understand how it controls AAV replication. In particular, it was shown that the Mre11-Rad50-Nbs1 (MRN) complex, a major player of the DDR induced by double-stranded DNA breaks and stalled replication forks, could negatively regulate AdV and AAV replication during co-infection. In contrast, MRN favors HSV-1 replication and is recruited to AAV replication compartments that are induced in the presence of HSV-1. In this study we examined the role of MRN during AAV replication induced by HSV-1. Our results indicated that knockdown of MRN significantly reduced AAV DNA replication after co-infection with polymerase deleted or wild type (wt) HSV-1. This effect was specific of wt AAV since it did not occur with recombinant AAV vectors. Positive regulation of AAV replication by MRN was dependent on its DNA tethering activity but did not require its nuclease activities. Importantly, knockdown of MRN also negatively regulated AAV integration within the human AAVS1 site, both in the presence and in the absence of HSV-1. Altogether, this work identifies a new function of MRN during integration of the AAV genome and demonstrates that this DNA repair complex positively regulates AAV replication in the presence of HSV-1.

    Importance Viral DNA genomes trigger a DNA damage response (DDR) which can be either detrimental or beneficial for virus replication. Adeno-Associated Virus (AAV) is a defective parvovirus that requires the help of an unrelated virus such as adenovirus (AdV) or herpes simplex virus type 1 (HSV-1) for productive replication. Previous studies have demonstrated that the cellular Mre11-Rad50-Nbs1 (MRN) complex, a sensor and regulator of the DDR, negatively regulates AAV replication during co-infection with AdV which counteracts this effect by inactivating the complex. Here, we demonstrate that MRN positively regulates AAV replication during co-infection with HSV-1. Importantly, our study also indicates that MRN in addition favors integration of AAV genomes within the human AAVS1 site. Altogether, this work indicates that MRN differentially regulates AAV replication depending on the helper virus which is present and identifies a new function of this DNA repair complex during AAV integration.

  • Mutation and recombination frequencies reveal a biological contrast within strains of Cucumber mosaic virus [PublishAheadOfPrint]

  • Recent in planta studies have shown that strains Fny and LS of Cucumber mosaic virus (CMV) display differential genetic diversities, with Fny- and LS-CMV having higher and lower mutation frequencies, respectively (Justin S. Pita and Marilyn J. Roossinck, J. Virol. 87 (2): 790-797, 2012). In this paper, we show that these virus strains have differential recombination frequencies as well. However, the high-diversity Fny strain is a low-recombination virus whereas the very low-diversity LS strain is instead a high-recombination virus. Unlike mutation frequency that was determined by both RNAs 1 and 2, the control elements of recombination frequency reside predominantly within RNA 2, specifically within the 2a gene.

    Importance to Field Recombination is an important mechanisms in virus evolution that can lead to increased or decreased variation, and is a major player in virus speciation events that can lead to emerging viruses. Although viral genomes show very frequent evidence of recombination, details of the mechanism involved in these events are still poorly understood. We show here that the reciprocal effects of high mutation frequency and low recombination frequency (and vice versa) involve the RNA dependent RNA polymerase of the virus, and we speculate that these evolutionary events are related to difference in processivity for two strains of the same virus.

  • Immunogenic display of purified chemically crosslinked HIV-1 spikes [PublishAheadOfPrint]

  • HIV-1 envelope glycoprotein (Env) spikes are prime vaccine candidates, at least in principle, but suffer from instability, molecular heterogeneity and a low copy number on virions. We anticipated that chemical crosslinking of HIV-1 would allow purification and molecular characterization of trimeric Env spikes as well as high copy number immunization. Broadly neutralizing antibodies bound tightly to all major quaternary epitopes on crosslinked spikes. Covalent crosslinking of the trimer also stabilized broadly neutralizing epitopes, although surprisingly some individual epitopes were still somewhat sensitive to heat or reducing agent. Immunodepletion using non-neutralizing antibodies to gp120 and gp41 was an effective method for removing non-native-like Env. Crosslinked spikes, purified via an engineered C-terminal tag, were shown by negative stain EM to have well-ordered, tri-lobed structure. An immunization was performed comparing a boost with Env spikes on virions to spikes crosslinked and captured onto nanoparticles, each following a gp160 DNA prime. Though differences in neutralization did not reach statistical significance, crosslinked Env spikes elicited a more diverse, and sporadically neutralizing antibody response against Tier 1b and 2 isolates when displayed on nanoparticles, despite attenuated binding titers to gp120 and V3 crown peptides. Our study demonstrates display of crosslinked trimeric Env spikes on nanoparticles, while showing a level of control over antigenicity, purity and density of virion-associated Env, which may have relevance for Env based vaccine strategies for HIV-1.

    Importance The envelope spike (Env) is the target of HIV-1 neutralizing antibodies, which a successful vaccine will need to elicit. However, native Env on virions is innately labile, as well as heterogeneously and sparsely displayed. We therefore stabilized Env spikes using a chemical crosslinker and removed non-native Env by immunodepletion with non-neutralizing antibodies. Fixed native spikes were recognized by all classes of known broadly neutralizing antibodies but not by non-neutralizing antibodies, and displayed on nanoparticles in high copy number. An immunization experiment in rabbits revealed that crosslinking Env reduced its overall immunogenicity; however, high copy display on nanoparticles enabled boosting of antibodies that sporadically neutralized some relatively resistant HIV-1 isolates, albeit at low titer. This study describes the purification of stable and antigenically correct Env spikes from virions that can be used as immunogens.

  • Epstein Barr-Virus Latent Membrane Protein 2 Induces Autophagy to Prevent Cell Death [PublishAheadOfPrint]

  • The Epstein-Barr virus protein latent membrane protein 2 induces many characteristics of carcinoma including transformation, migration, invasion, and impaired differentiation. The MCF10A cell line differentiates to form hollow acini when grown in Matrigel and expression of LMP2A inhibited differentiation and anoikis induced by loss of matrix attachment. The LMP2A cells formed large, lobular structures rather than hollow acini. Autophagy inhibitors impaired the abnormal growth and induced caspase 3 activation and acini formation. LMP2 also increased autophagosome formation and expression of proteins in the autophagosome pathway. These findings suggest that LMP2A may inhibit anoikis and luminal clearance in acini through induction of autophagy.

  • Therapeutic Immunization with a Mixture of Herpes Simplex Virus Type 1 Glycoprotein D Derived "Asymptomatic" Human CD8+ T-cell Epitopes Decreases Spontaneous Ocular Shedding in Latently Infected HLA Transgenic Rabbits: Association with Low Frequency of Local PD-1+TIM-3+CD8+ Exhausted T Cells [PublishAheadOfPrint]

  • Most blinding ocular herpetic disease is due to reactivation of herpes simplex virus type 1 (HSV-1) from latency rather than to primary acute infection. No herpes simplex vaccine is currently available for use in humans. In this study, we used the HLA-A*02:01 transgenic (HLA Tg) rabbit model of ocular herpes to assess the therapeutic efficacy of a therapeutic vaccine based on HSV-1 gD epitopes that are mainly recognized by CD8+ T cells from "naturally" protected HLA-A*02:01 positive, HSV-1 seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease). Three ASYMP CD8+ T cell epitopes (gD53-61, gD70-78 and gD278-286) were linked with a promiscuous CD4+ T-cell epitope (gD287-317) to create 3 separate pairs of CD4-CD8 peptides, which were then each covalently coupled to an N-palmitoyl-lysine moiety, a toll like receptor 2 (TLR-2) ligand. This resulted in the construction of 3 CD4-CD8 lipopeptide vaccines. Latently infected HLA-Tg rabbits were immunized with a mixture of these 3 ASYMP lipopeptide vaccines, delivered as eye drops in sterile PBS. The ASYMP therapeutic vaccination: (i) induced HSV-specific CD8+ T cells that prevent HSV-1 reactivation ex vivo from latently infected explanted trigeminal ganglia (TG); (ii) significantly reduced HSV-1 shedding detected in tears; (iii) boosted the number and function of HSV-1 gD epitopes-specific CD8+ T cells in draining lymph nodes (DLN), conjunctiva, and TG; and (iv) was associated with fewer exhausted HSV-1 gD-specific PD-1+TIM-3+CD8+ T-cells. The results underscore the potential of an ASYMP CD8+ T cell epitope-based therapeutic vaccine strategy against recurrent ocular herpes.

    IMPORTANCE Seventy to 90% of adults harbor herpes simplex virus type 1 (HSV-1) infections, which it establishes lifelong latency in sensory neurons of the trigeminal ganglia. This latent state sporadically switches to spontaneous reactivation resulting in viral shedding in tears. Most blinding herpetic disease in humans is due to reactivation of HSV-1 from latency rather than to primary acute infection. To date, there is no licensed therapeutic vaccine that can effectively stop or reduce HSV-1 reactivation from latently infected sensory ganglia and the subsequent shedding in tears. In the present study, we demonstrated that topical ocular therapeutic vaccination of latently infected HLA transgenic rabbits with a lipopeptide vaccine, that exclusively contains human "asymptomatic" CD8+ T cell epitopes, successfully decreased spontaneous HSV-1 reactivation, as judged by a significant reduction in spontaneous shedding in tears. The findings should guide the clinical development of a safe and effective T-cell-based therapeutic herpes vaccine.

  • Lassa-VSV chimeric virus safely destroys brain tumors [PublishAheadOfPrint]

  • High grade tumors in the brain are among the deadliest of cancers. Here, we took a promising oncolytic virus, vesicular stomatitis virus (VSV), and tested the hypothesis that the neurotoxicity associated with the virus could be eliminated without blocking its oncolytic potential in the brain by replacing the neurotropic VSV glycoprotein with the glycoproteins from one of five different viruses including Ebola, Marburg, LCMV, rabies, and Lassa. Based on in vitro infections of normal and tumor cells, we selected two viruses to test in vivo. Wild type VSV was lethal when injected directly into the brain. In contrast, a novel chimeric virus (VSV-LASV-GPC) containing genes from both Lassa and VSV showed no adverse actions within or outside the brain, and targeted and completely destroyed brain cancer, including high grade glioblastoma and melanoma, even in metastatic cancer models. When mice had two brain tumors, intratumoral VSV-LASV-GPC injection in one tumor (glioma or melanoma) led to complete tumor destruction; importantly, the virus moved contralaterally within the brain to selectively infect the second non-injected tumor. A chimeric virus combining VSV genes with the gene coding for the Ebola glycoprotein was safe in the brain, and also selectively targeted brain tumors, but was substantially less effective in destroying brain tumors and prolonging survival of tumor-bearing mice. A tropism for multiple cancer types combined with an exquisite tumor-specificity opens a new door to widespread application of VSV-LASV-GPC as a safe and efficacious oncolytic chimeric virus within the brain.

    IMPORTANCE. Many viruses have been tested for their ability to target and kill cancer cells. Vesicular stomatitis virus (VSV) has shown substantial promise, but a key problem is that if it enters the brain, it can generate adverse neurologic consequences including death. We tested a series of chimeric viruses containing genes coding for VSV, together with a gene coding for the glycoprotein from other viruses including Ebola, Lassa, LCMV, rabies, and Marburg which was substituted for the VSV glycoprotein gene. Ebola and Lassa chimeric viruses were safe in the brain, and targeted brain tumors. Lassa-VSV was particularly effective, showed no adverse side effects even when injected directly into the brain, and targeted and destroyed two different types of deadly brain cancer including glioblastoma and melanoma.

  • Structural basis for 2' -5' -oligoadenylate binding and enzyme activity of a viral RNase L antagonist [PublishAheadOfPrint]

  • Synthesis of 2rrsquo; -5rrsquo; -oligoadenylates (2-5A) by oligoadenylate synthetase (OAS) is an important innate cellular response that limits viral replication by activating the latent cellular ribonuclease, RNase L, to degrade single-stranded RNA. Some rotaviruses and coronaviruses antagonize the OAS/RNase L pathway through the activity of an encoded 2H phosphoesterase domain that cleaves 2-5A. These viral 2H phosphoesterases are phylogenetically related to the cellular A-kinase anchoring protein 7 (AKAP7) and share a core structure and an active site that contains two well-defined H(S/T) motifs, but their mechanism of substrate binding is unknown. Here we report the structures of a viral 2H phosphoesterase, the C-terminal domain (CTD) of the group A rotavirus VP3 protein, both alone and in complex with 2-5A. The domain forms a compact fold, with a concave bbeta;-sheet that contains the catalytic cleft, but it lacks two aalpha;-helical regions and two bbeta;-strands observed in AKAP7 and other 2H phosphoesterases. The co-crystal structure shows significant conformational changes in the "R-loop" upon ligand binding. Bioinformatics and biochemical analyses reveal that conserved residues and residues required for catalytic activity and substrate binding comprise the catalytic motifs and a region on one side of the binding cleft. We demonstrate that the VP3 CTD of group B rotavirus, but not that of group G, cleaves 2-5A. These findings suggest that the VP3 CTD is a streamlined version of a 2H phosphoesterase with a ligand-binding mechanism that is shared among 2H phosphodiesterases that cleave 2-5A.

    Importance The C-terminal domain (CTD) of rotavirus VP3 is a 2H phosphoesterase that cleaves 2rrsquo; -5rrsquo; -oligoadenylates (2-5A), potent activators of an important innate cellular antiviral pathway. 2H phosphoesterase superfamily proteins contain two conserved catalytic motifs and a proposed core structure. Here, we present structures of a viral 2H phosphoesterase, the rotavirus VP3 CTD, alone and in complex with its substrate, 2-5A. The domain lacks two aalpha;-helical regions and bbeta;-strands present in other 2H phosphoesterases. A loop of the protein undergoes significant structural changes upon substrate binding. Together with our bioinformatics and biochemical findings, the crystal structures suggest that the RVA VP3 CTD domain is a streamlined version of a cellular enzyme that shares a ligand-binding mechanism with other 2H phosphodiesterases that cleave 2-5A, but differs from those of 2H phosphodiesterases that cleave other substrates. These findings may aid in the future design of antivirals targeting viral phosphodiesterases with cleavage specificity for 2-5A.

  • Dynasore disrupts trafficking of HSV proteins [PublishAheadOfPrint]

  • Dynasore, a small molecule inhibitor of the GTPase activity of dynamin, inhibits entry of several viruses including herpes simplex virus (HSV), but its impact on other steps in the viral life cycle has not been delineated. The current study was designed to test the hypothesis that dynamin is required for viral protein trafficking and thus has pleiotropic inhibitory effects on HSV infection. Dynasore inhibited HSV-1 and HSV-2 Infection of human epithelial and neuronal cells including primary genital tract cells and human fetal neurons and astrocytes. Similar results were obtained when cells were transfected with a plasmid expressing dominant negative dynamin. Kinetic studies demonstrated that dynasore reduced the number of viral capsids reaching the nuclear pore if added at the time of viral entry and, when added as late as 8 hours post-entry, blocked the transport of newly synthesized viral proteins from the nucleus to the cytosol. Proximity ligation assays demonstrated that treatment with dynasore prevented colocalization of VP5 and dynamin. This resulted in a reduction in the number of viral capsids isolated from sucrose gradients. A paucity of capsids was observed by electron microscopy in dynasore compared to control treated cells. There was also a reduction in infectious progeny released into culture supernatants and a reduction in cell-to-cell spread. Together, these findings suggest that targeting dynamin-HSV interactions may provide new strategy for HSV treatment and prevention.

    IMPORTANCE HSV infections remain a global health problem associated with significant morbidity, particularly in neonates and immunocompromised hosts, highlighting the need for novel approaches to treatment and prevention. The current studies indicate that dynamin plays a role in multiple steps in the viral life cycle and provides a new target for antiviral therapy. Dynasore, a small molecule inhibitor of dynamin, has pleiotropic effects on HSV-1 and HSV-2 infection and impedes viral entry, trafficking of viral proteins and capsid formation.

  • Stable Phenotypic Changes of the Host T Cells are Essential to the Long-term Stability of Latent HIV-1 Infection [PublishAheadOfPrint]

  • The extreme stability of the latent HIV-1 reservoir in the CD4+ memory T cell population prevents viral eradication with current antiretroviral therapy. It has been demonstrated that homeostatic T cell proliferation and clonal expansion of latently infected T cells due to viral integration into specific genes contribute to this extraordinary reservoir stability. Nevertheless, given the constant exposure of the memory T cell population to specific antigen or by-stander activation, this reservoir stability seems remarkable, unless it is assumed that latent HIV-1 resides exclusively in memory T cells that recognize rare antigens. Another explanation for the stability of the reservoir could be that the latent HIV-1 reservoir is associated with an unresponsive T cell phenotype. We here demonstrate that host-cells of latent HIV-1 infection events were functionally altered in ways that are consistent with the idea of an anergic, unresponsive T cell phenotype. Manipulations that induced or mimicked an anergic T cell state promoted latent HIV-1 infection. Kinome analysis data reflected this altered host-cell phenotype at a system-wide level and revealed how the observed stable kinase activity changes network to stabilize latent HIV-1 infection. Protein-protein interaction networks generated from kinome data could further be used to guide targeted genetic or pharmacological manipulations that altered the stability of latent HIV-1 infection. In summary, our data demonstrate that stable changes to the signal transduction and transcription factor network of latently HIV-1 infected host-cells are essential to the ability of HIV-1 to establish and maintain the latent HIV-1 infection status.

    IMPORTANCE The extreme stability of the latent HIV-1 reservoir allows the infection to persist for the lifetime of a patient, despite completely suppressive anti-retroviral therapy. This extreme reservoir stability is somewhat surprising, as the latently HIV-1 infected CD4+ memory T cells that form the structural basis of the viral reservoir should be exposed to cognate antigen over time. Antigen exposure would trigger a recall response and should deplete the reservoir, likely over a relatively short period of time. Our data demonstrate that stable and system-wide phenotypic changes to the host cells of latent HIV-1 infection events are a prerequisite for the establishment and maintenance of latent HIV-1 infection events. The observed changes are consistent with an unresponsive, anergy-like T cell phenotype of the latently HIV-1 infected host cells. An anergy-like, unresponsive state of the host cells of latent HIV-1 infection events would explain the HIV-1 reservoir stability in the face of continuous antigen exposure.

  • The vesicle-forming 6K2 protein of turnip mosaic virus interacts with the COPII coatomer Sec24a for viral systemic infection [PublishAheadOfPrint]

  • Positive sense RNA viruses remodel host cell endomembranes to generate quasi-organelles known as viral factories to coordinate diverse viral processes such as genome translation and replication. It is also becoming clear that enclosing viral RNA complexes within membranous structures is important for virus cell-to-cell spread throughout the host. In plant cells infected by turnip mosaic virus (TuMV), a member of the family Potyviridae, peripheral motile endoplasmic reticulum (ER)-derived viral vesicles are produced that carry the viral RNA to plasmodesmata for delivery into adjacent non-infected cells. The viral protein 6K2 is responsible for the formation of these vesicles but how 6K2 is involved in their biogenesis is unknown. We show here that 6K2 is associated with cellular membranes. Deletion mapping and site-directed mutagenesis experiments defined a soluble N-terminal 12 amino acid stretch, in particular a potyviral highly conserved tryptophan residue and two lysine residues that were important for vesicle formation. When the tryptophan residue was changed into an alanine in the viral polyprotein, virus replication still took place, albeit at a reduced level, but cell-to-cell movement of the virus was abolished. Yeast two-hybrid and co-immunoprecipitation experiments showed that 6K2 interacted with Sec24a, a COPII coatomer component. Appropriately, TuMV systemic movement was delayed in an Arabidopsis thaliana mutant line defective in Sec24a. Intercellular movement of TuMV replication vesicles thus requires ER export of 6K2, which is mediated by the interaction of the N-terminal domain of the viral protein with Sec24a.

    Importance Many plant viruses remodel the endoplasmic reticulum (ER) for generating vesicles that are associated with the virus replication complex. The viral protein 6K2 of Turnip mosaic virus (TuMV) is known to induce ER-derived vesicles that contain vRNA as well as viral and host proteins required for vRNA synthesis. These vesicles not only sustain vRNA synthesis, they are also involved in the intercellular trafficking of vRNA. In this investigation, we found that the N-terminal soluble domain of 6K2 is required for ER export of the protein and for the formation of vesicles. ER export is not absolutely required for vRNA replication, but is necessary for virus cell-to-cell movement. Furthermore, we found that 6K2 physically interacts with the COPII coatomer Sec24a and that an Arabidopsis thaliana mutant line with a defective Sec24a shows a delay in the systemic infection by TuMV.

  • Novel CD4-Based Bispecific Chimeric Antigen Receptor Designed for Enhanced Anti-HIV Potency and Absence of HIV Entry Receptor Activity [PublishAheadOfPrint]

  • Adoptive transfer of CD8 T cells genetically engineered to express chimeric antigen receptors (CARs) represents a potential approach toward an HIV "functional cure" whereby durable virologic suppression is sustained after discontinuation of antiretroviral therapy. We describe a novel bispecific CAR in which a CD4 segment is linked to a single chain variable fragment of the 17b human monoclonal antibody recognizing a highly conserved CD4-induced epitope on gp120 involved in coreceptor binding. We compared a standard CD4 CAR with CD4-17b CARs wherein the polypeptide linker between the CD4 and 17b moieties is sufficiently long (CD4-35-17b CAR) versus too short (CD4-10-17b) to permit simultaneous binding of both moieties to a single gp120 subunit. When transduced into PBMC or T cells thereof, all three CD4-based CARs displayed specific functional activities against HIV-1 Env-expressing target cells including stimulation of interferon- release, specific target cell killing, and suppression of HIV-1 pseudovirus production. In PBMC spreading infection assays with genetically diverse HIV-1 primary isolates, the CD4-10-17b CAR displayed enhanced potency compared to the CD4 CAR whereas the CD4-35-17b CAR displayed diminished potency. Importantly, both CD4-17b CARs were devoid of a major undesired activity observed with the CD4 CAR, namely rendering the transduced CD8+ T cells susceptible to HIV-1 infection. Likely mechanisms for the superior potency of the CD4-10-17b CAR over the CD4-35-17b CAR include the greater potential of the former to engage in serial antigen binding required for efficient T cell activation, and the ability of two CD4-10-17b molecules to simultaneously bind a single gp120 subunit.

    Importance HIV research has been energized by prospects for a cure for HIV infection, or at least a "functional cure" whereby antiretroviral therapy can be discontinued without virus rebound. This study describes a novel CD4-based "chimeric antigen receptor" (CAR) which, when genetically engineered into T cells, gives them the capability to selectively respond to and kill HIV-infected cells. This CAR displays enhanced features compared to previously described CD4-based CARs, namely increased potency and avoidance of the undesired rendering the genetically modified CD8 T cells susceptible to HIV infection. When adoptively transferred back to the individual, the genetically modified T cells will hopefully provide durable killing of infected cells and sustained virus suppression without continued antiretroviral therapy, i.e. a functional cure.

  • Mutation of Putative N-glycosylation Sites on Dengue NS4B Decreases RNA Replication [PublishAheadOfPrint]

  • Dengue virus (DENV) nonstructural protein 4B (NS4B) is an endoplasmic reticulum (ER) membrane-associated protein, and mutagenesis studies have revealed its significance in viral genome replication. In this work, we demonstrated that NS4B is an N-glycosylated protein in virus-infected cells as well as in recombinant protein expression. NS4B is N-glycosylated at residues 58 and 62 and exists in two forms: glycosylated and unglycosylated. We manipulated full-length infectious RNA clones and sub-genomic replicons to generate N58Q, N62Q and N58QN62Q mutants. Each of the single mutants had distinct effects, but the N58QN62Q mutation resulted in dramatic reduction of viral production efficiency without affecting secretion or infectivity of the virion in mammalian and mosquito C6/36 hosts. Real time-qPCR, sub-genomic replicon and trans-complementation assays indicated that N58QN62Q mutation affected RNA replication possibly by the loss of glycans. In addition, four intragenic mutations (S59Y, S59F, T66A and A137T) were obtained from mammalian and/or mosquito C6/36 cell culture systems. All of these second site mutations compensated for the replication defect of N58QN62Q mutant without creating novel glycosylation sites. In vivo protein stability analyses revealed that N58QN62Q mutation alone or plus a compensatory mutation did not affect the stability of NS4B. Overall, our findings indicated that mutation of putative N-glycosylation sites affected the biological function of NS4B in the viral replication complex.

    Importance of findings This is the first report to identify and reveal the biological significance of DENV NS4B post-translation N-glycosylation to the virus life cycle. The study demonstrated that NS4B is N-glycosylated in virus-infected cells and in recombinant protein expression. NS4B is modified by glycans at Asn-58 and Asn-62. Functional characterization implied that DENV NS4B utilizes the glycosylation machinery in both mammalian and mosquito hosts. Four intragenic mutations were found to compensate for replication and subsequent viral production deficiencies without creating novel N-glycosylation sites or modulating stabilities of protein, suggesting that glycans may be involved in maintaining the NS4B protein conformation. NS4B glycans may be necessary elements of the viral life cycle, but compensatory mutations can circumvent their requirement. This novel finding may have broader implications in flaviviral biology as the most potential glycan at Asn-62 of NS4B is conserved in DENV serotypes and in some related flaviviruses.

  • Timing Is Everything: Coordinated Control of Host Shutoff by Influenza A Virus NS1 and PA-X Proteins [PublishAheadOfPrint]

  • Like all viruses, influenza viruses (IAVs) use host translation machinery to decode viral mRNAs. IAVs ensure efficient translation of viral mRNAs through host shutoff, a process whereby viral proteins limit the accumulation of host proteins through subversion of their biogenesis. Despite its small genome, the virus deploys multiple host shutoff mechanisms at different stages of infection, thereby ensuring successful replication while limiting the communication of host antiviral responses. In this Gem, we review recent data on IAV host shutoff proteins, frame the outstanding questions in the field, and propose a temporally coordinated model for IAV host shutoff.

  • EFTUD2 is a novel innate immune regulator restricting hepatitis C virus infection through the RIG-I/MDA5 pathway [PublishAheadOfPrint]

  • The Elongation factor Tu GTP binding domain containing 2 (EFTUD2) was identified as an anti-HCV host factor in our recent genome-wide siRNA screen. In this study, we sought to further determine EFTUD2's role in HCV infection and investigate the interaction between EFTUD2 and other regulators involved in HCV innate immune (RIG-I, MDA5, TBK1, and IRF3) and JAK/STAT1 pathways. We found that HCV infection decreased the expression of EFTUD2 and the viral RNA sensors RIG-I and MDA5 in HCV-infected Huh7 and Huh7.5.1 cells and in liver tissue from in HCV-infected patients, suggesting that HCV infection downregulated EFTUD2 expression to circumvent the innate immune response. EFTUD2 inhibited HCV infection by inducing expression of the interferon-stimulated genes (ISGs) in Huh7 cells. However, its impact on HCV infection was absent in both RIG-I knockdown Huh7 cells and RIG-I defective Huh7.5.1 cells, indicating that the antiviral effect of EFTUD2 is dependent on RIG-I. Furthermore, EFTUD2 upregulated the expression of the RIG-I like receptors (RLR) RIG-I and MDA5 to enhance innate immune response by gene splicing. Functional experiments revealed that EFTUD2-induced expression of ISGs was mediated through interaction of the EFTUD2 downstream regulators RIG-I, MDA5, TBK1 and IRF3. Interestingly, the EFTUD2-induced antiviral effect was independent of the classical IFN-induced JAK-STAT pathway. Our data demonstrate that EFTUD2 restricts HCV infection mainly through a RIG-I/MDA5-mediated, JAK-STAT-independent pathway, thereby revealing the participation of EFTUD2 as a novel innate immune regulator and suggesting a potentially targetable antiviral pathway.

    Importance Innate immunity is the first line defense against HCV and determines the outcome of HCV infection. Based on a recent high throughput whole-genome siRNA library screen revealing a network of host factors mediating antiviral effects against HCV, we identified EFTUD2 as a novel innate immune regulator against HCV in the infectious HCV cell culture model and confirmed that its expression in HCV-infected liver tissue is inversely related to HCV infection. Furthermore, we elucidated that EFTUD2 exerts its antiviral activity mainly through governing its downstream regulators RIG-I and MDA5 by gene splicing to activate IRF3 and induce classical ISGs expression independent of JAT-STAT signaling pathway. This study broadens our understanding of the HCV innate immune response and provides a possible new antiviral strategy targeting this novel regulator of the innate response.

  • Unravelling the role of the F55 regulator in the transition from lysogeny to UV induction of Sulfolobus spindle-shaped virus 1 [PublishAheadOfPrint]

  • Sulfolobus spindle-shaped virus 1 represents a model for studying virus-host interaction in harsh environments and it is so far the only member of the Fuselloviridae family that shows a UV-inducible life cycle. Although the virus has been extensively studied, mechanisms underpinning the maintenance of lysogeny as well as those regulating the UV induction have received little attention. Recently, a novel SSV1 transcription factor, F55, was identified. This factor was able to bind in vitro to several sequences derived from the early and UV-inducible promoters of the SSV1 genome. The location of these binding sites together with the differential affinity of F55 towards these sequences, led to the hypothesis that this protein might be involved in the maintenance of the SSV1 lysogeny. Herein, it is reported an in vivo survey of the molecular events occurring at the UV-inducible region of the SSV1 genome, with a focus on the binding profile of F55 before and after the UV irradiation. The binding of F55 to the target promoters correlates with transcription repression, whereas its dissociation is paralleled by transcription activation. Therefore, we propose that F55 acts as molecular switch for the transcriptional regulation of the early viral genes.

    Importance Functional genomic studies of SSV1 proteins have been hindered by the lack of similarity with other characterized proteins. As a result, only few insights into their in vivo roles have been gained throughout the last three decades. Herein, we report the first in vivo investigation of a SSV1 transcription regulator, F55, that exerts a key role in the transition from the lysogenic to the induced state of SSV1. We show that F55 regulates the expression of the UV-inducible as well as of the early genes. Moreover, the differential affinity of this transcription factor towards these targets allows a fine-tuned and temporal coordinated regulation of transcription of viral genes.

  • RNA polymerase slippage as a mechanism for the production of frameshift gene products in plant viruses of the Potyviridae family [PublishAheadOfPrint]

  • Modifications of RNA sequences by nucleotide insertions, deletions or substitutions can result in expression of multiple proteins in overlapping open reading frames (ORF)....

  • Human cytomegalovirus UL135 and UL136 genes are required for post-entry tropism in endothelial cells [PublishAheadOfPrint]

  • Endothelial cells (ECs) are a critical target of viruses and infection of the endothelium represents a defining point in viral pathogenesis. Human cytomegalovirus (HCMV), the prototypical bbeta;-herpesvirus, encodes proteins specialized for entry into and delivery of the genome to the nucleus in ECs. Virus strains competent to enter ECs replicate with differing efficiencies suggesting that virus encodes genes for post-entry tropism in ECs. We previously reported a specific requirement for the UL133/8 locus of HCMV for replication in ECs. The UL133/8 locus encodes four genes, UL133, UL135, UL136, and UL138. In this study, we find that while UL133 and UL138 are dispensable for replication in ECs, both UL135 and UL136 are important. These genes are not required for virus entry or the expression of viral genes. The phenotypes associated with disruption of either gene reflect phenotypes observed for the UL133/8NULL virus lacking the entire UL133/8 locus, but are largely distinct from one another. Viruses lacking UL135 fail to properly envelop capsids in the cytoplasm, produce fewer dense bodies (DB), and are unable to incorporate viral products into multivesicular bodies (MVB) relative to wild-type (WT) infection. Viruses lacking UL136 also fail to properly envelop virions and produce enlarged dense bodies relative to WT infection. Our results indicate roles for the UL135 and UL136 proteins in commandeering host membrane trafficking pathways for virus maturation. UL135 and UL136 represent the first HCMV genes crucial for early to late stage tropism in ECs.

    IMPORTANCE Human cytomegalovirus (HCMV) persists in the majority of the world's population. While typically asymptomatic in healthy hosts, HCMV can cause significant morbidity and mortality in immune compromised or naiiuml;ve individuals, particularly transplant patients and in congenital infection, respectively. Life-long persistence of the virus may also contribute to age related pathologies, such as vascular disease. One aspect of HCMV infection contributing to complex and varied pathogenesis is the diverse array of cell types this virus infects in the host. The vascular endothelium is a particularly important target of infection, contributing to viral dissemination, and likely leading to CMV complications following transplantation. In this work, we identify two viral gene products required for post-entry tropism in endothelial cells. Identifying tropism factors required for replication in critical cell targets of infection is important to understanding the mechanisms restricting virus tropism and to developing strategies to restrict virus replication.

  • Glycan microheterogeneity at the PGT135 antibody recognition site on HIV-1 gp120 reveals a molecular mechanism for neutralization resistance [PublishAheadOfPrint]

  • Broadly neutralizing antibodies have been isolated which bind the glycan shield of the HIV-1 envelope spike. One such antibody, PGT135, contacts the intrinsic mannose patch of gp120 at the Asn332, Asn392 and Asn386 glycosylation sites. Here, site-specific glycosylation analysis of recombinant gp120 revealed glycan microheterogeneity sufficient to explain the existence of a minor population of virions resistant to PGT135 neutralization. Target microheterogeneity and antibody glycan specificity are therefore important parameters in HIV-1 vaccine design.

  • Herpes simplex virus-1 US3 phosphorylates cellular KIF3A to down-regulate CD1d expression [PublishAheadOfPrint]

  • Herpes simplex virus-1 (HSV-1) is one of the most prevalent herpesviral infections in humans and is the leading etiological agent for viral encephalitis and eye infections. Our understanding of how HSV-1 interacts with the host at the cellular level and organismal level is still limited. We and others previously reported that upon infection, HSV-1 rapidly and efficiently down-regulates CD1d cell surface expression and suppresses the function of NKT cells, a group of innate T cells with critical immune-regulatory function. The viral protein kinase US3 plays a major role in this immune evasion mechanism and its kinase activity is required for this function. In this report, we investigated the cellular substrate(s) phosphorylated by US3 and how it mediates the US3 suppression of CD1d recycling. We identified a type II kinesin motor protein KIF3A as a critical kinesin factor for the cell surface expression of CD1d. Interestingly, KIF3A is phosphorylated by US3 both in vitro and in infected cells. Mass spectrometry analysis of purified KIF3A showed that it is predominantly phosphorylated at serine 687 by US3. Ablation of this phosphorylation abolished US3-mediated down-regulation of CD1d expression, suggesting that phosphorylation of KIF3A is the primary mechanism for HSV-1 suppression of CD1d expression by US3 protein. Understanding of the precise mechanism in viral modulation of CD1d expression will help to develop more efficient vaccines in the future to boost host NKT cell-mediated immune responses against herpesviruses.

    IMPORTANCE Herpes simplex virus-1 (HSV-1) is among the most common human pathogens. Little is know regarding the exact mechanism how this virus evades human immune system, particularly the innate immune system. We previously reported that HSV-1 employs its protein kinase US3 to modulate the expression of the key antigen-presenting molecule, CD1d, to evade the antiviral function of NKT cells. Here we identified the key cellular motor protein, KIF3A, as a cellular substrate phosphorylated by US3 and this phosphorylation event mediates the US3-induced immune evasion.

  • Antibody Escape Kinetics of EIAV Infection of Horses [PublishAheadOfPrint]

  • Lentivirus escape from neutralizing antibodies (NAbs) is not well-understood. In this work we quantify antibody escape of a lentivirus, using antibody escape data from horses infected with equine infectious anemia virus. We calculate antibody blocking rates of wild-type virus, fitness costs of mutant virus, and growth rates of both viruses. These quantitative kinetic estimates of antibody escape are important for understanding lentiviral control by antibody neutralization and in developing NAb-eliciting vaccine strategies.

  • Faustovirus, an asfarvirus-related new lineage of giant viruses infecting amoebae [PublishAheadOfPrint]

  • Giant viruses are protist-associated viruses belonging to the proposed order Megavirales; almost all have been isolated on Acanthamoeba sp. Their isolation in humans suggests that they are part of the human virome. Using a high-throughput strategy to isolate new giant viruses on their original protozoa hosts, we obtained eight isolates of a new giant viral lineage on Vermamoeba vermiformis, the most common free-living protist found in human environments. This new lineage was proposed to be a Faustovirus. The prototype member Faustovirus E12 forms icosahedral virions of 200 nm in size that are devoid of fibrils and that encapsidate a 466 kilobase pair-long genome encoding 451 predicted proteins. Of these, 164 are found in the virion. The phylogenetic analysis of core viral genes shows that Faustovirus is distantly related to the mammalian pathogen African swine fever virus, but encodes 3 times more mosaic gene complements. About two-thirds of these genes do not show significant similarity to any known proteins. These findings show that expanding the panel of protists to discover new giant viruses is a fruitful strategy.

    Importance. By using for the first time Vermamoeba, a protist living in humans and their environment, we isolated eight strains of a new giant virus we named Faustovirus. The genomes of these strains were sequenced, showing that the faustoviruses are related but different from the vertebrate pathogen African swine fever virus (ASFV), which belongs to the family Asfarviridae. Moreover, the Faustovirus gene repertoire is 3 times larger than that of ASFV and comprises two-thirds ORFans.

  • In vitro and in vivo characterizations of the Pichinde viral NP exoribonuclease function [PublishAheadOfPrint]

  • Arenaviruses cause severe hemorrhagic fever diseases in humans with limited preventative and therapeutic measures. Previous structural and functional analyses of arenavirus nucleoproteins (NP) have revealed a conserved DEDDH exoribonuclease (RNase) domain that is important for type I interferon (IFN) suppression, but the biological roles of the NP RNase in viral replication and host immune suppression have not been well characterized. Infection of guinea pigs with Pichinde virus (PICV), a prototype arenavirus, can serve as a surrogate small animal model for arenavirus hemorrhagic fevers. In this report, we show that mutations of each of the five RNase catalytic residues of PICV NP diminish IFN suppression activity and slightly reduce viral RNA replication activity. Recombinant PICV with RNase catalytic mutations can induce high levels of IFNs and barely grow in the IFN-competent A549 cells, in sharp contrast to the wild-type (WT) virus. While in the IFN-deficient Vero cells, both WT and mutant viruses can replicate at relatively high levels. Upon infection of guinea pigs, the RNase mutant viruses stimulate strong IFN responses, fail to replicate productively, and can become WT revertants. Serial passages of the RNase mutants in vitro can also generate WT revertants. Thus, the NP RNase function is essential for innate immune suppression that allows the establishment of a productive early viral infection and may be partly involved in the process of viral RNA replication.

    Importance Arenaviruses such as Lassa, Lujo, and Machupo can cause severe and deadly hemorrhagic fever diseases in humans with limited preventative and treatment options. Development of broad-spectrum antiviral drugs depends on a better mechanistic understanding of the conserved arenavirus proteins in viral infection. The nucleoprotein (NP) of all arenaviruses encodes a unique exoribonuclease (RNase) domain that has been shown to be critical for the suppression of type I interferons. However, the functional roles of the NP RNase in arenavirus replication and host immune suppression have not been systematically characterized. Using a prototype arenavirus, Pichinde virus (PICV), we have characterized the recombinant RNase-defective mutants in viral growth and innate immune suppression in both cell culture and guinea pig models. Our study suggests that the NP RNase plays an essential role in the suppression of host innate immunity and possibly in viral RNA replication and that it can serve as a novel target for developing antiviral drugs against arenavirus pathogens.

  • Transformation with Oncogenic Ras and the Simian Virus 40 T antigens induces caspase-dependent sensitivity to fatty acid biosynthetic inhibition [PublishAheadOfPrint]

  • Oncogenesis is frequently accompanied by the activation of specific metabolic pathways. One such pathway is fatty acid biosynthesis, whose induction is observed upon transformation of a wide variety of cell types. Here we explored how defined oncogenic alleles, specifically the simian virus 40 (SV40) T antigens, and oncogenic Ras12V impact fatty acid metabolism. Our results indicate that SV40/Ras12V-mediated-transformation of fibroblasts induces fatty acid biosynthesis in the absence of significant changes to the concentration of fatty acid biosynthetic enzymes. This oncogene-induced activation of fatty acid biosynthesis was found to be mTOR dependent, as it was attenuated by rapamycin treatment. Furthermore, SV40/Ras12V-mediated transformation induced sensitivity to treatment with fatty acid biosynthetic inhibitors. Pharmaceutical inhibition of acetyl-CoA carboxylase (ACC), a key fatty acid biosynthetic enzyme, induced caspase-dependent cell death in oncogene-transduced cells. In contrast, isogenic non-transformed cells were resistant to fatty acid biosynthetic inhibition. This oncogene-induced sensitivity to fatty acid biosynthetic inhibition was independent of growth rate, and could be attenuated by media supplementation with unsaturated fatty acids. Both the activation of fatty acid biosynthesis, and the sensitivity to fatty acid biosynthetic inhibition could be conveyed to non-transformed breast epithelial cells through transduction with oncogenic Ras12V. Similar to what was observed in the transformed fibroblasts, the Ras12V-induced sensitivity to fatty acid biosynthetic inhibition was independent of proliferative status, and could be attenuated by media supplementation with unsaturated fatty acids. Combined, our results indicate that specific oncogenic alleles can directly confer sensitivity to inhibitors of fatty acid biosynthesis.

    Importance Viral oncoproteins and cellular mutations drive the transformation of normal cells to the cancerous state. These oncogenic alterations induce metabolic changes and dependencies that can be targeted to kill cancerous cells. Here we find that the cellular transformation resulting from combined expression of the SV40 early region with an oncogenic Ras allele, is sufficient to induce cellular susceptibility to fatty acid biosynthetic inhibition. Inhibition of fatty acid biosynthesis in these cells resulted in programmed cell death which could be rescued by media supplementation with non-saturated fatty acids. Similar results were observed with expression of oncogenic Ras in non-transformed breast epithelial cells. Combined, our results suggest that specific oncogenic alleles induce metabolic dependencies that can be exploited to selectively kill cancerous cells.

  • Regulation of hepatitis C virus genome replication by Xrn1, and microRNA-122 binding to individual sites in the 5' UTR [PublishAheadOfPrint]

  • miR-122 is a liver-specific miRNA that binds to two sites (S1 and S2) on the 5rrsquo; UTR of the hepatitis C virus (HCV) genome and promotes the viral life cycle. It positively affects viral RNA stability, translation, and replication, but the mechanism is not well understood. To unravel the roles of miR-122 binding at each site alone or in combination, we employed miR-122 binding-site mutant viral RNAs, Hep3B cells (which lack detectable miR-122), and complementation with wild-type miR-122, a miR-122 with the matching mutation, or both. We found that miR-122 binding at either site alone increased replication equally, while binding at both sites had a co-operative effect. Xrn1 depletion rescued miR-122-unbound full-length RNA replication to detectable levels, but not to miR-122-bound levels, confirming that miR-122 protects HCV RNA from Xrn1, a cytoplasmic 5rrsquo; to 3rrsquo; exoribonuclease, but also has additional functions. In cells depleted of Xrn1, replication levels of S1-bound HCV RNA were slightly higher than S2-bound, suggesting that both sites contribute, but their contributions may be unequal when the need for protection from Xrn1 is reduced. miR-122 binding at S1 or S2 also increased translation equally, but the effect was abolished by Xrn1 knockdown, suggesting that the influence of miR-122 on HCV translation reflects protection from Xrn1 degradation. Our results show that occupation of each miR-122 binding site contributes equally and co-operatively to HCV replication, but suggest somewhat unequal contributions of each site to Xrn1 protection and additional functions of miR-122.

    Importance The functions of miR-122 in promotion of the HCV life cycle are not fully understood. Here we show that binding of miR-122 to each of the two binding sites in the HCV 5rrsquo; UTR contributes equally to HCV replication, and that binding to both sites can function co-operatively. This suggests that active Ago2/miR-122 complexes assemble at each site and can co-operatively promote the association and/or function of adjacent complexes, similar to what has been proposed for translation suppression by adjacent miRNA binding sites. We also confirm a role for miR-122 in protection from Xrn1 and provide evidence that miR-122 has additional functions in the HCV life cycle unrelated to Xrn1. Finally, we show that each binding site may contribute unequally to Xrn1 protection and other miR-122 functions.

  • Zinc binding activity of human metapneumovirus M2-1 protein is indispensable for viral replication and pathogenesis in vivo [PublishAheadOfPrint]

  • Human metapneumovirus (hMPV) is a member of the subfamily Pneumovirinae in the Paramyxoviridae family that causes respiratory tract infections in humans. Unlike members of the Paramyxovirinae subfamily, the polymerase complex of pneumoviruses requires an additional cofactor, the M2-1 protein, which functions as a transcriptional anti-termination factor. The M2-1 protein was found to incorporate zinc ions, although the specific role(s) of the zinc binding activity in viral replication and pathogenesis remains unknown. In this study, we found that the third cysteine (C21) and the last histidine (H25) in the zinc binding motif (CCCH) of hMPV M2-1 were essential for zinc binding activity, whereas the first two cysteines (C7 and C15) play only minor or redundant roles in zinc binding. In addition, the zinc binding motif is essential for oligomerization of M2-1. Subsequently, recombinant hMPVs carrying mutations in the zinc binding motif were recovered. Interestingly, rhMPV-C21S and H25L, which lacked zinc binding activity, had delayed replication in cell culture and were highly attenuated in cotton rats. In contrast, rhMPV-C7S and C15L, which retained 60% of zinc binding activity, replicated as efficiently as rhMPV in cotton rats. Importantly, rhMPVs lacked zinc binding activity triggered high levels of neutralizing antibody and provided a complete protection against challenge with rhMPV. Taken together, these results demonstrate that zinc binding activity is indispensable for viral replication and pathogenesis in vivo. These results also suggest that inhibition of zinc binding activity may serve as a novel approach to rationally attenuate hMPV and perhaps other pneumoviruses for vaccine purposes.

    Importance The pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the leading causes of acute respiratory tract infection in infants and children. Despite major efforts, there is no antiviral or vaccine to combat these diseases. All known pneumoviruses encode a zinc binding protein, M2-1, which is a transcriptional anti-termination factor. In this work, we found that the zinc binding activity of M2-1 is essential for virus replication and pathogenesis in vivo. Recombinant hMPVs lacked zinc binding activity were not only defective in replication in the upper and lower respiratory tract, but also triggered a strong protective immunity in cotton rats. Thus, inhibition of M2-1 zinc binding activity can lead to the development of novel, live attenuated vaccines, as well as antiviral drugs for pneumoviruses.

  • Interaction between Shadoo and PrP affects the PrP folding pathway. [PublishAheadOfPrint]

  • Prion diseases are characterised by conformational changes of a cellular prion protein (PrPC) into a bbeta;-sheet-enriched and aggregated conformer (PrPSc). Shadoo (Sho), a member of the prion protein family, is expressed in the central nervous system (CNS) and is highly conserved among vertebrates. Based on histo-anatomical co-localisation and sequence similarities, it is suspected that Sho and PrP may be functionally related. The down-regulation of Sho expression during prion pathology and the direct interaction between Sho and PrP, as revealed by the two-hybrid analysis, suggest a relationship between Sho and prion replication. Using biochemical and biophysical approaches, we demonstrate that Sho forms a 1:1 complex with full-length PrP, with a Kd in the micromolar range, and this interaction consequently modifies the PrP folding pathway. Using a truncated PrP that mimics the C1 fragment, an allosteric binding behaviour with a Hill number of 4 was observed, suggesting that at least a tetramerisation state occurs. Cell-based prion titration assay performed with different concentrations of Sho revealed an increase in the PrPSc conversion rate in the presence of Sho. Collectively, our observations suggest that Sho can affect the prion replication process by 1) acting as a holdase and 2) interfering with the dominant-negative inhibitor effect of the C1 fragment.

    IMPORTANCE Since the inception of the prion theory, the search for a cofactor involved in the conversion process has been an active field of research. Although the PrP interactome presents a broad landscape, candidates corresponding to specific criteria for cofactors are currently missing. Here, we describe for the first time that Sho can affect PrP structural dynamics and therefore increase the prion conversion rate. A biochemical characterisation of Sho:PrP indicates that Sho acts as an ATP-independent holdase.

  • A single amino acid deletion in the matrix protein of porcine reproductive and respiratory syndrome virus confers resistance to a polyclonal swine antibody with broadly neutralizing activity. [PublishAheadOfPrint]

  • Assessment of virus neutralization (VN) activity in 176 pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV) identified one pig with broadly neutralizing activity. A Tyr-10 deletion in the matrix protein provided escape from broad neutralization without affecting homologous neutralizing activity. The role of the Tyr-10 deletion was confirmed through a Tyr-10 deleted infectious clone. The results demonstrate differences in the properties and specificities of VN responses elicited during PRRSV infection.

  • The Interaction between TIM-1 and NPC1 Is Important for the Cellular Entry of Ebola Virus [PublishAheadOfPrint]

  • Multiple host molecules are known to be involved in the cellular entry of filoviruses, including Ebola virus (EBOV); T-cell immunoglobulin and mucin domain 1 (TIM-1) and Niemann-Pick C1 (NPC1) have been identified as attachment and fusion receptors, respectively. However, the molecular mechanisms underlying the entry process have not been fully understood. We found that TIM-1 and NPC1 colocalized and interacted in the intracellular vesicles where EBOV glycoprotein (GP)-mediated membrane fusion occurred. Interestingly, a TIM-1-specific monoclonal antibody (MAb), M224/1, prevented GP-mediated membrane fusion and also interfered with the binding of TIM-1 to NPC1, suggesting that the interaction between TIM-1 and NPC1 is important for the filovirus membrane fusion. Moreover, MAb M224/1 efficiently inhibited the cellular entry of viruses from all known filovirus species. These data suggest a novel mechanism underlying filovirus membrane fusion and provide a potential cellular target for antiviral compounds that can be universally used against filovirus infections.

    Importance Filoviruses, including Ebola and Marburg viruses, cause rapidly fatal diseases in humans and nonhuman primates. There are currently no approved vaccines or therapeutics for filovirus diseases. In general, the cellular entry step of viruses is one of the key mechanisms to develop antiviral strategies. However, the molecular mechanisms underlying the entry process of filoviruses have not been fully understood. In this study, we demonstrate that TIM-1 and NPC1, which serve as attachment and fusion receptors for filovirus entry, interact in the intracellular vesicles where Ebola virus GP-mediated membrane fusion occurs and that this interaction is important for filovirus infection. We found that filovirus infection and GP-mediated membrane fusion in cultured cells were remarkably suppressed by treatment with a TIM-1-specific monoclonal antibody that interfered with the interaction between TIM-1 and NPC1. Our data provide new insights for the development of antiviral compounds that can be universally used against filovirus infections.

  • HIV Blocks Interferon Induction in Human Dendritic cells and Macrophages by Dysregulation of TBK1 [PublishAheadOfPrint]

  • Dendritic cells (DC) and macrophages are present in the tissues of the anogenital tracts where HIV-1 transmission occurs in almost all cases. These cells are both target cells for HIV-1 and represent the first opportunity for the virus to interfere with innate recognition. Previously we have shown that both cell types fail to produce type I interferons (IFN) in response to HIV-1 but that, unlike T cells, the virus does not block IFN induction by targeting IFN regulatory factor 3 (IRF3) for cellular degradation. Thus, either HIV-1 inhibits IFN induction by an alternate mechanism or, less likely, these cells fail to sense HIV-1. Here we show that HIV-1 (but not HSV-2 or Sendai virus) exposed DCs and macrophages fail to induce the expression of all known type I and III IFN genes. These cells do sense the virus and pattern recognition receptor (PRR) induced signalling pathways are triggered. The precise stage in the IFN inducing signalling pathway that HIV-1 targets to block IFN induction was identified; phosphorylation but not K63 polyubiquitination of TANK-binding kinase 1 (TBK1) was completely inhibited. Two HIV-1 accessory proteins, Vpr and Vif, were shown to bind to TBK1, and their individual deletion partly restored IFNbbeta; expression. Thus the inhibition of TBK1 autophosphorylation by binding of these proteins appears to be the principal mechanism by which HIV-1 blocks type I and III IFN induction in myeloid cells.

    Importance Dendritic cells (DCs) and macrophages are key HIV target cells. Therefore definition of how HIV impairs innate immune responses to initially establish infection is essential to design preventative interventions, especially by restoring initial interferon production. Here we demonstrate how HIV-1 blocks interferon induction by inhibiting the function of a key kinase in the interferon signalling pathway, TBK1, via two different viral accessory proteins. Other viral proteins have been shown to target the general effects of TBK1 but this precise targeting between ubiquitination and phosphorylation of TBK1 is novel.

  • Intercontinental Spread of Asian-origin H5N8 to North America through Beringia by Migratory Birds [PublishAheadOfPrint]

  • Phylogenetic network analysis and understanding of waterfowl migration patterns suggest the Eurasian H5N8 clade 2.3.4.4 avian influenza virus emerged in late 2013 in China, spread in early 2014 to South Korea and Japan, and reached Siberia and Beringia by summer 2014 via migratory birds. Three genetically distinct subgroups emerged and subsequently spread along different flyways during fall 2014 into Europe, North America, and East Asia, respectively. All three subgroups reappeared in Japan, a wintering site for waterfowl from Eurasia and parts of North America.

  • The SRSF1 RNA Recognition Motifs are strong inhibitors of HIV-1 replication [PublishAheadOfPrint]

  • Replication of the integrated HIV-1 genome is tightly regulated by a series of cellular factors. In previous work we showed that trans-activation of the HIV-1 promoter is regulated by the cellular splicing factor SRSF1. Here we report that SRSF1 can down-regulate replication of B, C and D subtype viruses by over 200 fold in a cell culture system. We show that viral transcription and splicing are inhibited by SRSF1 expression. Furthermore, SRSF1 deletion mutants containing the protein RNA binding domains (RBDs) but not the arginine serine rich activator domain can down-regulate viral replication by over 2,000 fold with minimal impact on cell viability and apoptosis. These data suggest a therapeutic potential for SRSF1 and its RNA binding domains.

    IMPORTANCE. Most drugs utilized to treat the HIV-1 infection are based on compounds that directly target proteins encoded by the virus. However, given the high viral mutation rate, the appearance of novel drug resistant viral strains is common. Thus, the need for novel therapeutics with diverse mechanisms of action. In this work we haven shown that the cellular protein SRSF1 is a strong inhibitor of viral replication. Furthermore, expression of the SRSF1 RNA binding domains alone can inhibit viral replication by over 2,000 fold in multiple viral strains without impacting cell viability. Given the strong antiviral properties of this protein RNA binding domains and the minimal effects observed on cell metabolism further studies are warranted to assert the therapeutic potential of peptides derived from these sequences.

  • Murine gammaherpesvirus 68 pathogenesis is independent of Caspase-1/11 in mice and impairs IL-1{beta} production upon extrinsic stimulation in culture. [PublishAheadOfPrint]

  • Gammaherpesviruses establish life-long infections that are associated with the development of cancer. These viruses subvert many aspects of the innate and adaptive immune response of the host. The inflammasome, a macromolecular protein complex that controls inflammatory responses to intracellular danger signals generated by pathogens, is both activated and subverted during human gammaherpesvirus infection in culture. The impact of the inflammasome response on gammaherpesvirus replication and latency in vivo is not known. Caspase-1 is the inflammasome effector protease that cleaves the proinflammatory cytokines IL-1bbeta; and IL-18. We infected mice deficient in caspase-1 with murine gammaherpesvirus 68 (MHV68) and observed no impact on acute replication in the lung or latency and reactivation from latency in the spleen. This led us to examine the effect of viral infection on inflammasome responses in bone marrow-derived macrophages. We determined that infection of macrophages with MHV68 led to a robust interferon response, but failed to activate caspase-1 or induce secretion of IL-1bbeta;. In addition, MHV68 infection led to a reduction in IL-1bbeta; production after extrinsic LPS stimulation or upon co-infection with Salmonella enterica Serovar Typhimurium. Interestingly, this impairment occurred at the proIL-1bbeta; transcript level and was independent of the lytic viral transcriptional activator RTA. Taken together, MHV68 impairs the inflammasome response by inhibiting IL-1bbeta; production during the initial stages of infection.

    IMPORTANCE Gammaherpesviruses persist for the lifetime of the host. To accomplish this, they must evade recognition and clearance by the immune system. The inflammasome consists of proteins that detect foreign molecules in the cell and respond by secreting proinflammatory signaling proteins that recruit immune cells to clear the infection. Unexpectedly, we found that murine gammaherpesvirus pathogenesis was not enhanced in mice lacking caspase-1, a critical inflammasome component. This led us to investigate if the virus actively impairs the inflammasome response. We found that the inflammasome was not activated upon macrophage cell infection with murine gammaherpesvirus 68. Infection also prevented the host cell inflammasome response to other pathogen-associated molecular patterns, indicated by reduced production of the proinflammatory cytokine IL-1bbeta; upon bacterial co-infection. Taken together, murine gammaherpesvirus impairment of the inflammatory cytokine IL-1bbeta; in macrophages identifies one mechanism by which the virus may inhibit caspase-1-dependent immune responses in the infected animal.

  • Temperature-sensitive mutants in the influenza A virus RNA polymerase: alterations in the PA linker reduce nuclear targeting of the PB1-PA dimer and hence result in viral attenuation [PublishAheadOfPrint]

  • The influenza virus RNA-dependent RNA polymerase catalyses genome replication and transcription within the cell nucleus. Efficient nuclear import and assembly of the polymerase subunits, PB1, PB2, and PA are critical steps in virus cycle. We investigated the structure and function of the PA linker (residues 197-256), located between its N-terminal endonuclease domain and its C-terminal structured domain that binds PB1, the polymerase core. Circular dichroiiuml;sm experiments revealed that the PA linker by itself is structurally disordered. A large series of PA linker mutants exhibited a temperature-sensitive (ts) phenotype (reduced viral growth at 39.5ddeg;C vs. 37ddeg;C/33ddeg;C), suggesting an alteration of folding kinetic parameters. The ts-phenotype was associated to a reduced efficiency of replication/transcription of a pseudo-viral reporter RNA in a minireplicon assay. Using a fluorescent-tagged PB1, we observed that ts- and lethal PA mutants do not efficiently recruit PB1 to reach the nucleus at 39.5ddeg;C. A protein complementation assay using PA mutants, PB1 and bbeta;-importin IPO5 tagged with fragments of the Gaussia princeps luciferase showed that increasing the temperature negatively modulates the PA-PB1 and the PA-PB1-IPO5 interactions or complex stability. The selection of revertant viruses allowed the identification of different types of compensatory mutations located in one or the other of the three polymerase subunits. Two ts-mutants were shown to be attenuated and able to induce antibodies in mice. Taken together, our results identified a PA domain as being critical of PB1-PA nuclear import and as a "hot spot" to engineer ts-mutants that could be used to design novel attenuated vaccines.

    Importance By targeting a discrete domain of the PA polymerase subunit of influenza virus, we were able to identify a series of 9 amino acid positions that are appropriate to engineer temperature-sensitive (ts) mutants. That is the first time that a large number of ts-mutations were engineered in such a short domain, demonstrating that rationale design of ts-mutants can be achieved. We were able to associate this phenotype to a defect of transport of the PA-PB1 complex into the nucleus. Reversion substitutions restore the ability of the complex to move to the nucleus. Two of these ts-mutants were shown attenuated and able to produce antibodies in mice. These results are of high interest to design novel attenuated vaccines and to elaborate antiviral drugs.

  • Increased Affinity of JC Polyomavirus Capsid for LSTc Over Other Sialylated Glycans Is a Major Determinant of Infectivity [PublishAheadOfPrint]

  • The human JC polyomavirus (JCPyV) establishes an asymptomatic, persistent infection in the kidney of the majority of the population, and is the causative agent of the fatal, demyelinating disease progressive multifocal leukoencephalopathy (PML) in immunosuppressed individuals. The Mad-1 strain of JCPyV, a brain isolate, was shown earlier to require aalpha;2,6-linked sialic acid on the LSTc glycan for attachment to host cells. In contrast, a JCPyV kidney isolate Type 3 strain, "WT3", has been reported to interact with sialic acid-containing gangliosides, but the role of these glycans in JCPyV infection has remained unclear. To help rationalize these findings and probe the effects of strain-specific differences on receptor binding, we performed a comprehensive analysis of the glycan receptor specificities of these two representative JCPyV strains using high-resolution X-ray crystallography and NMR spectroscopy, and correlated these data with infectivity assays. We show here that capsid proteins of Mad-1 and WT3 JCPyV can both engage LSTc as well as multiple sialylated gangliosides. However, the binding affinities exhibit subtle differences, with the highest affinity observed for LSTc. Engagement of LSTc is a prerequisite for functional receptor engagement while the weaker-binding gangliosides are not required for productive infection. Our findings highlight the complexity of virus-carbohydrate interactions and demonstrate that subtle differences in binding affinities, rather than the binding event alone, help determine tissue tropism and viral pathogenesis.

    IMPORTANCE Viral infection is initiated by attachment to receptors on host cells, and this event plays an important role in viral disease. We investigated the receptor-binding properties of human JC polyomavirus (JCPyV), a virus that resides in the kidney of the majority of the population and can cause the fatal, demyelinating disease progressive multifocal leukoencephalopathy (PML) in the brain of immunosuppressed individuals. JCPyV has been reported to interact with multiple carbohydrate receptors, and we sought to clarify how the interactions between JCPyV and cellular carbohydrate receptors influenced infection. Here we demonstrate that JCPyV can engage numerous sialylated carbohydrate receptors. However, the virus displays preferential binding to LSTc, and only LSTc mediates a productive infection. Our findings demonstrate that subtle differences in binding affinity, rather than receptor engagement alone, are a key determinant of viral infection.

  • Investigating Influenza A virus infection: Tools to track infection and tropism [PublishAheadOfPrint]

  • Influenza A viruses display a broad cellular tropism within the reparatory tract of mammalian hosts. Uncovering the relationship between tropism and virus immunity, pathogenesis, and transmission will be critical for the development of therapeutic interventions. Here we discuss recent developments of several recombinant strains of influenza A virus. These viruses have inserted reporters to track tropism, microRNA target sites to restrict tropism or barcodes to assess transmission dynamics, expanding our understanding of pathogen host interactions.

  • Genetic confirmation that the H5 protein is required for vaccinia virus DNA replication [PublishAheadOfPrint]

  • The duplication of the poxvirus dsDNA genome occurs in cytoplasmic membrane-delimited factories. This physical autonomy from the host nucleus suggests that poxviruses encode the full repertoire of proteins committed for genome replication. Biochemical and genetic analyses have confirmed that six viral proteins are required for efficient DNA synthesis; indirect evidence has suggested that the multifunctional H5 protein may also have a role. Here we show that H5 localizes to replication factories as visualized by immunofluorescence and immunoelectron microscopy, and can be retrieved upon purification of the viral polymerase holoenzyme complex. The temperature-sensitive mutant Dts57, which was generated by chemical mutagenesis and has a lesion in H5, exhibits defects in DNA replication and morphogenesis under non-permissive conditions, depending upon the experimental protocol. The H5 variant encoded by this mutant is ts for function, not stability. For a more precise investigation of how H5 contributes to DNA synthesis, we placed the ts57-H5 allele in an otherwise WT viral background, and also performed siRNA-mediated depletion of H5. Finally, we generated a complementing CV1-H5 cell line which allowed us to generate a viral recombinant in which the H5 ORF was deleted and replaced with mCherry (vH5). Analysis of vH5 allowed us to demonstrate conclusively that viral DNA replication is abrogated in the absence of H5. The loss of H5 does not compromise the accumulation of other early viral replication proteins or the uncoating of the virion core, suggesting that H5 plays a direct and essential role in facilitating DNA synthesis.

    IMPORTANCE Variola virus, the causative agent of smallpox, is the most notorious member of the Poxvirus family. Poxviruses are unique among DNA viruses that infect mammalian cells in that their replication is restricted to the cytoplasm of the cell. This physical autonomy from the nucleus has both cell biological and genetic ramifications. Poxviruses must establish cytoplasmic niches that support replication, and must encode the repertoire of proteins necessary for genome synthesis. Here we focus on H5, a multifunctional and abundant viral protein. We confirm that H5 associates with the DNA polymerase holoenzyme and localizes to the sites of DNA synthesis. By generating an H5-expressing cell line, we have been able to isolate a deletion virus that lacks the H5 gene, and have shown definitively that genome synthesis does not occur in the absence of H5. These data support the hypothesis that H5 is a crucial participant in cytoplasmic poxvirus genome replication.

  • IL-2 from adaptive T cells enhances natural killer cell activity against human cytomegalovirus infected macrophages [PublishAheadOfPrint]

  • Control of human cytomegalovirus (HCMV) requires a continuous immune surveillance, thus HCMV is the most important viral pathogen in severely immunocompromised individuals. Both innate and adaptive immunity contribute to the control of HCMV. Here, we report that peripheral blood natural killer cells (PBNKs) from HCMV seropositive donors showed an enhanced activity towards HCMV infected autologous macrophages. However, this enhanced response was abolished when purified NK cells were applied as effectors. We demonstrate that this enhanced PBNKs activity was dependent on the IL-2 secretion of CD4+ T cells when re-exposed to the virus. Purified T cells enhanced the activity of purified NK cells in response to HCMV infected macrophages. This effect could be suppressed by IL-2 blocking. Our findings not only extend the knowledge on the immune surveillance in HCMV, namely that NK cell mediated innate immunity can be enhanced by a preexisting T cell antiviral immunity, but also indicate a potential clinical implication for patients at risk for severe HCMV manifestations due to immunosuppressive drugs, which mainly suppress IL-2 production and T cell responsiveness.

    Importance Human cytomegalovirus (HCMV) is never cleared by the host after primary infection but instead establishes a lifelong latent infection with possible reactivations when the host's immunity becomes suppressed. Both, innate and adaptive immunity are important for the control of viral infections. Natural killer (NK) cells are main innate effectors providing a rapid response to virus-infected cells. Virus specific T cells are main adaptive effectors, which are critical for the control of the latent infection and limitation of re-infection. In this study, we found that IL-2 secreted by adaptive CD4+ T cells after re-exposure to HCMV enhance the activity of NK cells in response to HCMV infected target cells. This is the first direct evidence that the adaptive T cells can help NK cells to act against HCMV infection.

  • Genomic, Phylogenetic, and Recombinational Characterization of Herpes Simplex Virus Type 2 Strains [PublishAheadOfPrint]

  • Herpes simplex virus type 2 (HSV-2) is a major global pathogen, infecting 16% of people, 15-49 years old worldwide, causing recurrent, genital ulcers. Little is known about viral factors contributing to virulence, and there are currently only two genomic sequences available. In this study we determined nearly complete genomic sequences of six additional HSV-2 isolates, using the Illumina MiSeq. We report that HSV-2 has genomic overall mean distance of 0.2355%, which is lower than HSV-1. There were approximately 100 amino acid encoding SNPs/INDELs per genome. Microsatellite mapping found a bias towards intergenic regions in the non-conserved microsatellites, and a genic bias in all detected tandem repeats. Extensive recombination between the HSV-2 strains was also strongly implied. This is the first study to analyze multiple HSV-2 sequences, and will be valuable in future evolutionary, virulence, and structure-function studies.

    Importance Herpes simplex virus type 2 (HSV-2) is an significant worldwide pathogen, causing recurrent, genital ulcers. Here we present six nearly complete HSV-2 genomic sequences, and with the addition of two previously sequenced strains, for the first time genomic, phylogenetic, and recombination analysis was performed on multiple HSV-2 genomes. Our results show that microsatellite mapping found a bias towards intergenic regions in the non-conserved microsatellites, a genic bias in all detected tandem repeats, confirm that ChHV-1 is a separate species, and that each of the HSV-2 strains is genomic mosaic.

  • Comparison of immunogenicity in rhesus macaques of transmitted-founder, HIV-1 group M consensus and trivalent mosaic Envelope vaccines formulated as a DNA prime, NYVAC and Envelope protein boost [PublishAheadOfPrint]

  • An effective HIV-1 vaccine must induce protective antibody responses, as well as CD4+ and CD8+ T cell responses, that can be effective despite extraordinary diversity of HIV-1. The consensus and mosaic immunogens are complete but artificial proteins, computationally designed to elicit immune responses with improved cross-reactive breadth, to attempt to overcome the challenge of global HIV diversity. In this study, we have compared the immunogenicity of a transmitted-founder (T/F) B clade Env (B.1059), a global group M consensus Env (Con-S), and a global trivalent mosaic Env protein in rhesus macaques. These antigens were delivered using a DNA prime-rNYVAC vector and Env protein boost vaccination strategy. While Con-S Env was a single sequence, mosaic immunogens were a set of three Envs optimized to include the most common forms of potential T cell epitopes. Both Con-S and mosaic sequences retained common amino acids encompassed by both antibody and T cell epitopes, and were central to globally circulating strains. Mosaics and Con-S Envs expressed as full-length proteins bound well to a number of neutralizing antibodies with discontinuous epitopes. Also both consensus and mosaic immunogens induced significantly higher IFN- ELISpot responses than B.1059 immunogen. Immunization with these proteins, particularly Con-S, also induced significantly higher neutralizing antibodies to viruses than B.1059 Env, primarily to Tier 1 viruses. Both Con-S and mosaics stimulated more potent CD8-T cell responses against heterologous Envs than did B.1059. Both antibody and cellular data from this study strengthen the concept of using in silico designed centralized immunogens for global HIV-1 vaccine development strategies.

    IMPORTANCE There is an increasing appreciation for the importance of vaccine-induced anti-Env antibody responses for preventing HIV-1 acquisition. This nonhuman primate study demonstrates that in silico designed global HIV-1 immunogens, designed for a human clinical trial, are capable of eliciting not only T lymphocyte responses but also potent anti-Env antibody responses.

  • Influenza A virus protein PA-X contributes to viral growth and suppression of the host antiviral and immune responses [PublishAheadOfPrint]

  • Influenza virus infection causes global inhibition of host protein synthesis in infected cells. This host shutoff is thought to allow viruses to escape from the host antiviral response, which restricts virus replication and spread. Although the mechanism of host shutoff is unclear, a novel viral protein expressed by ribosomal frameshifting PA-X, was found to play a major role in influenza virus-induced host shutoff. However, little is known about the impact of PA-X expression on currently circulating influenza A virus pathogenicity and the host antiviral response. In this study, we rescued a recombinant influenza A virus A/California/04/09 (H1N1, Cal) containing mutations at the frameshift motif in the PA gene (Cal PA-XFS). Cal PA-XFS expressed significantly less PA-X than Cal wild type (WT). Cal WT, but not Cal PA-XFS, induced degradation of host bbeta;-actin mRNA and suppressed host protein synthesis, supporting the idea that PA-X induces host shutoff via mRNA decay. Moreover, Cal WT inhibited IFN-bbeta; expression and replicated more rapidly than Cal PA-XFS in human respiratory cells. Mice infected with Cal PA-XFS had significantly lower levels of virus growth and enhanced expression of IFN-bbeta; mRNA in their lungs than mice infected with Cal WT. Importantly, more anti-hemagglutinin and neutralizing antibodies were produced in Cal PA-XFS-infected mice than Cal WT-infected mice, despite the lower level of virus replication in the lungs. Our data indicate that PA-X of the pandemic H1N1 virus has a strong impact on viral growth and the host innate and acquired immune response to influenza virus.

    Importance Virus-induced host protein shutoff is considered to be a major factor to allow viruses to evade innate and acquired immune recognition. We provide evidence that the 2009 H1N1 influenza A virus protein PA-X plays a role in virus replication and inhibition of host antiviral response by means of its host protein synthesis shutoff activity both in vitro and in vivo. We also demonstrated that while growth of Cal PA-XFS was attenuated in the lungs of infected animals, this mutant induced a stronger humoral response than Cal WT. Our findings clearly highlight the importance of PA-X in counteracting the host innate and acquired immune response to influenza virus, an important global pathogen. This work demonstrates that inhibition of PA-X expression in influenza virus vaccine strains may provide a novel way of safely attenuating viral growth while inducing a more robust immune response.

  • The molecular chaperone Hsp90 is a therapeutic target for noroviruses [PublishAheadOfPrint]

  • Human noroviruses (HuNoV) are a significant cause of acute gastroenteritis in the developed world, yet our understanding of the molecular pathways involved in norovirus replication and pathogenesis has been limited by the inability to efficiently culture these viruses in the laboratory. Using the murine norovirus (MNV) model, we have recently identified a network of host factors that interact with the 5rrsquo; and 3rrsquo; extremities of the norovirus RNA genome. In addition to a number of well-known cellular RNA binding proteins, the molecular chaperone Hsp90 was identified as a component of the ribonucleoprotein complex. Here, we show that the inhibition of Hsp90 activity negatively impacts norovirus replication in cell culture. Small molecule-mediated inhibition of Hsp90 activity using 17-DMAG, revealed that Hsp90 plays a pleiotropic role in the norovirus life cycle, but that the stability of the viral capsid protein is integrally linked to Hsp90 activity. Furthermore, we demonstrate that both the MNV-1 and HuNoV capsid proteins require Hsp90 activity for their stability and that targeting Hsp90 in vivo can significantly reduce virus replication. In summary, we demonstrate that targeting cellular proteostasis can inhibit norovirus replication, identifying a potential novel therapeutic target for the treatment of norovirus infections.

    Importance (non-technical explanation) HuNoV are a major cause of acute gastroenteritis around the world. RNA viruses, including noroviruses, rely heavily on host cell proteins and pathways for all aspects of their life cycle. Here we identify one such protein, the molecular chaperone Hsp90, as an important factor required during the norovirus life cycle. We demonstrate that both murine and human noroviruses require the activity of Hsp90 for the stability of their capsid proteins. Furthermore, we demonstrate that targeting Hsp90 activity in vivo using small molecule inhibitors also reduces infectious virus production. Given the considerable interest in the development of Hsp90 inhibitors for use in cancer therapeutics, this work identifies a new target that could be explored for the development of antiviral strategies to control norovirus outbreaks and treat chronic norovirus infection in immunosuppressed patients.

  • Creation of Matrix Protein Gene Variants of Two Serotypes of VSV as Prime-Boost Vaccine Vectors [PublishAheadOfPrint]

  • To take advantage of live rVSVs as vaccine vectors for their high yield and for their induction of strong and long-lasting immune responses, it is necessary to make live vaccine vectors safe for use without losing their immunogenicity. We have generated safer and highly efficient recombinant VSV vaccine vectors by combining the M51R mutation in the M gene of VSVInd with a temperature sensitive mutation of the VSVInd Orsay tsO23. In addition, we have also generated two new VSVNJ vaccine vectors by combining M48R/M51R mutations with G22E and L110F mutations in the M gene, rVSVNJ(G22E/M48R/M51R; GMM) and VSVNJ(G22E/M48R/M51R/L110F; GMML). The combined mutations, G21E/M51R/L111F in the M protein of VSVInd significantly reduced the burst size of the virus by up to 10,000 fold at 37ddeg;C without affecting the level of protein expression. The BHK21 cells and SH-SY5Y human neuroblastoma cells infected with rVSVInd(GML), rVSVNJ(GMM), and rVSVNJ(GMML) showed significantly reduced cytopathic effects in vitro at 37ddeg;C, and mice injected with one million infectious virus particles of these mutants into the brain showed no neurological dysfunctions or any other adverse effects. In order to increase the stability of the temperature sensitive mutant, we have substituted the phenylalanine by alanine. This will change all three nucleotides from UUG (leucine) to GCA (alanine). The resulting mutant, L111A, showed a temperature sensitive phenotype of rVSVInd(GML) and increased stability. Twenty consecutive passages of the rVSVInd(GML) with an L111A mutation did not convert back to leucine (UUG) at position 111 in the M protein gene.

    Importance Recombinant vesicular stomatitis viruses, as live vaccine vectors, are very effective in expressing foreign genes and inducing adaptive T cell and B cell immune responses. As with any other live viruses in humans or animals, using live rVSVs as vaccine vectors demands the utmost safety in usage. Our strategy to attenuate the rVSVInd by utilizing a temperature-sensitive assembly-defective mutation of L111A and combining it with an M51R mutation in the M protein of rVSVInd, significantly reduced the pathogenicity of the virus while maintaining highly effective virus production. We believe our new temperature sensitive M gene mutant of the rVSVInd(GML) and M gene mutants of rVSVNJ(GMM) and VSVNJ(GMM) add excellent vaccine vectors to the pool of live viral vectors.

  • Genetic Determinants of Japanese Encephalitis Virus Vaccine Strain SA14-14-2 that Govern Attenuation of Virulence in Mice [PublishAheadOfPrint]

  • Safety and efficacy of the live-attenuated Japanese encephalitis virus (JEV) SA14-14-2 vaccine is attributed to mutations that accumulated in the viral genome during its derivation. However, little is known about the contribution that is made by most of these mutations to virulence attenuation and vaccine immunogenicity. Here, we generated recombinant JEV (rJEV) strains containing JEV SA14-14-2 vaccine-specific mutations that are located in the untranslated regions (UTRs) and seven protein genes or are introduced from PCR-amplified regions of the JEV SA14-14-2 genome. The resulting mutant viruses were evaluated in tissue culture and in mice. The authentic JEV SA14-14-2 E protein, with amino acid substitutions L107F, E138K, I176V, T177A, E244G, Q264H, K279M, A315V, S366A, and K439R relative to the wild-type rJEV clone, was essential and sufficient for complete attenuation of neurovirulence. Individually, nucleotide substitution T39A in the 5rrsquo; UTR, capsid (C) protein amino acid substitution L66S, and the complete NS1/2A genome region containing ten mutations, each significantly reduced virus neuroinvasion but not neurovirulence. The levels of peripheral virulence attenuation imposed by mutations 5rrsquo; UTR-T39A and C-L66S, individually, was somewhat mitigated in combination with other vaccine strain-specific mutations, which might be compensatory, and together did not affect immunogenicity. However, a marked reduction in immunogenicity was observed with the addition of the NS1/2A and NS5 vaccine virus genome regions. These results suggest that a second-generation recombinant vaccine can be rationally engineered to maximize levels of immunogenicity without compromising safety.

    Importance The live-attenuated JEV SA14-14-2 vaccine has been vital for controlling the incidence of disease caused by JEV, particularly in rural areas of Asia where it is endemic. The vaccine was developed more than 25 years ago by passaging wild-type JEV strain SA14 in tissue culture and rodents, with intermittent tissue culture plaque purifications, to produce a virus clone that had adequate levels of attenuation and immunogenicity. The vaccine and parent virus sequences were later compared and mutations were identified throughout the vaccine virus genome, but their contributions to its attenuation were never fully elucidated. Here, using reverse genetics, we comprehensively defined the impact of JEV SA14-14-2 mutations on attenuation of virulence and immunogenicity in mice. These results are relevant for quality control of new lots of the current live-attenuated vaccine and provide insight for the rational design of second-generation, live-attenuated, recombinant JEV vaccine candidates.

  • Mutations in coronavirus nonstructural protein 10 decrease virus replication fidelity. [PublishAheadOfPrint]

  • Coronaviruses (CoVs) are unique in encoding a 3rrsquo; -ggt;5rrsquo; exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is required for high-fidelity replication, likely via proofreading. Nsp14 associates with the CoV RNA-dependent RNA polymerase (nsp12-RdRp), and nsp14-ExoN activity is enhanced by binding nsp10, a small non-enzymatic protein. However it is not known whether nsp10 functions in the regulation of CoV replication fidelity. To test this, we engineered single and double alanine substitution mutations into the genome of murine hepatitis virus (MHV-A59) containing ExoN activity [ExoN(+)] at positions within nsp10 known to disrupt the nsp10-nsp14 interaction in vitro. We show that an nsp10 mutant, R80A/E82A-ExoN(+), was five-to-ten times more sensitive to treatment with the RNA mutagen 5-FU than WT-ExoN(+), suggestive of decreased replication fidelity. This decreased fidelity phenotype was confirmed using two additional nucleoside analogs, 5-azacytidine and ribavirin. R80A/E82A-ExoN(+) reached similar peak titer and demonstrated comparable RNA synthesis kinetics as compared to WT-ExoN(+). No change in 5-FU sensitivity was observed for R80A/E82A-ExoN(-), relative to MHV-ExoN(-), indicating that the decreased fidelity phenotype of R80A/E82A-ExoN(-) is linked to the presence of ExoN activity. Our results demonstrate that nsp10 is important for CoV replication fidelity, and support the hypothesis that nsp10 functions to regulate nsp14-ExoN activity during virus replication.

    Importance The adaptive capacity of CoVs, as well as all other RNA viruses, is partially attributed to the presence of extensive population genetic diversity. However, decreased fidelity is detrimental to CoV replication and virulence; mutant CoVs with decreased replication fidelity are attenuated and more sensitive to inhibition by RNA mutagens. Thus, identifying the viral protein determinants of CoV fidelity is important for understanding CoV replication, pathogenesis, and virulence. In this study we show that nsp10, a small, non-enzymatic viral protein, contributes to CoV replication fidelity. Our data support the hypothesis that CoVs have evolved multiple proteins, in addition to nsp14-ExoN, that are responsible for maintaining the integrity of the largest known RNA genomes.

  • CD8+ T Cells Can Mediate Short-term Protection Against Heterotypic Dengue Reinfection in Mice [PublishAheadOfPrint]

  • Dengue virus (DENV) is a major public health threat worldwide. Infection with one of the four serotypes of DENV results in a transient period of protection against reinfection with all serotypes (cross-protection), followed by life-long immunity to the infecting serotype. While a protective role for neutralizing antibody responses is well established, the contribution of T cells to reinfection is less clear, especially during heterotypic reinfection. This study investigates the role of T cells during homotypic and heterotypic DENV reinfection. Mice were sequentially infected with homotypic or heterotypic DENV serotypes, and T cell subsets were depleted before the second infection to assess the role of DENV-primed T cells during reinfection. Mice primed non-lethally with DENV were protected against reinfection with either a homotypic or a heterotypic serotype two weeks later. Homotypic priming induced a robust neutralizing antibody response, whereas heterotypic priming elicited binding, but non-neutralizing antibodies. CD8+ T cells were required for protection against heterotypic, but not homotypic, reinfection. These results suggest that T cells can contribute crucially to protection against heterotypic reinfection in situations where humoral responses alone may not be protective. Our findings have important implications for vaccine design, as they suggest that inducing both humoral and cellular responses during vaccination may maximize protective efficacy across all DENV serotypes.

    Importance Dengue virus is present in over 120 countries in tropical and sub-tropical regions. Infection with dengue virus can be asymptomatic, but can also progress into the potentially lethal severe dengue disease. There are four closely related dengue serotypes. Infection with one serotype results in a transient period of resistance against all serotypes (cross-protection), followed by life-long resistance to the infecting serotype, but not the other ones. The duration and mechanisms of the transient cross-protection period remain elusive. This study investigates the contribution of cellular immunity to cross-protection using mouse models of DENV infection. Our results demonstrate that cellular immunity is crucial to mediate cross-protection against reinfection with a different serotype, but not for protection against reinfection with the same serotype. A better understanding of the mediators responsible for the cross-protection period is important for vaccine design, as an ideal vaccine against dengue virus should efficiently protect against all serotypes.

  • Genetics, receptor binding, and virulence in mice of H10N8 influenza viruses isolated from ducks and chickens in live poultry markets in China [PublishAheadOfPrint]

  • We analyzed eight H10N8 viruses isolated from ducks and chickens in live poultry markets from 2009 to 2013 in China. These viruses showed distinct genetic diversity and formed five genotypes: the four duck isolates formed four different genotypes, whereas the four chicken viruses belong to a single genotype. The viruses bound to both human- and avian-type receptors, and four of the viruses caused 12.7% nndash; 22.5% body weight loss in mice.

  • Detection of SIV in semen, urethra and male reproductive organs during efficient HAART [PublishAheadOfPrint]

  • A number of men receiving prolonged suppressive HAART still shed HIV in semen. To investigate whether this seminal shedding may be due to poor drug penetration and/or viral production by long-lived cells within male genital tissues, we analyzed semen and reproductive tissues from macaques chronically infected with SIVmac251 submitted to HAART for 4 months, intensified over the last seven weeks with an integrase inhibitor. We showed that a subset of treated animals continued shedding SIV in semen despite efficient HAART. This shedding was not associated with low antiretroviral drug concentrations in semen, or in testis, epididymis, seminal vesicles and prostate. HAART had no significant impact on SIV RNA in the urethra, whereas it drastically reduced SIV RNA levels in the prostate and vas deferens, and to a lesser extent in the epididymis and seminal vesicle. The only detectable SIV RNA positive cells within the male genital tract after HAART were urethral macrophages. SIV DNA levels in genital tissues were not decreased by HAART, suggesting the presence throughout the male genital tract of non-productively infected cells. In conclusion, our results demonstrate that 4 months of HAART induced variable and limited control of viral infection in the male reproductive organs, particularly the urethra, and suggest that infected long-lived cells in the male genital tract may be involved in persistent seminal shedding during HAART. These results pave the way for further investigations of male genital organs infection in long term treated infected individuals.

    Importance A substantial subset of men receiving prolonged suppressive HAART in the blood still harbor HIV in semen, and cases of sexual transmission have been reported. To understand the origin of this persistence, we analyzed the semen and male reproductive tissues from SIV-infected macaques treated by HAART. We demonstrated that persistent seminal shedding was not linked to poor drug penetration in semen or semen-producing prostate, seminal vesicle, epididymis and testis. We revealed that HAART decreased SIV RNA to various extents in all male genital organs, with the exception of the urethra, in which SIV RNA+ macrophages were observed despite HAART. Importantly also, HAART did not impact SIV DNA levels in the male genital organs. These results suggest that infection of male genital organs, and particularly the urethra, could be involved in the release of virus in semen during HAART.

  • Axonal sorting and transport of the pseudorabies virus glycoprotein gM independent of Us9 [PublishAheadOfPrint]

  • Axonal sorting and transport of fully assembled pseudorabies virus virions is dependent on the viral protein Us9. Here we identify a Us9-independent mechanism for axonal localization of viral glycoprotein M (gM). We detected gM-mCherry assemblies transporting in the anterograde direction in axons. Furthermore, unlabeled gM, but not glycoprotein B, was detected by western blot in isolated axons during Us9-null PRV infection. These results suggest that gM differs from other viral proteins regarding axonal transport properties.

  • Lymphocytic choriomeningitis virus differentially affects virus-induced type I IFN response and mitochondrial apoptosis mediated by RIG-I/MAVS [PublishAheadOfPrint]

  • Arenaviruses are important emerging human pathogens maintained by non-cytolytic persistent infection in their rodent reservoir hosts. Despite high levels of viral replication, persistently infected carrier hosts show only mildly elevated levels of type I interferon (IFN-I). Accordingly, the arenavirus nucleoprotein (NP) has been identified as a potent IFN-I antagonist capable of blocking activation of interferon regulatory factor (IRF)-3 via the retinoic acid inducible gene (RIG)-I/mitochondrial antiviral signaling (MAVS) pathway. Another important mechanism of host innate anti-viral defense is represented by virus-induced mitochondrial apoptosis via RIG-I/MAVS and IRF3. In the present study, we investigated the ability of the prototypic Old World arenavirus lymphocytic choriomeningitis virus (LCMV) to interfere with RIG-I/MAVS-dependent apoptosis. We found that LCMV does not induce apoptosis at any time during infection. While LCMV efficiently blocked induction of IFN-I via RIG-I/MAVS in response to super-infection with cytopathic RNA viruses, virus-induced mitochondrial apoptosis remained fully active in LCMV infected cells. Notably, in LCMV infected cells, RIG-I was dispensable for virus-induced apoptosis via MAVS. Our study reveals that LCMV infection efficiently suppresses induction of IFN-I, but does not interfere with the cell's ability to undergo virus-induced mitochondrial apoptosis as a strategy of innate anti-viral defense. The RIG-I-independence of mitochondrial apoptosis in LCMV infected cells provides first evidence that arenaviruses can reshape apoptotic signaling according to their needs.

    IMPORTANCE Arenaviruses are important emerging human pathogens that are maintained in their rodent hosts by persistent infection. Persistent virus is able to subvert the cellular interferon response, a powerful branch of innate anti-viral defense. Here we investigated the ability of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) to interfere with the induction of programmed cell death or apoptosis in response to super-infection with cytopathic RNA viruses. Upon viral challenge, persistent LCMV efficiently blocked induction of interferons, whereas virus-induced apoptosis remained fully active in LCMV infected cells. Our studies reveal that the persistent virus is able to reshape innate apoptotic signaling in order to prevent interferon production while maintaining programmed cell death as a strategy for innate defense. The differential effect of persistent virus on the interferon response versus apoptosis appears as a subtle strategy to guarantee sufficiently high viral loads for efficient transmission while maintaining apoptosis as a mechanism of defense.

  • In Vivo Activation of Human NK cells by Treatment with an IL-15 Superagonist Potently Inhibits Acute In Vivo HIV-1 Infection in Humanized Mice [PublishAheadOfPrint]

  • Natural killer (NK) cells with anti-HIV-1 activity may inhibit HIV-1 replication and dissemination during acute HIV-1 infection. We hypothesized that the NK cell capacity to suppress acute in vivo HIV-1 infection would be augmented by activating them via treatment with an IL-15 superagonist, IL-15 bound to soluble IL-15Raalpha;, an approach that potentiates human NK cell-mediated killing of tumor cells. In vitro stimulation of human NK cells with a recombinant IL-15 superagonist significantly induced their expression of cytotoxic effector molecules granzyme B and perforin, their degranulation upon exposure to K562 cells as indicated by cell-surface expression of CD107a and their capacity to lyse K562 cells and HIV-1-infected T cells. The impact of IL-15 superagonist-induced activation of human NK cells on acute in vivo HIV-1 infection was investigated using hu-spl-PBMC-NSG mice, NOD-SCID-IL2r-/- mice intrasplenically injected with human PBMCs, which develop productive in vivo infection after intrasplenic inoculation with HIV-1. IL-15 superagonist treatment potently inhibited acute HIV-1 infection in hu-spl-PBMC-NSG mice even when delayed until three days after intrasplenic HIV-1 inoculation. Removal of NK cells from the human PBMCs prior to intrasplenic injection into NSG mice completely abrogated IL-15 superagonist-mediated suppression of in vivo HIV-1 infection. Thus, the in vivo activation of NK cells, integral mediators of the innate immune response, by treatment with an IL-15 superagonist increases their anti-HIV activity and enables them to potently suppress acute in vivo HIV-1 infection. These results indicate that in vivo activation of NK cells may represent a new immunotherapeutic approach to suppress acute HIV-1 infection.

    Importance Epidemiological studies have indicated that NK cells contribute to the control of HIV-1-infection and in vitro studies have demonstrated that NK cells can selectively kill HIV-1-infected cells. We demonstrated that in vivo activation of NK cells by treatment with an IL-15 superagonist that potently stimulates the anti-tumor activity of NK cells markedly inhibited acute HIV-1 infection in humanized mice, even when activation of the NK cells by IL-15 superagonist treatment is delayed until 3 days after HIV-1 inoculation. NK cell-depletion from the PBMCs prior to their intrasplenic injection abrogated the suppression of in vivo HIV-1-infection observed in humanized mice treated with the IL-15 superagonist, demonstrating that the activated human NK cells were mediating IL-15 superagonist-induced inhibition of acute HIV-1 infection. Thus, in vivo immunostimulation of NK cells, a promising therapeutic approach for cancer therapy, may represent a new treatment modality for HIV-1-infected individuals particularly in the earliest stages of infection.

  • Artificial recruitment of UAF1-USP complexes by a PHLPP1-E1 chimeric helicase enhances HPV DNA replication [PublishAheadOfPrint]

  • The E1 helicase from anogenital HPV types interacts with the cellular WD-repeat-containing protein UAF1 in complex with the deubiquitinating enzyme USP1, USP12 or USP46. This interaction stimulates viral DNA replication and is required for maintenance of the viral episome in keratinocytes. E1 associates with UAF1 through a short UAF1-binding site (UBS) located within the N-terminal 40 residues of the protein. Here, we investigated if the E1 UBS could be replaced by the analogous domain from an unrelated protein, the PHLPP1 phosphatase. We found that PHLPP1 and E1 interact with UAF1 in a mutually exclusive manner and mapped the minimal PHLPP1 UBS (PUBS) to a 100 amino acid region sufficient for assembly into UAF1-USP complexes. Similarly to the E1 UBS, overexpression of PUBS in trans inhibited HPV DNA replication, albeit less efficiently. Characterization of a PHLPP1-E1 chimeric helicase revealed that PUBS could partially substitute for the E1 UBS in enhancing viral DNA replication and that the stimulatory effect of PUBS likely involves recruitment of UAF1-USP complexes as it was abolished by mutations that weaken UAF1-binding and by overexpression of catalytically-inactive USPs. Although functionally similar to the E1 UBS, PUBS is larger in size and requires both the WD-repeat region and C-terminal SUMO-like domain of UAF1 for interaction, in contrast to E1 which does not contact the latter. Overall, this comparison of two heterologous UBSs indicate that these domains function as transferable protein-interaction modules and provide further evidence that the association of E1 with UAF1-containing deubiquitinating complexes stimulates HPV DNA replication.

    IMPORTANCE The E1 protein from anogenital HPV types interact with the UAF1-associated deubiquitinating enzymes USP1, 12 and 46 to stimulate replication of the viral genome. Little is known about the molecular nature of the E1-UAF1 interaction and, more generally, how UAF1-USP complexes recognize their substrate proteins. To address this question, we characterized the UAF1-binding site (UBS) of the PHLPP1 phosphatase, a protein unrelated to E1. Using a PHLPP1-E1 chimeric helicase, we show that the PHLPP1 UBS (PUBS) can partially substitute for the E1 UBS in stimulating HPV DNA replication. This stimulation required conserved sequences in PUBS that meditate its interaction with UAF1, including a motif common to the E1 UBS. These results indicate that UAF1-binding sequences function as transferable protein-interaction modules and provide further evidence that UAF1-USP complexes stimulate HPV DNA replication.

  • Noncapped Alphavirus genomic RNAs and their role during infection [PublishAheadOfPrint]

  • Alphaviruses are enveloped positive-sense RNA viruses that exhibit a wide host range consisting of vertebrate and invertebrate species. Previously we have reported that the infectivity of Sindbis virus (SINV), the model alphavirus, was largely a function of the cell line producing the viral particles. Mammalian derived SINV particles on average exhibit a higher particle-to-PFU ratio than mosquito derived SINV particles. Nevertheless, the outcome of nonproductive infection, the molecular traits that determine particle infectivity and the biological importance of noninfectious particles were, prior to this study, unknown. In this study we report that the incoming genomic RNAs of noninfectious SINV particles undergo rapid degradation following infection. Moreover, these studies have led to the identification of the absence of the 5rrsquo; cap structure as a primary molecular determinant of particle infectivity. We show that the genomic RNAs of alphaviruses are not universally 5rrsquo; capped with a significant number of noncapped genomic RNA produced early in infection. The production of noncapped viral genomic RNAs, is important to the establishment and maintenance of alphaviral infection.

    Importance This report is of importance to the field of virology for three reasons. First, these studies demonstrate that noncapped Sindbis virus particles are produced as a result of viral RNA synthesis. Second, this report is, to our knowledge, the first instance of the direct measurement of the RNA half-life of an incoming genomic RNA from a positive sense RNA virus. Finally, these studies indicate that alphaviral infection is likely a concerted effort between infectious and noninfectious viral particles.

  • Determinants of dengue virus NS4A protein oligomerization [PublishAheadOfPrint]

  • Flavivirus NS4A protein induces host membrane rearrangement and functions as a replication complex component. The molecular details of how flavivirus NS4A exerts these functions remain elusive. Here, we used dengue virus (DENV) as a model to characterize and demonstrate the biological relevance of flavivirus NS4A oligomerization. DENV-2 NS4A protein forms oligomers in infected cells or when expressed alone. Deletion mutagenesis mapped amino acids 50-76 (spanning the first transmembrane domain [TMD1]) of NS4A as the major determinant for oligomerization, while the N-terminal 50 residues only slightly contribute to the oligomerization. NMR analysis of NS4A amino acids 17-80 suggests that residues L31, L52, E53, G66, and G67 could participate in oligomerization. Ala-substitution of fifteen flavivirus-conserved NS4A residues revealed that these amino acids are important for viral replication. Among the fifteen mutated NS4A residues, two amino acids (E50A and G67A) are located within TMD1. Both E50A and G67A attenuated viral replication, decreased NS4A oligomerization, and reduced NS4A protein stability. In contrast, NS4A oligomerization was not affected by the replication-defective mutations (R12A, P49A, and K80A) located outside TMD1. Trans complementation experiments showed that expression of wild-type NS4A alone was not sufficient to rescue the replication-lethal NS4A mutants. However, the presence of DENV-2 replicons could partially restore the replication defect of some lethal NS4A mutant (L26A and K80A), but not others (L60A and E122A), suggesting an unidentified mechanism governing the outcome of complementation in a mutant-dependent manner. Collectively, the results have demonstrated the importance of TMD1-mediated NS4A oligomerization in flavivirus replication.

    IMPORTANCE We report that DENV NS4A forms oligomers. Such NS4A oligomerization is mediated mainly through amino acids 50-76 (spanning the first transmembrane domain [TMD1]). The biological importance of NS4A oligomerization is demonstrated by the results that mutations of flavivirus-conserved residues (E50A and G67A located within the TMD1) reduced the oligomerization and stability of NS4A protein, leading to attenuated viral replication. A systematic mutagenesis analysis demonstrates that flavivirus-conserved NS4A residues are important for DENV replication. A successful trans complementation of replication-lethal NS4A mutant virus requires the wild-type NS4A in the context of viral replication complex. The wild-type NS4A alone is not sufficient to rescue the replication defect of NS4A mutants. Intriguingly, distinct NS4A mutants yielded different complementation outcome in the replicon-containing cells. Overall, the study has enhanced our understanding of flavivirus NS4A at molecular details. The results also suggest that inhibitor blocking NS4A oligomerization could be explored for antiviral drug discovery.

  • Corona and Influenza Virus Proteolytic Priming takes place in Tetraspanin-Enriched Membrane Microdomains [PublishAheadOfPrint]

  • Coronaviruses (CoVs) and low-pathogenicity influenza A viruses (LP IAVs) depend on target cell proteases to cleave their viral glycoproteins and prime them for virus-cell membrane fusion. Several proteases cluster into tetraspanin-enriched microdomains (TEMs), suggesting that TEMs are preferred virus entry portals. Here we found that several CoV receptors and virus-priming proteases were indeed present in TEMs. Isolated TEMs, when mixed with CoV and LP IAV pseudo-particles, cleaved viral fusion proteins to fusion-primed fragments and potentiated viral transductions. That entering viruses utilize TEMs as a protease source was further confirmed using tetraspanin antibodies and tetraspanin shRNAs. Tetraspanin antibodies inhibited CoV and LP IAV infections, but their virus-blocking activities were overcome by expressing excess TEM-associated proteases. Similarly, cells with reduced levels of the tetraspanin CD9 resisted CoV pseudo-particle transductions, but were made susceptible by overproducing TEM-associated proteases. These findings indicated that antibodies and CD9 depletions interfere with viral proteolytic priming, in ways that are overcome by surplus proteases. TEMs appear to be exploited by some CoVs and LP IAVs for appropriate co-engagement with cell receptors and proteases.

    IMPORTANCE Enveloped viruses use their surface glycoproteins to catalyze membrane fusion, an essential cell entry step. Host cell components prime these viral surface glycoproteins to catalyze membrane fusion at specific times and places during virus-cell entry. Amongst these priming components are proteases, which cleave viral surface glycoproteins, unleashing them to refold in ways that catalyze virus-cell membrane fusions. For some enveloped viruses, these proteases are known to reside on target cell surfaces. This research focuses on corona- and influenza A- virus-cell entry, and identifies TEMs as sites of viral proteolysis, thereby defining subcellular locations of virus priming with greater precision. Implications of these findings extend to the use of virus entry antagonists, such as protease inhibitors, which might be most effective when localized to these microdomains.

  • Interregional coevolution analysis revealing functional/structural interrelatedness between different genomic regions in Human mastadenovirus D [PublishAheadOfPrint]

  • Human mastadenovirus D (HAdV-D) is exceptionally rich in type among the seven human adenovirus species. This feature is attributed to frequent intertypic recombination events that have reshuffled orthologous genomic regions between different HAdV-D types. However, this trend appears to be paradoxical, as it has been demonstrated that the replacement of some of the interacting proteins for a specific function with other orthologues causes malfunction, indicating that intertypic recombination events may be deleterious. In order to understand why the paradoxical trend has been possible in HAdV-D evolution, we conducted an inter-regional coevolution analysis between different genomic regions of 45 different HAdV-D types and found that ca. 70% of the genome has coevolved, even though these are fragmented into several pieces via short intertypic recombination hotspot regions. Since it is statistically and biologically unlikely that all of the coevolving fragments have synchronously recombined between different genomes, it is probable that these regions have stayed in their original genomes during evolution as a platform for frequent intertypic recombination events in limited regions. It is also unlikely that the same genomic regions have remained almost untouched during frequent recombination events, independently, in all different types, by chance. In addition, the coevolving regions contain the coding regions of physically interacting proteins for important functions. Therefore, the coevolution of these regions should be attributed at least in part to natural selection due to common biological constraints operating on all types, including protein-protein interactions for essential functions. Our results predict additional unknown protein interactions.

    Importance Human mastadenovirus D, an exceptionally type-rich human adenovirus species and causative agent of different diseases in a wide variety of tissues, including that of ocular region and digestive tract, as well as an opportunistic infection in immune-compromised patients, is known to have highly diverged through frequent intertypic recombination events; however, it has also been demonstrated that the replacement of a component protein of a multi-protein system with a homologous protein causes malfunction. The present study solved this apparent paradox by looking at which genomic parts have coevolved using a newly developed method. The results revealed that intertypic recombination events have occurred in limited genomic regions and been avoided in the genomic regions encoding proteins that physically interact for a given function. This approach detects purifying selection against recombination events causing the replacement of partial components of multi-protein systems and therefore predicts physical/functional interactions between different proteins and/or genomic elements.

  • Continual Re-Introduction of Human Pandemic H1N1 Influenza A Viruses into US Swine, 2009-2014 [PublishAheadOfPrint]

  • The diversity of influenza A viruses in swine (swIAVs) presents an important pandemic threat. Knowledge of the human-swine interface is particularly important for understanding how viruses with pandemic potential evolve in swine hosts. Through phylogenetic analysis of contemporary swIAVs in the United States, we demonstrate that human-to-swine transmission of pandemic H1N1 viruses (pH1N1) has occurred continuously in the years following the 2009 H1N1 pandemic and has been an important contributor to the genetic diversity of US swIAVs. Although pandemic H1 and N1 segments were largely removed from the US swine population by 2013 via reassortment with other swIAVs, these antigens re-emerged following multiple human-to-swine transmission events during the 2013-2014 seasonal epidemic. These findings indicate that the six internal gene segments from pH1N1 viruses are likely to be sustained long-term in the US swine population, with periodic re-emergence of pandemic HA and NA segments in association with seasonal pH1N1 epidemics in humans. Vaccinating US swine workers may reduce infection of both humans and swine, and in turn limit the role of humans as sources of influenza virus diversity in pigs.

    Importance Swine are important hosts in the evolution of influenza A viruses with pandemic potential. Here we analyze influenza virus sequence data generated by the US Department of Agriculture's national surveillance system to identify the central role of humans in the re-emergence of pandemic H1N1 influenza viruses (pH1N1) in US swine herds in 2014. These findings emphasize the important role of humans as continuous sources of influenza virus diversity in swine, and indicate that influenza viruses with pandemic HA and NA segments are likely to continue to re-emerge US swine in association with seasonal pH1N1 epidemics in humans.

  • Crystal structures of yeast-produced enterovirus 71 and enterovirus 71/coxsackievirus A16 chimeric virus-like particles provide structural basis for novel vaccine design against hand-foot-and-mouth disease [PublishAheadOfPrint]

  • Human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the two major causative agents for hand-foot-and-mouth diseases (HFMD). Previously we demonstrated that a virus-like particle (VLP) for EV71 produced from Saccharomyces cerevisiae is a potential vaccine candidate against EV71 infection, and an EV71/CVA16 chimeric VLP can elicit protective immune response against both virus infections. Here we presented the crystal structures of both VLPs, showing that both the linear and conformational neutralization epitopes identified in EV71 are mostly preserved on both VLPs. The substitution of only 4 residues in the VP1 GH loop converted strongly negative charged surface patches formed by portions of the SP70 epitope in EV71 VLP into a relatively neutral surface in the chimeric VLP, which likely accounted for the additional neutralization capability of the chimeric VLP against CVA16 infection. Such local variations in the amino acid sequences and the surface charge potential are also present in different types of polioviruses. In comparison to EV71 VLP, the chimeric VLP exhibits structural changes at the local site of amino acid replacement and the surface loops of all capsid proteins. This is consistent with the observation that the VP1 GH loop located near the pseudo 3-fold junction is involved in extensive interactions with other capsid regions. Furthermore, portions of VP0 and VP1 in EV71 VLP are at least transiently exposed, revealing the structural flexibility of the VLP. Together, our structural analysis provided insights into the structural basis of enterovirus neutralization and novel vaccine design against HFMD and other enterovirus-associated diseases.

    Importance Our previous studies demonstrated that the EV71 VLP produced from yeast is a vaccine candidate against EV71 infection and a chimeric EV71/CVA16 VLP with the substitution of 4 amino acids in the VP1 GH loop can confer protection against both EV71 and CVA16 infections. This study reported the crystal structures of both the EV71 VLP and the chimeric EV71/CVA16 VLP, and revealed that the major neutralization epitopes of EV71 are mostly preserved in both VLPs. In addition, the mutated VP1 GH loop in the chimeric VLP is well exposed on the particle surface and exhibits different surface charge potential from that contributed by the original VP1 GH loop in EV71 VLP. Together, this study provided insights into the structural basis of enterovirus neutralization and evidence that the yeast-produced VLPs can be developed into novel vaccines against HFMD and other enterovirus-associated diseases.

  • Morphogenesis of the ER membrane-invaginated vesicles during Beet black scorch virus infection: The role of auxiliary replication protein and new implications of three-dimensional architecture [PublishAheadOfPrint]

  • All well-characterized positive-strand RNA viruses induce the formation of host membrane-bound viral replication complexes (VRCs), yet the underlying mechanism and machinery for VRC formation remains elusive. We report here the biogenesis and topology of the Beet black scorch virus (BBSV) replication complex. Distinct cytopathological changes typical of endoplasmic reticulum (ER) aggregation and vesiculation were observed in BBSV-infected Nicotiana benthamiana cells. Immunogold labeling of the auxiliary replication protein p23 and dsRNA revealed that the ER-derived membranous spherules provide the site for BBSV replication. Further studies indicated that the p23 plays a crucial role in mediating the ER rearrangement. Three-dimensional electron tomographic analysis revealed the formation of multiple ER-originated vesicle packets. Each vesicle packet enclosed few to hundreds of independent spherules that were invaginations of the ER membranes into the lumen. Strikingly, these vesicle packets were connected to each other via tubule, a rare rearrangement event among other virus-induced membrane reorganizations. Fibrillar contents within the spherules were also reconstructed by electron tomography, which shows diverse structures. Our results provide the first, to our knowledge, three-dimensional ultrastructural analysis of membrane-bound VRCs of a plant (+)RNA virus and should aid a better mechanistic understanding of the organization and microenvironment of plant (+)RNA viruses replication complexes.

    Importance Assembly of virus replication complexes for all known positive-strand RNA viruses depends on the extensive remodeling of host intracellular membranes. Beet black scorch virus, a necrovirus in the Tombusviridae, invaginates the endoplasmic reticulum (ER) membranes to form spherules in infected cells. Double-stranded RNAs, the viral replication intermediate, and the viral auxiliary replication protein p23 (p23) are all localized within such viral spherules, indicating that these are the sites for generating progeny viral RNAs. Furthermore, BBSV p23 protein could to some extent reorganize the ER when transiently expressed in N. benthamiana. Electron tomographic analysis resolves the three-dimensional (3D) architecture of such spherules, which are connected to the cytoplasm via a neck-like structure. Strikingly, different numbers of spherules are enclosed in ER-originated vesicle packets that are interconnected to each other via tubule-like structures. Our results provide significant implications for further understanding the mechanisms underlying the replication of positive-strand RNA viruses.

  • SIVsab Infection of Rhesus Macaques as a Model of Complete Immunological Suppression with Persistent Reservoirs of Replication-Competent Virus: Implications for Cure Research [PublishAheadOfPrint]

  • SIVsab infection is completely controlled in rhesus macaques (RMs) through functional immune responses. We report that in SIVsab-infected RMs: (i) viral replication is controlled to llt;0-3 copies/ml; (ii) about 1/3 of the virus strains in reservoirs are replication-incompetent; (iii) rebounding virus after CD8+ cell depletion is replication-competent and genetically similar to the original virus stock, suggesting early reservoir seeding. This model permits assessment of strategies aimed at depleting the reservoir without multidrug antiretroviral therapy.

  • Equine herpesvirus type 1 (EHV-1) multiply transmembrane protein pUL43 cooperates with pUL56 in downregulation of cell surface MHC class I [PublishAheadOfPrint]

  • Herpesviruses have evolved an array of strategies to counteract antigen presentation by major histocompatibility complex class I (MHC-I). Previously, we have identified pUL56 of equine herpesvirus type 1 (EHV-1) as one major determinant of downregulation of cell surface MHC-I (G. Ma, S. Feineis, N. Osterrieder, and G. R. Van de Walle, J. Virol. 86:3554nndash;3563, 2012, doi:10.1128/JVI.06994-11; T. Huang, M. J. Lehmann, A. Said, G. Ma, and N. Osterrieder, J. Virol. 88:12802-12815, 2014, doi:10.1128/JVI.02079-14). Since pUL56 was able to exert its function only in the context of virus infection, we hypothesized that pUL56 cooperates with another viral protein. Here, we generated and screened a series of EHV-1 single gene deletion mutants and found that the pUL43 orthologue was required for downregulation of cell surface MHC-I expression at the same time of infection when pUL56 exerts its function. We demonstrate that the absence of pUL43 was not deleterious to virus growth and that expression of pUL43 was detectable from 2 h post-infection (h p.i.), but decreased after 8 h p.i due to lysosomal degradation. pUL43 localized within Golgi vesicles and required a unique hydrophilic N-terminal domain to function properly. Finally, co-expression of pUL43 and pUL56 in transfected cells reduced cell surface expression of MHC-I. This process was dependent on PPxY motifs present in pUL56, suggesting that late domains are required for pUL43- and pUL56-dependent sorting of MHC class I for lysosomal degradation.

    IMPORTANCE We describe here that the poorly characterized herpesviral pUL43 is involved in downregulation of cell surface MHC-I. pUL43 is an early protein and degraded in lysosomes. pUL43 resides in the Golgi and needs an intact N-terminus to induce MHC-I downregulation in infected cells. Importantly, pUL43 and pUL56 cooperate to reduce MHC-I expression on the surface of transfected cells. Our results suggest a model for MHC-I downregulation in which late domains in pUL56 are required for rerouting of vesicles containing MHC-I, pUL56 and pUL43 to the lysosomal compartment.

  • Quantification of HCV cell-to-cell spread using a stochastic modeling approach [PublishAheadOfPrint]

  • It has been proposed that viral cell-to-cell transmission plays a role in establishing and maintaining chronic infections. Thus, understanding the mechanisms and kinetics of cell-to-cell spread are fundamental to elucidating the dynamics of infection and may provide insight into factors that determine chronicity. Because hepatitis C virus (HCV) spreads cell-to-cell and HCV has a chronicity rate of up to 80% in exposed individuals, we examined the dynamics of HCV cell-to-cell spread in vitro and quantified the effect of inhibiting individual host factors. Using a multidisciplinary approach, we performed HCV spread assays and assessed the appropriateness of different stochastic models for describing HCV foci expansion. To evaluate the effect of blocking specific host cell factors on HCV cell-to-cell transmission, assays were performed in the presence of blocking antibodies and/or small molecule inhibitors targeting different cellular HCV entry factors. In all experiments, HCV positive cells were identified by immunohistochemical staining and the number of HCV-positive cells per focus was counted to determine focus size. We found that HCV foci expansion can best be explained by mathematical models assuming foci size-dependent growth. Consistent with previous reports suggesting some factors impact HCV cell-to-cell spread to different extents, modeling results estimate a hierarchy of efficacies for blocking HCV cell-to-cell spread when targeting different host factors (e.g. CLDN1 ggt; NPC1L1 ggt; TfR1). This approach can be adapted to describe foci expansion dynamics under a variety of experimental conditions as a means to quantify cell-to-cell transmission and assess the impact of cellular factors, viral factors and antivirals.

    Importance: The ability of viruses to efficiently spread by direct cell-to-cell transmission is thought to play an important role the establishment and maintenance of viral persistence. As such, elucidating the dynamics of cell-to-cell spread and quantifying the effect of blocking the factors involved has important implications for the design of potent antiviral strategies and controlling viral escape. Mathematical modeling has been widely used to understand HCV infection dynamics and treatment response, however these models typically assume only cell-free virus infection mechanisms. Here, we used stochastic models describing foci expansion as a means to understand and quantify the dynamics of HCV cell-to-cell spread in vitro and determined the degree to which cell-to-cell spread is reduced when individual HCV entry factors are blocked. The results presented demonstrate the ability of this approach to recapitulate and quantify cell-to-cell transmission, as well as the impact of specific factors and potential antivirals.

  • Virus-like particle system identifies the endonuclease domain of Crimean-Congo hemorrhagic fever virus [PublishAheadOfPrint]

  • Crimean-Congo Hemorrhagic Fever virus (CCHFV; genus Nairovirus) is an extremely pathogenic member of the Bunyaviridae family. Since handling of the virus requires a BSL-4 facility, little is known about pathomechanisms and host interactions. Here, we describe the establishment of a transcriptionally competent (tc)-VLP system for CCHFV. Recombinant polymerase (L), nucleocapsid protein (N) and a reporter minigenome expressed in human HuH-7 cells resulted in formation of transcriptionally active nucleocapsids that could be packaged by co-expressed CCHFV glycoproteins into tc-VLPs. The tc-VLPs resembled authentic virus particles in their protein composition and neutralization sensitivity to anti-CCHFV antibodies, and could recapitulate all steps of the viral replication cycle. Particle attachment, entry and primary transcription were modelled by infection of naive cells. The subsequent steps of genome replication, secondary transcription, and particle assembly and release can be obtained upon passaging the tc-VLPs on cells expressing CCHFV structural proteins. The utility of the VLP system was demonstrated by showing that the endonuclease domain of L is located around amino acid D693, as it was predicted in silico by Morin et al. (PLoS Pathogens 6: e1001038). The tc-VLP system will greatly facilitate studies and diagnostics of CCHFV under non-BSL-4 conditions.

    Importance Crimean-Congo Hemorrhagic Fever virus (CCHFV) is an extremely virulent pathogen of humans. Since the virus can only be handled at the highest biosafety level, research is restricted to a few specialized laboratories. We developed a plasmid-based system to produce virus-like particles with the ability to infect cells and transcribe a reporter genome. Due to the absence of viral genes, the virus-like particles are unable to spread or cause disease, thus allowing to study aspects of CCHFV biology under relaxed biosafety conditions.

  • Murine Norovirus replication induces a G0/G1 cell cycle arrest in asynchronous growing cells. [PublishAheadOfPrint]

  • Many viruses replicate most efficiently in specific phases of the cell cycle, establishing or exploiting favourable conditions for viral replication, although little is known about the relationship between caliciviruses and the cell cycle. Microarray and Western blot analysis of Murine norovirus-1 (MNV-1) infected cells showed changes in cyclin transcript and protein levels indicative of a G1 phase arrest. Cell cycle analysis confirmed that MNV-1 infection caused a prolonging of the G1 phase and an accumulation of cells in the G0/G1 phase. The accumulation in G0/G1 phase was caused by a reduction in cell cycle progression through the G1/S restriction point, with MNV-1 infected cells released from a G1 arrest showing reduced cell cycle progression compared to mock infected cells. MNV-1 replication was compared in populations of cells synchronised into specific cell cycle phases or in asynchronously growing cells. Cells actively progressing through the G1 phase had a 2-fold or higher increase in virus progeny and capsid protein expression over cells in other phases of the cell cycle or in unsynchronised populations. These findings suggest that MNV-1 infection leads to prolonging of the G1 phase and a reduction in S phase entry in host cells, establishing favourable conditions for viral protein production and viral replication. There is limited information on the interactions between noroviruses and the cell cycle and this observation of increased replication in the G1 phase may be representative of other members of the Caliciviridae.

    IMPORTANCE Noroviruses have proven recalcitrant to growth in cell culture, limiting our understanding of the interaction between these viruses and the infected cell. In this study we use the cell culturable MNV-1 to show that infection of murine macrophages affects the G1/S cell cycle phase transition, leading to an arrest in cell cycle progression and an accumulation of cells in the G0/G1 phase. Furthermore, we show that MNV replication is enhanced in the G1 phase compared to other stages of the cell cycle. Manipulating the cell cycle or adapting to cell cycle responses of the host cell is a mechanism to enhance virus replication. To the best of our knowledge this is the first report of a norovirus interacting with the host cell cycle and exploiting the favourable conditions of the G0/G1 phase for RNA virus replication.

  • Early kinetics of HLA class I-associated peptidome of MVA.HIVconsv-infected cells [PublishAheadOfPrint]

  • Cytotoxic T cells substantially contribute to the control of intracellular pathogens such as human immunodeficiency virus type 1 (HIV-1). Here, we evaluated the immunopeptidome of Jurkat cells infected with the vaccine candidate MVA.HIVconsv which delivers HIV-1 conserved antigenic regions using modified vaccinia Ankara (MVA). We employed liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify 6,358 unique peptides associated with class I human leukocyte antigen (HLA), of which 98 peptides were derived from the MVA vector and 7 from the HIVconsv immunogen. Human vaccine recipients responded to the peptide sequences identified by LC-MS/MS. Peptides derived from the conserved HIV-1 regions were readily detected as early as 1.5 hour after the MVA.HIVconsv infection. Four of the 7 conserved peptides were followed between 0 and 3.5 hours of infection using quantitative mass spectrometry (Q-MS) and their abundance in HLA class I association reflected levels of the whole HIVconsv protein in the cell. While immunopeptides delivered by the incoming MVA vector proteins could be detected, all early HIVconsv-derived immunopeptides were likely synthesized de novo. MVA.HIVconsv infection generally altered the composition of HLA class I-associated human (self) peptides, but these changes corresponded only partially to changes in the whole cell host protein abundance.

    IMPORTANCE The vast changes in cellular antigen presentation after infection of cells with a vectored vaccine, as shown here for MVA.HIVconsv, highlight the complexity of factors that need to be considered for efficient antigen delivery and presentation. Identification and quantitation of HLA class I-associated peptides by Q-MS will not only find broad application in T-cell epitope discovery, but also inform vaccine design and allow evaluation of efficient epitope presentation using different delivery strategies.

  • Influenza Virus-Induced Caspase-Dependent Enlargement of Nuclear Pores Promotes Nuclear Export of Viral Ribonucleoprotein Complexes [PublishAheadOfPrint]

  • Influenza A virus (IAV) replicates its segmented RNA genome in the nucleus of infected cells and utilizes caspase-dependent nucleocytoplasmic export mechanisms to transport newly formed ribonucleoprotein complexes (RNPs) to the site of infectious virion release at the plasma membrane. In this study, we obtained evidence that apoptotic caspase activation in IAV-infected cells is associated with the degradation of the nucleoporin Nup153, an integral subunit of the nuclear pore complex. Transmission electron microscopy studies revealed a distinct enlargement of nuclear pores in IVA-infected cells. Transient expression and subcellular accumulation studies of multimeric marker proteins in virus-infected cells provided additional evidence for increased nuclear pore diameters facilitating the translocation of large protein complexes across the nuclear membrane. Furthermore, caspase 3/7 inhibition data obtained in this study suggest that active, Crm1-dependent IAV RNP export mechanisms are increasingly complemented by passive, caspase-induced export mechanisms at later stages of infection.

    Importance In contrast to most other RNA viruses, influenza virus genome replication occurs in the nucleus (rather than cytoplasm) of infected cells. Therefore, completion of the viral replication cycle critically depends on intracellular transport mechanisms that ensure the translocation of viral ribonucleoprotein (RNP) complexes across the nuclear membrane. Here, we demonstrate that virus-induced cellular caspase activities cause a widening of nuclear pores, thereby facilitating nucleocytoplasmic translocation processes and, possibly, promoting nuclear export of newly synthesized RNPs. These passive transport mechanisms are suggested to complement Crm1-dependent RNP export mechanisms known to occur at early stages of the replication cycle and may contribute to highly efficient production of infectious virus progeny at late stages of the viral replication cycle. The study provides an intriguing example of how influenza virus exploits cellular structures and regulatory pathways, including intracellular transport mechanisms, to complete its replication cycle and maximize the production of infectious virus progeny.

  • Deep Sequencing and Phylogenetic Analysis of Variants Resistant to Interferon-Based Protease Inhibitor Therapy in Chronic Hepatitis Induced by Genotype-1b Hepatitis C Virus [PublishAheadOfPrint]

  • Because of recent advances in deep sequencing technology, detailed analysis of hepatitis C virus (HCV) quasispecies and its dynamic change in response to direct antiviral agents (DAAs) became possible although the role of quasispecies is not fully understood. In this study, to clarify the evolution of viral quasispecies and the origin of drug-resistant mutations induced by interferon-based protease inhibitor therapy, the nonstructural-3 (NS3) region of genotype 1b HCV in 34 chronic hepatitis patients treated with telaprevir (TVR)/pegylated-interferon (PEG-IFN)/ribavirin (RBV) was subjected to a deep sequencing study coupled with phylogenetic analysis. Twenty-six patients (76.5%) achieved a sustained viral response (SVR) while 8 patients did not (non-SVR, 23.5%). When the complexity of the quasispecies was expressed as mutation frequency or Shannon entropy, a significant decrease in the IFNL3 (rs8099917) TT group and a marginal decrease in the SVR group were found soon (12 h) after the introduction of treatment, whereas there was no decrease in the non-SVR group and no significant decrease in mutation frequency in the IFNL3 TG/GG group. In the analysis of viral quasispecies composition in non-SVR patients, major populations greatly changed accompanied by the appearance of resistance and the compositions were unlikely to return to the pretreatment composition even after the end of therapy. Clinically TVR-resistant variants were observed in 5 non-SVR patients (5/8, 62.5%), all of which were suspected to have acquired resistance by mutations through phylogenetic analysis. In conclusion, results of the study have important implications for treatment response and outcome in interferon-based protease inhibitor therapy.

    IMPORTANCE In the host, hepatitis C virus (HCV) consists of a variety of populations (quasispecies), and it is supposed that dynamic changes in quasispecies are closely related to pathogenesis although it is poorly understood. In this study, recently developed deep sequencing technology was introduced and changes in quasispecies associated with telaprevir (TVR)/pegylated interferon (PEG-IFN)/ribavirin (RBV) triple therapy and their clinical significance were investigated extensively by phylogenetic tree analysis. Through this study, the associations among treatment response, changes in viral quasispecies complexity in the early stage of treatment, changes in the quasispecies composition, and origin of TVR-resistant variant HCV were elucidated.

  • Pandemic swine H1N1 influenza viruses with almost undetectable neuraminidase activity do not transmit via aerosols in ferrets and are inhibited by human mucus, but not swine mucus. [PublishAheadOfPrint]

  • A balance between the functions of the influenza virus surface proteins hemagglutinin (HA) and neuraminidase (NA) is thought to be important for the transmission of viruses between humans. Here we describe two pandemic H1N1 viruses, A/swine/Virginia/1814-1/2012 and A/swine/Virginia/1814-2/2012 (pH1N1low-1 and -2, respectively) that were isolated from swine symptomatic for influenza. The enzymatic activity of the NA of these viruses was almost undetectable whilst the HA binding affinity for aalpha;2,6 sialic acids was greater than that of highly homologous pH1N1 viruses, A/swine/Pennsylvania/2436/2012 and A/swine/Minnesota/2499/2012 (pH1N1-1 and -2), which exhibited better balanced HA and NA activities. In vitro growth kinetics of pH1N1low and pH1N1 viruses were similar but aerosol transmission of pH1N1low-1 was abrogated and transmission via direct contact was significantly impaired in ferrets compared to pH1N1-1, which transmitted by direct and aerosol contact. In normal human bronchial epithelial cells, pH1N1low-1 was significantly inhibited by mucus but pH1N1-1 was not. In Madin-Darby canine kidney cell cultures overlaid with human or swine mucus, human mucus inhibited pH1N1low-1 but swine mucus did not. These data shows that the interaction between viruses and mucus may be an important factor in viral transmissibility and could be a barrier for interspecies transmission between humans and swine for influenza viruses.

    Importance A balance between the function of the influenza virus surface proteins hemagglutinin (HA) and neuraminidase (NA) is thought to be important for transmission of viruses from swine to humans. Here we show that a swine virus with extremely functionally mismatched HA and NAs (pH1N1low-1) cannot transmit via aerosol in ferrets, whilst another highly homologous virus with HA and NAs that are better matched functionally (pH1N1-1) can transmit via aerosol. These viruses show similar growth kinetics in Madin Darby canine kidney (MDCK) cells but pH1N1low-1 is significantly inhibited by mucus in normal human bronchial epithelial cells whilst pH1N1-1 is not. Further, human mucus could inhibit these viruses but swine mucus could not. These data shows that the interaction between viruses and mucus may be an important factor in viral transmissibility and could be a species barrier between humans and swine for influenza viruses.

  • Decoding viral infection by ribosome profiling [PublishAheadOfPrint]

  • Ribosome profiling is an emerging technique that uses deep sequencing to monitor translation in live cells. Studies using ribosome profiling have already provided novel insights into the identity and the amount of proteins that are being produced in cells, as well as novel insights into the mechanism of protein synthesis and translation regulation. Application of ribosome profiling on cells infected with HCMV and KSHV revealed an unanticipated complexity in herpesviruses coding capacity. Here, I discuss these results and how application of ribosome profiling to cell infected with other viruses can reveal novel insights into the process of infection.

  • Asymptomatic Middle East Respiratory Syndrome Coronavirus Infection in Rabbits [PublishAheadOfPrint]

  • The ability of Middle East respiratory syndrome coronavirus (MERS-CoV) to infect small animal species may be restricted given the fact that mice, ferrets and hamsters were shown to resist MERS-CoV infection. We inoculated rabbits with MERS-CoV. Although virus was detected in the lungs, neither significant histopathological changes nor clinical symptoms were observed. Infectious virus, however, was excreted from the upper respiratory tract, indicating a potential route of MERS-CoV transmission in some animal species.

  • African swine fever virus Georgia isolate harboring deletions of MGF360 and MGF505 genes is attenuated in swine and confers protection against challenge with the virulent parental virus [PublishAheadOfPrint]

  • African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The control of African Swine Fever (ASF) has been hampered by the unavailability of vaccines. Experimental vaccines have been developed using genetically modified-live attenuated ASFVs where viral genes involved in virus virulence were removed from the genome. Multigene families (MGF) 360 and 505 represent a group of genes sharing partial sequence and structural identities that have been connected with ASFV host range specificity, blocking of the host innate response, and virus virulence. Here we report the construction of a recombinant virus (ASFV-G-MGF) derived from the highly virulent ASFV Georgia 2007 isolate (ASFV-G) by specifically deleting six genes belonging to MGF360/MGF505: MGF505-1R, MGF360-12L, MGF360-13L, MGF-14L, MGF505-2R and MGF505-3R. ASFV-G-MGF replicates as efficiently in primary swine macrophage cell cultures as the parental virus. In vivo, ASFV-G-MGF is completely attenuated in swine, since pigs inoculated intramuscularly with either 102 or 104 HAD50 remained healthy without signs of the disease. Importantly, when these animals were subsequently exposed to highly virulent parental ASFV-G, no signs of the disease were observed although a proportion of these animals harbor the challenge virus. This is the first report demonstrating the role of MGF genes acting as independent determinants of ASFV virulence. Additionally, ASFV-G-MGF is the first experimental vaccine reported to induce protection when challenged with the highly virulent and epidemiologically relevant ASFV-G.

    Importance The main problem for controlling ASF is the lack of vaccines. Studies focusing on understanding ASFV virulence lead to the production of genetically modified recombinant viruses that while attenuated they are able to confer protection in pigs when challenged with homologous viruses. Here we have produced an attenuated recombinant ASFV derived from highly virulent ASFV strain Georgia (ASFV-G) lacking only six of the multi-gene family (MGF) 360 and 505 genes (ASFV-G-MGF). It is demonstrated, by first time, that deleting specific MGF genes alone can completely attenuated a highly virulent field ASFV isolate. The recombinant virus ASFV-G-MGF effectively confers protection in pigs against challenge with ASFV-G when delivered once via IM. The protection against ASFV-G is highly effective by 28 days post vaccination. This is the first report of an experimental vaccine that induces a solid protection against virulent ASFV-G.

  • The Function of the Herpes Simplex Virus 1 Small Capsid Protein VP26 is Regulated by Phosphorylation at a Specific Site. [PublishAheadOfPrint]

  • Replacement of the herpes simplex virus 1 small capsid protein VP26 phosphorylation site Thr-111 with alanine reduced viral replication and neurovirulence to levels observed with the VP26 null-mutation. This mutation reduced VP26 expression and mis-localized VP26 and its binding partner, the major capsid protein VP5, in the nucleus. VP5 mis-localization was also observed with the VP26 null-mutation. Thus we postulate that phosphorylation of VP26 at Thr-111 regulates VP26 function in vitro and in vivo.

  • The 5' untranslated region of a novel infectious molecular clone of the dicistrovirus Cricket paralysis virus modulates infection [PublishAheadOfPrint]

  • Dicistroviridae are a family of RNA viruses that possesses a single-stranded positive-sense RNA genome containing two distinct open reading frames (ORFs) each preceded by an internal ribosome entry site that drives translation of the viral structural and non-structural proteins, respectively. The type species, Cricket paralysis virus (CrPV), has served as a model for studying host-virus interactions; however, investigations into the molecular mechanisms of CrPV and other dicistroviruses have been limited, as an established infectious clone was elusive. Here, we report the construction of an infectious molecular clone of CrPV. Transfection of in vitro transcribed RNA from the CrPV clone into Drosophila S2 cells resulted in cytopathic effects, viral RNA accumulation, detection of negative sense viral RNA and expression of viral proteins. Transmission electron microscopy, viral titres and immunofluorescence-coupled transwell assays demonstrated that infectious viral particles are released from transfected cells. In contrast, mutant clones containing stop codons in either ORFs decreased virus infectivity. Injection of adult Drosophila flies with virus derived from CrPV clones but not UV-inactivated clones resulted in mortality. Molecular analysis of the CrPV clone revealed a 196-nucleotide duplication within its 5rrsquo; UTR that stimulated translation of reporter constructs. In cells infected with the CrPV clone, the duplication inhibited viral infectivity yet did not affect viral translation nor RNA accumulation, suggesting an effect on viral packaging or entry. The generation of the CrPV infectious clone provides a powerful tool for investigating the viral life cycle and pathogenesis of dicistroviruses and further understanding of fundamental host-virus interactions in insect cells.

    IMPORTANCE Dicistroviridae, which are RNA viruses that infect arthropods, have served as a model to gain insights into fundamental host-virus interactions in insect cells. Further insights into the viral molecular mechanisms are hampered due to a lack of an established infectious clone. We report the construction of the first infectious clone of the dicistrovirus, Cricket paralysis virus (CrPV). We show that transfection of the CrPV clone RNA into Drosophila cells led to production of infectious particles that resemble natural CrPV virions and result in cytopathic effects, expression of CrPV proteins and RNA in infected cells. The CrPV clone should provide insights into the dicistrovirus life cycle and host-virus interactions in insect cells. Using this clone, we find that a 196-nucleotide duplication within the 5rrsquo; untranslated region of the CrPV clone increases viral translation in reporter constructs but decreased virus infectivity, thus revealing a balance that interplays between viral translation and replication.

  • Borna disease virus phosphoprotein modulates epigenetic signaling in neurons to control viral replication [PublishAheadOfPrint]

  • Understanding the modalities of interaction of neurotropic viruses with their target cells represents a major challenge that may improve our knowledge of many human neurological disorders for which viral origin is suspected. Borna disease virus (BDV) represents an ideal model to analyze the molecular mechanisms of viral persistence in neurons and its consequences for neuronal homeostasis. It is now established that BDV ensures its long-term maintenance in infected cells through a stable interaction of viral components with the host cell chromatin, in particular with core histones. This has led to our hypothesis that such an interaction may trigger epigenetic changes in the host cell. Here, we focused on histone acetylation, which play key roles in epigenetic regulation of gene expression, notably for neurons. We performed a comparative analysis of histone acetylation patterns of neurons infected or not by BDV, which revealed that infection decreases histone acetylation on selected lysine residues. We showed that the BDV phosphoprotein (P) is responsible for these perturbations, even when expressed alone independently of the viral context, and that this action depends on its phosphorylation by protein kinase C. We also demonstrated that BDV P inhibits cellular histone acetyl transferase activities. Finally, by pharmacologically manipulating cellular acetylation levels, we observed that inhibiting cellular acetyl transferases reduces viral replication in cell culture. Our findings reveal that manipulation of cellular epigenetics by BDV could be a mean to modulate viral replication and thus illustrate a fascinating example of virus/host cell interaction.

    Importance Persistent DNA viruses often subvert the mechanisms that regulate cellular chromatin dynamics, thereby benefitting from the resulting epigenetic changes to create a favorable milieu for their latent/persistent states. Here, we reasoned that Borna Disease Virus (BDV), the only RNA virus known to durably persist in the nucleus of infected cells, notably neurons, might employ a similar mechanism. In this study, we uncover a novel modality of virus/cell interaction in which BDV phosphoprotein inhibits cellular histone acetylation by interfering with histone acetyl transferase activities. Manipulation of cellular histone acetylation is accompanied by a modulation of viral replication, revealing the perfect adaptation of this "ancient" virus to its host that may favor neuronal persistence and limit cellular damage.

  • Structure of serotype 1 reovirus attachment protein {sigma}1 in complex with JAM-A reveals a conserved serotype-independent binding epitope [PublishAheadOfPrint]

  • Mammalian orthoreoviruses use glycans and junctional adhesion molecule-A (JAM-A) as attachment receptors. We determined the structure of serotype 1 reovirus attachment protein 1 alone and in complex with JAM-A. Comparison with the structure of serotype 3 reovirus 1 bound to JAM-A reveals that both 1 proteins engage JAM-A with similar affinities and via conserved binding epitopes. Thus, 1-JAM-A interactions are unlikely to explain the differences in pathogenesis displayed by these reovirus serotypes.

  • Increased Infectivity of Anchorless Mouse Scrapie Prions in Transgenic Mice Overexpressing Human Prion Protein [PublishAheadOfPrint]

  • Prion protein (PrP) is found in all mammals mostly as a glycoprotein anchored to the plasma membrane by a C-terminal glycophosphatidylinositol (GPI) linkage. Following prion infection, host protease-sensitive prion protein (PrPsen or PrPC) is converted into an abnormal, disease-associated, protease-resistant form (PrPres). Biochemical characteristics such as the PrP amino acid sequence and post-translational modifications such as glycosylation and GPI anchoring, can affect the transmissibility of prions as well as the biochemical properties of the PrPres generated. Previous in vivo studies on the effects of GPI anchoring on prion infectivity have not examined cross-species transmission. Here we tested the effect of lack of GPI anchoring on a species barrier model using mice expressing human PrP. In this model, anchorless 22L prions derived from tg44 mice were more infectious than 22L prions derived from C57BL/10 mice when tested in tg66 transgenic mice, which expressed wild-type anchored human PrP at 8-16 fold above normal. Thus the lack of the GPI anchor on the PrPres from tg44 mice appeared to reduce the effect of the mouse-human PrP species barrier. In contrast, neither source of prions induced disease in tgRM transgenic mice which expressed human PrP at 2-4 fold above normal.

    Importance Prion protein (PrP) is found in all mammals, usually attached to cells by an anchor molecule, called GPI. Following prion infection, PrP is converted into a disease-associated form (PrPres). While most prion diseases are species-specific, this finding is not consistent, and species barriers differ in strength. The amino acid sequence of PrP varies among species, and this variability affects prion species barriers. However, other PrP modifications, including glycosylation and GPI-anchoring, may also influence cross-species infectivity. We studied the effect of PrP GPI-anchoring using a mouse to human species barrier model. Experiments showed that prions produced by mice expressing only anchorless PrP were more infectious than prions produced in mice expressing anchored PrP. Thus, the lack of the GPI anchor on prions reduced the effect of the mouse-human species barrier. Our results suggest that prion diseases that produce higher levels of anchorless PrP may pose an increased risk for cross-species infection.

  • What do chaotrope-based avidity assays for antibodies to HIV-1 envelope glycoproteins measure? [PublishAheadOfPrint]

  • When HIV-1 vaccine candidates that include soluble envelope glycoproteins (Env) are tested in humans and other species, the resulting antibody responses to Env are sifted for correlates of protection or risk. One frequently used assay measures the reduction in antibody binding to Env antigens by an added chaotrope (such as thiocyanate). Based on that assay, an avidity index was devised for assessing the affinity maturation of antibodies of unknown concentration in polyclonal sera. Since a high avidity index was linked to protection in animal models of HIV-1 infection, it has become a criterion for evaluating antibody responses to vaccine candidates. But what does the assay measure and what does an avidity index mean? Here, we have used a panel of monoclonal antibodies to well-defined epitopes on Env (gp120, gp41, and SOSIP.664 trimers) to explore how the chaotrope acts. We conclude that the chaotrope sensitivity of antibody binding to Env depends on several properties of the epitopes (continuity vs. tertiary- and quaternary-structural dependence), and that the avidity index has no simple relationship to antibody affinity for functional Env spikes on virions. We show that the binding of broadly neutralizing antibodies against quaternary-structural epitopes is particularly sensitive to chaotrope treatment, whereas antibody binding to epitopes in variable loops and to non-neutralization epitopes in gp41 is generally resistant. As a result of such biases, the avidity index may at best be a mere surrogate for undefined antibody or other immune responses that correlate weakly with protection.

    Importance An effective HIV-1 vaccine is an important goal. Such a vaccine will probably need to induce antibodies that neutralize typically transmitted variants of HIV-1, i.e., preventing them from infecting target cells. Vaccine candidates have so far failed to induce such antibody responses, although some do protect weakly against infection in animals and possibly humans. In the search for responses associated with protection, an avidity assay based on chemical disruption is often used to measure the strength of antibody binding. We have analyzed this assay mechanistically and found that the epitope specificity of an antibody has a greater influence on the outcome than does its affinity. As a result, the avidity assay is biased towards the detection of some antibody specificities while disfavoring others. We conclude that the assay may yield merely indirect correlations with weak protection, specifically when Env vaccination has failed to induce broad neutralizing responses.

  • CpG Oligodeoxynucleotides Facilitate Delivery of Whole Inactivated H9N2 Influenza Virus via Transepithelial Dendrites of Dendritic Cells in Nasal Mucosa [PublishAheadOfPrint]

  • The spread of the low pathogenic avian H9N2 influenza virus has seriously increased the risk of a new influenza pandemic. Although whole inactivated virus (WIV) vaccine via intranasal pathway is the effective method of blocking virus transmission, mucosal barrier seems to a major development-hampering factor. CpG oligodeoxynucleotides (CpG ODN), a known adjuvant, can target downstream dendritic cells (DCs) and effectively enhance the mucosal and systemic immune responses. However, the ability of CpG ODN to assist H9N2 WIV in transepithelial transport remains unknown. Here, in vitro and in vivo, we showed that CpG ODN provided assistance for H9N2 WIV in recruiting DCs to the nasal epithelial cells (ECs), and forming transepithelial dendrites (TEDs) to capture luminal viruses. CD103+ DCs participated in this process. Chemokine CCL20 from nasal ECs played a key role in driving DCs recruitment and TEDs formation. Viruses-loaded DCs quickly migrated into the draining cervical lymph nodes (CLNs) for antigen presentation. In addition, the competence of CpG ODN was independent of direct epithelial transport via transcellular or paracellular pathway. Taken together, our data demonstrated that CpG ODN enhanced the transport of H9N2 WIV via TEDs of nasal DCs, which might be a novel mechanism for the optimal adaptive immune responses.

    Importance This paper demonstrates both in vivo and in vitro coculture model that CpG ODN, a known adjuvant that generally target downstream immune responses, also are crucial for transport of H9N2 WIV across nasal epithelial cells (ECs) via transepithelial dendrites (TEDs) uptake. Our results prove for the first time to our knowledge that the immune-potentiating mechanism of CpG ODN is based on to strengthen transepithelial uptake of H9N2 WIV in nasal mucosa. These findings provide a fresh perspective for further improvement of intranasal influenza vaccines, which are urgently needed in the face of the potential threat of H9N2 influenza.

  • The association of Kaposi's sarcoma-associated herpesvirus ORF31 with ORF34 and ORF24 is critical for late gene expression [PublishAheadOfPrint]

  • Transcription of herpesvirus late genes depends on several virus-encoded proteins whose function is not completely understood. Here, we identify a viral trimeric complex of Kaposi's sarcoma-associated herpesvirus (KSHV) ORF31, ORF24 and ORF34 that is required for late gene expression but not viral DNA replication. We found that (i) ORF34 bridges the interaction between ORF31 and ORF24, (ii) the amino-terminal cysteine-rich and carboxyl-terminal basic domains of ORF31 mediate ORF31-ORF34 interaction required for late gene expression, and (iii) a complex consisting of ORF24, ORF31 and ORF34 specifically binds to the K8.1 late promoter. Together, our results support the model that a subset of lytic viral proteins assembles into a transcriptional activator complex to induce expression of late genes.

  • Influenza A Virus Panhandle Structure is Directly Involved in RIG-I Activation and IFN Induction [PublishAheadOfPrint]

  • Retinoic acid-inducible gene I (RIG-I) is an important innate immune sensor that recognizes viral RNA in the cytoplasm. Its non-self recognition largely depends on the unique RNA structures imposed by viral RNA. The panhandle structure residing in the influenza A virus (IAV) genome, whose primary function is to serve as the viral promoter for transcription and replication, has been proposed to be a RIG-I agonist. However, it has never been proved experimentally. Here, we employed multiple approaches to determine if the IAV panhandle structure is directly involved in RIG-I activation and type I interferon (IFN) induction. First, in porcine alveolar macrophages, we demonstrated that viral genomic coding region is dispensable for RIG-I-dependent IFN induction. Second, using in vitro synthesized hairpin RNA, we showed that the IAV panhandle structure could directly bind to RIG-I and stimulate IFN production. Furthermore, we investigated the contribution of the wobble base pairs, mismatch, and unpaired nucleotide within the wild-type panhandle structure to RIG-I activation. Elimination of these destabilizing elements within the panhandle structure promoted RIG-I activation and IFN induction. Given the function of the panhandle structure as the viral promoter, we further monitored the promoter activity of these panhandle variants and found that the viral replication was moderately affected whereas the viral transcription was impaired dramatically. In all, our results indicate that the IAV panhandle promoter region adopts a nucleotide composition that is optimal for balanced viral RNA synthesis and suboptimal for RIG-I activation.

    IMPORTANCE The IAV genomic panhandle structure has been proposed to be the RIG-I agonist due to its partial complementarity; however, it has not been experimentally confirmed. Here, we provided direct evidence that the IAV panhandle structure is competent in, and sufficient for RIG-I activation and IFN induction. By constructing panhandle variants with increased complementarity, we demonstrated that wild-type panhandle structure could be modified to enhance RIG-I activation and IFN induction. These panhandle variants posed moderate influence on viral replication but dramatic impairment on viral transcription. These results indicate that the IAV panhandle promoter region adopts a nucleotide composition to achieve optimal balance of viral RNA synthesis and suboptimal RIG-I activation. Our results highlight the multifunctional role of the IAV panhandle promoter region in the virus life cycle, and offer novel insights into the development of antiviral agents aiming to boost RIG-I signaling or virus attenuation by manipulating this conserved region.

  • Dissection of Amino-Terminal Functional Domains of Murine Coronavirus Nonstructural Protein 3 [PublishAheadOfPrint]

  • Coronaviruses, the largest RNA viruses, have a complex program of RNA synthesis that entails genome replication and transcription of subgenomic mRNAs. RNA synthesis by the prototype coronavirus mouse hepatitis virus (MHV) is carried out by a replicase-transcriptase composed of 16 nonstructural protein (nsp) subunits. Among these, nsp3 is the largest and the first to be inserted into the endoplasmic reticulum. Nsp3 comprises multiple structural domains, including two papain-like proteases (PLPs) and a highly conserved ADP-ribose-1"-phosphatase (ADRP) macrodomain. We have previously shown that the ubiquitin-like domain at the amino terminus of nsp3 is essential and participates in a critical interaction with the viral nucleocapsid protein early in infection. In the current study, we exploited atypical expression schemes to uncouple PLP1 from the processing of nsp1 and nsp2 in order to investigate the requirements of nsp3 domains for viral RNA synthesis. In the first strategy a mutant was created in which replicase polyprotein translation initiated with nsp3, thereby establishing that complete elimination of nsp1 and nsp2 does not abolish MHV viability. In the second strategy a picornavirus autoprocessing element was used to separate a truncated nsp1 from nsp3. This provided a platform for further dissection of amino-terminal domains of nsp3. From this we found that catalytic mutation of PLP1 or complete deletion of PLP1 and the adjacent ADRP domain was tolerated by the virus. These results showed that neither the PLP1 or ADRP domains of nsp3 provide integral activities essential for coronavirus genomic or subgenomic RNA synthesis.

    IMPORTANCE The largest component of the coronavirus replicase-transcriptase complex, nsp3, contains multiple modules, many of which do not have clearly defined functions in genome replication or transcription. These domains may play direct roles in RNA synthesis, or they may have evolved for other purposes, such as to combat host innate immunity. We initiated a dissection of MHV nsp3 aimed at identifying those activities or structures in this huge molecule that are essential to replicase activity. We found that both PLP1 and ADRP could be entirely deleted, provided that the requirement for proteolytic processing by PLP1 was offset by an alternative mechanism. This demonstrated that neither PLP1 nor ADRP plays an essential role in coronavirus RNA synthesis.

  • Maintenance of Epstein-Barr viral latent status by a novel mechanism, LMP1-induced Interleukin-32 via the protein kinase C{delta} pathway [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV), an oncogenic herpesvirus, has the potential to immortalize primary B cells into lymphoblastoid cell lines (LCLs) in vitro. During immortalization, several EBV products induce cytokines or chemokines and most of these are required for the proliferation of LCLs. IL-32, a recently discovered proinflammatory cytokine, is upregulated after EBV infection and this upregulation is detectable in all LCLs tested. EBV latent membrane protein 1 (LMP1) is responsible for inducing IL-32 expression at the mRNA and protein levels. Mechanistically, we showed that this LMP1-induction is provided by the p65 subunit of NF-B, which binds to and activates the IL-32 promoter. Furthermore, the shRNA-mediated depletion of endogenous LMP1 and p65 in LCLs suppressed IL-32 expression, further suggesting that LMP1 is the key factor that stimulates IL-32 in LCLs via the NF-B p65 pathway. Functionally, knockdown of IL-32 in LCLs elicits viral reactivation and affects cytokine expression, but has no impact on cell proliferation and apoptosis. Of note, we reveal the mechanism whereby IL-32 is involved in the maintenance of EBV viral latency by inactivation of Zta promoter activity. This atypical cytoplasmic IL-32 hijacks the Zta activator PKC and inhibits its translocation from the cytoplasm to the nucleus, where PKC binds to the Zta promoter and activates lytic cycle progression. These novel findings reveal that IL-32 is involved in the maintenance of EBV latency in LCLs. This finding may provide new information to explain how EBV maintains latency, in addition to viral chromatin structure and epigenetic modification.

    Importance EBV persists in two states, latency and lytic replication, which is a unique characteristic of human infections. So far, little is known about how herpesviruses maintain latency in particular tissues or cell types. EBV is an excellent model to study this question because more than 90% of people are latently infected. EBV can immortalize primary B cells into lymphoblastoid cell lines in vitro. Expression of IL-32, a novel atypical cytoplasmic proinflammatory cytokine, increased after infection. The expression of IL-32 was controlled by LMP1. In investigating the regulatory mechanism, we demonstrated that the p65 subunit of NF-B is required for this upregulation. Of note, the important biological activity of IL-32 was to trap protein kinase C in the cytoplasm and prevent it binding to the Zta promoter, which is the key event for EBV reaction. So, the expression of LMP1-induced IL-32 plays a role in the maintenance of EBV latency.

  • The glycoprotein precursor gene of Junin virus determines the virulence of Romero strain and attenuation of Candid #1 strain in a representative animal model of Argentine Hemorrhagic Fever [PublishAheadOfPrint]

  • The New World arenavirus Junin (JUNV) is the causative agent of the Argentine hemorrhagic fever (AHF), a potentially deadly disease endemic to central regions of Argentina. The live-attenuated Candid #1 strain of JUNV is currently used to vaccinate the human population at risk. However, the mechanism of attenuation of this strain is still largely unknown. Therefore, the identification and functional characterization of viral genetic determinants dictating JUNV virulence or attenuation would significantly improve the understanding of the mechanisms underlying AHF and facilitate the development of novel, more effective and safer vaccines. Here, we utilized a reverse genetics approach to generate recombinant JUNV (rJUNV) encoding different gene combinations of the pathogenic Romero (Rom) and attenuated Candid #1 (Can) strains of JUNV. All rJUNV exhibited similar in vitro growth kinetics to their parental counterparts. Analysis of virulence of the rJUNV in a guinea pigs model of lethal infection that closely reproduces the features of AHF, identified the envelope glycoproteins (GPs) as the major determinants of pathogenesis and attenuation of JUNV. Accordingly, rJUNV expressing the full-length GPs of Rom and Can exhibited virulent and attenuated phenotypes, respectively, in guinea pigs. Mutation F427I in the transmembrane region of JUNV envelope glycoprotein GP2 has been shown to attenuate the neurovirulence of JUNV in suckling mice. We document that in the guinea pig model of AHF, mutation F427I in GP2 is also highly attenuating but insufficient to prevent virus dissemination and development of mild clinical and pathological symptoms, indicating that complete attenuation of JUNV requires additional mutations present in Can GPC.

    IMPORTANCE Development of antiviral strategies against viral hemorrhagic fevers, including AHF, is one of the top priorities within the Implementation Plan of the U.S. Department of Health and Human Services Public Health Emergency Medical Countermeasures Enterprise. Live-attenuated Candid #1 strain, derived from the 44th mouse brain passage of the prototype XJ strain of JUNV has been demonstrated to be safe, immunogenic, and highly protective, and is currently licensed for human use in Argentina. However, the bases for the attenuated phenotype of Candid #1 have not been established. Therefore, the identification and functional characterization of viral genetic factors implicated in JUNV pathogenesis and attenuation would significantly improve the understanding of the molecular mechanisms underlying AHF and facilitate the development of novel antiviral strategies.

  • Emergence of highly pathogenic avian influenza A(H5N1) virus PB1-F2 variants and their virulence in BALB/c mice. [PublishAheadOfPrint]

  • Influenza A viruses (IAV) express the PB1-F2 protein from an alternate reading frame within the PB1 gene segment. The roles of PB1-F2 are not well understood, but appear to involve modulation of host cell responses. As shown in previous studies, we find that PB1-F2 of mammalian IAV frequently have premature stop codons that are expected to cause truncations of the protein, whereas avian IAV usually express a full-length 90 amino acid PB1-F2. However, in contrast to other avian IAV, recent isolates of highly pathogenic H5N1 influenza viruses had a high proportion of PB1-F2 truncations (15% since 2010; 61% of isolates in 2013) due to several independent mutations that have persisted and expanded in circulating viruses. One natural H5N1 IAV containing a mutated PB1-F2 start codon (i.e., lacking ATG) was 1000-fold more virulent for BALB/c mice than a closely-related H5N1 containing intact PB1-F2. In vitro, we detected expression of an in-frame protein (C-terminal PB1-F2) from downstream ATGs in PB1-F2 plasmids lacking the well-conserved ATG start codon. Transient expression of full-length, truncated (25 amino acids), and PB1-F2 lacking the initiating ATG in mammalian and avian cells had no effect on cell apoptosis or interferon expression in human lung epithelial cells. Full length and C-terminal PB1-F2 mutants co-localized with mitochondria in A549 cells. Close monitoring of alterations of PB1-F2 and their frequency in contemporary avian H5N1 viruses should continue, as such changes may be markers for mammalian virulence.

    IMPORTANCE Although most avian influenza viruses are harmless for humans, some (such as highly pathogenic H5N1 avian influenza viruses) are capable of infecting humans and causing severe disease with a high mortality rate. A number of risk factors potentially associated with adaptation to mammalian infection have been noted. Here we demonstrate that the protein PB1-F2 is frequently truncated in recent isolates of highly pathogenic H5N1 viruses. Truncation of PB1-F2 has been proposed to act as an adaptation to mammalian infection. We show that some forms of truncation of PB1-F2 may be associated with increased virulence in mammals. Our data support the assessment of PB1-F2 truncations for genomic surveillance of influenza viruses.

  • What is the Reservoir of Emergent Human Norovirus Strains? [PublishAheadOfPrint]

  • Since 2002, there have been five human norovirus pandemics. All the pandemic strains are genetically related, segregating in the genogroup II, genotype 4 (GII.4) cluster within the norovirus genus. Evidence indicates that these strains are closely related but antigenically distinct, supporting immune-driven viral evolution. Thus norovirus vaccines will likely require periodic reformulation to protect from newly emergent strains. A major obstacle is that the reservoir of emergent strains is unknown. Noroviruses display tight species specificity and there is no evidence supporting zoonotic transmission so an animal reservoir is considered unlikely. Moreover, available data indicate minimal viral diversity in most natural human infections. In this Gem, we discuss the widely speculated idea that chronically infected immunocompromised individuals are norovirus reservoirs and provide a rationale for the theory that elderly and malnourished hosts may also represent norovirus reservoirs.

  • Passive Immunotherapy With Dromedary Immune Serum In An Experimental Animal Model For MERS Coronavirus Infection. [PublishAheadOfPrint]

  • The Middle East Respiratory Syndrome (MERS) is a highly lethal pulmonary infection. Sera from MERS convalescent patients may provide some benefit but is not readily available. In contrast, nearly all camels in the Middle East were previously infected with MERS-CoV. Here, we show that sera obtained from MERS immune camels augment the kinetics of MERS-CoV clearance, and reduce the severity of pathological changes in infected lungs, with efficacy proportional to MERS-CoV neutralizing serum antibody titers.

    Importance. The Middle East Respiratory Syndrome, caused by a coronavirus, is highly lethal with a case-fatality rate of 35-40%. No specific therapy is available and care is generally supportive. One promising approach is passive administration of sera from MERS convalescent humans or other animals to exposed or infected patients. The vast majority if not all camels in the Middle East were previously infected with MERS-CoV and some contain high titers of antibody to the virus. Here, we show that this antibody is protective if delivered either prophylactically or therapeutically to mice infected with MERS-CoV, indicating that this may be a useful intervention in infected patients.

  • Receptor usage and cell entry of porcine epidemic diarrhea coronavirus [PublishAheadOfPrint]

  • Porcine epidemic diarrhea coronavirus (PEDV) has significantly damaged America's pork industry. Here we investigated the receptor usage and cell entry of PEDV. PEDV recognizes protein receptor aminopeptidase N from pig and human, and sugar co-receptor N-acetylneuraminic acid. Moreover, PEDV infects cells from pig, human, monkey, and bat. These results support bats as an evolutionary origin for PEDV, implicate PEDV as a potential threat to other species, and suggest antiviral strategies to control its spread.

  • Highly divergent hepaciviruses from African cattle [PublishAheadOfPrint]

  • The hepatitis C virus (HCV; genus Hepacivirus) is a highly relevant human pathogen. Unique hepaciviruses (HV) were discovered recently in animal hosts. The direct ancestor of HCV has not been found, but the genetically most closely related animal HVs exist in horses. To investigate whether other peri-domestic animals also carry HVs, we analyzed sera from Ghanaian cattle for HVs by RT-PCR. Nine of 106 specimens from different sampling sites contained HV RNA (8.5%) at median viral loads of 1.6x105 copies/mL. Infection was unrelated to cattle age and gender. Near-full genome sequencing of five representative viruses confirmed taxonomic classifications. Cattle HVs formed two distinct phylogenetic lineages that differed by up to 17.7% on nt level in the polyprotein-encoding region, suggesting existence of different virus subtypes. A conserved micro-RNA122 binding site in the 5rrsquo; -internal ribosomal entry site suggested liver tropism of cattle HVs. Phylogenetic analyses suggested circulation of HVs in cattle for several centuries. Cattle HVs were genetically highly divergent from all other HVs, including HCV. HVs from genetically related equine and bovine hosts were not monophyletic, corroborating host shifts during the evolution of the genus Hepacivirus. Similar to equine HVs, the genetic diversity of cattle HVs was low compared to HCV genotypes. This may suggest an influence of the human-modified ecology of peri-domestic animals on virus diversity. Further studies should investigate the occurrence of cattle HVs in other geographic areas and breeds, virus pathogenicity in cattle and the potential exposure of human risk groups such as farmers, butchers and abattoir workers.

    Importance The hepatitis C virus (HCV; genus Hepacivirus) is a major human pathogen causing liver failure and cancer. Unique hepaciviruses (HVs) were discovered over the last years in animals, but the direct ancestor of HCV has not been found. The animal HV so most closely related to HCV so far originated from horses, suggesting that other livestock animals may also harbor HVs. Therefore, we investigated African cattle and discovered previously unknown HVs at high prevalence and viral loads. Because of the agricultural importance of cattle, it may be relevant to investigate HV pathogenicity. The frequent exposure of humans to cattle may also warrant investigations of the zoonotic potential of these viruses. Evolutionary analyses suggested that cattle HVs have existed for centuries. Despite the genetic relatedness of their animal hosts, HVs from cattle and horses were not phylogenetically related, corroborating frequent host shifts during the evolution of the genus Hepacivirus.

  • Mechanism of HIV-1 Resistance to Short Peptide Fusion Inhibitors Targeting the Gp41 Pocket [PublishAheadOfPrint]

  • The deep hydrophobic pocket on the N-trimer of HIV-1 gp41 has been considered as an ideal drug target. On the basis of the M-T hook structure, we recently developed short peptide-based HIV-1 fusion inhibitors (MTSC22 and HP23), which mainly target the pocket site and possess highly potent antiviral activity. In this study, we focused to investigate their resistance pathway and mechanisms by selected escape HIV-1 mutants to SC22EK, a template peptide for MTSC22 and HP23. Two substitutions, E49K and N126K located respectively at the N- and C-heptad repeat regions of gp41, were identified to confer high resistance to the inhibitors targeting the pocket and cross-resistance to the first-generation fusion inhibitor Enfuvirtide (T20) and the next-generation fusion inhibitor Sifuvirtide (SFT). The underlying mechanisms of SC22EK-induced resistance include: (i) the significantly reduced binding affinity of inhibitors; (ii) the dramatically enhanced interaction of viral 6-helix bundle; and (iii) the severely damaged functionality of viral Env omplex. Our data have provided important information for the structure-function relationship of gp41 and the structure-activity relationship of viral fusion inhibitors.

    Importance Enfuvirtide (T20) is the only HIV-1 fusion inhibitor in clinic but the problem of resistance significantly limits its use, calling for new strategies or concepts to develop next-generation drugs. On the basis of the M-T hook structure, short peptide HIV-1 fusion inhibitors specifically targeting the gp41 pocket site exhibit high binding and antiviral activities. Here, we investigated Herethe molecular pathway of HIV-1 resistance to the short inhibitors by selecting and mapping the escape mutants. The key substitutions for resistance and the underlying mechanisms have been finely characterized. The data provide important information for the structure-function relationship of gp41 and its inhibitors and will definitely help our future development for novel drugs that block gp41-dependent fusion.

  • A unique amino acid substitution in NS2A protein of Japanese encephalitis virus affects virus propagation in vitro but not in vivo [PublishAheadOfPrint]

  • We identified a unique amino acid of NS2A113 phenylalanine that affects the efficient propagation of two Japanese encephalitis virus strains, JaTH160 and JaOArS982 in neuroblastoma Neuro-2a cells but not in cell lines of extraneural origin. This amino acid did not affect viral loads in the brain nor survival curves in mice. These findings suggest that virus propagation in vitro may not reflect the level of virus neuroinvasiveness in vivo.

  • A Japanese Encephalitis virus genotype 5 molecular clone is highly neuropathogenic in a mouse model: implication of the structural proteins region in virulence [PublishAheadOfPrint]

  • Japanese Encephalitis virus (JEV) can be separated into 5 genotypes (g1 to g5) based on sequence similarity. JEV g5 strains have been rarely isolated, and are poorly characterized. We report here the full characterization of a g5 virus generated using a cDNA-based technology, and its comparison with a widely studied g3 strain. We did not observe any major differences between those viruses when their infectious cycles was studied in various cell linesin vitro. Interestingly, the JEV g5 strain was highly pathogenic when inoculated to BALB/c mice, which are known to be largely resistant to JEV g3 infection. The study of chimeric viruses between JEV g3 and g5 showed that there was a poor viral clearance ofviruses that express JEV g5 structural proteins in BALB/c mice blood, that correlated with viral invasion of the central nervous system, and encephalitis. In addition, using an in vitro model of the blood-brain barrier, we were able to show that JEV g5 does not have an enhanced capacity at entering the central nervous system, when compared to JEV g3. Overall,in addition to providing a first characterization of the understudied JEV g5, our work highlights the importance of sustaining an early viremia inthe development of JEV encephalitis.

    Importance Genotype 5 viruses are genetically and serologically distinct from other JEV genotypes and can been associated with human encephalitis, which warrants the need for their characterization. In this study, we characterized the in vitro and in vivo properties of a JEV g5 strain, and showed that it was more neuropathogenic in a mouse model than a well-characterized JEV g3 strain. The enhanced virulence of JEV g5 was associated with poor viral clearance but not with enhanced crossing of the blood-brain barrier, thus providing new insights into JEV pathogenesis.

  • Diversity of viruses infecting the green micro-alga Ostreococcus lucimarinus [PublishAheadOfPrint]

  • The functional diversity of eukaryotic viruses infecting a single host strain from seawater samples originating from distant marine locations is unknown. To estimate this diversity, we used lysis plaque assays to detect viruses that infect the widespread species Ostreococcus lucimarinus, which is found in coastal and mesotrophic systems, and O. tauri, which was isolated from coastal and lagoon sites from the NW Mediterranean Sea. Detection of viral lytic activities against O. tauri was not observed using seawater from most sites, except those close to the area where the host strain was isolated. In contrast, the more cosmopolitan O. lucimarinus species recovered viruses from locations in the Atlantic and Pacific Oceans and the Mediterranean Sea. Six new O. lucimarinus viruses (OlVs) were then characterized and their genomes sequenced. Two subgroups of OlVs were distinguished based on their genetic distances and on the inversion of a central 32 kb long DNA fragment, but overall their genomes displayed a high level of synteny. The two groups did not correspond to proximity of isolation sites and the phylogenetic distance between these subgroups was higher than the distances observed among viruses infecting O. tauri. Our study demonstrates that viruses originating from very distant sites are able to infect the same algal host strain and can be more diverse than those infecting different species of the same genus. Finally, distinctive features and evolutionary distances between these different viral subgroups does not appear to be linked to biogeography of the viral isolates.

    Importance Marine eukaryotic phytoplankton virus diversity has yet to be addressed and, more specifically, it is unclear whether diversity is connected to geographical distance and whether differential infection and lysis patterns exist among such viruses that infect the same host strain. Here, we assessed the genetic distance of geographically segregated viruses that infect the ubiquitous green microalga Ostreococcus. This study provides the first glimpse into the diversity of predicted gene functions in Ostreococcus viruses originating from distant sites and provide new insights to potential host-distributions and restrictions in the world oceans.

  • Dengue virus infection with highly-neutralizing levels of cross-reactive antibodies cause acute lethal small intestinal pathology without high level of viremia in mice. [PublishAheadOfPrint]

  • Severe dengue virus (DENV)-associated diseases can occur in patients who have pre-existing DENV antibodies (Abs) through antibody-dependent enhancement (ADE) of infection. It is well established that during ADE, DENV-antibody immune complexes (ICs) infect Fc receptor-bearing cells, and increase the systemic viral burden that can be measured in the blood. For protection against DENV serotype 1-4 infection, strongly neutralizing Abs must be elicited to overcome the effect of ADE. Clinical observations in infant who have maternal DENV Abs or recent Phase II/III clinical trials with a leading tetravalent dengue vaccine suggested a lack of correlation between Abs neutralization and in vivo disease prevention. In addressing this gap in knowledge, we found that inoculation of ICs formed with serotype cross-reactive Abs that are more than 98% neutralized in vitro promote high mortality in AG129 mice even though peak viremia was lower than direct virus infection. This suggests that serum viremia level is not always associated with disease severity. We further demonstrated that infection with the ICs resulted in increased vascular permeability specifically in the small intestine (S. intestine) accompanied with the increased tissue viral load and cytokine production, which can be suppressed by anti-TNFaalpha; Abs. Flow cytometric analysis identified increased infection in CD11bintCD11cint/hiCD103- antigen-presenting cells by ICs inoculation, suggesting that these infected cells may be responsible for the increase in TNFaalpha; production and vascular permeability in S. intestine that lead to mortality in the mice. Our finding may have important implications for dengue therapeutics development.

    Importance We examined the relationship between neutralizing level of Ab at the time of infection and subsequent disease progression in a mouse model in order to understand why the patients who are suggested to have a neutralizing quantity of Abs still allow sufficient DENV replication to induce severe dengue manifestations, which sometime does not correlate with viremia level. Strikingly, we found that high mortality was induced in AG129 mice by the increase in TNFaalpha;-induced vascular permeability accompanied with increased viral load specifically in small intestine, even when initial infection level is suppressed to less than 5% and the peak viremia level is not enhanced. This suggests that the ADE overcomes the protective efficacy of Abs in a tissue-dependent manner that leads to severe small intestinal pathology. Our finding may serve to address the pathogenic role of Abs on severe dengue disease and also help to develop safe Ab-based therapeutic strategies.

  • Sustained delivery of a broadly neutralizing antibody in non-human primates confers long-term protection against SHIV infection [PublishAheadOfPrint]

  • Pathogen-specific neutralizing antibodies protect against many viral infections and can potentially prevent HIV transmission in humans. However, neutralizing antibodies have so far only been shown to protect non-human primates (NHP) against lentiviral infection when given shortly before challenge. Thus, the clinical utility and feasibility of passive antibody transfer to confer long-term protection to HIV-1 is still debated. Here, we investigate the potential of a broadly neutralizing HIV-1 antibody to provide long-term protection in a NHP model of HIV-1 infection. A human antibody was simianized to avoid immune rejection and used to sustain therapeutic levels for ~5 months. Two months after the final antibody administration, animals were completely protected against viral challenge. These findings demonstrate the feasibility and potential of long-term passive antibody for protection against HIV-1 in humans, and provides a model to test antibody therapies for other diseases in NHP.

    Importance: Antibodies against HIV are potential drugs that may be able to prevent HIV infection in humans. However the long-term protective capacity of antibodies against HIV has not been assessed. Here we repetitively administered a macaque version of a human anti-HIV antibody to monkeys after which the antibody persisted in the blood for more than five months. Moreover, the antibody could be sustained at protective levels for 108 days conferring protection 52 days after the last dose in a monkey model of HIV infection. Thus, antibody passive transfer can provide durable protection against infection by viruses that cause AIDS in primates.

  • Phosphorylation controls the nuclear-cytoplasmic shuttling of influenza A virus nucleoprotein [PublishAheadOfPrint]

  • The nucleoprotein (NP) is a major component of the viral ribonucleoprotein (vRNP) complex. During the replication of influenza virus, the vRNP complex undergoes nuclear-cytoplasmic shuttling, during which NP serves as one of the determinants. To date, many phosphorylation sites on NP have been identified, but the biological functions of many of these phosphorylation sites remain unknown. In the present study, the functions of phosphorylation sites S9, Y10, and Y296 were characterized. These residues are highly conserved, and their phosphorylation was essential for virus growth in cell culture and a mouse model by regulating the activity of the viral polymerase and the nuclear-cytoplasmic shuttling of NP. The phosphorylation and dephosphorylation of S9 and Y10 controlled nuclear import of NP by affecting the binding affinity between NP and different isoforms of importin-aalpha;. In addition, the phosphorylation of Y296 caused nuclear retention of NP by reducing the interaction between NP and CRM1. Furthermore, tyrosine phosphorylation of NP during the early stage of virus infection was ablated when Y296 was mutated to F. However, at later stages of infection, it was weakened by the Y10F mutation. Taken together, the present data indicate that the phosphorylation and dephosphorylation of NP control the shuttling of NP between the nucleus and cytoplasm during virus replication.

    Importance It is well known that phosphorylation regulates the functions of viral proteins and the life cycle of influenza A virus. As the most abundant protein in the vRNP complex of influenza A virus, several phosphorylation sites on NP were identified. However, the functions of these phosphorylation sites were unknown. The present study demonstrated that the phosphorylation status of these sites on NP can mediate its nuclear-cytoplasmic shuttling, which drives the trafficking of vRNP complexes in infected cells. The present data suggest that the phosphorylated residues of NP are multi-step controllers for the virus life cycle and new targets for designing anti-flu drugs.

  • Macrophages are phenotypically and functionally diverse across tissues in SIV-infected and uninfected Asian macaques [PublishAheadOfPrint]

  • Macrophages regulate tissue immunity, orchestrating the initiation and resolution of antimicrobial immune responses and repair of damaged tissue architecture. Their dysfunction can, thus, manifest in either pro- and anti-inflammatory responses. Indeed, despite the importance of macrophage function in health and disease, the role of tissue-resident macrophages in HIV disease progression remains incompletely defined. Here, we use flow cytometry to assess the phenotype and function of macrophages with macrophages isolated from the spleen, axillary lymph nodes, colon, jejunum and liver of healthy and chronically SIV-infected Asian macaques, the prominent non-human primate model for HIV-infection. Our data demonstrate that macrophages from healthy animals exhibit considerable phenotypic and functional heterogeneity across tissues and across a variety of stimuli. Further, our analysis reveals changes in the LPS responsiveness of macrophages isolated from SIV-infected animals. We anticipate that our findings will inform future research into macrophage-directed immunity across a variety of primate diseases.

    IMPORTANCE These findings highlight the functional and phenotypic heterogeneity of tissue macrophages in different anatomical sites and as a result of SIV infection. We believe our data will lead to novel therapeutic interventions aimed at altering the pro-inflammatory capacity of tissue macrophages in progressively HIV-infected individuals.

  • Structure and dynamics of the native HIV-1 Env trimer [PublishAheadOfPrint]

  • HIV-1/AIDS remains one of the worst pandemics in human history. Despite tremendous efforts, no effective vaccine has been found. Two recent reports give new insights into the structure and dynamics of the HIV-1 Env trimer and renew hopes that a better understanding of Env will translate into new vaccine candidates and more effective antiretroviral therapies.

  • Murine gammaherpesvirus 68 ORF48 is an RTA responsive gene product and functions in both viral lytic replication and latency during in vivo infection [PublishAheadOfPrint]

  • Replication and transcription activator (RTA) of gammaherpesvirus is an immediate-early gene product and regulates the expression of many downstream viral lytic genes. ORF48 is also conserved among gammaherpesviruses; however, its expression regulation and function remained largely unknown. In this study, we characterized the transcription unit of ORF48 from murine gammaherpesvirus 68 (MHV-68) and analyzed its transcriptional regulation. We showed that RTA activates the ORF48 promoter via a RTA responsive element (48pRRE). RTA binds to 48pRRE directly in vitro, and also associates with ORF48 promoter in vivo. Mutagenesis of 48pRRE in the context of viral genome demonstrated that the expression of ORF48 is activated by RTA through 48pRRE during de novo infection. Through site-specific mutagenesis, we generated an ORF48-null virus and examined the function of ORF48 in vitro and in vivo. The ORF48-null mutation remarkably reduced the viral replication efficiency in culturing cells. Moreover, through intranasal or intraperitoneal infection of laboratory mice, we showed that ORF48 is important for viral lytic replication in the lung, establishment of latency in the spleen, as well as viral reactivation from latency. Collectively, our study identified ORF48 as a RTA responsive gene and showed that ORF48 is important for MHV-68 replication both in vitro and in vivo.

    IMPORTANCE The Replication and Transcription Activator (RTA), conserved among gammaherpesviruses, serves as a "molecular switch" for virus life cycle. It works as a transcriptional regulator to activate the expression of many viral lytic genes. However, only a limited number of such downstream genes have been uncovered for MHV-68. In this study, we identified ORF48 as an RTA responsive gene of MHV-68 and mapped the cis-element involved. By constructing a mutant virus that is deficient in ORF48 expression and through infection of laboratory mice, we showed that ORF48 plays important roles in different stages of viral infection in vivo. Our study provides insights into the transcriptional regulation and protein function of MHV-68, a desired model for studying gammaherpesviruses.

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