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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]

  • Decoding Viral Infection by Ribosome Profiling [Gems]

  • 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 identities and amounts of the proteins being produced in cells, as well as novel insights into the mechanism of protein synthesis and translation regulation. Application of ribosome profiling to cells infected with human cytomegalovirus and Kaposi's sarcoma-associated herpesvirus revealed unanticipated complexity in the coding capacity of herpesviruses. Here, I discuss these results and how the application of ribosome profiling to cells infected with other viruses can reveal novel insights into the process of infection.

  • Investigating Influenza A Virus Infection: Tools To Track Infection and Limit Tropism [Gems]

  • Influenza A viruses display a broad cellular tropism within the respiratory tracts 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.

  • Determinants of Dengue Virus NS4A Protein Oligomerization [Structure and Assembly]

  • 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 type 2 (DENV-2) NS4A protein forms oligomers in infected cells or when expressed alone. Deletion mutagenesis mapped amino acids 50 to 76 (spanning the first transmembrane domain [TMD1]) of NS4A as the major determinant for oligomerization, while the N-terminal 50 residues contribute only slightly to the oligomerization. Nuclear magnetic resonance (NMR) analysis of NS4A amino acids 17 to 80 suggests that residues L31, L52, E53, G66, and G67 could participate in oligomerization. Ala substitution for 15 flavivirus conserved NS4A residues revealed that these amino acids are important for viral replication. Among the 15 mutated NS4A residues, 2 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 mutants (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 to 76 (spanning the first transmembrane domain [TMD1]). The biological importance of NS4A oligomerization is demonstrated by results showing that mutations of flavivirus conserved residues (E50A and G67A located within TMD1) reduced the oligomerization and stability of the NS4A protein, leading to attenuated viral replication. A systematic mutagenesis analysis demonstrated that flavivirus conserved NS4A residues are important for DENV replication. A successful trans complementation of replication-lethal NS4A mutant virus requires wild-type NS4A in the context of the viral replication complex. The wild-type NS4A protein alone is not sufficient to rescue the replication defect of NS4A mutants. Intriguingly, distinct NS4A mutants yielded different complementation outcomes in the replicon-containing cells. Overall, the study has enhanced our understanding of flavivirus NS4A at the molecular level. The results also suggest that inhibitor blocking of NS4A oligomerization could be explored for antiviral drug discovery.

  • Morphogenesis of Endoplasmic Reticulum Membrane-Invaginated Vesicles during Beet Black Scorch Virus Infection: Role of Auxiliary Replication Protein and New Implications of Three-Dimensional Architecture [Spotlight]

  • All well-characterized positive-strand RNA viruses[(+)RNA viruses] induce the formation of host membrane-bound viral replication complexes (VRCs), yet the underlying mechanism and machinery for VRC formation remain 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 double-stranded RNA (dsRNA) revealed that the ER-derived membranous spherules provide the site for BBSV replication. Further studies indicated that 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 a 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 tubules, a rearrangement event that is rare among other virus-induced membrane reorganizations. Fibrillar contents within the spherules were also reconstructed by electron tomography, which showed 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 help to achieve a better mechanistic understanding of the organization and microenvironment of plant (+)RNA virus 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 family 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 are all localized within such viral spherules, indicating that these are the sites for generating progeny viral RNAs. Furthermore, the 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 connected to each other via tubule-like structures. Our results have significant implications for further understanding the mechanisms underlying the replication of positive-strand RNA viruses.

  • Crystal Structures of Yeast-Produced Enterovirus 71 and Enterovirus 71/Coxsackievirus A16 Chimeric Virus-Like Particles Provide the Structural Basis for Novel Vaccine Design against Hand-Foot-and-Mouth Disease [Structure and Assembly]

  • Human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the two major causative agents for hand-foot-and-mouth disease (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 responses 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 replacement of only 4 residues in the VP1 GH loop converted strongly negatively 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 enterovirus 71 (EV71) virus-like particle (VLP) produced from yeast is a vaccine candidate against EV71 infection and that a chimeric EV71/coxsackievirus A16 (CVA16) VLP with the replacement 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 a surface charge potential different 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 hand-foot-and-mouth disease (HFMD) and other enterovirus-associated diseases.

  • Interregional Coevolution Analysis Revealing Functional and Structural Interrelatedness between Different Genomic Regions in Human Mastadenovirus D [Genetic Diversity and Evolution]

  • 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 interregional 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 hot spot 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 immunocompromised 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 multiprotein 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 multiprotein systems and therefore predicts physical and functional interactions between different proteins and/or genomic elements.

  • Continual Reintroduction of Human Pandemic H1N1 Influenza A Viruses into Swine in the United States, 2009 to 2014 [Genetic Diversity and Evolution]

  • 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 (pH1N1) viruses has occurred continuously in the years following the 2009 H1N1 pandemic and has been an important contributor to the genetic diversity of U.S. swIAVs. Although pandemic H1 and N1 segments had been largely removed from the U.S. swine population by 2013 via reassortment with other swIAVs, these antigens reemerged 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 U.S. swine population, with periodic reemergence of pandemic hemagglutinin (HA) and neuraminidase (NA) segments in association with seasonal pH1N1 epidemics in humans. Vaccinating U.S. 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 U.S. Department of Agriculture's national surveillance system to identify the central role of humans in the reemergence of pandemic H1N1 (pH1N1) influenza viruses in U.S. 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 reemerge in U.S. swine in association with seasonal pH1N1 epidemics in humans.

  • Artificial Recruitment of UAF1-USP Complexes by a PHLPP1-E1 Chimeric Helicase Enhances Human Papillomavirus DNA Replication [Genome Replication and Regulation of Viral Gene Expression]

  • The E1 helicase from anogenital human papillomavirus (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 pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1). 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 ubiquitin-like domain of UAF1 for interaction, in contrast to E1, which does not contact the latter. Overall, this comparison of two heterologous UBSs indicates 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 interacts with the UAF1-associated deubiquitinating enzymes USP1, USP12, and USP46 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 PHLPP1, 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.

  • Lymphocytic Choriomeningitis Virus Differentially Affects the Virus-Induced Type I Interferon Response and Mitochondrial Apoptosis Mediated by RIG-I/MAVS [Virus-Cell Interactions]

  • Arenaviruses are important emerging human pathogens maintained by noncytolytic 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 3 (IRF3) via the retinoic acid inducible gene (RIG)-I/mitochondrial antiviral signaling (MAVS) pathway. Another important mechanism of host innate antiviral 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 superinfection 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 antiviral defense. The RIG-I independence of mitochondrial apoptosis in LCMV-infected cells provides the 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 the innate antiviral 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 superinfection 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 its effect on apoptosis appears as a subtle strategy to guarantee sufficiently high viral loads for efficient transmission while maintaining apoptosis as a mechanism of defense.

  • Equine Herpesvirus 1 Multiply Inserted Transmembrane Protein pUL43 Cooperates with pUL56 in Downregulation of Cell Surface Major Histocompatibility Complex Class I [Pathogenesis and Immunity]

  • Herpesviruses have evolved an array of strategies to counteract antigen presentation by major histocompatibility complex class I (MHC-I). Previously, we identified pUL56 of equine herpesvirus 1 (EHV-1) as one major determinant of the downregulation of cell surface MHC-I (G. Ma, S. Feineis, N. Osterrieder, and G. R. Van de Walle, J. Virol. 86:3554nndash;3563, 2012, http://dx.doi.org/10.1128/JVI.06994-11; T. Huang, M. J. Lehmann, A. Said, G. Ma, and N. Osterrieder, J. Virol. 88:12802nndash;12815, 2014, http://dx.doi.org/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 as 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 postinfection (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, coexpression of pUL43 and pUL56 in transfected cells reduced the 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 protein pUL43 is involved in downregulation of cell surface MHC-I. pUL43 is an early protein and degraded in lysosomes. pUL43 resides in the Golgi vesicles 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 the rerouting of vesicles containing MHC-I, pUL56, and pUL43 to the lysosomal compartment.

  • In Vivo Activation of Human NK Cells by Treatment with an Interleukin-15 Superagonist Potently Inhibits Acute In Vivo HIV-1 Infection in Humanized Mice [Pathogenesis and Immunity]

  • 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 capacity of NK cells to suppress acute in vivo HIV-1 infection would be augmented by activating them via treatment with an interleukin-15 (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 the 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 by using hu-spl-PBMC-NSG mice, NOD-SCID-IL2rnndash;/nndash; (NSG) mice intrasplenically injected with human peripheral blood mononuclear cells (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 3 days after intrasplenic HIV-1 inoculation. Removal of NK cells from 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 antitumor activity of NK cells markedly inhibited acute HIV-1 infection in humanized mice, even when activation of NK cells by IL-15 superagonist treatment is delayed until 3 days after HIV-1 inoculation. NK cell depletion from 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 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.

  • SRSF1 RNA Recognition Motifs Are Strong Inhibitors of HIV-1 Replication [Virus-Cell Interactions]

  • Replication of the integrated HIV-1 genome is tightly regulated by a series of cellular factors. In previous work we showed that transactivation of the HIV-1 promoter is regulated by the cellular splicing factor SRSF1. Here we report that SRSF1 can downregulate the replication of B, C, and D subtype viruses by ggt;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 but not the arginine serine-rich activator domain can downregulate viral replication by ggt;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, there is a need for novel therapeutics with diverse mechanisms of action. In this study, we show 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 ggt;2,000-fold in multiple viral strains without impacting cell viability. Given the strong antiviral properties of this protein, the RNA-binding domains, and the minimal effects observed on cell metabolism, further studies are warranted to assess the therapeutic potential of peptides derived from these sequences.

  • Interaction between Shadoo and PrP Affects the PrP-Folding Pathway [Prions]

  • Prion diseases are characterized 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. On the basis of histoanatomical colocalization and sequence similarities, it is suspected that Sho and PrP may be functionally related. The downregulation of Sho expression during prion pathology and the direct interaction between Sho and PrP, as revealed by 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 dissociation constant in the micromolar range, and this interaction consequently modifies the PrP-folding pathway. Using a truncated PrP that mimics the C-terminal C1 fragment, an allosteric binding behavior with a Hill number of 4 was observed, suggesting that at least a tetramerization state occurs. A 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 (i) acting as a holdase and (ii) 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 characterization of Sho-PrP indicates that Sho acts as an ATP-independent holdase.

  • Regulation of Hepatitis C Virus Genome Replication by Xrn1 and MicroRNA-122 Binding to Individual Sites in the 5' Untranslated Region [Spotlight]

  • miR-122 is a liver-specific microRNA (miRNA) that binds to two sites (S1 and S2) on the 5' untranslated region (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, an 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 cooperative 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 5'-to-3' exoribonuclease, but also has additional functions. In cells depleted of Xrn1, replication levels of S1-bound HCV RNA were slightly higher than S2-bound RNA levels, 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 cooperatively 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 the 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 5' UTR contributes equally to HCV replication and that binding to both sites can function cooperatively. This suggests that active Ago2nndash;miR-122 complexes assemble at each site and can cooperatively 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.

  • Genetic Confirmation that the H5 Protein Is Required for Vaccinia Virus DNA Replication [Genome Replication and Regulation of Viral Gene Expression]

  • The duplication of the poxvirus double-stranded DNA genome occurs in cytoplasmic membrane-delimited factories. This physical autonomy from the host nucleus suggests that poxvirus genomes 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 (ts) mutant Dts57, which was generated by chemical mutagenesis and has a lesion in H5, exhibits defects in DNA replication and morphogenesis under nonpermissive conditions, depending upon the experimental protocol. The H5 variant encoded by the genome of this mutant is ts for function but not stability. For a more precise investigation of how H5 contributes to DNA synthesis, we placed the ts57 H5 allele in an otherwise wild-type viral background and also performed small interfering RNA-mediated depletion of H5. Finally, we generated a complementing cell line, CV-1nndash;H5, which allowed us to generate a viral recombinant in which the H5 open reading frame 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 Poxviridae 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 the genomes 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 were able to isolate a deletion virus that lacks the H5 gene and show 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.

  • Genetic Determinants of Japanese Encephalitis Virus Vaccine Strain SA14-14-2 That Govern Attenuation of Virulence in Mice [Vaccines and Antiviral Agents]

  • The safety and efficacy of the live-attenuated Japanese encephalitis virus (JEV) SA14-14-2 vaccine are 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, the nucleotide substitution T39A in the 5' UTR (5'-UTR-T39A), the capsid (C) protein amino acid substitution L66S (C-L66S), and the complete NS1/2A genome region containing 10 mutations each significantly reduced virus neuroinvasion but not neurovirulence. The levels of peripheral virulence attenuation imposed by the 5'-UTR-T39A and C-L66S mutations, individually, were 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 ggt;25 years ago by passaging wild-type JEV strain SA14 in tissue cultures 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 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.

  • Creation of Matrix Protein Gene Variants of Two Serotypes of Vesicular Stomatitis Virus as Prime-Boost Vaccine Vectors [Vaccines and Antiviral Agents]

  • To take advantage of live recombinant vesicular stomatitis viruses (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 serotype VSV-Indiana (VSVInd) with a temperature-sensitive mutation (tsO23) of the VSVInd Orsay strain. In addition, we have generated two new serotype VSV-New Jersey (VSVNJ) vaccine vectors by combining M48R and M51R mutations with G22E and L110F mutations in the M gene, rVSVNJ(G22E M48R M51R) [rVSVNJ(GMM)] and VSVNJ(G22E M48R M51R L110F) [rVSVNJ(GMML)]. The combined mutations G21E, M51R, and 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. 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 1 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 replaced the phenylalanine with alanine. This will change all three nucleotides from UUG (leucine) to GCA (alanine). The resulting L111A mutant showed the temperature-sensitive phenotype of rVSVInd(GML) and increased stability. Twenty consecutive passages of 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, the use of live rVSVs as vaccine vectors demands the utmost safety. Our strategy to attenuate 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 rVSVInd(GML) and M gene mutants of rVSVNJ(GMM) and rVSVNJ(GMML) add excellent vaccine vectors to the pool of live viral vectors.

  • Molecular Chaperone Hsp90 Is a Therapeutic Target for Noroviruses [Spotlight]

  • Human noroviruses (HuNoV) are a significant cause of acute gastroenteritis in the developed world, and 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 5' and 3' 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 (17-dimethylaminoethylamino-17-demethoxygeldanamycin) 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 the 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 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, we identify here 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.

  • The Greater Affinity of JC Polyomavirus Capsid for {alpha}2,6-Linked Lactoseries Tetrasaccharide c than for Other Sialylated Glycans Is a Major Determinant of Infectivity [Structure and Assembly]

  • The human JC polyomavirus (JCPyV) establishes an asymptomatic, persistent infection in the kidneys 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 lactoseries tetrasaccharide c (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 nuclear magnetic resonance (NMR) spectroscopy, and correlated these data with the results of 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 more weakly 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 kidneys of the majority of the population and can cause the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) in the brains 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.

  • Temperature-Sensitive Mutants in the Influenza A Virus RNA Polymerase: Alterations in the PA Linker Reduce Nuclear Targeting of the PB1-PA Dimer and Result in Viral Attenuation [Structure and Assembly]

  • The influenza virus RNA-dependent RNA polymerase catalyzes 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 the virus life cycle. We investigated the structure and function of the PA linker (residues 197 to 256), located between its N-terminal endonuclease domain and its C-terminal structured domain that binds PB1, the polymerase core. Circular dichroism 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 versus 37ddeg;C/33ddeg;C), suggesting an alteration of folding kinetic parameters. The ts phenotype was associated with a reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using a fluorescent-tagged PB1, we observed that ts and lethal PA mutants did 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 modulated 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 identify a PA domain critical for PB1-PA nuclear import and that is 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. This is the first time that a large number of ts mutations were engineered in such a short domain, demonstrating that rational design of ts mutants can be achieved. We were able to associate this phenotype with a defect of transport of the PA-PB1 complex into the nucleus. Reversion substitutions restored the ability of the complex to move to the nucleus. Two of these ts mutants were shown to be attenuated and able to produce antibodies in mice. These results are of high interest for the design of novel attenuated vaccines and to develop new antiviral drugs.

  • Zinc Binding Activity of Human Metapneumovirus M2-1 Protein Is Indispensable for Viral Replication and Pathogenesis In Vivo [Pathogenesis and Immunity]

  • Human metapneumovirus (hMPV) is a member of the Pneumovirinae subfamily 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 antitermination 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 the oligomerization of M2-1. Subsequently, recombinant hMPVs (rhMPVs) carrying mutations in the zinc binding motif were recovered. Interestingly, rhMPV-C21S and -H25L mutants, which lacked zinc binding activity, had delayed replication in cell culture and were highly attenuated in cotton rats. In contrast, rhMPV-C7S and -C15S strains, which retained 60% of the zinc binding activity, replicated as efficiently as rhMPV in cotton rats. Importantly, rhMPVs that lacked zinc binding activity triggered high levels of neutralizing antibody and provided 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 antitermination 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 that 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.

  • Transformation with Oncogenic Ras and the Simian Virus 40 T Antigens Induces Caspase-Dependent Sensitivity to Fatty Acid Biosynthetic Inhibition [Transformation and Oncogenesis]

  • 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, affect fatty acid metabolism. Our results indicate that SV40/Ras12V-mediated transformation of fibroblasts induces fatty acid biosynthesis in the absence of significant changes in the concentration of fatty acid biosynthetic enzymes. This oncogene-induced activation of fatty acid biosynthesis was found to be mammalian target of rapamycin (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-coenzyme A (CoA) carboxylase (ACC), a key fatty acid biosynthetic enzyme, induced caspase-dependent cell death in oncogene-transduced cells. In contrast, isogenic nontransformed cells were resistant to fatty acid biosynthetic inhibition. This oncogene-induced sensitivity to fatty acid biosynthetic inhibition was independent of the cells' growth rates and could be attenuated by supplementing the medium with unsaturated fatty acids. Both the activation of fatty acid biosynthesis and the sensitivity to fatty acid biosynthetic inhibition could be conveyed to nontransformed 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 the proliferative status and could be attenuated by supplementing the medium 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 supplementing the medium with nonsaturated fatty acids. Similar results were observed with the expression of oncogenic Ras in nontransformed breast epithelial cells. Combined, our results suggest that specific oncogenic alleles induce metabolic dependencies that can be exploited to selectively kill cancerous cells.

  • Mutations in Coronavirus Nonstructural Protein 10 Decrease Virus Replication Fidelity [Genome Replication and Regulation of Viral Gene Expression]

  • Coronaviruses (CoVs) are unique in encoding a 3'-ggt;5' 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 nonenzymatic 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-fluorouracil (5-FU) than wild-type (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 a peak titer similar to and demonstrated RNA synthesis kinetics comparable to those seen with WT-ExoN(+). No change in 5-FU sensitivity was observed for R80A/E82A-ExoN(nndash;) relative to MHV-ExoN(nndash;), indicating that the decreased-fidelity phenotype of R80A/E82A-ExoN(nndash;) 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 report, we show that nsp10, a small, nonenzymatic 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.

  • Genomic, Phylogenetic, and Recombinational Characterization of Herpes Simplex Virus 2 Strains [Genetic Diversity and Evolution]

  • Herpes simplex virus 2 (HSV-2) is a major global pathogen, infecting 16% of people 15 to 49 years old worldwide and 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 Illumina MiSeq. We report that HSV-2 has a genomic overall mean distance of 0.2355%, which is less than that of HSV-1. There were approximately 100 amino-acid-encoding and indels per genome. Microsatellite mapping found a bias toward intergenic regions in the nonconserved microsatellites and a genic bias in all detected tandem repeats. Extensive recombination between the HSV-2 strains was also strongly implied. This was the first study to analyze multiple HSV-2 sequences, and the data will be valuable in future evolutionary, virulence, and structure-function studies.

    IMPORTANCE HSV-2 is a 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 toward intergenic regions in the nonconserved microsatellites and a genic bias in all detected tandem repeats and confirm that chimpanzee herpesvirus 1 (ChHV-1) is a separate species and that each of the HSV-2 strains is a genomic mosaic.

  • Interleukin-2 from Adaptive T Cells Enhances Natural Killer Cell Activity against Human Cytomegalovirus-Infected Macrophages [Virus-Cell Interactions]

  • 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 toward HCMV-infected autologous macrophages. However, this enhanced response was abolished when purified NK cells were applied as effectors. We demonstrate that this enhanced PBNK activity was dependent on the interleukin-2 (IL-2) secretion of CD4+ T cells when reexposed 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 HCMVmmdash;namely, that NK cell-mediated innate immunity can be enhanced by a preexisting T cell antiviral immunitymmdash;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 immunity 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 the main adaptive effectors that are critical for the control of the latent infection and limitation of reinfection. In this study, we found that IL-2 secreted by adaptive CD4+ T cells after reexposure to HCMV enhances 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.

  • Influenza A Virus Protein PA-X Contributes to Viral Growth and Suppression of the Host Antiviral and Immune Responses [Virus-Cell Interactions]

  • 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 polymerase 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 beta interferon (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 viral growth and greater expression of IFN-bbeta; mRNA in their lungs than mice infected with Cal WT. Importantly, more antihemagglutinin and neutralizing antibodies were produced in Cal PA-XFS-infected mice than in 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 responses to influenza virus.

    IMPORTANCE Virus-induced host protein shutoff is considered to be a major factor allowing 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 the 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 responses 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.

  • Unravelling the Role of the F55 Regulator in the Transition from Lysogeny to UV Induction of Sulfolobus Spindle-Shaped Virus 1 [Genome Replication and Regulation of Viral Gene Expression]

  • 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 family Fuselloviridae 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 for these sequences led to the hypothesis that this protein might be involved in the maintenance of the SSV1 lysogeny. Here, we report 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 a 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, few insights into their in vivo roles have been gained throughout the last 3 decades. Here, we report the first in vivo investigation of an SSV1 transcription regulator, F55, that plays 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 the early genes. Moreover, the differential affinity of this transcription factor for these targets allows a fine-tuned and temporal coordinated regulation of transcription of viral genes.

  • 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 [Vaccines and Antiviral Agents]

  • An effective human immunodeficiency virus type 1 (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-recombinant NYVAC (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 gamma interferon (IFN-) enzyme-linked immunosorbent spot assay (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.

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

  • 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 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.

  • CD8+ T Cells Can Mediate Short-Term Protection against Heterotypic Dengue Virus Reinfection in Mice [Pathogenesis and Immunity]

  • 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 lifelong 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 nonlethally with DENV were protected against reinfection with either a homotypic or heterotypic serotype 2 weeks later. Homotypic priming induced a robust neutralizing antibody response, whereas heterotypic priming elicited binding, but nonneutralizing 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 more than 120 countries in tropical and subtropical regions. Infection with dengue virus can be asymptomatic, but it can also progress into the potentially lethal severe dengue disease. There are four closely related dengue virus serotypes. Infection with one serotype results in a transient period of resistance against all serotypes (cross-protection), followed by lifelong 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 [Genetic Diversity and Evolution]

  • 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% to 22.5% body weight loss in mice.

  • Us9-Independent Axonal Sorting and Transport of the Pseudorabies Virus Glycoprotein gM [Spotlight]

  • Axonal sorting and transport of fully assembled pseudorabies virus (PRV) 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 blotting in isolated axons during Us9-null PRV infection. These results suggest that gM differs from other viral proteins regarding axonal transport properties.

  • 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 [Pathogenesis and Immunity]

  • 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 an infectious clone with a Tyr-10 deletion. The results demonstrate differences in the properties and specificities of VN responses elicited during PRRSV infection.

  • Intercontinental Spread of Asian-Origin H5N8 to North America through Beringia by Migratory Birds [Genetic Diversity and Evolution]

  • Phylogenetic network analysis and understanding of waterfowl migration patterns suggest that 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.

  • Correction for Doores et al., Two Classes of Broadly Neutralizing Antibodies within a Single Lineage Directed to the High-Mannose Patch of HIV Envelope [Author Corrections]

  • Correction for Ingale et al., Hyperglycosylated Stable Core Immunogens Designed To Present the CD4 Binding Site Are Preferentially Recognized by Broadly Neutralizing Antibodies [Author Corrections]

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    JVI Accepts: Articles Published Ahead of Print

  • Rethinking the Response to Emerging Microbes: Vaccines and Therapeutics in the Ebola Era - A Conference at Harvard Medical School [PublishAheadOfPrint]

  • Harvard Medical School convened a meeting of biomedical and clinical experts on March 5, 2015 on the topic of "Rethinking the Response to Emerging Microbes: Vaccines and Therapeutics in the Ebola Era," with the goals of discussing the lessons from the recent Ebola outbreak and using those lessons as a case study to aid preparations for future emerging infections. The speakers and audience discussed the special challenges in combatting an infectious agent that causes sporadic outbreaks in resource-poor countries. The meeting led to a call for improved basic medical care for all and continued support of basic discovery research to provide the foundation for preparedness for future outbreaks in addition to the targeted emergency response to outbreaks and targeted research programs against Ebola and other specific emerging pathogens.

  • Tick saliva enhances Powassan virus transmission to the host influencing its dissemination and the course of disease [PublishAheadOfPrint]

  • Powassan virus (POWV) is an encephalitic tick-borne flavivirus which can result in serious neuroinvasive disease with up to 10% case fatality rate. The study objective was to determine whether the salivary gland extract (SGE) from I. scapularis ticks facilitates the transmission and dissemination of POWV in a process known as saliva-activated transmission. Groups of BALB/c mice were footpad inoculated with one of the following: high dose of POWV with and without SGE, or low dose of POWV with and without SGE. Mice from each group were sacrificed daily. Organ viral loads and gene expression profiles were evaluated by quantitative real-time PCR. Both groups of mice infected with POWV high dose showed severe neurological signs of disease preceding death. The presence of SGE did not affect POWV transmission or disease outcome for mice infected with the high dose of POWV. Neuroinvasion, paralysis, and death occurred for all mice infected with the low dose of POWV plus SGE; however, for mice infected with the low dose of POWV in the absence of SGE, there were no clinical signs of infection nor did any mice succumb to disease. Although this group displayed low-level viremias, all mice were completely healthy and it was the only group in which POWV was cleared from the lymph nodes. We conclude that saliva-activated transmission occurs in mice infected with a low dose of POWV. Our study is the first to demonstrate virus dose-dependent saliva-activated transmission, warranting further investigation of the specific salivary factors responsible for enhancing POWV transmission.

    IMPORTANCE. Powassan virus (POWV) is a tick-borne flavivirus that continues to emerge in the United States, as is evident by the surge in number and expanding geographic range of confirmed cases in the past decade. This neuroinvasive virus is transmitted to humans by infected tick bites. Successful tick feeding is facilitated by a collection of pharmacologically active factors in tick saliva. In a process known as saliva-activated transmission, tick bioactive salivary molecules are thought to modulate the host environment making it more favorable for the transmission and establishment of a pathogen. This phenomenon has been demonstrated for several tick-borne pathogens; however, a systematic investigation of the role of tick saliva on dissemination and pathogenesis of a tick-borne viral disease has never been attempted before. This study will fill that gap by systematically examining whether the presence of tick saliva contributes to the transmission and dissemination of POWV in mice.

  • HIV-1 is Restricted Prior to Integration in Primary Cord-Derived Human CD34+ Cells [PublishAheadOfPrint]

  • Certain cells have the ability to block retroviral infection at specific stages of the viral cycle by the activities of well-characterized factors and transcriptional silencing machinery. Infection of murine stem cells (MSCs) by the murine leukemia viruses (MLVs) is profoundly blocked post-integration by transcriptional silencing. Here we show that a dominant point of restriction of HIV-1 in human CD34+ cells is prior to integration, and that HIV-1 restriction by human CD34+ cells is fundamentally different.

  • Expanding regulatory T cells alleviates chikungunya virus-induced pathology in mice [PublishAheadOfPrint]

  • Chikungunya virus (CHIKV) infection is a re-emerging pandemic human arboviral disease. CD4+ T cells were previously shown to contribute to joint inflammation in the course of CHIKV infection in mice. The JES6-1 anti-IL-2 antibody selectively expands mouse regulatory T cells (Tregs) by forming a complex with IL-2. In this study, we show that the IL-2 JES6-1-mediated expansion of Tregs ameliorates CHIKV-induced joint pathology. It does so by inhibiting the infiltration of CD4+ T cells due to the induction of anergy in CHIKV-specific CD4+ effector T cells. These findings suggest that activation of Tregs could also become an alternative approach to control CHIKV-mediated disease.

    Importance Chikungunya virus (CHIKV) has re-emerged as a pathogen of global significance. Patients infected with CHIKV suffer from incapacitating joint pain that severely affects their daily functioning. Despite the best efforts, effective treatment is still inadequate. While T cells-mediated immunopathology in CHIKV infections has been reported, the role of regulatory T cells (Tregs) has not been explored. The JES6-1 anti-IL-2 antibody has been demonstrated to selectively expand mouse Tregs by forming a complex with IL-2. We reveal here that IL-2 JES6-1-mediated expansion of Tregs ameliorates the CHIKV-induced joint pathology in mice by neutralizing virus-specific CD4+ effector T (Teff) cells. We show that this treatment abrogates the infiltration of pathogenic CD4+ T cells through induction of anergy in CHIKV-specific CD4+ Teff cells. This is the first evidence where the role of Tregs is demonstrated in CHIKV pathogenesis and its expansion could control virus-mediated immunopathology.

  • Repeated Low-dose Influenza Virus Infection Causes Severe Disease in Mice: a Model for Vaccine Evaluation [PublishAheadOfPrint]

  • Influenza infection causes severe disease and death in humans. In traditional vaccine research and development, a single high-dose virus challenge of animals is used to evaluate vaccine efficacy. This type of challenge model may have limitations. In the present study, we developed a novel challenge model by infecting mice repeatedly in short intervals with low-doses of influenza A virus. Our results show that compared to a single high-dose infection, mice that received repeated low-dose challenges showed earlier morbidity and mortality and more severe disease. They developed higher vial loads, more severe lung pathology, greater inflammatory responses, and generated only limited influenza A virus-specific B and T cell responses. A commercial trivalent influenza vaccine protected mice against a single high and lethal dose of influenza A virus but was ineffective against repeated low-dose virus challenges. Overall our data show that the repeated low-dose influenza A virus infection mouse model is more stringent and may thus be more suitable to select for highly efficacious influenza vaccines.

    Importance Influenza epidemics and pandemics pose serious threats to public health. Animal models are crucial for evaluating the efficacy of influenza vaccines. Traditional models based on a single high-dose virus challenge may have limitations. Here, we describe a new mouse model based on repeated low dose influenza A virus challenges given within a short period. Repeated low-dose challenges caused more severe disease in mice associated with higher viral loads and increased lung inflammation and reduced influenza A virus-specific B and T cell responses. A commercial influenza vaccine that was shown to protect mice from high-dose challenge was ineffective against repeated low-dose challenges. Overall, our results show that the low-dose repeated challenge model is more stringent and may therefore be better suited for pre-clinical vaccine efficacy studies.

  • A transgenic Drosophila melanogaster model to study Human T-Lymphotropic Virus oncoprotein Tax-1-driven transformation in vivo [PublishAheadOfPrint]

  • HTLV-1-induced adult T-cell leukemia/lymphoma is an aggressive malignancy. HTLV-2 is genetically related to HTLV-1 but does not cause any malignant disease. HTLV-1 Tax transactivator (Tax-1) contributes to leukemogenesis via NF-B. We describe transgenic Drosophila models expressing Tax in the compound eye and plasmatocytes. We demonstrate that Tax-1 but not Tax-2 induces ommatidia perturbation and increased plasmatocyte proliferation and that the eye phenotype is dependent on Kenny (IKK/NEMO), thus validating this new in vivo model.

  • Caribbean and La Reunion Chikungunya virus isolates differ in their capacity to induce pro-inflammatory Th1 and NK cell responses and acute joint pathology [PublishAheadOfPrint]

  • Chikungunya virus (CHIKV) is a mosquito-borne arthralgic alphavirus that has garnered international attention as an important emerging pathogen since 2005. More recently, it invaded the Caribbean islands and the western hemisphere. Intriguingly, the current CHIKV outbreak in the Caribbean is caused by the Asian CHIKV genotype, which differs from the La Reeacute;union LR2006 OPY1 isolate belonging to the Indian Ocean lineage. Here, we adopted a systematic and comparative approach against LR2006 OPY1 to characterize the pathogenicity of the Caribbean CNR20235 isolate and consequential host immune responses in mice. Ex vivo infection using primary mouse-tail fibroblasts revealed weaker replication efficiency by CNR20235 isolate. In the CHIKV mouse model, CNR20235 infection induced an enervated joint pathology characterized by moderate edema and swelling, independent of mononuclear cell infiltration. Based on systemic cytokine analysis, localized immune-phenotyping and gene expression profiles in the popliteal lymph node and inflamed joints, two pathogenic phases were defined for CHIKV infection: early acute (2-3 dpi) and late acute (6-8 dpi). Reduced joint pathology during early acute phase of CNR20235 infection was associated with a weaker pro-inflammatory Th1 response and natural killer (NK) cells activity. The pathological role of NK cells was further demonstrated as depletion of NK cells reduced joint pathology in LR2006 OPY1. Taken together, this study provides evidence that the Caribbean CNR20235 isolate has an enfeebled replication and induces a less pathogenic response in the mammalian host.

    IMPORTANCE The introduction of CHIKV in the Americas has heightened the risk of large-scale outbreaks due to the close proximity between the United States and the Caribbean. Immune-pathogenicity of the circulating Caribbean CHIKV isolate was explored, where it was demonstrated to exhibit reduced infectivity resulting in a weakened joint pathology. Analysis of serum cytokine levels, localized immune-phenotyping, and gene expression profile in the organs revealed that a limited Th1 response and reduced NK cells activity could underlie the reduced pathology in the host. Interestingly, higher asymptomatic infections were observed in the Caribbean compared to the La Reeacute;union outbreaks in 2005-2006. This is the first study that showed an association between key pro-inflammatory factors and pathology-mediating leukocytes with a less severe pathological outcome in Caribbean CHIKV infection. Given the limited information regarding the sequela of Caribbean CHIKV infection, our study is timely and will aid the understanding of this increasingly important disease.

  • Activated Nrf2 Interacts with Kaposi's Sarcoma-Associated Herpesvirus Latency Protein LANA-1 and Host Protein KAP1 to Mediate Global Lytic Gene Repression [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman's disease. We have previously shown that KSHV utilizes the host transcription factor Nrf2 to aid in infection of endothelial cells and oncogenesis. Here, we investigate the role of Nrf2 in PEL and PEL-derived cell lines and show that KSHV latency induces Nrf2 protein levels and transcriptional activity through the COX-2/PGE2/EP4/PKC axis. Next-generation sequencing of KSHV transcripts in the PEL-derived BCBL-1 cell line reveals that knockdown of this activated Nrf2 results in global elevation of lytic genes. Nrf2 inhibition by the chemical Brusatol also induces lytic gene expression. Both Nrf2 knockdown and Brusatol-mediated inhibition induced KSHV lytic reactivation in BCBL-1 cells. In a series of follow-up experiments, we characterized the mechanism of Nrf2-mediated regulation of KSHV lytic repression during latency. Biochemical assays show that Nrf2 interacted with the KSHV latency-associated nuclear antigen (LANA-1) and the host transcriptional repressor KAP1, which together have been shown to repress lytic gene expression. Promoter studies show that although Nrf2 alone induces the ORF50 promoter, its association with LANA-1 and KAP1 abrogates this effect. Interestingly, LANA-1 is crucial for efficient KAP1/Nrf2 association, while Nrf2 is essential for LANA-1 and KAP1 recruitment to the ORF50 promoter and its repression. Overall, these results suggest that activated Nrf2, LANA-1 and KAP1 assemble on the ORF50 promoter in a temporal fashion. Initially, Nrf2 binds to and activates the ORF50 promoter during early de novo infection, an effect that is exploited during latency by LANA-1-mediated recruitment of the host transcriptional repressor KAP1 on Nrf2. Cell death assays further show that Nrf2 and KAP1 knockdown induce significant cell death in PEL cell lines. Our studies suggest that Nrf2 modulation through available oral agents is a promising therapeutic approach in the treatment of KSHV-associated malignancies.

    Importance KS and PEL are aggressive KSHV-associated malignancies with moderately effective, highly toxic chemotherapies. Besides Ganciclovir and IFN-aalpha; prophylaxis, no KSHV-associated chemotherapy targets the underlying infection, a major oncogenic force. Hence, drugs that selectively target KSHV infection are necessary to eradicate the malignancy while sparing healthy cells. We recently showed that KSHV infection of endothelial cells activates the transcription factor Nrf2 to promote an environment conducive to infection and oncogenesis. Nrf2 is modulated through several well-tolerated oral agents, and may be an important target in KSHV biology. Here, we investigate the role of Nrf2 in PEL, and demonstrate that Nrf2 plays an important role in KSHV gene expression, lytic reactivation and cell survival by interacting with the host transcriptional repressor KAP1 and the viral latency-associated protein LANA-1 to mediate global lytic gene repression and thus cell survival. Hence, targeting Nrf2 with available therapies is a viable approach in the treatment of KSHV malignancies.

  • Naturally-occurring capsid protein variants of human papillomavirus genotype 31 represent a single L1 serotype. [PublishAheadOfPrint]

  • We investigated naturally-occurring variation within the major (L1) and minor (L2) capsid proteins of oncogenic human papillomavirus (HPV) genotype HPV31 to determine the impact on capsid antigenicity. L1L2 pseudoviruses (PsV) representing the three HPV31 variant lineages A, B and C exhibited comparable particle to infectivity ratios and morphology. Lineage-specific L1L2 PsV demonstrated subtle differences in susceptibility to neutralization by antibodies elicited following vaccination, pre-clinical L1 VLP immunization or by monoclonal antibodies; however, these differences were generally of a low magnitude. These data indicate diagnostic lineage-specific single nucleotide polymorphisms within the HPV31 capsid genes have a limited effect on L1 antibody mediated neutralization and that the three HPV31 variant lineages belong to a single L1 serotype. These data contribute to our understanding of HPV L1 variant antigenicity.

    Importance The virus coat (capsid) of the human papillomavirus contains major (L1) and minor (L2) capsid proteins. These proteins facilitate host cell attachment and viral infectivity and are the targets for antibodies which interfere with these events. In this study we investigated the impact of naturally-occurring variation within these proteins upon susceptibility to viral neutralization by antibodies induced by L1 VLP immunization. We demonstrate that HPV31 L1 and L2 variants exhibit similar susceptibility to antibody-mediated neutralization and that for the purposes of L1 VLP-based vaccines these variant lineages represent a single serotype.

  • A novel Leu92 mutant of HIV-1 reverse transcriptase with a selective deficiency in strand transfer causes a loss of viral replication [PublishAheadOfPrint]

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

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

  • PI(4,5)P2 acyl chains differentiate membrane binding of HIV-1 Gag from that of the phospholipase C{delta}1 PH domain [PublishAheadOfPrint]

  • HIV-1 Gag, which drives virion assembly, interacts with a plasma-membrane(PM)-specific phosphoinositide, phosphatidylinositol- (4, 5)-bisphosphate [PI(4,5)P2]. While cellular acidic-phospholipid-binding proteins/domains, such as the PI(4,5)P2-specific pleckstrin homology domain of phospholipase C1 (PHPLC1), mediate headgroup-specific interactions with corresponding phospholipids, the exact nature of the Gag-PI(4,5)P2 interaction remains undetermined. In this study, we used giant unilamellar vesicles (GUVs) to examine how PI(4,5)P2 with unsaturated or saturated acyl chains affect membrane binding of PHPLC1 and Gag. Both unsaturated dioleoyl-PI(4,5)P2 [DO-PI(4,5)P2] and saturated dipalmitoyl-PI(4,5)P2 [DP-PI(4,5)P2] successfully recruited PHPLC1 to membranes of single-phase GUVs. In contrast, DO-PI(4,5)P2 but not DP-PI(4,5)P2 recruited Gag to GUVs, indicating that PI(4,5)P2 acyl chains contribute to stable membrane binding of Gag. GUVs containing PI(4,5)P2, cholesterol, and dipalmitoyl phosphatidylserine separated into two coexisting phases: one was a liquid phase, and the other appeared to be a phosphatidylserine-enriched gel phase. In these vesicles, the liquid phase recruited PHPLC1 regardless of PI(4,5)P2 acyl chains. Likewise, Gag bound to the liquid phase when PI(4,5)P2 has DO-acyl chains. DP-PI(4,5)P2-containing GUVs showed no detectable Gag binding to the liquid phase. Unexpectedly, however, DP-PI(4,5)P2 still promoted recruitment of Gag, but not PHPLC1, to the dipalmitoyl-phosphatidylserine-enriched gel phase of these GUVs. Altogether, these results revealed different roles for PI(4,5)P2 acyl chains in membrane binding of two PI(4,5)P2-binding proteins, Gag and PHPLC1. Notably, we observed that non-myristylated Gag retains the preference for PI(4,5)P2 containing an unsaturated acyl chain over DP-PI(4,5)P2, suggesting that Gag sensitivity to PI(4,5)P2-acyl-chain saturation is determined directly by the MA-PI(4,5)P2 interaction, rather than indirectly by a myristate-dependent mechanism.

    Importance Binding of HIV-1 Gag to the plasma membrane is promoted by its interaction with a plasma-membrane-localized phospholipid PI(4,5)P2. Many cellular proteins are also recruited to the plasma membrane via PI(4,5)P2-interacting domains represented by PHPLC1. However, differences and/or similarities between these host proteins and viral Gag protein in the nature of their PI(4,5)P2 interactions, especially in the context of membrane binding, remain to be determined. Using a novel giant-unilamellar-vesicle-based system, we found that PI(4,5)P2 with an unsaturated acyl chain recruits PHPLC1 and Gag similarly, whereas PI(4,5)P2 with saturated acyl chains either recruits PHPLC1 but not Gag or sorts these proteins to different phases of vesicles. To our knowledge, this is the first study to show that PI(4,5)P2 acyl chains differentially modulate membrane binding of PI(4,5)P2-binding proteins. Since Gag membrane binding is essential for progeny virion production, the PI(4,5)P2 acyl chain property may serve as a potential target for anti-HIV therapeutic strategies.

  • Prolonged antiretroviral therapy preserves HIV-1-specific CD8 T cells with stem cell-like properties [PublishAheadOfPrint]

  • HIV-1-specific CD8 T cells can influence HIV-1 disease progression during untreated HIV-1 infection, but the functional and phenotypic properties of HIV-1-specific CD8 T cells in individuals treated with suppressive antiretroviral therapy remain less well understood. Here we show that a subgroup of HIV-1-specific CD8 T cells with stem cell-like properties (CD8 TSCM) is enriched in patients receiving suppressive antiretroviral therapy, as opposed to untreated progressors or controllers. In addition, prolonged duration of antiretroviral therapy was associated with a progressive increase in the relative proportions of these stem cell-like HIV-1-specific CD8 T cells. Interestingly, proportions of HIV-1-specific CD8 TSCM and total HIV-1-specific CD8 TSCM cells were associated with CD4 T cell counts during treatment with antiretroviral therapy, but not with CD4 T cell counts, viral loads or immune activation parameters in untreated patients, including controllers. HIV-1-specific CD8 TSCM cells seemed to preferentially secrete IL-2 in response to viral stimulation, while secretion of IFN- was more limited; however, only proportions of IFN--secreting HIV-1-specific CD8 TSCM were associated with CD4 T cell counts during antiretroviral therapy. Together, these data suggest that HIV-1-specific CD8 TSCM cells represent a long-lasting component of the cellular immune response to HIV-1 that persists in an antigen-independent fashion during antiretroviral therapy, but seems unable to survive and expand under conditions of ongoing viral replication during untreated infection.

    Importance Memory CD8 T cells that imitate functional properties of stem cells to maintain life-long cellular immunity have been hypothesized for many years, but only recently have such cells, termed T memory stem cells (TSCM), been physically identified and isolated in humans, mice and non-human primates. Here, we investigated whether cellular immune responses against HIV-1 include such T memory stem cells. Our data show that HIV-1-specific CD8 T memory stem cells are detectable during all stages of HIV-1 infection, but occur most visibly at times of prolonged viral antigen suppression by antiretroviral combination therapy. These cells may therefore be particularly relevant for designing antiviral immune defense strategies against the residual reservoir of HIV-1 infected cells that persists despite treatment and leads to viral rebound upon treatment discontinuation.

  • Inhibitory effect of individual or combinations of broadly neutralizing antibodies and antiviral reagents against cell-free and cell-to-cell HIV-1 transmission [PublishAheadOfPrint]

  • To date, most therapeutic and vaccine candidates for HIV-1 are evaluated pre-clinically for efficacy against cell-free viral challenges. However, cell-associated HIV-1 is suggested to be a major contributor to sexual transmission by mucosal routes. To determine if neutralizing antibodies or inhibitors block cell-free and cell-associated virus transmission of diverse HIV-1 strains with different efficiencies, we tested twelve different antibodies and five inhibitors against four GFP-labeled HIV-1 envelope (Env) variants from transmitted/founder or chronic isolates. We evaluated antibody/inhibitor-mediated virus neutralization using either TZM-bl target cells, in which infectivity was determined by virus-driven luciferase expression, or A3R5 lymphoblastoid target cells, in which infectivity was evaluated by GFP expression. In both the TZM-bl and A3R5 assay, cell-free virus or infected CD4+ lymphocytes were used as targets for neutralization. We further hypothesized that the combined use of specific neutralizing antibodies targeting HIV-1 Env would more effectively prevent cell-associated virus transmission than the use of individual antibodies. The tested antibody combinations included two gp120-directed antibodies, VRC01 and PG9, or VRC01 with the gp41-directed antibody, 10E8. Our results demonstrated that cell-associated virus was less sensitive to neutralizing antibodies and inhibitors, particularly using the A3R5 neutralization assay, and potencies of these neutralizing agents differed among Env variants. A combination of different neutralizing antibodies that target specific sites on gp120 led to a significant reduction in cell-associated virus transmission. These assays will help identify ideal combinations of broadly neutralizing antibodies to use for passive preventive antibody administration and further characterize targets for the most effective neutralizing antibodies/inhibitors.

    Importance: Preventing the transmission of human immunodeficiency virus-1 (HIV-1) remains a prominent goal of HIV research. The relative contribution of HIV-1 within an infected cell versus cell-free HIV-1 to virus transmission remains debated. It has been suggested that cell-associated virus is more efficient at transmitting HIV-1 and more difficult to neutralize than cell-free virus. Several broadly neutralizing antibodies and retroviral inhibitors are currently being studied as potential therapies against HIV-1 transmission. The present study demonstrates a decrease in neutralizing antibody and inhibitor efficiencies against cell-associated compared to cell-free HIV-1 transmission among different strains of HIV-1. We also observed a significant reduction in virus transmission using a combination of two different neutralizing antibodies that target specific sites on the outermost region of HIV-1, the virus envelope. Therefore, our findings support the use of antibody combinations against both cell-free and cell-associated virus in future candidate therapy regimens.

  • Pseudorabies virus fast-axonal transport occurs by a pUS9-independent mechanism [PublishAheadOfPrint]

  • Reactivation from latency results in transmission of neurotropic herpesviruses from the nervous system to body surfaces, referred to as anterograde axonal trafficking. The virus-encoded protein, pUS9, promotes axonal dissemination by sorting virus particles into axons, but whether it is also an effector of fast axonal transport within axons is unknown. To determine the role of pUS9 in anterograde trafficking, we analyzed the axonal transport of pseudorabies virus in the presence and absence of pUS9.

  • IFN{beta} and IFN{alpha}2 differentially protect head and neck cancer cells from VSV-induced oncolysis [PublishAheadOfPrint]

  • Oncolytic viruses (OV) preferentially kill cancer cells due in part to defects in their antiviral responses upon exposure to type I interferons (IFNs). However, IFN responsiveness of some tumor cells confers resistance to OV treatment. The human type I IFNs include one IFNbbeta; and multiple IFNaalpha; subtypes that share the same receptor but are capable of differentially inducing biological responses. The role of individual IFN subtypes in promoting tumor cell resistance to OV is addressed here. Two human IFNs which have been produced for clinical use, IFNaalpha;2a and IFNbbeta;, were compared for activity in protecting human head and neck squamous cell carcinoma (HNSCC) lines from oncolysis by vesicular stomatitis virus (VSV). Susceptibility of HNSCC lines to killing by VSV varied. VSV infection induced increased production of IFNbbeta; in resistant HNSCC cells. When added exogenously, IFNbbeta; was significantly more effective at protecting HNSCC cells from VSV oncolysis than was IFNaalpha;2a. In contrast, normal keratinocytes and endothelial cells were equivalently protected by both IFN subtypes. Differential responsiveness of tumor cells to IFNs aalpha; and bbeta; was further supported by the finding that autocrine IFNbbeta; but not IFNaalpha; promoted survival of HNSCC cells during persistent VSV infection. Therefore, IFNs aalpha; and bbeta; differentially affect VSV oncolysis, justifying the evaluation and comparison of IFN subtypes for use in combination with VSV therapy. Pairing VSV with IFNaalpha;2a may enhance selectivity of oncolytic VSV therapy for HNSCC by inhibiting VSV replication in normal cells without a corresponding inhibition in cancer cells.

    Importance There has been a great deal of progress in the development of oncolytic viruses. However, a major problem is that individual cancers vary in their sensitivity to oncolytic viruses. In many cases this is due to differences in their production and response to interferons (IFNs). The experiments described here compared the responses of head and neck squamous cell carcinoma cell lines to two IFN subtypes, IFNaalpha;2a and IFNbbeta;, in protection from oncolytic vesicular stomatitis virus. We found that IFNaalpha;2a was significantly less protective for cancer cells than was IFNbbeta;, whereas normal cells were equivalently protected by both IFNs. These results suggest that from a therapeutic standpoint, selectivity for cancer versus normal cells may be enhanced by pairing VSV with IFNaalpha;2a.

  • Herpes simplex virus 1 recruits CD98 heavy chain and {beta}1 integrin to the nuclear membrane for viral de-envelopment [PublishAheadOfPrint]

  • Herpesviruses have evolved a unique mechanism for nucleocytoplasmic transport of nascent nucleocapsids: the nucleocapsids bud through the inner nuclear membrane (INM) (primary envelopment), and the enveloped nucleocapsids then fuse with the outer nuclear membrane (de-envelopment). Little is known about the molecular mechanism of herpesviral de-envelopment. Here we showed that knockdown of both CD98 heavy chain (CD98hc) and its binding partner bbeta;1 integrin induced membranous structures containing enveloped herpes simplex virus 1 (HSV-1) virions that are invaginations of the INM into the nucleoplasm, and induced aberrant accumulation of enveloped virions in the perinuclear space and in the invagination structures. These effects were similar to those of the previously reported mutation(s) in HSV-1 proteins gB, gH, UL31 and/or Us3, which were shown here to form a complex(es) with CD98hc in HSV-1-infected cells. These results suggested that cellular proteins CD98hc and bbeta;1 integrin synergistically or independently regulated HSV-1 de-envelopment, probably by interacting directly and/or indirectly with these HSV-1 proteins.

    IMPORTANCE Certain cellular and viral macromolecular complexes, such as Drosophila large ribonucleoprotein complexes and herpesvirus nucleocapsids, utilize a unique vesicle-mediated nucleocytoplasmic transport: the complexes acquire primary envelopes by budding through the inner nuclear membrane into the space between the inner and outer nuclear membranes (primary envelopment), and the enveloped complexes then fuse with the outer nuclear membrane to release de-enveloped complexes into the cytoplasm (de-envelopment). However, there is a lack of information on the molecular mechanism of de-envelopment fusion. We report here that HSV-1 recruited cellular fusion regulatory proteins CD98hc and bbeta;1 integrin to the nuclear membrane for viral de-envelopment fusion. This is the first report of cellular proteins required for efficient de-envelopment of macromolecular complexes during their nuclear egress.

  • FUSE Binding Protein1 Facilitates Persistent Hepatitis C Virus Replication in Hepatoma Cells by Regulating Tumor Suppressor p53 [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) is a leading cause of chronic hepatitis C (CHC), liver cirrhosis, and hepatocellular carcinoma (HCC). Immunohistochemistry of archived HCC tumors showed abundant FBP1 expression in HCC tumors with CHC background. Oncomine data analysis of normal versus HCC tumors with CHC background indicated a 4-fold increase in FBP1 expression with a concomitant 2.5-fold decrease in the expression of p53. We found that FBP1 promotes HCV replication by inhibiting p53, and regulating BCCIP and TCTP, which, respectively, are positive and negative regulators of p53. The severe inhibition of HCV replication in FBP1-knockdown Huh7.5 cells was restored to a normal level by downregulation of either p53 or BCCIP. Although p53 in Huh7.5 cells is transcriptionally inactive as a result of Y220C mutation, we found that activation and DNA binding ability of Y220C p53 were strongly suppressed by FBP1, but significantly activated upon knockdown of FBP1. Transient expression of FBP1 in FBP1-knockdown cells fully restored the control phenotype in which the DNA binding ability of p53 was strongly suppressed. Using EMSA and ITC, we found no significant difference in in-vitro target DNA binding affinity of recombinant wild-type p53 and its Y220C mutant p53. However, in the presence of recombinant FBP1, DNA binding ability of p53 is strongly inhibited. We confirmed that FBP1 downregulates BCCIP, p21, and p53, and upregulates TCTP under radiation-induced stress. Since FBP1 is overexpressed in most HCC tumors with HCV background, it may have a role in promoting persistent virus infection and tumorigenesis.

    Importance It is our novel finding that Fuse binding protein1 (FBP1) strongly inhibits the function of tumor suppressor p53 and is an essential host-cell factor required for HCV replication. Oncomine data analysis on a large number of samples has revealed that overexpression of FBP1 in most HCC tumors with chronic hepatitis C is significantly linked with the decreased expression level of p53. The most significant finding is that FBP1 not only physically interacts with p53 and interferes with its binding to the target DNA but also functions as a negative regulator of p53 under cellular stress. FBP1 is barely detectable in normal differentiated cells; its overexpression in HCC tumors with CHC background suggests that FBP1 may have an important role in promoting HCV infection and HCC tumors by suppressing p53.

  • RESPIRATORY SYNCYTIAL VIRUS INHIBITOR AZ-27 DIFFERENTIALLY INHIBITS DIFFERENT POLYMERASE ACTIVITIES AT THE PROMOTER [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) is the leading cause of pediatric respiratory disease. RSV has an RNA dependent RNA polymerase that transcribes and replicates the viral negative sense RNA genome. The large polymerase subunit (L) has multiple enzymatic activities, having the capability to synthesize RNA, and add and methylate a cap on each of the viral mRNAs. Previous studies had identified a small molecule inhibitor, AZ-27, that targets the L protein. In this study, we examined the effect of AZ-27 on different aspects of RSV polymerase activity. AZ-27 was found to equally inhibit both mRNA transcription and genome replication in cell-based minigenome assays, indicating that it inhibits a step common to both these RNA synthesis processes. Analysis in an in vitro transcription run-on assay, containing RSV nucleocapsids, showed that AZ-27 inhibits synthesis of transcripts from the 3rrsquo; end of the genome to a greater extent that those from the 5rrsquo; end, indicating that it inhibits transcription initiation. Consistent with this finding, experiments that assayed polymerase activity on the promoter showed that AZ-27 inhibited transcription and replication initiation. The RSV polymerase can also utilize the promoter sequence to perform a back-priming reaction. Interestingly, addition of AZ-27 had no effect on addition of up to three nucleotides by back-priming, but inhibited further extension of the back-primed RNA. These data provide new information regarding the mechanism of inhibition by AZ-27. They also suggest that the RSV polymerase adopts different conformations to perform its different activities at the promoter.

    IMPORTANCE Currently, there are no effective antiviral drugs to treat RSV infection. The RSV polymerase is an attractive target for drug development, but this large enzymatic complex is poorly characterized, hampering drug development efforts. AZ-27 is a small molecule inhibitor previously shown to target the RSV large polymerase subunit, but its inhibitory mechanism was unknown. Understanding this would be valuable both for characterizing the polymerase and for further development of inhibitors. Here we show that AZ-27 inhibits an early stage in mRNA transcription, as well as genome replication, by inhibiting initiation of RNA synthesis from the promoter. However, the compound does not inhibit back-priming, another RNA synthesis activity of the RSV polymerase. These findings provide insight into the different activities of the RSV polymerase and will aid further development of antiviral agents against RSV.

  • CONSERVATION OF G PROTEIN EPITOPES IN RESPIRATORY SYNCYTIAL VIRUS (GROUP A) DESPITE BROAD GENETIC DIVERSITY: IS ANTIBODY SELECTION INVOLVED IN VIRUS EVOLUTION? [PublishAheadOfPrint]

  • Worldwide G glycoprotein phylogeny of human respiratory syncytial virus (hRSV) group A sequences revealed diversification in major clades and genotypes over more than fifty years of recorded history. Multiple genotypes co-circulated during prolonged periods of time but recent dominance of the GA2 genotype was noticed in several studies and it is highlighted here with sequences from viruses circulating recently in Spain and Panama. Reactivity of group A viruses with MAbs that recognize strain-variable epitopes of the G glycoprotein failed to correlate genotype diversification with antibody reactivity. Additionally, no clear correlation was found between changes in strain-variable epitopes and predicted sites of positive selection, despite both traits being associated to the C-terminal third of the G glycoprotein. Hence, our data do not lend support to the proposed antibody-driven selection of variants as major determinant of hRSV evolution. Other alternative mechanisms are considered to account for the high degree of hRSV_G variability.

    IMPORTANCE An unusual characteristic of the G glycoprotein of human respiratory syncytial virus (hRSV) is the accumulation of non-synonymous (N) changes at higher rates than synonymous (S) changes, reaching dN/dS values at certain sites predictive of positive selection. Since these sites cluster preferentially in the C-terminal third of the G protein, as certain epitopes recognized by murine antibodies, it was proposed that immune-(antibody) selection might be driving the apparent positive selection, analogous to the antigenic drift observed in the influenza virus hemagglutinin (HA). However, careful antigenic and genetic comparison of the G glycoprotein does not provide evidence of antigenic drift in the G molecule, in agreement with recent published data which did not sense antigenic drift in the G protein with human sera. Alternative explanations to the immune driven selection hypothesis are offered to account for the high level of G protein genetic diversity highlighted in this study.

  • Varicella Zoster Virus and Herpes Simplex Virus-1 Differentially Modulate NKG2D Ligand Expression During Productive Infection [PublishAheadOfPrint]

  • Natural killer (NK) cell deficient patients are particularly susceptible to severe infection with herpesviruses, especially varicella zoster virus (VZV) and herpes simplex virus (HSV-1). The critical role that NK cells play in controlling these infections denotes an intricate struggle for dominance between virus and NK cell antiviral immunity; however, research in this area has remained surprisingly limited. Our study addressed this absence of knowledge and found that infection with VZV was not associated with enhanced NK cell activation, suggesting that the virus uses specific mechanisms to limit NK cell activity. Analysis of viral regulation of ligands for NKG2D, a potent activating receptor ubiquitously expressed on NK cells, revealed that VZV differentially modulates expression of the NKG2D ligands, MICA, ULBP2 and ULBP3, by upregulating MICA expression while reducing ULBP2 and ULBP3 expression on the surface of infected cells. Despite being closely related to VZV, infection with HSV-1 produced a remarkably different effect on NKG2D ligand expression. A significant decrease in MICA, ULBP2 and ULBP3 was observed with HSV-1 infection at a total cellular protein level, as well as on the cell surface. We also demonstrate that HSV-1 differentially regulates expression of an additional NKG2D ligand, ULBP1, by reducing cell-surface expression while total protein levels are unchanged. Our findings illustrate both a striking point of difference between two closely related alphaherpesviruses, as well as suggest a powerful capacity for VZV and HSV-1 to evade antiviral NK cell activity through novel modulation of NKG2D ligand expression.

    IMPORTANCE Patients with deficiencies in NK cell function experience an extreme susceptibility to infection with herpesviruses, in particular, VZV and HSV-1. Despite this striking correlation, research into understanding how these two alphaherpesviruses interact with NK cells is surprisingly limited. Through examination of viral regulation of ligands to the activating NK cell receptor, NKG2D, we reveal patterns of modulation by VZV, which were unexpectedly varied to regulation by HSV-1 infection. Our study begins to unravel the undoubtedly complex interactions that occur between NK cells and alphaherpesvirus infection by providing novel insights into how VZV and HSV-1 manipulate NKG2D ligand expression to modulate NK cell activity, while also illuminating a distinct variation between two closely related alphaherpesviruses.

  • Cytokine-mediated activation of NK cells during viral infection [PublishAheadOfPrint]

  • Natural killer cells provide a first line of defense against infection via the production of antiviral cytokines and direct lysis of target cells. Cytokines such as IL-12 and IL-18 are critical regulators of NK cell activation, but much remains to be learned about how cytokines interact to regulate NK cell function. Here, we have examined cytokine-mediated activation of NK cells during infection with two natural mouse pathogens: lymphocytic choriomenengitis virus (LCMV) and murine cytomegalovirus (MCMV). Using a systematic screen of 1,849 cytokine pairs, we identified the most potent combinations capable of eliciting IFN production in NK cells. We observed that NK cell responses to cytokine stimulation were reduced at 8 days after acute LCMV infection, but recovered to pre-infection levels by 60 days post-infection. In contrast, during MCMV infection NK cell responses to cytokines remained robust at all time points examined. Ly49H+ NK cells recognizing the viral ligand, m157, showed preferential proliferation during early MCMV infection. A population of these cells remained detectable beyond 60 days post-infection, but these divided cells did not demonstrate enhanced IFN production in response to innate cytokine stimulation. Instead, the maturation state of the NK cells (as determined by CD11b/CD27 surface phenotype) was predictive of responsiveness to cytokines, regardless of Ly49H expression. These results help define cytokine interactions that regulate NK cell activation, and highlight variations in NK cell function during two unrelated viral infections.

    Importance Natural killer cells play an important role in immunity to many viral infections. From an initial screen of 1,849 cytokine pairs, we identified the most stimulatory cytokine combinations capable of inducing IFN production by NK cells. Ly49H+ NK cells, which can be directly activated by the MCMV protein, m157, preferentially proliferated during MCMV infection but did not show enhanced IFN production following direct ex vivo cytokine stimulation. Instead, mature CD11b+ and/or CD27+ NK cells responded similarly to innate cytokine stimulation regardless of Ly49H expression. Collectively, our data provide a better foundation for understanding cytokine-mediated NK cell activation during viral infection.

  • Adaptive mutations enhance assembly and cell-to-cell transmission of a high-titer hepatitis C virus genotype 5a Core-NS2 JFH1-based recombinant [PublishAheadOfPrint]

  • Recombinant hepatitis C virus (HCV) clones propagated in human hepatoma cell cultures yield relatively low infectivity titers. Here we adapted the JFH1-based Core-NS2 recombinant SA13/JFH1C3405G,A3696G (termed SA13/JFH1orig), of the poorly characterized genotype 5a, to Huh7.5 cells, yielding a virus with greatly improved spread kinetics and an infectivity titer of 6.7 log10 focus forming units (FFU)/mL. We identified several putative adaptive amino acid changes. In head-to-head infections at fixed multiplicities of infection, one SA13/JFH1orig mutant termed SA13/JFH1Core-NS5B, containing 13 amino acid changes (R114W, V187A [Core]; V235L [E1]; T385P [E2]; L782V [p7]; Y900C [NS2]; N2034D, E2238G, V2252A, L2266P, I2340T [NS5A]; A2500S, V2841A [NS5B]), displayed fitness comparable to the polyclonal high-titer adapted virus. Single-cycle virus production assays in CD81-deficient Huh7-derived cells demonstrated that these changes did not affect replication, but increased HCV assembly and specific infectivity as early as 24 hours post-transfection. Infectious co-culture assays in Huh7.5 cells showed a significant increase in cell-to-cell transmission for SA13/JFH1Core-NS5B viruses as well as viruses with only p7 and nonstructural protein mutations. Interestingly, the E2 HVR1 mutation T385P caused (i) increased sensitivity to neutralizing patient IgG and human monoclonal antibodies AR3A and AR4A and (ii) increased accessibility to the CD81 binding site without affecting the usage of CD81 and SR-BI. We finally demonstrated that SA13/JFH1orig and SA13/JFH1Core-NS5B, with and without the E2 mutation T385P, displayed similar biophysical properties following iodixanol gradient ultracentrifugation. This study has implications for investigations requiring high virus concentrations, such as studies of HCV particle composition and development of whole virus vaccine antigens.

    Importance Hepatitis C virus (HCV) is a major global healthcare burden, affecting more than 150 million people worldwide. These individuals are at high risk of developing severe end-stage liver diseases. No vaccine exists. While it is possible to produce HCV particles resembling isolates of all HCV genotypes in human hepatoma cells (HCVcc), production efficacy varies. Thus, for several important studies, including vaccine development, in vitro systems enabling high titer production of diverse HCV strains would be advantageous. Our study offers important functional data on how cell culture adaptive mutations identified in a genotype 5a JFH1-based HCVcc permit high-titer culture by affecting HCV genesis through increasing virus assembly and HCV fitness by enhancing the virus specific infectivity and cell-to-cell transmission ability, without influencing the biophysical particle properties. High-titer HCVcc like the one described in this study may be pivotal in future vaccine-related studies where large quantities of infectious HCV particles are necessary.

  • Different temporal effects of Ebola virus VP35 and VP24 proteins on the global gene expression in human dendritic cells [PublishAheadOfPrint]

  • Ebola virus (EBOV) causes a severe hemorrhagic fever with a deficient immune response, lymphopenia and lymphocyte apoptosis. Dendritic cells (DC), which trigger the adaptive response, do not mature despite EBOV infection. We recently demonstrated that DC maturation is unblocked by disabling the innate response antagonizing domains (IRADs) in EBOV VP35 and VP24 by mutations R312A and K142A, respectively. Here we analyzed the effects of VP35 and VP24 with the IRADs disabled on global gene expression in human DC. Human monocyte-derived DC were infected by wild-type EBOV (wt EBOV), or EBOVs carrying the mutation in VP35 (EBOV/VP35m), VP24 (EBOV/VP24m), or both (EBOV/VP35m/VP24m). Global gene expression at 8 and 24 hrs was analyzed by deep sequencing, and the expression of interferon (IFN) subtypes up to 5 days post-infection was analyzed by quantitative RT-PCR (qRT-PCR). Wt EBOV induced a weak global gene expression response, including markers of DC maturation, cytokines, chemokines, chemokine receptors, and multiple IFN. The VP35 mutation unblocked the expression resulting in a dramatic increase in expression of these transcripts at 8 and 24 hrs. Surprisingly, DC infected with EBOV/VP24m expressed lower levels of many of these transcripts at 8 hrs after infection, compared to wt EBOV. In contrast, at 24 hrs, expression of the transcripts increased in DC infected with any of the three mutants, as compared to wt EBOV. Moreover, sets of genes affected by the two mutations only partially overlapped. Pathway analysis demonstrated that the VP35 mutation unblocked pathways involved in antigen processing and presentation, and IFN signaling. These data suggest EBOV IRADs have profound effects on the host adaptive immune response through massive transcriptional downregulation of DC.

    IMPORTANCE. The study shows that infection of DC with EBOV, but not its mutant forms with VP35 and/or VP24 IRADs disabled, causes the global block in expression of host genes. The temporal effects of mutations disrupting the two IRADs differ, and the lists of affected genes only partially overlap such that VP35 and VP24 IRADs each have profound effects on antigen presentation by exposed DC. The global modulation of DC gene expression and the resulting lack of their maturation represent a major mechanism by which EBOV disables the T cell response, and suggests that these suppressive pathways are a therapeutic target that may unleash the T cell responses during EBOV infection.

  • The ability of the encephalitic arbovirus Semliki Forest virus to cross the blood brain barrier is determined by the charge of the E2 glycoprotein [PublishAheadOfPrint]

  • Semliki Forest virus (SFV) provides a well-characterized model system to study the pathogenesis of virus encephalitis. Several studies have used virus derived from the molecular clone SFV4. SFV4 virus does not have the same phenotype as the closely related L10 or the prototype virus from which its molecular clone was derived. In mice, L10 generates a high titer plasma viremia, is efficiently neuroinvasive and produces a fatal panencephalitis. Whereas, low dose SFV4 produces a low titer viremia, rarely enters the brain and is generally avirulent. To determine the genetic differences responsible, the consensus sequence of L10 was determined and compared to SFV4. There were twelve nucleotide differences, six were non-synonymous; these were engineered into a new molecular clone, termed SFV6. The derived virus, SFV6, generated a high titer viremia and was efficiently neuroinvasive and virulent. The phenotypic difference mapped to a single aa residue at position 162 in the E2 envelope glycoprotein; lysine in SFV4, glutamic acid in SFV6. Analysis of the L10 virus showed it contained different plaque phenotypes which differed in virulence. A lysine at E2 247 conferred a small plaque avirulent phenotype and glutamic acid a large plaque virulent phenotype. Viruses with a positively charged lysine at E2 162 or 247 were more reliant on glycosaminoglycans (GAGs) to enter cells and were selected for by passage in BHK-21 cells. Interestingly, viruses with the greatest reliance on binding to GAGs replicated to higher titers in the brain and more efficiently crossed an in vitro blood-brain barrier (BBB).

    Importance Virus encephalitis is a major disease and alphaviruses, as highlighted by the recent epidemic of chikungunya virus (CHIKV), are medically important pathogens. In addition, alphaviruses provide well-studied experimental systems with extensive literature, many tools and easy genetic modification. In this study we elucidate the genetic basis for the difference in phenotype between SFV4 and the virus stocks from which it was derived and correct this by engineering a new molecular clone. We then use this in one comprehensive study to demonstrate that positively charged aa residues on the surface of the E2 glycoprotein, mediated by binding to GAGs, determine selective advantage and plaque size in BHK-21 cells, level of viremia in mice, ability to cross an artificial BBB, efficiency of replication in the brain and virulence. Together with studies on Sindbis virus (SINV), this study provides an important advance in understanding alphavirus, and probably other virus, encephalitis.

  • A Rhesus Rhadinovirus Viral Interferon Regulatory Factor is Virion-associated and Inhibits the Early IFN Antiviral Response [PublishAheadOfPrint]

  • The interferon (IFN) response is the earliest host immune response dedicated to combating viral infection. As such, viruses have evolved strategies to subvert this potent antiviral response. Two closely related gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) and rhesus macaque rhadinovirus (RRV), are unique in that they express viral homologues to cellular interferon regulatory factors (IRFs), deemed viral IRFs (vIRFs). Cellular IRFs are a family of transcription factors that are particularly important for the transcription of type I IFNs. Here, we demonstrate a strategy employed by RRV to ensure rapid inhibition of virus-induced type I IFN induction. We found that RRV vIRF R6, when expressed ectopically, interacts with transcriptional coactivator, CREB-binding protein (CBP), in the nucleus. As a result, phosphorylated IRF3, an important transcriptional regulator in IFNbbeta; transcription, fails to effectively bind to the IFNbbeta; promoter, thus inhibiting the activation of IFNbbeta; genes. In addition, we found R6 within RRV virion particles via immunoelectron microscopy and furthermore, that, virion-associated R6 is capable of inhibiting the type I IFN response by preventing efficient binding of IRF3/CBP complexes to the IFNbbeta; promoter in the context of infection. The work shown here is the first example of a vIRF being associated with either the KSHV or RRV virion. The presence of this immunomodulatory protein in the RRV virion provides the virus with an immediate mechanism to evade the host IFN response, thus enabling the virus to effectively establish an infection within the host.

    IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related rhesus macaque rhadinovirus (RRV) are the only known viruses to encode viral homologues to cellular interferon regulatory factors (IRFs) known as vIRFs. In KSHV, these proteins have been shown to play major roles in a variety of cellular processes and are particularly important in the evasion of the host type I interferon (IFN) response. In this study, we delineate the immunomodulatory mechanism of an RRV vIRF and its ability to assist the virus in rapid immune evasion by being prepackaged within the virion, thus providing evidence, for the first time, of a virion-associated vIRF. This work further contributes to our understanding of the mechanisms behind the immunomodulation by the RRV vIRFs during infection.

  • Bile acids reduce prion conversion, reduce neuronal loss, and prolong male survival in models of prion disease. [PublishAheadOfPrint]

  • Prion diseases are fatal neurodegenerative disorders associated with the conversion of cellular prion protein (PrPC) into its aberrant infectious form (PrPSc). There is no treatment available for these diseases. The bile acids tauroursodeoxycholic acid (TUDCA) and ursodeoxycholic acid (UDCA) have been recently shown to be neuroprotective in other protein misfolding disease models including Parkinson's, Huntington's and Alzheimer's diseases, and also in humans with amyotrophic lateral sclerosis (ALS). Here we studied the therapeutic efficacy of these compounds in prion disease. We demonstrated that TUDCA and UDCA substantially reduced PrP conversion in cell-free aggregation assays as well as in chronically and acutely infected cell cultures. This effect was mediated through reduction of PrPSc seeding ability, rather than an effect on PrPC. We also demonstrated the ability of TUDCA and UDCA to reduce neuronal loss in prion infected cerebellar slice cultures. UDCA treatment reduced astrocytosis and prolonged survival in RML prion-infected mice. Interestingly, these effects were limited to the males, implying a gender-specific difference in drug metabolism. Beyond effects on PrPSc, we found that levels of phosphorylated eIF2aalpha; were increased at early time points, with correlated reductions in PSD-95. As demonstrated for other neurodegenerative diseases, we now show that TUDCA and UDCA may have a therapeutic role in prion diseases, with effects on both prion conversion and neuroprotection. Our findings, together with the fact that these natural compounds are orally bioavailable, permeable to the blood-brain barrier and FDA-approved for use in humans, make these compounds promising alternatives for the treatment of prion diseases.

    IMPORTANCE Prion diseases are fatal neurodegenerative diseases that are transmissible to humans and other mammals. There are no disease-modifying therapies available, despite decades of research. Treatment targets have included inhibition of protein accumulation, clearance of toxic aggregates, and prevention of downstream neurodegeneration. No one target may be sufficient; rather, compounds which have a multi-modal mechanism, acting on different targets, would be ideal. TUDCA and UDCA are bile acids that may fulfill this dual role; previous studies have demonstrated their neuroprotective effects in several neurodegenerative disease models, and we now demonstrate that this effect occurs in prion disease, with an added mechanistic target of upstream prion seeding. Importantly, these are natural compounds which are orally bioavailable, permeable to the blood-brain barrier and FDA-approved for use in humans with primary biliary cirrhosis. They have recently been proven efficacious in human ALS. Therefore, these compounds are promising options for the treatment of prion diseases.

  • Isolation and analysis of rare norovirus recombinants from co-infected mice using drop-based microfluidics [PublishAheadOfPrint]

  • Human noroviruses (HuNoVs) are positive-sense RNA viruses that can cause severe, highly infectious gastroenteritis. HuNoV outbreaks are frequently associated with recombination between circulating strains. Strain genotyping and phylogenetic analyses show that noroviruses often recombine in a highly conserved region near the junction of the viral polyprotein (ORF1) and capsid (ORF2) genes and occasionally within the RNA-dependent RNA polymerase (RdRP) gene. Although genotyping methods are useful for tracking changes in circulating viral populations, they only report the dominant recombinant strains and do not elucidate the frequency or range of recombination events. Furthermore, the relatively low frequency of recombination in RNA viruses has limited studies to cell culture or in vitro systems that do not reflect the complexities and selective pressures present in an infected organism. Using two murine norovirus (MNV) strains to model co-infection, we developed a microfluidic platform to amplify, detect, and recover individual recombinants following in vitro and in vivo co-infection. One-step RT-PCR was performed in picoliter drops with primers that identified the wild-type and recombinant progenies and scanned for recombination breakpoints at approximately 1-kb intervals. We detected recombination between MNV strains at multiple loci spanning the viral protease, RdRP, and capsid ORFs and isolated individual recombinant RNA genomes that were present at a frequency of 1/300,000 or greater. This study is the first to examine norovirus recombination following co-infection of an animal and suggests that the exchange of RNA among viral genomes in the infected host occurs in multiple locations and is an important driver of genetic diversity.

    IMPORTANCE RNA viruses increase diversity and escape host immune barriers by genomic recombination. Studies from a number of viral systems indicate that recombination occurs via template switching by the virus-encoded RNA-dependent RNA polymerase (RdRP). However, factors that govern the frequency and positions of recombination in an infected organism remain largely unknown. This work leverages advances in the applied physics of drop-based microfluidics to isolate and sequence rare recombinants arising from the co-infection of mice with two distinct strains of murine norovirus. This report is the first to detect and analyze norovirus recombination in an animal model.

  • Conservation of the STING-mediated cytosolic DNA sensing pathway in zebrafish [PublishAheadOfPrint]

  • Zebrafish (Danio rerio) is a unique and potential model animal for dissecting innate immune signalings. Here, we demonstrate that herpes simplex virus type -1(HSV-1) could infect zebrafish at its different developmental stages, and trigger the expression of type I interferons (IFNs) as well as ISGs in zebrafish larva. Silencing of zSTING, but not zMAVS, markedly attenuates the DNA-virus induced antiviral responses. Notably, a conserved serine residue (S373) is essential for the action of zSTING. Unexpectedly, zebrafish cGAS is dispensable for the STING signaling, whereas zDHX9 and zDDX41 are potential sensors for HSV-1 infection in vivo. Taken together, this proof-of-concept study establishes the zebrafish larva as a feasible model for investigating the cytosolic DNA sensing mechanism, shedding light on the conservation of the STING antiviral signaling pathway.

    Importance The zebrafish larva provides technical advantages for understanding host-pathogen interactions. In this study, we establish the zebrafish larva as a useful model for studying HSV-1 infection. HSV-1 infection triggers strong type I interferon production, which depends on STING expression. In addition, STING-mediated antiviral signaling is conserved in zebrafish. Interestingly, zDHX9 and zDDX41 are indispensable for detecting HSV-1 while cGAS is dispensable. This proof-of-concept study indicates that the zebrafish represents an amenable model for the investigation of cytosolic DNA sensing mechanisms.

  • Historical Outbreaks of Simian Hemorrhagic Fever in Captive Macaques Were Caused by Distinct Arteriviruses [PublishAheadOfPrint]

  • Simian hemorrhagic fever (SHF) is lethal for macaques. Based on clinical presentation and serological diagnosis, all reported SHF outbreaks were thought to be caused by different strains of the same virus, simian hemorrhagic fever virus (SHFV; Arteriviridae). Here we show that SHF outbreaks in Sukhumi in 1964 and in Alamogordo in 1989 were not caused by SHFV, but by two novel, divergent arteriviruses. Our results indicate that multiple, divergent simian arteriviruses can cause SHF.

  • Tuberous Sclerosis Complex Protein 2 (TSC2)-Independent Activation of mTORC1 by Human Cytomegalovirus pUL38 [PublishAheadOfPrint]

  • The mammalian target of rapamycin complex 1 (mTORC1) controls cell growth and anabolic metabolism and is a critical host factor activated by human cytomegalovirus (HCMV) for successful infection. The multi-functional HCMV protein pUL38 has previously been reported to activate mTORC1 by binding to and antagonizing tuberous sclerosis complex protein 2 (TSC2). pUL38 also plays a role in blocking endoplasmic reticulum stress-induced cell death during HCMV infection. In this study, we showed that a mutant pUL38 lacking the N-terminal 24 amino acids (pHA-UL3825-331) was fully functional in suppressing cell death during infection. Interestingly, pHA-UL3825-331 lost the ability to interact with TSC2 but it retained the ability to activate mTORC1, although to a lesser extent than full-length pHA-UL38. Recombinant virus expressing pHA-UL3825-331 replicated with ~10 fold less efficiency than the wild type virus at a low multiplicity of infection (MOI), but it grew similarly well at a high MOI, suggesting an MOI dependent importance of pUL38-TSC2 interaction in supporting virus propagation. Site-directed mutational analysis identified a TQ motif at amino acid residues 23-24 as critical for pUL38 interaction with TSC2. Importantly, when expressed in isolation, the TQ/AA substitution mutant pHA-UL38 TQ/AA was capable of activating mTORC1 just like pHA-UL3825-331. We also created TSC2-null U373-MG cell lines by CRISPR genome editing and showed that pUL38 was capable of further increasing mTORC1 activity in TSC2-null cells. This study therefore identified the residues important for pUL38-TSC2 interaction and demonstrated that pUL38 can activate mTORC1 in both TSC2-dependent and -independent manners.

    Importance Human cytomegalovirus (HCMV), like other viruses, depends exclusively on its host cell to propagate. It has therefore developed methods to protect against host stress responses and to usurp cellular processes to complete its life cycle. Mammalian target of rapamycin complex 1 (mTORC1) is believed to be important for virus replication, and HCMV maintains high mTORC1 activity despite the stressful cellular environment associated with infection. mTORC1 inhibitors suppressed HCMV replication in vitro and reduced the incidence of HCMV reactivation in transplant recipients. We demonstrated that mTORC1 was activated by HCMV protein pUL38 in both tuberous sclerosis complex protein 2 (TSC2)-dependent and TSC2-independent manners. pUL38-independent mode of mTORC1 activation has also been reported. These novel findings suggest the evolution of sophisticated approaches whereby HCMV activates mTORC1, indicating its importance in the biology and pathogenesis of HCMV.

  • Measles Virus Defective-interfering RNAs are Generated Frequently and Early in the Absence of C Protein and can be Destabilized by Adenosine Deaminase Acting on RNA 1-like Hypermutations [PublishAheadOfPrint]

  • Defective-interfering viral genome RNAs (DI-RNAs) can form during infections of negative strand RNA viruses and outgrow full-length viral genomes, thereby modulating the severity and duration of infection. Here we document frequent de novo generation of copyback DI-RNAs from independent rescue events both for vaccine (vac2) and wild-type (IC323) measles viruses as early as passage 1 after virus rescue. Moreover, vaccine and wild-type C protein-deficient (CKO) measles viruses generated about ten times more DI-RNAs than parental virus, suggesting that C enhances processivity of the viral polymerase. We obtained nucleotide sequences of 65 individual DI-RNAs, identified breakpoints and re-initiation sites and predicted their structural features. Several DI-RNAs possessed clusters of A-to-G or U-to-C transitions. Sequences flanking these mutation sites were characteristic of those favored by adenosine deaminase acting on RNA 1 (ADAR1), which catalyzes in double-stranded RNA the C6 deamination of adenosine to produce inosine that is recognized as guanosine, a process known as A-to-I RNA editing. In individual DI-RNAs the transitions were of the same type and occurred on both sides of the breakpoint. These patterns of mutations suggest that ADAR1 edits unencapsidated DI-RNAs that formed double strand RNA structures. Encapsidated DI-RNAs were incorporated into virus particles, which reduced infectivity of virus stocks. The CKO-phenotype was dominant: DI-RNAs derived from vac2-CKO suppressed replication of vac2, as shown by co-infections of interferon-incompetent lymphatic cells with viruses expressing different fluorescent reporter proteins. In contrast, co-infection with a C protein-expressing virus did not counteract the suppressive phenotype of DI-RNAs.

    Importance Recombinant measles viruses (MV) are in clinical trials as cancer therapeutics and as vectored vaccines for HIV-AIDS and other infectious diseases. Efficacy of MV-based vectors depends on their replication proficiency and immune activation capacity. Here we document that copyback defective-interfering RNAs (DI-RNAs) are generated by recombinant vaccine and wild-type MVs immediately after rescue. The MV C protein interferes with DI-RNA generation and may enhance processiviry of the viral polymerase. We frequently detected clusters of A-to-G or U-to-C transitions and noted that sequences flanking individual mutations contain motifs favoring recognition by the adenosine deaminase acting on RNA 1 (ADAR1). The consistent type of transitions on the DI-RNAs indicates that these were direct substrates for editing by ADAR1. The ADAR1-mediated biased hypermutation events are consistent with the protein kinase R (PKR)-ADAR1 balancing model of innate immunity activation. We show by co-infection that the C-defective phenotype is dominant.

  • Different effects of NNRTI-resistance mutations on CTL recognition between HIV-1 subtype B and subtype A/E infections [PublishAheadOfPrint]

  • The effect of antiretroviral drug-resistance mutations on CTL recognition has been analyzed in HIV-1 subtype B infections, but it remains unclear in other HIV-1 subtype infections that are epidemic in countries where antiretroviral drugs are not effectively used. We investigated the effect of non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistance mutations (Y181C, Y181I, and 181V) on epitope recognition by CTLs specific for 3 different HIV-1 epitopes (HLA-A*02:01-restricted IV10, HLA-B*35:01-restricted NY9, and HLA-C*12:02-restricted KY9) in subtype B and subtype A/E infections and the accumulation of these mutations in treatment-naiiuml;ve Japanese and Vietnamese. These NNRTI-resistance mutations critically affected NY9-specific and KY9-specific T cell responses in the subtype B infections, whereas they showed a different effect on IV10-specific T cell responses among the subtype B-infected individuals. These mutations affected IV10-specific T cell responses but weakly affected NY9-specific T cell responses in the subtype A/E infections. The substitution at position 3 of NY9 epitope, which was found in the subtype A/E virus, differently influenced the peptide binding to HLA-B*35:01, suggesting that the different peptide binding may result in the different T cell recognition between the subtype B virus and A/E virus infections. The Y181C mutation was found to be accumulating in treatment-naiiuml;ve Vietnamese infected with the subtype A/E virus. The present study demonstrated different effect of NNRTI-resistance RT181 mutations on CTL responses between the 2 subtype infections. The Y181C mutation may influence HIV-1 control by the CTLs in Vietnam, since this mutation has been accumulating in treatment-naiiuml;ve Vietnamese.

    Importance Antiretroviral therapy leads to the emergence of drug resistance HIV-1, resulting in virological and clinical failures. Though HIV-1-specific CTLs play a critical role in HIV-1 infection, some of drug-resistance mutations located in CTL epitopes are known to affect HIV-1-specific CTL responses. Non-nucleoside reverse transcriptase inhibitor (NNRTIs)-resistance RT181 mutations is frequently observed in patients treated with NNRTIs. Such drug resistance mutations may influence on immune control by HIV-1-specific CTLs, especially in countries where antiretroviral drugs are not effectively used. We here investigated the effect of three NNRTI resistance RT181 mutations on immune responses by HIV-1-specific CTLs and the accumulation of this mutation in the recent treatment-naiiuml;ve Vietnamese infected with HIV-1 subtype A/E virus. RT181 mutations affected the CTL recognitions in both subtype A/E and B infections while RT Y181C mutation has been accumulating in treatment-naiiuml;ve Vietnamese. The results suggest that Y181C mutation may influence HIV-1 control by the CTLs in Vietnam.

  • Regulation of DNA Damage Signaling and Cell Death Responses by Epstein-Barr virus Latent Membrane Proteins (LMP) 1 and LMP2A in Nasopharyngeal Carcinoma Cells [PublishAheadOfPrint]

  • Nasopharyngeal carcinoma (NPC) is closely associated with latent Epstein-Barr virus (EBV) infection. Although EBV infection of pre-neoplastic epithelial cells is not immortalizing, EBV can modulate oncogenic and cell death mechanisms. The viral latent membrane proteins (LMP) 1 and LMP2A are consistently expressed in NPC, and can co-operate in bi-transgenic mice expressed from the keratin-14 promoter to enhance carcinoma development in an epithelial chemical carcinogenesis model. In this study, LMP1 and LMP2A were co-expressed in the EBV-negative NPC cell line HK1, and examined for combined effects in response to genotoxic treatments. In response to DNA damage activation, LMP1 and LMP2A co-expression reduced H2AX (S139) phosphorylation and caspase cleavage induced by a lower dose (5mmu;M) of the topoisomerase II inhibitor etoposide. Regulation of H2AX occurred before the onset of caspase activation, without modulation of other DNA damage signaling mediators including ATM, Chk1 or Chk2, and was additionally suppressed by inducers of DNA single strand breaks (SSBs) and replication stress. Despite reduced DNA damage repair signaling, LMP1-2A co-expressing cells recovered from cytotoxic doses of etoposide, however LMP1 expression was sufficient for this effect. LMP1 and LMP2A co-expression did not enhance cell growth, with moderate increase of cell motility to fibronectin. This study supports that LMP1 and LMP2A jointly regulate DNA repair signaling and cell death activation with no further enhancement in the growth properties of neoplastic cells.

    Importance NPC is characterized by clonal EBV infection and accounts for ggt;78,000 annual cancer cases with increased incidence in endemic regions such as Southeast Asia. The latent proteins LMP1 and LMP2A co-expressed in NPC can individually enhance growth or survival properties in epithelial cells, but their combined effects and potential regulation of DNA repair and checkpoint mechanisms are relatively undetermined. In this study, LMP1-2A co-expression suppressed activation of the DNA damage response (DDR) protein H2AX induced by selective genotoxins that promote DNA replication stress or SSBs. Expression of LMP1 was sufficient to recover cells, resulting in outgrowth of LMP1 and LMP1-2A co-expressing cells and indicating distinct LMP1-dependent effects in restoration of replicative potential. These findings demonstrate novel properties for LMP1 and LMP2A in the co-operative modulation of DDR and apoptotic signaling pathways, further implicating both proteins in the progression of NPC and epithelial malignancies.

  • HIV-1 Env and Nef cooperatively contribute to pDCs activation via CD4-dependent mechanisms [PublishAheadOfPrint]

  • Plasmacytoid dendritic cells (pDCs) are the major source of type I IFN (IFN-I) in response to Human Immunodeficiency Virus type-1 (HIV-1) infection. pDCs are rapidly activated during HIV-1 infection and are implicated in both reducing early viral load as well as contributing to HIV-1-induced pathogenesis. However, most cell-free HIV-1 isolates are inefficient in activating human pDCs, and the mechanisms of HIV-1 recognition by pDCs and pDC activation are not clearly defined. In this study we report that two genetically similar HIV-1 variants (R3A and R3B) isolated from a rapid progressor differentially activated pDCs to produce IFNaalpha;. The highly pathogenic R3A efficiently activated pDCs to induce robust IFNaalpha; production, while the less pathogenic R3B did not. The viral determinant for efficient pDC activation was mapped to the V1V2 region of R3A Env, which also correlated with enhanced CD4 binding activity. Furthermore, we showed that the Nef protein was also required for the activation of pDCs by R3A. Analysis of a panel of R3A Nef functional mutants demonstrated that Nef domains involved in CD4 down-regulation were necessary for R3A to activate pDCs. Our data indicate that R3A-induced pDC activation depends on (1) the high affinity of R3A Env to bind CD4 receptor and (2) Nef activity, which is involved in CD4 down-regulation. Our findings provide new insights into the mechanism by which HIV-1 induces IFNaalpha; in pDCs, which contributes to pathogenesis.

    Importance Plasmacytoid dendritic cells (pDCs) are the major type I interferon (IFN-I) producing cells, and IFN-Is actually contribute to pathogenesis during chronic viral infections. How HIV-1 activates pDCs and the role of pDC/IFN-I in HIV-1 pathogenesis remain unclear. We report here the highly pathogenic HIV-R3A efficiently activated pDCs to induce IFNaalpha; production, while most HIV-1 isolates are inefficient in activating pDCs. We have discovered that R3A-induced pDC activation depends on (1) the high affinity of R3A Env to bind CD4 receptor and (2) Nef activity, which is involved in CD4 down-regulation. Our findings thus provide new insights into the mechanism by which HIV-1 induces IFNaalpha; in pDCs and contributes to HIV-1 pathogenesis. These novel findings will be of great interest to those working on the role of IFN and pDCs in HIV-1 pathogenesis in general, and on the interaction of HIV-1 with pDCs in particular.

  • Molecular mechanism for LAMP1 recognition by Lassa Virus [PublishAheadOfPrint]

  • Lassa virus is a notorious human pathogen that infects many thousands of people each year in West Africa, causing severe viral hemorrhagic fevers and significant mortality. The surface glycoprotein of Lassa virus mediates receptor recognition through its GP1 subunit. Here we report the crystal structure of GP1 from Lassa virus, which is the first representative GP1 structure for Old World arenaviruses. We identify a unique triad of histidines that forms a binding site for LAMP1, a known lysosomal protein recently discovered to be a critical receptor for internalized Lassa virus at acidic pH. We demonstrate that mutation of this histidine-triad, which is highly conserved among Old World arenaviruses, impairs LAMP1 recognition. Our biochemical and structural data further suggest that GP1 from Lassa may undergo irreversible conformational changes that could serve as an immunological decoy mechanism. Together with a variable region that we identify on the surface of GP1, those could be two distinct mechanisms that Lassa virus utilizes to avoid antibody-based immune response.

    IMPORTANCE Structural data at atomic resolution for viral proteins is key for understanding their function at the molecular level and can facilitate novel avenues for combating viral infections. Here we use X-ray protein crystallography to decipher the crystal structure of the receptor-binding domain (GP1) from Lassa virus. This is a pathogenic virus that causes significant illness and mortality in West Africa. This structure reveals the overall architecture of GP1 domains from the group of viruses known as the Old World arenaviruses. Using this structural information we elucidate the pH switch and binding mechanisms of Lassa virus to LAMP1, a recently identified host receptor that is critical for successful infection. Lastly, our structural analysis suggests two novel immune evasion mechanisms that Lassa virus may utilize to escape antibody-based immune response.

  • HBV-infected HepG2hNTCP cells serve as a novel immunological tool to analyze the antiviral efficacy of CD8+ T cells in vitro [PublishAheadOfPrint]

  • CD8+ T cells are main effector lymphocytes in the control of hepatitis B virus (HBV) infection. However, limitations of model systems such as low infection rates restricted mechanistic studies of HBV-specific CD8+ T cells. Here, we established a novel immunological cell culture model based on HBV-infected HepG2hNTCP cells that endogenously processed and presented viral antigens to HBV-specific CD8+ T cells. This induced cytolytic and non-cytolytic CD8+ T-cell effector functions and reduction of viral loads.

  • High-throughput analysis of human cytomegalovirus genome diversity highlights the widespread occurrence of gene-disrupting mutations and pervasive recombination [PublishAheadOfPrint]

  • Human cytomegalovirus is a widespread pathogen of major medical importance. It causes significant morbidity and mortality in the immunocompromised and congenital infections can result in severe disabilities or stillbirth. Development of a vaccine is prioritized, but no candidate is close to release. Although correlations of viral genetic variability with pathogenicity are suspected, knowledge about strain diversity of the 235kb genome is still limited. In this study, 96 full-length human cytomegalovirus genomes from clinical isolates were characterized, quadrupling the available information for full-genome analysis. These data provide the first high-resolution map of human cytomegalovirus interhost diversity and evolution. We show that cytomegalovirus is significantly more divergent than all other human herpesviruses and highlight hotspots of diversity in the genome. Importantly, 75% of strains are not genetically intact, but contain disruptive mutations in a diverse set of 26 genes, including immunomodulative genes UL40 and UL111A. These mutants are independent from culture passaging artifacts and circulate in natural populations. Pervasive recombination, which is linked to the widespread occurrence of multiple infections, was found throughout the genome. Recombination density was significantly higher than in other human herpesviruses and correlated with strain diversity. While the overall effects of strong purifying selection on virus evolution are apparent, evidence of diversifying selection was found in several genes encoding proteins that interact with the host immune system, including UL18, UL40, UL142 and UL147. These residues may present phylogenetic signatures of past and ongoing virus-host interactions.

    IMPORTANCE Human cytomegalovirus has the largest genome of all viruses that infect humans. Currently, there is a great interest in establishing associations between genetic variants and strain pathogenicity of this herpesvirus. Since the number of publicly available full-genome sequences is limited, knowledge about strain diversity is highly fragmented and biased towards a small set of loci. Combined with our previous work, we have now contributed 101 complete genome sequences. We have used these data to conduct the first high-resolution analysis of interhost genome diversity, providing an unbiased and comprehensive overview of cytomegalovirus variability. These data are of major value to the development of novel antivirals and a vaccine and to identify potential targets for genotype-phenotype experiments. Furthermore, they have enabled a thorough study of the evolutionary processes that have shaped cytomegalovirus diversity.

  • Vinexin {beta} interacts with Hepatitis C Virus NS5A modulating its hyperphosphorylation to regulate viral propagation [PublishAheadOfPrint]

  • The hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is essential for HCV genome replication and virion production, and is involved in regulating multiple host signaling pathways. As a proline-rich protein, NS5A is capable of interacting with various host proteins containing Src homology 3 (SH3) domain. Previous studies have suggested that Vinexin, a member of Sorbin Homology (SoHo) adaptor family, might be a potential binding partner of NS5A by yeast two-hybrid screening. However, firm evidence for this interaction is lacking, and the significance of Vinexin to HCV life cycle remains unclear. In this study, we demonstrated that the endogenously and exogenously expressed Vinexin bbeta; co-immunoprecipitated with NS5A derived from different HCV genotypes. Two residues-tryptophan (W307) and tyrosine (Y325) in third SH3 domain of Vinexin bbeta; and conserved Pro-X-X-Pro-X-Arg motifs at C-terminus of NS5A were indispensable for the Vinexin-NS5A interaction. Furthermore, down-regulation of endogenous Vinexin bbeta; significantly suppressed NS5A hyperphosphorylation and decreased HCV replication which could be rescued by expressing a Vinexin bbeta; shRNA resistant mutant. We also found that Vinexin bbeta; modulated hyperphosphorylation of NS5A in a casein kinase 1aalpha; (CK1aalpha;)-dependent on manner. Taken together, our findings suggest that Vinexin bbeta; modulates NS5A phosphorylation via its interaction with NS5A thereby regulating HCV replication, implicating the role of Vinexin bbeta; in viral life cycle.

    Importance The hepatitis C virus (HCV) nonstructural protein NS5A is a phosphoprotein, and its phosphorylation states are usually modulated by host kinases and other viral non-structural elements. Additionally cellular factors containing Src homology 3 (SH3) domain have been reported to interact with proline-rich regions of NS5A. However, it is unclear whether there are any relationships between NS5A phosphorylation and NS5A-SH3 interaction, and little is known about the significance of this interaction in the HCV life cycle. In this work, we demonstrate that Vinexin bbeta; modulates NS5A hyperphosphorylation through NS5A-Vinexin bbeta; interaction. Hyperphosphorylated NS5A induced by Vinexin bbeta; is casein kinase 1aalpha;-dependent, and is also crucial for HCV propagation. Overall, our findings not only elucidate the relationships between NS5A phosphorylation and NS5A-SH3 interaction, but also shed new mechanistic insight on Flaviviridae NS5A (NS5) phosphorylation. We believe that our results may afford the potential to offer an antiviral therapeutic strategy.

  • Cooperativity of HIV-specific cytolytic CD4+ T cells and CD8+ T cells in control of HIV viremia [PublishAheadOfPrint]

  • CD4+ T cells play a pivotal role in the control of chronic viral infections. Recently, non-traditional CD4+ T cell functions beyond helper effects have been described and a role of cytolytic CD4+ T cells in the control of HIV infection suggested. Here we define the transcriptional, phenotypic, and functional profiles of HIV-specific cytolytic CD4+ T cells. Fluidigm BioMark and multiparameter flow cytometric analysis of HIV-specific cytolytic CD4+ T cells revealed a distinct transcriptional signature compared to Th1 CD4+ cells, but shared similar features with HIV-specific cytolytic CD8+ T cells. Furthermore, HIV-specific cytolytic CD4+ T cells showed comparable killing activity relative to HIV-specific CD8+ T cells and worked cooperatively in the elimination of virally infected cells. Interestingly, we found that cytolytic CD4+ T cells emerge early during acute HIV infection and tightly follow acute viral load trajectory. This emergence was associated to the early viral set point, suggesting an involvement in early control, in spite of their susceptibility to HIV infection. Our data suggest cytolytic CD4+ T cells as an independent subset distinct from Th1 cells that show combined activity with CD8+ T cells in the long-term control of HIV infection.

    Importance The ability of the immune system to control chronic HIV infection is of critical interest to both vaccine design and therapeutic approaches. Much research has focused on the effect of the ability of CD8+ T cells to control the virus, while CD4+ T cells have been overlooked as effectors in HIV control due to the fact that they are preferentially infected. Here we show that a subset of HIV-specific CD4+ T cells cooperate in the cytolytic control of HIV viral replication. Moreover, these cells represent a distinct subset of CD4+ T cells showing significant transcriptional and phenotypical differences to HIV-specific Th1 cells, but have similarities to CD8+ T cells. These findings are important for our understanding of HIV immunopathology.

  • Presence of a trs-like Motif Promotes Rep-mediated wtAAV2 Integration [PublishAheadOfPrint]

  • High-troughput integration site (IS) analysis of wild type (wt) AAV2 in human dermal fibroblasts (HDF) and HeLa cells revealed that juxtaposition of a Rep binding site (RBS) and terminal resolution site (trs)-like motif leads to a 4-fold increased probability of wtAAV integration. EMSA assays confirmed binding of Rep to off-target RBS. For the first time, we show Rep protein off-target nicking activity, highlighting the importance of the nicking substrate for Rep-mediated integration.

  • Comparable antigenicity and immunogenicity of oligomeric forms of a novel, acute HIV-1 subtype C gp145 envelope for use in preclinical and clinical vaccine research [PublishAheadOfPrint]

  • Eliciting broadly reactive functional antibodies remains a challenge in HIV-1 vaccine development, complicated by variations in envelope (Env) subtype and structure. The majority of new global HIV-1 infections are subtype C and novel antigenic properties have been described for C Envs. Thus, an HIV-1 subtype C Env protein (CO6980v0c22) from an acutely infected (Fiebig stage I/II) subject was developed as a research reagent and candidate immunogen. The gp145 envelope is a novel immunogen with a fully intact membrane proximal external region (MPER), extended by a poly-lysine tail. Soluble gp145 was enriched for trimers that yielded the expected "fan-blade" motifs when visualized by cryo-electron microscopy. The CO6980v0c22 gp145 reacts with the 4E10, PG9, PG16 and VRC01 HIV-1 neutralizing monoclonal antibodies (mAbs), as well as the V1/V2 specific PGT121, 697, 2158 and 2297 mAbs. Different gp145 oligomers were tested for immunogenicity in rabbits, and purified dimers, trimers and larger multimers elicited similar levels of cross-clade binding and neutralizing antibodies to tier 1, and some tier 2 viruses. Immunized rabbit sera did not neutralize the highly resistant CO6980v0c22 pseudovirus, but did inhibit the homologous infectious molecular clone (IMC) in a PBMC assay. This Env is currently in GMP production to be made available for use as a clinical research tool and further evaluation as a candidate vaccine.

    IMPORTANCE At present, the product pipeline for HIV vaccines is insufficient and is limited by inadequate capacity to produce large quantities of vaccine to standards required for human clinical trials. Such products are required to evaluate critical questions of vaccine formulation, route, dosing and schedule as well as to establish vaccine efficacy. The gp145 Env protein presented in this study forms physical trimers, binds to many of the well-characterized broad neutralizing monoclonal antibodies that target conserved Env epitopes, and induce cross-subtype neutralizing antibodies as measured in both cell line and primary cell assays. This subtype C Env gp145 protein is currently undergoing GMP production for use as a reagent for preclinical studies and for human clinical research. This product will serve as a reagent for comparative studies and may represent a next generation, candidate HIV immunogen.

  • A Two-year Follow-up of Macaques Developing Intermittent Control of the HIV Homolog SIVmac251 in the Chronic Phase of the Infection [PublishAheadOfPrint]

  • Off-therapy control of viremia by HIV-infected individuals has been associated with two likely players: a restricted viral reservoir and an efficient cell-mediated immune response. We previously showed that a combination of highly suppressive antiretroviral therapy, and two experimental drugs, i.e. auranofin and buthionine sulfoximine, was able to reduce the viral reservoir, elicit efficient cell-mediated antiviral responses, and induce intermittent post-therapy viral load control in chronically SIVmac251-infected macaques. We here show that the macaques that had received this drug combination and then stopped antiretroviral therapy were also able to maintain low numbers of activated CD4+ T-cells at viral rebound. Moreover, these macaques consistently displayed low-level SIV diversity, which was in line with the strong and broadly reactive cell-mediated immune responses against conserved Gag antigens. Extended follow-up showed that the two macaques that had received the complete drug combination remained healthy and did not develop AIDS in two years of follow-up after therapy suspension. This disease-free survival is longer than twice the average time of progression to AIDS in SIVmac251-infected rhesus macaques. These results suggest that limited numbers of activated T-cells at viral rebound and subsequent development of broadly reactive cell-mediated responses may be interrelated in reducing the viral reservoir.

    Importance The HIV reservoir in CD4+ T-cells represents one main obstacle to HIV eradication. Recent studies, however, show that a drastic reduction of this reservoir is insufficient for inducing a functional cure of AIDS. In the present report, we thoroughly studied and subjected to long-term follow-up two macaques showing intermittent control of the virus following suspension of antiretroviral therapy plus an experimental anti-reservoir treatment, i.e. the gold salt auranofin and the investigational chemotherapeutic agent buthionione sulfoximine (BSO). We found that these drugs were able to decrease the number of activated CD4+ T-cells, which are preferential targets for HIV infection. Then, efficient immune responses against the virus were developed in the macaques, which remained healthy during two years of follow-up. This result may furnish another building block to future attempts to cure HIV/AIDS.

  • Phage therapy: a step forward in the treatment of Pseudomonas aeruginosa infections [PublishAheadOfPrint]

  • Antimicrobial resistance constitutes one of the major worldwide public health concerns. Bacteria are becoming resistant to the vast majority of antibiotics and nowadays, a common infection can be fatal. To revert this situation, the use of phages for the treatment of bacterial infections has been extensively studied as an alternative therapeutic strategy. Since P. aeruginosa is one of the most common causes of healthcare-associated infections, many studies have reported the in vitro and in vivo antibacterial efficacy of phage therapy against this bacterium. This review collects data of all the P. aeruginosa phages sequenced to date, providing a better understanding about their biodiversity. This review will further address the in vitro and in vivo results obtained by using phages to treat or prevent P. aeruginosa infections as well as the major hurdles associated with this therapy.

  • A single point mutation creating a furin cleavage site in the spike protein renders porcine epidemic diarrhea coronavirus trypsin-independent for cell entry and fusion. [PublishAheadOfPrint]

  • The emerging porcine epidemic diarrhea virus (PEDV) requires trypsin supplementation to activate its S protein for membrane fusion and virus propagation in cell culture. By substitution of a single amino acid in the S protein we created a recombinant PEDV (PEDV-SFCS) with an artificial furin protease cleavage site N-terminal of the putative fusion peptide. PEDV-SFCS exhibited trypsin-independent cell-cell fusion and was able to replicate in culture cells independent of trypsin, though to low titer.

  • Respiratory syncytial virus infection upregulates NLRC5 and MHC I expression through RIG-I induction in airway epithelial cells [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) is the leading cause of acute respiratory tract viral infection in infants, causing bronchiolitis and pneumonia. The host antiviral response to RSV acts via retinoic acid inducible gene I (RIG-I). We show here that RSV infection upregulates MHC I expression through the induction of NLRC5, a NOD-like, CARD domain-containing intracellular protein that has recently been identified as a class I MHC transactivator (CITA). RSV infection of A549 cells promotes upregulation of NLRC5 via IFNbbeta; production since the NLRC5-inducing activity in a conditioned medium from RSV-infected A549 cells was removed by antibody to IFNbbeta;, but not to IFN. RSV infection resulted in RIG-I upregulation, induction of NLRC5, and MHC I. Suppression of RIG-I induction significantly blocked NLRC5 as well as MHC I upregulation and diminished IRF3 activation. Importantly, Vero cells deficient in interferon production still upregulated MHC I following the introduction of RSV genome by infection or transfection, further supporting a key role for RIG-I. A model is therefore formed in which the host upregulates MHC I expression during RSV infection directly via the induction of RIG-I and NLRC5 expression. Since elevated expression of MHC I molecules can sensitize host cells to T lymphocyte-mediated cytotoxicity or immunopathologic damage, the results have significant implications for the modification of immunity in RSV disease.

    Importance Human respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in infants and young children worldwide. Early life infection is linked to persistent wheezing and allergic asthma in later life, possibly related to upregulation of major histocompatibility class I on the cell surface which facilitates molecules cytotoxic T cell activation and antiviral immunity. Here we show that RSV infection of lung epithelial cells induces expression of RIG-I, resulting in induction of a class I MHC transactivator NLRC5 and subsequent upregulation of MHC I. Suppression of RIG-I induction blocked RSV-induced NLRC5 expression and MHC I upregulation. Increased MHC I expression may exacerbate the disease condition of RSV condition due to immunopathologic damage, linking innate immune response to RSV disease.

  • Post-translational Modifications of Baculovirus Protamine-like Protein P6.9 and the Significance of Its Hyperphosphorylation for Viral Very Late Gene Hyperexpression [PublishAheadOfPrint]

  • Many viruses utilize viral or cellular chromatin machinery for efficient infection. Baculoviruses encode a conserved protamine-like protein P6.9. This protein plays essential roles in various viral physiological processes during infection. However, the mechanism by which P6.9 regulates transcription remains unknown. In this study, 7 phosphorylated species of P6.9 were resolved in Sf9 cells infected with the baculovirus type species Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Mass spectrometry identified 22 phosphorylation and 10 methylation sites but no acetylation sites in P6.9. Immunofluorescence demonstrated that the P6.9 and virus-encoded serine/threonine kinase PK1 exhibited a similar distribution pattern in infected cells, and co-immunoprecipitation confirmed the interaction between them. Upon pk1 deletion, nucleocapsid assembly and polyhedron formation were interrupted and the transcription of viral very late genes were down-regulated. Interestingly, we found that the 3 most phosphorylated P6.9 species vanished in Sf9 cells transfected with the pk1-deletion mutant, suggesting that PK1 is involved in the hyperphosphorylation of P6.9. Mass spectrometry suggested that the phosphorylation of the 7 Ser/Thr and 5 Arg residues in P6.9 was PK1-dependent. Substitution of the 7 Ser/Thr residues with Ala resulted in a P6.9 phosphorylation pattern similar to that of the pk1-deletion mutant. Importantly, the decreases in the transcription level of viral very late genes and the viral infectivity were consistent. Our findings reveal that P6.9 hyperphosphorylation is a precondition for the maximal hyperexpression of baculovirus very late genes and provide the first experimental insight into the function of the baculovirus protamine-like protein and the related protein kinase in epigenetics.

    IMPORTANCE Diverse post-translational modifications (PTMs) of histones constitute a code that creates binding platforms that recruit transcription factors to regulate gene expression. Many viruses also utilize host- or virus-induced chromatin machinery to promote efficient infections. Baculoviruses encode a protamine-like protein P6.9, which is required for a variety of processes in the infection cycle. Currently, P6.9's PTM sites and its regulating factors remain unknown. Here, we found that P6.9 could be categorized as unphosphorylated, hypophosphorylated, and hyperphosphorylated species and that a virus-encoded serine/threonine kinase PK1 was essential for P6.9 hyperphosphorylation. Abundant PTM sites on P6.9 were identified, among which 7 Ser/Thr phosphorylated sites were PK1-dependent. Mutation of these Ser/Thr sites reduced very late viral genes transcription and viral infectivity, indicating that the PK1-mediated P6.9 hyperphosphorylation contributes to viral proliferation. These data suggest that a potential "code" might exist in the sophisticated PTM of viral protamine-like proteins and participate in viral gene transcription.

  • Arenavirus genome rearrangement for the development of live-attenuated vaccines [PublishAheadOfPrint]

  • Several members of the Arenaviridae family cause hemorrhagic fever disease in humans and pose serious public health problems in their endemic geographic regions, as well as a credible biodefense threat. To date, there are no FDA-approved arenavirus vaccines and current anti-arenaviral therapy is limited to an off-label use of ribavirin that is only partially effective. Arenaviruses are enveloped viruses with a bi-segmented negative-stranded RNA genome. Each genome segment uses an ambisense coding strategy to direct the synthesis of two viral polypeptides in opposite orientation, separated by a non-coding intergenic region. Here we have used minigenome-based approaches to evaluate expression levels of reporter genes from the NP and GPC loci within the S segment of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). We found that reporter genes are expressed to higher levels from the NP than GPC locus. Differences in reporter gene expression levels from the NP and GPC loci were confirmed with recombinant tri-segmented LCM viruses. We then used reverse genetics to rescue a recombinant LCMV containing a translocated viral S segment (rLCMV/TransS), where the viral NP and GPC open reading frames replaced one another. The rLCMV/TransS showed slower growth kinetics in cultured cells and was highly attenuated in vivo in a mouse model of lethal LCMV infection, but immunization with rLCMV/TransS conferred complete protection against a lethal challenge with wild type LCMV. Attenuation of rLCMV/TransS was associated with reduced NP expression levels. These results open a new avenue for the development of arenavirus live-attenuated vaccines based on rearrangement of their viral genome.

    IMPORTANCE Several arenaviruses cause severe hemorrhagic fever in humans and pose also a credible bioterrorism threat. Currently, no FDA-licensed vaccines are available to combat arenavirus infections and anti-arenaviral therapy is limited to the off-label use of ribavirin that is only partially effective and associated with side effects. Here we describe, for the first time, the generation of a recombinant LCMV where the viral protein products encoded by the S RNA segment (NP and GPC) were swapped between themselves to generate rLCMV/TransS. The rLCMV/TransS exhibited reduced viral multiplication in cultured cells and was highly attenuated in vivo, while conferred protection, upon a single immunization dose, against a lethal challenge with wild-type LCMV. Our studies provide proof of concept for the rational development of safe and protective live-attenuated vaccine candidates based on genome reorganization for the treatment of pathogenic arenavirus infections in humans.

  • Transcriptome profiling of the virus-induced innate immune response in Pteropus vampyrus and its attenuation by Nipah virus interferon antagonist functions [PublishAheadOfPrint]

  • Bats are important reservoirs for several viruses, many of which cause lethal infections in humans but have reduced pathogenicity in bats. As the innate immune response is critical for controlling viruses, the nature of this response in bats, and how it may differ from other mammals, is of great interest. Using next generation mRNAseq, we profiled the transcriptional response of Pteropus vampyrus bat kidney (PVK) cells to Newcastle disease virus (NDV), an avian paramyxovirus known to elicit a strong innate immune response in mammalian cells. This bat species is a known reservoir of Nipah virus (NiV) and Hendra virus (HeV). Analysis of the 200-300 regulated genes showed that interferon (IFN) and antiviral pathways are highly upregulated in NDV infected PVK cells, including genes such as IFN bbeta;, RIGI, MDA5, ISG15, and IRF1. NDV infected cells also upregulated several genes not previously characterized as antiviral such as RND1, SERTAD1, CHAC1, and MORC3. In fact, we show that MORC3 is induced by both IFN and NDV infection in PVK cells, but by neither stimulus in human A549 cells. In contrast to NDV, HeV and NiV infection of PVK cells failed to induce these innate immune genes. Likewise, an attenuated response was observed in PVK cells infected with recombinant NDVs expressing the NiV IFN antagonist proteins V and W. This study provides the first global profile of a robust virus-induced innate immune response in bats and indicates that henipavirus IFN antagonist mechanisms are likely active in bat cells.

    Importance Bats are the reservoir host for many highly pathogenic human viruses, including henipaviruses, lyssaviruses, SARS coronavirus, and filoviruses, and many other viruses have also been isolated from bats. Viral infections are reportedly asymptomatic or heavily attenuated in bat populations. Despite their ecological importance to viral maintenance, research into their immune system and mechanisms for viral control has only recently begun. Nipah virus and Hendra virus are two paramyxoviruses associated with high mortality rates in humans and whose reservoir is the Pteropus genus of bats. Greater knowledge of the innate immune response of P. vampyrus to viral infection may elucidate how bats serve as a reservoir for so many viruses.

  • Complexity of neutralization antibodies against multiple dengue viral serotypes after heterotypic immunization and secondary infection revealed by in-depth analysis of cross-reactive antibodies [PublishAheadOfPrint]

  • The four serotypes of dengue virus (DENV) cause the most important and rapidly emerging arboviral diseases in humans. The recent Phase 2b and 3 studies of a tetravalent dengue vaccine reported a moderate efficacy despite the presence of neutralizing antibodies, highlighting the need for better understanding of neutralizing antibodies in polyclonal human sera. Certain type specific (TS) antibodies were recently discovered to account for the monotypic neutralizing activity and protection after primary DENV infection. The nature of neutralizing antibodies after secondary DENV infection remains largely unknown. In this study, we examined sera from 10 vaccinees with well-documented first and second DENV serotypes exposed through heterotypic immunization of live-attenuated vaccines. Higher serum IgG avidity to both exposed and non-exposed serotypes was found after secondary immunization compared with primary immunization. Using a two-step depletion protocol to remove different anti-envelope antibodies including group-reactive (GR) and complex-reactive (CR) antibodies separately, we found GR and CR antibodies together contribute to more than 50% neutralizing activities against multiple serotypes after secondary immunization. Similar findings were demonstrated in patients after secondary infection. Anti-envelope antibodies recognizing previously exposed serotypes consist of a large proportion of GR antibodies, CR antibodies and a small proportion of TS antibodies, whereas those recognizing non-exposed serotypes consist of GR and CR antibodies. These findings have implications for sequential heterotypic immunization or primary immunization of DENV-primed individuals as alternative strategies for DENV vaccination. The complexity of neutralizing antibodies after secondary infection provides new insights into the difficulty of their application as surrogates of protection.

    IMPORTANCE The four serotypes of dengue virus (DENV) are the leading cause of arboviral diseases in humans. Despite the presence of neutralizing antibodies, a moderate efficacy was recently reported in Phase 2b and 3 trials of a dengue vaccine; a better understanding of neutralizing antibodies in polyclonal human sera is urgently needed. We studied vaccinees who received heterotypic immunization of live-attenuated vaccines as cases with known first and second DENV serotypes exposed. We found anti-envelope antibodies consist of group-reactive (GR), complex-reactive (CR) and type-specific (TS) antibodies; both GR and CR antibodies contribute significantly to multitypic neutralizing activities after secondary DENV immunization. These findings have implications for alternative strategies for DENV vaccination. Certain TS antibodies were recently discovered to contribute to the monotypic neutralizing activity and protection after primary DENV infection; our findings of the complexity of neutralizing activities after secondary immunization/infection provide new insights into neutralizing antibodies as surrogates of protection.

  • The minor capsid protein VP11 of thermophilic bacteriophage P23-77 facilitates virus assembly using lipid-protein interactions [PublishAheadOfPrint]

  • Thermus thermophilus bacteriophage P23-77 is the type member of a new virus family of icosahedral, tailless, inner membrane-containing dsDNA viruses infecting thermophilic bacteria and halophilic archaea. Those viruses have a unique capsid architecture consisting of two major capsid proteins assembled in various building blocks. We analyzed the function of minor capsid protein VP11, which is the third known capsid component in bacteriophage P23-77. Our findings show that VP11 is a dynamically elongated dimer with predominantly aalpha;-helical secondary structure and a high thermal stability. A high proportion of basic amino acids in the protein enable electrostatic interaction with negatively charged molecules including nucleic acid and large unilamellar lipid vesicles (LUVs). The plausible biological function for VP11 is elucidated by demonstrating the interactions of VP11 with Thermus derived LUVs and with the major capsid proteins by means of dynamic light scattering technique. In particular, the major capsid protein VP17 was able to link VP11-complexed LUVs into larger particles, whereas the other P23-77 major capsid protein, VP16, was unable to link VP11-comlexed LUVs. Our results rule out a previously suggested penton function for VP11. Instead, the electrostatic membrane association of VP11 triggers binding of major capsid protein VP17, thus facilitating a controlled incorporation of the two different protein species into the assembling capsid.

    Importance The study of thermophilic viruses with inner membranes provides valuable insights into the mechanisms used for stabilization and assembly of protein-lipid systems at high temperatures. Our results reveal a novel way by which an internal membrane and outer capsid shell are linked in a virus that uses two different major protein species for capsid assembly. We show that a positive protein-charge is important to form electrostatic interactions with the lipid surface, thereby facilitating the incorporation of other capsid proteins on the membrane surface. This implies an alternative function for basic proteins present in the virions of other lipid-containing thermophilic viruses, whose proposed role in genome packaging is based on their capability to bind DNA. The unique minor capsid protein of bacteriophage P23-77 resembles in its characteristics the scaffolding proteins of tailed phages, though it constitutes a substantial part of the mature virion.

  • The Nuclear DNA Sensor IFI16 Acts as a Restriction Factor for Human Papillomavirus Replication through Epigenetic Modifications of the Viral Promoters [PublishAheadOfPrint]

  • The human interferon-inducible IFI16 protein, an innate immune sensor of intracellular DNA, was recently demonstrated to act as a restriction factor for human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) infection by inhibiting both viral DNA replication and transcription. Through the use of two distinct cellular models, this study provides strong evidence in support of the notion that IFI16 can also restrict HPV18 replication. In the first model, an immortalized keratinocyte cell line (NIKS) was used in which the IFI16 protein was knocked down through the use of siRNA technology, and overexpressed following transduction with the AdVIFI16 vector. The second model consisted of U2OS cells transfected by electroporation with HPV18 minicircles. In differentiated IFI16-silenced NIKS-HPV18 cells, viral load values were significantly increased compared with differentiated control cells. Consistent with this, IFI16 overexpression severely impaired HPV18 replication in both NIKS and U2OS cells, thus confirming its antiviral restriction activity. In addition to the inhibition of viral replication, IFI16 was also able to reduce viral transcription, as demonstrated by viral gene expression analysis in U2OS cells carrying episomal HPV18 minicircles and HeLa cells. We also provide evidence that IFI16 promotes the addition of heterochromatin marks and the reduction of euchromatin marks on viral chromatin at both early and late promoters, thus reducing both viral replication and transcription. Altogether, these results argue that IFI16 restricts chromatinised HPV DNA through epigenetic modifications and executes a broad surveillance role against viral DNA in the nucleus that is not restricted to Herpesviruses.

    IMPORTANCE Intrinsic immunity is mediated by cellular restriction factors that are constitutively expressed and active even before a pathogen enters the cell. The host nuclear factor IFI16 acts as sensors of foreign DNA and antiviral restriction factors, as recently demonstrated by our group for human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1). Here, we provide the first evidence that IFI16 inhibits HPV18 replication by repressing viral gene expression and replication. This antiviral restriction activity was observed in immortalized keratinocytes transfected with the religated genomes and in U2OS transfected with HPV18 minicircles suggesting that it is not cell-type specific. We also show that IFI16 promotes the assembly of heterochromatin on HPV DNA. These changes in viral chromatin structure lead to the generation of a repressive state at both early and late HPV18 promoters, thus implicating a role of this protein in the epigenetic regulation of HPV gene expression and replication.

  • Ecological drivers of virus evolution: Astrovirus as a case study [PublishAheadOfPrint]

  • Although RNA viruses exhibit a high frequency of host jumps, major differences exist among the different virus families. Astroviruses manifest the capacity to infect a wide range of hosts that exhorts both public health systems and economic production chains. Here we delineate the ecological and adaptive processes that drive the cross-species transmission of Astroviruses. We observe that distinct transmission zones determine the prevailing Astrovirus host and virus diversity, which in turn suggests that no single host group (e.g. bats) can be natural reservoirs, as illustrated through our phylogenetic analysis.

  • MAVS coordination of antiviral innate immunity [PublishAheadOfPrint]

  • RNA virus infection is sensed in the cytoplasm by the RIG-I-like receptors. These proteins signal through the host adaptor protein MAVS to trigger the antiviral innate immune response. Here, we describe how MAVS subcellular localization impacts its function and the regulation underlying MAVS signaling. We propose a model to describe how the coordination of MAVS function at the interface between the mitochondria and the mitochondrial-associated ER membrane programs antiviral signaling.

  • Human memory B cells producing potent cross-neutralizing antibodies against human parechovirus; implications for prevalence, treatment and diagnosis [PublishAheadOfPrint]

  • The family of Picornaviridae is a large and diverse group of positive sense RNA viruses, including the human enteroviruses (EVs) and human parechoviruses (HPeVs). The human immune response against EVs and HPeVs is thought to be mainly humoral and an insufficient neutralizing antibody (Ab) response during infection is a risk factor and can ultimately be life threatening. The accessibility of different antigenic sites and observed cross-reactivity makes HPeVs a good target for development of therapeutic human monoclonal antibodies (mAbs). In this study we generated two different human mAbs specific for HPeV by screening culture supernatants of Ab producing human B cell cultures for direct neutralization of HPeV1. Both mAbs showed HPeV1-specific neutralization, as well as neutralization of HPeV2. One antibody, AM18, cross-neutralized HPeV4, 5 and 6 and coxsackievirus A9 (CV-A9). VP1 capsid protein specific assays confirmed that AM18 bound VP1 of HPeV1, 2 and 4 with high affinity (11.5 pM). In contrast, the HPeV1specific mAb AM28, which neutralized HPeV1 even more efficiently compared to AM18, showed no cross-reactivity with HPeV3-6 or other EVs and did not bind any of the capsid proteins, suggesting AM28 is specific for a conformation dependent, non-linear epitope on the virus. The discovery of mAbs cross-reactive between HPeVs may help development of HPeV treatment options by antibodies and vaccine design based on epitopes recognized by these antibodies.

    Importance HPeV infections are widespread among young children and adults, causing a broad range of disease. Infections can be severe and life threatening, while no antiviral treatment is available. Given the absence of neutralizing Abs is a risk factor for severe disease in infants, treatment of picornavirus infections with mAbs would be a therapeutic option. To study antibody neutralization of HPeV in more detail, we generated two different HPeV1-specific human mAbs. Both mAbs show HPeV1-specific neutralization and cross-neutralized HPeV2. One mAb also cross-neutralized with other HPeVs. Surprisingly, this mAb also neutralized CV-A9. These mAbs provide a unique tool for further research and for diagnosis (antigen detection) and possible treatment of HPeV infections.

  • K1 and K15 of Kaposi sarcoma-associated herpes virus are partial functional homologues of latent membrane protein 2A of Epstein-Barr virus [PublishAheadOfPrint]

  • The human herpes viruses Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpes virus (KSHV) are associated with Hodgkin's lymphoma (HL) and Primary effusion lymphomas (PEL), respectively, which are B cell malignancies that originate from germinal center B cells. PEL cells but also a quarter of EBV-positive HL tumor cells do not express the genuine B cell receptor (BCR), a situation incompatible with survival of normal B cells. EBV encodes LMP2A, one of EBV's viral latent membrane proteins, which likely replaces the BCR's survival signaling in HL. Whether KSHV encodes a viral BCR mimic that contributes to oncogenesis is not known because an experimental model of KSHV-mediated B cell transformation is lacking. We addressed this uncertainty with mutant EBVs encoding the KSHV genes K1 or K15 in lieu of LMP2A and infected primary BCR-negative (BCRnndash;) human B cells with them. We confirmed that the survival of BCRnndash; B cells and their proliferation depended on an active LMP2A signal. Like LMP2A, expression of K1 and K15 led to the survival of BCRnndash; B cells prone to apoptosis, supported their proliferation and regulated a similar set of cellular target genes. K1 and K15 encoded proteins appear to have non-complementing, redundant functions in this model but our findings suggest that both KSHV proteins can replace LMP2A's key activities contributing to the survival, activation and proliferation of BCRnndash; PEL cells in vivo.

    Importance Several herpes viruses encode oncogenes that are receptor-like proteins. Often, they are constitutively active providing important functions to the latently infected cells. LMP2A of Epstein-Barr virus (EBV) is such a receptor that mimics an activated B cell receptor, BCR. K1 and K15, related receptors of Kaposi sarcoma-associated herpes virus (KSHV) expressed in virus-associated tumors, have less obvious functions. We found in infection experiments that both viral receptors of KSHV can replace LMP2A and deliver functions similar to the endogenous BCR. K1, K15, and LMP2A also control the expression of a related set of cellular genes in primary human B cells, the target cells of EBV and KSHV. The observed phenotypes as well as the known characteristics of these genes argue for their contributions to cellular survival, B cell activation, and proliferation. Our findings provide one possible explanation for the tumorigenicity of KSHV, which poses a severe problem in immunocompromised patients.

  • Novel Functions of Hendra Virus G N-Glycans and Comparisons to Nipah Virus [PublishAheadOfPrint]

  • Hendra virus (HeV) and Nipah virus (NiV) are reportedly the most deadly pathogens within the Paramyxoviridae family. These two viruses bind the cellular entry receptors ephrinB2 and/or ephrinB3 via the viral attachment glycoprotein (G), and the concerted efforts of G and the viral fusion glycoprotein F result in membrane fusion. Membrane fusion is essential for viral entry into host cells, and for cell-cell fusion, a hallmark of the disease pathobiology. HeV G is heavily N-glycosylated, but the N-glycan functions remain unknown. We disrupted eight predicted N-glycosylation sites in HeV G by conservative mutations (Asn to Gln) and found that six out of eight sites were actually glycosylated (G2-G7); one in the stalk (G2) and five in the globular head domain (G3-G7). We then tested the roles of individual and combined HeV G N-glycan mutants and found functions in the modulation of shielding against neutralizing antibodies, intracellular transport, G-F interactions, cell-cell fusion and viral entry. Between the highly conserved HeV and NiV G glycoproteins, similar trends in the effects of N-glycans on protein functions were observed, with differences in the levels at which some N-glycan mutants affected such functions. While the N-glycan in the stalk domain (G2) had very conserved roles between HeV and NiV G, individual N-glycans in the head affected the levels of several protein functions differently. Our findings are discussed in the context of their contribution to our understanding of HeV and NiV pathogenesis and immune responses.

    IMPORTANCE Viral envelope glycoproteins are important for viral pathogenicity and immune evasion. N-glycan shielding is one mechanism by which immune evasion can be achieved. In paramyxoviruses, viral attachment and membrane fusion are governed by the close interaction of the attachment protein H/HN/G and the fusion protein F. In this study we show that the attachment glycoprotein G of Hendra virus (HeV), a deadly paramyxovirus, is N-glycosylated at six sites (G2-G7) and that most of these sites have important roles in viral entry, cell-cell fusion, G-F interactions, G oligomerization, and immune evasion. Overall, we found that the N-glycan in the stalk domain (G2) had very conserved roles between HeV G and the closely related Nipah virus G, whereas individual N-glycans in the head quantitatively modulated several protein functions differently between both viruses.

  • LMP1-induced Sumoylation Influences the Maintenance of EBV Latency Through KAP1. [PublishAheadOfPrint]

  • As a herpesvirus, EBV establishes a latent infection that can periodically undergo reactivation, resulting in lytic replication and the production of new infectious virus. Latent membrane protein-1 (LMP1), the principal viral oncoprotein, is a latency-associated protein implicated in regulating viral reactivation and the maintenance of latency. We recently found that LMP1 hijacks the SUMO-conjugating enzyme, Ubc9, via its C-terminal activating region-3 (CTAR3), and induces the sumoylation of cellular proteins. Because protein sumoylation can promote transcriptional repression, we hypothesized that LMP1-induced protein sumoylation induces the repression of EBV lytic promoters and helps maintain the viral genome in its latent state. We now show that with inhibition of LMP1-induced protein sumoylation, the latent state becomes less stable or "leakier" in EBV-transformed LCLs. The cells are also more sensitive to viral reactivation induced by irradiation, which results in increased production and release of infectious virus, as well as increased susceptibility to ganciclovir treatment. We have identified a target of LMP1-mediated sumoylation that contributes to the maintenance of latency in this context: KRAB-associated protein-1 (KAP1). LMP1 CTAR3-mediated sumoylation regulates the function of KAP1. KAP1 also binds to EBV oriLyt and immediate early promoters in a CTAR3-dependent manner, and inhibition of sumoylation processes abrogates the binding of KAP1 to these promoters. These data provide an additional line of evidence that supports our findings that CTAR3 is a distinct functioning regulatory region of LMP1 and confirm that LMP1-induced sumoylation may help stabilize the maintenance of EBV latency.

    Importance Epstein-Barr virus (EBV) Latent Membrane Protein-1 (LMP1) plays an important role in the maintenance of viral latency. Previously, we documented that LMP1 targets cellular proteins to be modified by an ubiquitin-like protein (SUMO). We have now identified a function for this LMP1-induced modification of cellular proteins in the maintenance of EBV latency. Because latently infected cells have to undergo viral reactivation in order to be vulnerable to anti-viral drugs, these findings identify a new way to increase the rate of EBV reactivation, which increases cell susceptibility to anti-viral therapies.

  • The roles of phosphorylation of the nucleocapsid protein of mumps virus in regulating viral RNA transcription and replication [PublishAheadOfPrint]

  • Mumps virus (MuV) is a paramyxovirus with a negative sense non-segmented RNA genome. The viral RNA genome is encapsidated by the nucleocapsid protein (NP) to form the ribonucleoprotein (RNP), which serves as a template for transcription and replication. In this study, we investigated the roles of phosphorylation sites of NP in MuV RNA synthesis. Using radioactive labeling, we first demonstrated that NP was phosphorylated in MuV-infected cells. Using both liquid chromatography-mass spectrometry (LC-MS) and in silico modeling, we identified nine putative phosphorylated residues within NP. We mutated these nine residues to alanine. Mutation of the serine residue at position 439 to alanine (S439A) was found to reduce the phosphorylation of NP in transfected cells by over 90%. The effects of these mutations on the MuV mini-genome system were examined. S439A was found to have higher activity, four mutants had lower activity and four mutants had similar activity compared to wild-type NP. MuV containing the S439A mutation had reduced phosphorylation of NP by 90% and enhanced viral RNA synthesis and viral protein expression at early time point after infection, indicating that S439 is the major phosphorylation site of NP and its phosphorylation plays an important role in down-regulating viral RNA synthesis.

  • Adaptive mutations that occurred during 2009 H1N1 influenza virus pandemic circulation in humans enhance virulence in mice [PublishAheadOfPrint]

  • During the 2009 H1N1 influenza pandemic, infection attack rates were particularly high among young individuals who suffered from pneumonia with occasional death. Moreover, previously reported determinants of mammalian adaptation and pathogenicity were not present in 2009 pandemic H1N1 influenza A viruses. Thus, it was proposed that unknown viral factors might have contributed to disease severity in humans. In this study, we performed a comparative analysis of two clinical 2009 pandemic H1N1 strains that belong to very early and later phases of the pandemic. We identified mutations in the viral hemagglutinin (HA) and the nucleoprotein (NP) that have occurred during pandemic progression and mediate increased virulence in mice. Lethal disease outcome correlated with elevated viral replication in the alveolar epithelium, increased pro-inflammatory cytokine and chemokine response, pneumonia and lymphopenia in mice. These findings show that viral mutations that have occurred during pandemic circulation among humans are associated with severe disease in mice.

    Importance In this study, novel determinants of 2009 pandemic H1N1 influenza pathogenicity were identified in the viral hemagglutinin (HA) and the nucleoprotein (NP) genes. In contrast to highly pathogenic avian influenza viruses, increased virulence in mice did not correlate with enhanced but with reduced polymerase activity. Lethal 2009 pandemic H1N1 infection in mice correlated with lymphopenia and severe pneumonia. These studies suggest that molecular mechanisms that mediate 2009 pandemic H1N1 influenza pathogenicity are distinct from those that mediate avian influenza virus pathogenicity in mice.

  • Characterization of the Human Papillomavirus Type 16 E8 Promoter [PublishAheadOfPrint]

  • Persistent infections with certain human papillomaviruses (HPV) such as HPV16 are a necessary risk factor for the development of anogenital and oropharyngeal cancers. HPV16 genomes replicate as low copy number plasmids in the nucleus of undifferentiated keratinocytes which requires the viral E1 and E2 replication proteins. The HPV16 E8^E2C (or E8^E2) protein limits genome replication by repressing both viral transcription and the E1/E2-dependent DNA replication. How E8^E2C expression is regulated is not understood. Previous transcript analyses indicated that the spliced E8^E2C RNA is initiated at a promoter located in the E1 region upstream of the E8 gene. Deletion and mutational analyses of the E8 promoter region identify two conserved elements that are required for basal promotor activity in HPV-negative keratinocytes. In contrast, the transcriptional enhancer in the upstream regulatory region of HPV16 does not modulate basal E8 promoter activity. Co-transfection studies indicate that E8^E2C inhibits, whereas E2 weakly activates the E8 promoter. Interestingly, the co-transfection of E1 and E2 induces the E8 promoter much stronger than the major early promoter and this is partially dependent upon binding of E2 to Brd4. Mutation of E8 promoter elements in the context of HPV16 genomes results in an increased genome copy number and elevated levels of viral early and late transcripts. In summary, the promoter responsible for the expression of E8^E2C is both positively and negatively regulated by viral and cellular factors and this regulatory circuit may be crucial to maintain a low but constant copy number of HPV16 genomes in undifferentiated cells.

    Importance HPV16 replicates in differentiating epithelia and can cause cancer. How HPV16 maintains its genome in undifferentiated cells at a low but constant level is not well understood but may be relevant for the immunological escape of HPV16 in the basal layers of the infected epithelium. This study demonstrates that the expression of the viral E8^E2C protein, which is a potent inhibitor of viral replication in undifferentiated cells, is driven by a separate promoter. The E8 promoter is both positively and negatively regulated by viral proteins and thus most likely acts a sensor and modulator of viral copy number.

  • The human cytomegalovirus pUL47 modulates tegumentation and capsid accumulation at the viral assembly complex [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) tegument protein pUL47 is an interaction partner of pUL48 and highly conserved among herpesviruses. It is closely associated with the capsid and has an important function early in infection. Here, we report a specific role of pUL47 in tegumentation of capsids in the cytoplasm. A newly generated mutant virus (TB-47stop), in which expression of pUL47 is blocked, exhibited a severe impairment in cell-to-cell spread and release of infectivity from infected cells. Ultrastructural analysis of TB-47stop infected cells clearly showed cytoplasmic accumulations of non-enveloped capsids that were only partially tegumented, indicating that these capsids failed to complete tegumentation. Nevertheless, these accumulations were positive for HCMV inner tegument proteins pp150 and pUL48, suggesting that their attachment to capsids occurs independently of pUL47. Despite these morphological alterations, fully enveloped virus particles were found in the extracellular space and at the viral assembly complex (vAC) of TB-47stop infected cells, indicating that pUL47 is not essential for the generation of virions. We confirmed findings that incorporation of pUL48 into virions is impaired in absence of pUL47. Interestingly, pUL47 exhibited a strong nuclear localization in transfected cells whereas it was exclusively found at the vAC in the context of virus infection. Co-localization of pUL47 and pUL48 at the vAC is consistent with their interaction. We also found a shift to a more nuclear localization of pUL47 when the expression of pUL48 was reduced. Summarizing our results, we hypothesize that pUL48 directs pUL47 to the vAC to promote tegumentation and secondary envelopment of capsids.

    Importance Generation of infectious HCMV particles requires an organized and multistep process involving the action of several viral and cellular proteins as well as protein-protein interactions. A better understanding of these processes is important for understanding the biology of HCMV and may help to identify targets for antiviral intervention. Here, we identified tegument protein pUL47 to function in tegumentation and proper trafficking of capsids during late phases of infection. Although pUL47 is not essential for generation and release of infectious virions, its absence led to massive accumulations of partially tegumented capsids at the cell periphery. Detection of pUL48 at these accumulations indicated for a pUL47 independent attachment of pUL48 to the capsid. On the other hand, localization of pUL47 to the vAC during infection appeared to be dependent on tegument protein pUL48, which suggests an intricate interplay of these proteins for normal generation of infectious virus progeny.

  • Interferon-Gamma Prolongs Survival of Varicella Zoster Virus-Infected Human Neurons In Vitro [PublishAheadOfPrint]

  • Infection of human neurons in vitro with varicella-zoster virus (VZV) at low multiplicity of infection does not result in a cytopathic effect (CPE) within 14 days post-infection (dpi), despite production of infectious virus. We showed that by 28 dpi a CPE ultimately developed in infected neurons, and that interferon gamma not only inhibited the CPE, but also VZV DNA accumulation, transcription and virus production, thereby prolonging the life of VZV-infected neurons.

  • Cross-reactive neuraminidase-inhibiting antibodies elicited by immunization with recombinant neuraminidase proteins of H5N1 and pH1N1 influenza A viruses [PublishAheadOfPrint]

  • Neuraminidase (NA), an influenza virus envelope glycoprotein, removes sialic acid from receptors for virus release from infected cells. For this study we used a baculovirus-insect cell expression system to construct and purify H5N1-rNA (A/Vietnam/1203/2004) and pH1N1-rNA (A/Texas/05/2009) proteins. BALB/c mice immunized with these proteins had high titers of NA-specific IgG and NA-inhibiting (NI) antibodies against H5N1, pH1N1, H3N2 and H7N9 viruses. H5N1-rNA immunization resulted in higher quantities of NA-specific antibody-secreting B cells in spleen against H5N1 and heterologous pH1N1 viruses. H5N1-rNA and pH1N1-rNA immunizations both provided complete protection against homologous virus challenges, with H5N1-rNA immunization providing better protection against pH1N1 virus challenges. Cross-reactive NI antibodies were further dissected via pH1N1-rNA protein immunizations with I149V, N344Y and I365T/S366N NA mutations. The I365T/S366N mutation of pH1N1-rNA enhanced cross-reactive NI antibodies against H5N1, H3N2 and H7N9 viruses. It is our hope that these findings provide useful information for the development of a NA-based universal influenza vaccine.

    Importance Neuraminidase (NA) is an influenza virus enzymatic protein that cleaves sialic acid linkages on infected cell surfaces, thus facilitating viral release and contributing to viral transmission and mucus infection. In currently available inactivated or live-attenuated influenza vaccines based on the antigenic content of hemagglutinin proteins, vaccine efficacy can be partly contributed through NA-elicited immune responses. We investigated the NA immunity of different recombinant NA (rNA) proteins associated with pH1N1 and H5N1 viruses. Our results indicate that H5N1-rNA immunization induced more potent cross-protective immunity than pH1N1-rNA immunization, and three residues at I149V, I365T and S366N near the NA enzyme active site(s) are linked to enhanced cross-reactive NA-inhibiting antibodies against heterologous and heterosubtypic influenza A viruses. These findings provide useful information for the development of a NA-based universal influenza vaccine.

  • Inclusion of flagellin during vaccination against influenza enhances recall responses in nonhuman primate neonates [PublishAheadOfPrint]

  • Influenza virus can cause life-threatening infections in neonates and young infants. Although vaccination is a major countermeasure against influenza, current vaccines are not approved for use in infants less than 6 months of age, in part due to the weak immune response following vaccination. Thus, there is a strong need to develop new vaccines with improved efficacy for this vulnerable population. To address this issue, we established a neonatal African green monkey (AGM) nonhuman primate model that could be used to identify effective influenza vaccine approaches for use in young infants. We assessed the ability of flagellin, a toll like receptor 5 (TLR5) agonist, to serve as an effective adjuvant in this at risk population. Four to six day old AGM were primed and boosted with inactivated PR8 influenza virus (IPR8) adjuvanted with either wild type or mutant flagellin (m229), the latter of which is incapable of signaling through TLR5. Increased IgG responses were observed following boost, as well as at early times after challenge in infants vaccinated with flagellin adjuvanted IPR8. Inclusion of flagellin during vaccination also resulted in a significantly increased number of influenza-specific T cells following challenge compared to infants vaccinated with m229 adjuvant. Finally, infants vaccinated with IPR8+flagellin exhibited reduced pathology in the lungs following challenge compared to those that received IPR8+m229. This study provides the first evidence of flagellin mediated enhancement of vaccine responses in nonhuman primate neonates.

    Importance: Young infants are particularly susceptible to severe disease as a result of influenza virus infection. Compounding this is the lack of effective vaccines for use in this vulnerable population. Here we describe a vaccine approach that results in improved immune responses and protection in young infants. Incorporation of flagellin during vaccination resulted in increased antibody and T cell responses together with reduced disease following virus infection. These results suggest flagellin may serve as an effective adjuvant for vaccines targeted to this vulnerable population.

  • Regulation of LMP1 Signaling Through Interaction with Cytoskeletal Proteins [PublishAheadOfPrint]

  • Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) induces constitutive signaling in EBV infected cells to ensure the survival of the latently infected cells. LMP1 is localized to lipid raft domains to induce signaling. In the present study a genome-wide screen was performed based on bimolecular fluorescence complementation (BiFC) to identify LMP1-binding proteins. Several actin cytoskeleton-associated proteins were identified in the screen. Overexpression of these proteins affected LMP1-induced signaling. BiFC between the identified proteins and LMP1 was localized to lipid raft domains and was dependent on LMP1 signaling. Proximity biotinylation assays with LMP1 induced biotinylation of the actin-associated proteins which were shifted in molecular weight. Together this study suggests that LMP1 raft association is mediated at least in part through interactions with the actin cytoskeleton.

    IMPORTANCE LMP1 signaling requires oligomerization, lipid raft partitioning, and binding to cellular adaptors. The current study utilized a genome-wide screen to identify several actin-associated proteins as candidate LMP1-binding proteins. Interaction between LMP1 and these proteins was localized to lipid rafts and dependent on LMP1 signaling. This suggests that LMP1 raft association is mediated through interactions with actin-associated proteins.

  • Rift Valley fever virus MP-12 vaccine is fully attenuated by a combination of partial attenuations in the S-, M- and L-segments [PublishAheadOfPrint]

  • Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa, and characterized by high-rate abortion in ruminants and hemorrhagic fever, encephalitis or blindness in humans. RVF is caused by Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus), which has a tripartite negative-stranded RNA genome (S-, M- and L-segments). Further spread of RVF into non-endemic countries may affect the economy and public health, and vaccination is an effective approach to prevent the spread of RVFV. A live-attenuated MP-12 vaccine is one of the best characterized RVF vaccines for safety and efficacy, and is currently conditionally licensed for veterinary purpose in the U.S. Meanwhile, no other RVF vaccines are conditionally or fully licensed in the U.S., as of 2015. MP-12 strain is derived from wild-type pathogenic ZH548 strain, and encodes 23 mutations in the three genome segments. However, the attenuation mechanism of MP-12 remains unknown. We characterized the attenuation of wild-type pathogenic ZH501 strain encoding mutation(s) of MP-12 S-, M- or L-segment in the mouse model. Our results indicated that MP-12 is attenuated by all S-, M- and L-segment, while the M- and L-segment confer stronger attenuation than the S-segment. We identified a combination of 3 amino acid changes, Y259H (Gn), R1182G (Gc) and R1029K (L) was sufficient to attenuate ZH501. However, MP-12 encoding reversion mutations at those 3 sites was still highly attenuated. Our results indicated that MP-12 attenuation is supported by a combination of multiple partial attenuation mutations, and a single reversion mutation will less likely cause a reversion to virulence of MP-12 vaccine.

    IMPORTANCE Rift Valley fever (RVF) is a mosquito-transmitted viral disease endemic to Africa, which has the potential to spread into other countries. Vaccination is considered an effective way to prevent the disease, and the only available veterinary RVF vaccine in the U.S. is a live-attenuated MP-12 vaccine, which is conditionally licensed. MP-12 strain is unique to parental pathogenic RVFV strain by 23 mutations. This study determined the role of individual mutations in attenuation of MP-12 strain. We found that full attenuation of MP-12 occurs by a combination of multiple mutations. Our findings indicate a single reversion mutation will less likely cause a major reversion to virulence of MP-12 vaccine.

  • A human cytokinome analysis for interferon response [PublishAheadOfPrint]

  • Cytokines are a group of secreted small proteins that mediate a diverse range of immune and non-immune responses toward inflammatory and microbial stimuli. Only a few of these cytokines mount antiviral response, and include Type-I, -II, and -III interferons (IFN). During viral infections and inflammatory conditions, a number of cytokines and chemokines are co-produced with IFN; however, no systemic study exists on the interactions of the cytokine repertoire with IFN response. Here, we performed the largest cytokine and chemokine screen (the human cytokinome ggt;240 members) to investigate their modulation of Type-I and Type-II IFN responses in a cell line model. We evaluated the cytokine activities in both IFN-stimulated response element (ISRE) and IFN- activation sequence (GAS) reporter systems. Several cytokine clusters, that augment either or both ISRE- and GAS-mediated responses towards IFNs, were derived from the screen. We identified novel modulators of IFN response- betacellulin (BTC), IL11, and IL17F- which caused time-dependent induction of IFN response. The ability of inducing endogenous IFN-bbeta; and IFN-stimulated genes varies among these cytokines, and was largely dependent on Stat-1 as assessed by Stat-1 mutant fibroblasts. Certain cytokines appear to augment IFN-bbeta; response through NF-B pathway. The novel IFN-like cytokines augmented the antiviral activity of IFN-aalpha; against several RNA viruses, including encephalomyocarditis virus, vesicular stomatitis virus, and influenza virus in susceptible cell lines. Overall, the study reports a large-scale analysis of cytokines for enhancing the IFN response, and identified cytokines capable of enhancing Stat1, IFN-induced gene expression, and antiviral activities.

    Importance Innate immunity to viruses is an early defense system to ward off viruses. One mediator is interferon (IFN) which activates a cascade of biochemical events that aim to control the virus life cycle. In our work, we examined more than 200 cytokines, soluble mediators produced within the body as a result of infection, for their ability to enhance IFN action. We identified enhanced interactions with specific members of IFNs and cytokines. We also revealed that betacellulin, IL-17, and IL-11 cytokines, have the novel property in enhancing the antiviral action of IFN against several viruses. These results demonstrate that the human genome code for previously unknown proteins of unrelated functions that can augment the innate immunity to viruses. Knowing these interactions, not only help our understanding of immunity to viruses and emerging diseases but can also lead to devising possible new therapeutics by enhancing the mediator of antiviral action itself, IFN.

  • Inefficient codon usage impairs mRNA accumulation: the case of the v-FLIP gene of Kaposi's sarcoma-associated herpesvirus. [PublishAheadOfPrint]

  • Latent KSHV genomes encode a homolog of cellular FLICE-inhibitory proteins (termed v-FLIP) that activates NF[graphic]B and can trigger important proinflammatory and anti-apoptotic changes in latently infected cells. The protein is present at very low levels in infection and has generally been difficult to efficiently express in recombinant vectors. Here we show that codon usage in the vFLIP gene is strikingly suboptimal. Optimization of codon use in expression vectors, as expected, restores efficient protein expression. Surprisingly, however, it also dramatically increases the steady-state level of vFLIP mRNA, at least in part by increasing mRNA stability. When codon-optimized vFLIP sequences are reintroduced into intact KSHV genomes, the resulting virus expresses readily detectable monocistronic vFLIP mRNAs that are undetectable in WT infection by blot-hybridization nndash; suggesting that such RNAs are in fact transcribed in WT infection but fail to accumulate. The overexpression of vFLIP by codon-optimized latent genomes results in a 5 to 7-fold decrement in virus production following lytic induction, indicating that maximizing NFB signaling is deleterious to induction. These studies provide a clear explanation for the evolution of inefficient codon usage in this gene, and point to a strong connection between translational efficiency and RNA accumulation in mammalian cells.

    Importance: This study reports that inefficient codon usage in a herpesviral gene is strikingly correlated with the inability of its mRNA to accumulate in cells; correction of efficient translatability restores RNA abundance. A similar correlation has been reported in yeast, but the mechanisms operating in mammalian cells appear substantially different.

  • Heterogeneous nuclear ribonucleoprotein M facilitates enterovirus infection [PublishAheadOfPrint]

  • Picornavirus infection involves a dynamic interplay of host and viral protein interactions that modulates cellular processes to facilitate virus infection and evade host-antiviral defenses. Here, using a proteomics-based approach to identify protease-generated neo-N-termini peptides, we identify a novel target of the poliovirus 3C proteinase, the heterogeneous nuclear ribonucleoprotein M (hnRNP M), which is a nucleo-cytoplasmic shuttling RNA-binding protein that is primarily known for its role in pre-mRNA splicing. hnRNP M is cleaved in vitro by poliovirus and coxsackievirus B3 (CVB3) 3C proteinases and is targeted in poliovirus- and CVB3-infected HeLa cells and in hearts of CVB3-infected mice. hnRNP M relocalizes from the nucleus to cytoplasm during poliovirus infection. Finally, depletion of hnRNP M using siRNA knockdown approaches decreases poliovirus and CVB3 infections in HeLa cells and does not affect poliovirus IRES translation and viral RNA stability. We propose that cleavage of and subverting the function of hnRNP M is a general strategy utilized by picornaviruses to facilitate viral infection.

    IMPORTANCE Enteroviruses, a member of the picornavirus family, are RNA viruses that cause a range of diseases including respiratory ailments, dilated cardiomyopathy and paralysis. Although enteroviruses have been studied for several decades, the molecular basis of infection and the pathogenic mechanisms leading to disease are still poorly understood. Here, we have identified hnRNP M as a novel target of a viral proteinase. We demonstrate that the virus subverts the function of hnRNP M and redirects it to a step in the viral life cycle. We propose that cleavage of hnRNP M is a general strategy that picornaviruses use to facilitate in infection.

  • A semi-nomadic population in Bangladesh with extensive exposure to macaques does not exhibit high levels of zoonotic simian foamy virus infection [PublishAheadOfPrint]

  • Simian Foamy viruses (SVF) are ubiquitous in non-human primates (NHP). SFV can be zoonotically transmitted to humans, who either work with, or live commensally with NHP. We analyzed the blood of 45 Bangladeshi performing monkey owners (an ethnic group called the Bedey) for SFV infection. Surprisingly, PCR failed to detect SFV infection in any of these participants. This is in contrast to our previously reported infection rate of about 5% among Bangladeshi villagers.

  • Human plasmacytoid dendritic cells elicited different responses after infection with pathogenic or non-pathogenic Junin virus strains [PublishAheadOfPrint]

  • The Juniiacute;n virus (JUNV) arenavirus is the etiologic agent of Argentina Haemorrhagic Fever (AHF). We characterised the JUNV infection of human peripheral blood-derived plasmacytoid dendritic cells (hpDC), demonstrating that hpDC are susceptible to infection by the C#1 strain (attenuated), and even more, by the P (virulent) JUNV strain. However, hpDC elicited different responses in terms of viability, activation, maturation, and cytokine expressions after infection with both JUNV strains.

  • Recombination Analysis of Herpes Simplex Virus Type 1 Reveals a Bias towards GC Content and the Inverted Repeat Regions [PublishAheadOfPrint]

  • Herpes simplex virus type 1 (HSV-1) causes recurrent mucocutaneous ulcers, and is the leading cause of infectious blindness and sporadic encephalitis in the United States. HSV-1 has been shown to be highly recombinagenic, however to date there has been no genome wide analysis of recombination. To address this, we generated 40 recombinant HSV-1 viruses derived from two parental strains; OD4 and CJ994. The 40 OD4-CJ994 HSV-1 recombinants were sequenced using the Illumina sequencing system, and recombination breakpoints were determined for each of the recombinants using the Bootscan program. Breakpoints occurring in the terminal inverted repeats were excluded from analysis to prevent double counting, resulting in a total of 272 breakpoints in the dataset. By placing windows around the 272 breakpoints followed by Monte Carlo analysis comparing actual data to simulated data, we identified recombination bias towards both GC content and intergenic regions. A Monte Carlo analysis also suggested that recombination did not appear to be responsible for the generation of spontaneous nucleotide mutations detected following sequencing. Additionally, kernel density estimation analysis across the genome found that the large, inverted repeats comprise a recombination hotspot.

    Importance Herpes simplex virus type 1 (HSV-1) virus is the leading cause of sporadic encephalitis and blinding keratitis in developed countries. HSV-1 has been shown to be highly recombinagenic and recombination itself appears to be a significant component of genome replication. To date there has been no genome wide analysis of recombination. Here we present the first genome wide study of recombination by generating and sequencing 40 HSV-1 recombinants derived from the OD4 and CJ994 parental strains, followed by bioinformatics analysis. Recombination breakpoints were determined, yielding 272 in the full dataset. Kernel density analysis determined that the large inverted repeats constitute a recombination hotspot. Additionally, Monte Carlo analyses found biases towards GC content, intergenic regions, and repetitive regions.

  • A combined genetic-proteomic approach identifies residues within Dengue virus NS4B critical for interaction with NS3 and viral replication [PublishAheadOfPrint]

  • Dengue virus (DENV) infection causes the most prevalent arthropod-borne viral disease worldwide. Approved vaccines are not available and targets suitable for the development of antiviral drugs are lacking. One possible drug target is nonstructural protein (NS) 4B, because it is absolutely required for virus replication; however, its exact role for the DENV replication cycle is largely unknown. With the aim to map NS4B determinants critical for DENV replication, we performed a reverse genetic screening of 33 NS4B mutants in the context of an infectious DENV genome. While the majority of these mutations was lethal, for several of them we were able to select for second-site pseudoreversions, most often residing in NS4B and restoring replication competence. To identify all viral NS4B interaction partners, we engineered a fully viable DENV genome encoding an affinity tagged NS4B. Mass spectrometry-based analysis of the NS4B complex isolated from infected cells identified the NS3 protease/helicase as a major interaction partner of NS4B. By combining the genetic complementation map of NS4B with a replication-independent expression system, we identified the NS4B cytosolic loop, more precisely amino acid residue Q134, as critical determinant for NS4B-NS3 interaction. An alanine substitution at this site completely abrogated the interaction and DENV RNA replication and both were restored by pseudoreversions A69S and A137V. This strict correlation between the degree of NS4B-NS3 interaction and DENV replication provides strong evidence that this viral protein complex plays a pivotal role during the DENV replication cycle, hence representing a promising target for novel antiviral strategies.

    IMPORTANCE With no approved therapy or vaccine against dengue virus infection, the viral nonstructural protein (NS) 4B represents a possible drug target, because it is indispensable for virus replication. However, little is known about its precise structure and function. Here we established the first comprehensive genetic interaction map of NS4B, identifying amino acid residues that are essential for virus replication as well as second-site mutations compensating for these defects. Additionally, we determined the NS4B viral interactome in infected cells and identified the NS3 protease/helicase as a major interaction partner of NS4B. We mapped residues in the cytosolic loop of NS4B as critical determinants for interaction with NS3 as well as RNA replication. The strong correlation between NS3-NS4B interaction and RNA replication provides strong evidence that this complex plays a pivotal role for the viral replication cycle, hence representing a promising antiviral drug target.

  • HCoV-HKU1 Spike protein uses O-acetylated sialic acid as an attachment receptor determinant and employs HE protein as a receptor-destroying enzyme [PublishAheadOfPrint]

  • Human coronavirus (hCoV) HKU1 is one of six hCoVs identified to date and the only one with an unidentified cellular receptor. HCoV-HKU1 encodes a hemagglutinin-esterase (HE) protein that is unique to the betacoronaviruses group a (group 2a). The function of HKU1-HE remains largely undetermined. In this study, we examined binding of the S1 domain of hCoV-HKU1 spike to a panel of cells and found that the S1 could specifically bind on the cell surface of a human rhabdomyosarcoma cell line, RD. Pretreatment of RD cells with neuramidase (NA) and trypsin greatly reduced the binding, suggesting that the binding was mediated by sialic acids on glycoproteins. However, unlike other group 2a CoVs, e.g. hCoV-OC43 for which 9-O-acytylated sialic acid (9-O-Ac-Sia) servers as a receptor determinant, HKU1-S1 neither bound with 9-O-Ac-Sia containing glycoprotein(s), nor rat and mouse erythrocytes. Nonetheless, the HKU1-HE was similar to OC43-HE, also possessed sialate-O- acetylesterase activity and acted as a receptor-destroying enzyme (RDE) capable of eliminating the binding of HKU1-S1 to RD cells, whereas the O-acetylesterase inactive HKU1-HE mutant lost this capacity. Using primary human ciliated airway epithelial cell cultures (HAE), the only in vitro replication model for hCoV-HKU1 infection, we confirmed that pretreatment of HAE cells with HE but not the enzymatically inactive mutant blocked hCoV-HKU1 viral infection. These results demonstrate that hCoV-HKU1 exploits O-AC-Sia as a cellular attachment receptor determinant to initiate the infection of host cells, and its HE protein possesses the corresponding sialate-O-acetylesterase RDE activity.

    Importance statement Human coronaviruses (hCoV) are important human respiratory pathogens. Among the six hCoVs identified to date, only hCoV-HKU1 has no defined cellular receptor. It is also unclear whether hemagglutinin-esterase (HE) protein plays a role in viral entry. In this study, we found that, similar to other members of the group 2a CoVs, sialic acid moieties on glycoproteins are critical receptor determinants for the hCoV-HKU1 infection. Interestingly, the virus seems to employ different type of sialic acid than other group 2a CoVs. In addition, we determined that the HKU1-HE protein is an O-acetylesterase and acts as a receptor-destroying enzyme (RDE) for hCoV-HKU1. This is the first study to demonstrate that hCoV-HKU1 uses certain types of O-acytylated sialic acid residues on glycoproteins to initiate the infection of host cells, and HKU1-HE protein possesses sialate-O-acetylesterase RDE activity.

  • The nectin-4/afadin protein complex and intercellular membrane pores contribute to rapid spread of measles virus in primary human airway epithelia [PublishAheadOfPrint]

  • The discovery that measles virus (MV) uses the adherens junction protein nectin-4 as its epithelial receptor provides a new vantage point from which to characterize its rapid spread in the airway epithelium. We show here that in well-differentiated primary cultures of airway epithelial cells from human donors (HAE), MV infectious centers form rapidly and become larger than those of the other respiratory pathogens: human respiratory syncytial virus, parainfluenza virus 5, and Sendai virus. While visible syncytia do not form after MV infection of HAE, the cytoplasm of an infected cell suddenly flows into an adjacent cell, as visualized through wild type MV-expressed cytoplasmic green fluorescent protein (GFP). High-resolution video microscopy documents that GFP flows through openings that form on the lateral surfaces between columnar epithelial cells. To assess the relevance of the protein afadin, which connects nectin-4 to the actin cytoskeleton, we knocked down its mRNA. This resulted in more limited infectious center formation. We also generated a nectin-4 mutant without the afadin-binding site in its cytoplasmic tail. This mutant was less effective than wild-type human nectin-4 in promoting MV infection in primary cultures of porcine airway epithelia. Thus, in airway epithelial cells, MV spread requires the nectin-4/afadin complex, and is based on cytoplasm transfer between columnar cells. Since the viral membrane fusion apparatus may open the passages that allow cytoplasm transfer, we refer to them as intercellular membrane pores. Virus-induced intercellular pores may contribute to extremely efficient measles contagion by promoting rapid spread of the virus through the upper respiratory epithelium.

    IMPORTANCE Measles virus (MV), while targeted for eradication, still causes about 120,000 yearly deaths worldwide. Recent measles re-emergence in insufficiently vaccinated populations in Europe and North America reminds us that measles is extremely contagious, but the processes favoring its spread in the respiratory epithelium remain poorly defined. Here, we characterize wild type MV spread in well-differentiated primary cultures of human airway epithelial cells. We observed that viral infection promotes the flow of cytoplasmic contents from infected to proximal uninfected columnar epithelial cells. Cytoplasm flows through openings that form on the lateral surfaces. Infectious center growth is facilitated by afadin, a protein connecting the adherens junction and the actin cytoskeleton. The viral fusion apparatus may open intercellular pores and the cytoskeleton may stabilize them. Rapid homogenization of cytoplasmic contents in epithelial infectious centers may favor rapid spread and contribute to the extremely contagious nature of measles.

  • Design and Structure of an Engineered Disulfide-Stabilized Influenza Hemagglutinin Trimer [PublishAheadOfPrint]

  • We engineered a disulfide-stabilized influenza hemagglutinin (HA) trimer, termed HA3-SS, by introducing cysteine residues in the HA stem to covalently bridge the three protomers. HA3-SS has increased thermostability compared to wild-type HA and binding of head- and stem-targeted antibodies is preserved; only minor structural changes are found in the vicinity of the additional disulfide. This platform has been applied to H1 and H3 HAs and provides prospects for design of intact, stabilized influenza HA immunogens.

  • Immediate and ongoing detection of prions in the blood of hamsters and deer following oral, nasal, or blood inoculations [PublishAheadOfPrint]

  • Infectious prions traverse epithelial barriers to gain access to the circulatory system, yet the temporal parameters of transepithelial transport and persistence in the blood over time remains unknown. We used wbRT-QuIC to analyze whole blood collected from TSE-inoculated deer and hamsters throughout the entire incubation period for the presence of PrPC-conversion competent amyloid (PrPC-CCA). We observed PrPC-CCA in the blood of TSE-inoculated hosts throughout disease course from minutes post exposure to terminal disease.

  • A functional interaction between herpes simplex virus 1 glycoproteins gH/gL domains I-II and gD is defined using {alpha}-herpesvirus gH and gL chimeras [PublishAheadOfPrint]

  • Whereas most viruses require only a single protein to bind to and fuse with cells, herpesviruses use multiple glycoproteins to mediate virus entry and thus communication among these proteins is required. For most aalpha;-herpesviruses, the minimal set of viral proteins required for fusion with the host cell includes glycoproteins gD, gB, and a gH/gL heterodimer. In the current model of entry, gD binds to a cellular receptor and transmits a signal to gH/gL. This signal then triggers gB, the conserved fusion protein, to insert into the target membrane and refold to merge the viral and cellular membranes. We previously demonstrated that gB homologs from two aalpha;-herpesviruses, herpes simplex virus 1 (HSV-1) and saimiriine herpesvirus 1 (SaHV-1), were interchangeable. In contrast neither gD nor gH/gL functioned with heterotypic entry glycoproteins, indicating that gD and gH/gL exhibit an essential type-specific functional interaction. To map this homotypic interaction site on gH/gL, we generated HSV-1/SaHV-1 gH and gL chimeras. The functional interaction with HSV-1 gD mapped to the N-terminal domains I and II of the HSV-1 gH ectodomain. The core of HSV-1 gL that interacts with gH also was required for functional homotypic interaction. The N-terminal gH/gL domains I and II are the least conserved and may have evolved to support species-specific glycoprotein interactions.

    IMPORTANCE The first step of the herpesvirus lifecycle is entry into a host cell. A coordinated interaction among multiple viral glycoproteins is required to mediate fusion of the viral envelope with the cell membrane. The details of how these glycoproteins interact to trigger fusion are unclear. By swapping the entry glycoproteins of two aalpha;-herpesviruses (HSV-1 and SaHV-1), we previously demonstrated a functional homotypic interaction between gD and gH/gL. To define the gH and gL requirements for homotypic interaction, we evaluated the function of a panel of HSV-1/SaHV-1 gH and gL chimeras. We demonstrate that domains I-II of HSV-1 gH are sufficient to promote a functional, albeit reduced, interaction with HSV-1 gD. These findings contribute to our model of how the entry glycoproteins cooperate to mediate herpesvirus entry into the cell.

  • Differential expression of CX3CL1 in HBV-replicating hepatoma cells could affect the migration-activity of CX3CR1+ immune cells [PublishAheadOfPrint]

  • [Background and Aims]

    In addition to stellate cells and immune cells, inflamed hepatocytes and hepatoma cells express various kinds of chemokines that attract various kinds of immune cells. Previously, we reported that HBV replication could induce physiological stress. The aim of this study is to analyze the effect of chemokines produced by HBV-infected hepatocytes and hepatoma cells.

    [Methods]

    Realtime PCR array targeting genes related to chemokines and ELISA were carried out to detect the specific chemokines produced by Huh7 cells and HepG2 cells infected with various HBV genotypes. A migration assay, flow cytometry analysis and immunohistochemistry were carried out to analyze the candidate immune cells that could affect the immunopathogenesis of HBV infection.

    [Results]

    The expressions of CX3CL1 mRNA and protein were significantly different among the HBV genotypes A, B, C, and Mock (pllt;0.05). CD56+ NK cells and CD8+ T cells migrated to the hepatoma cells with HBV replication. Moreover, the migration activity of both immune cells was partially cancelled after the treatment of CX3CL1 neutralizing antibody. The expression level of NKG2D on CX3CR1+NK cells in HCC with HBV infection was significantly lower than that in HCC with HCV infection and chronic hepatitis B and C patients (pllt;0.05). On the other hand, the frequency of PD-1highCX3CR1+CD8+ T cells in HCC with HBV infection was significantly higher than that in HCC with HCV infection and chronic hepatitis B and C patients (pllt;0.05).

    [Conclusions]

    The expression of CX3CL1 in HBV-replicating hepatocytes and hepatoma cells could contribute to the immunopathogenesis of HBV infection.

    Importance The progression of the disease status is significantly different among HBV genotypes. However, it has not been clear that how different HBV genotypes could induce different inflammatory responses. In this report, we first reported that the expressions of CX3CL1 mRNA and protein were significantly different among the HBV genotypes A, B, C, and Mock. Not only the differential expression of CX3CL1 among the genotypes but also the phenotype of CX3CR1+ NK cells and T cells were gradually changed during the progression of the disease status. In addition to in vitro study, the analysis of immunohistochemistry with human samples and NOG mice with human lymphocytes and hepatoma cells support this phenomenon. The quantification of CX3CL1 could contribute to better understanding about the disease status of HBV infection. Moreover, modifying CX3CL1 might induce an immune response appropriate to the disease status of HBV infection.

  • The human adenovirus type 5 L4 promoter is negatively regulated by TFII-I and by L4-33K [PublishAheadOfPrint]

  • The late phase of adenovirus gene expression is controlled by proteins made in the intermediate phase, including L4 22K and 33K proteins that are expressed initially from the L4 promoter (L4P). L4P is activated by a combination of viral proteins and cellular p53 and is ultimately inhibited again by its own products. Here we have examined L4P of human adenovirus type 5 in detail and have defined its transcription start site, which our data suggest is positioned by a weak TATA box. Rather than contributing positively to promoter activity, a putative initiator element at the transcription start site acts as a target for negative regulation imposed on L4P by cellular TFII-I. We show that this TFII-I inhibition is relieved by one of the previously defined viral activators of L4P, E4 Orf3 protein, which alters the pool of TFII-I in the cell. We also explore further the negative regulation of L4P by its products, and show that the L4-33K protein is more significant in this process than L4-22K. It is the combined action of positive and negative factors that leads to the transient activation of L4P at the onset of the late phase of adenovirus gene expression.

    Importance The adenovirus replication cycle proceeds through multiple phases of gene expression in which a key step is the activation of late phase gene expression to produce proteins from which progeny particles can be formed. Working with human adenovirus type 5, we showed previously that two proteins expressed from the L4 region of the viral genome perform essential roles in moving the infection on into the late phase; these two proteins are produced by the action of a dedicated promoter, L4P, and without them the infection does not proceed successfully to progeny generation. In this new work, we delineate further aspects of L4P activity and regulation. Understanding how L4P works, and how it contributes to activation of the late phase of infection, is important to our understanding of natural infections by this virus, in which late gene expression can fail to occur, allowing the virus to persist.

  • New non-coding lytic transcripts derived from the Epstein Barr virus latency origin of replication oriP are hyper-edited, bind the paraspeckle protein, NONO/p54nrb, and support lytic viral transcription. [PublishAheadOfPrint]

  • We have previously shown that the Epstein-Barr virus (EBV) likely encodes hundreds of viral long non-coding RNAs (vlncRNAs) that are expressed during reactivation. Here we show that the EBV latency origin of replication (oriP) is transcribed bi-directionally during reactivation and that both leftward (oriPtLs) and rightward transcripts (oriPtRs) are largely localized in the nucleus. While the oriPtLs are most likely non-coding, at least some of the oriPtRs contain the BCRF1/vIL10 open reading frame. Nonetheless, oriPtR transcripts with long 5rrsquo; UTRs may partially serve non-coding functions. Both oriPtL and oriPtR transcripts are expressed with late kinetics and their expression is inhibited by phosphonoacetic acid. RNA-seq analysis showed that oriPtLs and oriPtRs exhibited extensive "hyper-editing" at their Family of Repeat (FR) regions. RNA secondary structure prediction revealed that the FR region of both oriPtLs and oriPtRs may form large evolutionarily conserved and thermodynamically stable hairpins. The double-stranded RNA-binding protein and RNA-editing enzyme ADAR was found to bind to oriPtLs, likely facilitating editing of the FR hairpin. Further, the multifunctional paraspeckle protein, NONO, was found to bind to oriPt transcripts suggesting that oriPts interacts with the paraspeckle-based innate anti-viral immune pathway. Knock-down and ectopic expression of oriPtLs showed that it contributes to global viral lytic gene expression and viral DNA replication. Together, these results show that these new vlncRNAs interact with cellular innate immune pathways and that they help facilitate progression of the viral lytic cascade.

    IMPORTANCE Recent studies have revealed that the complexity of lytic herpesviral transcriptomes is significantly greater than previously appreciated with hundreds of viral long non-coding RNAs (vlncRNAs) being recently discovered. Work on cellular lncRNAs over the past several years has just begun to give us an initial appreciation for the array of functions they play in complex formation and regulatory processes in the cell. The newly identified herpesvirus lncRNAs are similarly likely to play a variety of different functions though these functions are likely tailored to specific needs of the viral infection cycles. Here we describe novel transcripts derived from the EBV latency origin of replication. We show that they are hyper-edited, that they interact with a relatively newly appreciated anti-viral pathway, and that they play a role in facilitating viral lytic gene expression. These investigations are a starting point to unraveling the complex arena of vlncRNA function in herpesvirus lytic replication.

  • Human Endogenous Retroviruses Type-K (HERV-K) Virus Particles Package and Transmit HERV-K-Related Sequences [PublishAheadOfPrint]

  • Human Endogenous Retroviruses make up 8% of the human genome. While the youngest of these retroviruses, HERV-K (HML-2), termed HK2, is able to code for all viral proteins and produce virus-like particles, it is not known if these virus particles package and transmit HK2-related sequences. Here we analyzed the capacity of HK2 for packaging and transmitting HK2 sequences. We created a HK2 probe, termed Bogota, which can be packaged into HK2 viruses, and transfected it into cells that make HK2 particles. Supernatants of the transfected cells, which contained HK2 viral particles, were then added to target cells and the transmissibility of the HK2 Bogota reporter was tracked by G418 resistance. Our studies revealed that contemporary HK2 virions produced by some teratocarcinoma and breast cancer cell lines, as well as by peripheral blood lymphocytes from lymphoma patients, can package HK2 Bogota probes, and these viruses transmitted these probes to other cells. After transmission, HK2 Bogota transcripts undergo reverse transcription, a step impaired by antiretroviral agents or by introduction of mutations into the probe sequences required for reverse transcription. HK2 viruses were more efficiently transmitted in the presence of HK2 Rec or HIV-1 Tat and Vif. Transmitted Bogota probes formed episomes, but did not integrate into the cellular genome. Resistance to integration might explain the relatively low number of HK2 insertions that were acquired during the last 25 million years of evolution. Whether transient transmission of modern HK2 sequences, which encode two putative oncoproteins, can lead to disease remains to be studied.

    Importance Retroviruses have invaded the genome of human ancestors over millions of years, yet these viruses have been generally inactivated during evolution, with only remnants of these infectious sequences remaining in the human genome. One of these viruses, termed HK2, is still capable of producing virus particles, although these particles have been regarded as being non-infectious. Using a genetic probe derived from HK2, we have discovered that HK2 viruses produced in modern humans can package HK2 sequences and transmit them to various other cells. Furthermore, the genetic sequences packaged in HK2 undergo reverse transcription. The transmitted probe circularized in the cell and failed to integrate into the cellular genome. These findings suggest that modern HK2 viruses can package viral RNA and transmit it to other cells. Contrary to previous views, we provide evidence of an extracellular viral phase of modern HK2 viruses. We have no evidence of sustained, spreading infection.

  • Identification of a Novel Hepacivirus in Domestic Cattle from Germany [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) continues to represent one of the most significant threats to human health. In recent years, HCV-related sequences have been found in bats, rodents, horses and dogs indicating a widespread distribution of hepaciviruses among animals. By applying unbiased high-throughput sequencing, a novel virus of the genus Hepacivirus was discovered in a bovine serum sample. De novo assembly yielded a near full length genome coding for a polyprotein of 2,779 amino acids. Phylogenetic analysis confirmed that the virus represents a novel species within the genus hepacivirus. Viral RNA screening determined 1.6% (n=5) of 320 individual animals and 3.2% (n=5) of 158 investigated cattle herds in Germany positive for bovine hepacivirus. Repeated RT-PCR analyses of animals from one dairy herd proved that a substantial percentage of cows were infected, with some of them being viremic for over six months. Clinical and postmortem examination revealed no signs of disease including liver damage. Interestingly, quantitative RT-PCR from different organs and tissues together with the presence of a miR-122 binding site in the viral genome strongly suggest a liver tropism for bovine hepacivirus, making this novel virus a promising animal model for HCV infections in humans.

    Importance Livestock animals act as important source for emerging pathogens. In particular, their large herd size and the existence of multiple ways of direct and food-borne infection routes emphasize their role as virus reservoirs. Apart from searching for novel viruses, detailed characterization of these pathogens is indispensable concerning risk analysis. Here, we describe the identification of a novel HCV-like virus in cattle. Beyond, determination of the prevalence and the course of infection in cattle herds provide valuable insights into the biology of this novel virus. The results presented here form a basis for future studies targeting viral pathogenesis of bovine hepaciviruses and their potential to establish zoonotic infections.

  • Highly pathogenic New World and Old World human arenaviruses induce distinct interferon responses in human cells [PublishAheadOfPrint]

  • The arenavirus family includes several important pathogens that cause severe and sometimes fatal diseases in humans. The highly pathogenic Old World (OW) arenavirus Lassa fever virus (LASV) is the causative agent of Lassa fever (LF) disease in humans. LASV infections in severe cases are generally immunosuppressive without stimulating interferon (IFN) induction, pro-inflammatory response or T cell activation. However, the host innate immune responses to highly pathogenic New World (NW) arenaviruses are not well understood. We have previously shown that the highly pathogenic NW arenavirus, Juniiacute;n virus (JUNV), induced an IFN response in human A549 cells. Here we report that Machupo virus (MACV), another highly pathogenic NW arenavirus also induces an IFN response. Importantly, both pathogenic NW arenaviruses, in contrast to the OW highly pathogenic arenavirus LASV, readily elicited IFN in human primary dendritic cells and A549 cells. Co-infection experiments revealed that LASV could potently inhibit MACV-activated IFN responses even at 6 hr after MACV infection, while the replications of MACV and LASV were not affected by virus co-infection. Our results clearly demonstrated that although all viruses studied herein are highly pathogenic to humans, the host IFN responses toward infections with NW arenavirus JUNV and MACV are quite different from infections with OW arenavirus LASV, a discovery that needs to be further investigated in relevant animal models. This finding might help us better understand various interplays between the host immune system and highly pathogenic arenaviruses as well as distinct mechanisms underlying viral pathogenesis.

    Importance Infections of humans with the highly pathogenic OW LASV are accompanied by potent suppression of interferon or pro-inflammatory cytokine production. In contrast, infections with highly pathogenic NW arenavirus JUNV are associated with high levels of IFNs and cytokines in severe and fatal cases. Arenaviruses initially target macrophages and dendritic cells, which are potent IFN/cytokine-producers. In human macrophages, JUNV reportedly does not trigger IFN responses. We herein demonstrated that JUNV activated IFN responses in human dendritic cells. MACV, another highly pathogenic NW arenavirus, also activated IFN responses. LASV did not induce detectable IFN responses in spite of higher replications and blocked the MACV-triggered IFN response in a co-infection assay. Although these viruses are highly pathogenic to humans, our study highlights distinct innate immune responses to infections with NW arenavirus JUNV and MACV and to infection with OW arenavirus LASV, and provides important insights into virus-host interaction and pathogenesis.

  • Lymph node macrophages restrict murine cytomegalovirus dissemination [PublishAheadOfPrint]

  • Cytomegaloviruses (CMVs) establish chronic infections that spread from a primary entry site to secondary vascular sites such as the spleen, then to tertiary shedding sites such as the salivary glands. Human CMV (HCMV) is difficult to analyze because its spread precedes clinical presentation. Murine CMV (MCMV) offers a tractable model. It is hypothesized to spread from peripheral sites via vascular endothelial cells and associated monocytes. However viral luciferase imaging showed footpad-inoculated MCMV reaching first the popliteal lymph nodes (PLN). PLN colonization was rapid and further spread was slow, implying that LN infection can be a significant bottleneck. Most acutely infected PLN cells were CD169+ subcapsular sinus macrophages (SSM). Replication-deficient MCMV also reached them, indicating direct infection. Many SSM expressed viral reporter genes but few expressed lytic genes; SSM expressed CD11c, and MCMV with a cre-sensitive fluorochrome switch showed switched infected cells in PLN of CD11c-cre mice, but yielded little switched virus; and SSM depletion with liposomal clodronate or via a CD169-diphtheria toxin receptor transgene shifted infection to ER-TR7+ stromal cells, increased virus production and accelerated its spread to the spleen. Therefore MCMV disseminated via LN, and SSM slowed this spread by shielding permissive fibroblasts and supporting poorly viral lytic replication.

    Importance Human cytomegalovirus (HCMV) chronically infects most people, causing congenital disability and harming the immunocompromised. A major goal of vaccination is to prevent systemic infection. How this is established is unclear. Restriction to humans makes HCMV difficult to analyse. We show that peripheral Murine CMV (MCMV) infection spreads via lymph nodes. Here MCMV infected filtering macrophages, which supported virus replication poorly. When these macrophages were depleted, MCMV infected susceptible fibroblasts and spread faster. The capacity of filtering macrophages to limit MCMV spread argued that their infection is an important bottleneck in host colonization, and so might be a good a vaccine target.

  • Recombination of Globally Circulating Varicella Zoster Virus [PublishAheadOfPrint]

  • Varicella zoster virus (VZV) is a human herpesvirus, which during primary infection typically causes varicella (chickenpox) and establishes lifelong latency in sensory and autonomic ganglia. Later in life, the virus may reactivate to cause herpes zoster (HZ, shingles). To prevent these diseases, a live-attenuated heterogeneous vaccine preparation; vOka, is used routinely in many countries worldwide. Recent studies of another alphaherpesvirus, infectious laryngotracheitis virus, demonstrate that live-attenuated vaccine strains can recombine in vivo creating virulent progeny. These findings raised concerns about using attenuated herpesvirus vaccines under conditions that favor recombination. To investigate whether VZV may undergo recombination, which is a prerequisite for VZV vaccination to create such conditions, we here analyzed 115 complete VZV genomes. Our results demonstrate that recombination occurs frequently for VZV. It thus seems that VZV is fully capable of recombination if given the opportunity, which may have important implications for continued VZV vaccination. Although no interclade vaccine-wildtype recombinant strains were found, intraclade recombinants were frequently detected in clade 2, which harbors the vaccine strains, suggesting that the vaccine strains have already been involved in recombination events, either in vivo, or in vitro during passages in cell culture. Finally, previous part- and complete genomic studies have described strains that do not cluster phylogenetically to any of the five established clades. The additional VZV strains sequenced here, in combination with those previously published, have enabled us to formally define a novel sixth VZV clade.

    Importance Although genetic recombination has been demonstrated to frequently occur for other human alphaherpesviruses, herpes simplex virus type I and II, only a few ancient and isolated recent recombination events have hitherto been demonstrated for VZV. In the present study, we demonstrate that also VZV frequently undergoes genetic recombination, including strains belonging to the clade containing the vOKA strain.

  • Kallikrein-8 proteolytically processes Human papillomaviruses in the extracellular space to facilitate entry into host cells [PublishAheadOfPrint]

  • The entry of human papillomaviruses into host cells is a complex process. It involves conformational changes at the cell surface, receptor switching, internalization by a novel endocytic mechanism, uncoating in endosomes, trafficking of a subviral complex to the Golgi complex, and nuclear entry during mitosis. Here, we addressed how the stabilizing contacts in the virus capsid may be reversed to allow uncoating of the viral genome. Using biochemical and cell biological analysis, we determined that the major capsid protein L1 underwent proteolytic cleavage during entry. Besides a dispensable cathepsin-mediated proteolysis that occured likely after removal of capsomers from the subviral complex in endosomes, at least two further proteolytic cleavages of L1 were observed, one of which was independent of the low pH environment of endosomes. This cleavage occured extracellularly. Further analysis showed that the responsible protease was the secreted trypsin-like serine protease kallikrein-8 (KLK8) involved in epidermal homeostasis and wound healing. Required for infection, the cleavage was facilitated by prior interaction of viral particles with heparan sulfate proteoglycans. KLK8-mediated cleavage was crucial for further conformational changes exposing an important epitope of the minor capsid protein L2. Occuring independently of cyclophilins and of furin that mediate L2 exposure, KLK8-mediated cleavage of L1 likely facilitated access to L2 located in the capsid lumen, and potentially uncoating. Since HPV6 and HPV18 also required KLK8 for entry, we propose that the KLK8-dependent entry step is conserved.

    Importance Our analysis of the proteolytic processing of incoming HPV16, an etiological agent of cervical cancer, demonstrated that the capsid is cleaved extracellularly by a serine protease active during wound healing, and that this cleavage was crucial for infection. The cleavage of L1 is one of at least four structural alterations that prime the virus extracellularly for receptor switching, internalization, and possibly uncoating. This step was also important for HPV6 and HPV18, which may suggest that it is conserved among the papillomaviruses. This study advances the understanding of how HPV16 initially infects cells, strengthens the notion that wounding facilitates infection of epidermal tissue, and may help the development of anti-viral measures.

  • Enterovirus 71 Proteins 2A and 3D Antagonize the Antiviral Activity of IFN-{gamma} via Signaling Attenuation [PublishAheadOfPrint]

  • Enterovirus 71 (EV71) infection causes severe mortality involving multiple possible mechanisms including cytokine storm, brainstem encephalitis, and fulminant pulmonary edema. Interferon (IFN)- may confer anti-EV71 activity; however, the claim that disease severity is highly correlated to an increase in IFN- is controversial and would indicate an immune escape initiated by EV71. This study investigating the role of IFN- in EV71 infection using a murine model showed that IFN- was elevated. Moreover, IFN- receptor-deficient mice showed higher mortality and more severe disease progression with slower viral clearance than wild-type mice. In vitro results showed that IFN- pre-treatment reduced EV71 yield whereas EV71 infection caused IFN- resistance with attenuated IFN- signaling in IFN regulatory factor (IRF) 1 transactivation. To study the immunoediting ability of EV71 proteins on IFN- signaling, 11 viral proteins were stably expressed in cells without cytotoxicity; however, viral proteins 2A and 3D blocked IFN--induced IRF1 transactivation following a loss of signal transducer and activator of transcription (STAT) 1 nuclear translocation. Viral 3D attenuated IFN- signaling accompanied by a STAT1 decrease without interfering with IFN- receptor expression. Restoration of STAT1 or blocking 3D activity was able to rescue IFN- signaling. Interestingly, viral 2A attenuated IFN- signaling using another mechanism by reducing the serine phosphorylation of STAT1 following the inactivation of extracellular signal-regulated kinase without affecting STAT1 expression. These results demonstrate the anti-EV71 ability of IFN- and the immunoediting ability by EV71 2A and 3D, which attenuate IFN- signaling through different mechanisms.

    Importance Immunosurveillance by interferon (IFN)- may confer anti-enterovirus 71 (EV71) activity; however, the claim that disease severity is highly correlated to an increase in IFN- is controversial and would indicate an immune escape initiated by EV71. IFN- receptor-deficient mice showed higher mortality and more severe disease progression, indicating the anti-EV71 property of IFN-. However, EV71 infection caused cellular insusceptibility in response to IFN- stimulation. We used an in vitro system with viral protein expression to explore the novel IFN- inhibitory properties of the EV71 2A and 3D proteins through the different mechanisms. According to this study, targeting either 2A or 3D pharmacologically and/or genetically may sustain a cellular susceptibility in response to IFN-, particularly for IFN--mediated anti-EV71 activity.

  • SAMHD1 limits HIV-1 antigen presentation by monocyte-derived dendritic cells [PublishAheadOfPrint]

  • Monocyte-derived Dendritic cells (MDDC) stimulate CD8+ cytotoxic T lymphocytes (CTL) by presenting endogenous and exogenous viral peptides via MHC-I molecules. MDDC are poorly susceptible to HIV-1, in part due to the presence of SAMHD1, a cellular enzyme that depletes intracellular dNTPs and degrades viral RNA. Vpx, an HIV-2/SIVsm protein absent in HIV-1, antagonizes SAMHD1 by inducing its degradation. The impact of SAMHD1 on the adaptive cellular immune response remains poorly characterized. Here, we asked whether SAMHD1 modulates MHC-I-restricted HIV-1 antigen presentation. MDDC pre-treated or not with Vpx, were exposed to HIV-1 and antigen presentation was examined by monitoring the activation of an HIV-1 Gag-specific CTL clone. SAMHD1 depletion strongly enhanced productive infection of MDDC as well as endogenous HIV-1 antigen presentation. Time-lapse microscopy analysis demonstrated that in the absence of SAMHD1, the CTL rapidly killed infected MDDC. We also report that various transmitted/founder (T/F) HIV-1 poorly infected MDDCs and as a consequence, did not stimulate CTL. VSV-G pseudotyping of T/F alleviated a block in viral entry and induced antigen presentation only in the absence of SAMHD1. Furthermore, by using another CTL clone that mostly recognizes incoming HIV-1 antigens, we demonstrate that SAMHD1 does not influence exogenous viral antigen presentation. Altogether, our results demonstrate that the antiviral activity of SAMHD1 impacts antigen presentation by DC, highlighting the link that exists between restriction factors and adaptive immune responses.

    IMPORTANCE Upon viral infection, Dendritic cells (DC) may present antigens derived from incoming viral material, in the absence of productive infection of DCs, or from newly synthesized viral proteins. In the case of HIV, productive infection of DCs is blocked at an early post-entry step. This is due to the presence of SAMHD1, a cellular enzyme that depletes intracellular levels of dNTPs and inhibits viral reverse transcription. We show that the depletion of SAMHD1 in DCs strongly stimulates presentation of viral antigens derived from newly produced viral proteins, leading to activation of HIV-1-specific cytotoxic T lymphocytes (CTL). We further show in real time that the enhanced activation of CTL leads to killing of infected DCs. Our results indicate that the antiviral activity of SAMHD1 not only impacts HIV replication, but also impacts antigen presentation by DCs. They highlight the link that exists between restriction factors and adaptive immune responses.

  • Newcastle disease virus-vectored H7 and H5 live vaccines protect chickens from challenge with H7N9 or H5N1 avian influenza viruses [PublishAheadOfPrint]

  • Sporadic human infections by a novel H7N9 virus occurred over a large geographic region in China. In this study, we show that NDV-vectored H7 and NDV-H5 vaccines are able to induce high HI titer antibodies and completely protect chickens from challenge with the novel H7N9 or highly pathogenic H5N1 viruses, respectively. Notably, a baculovirus expressed H7 protein failed to protect chicken from H7N9 infection.

  • 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.

  • 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.

  • 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.

  • 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.

  • 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.

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