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Jean-Yves Sgro
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Table of Contents for this page:

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  • Current Issue of Journal of Virology

    JVI Current Issue

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  • Interaction of Human Cytomegalovirus Tegument Proteins ppUL35 and ppUL35A with Sorting Nexin 5 Regulates Glycoprotein B (gpUL55) Localization [Structure and Assembly]

  • Human cytomegalovirus (HCMV) is a widespread human pathogen that causes asymptomatic infection in healthy individuals but poses a serious threat to immunocompromised patients. During the late phase of HCMV infection, the viral capsid is transported to the cytoplasmic viral assembly center (cVAC), where it is enclosed by the tegument protein layer and the viral envelope. The cVAC consists of circularly arranged vesicles from the trans-Golgi and endosomal networks. The HCMV gene UL35 encodes ppUL35 and its shorter form, ppUL35A. We have previously shown that the UL35 gene is involved in HCMV assembly, but it is unknown how UL35 proteins regulate viral assembly. Here we show that sorting nexin 5 (SNX5), a component of the retromer and part of the retrograde transport pathway, interacts with UL35 proteins. Expression of wild-type proteins but not mutants defective in SNX5 binding resulted in the cellular redistribution of the cation-independent mannose-6-phosphate receptor (CI-M6PR), indicating that UL35 proteins bind and negatively regulate SNX5 to modulate cellular transport pathways. Furthermore, binding of UL35 proteins to SNX5 was required for efficient viral replication and for transport of the most abundant HCMV glycoprotein B (gB; gpUL55) to the cVAC. These results indicate that ppUL35 and ppUL35A control the localization of the essential gB through the regulation of a retrograde transport pathway. Thus, this work is the first to define a molecular interaction between a tegument protein and a vesicular transport factor to regulate glycoprotein localization.

    IMPORTANCE Human cytomegalovirus is ubiquitously present in the healthy population, but reactivation or reinfection can cause serious, life-threatening infections in immunocompromised patients. For completion of its lytic cycle, human cytomegalovirus induces formation of an assembly center where mature virus particles are formed from multiple viral proteins. Viral glycoproteins use separate vesicular pathways for transport to the assembly center, which are incompletely understood. Our research identified a viral structural protein which affects the localization of one of the major glycoproteins. We could link this change in glycoprotein localization to an interaction of the structural protein with a cellular protein involved in regulation of vesicle transport. This increases our understanding of how the virus intersects into cellular regulatory pathways to enhance its own replication.

  • Downregulation of MicroRNA eca-mir-128 in Seminal Exosomes and Enhanced Expression of CXCL16 in the Stallion Reproductive Tract Are Associated with Long-Term Persistence of Equine Arteritis Virus [Pathogenesis and Immunity]

  • Equine arteritis virus (EAV) can establish long-term persistent infection in the reproductive tract of stallions and is shed in the semen. Previous studies showed that long-term persistence is associated with a specific allele of the CXCL16 gene (CXCL16S) and that persistent infection is maintained despite the presence of a local inflammatory and humoral and mucosal antibody responses. In this study, we demonstrated that equine seminal exosomes (SEs) are enriched in a small subset of microRNAs (miRNAs). Most importantly, we demonstrated that long-term EAV persistence is associated with the downregulation of an SE-associated miRNA (eca-mir-128) and with an enhanced expression of CXCL16 in the reproductive tract, a putative target of eca-mir-128. The findings presented here suggest that SE eca-mir-128 is implicated in the regulation of the CXCL16/CXCR6 axis in the reproductive tract of persistently infected stallions, a chemokine axis strongly implicated in EAV persistence. This is a novel finding and warrants further investigation to identify its specific mechanism in modulating the CXCL16/CXCR6 axis in the reproductive tract of the EAV long-term carrier stallion.

    IMPORTANCE Equine arteritis virus (EAV) has the ability to establish long-term persistent infection in the stallion reproductive tract and to be shed in semen, which jeopardizes its worldwide control. Currently, the molecular mechanisms of viral persistence are being unraveled, and these are essential for the development of effective therapeutics to eliminate persistent infection. Recently, it has been determined that long-term persistence is associated with a specific allele of the CXCL16 gene (CXCL16S) and is maintained despite induction of local inflammatory, humoral, and mucosal antibody responses. This study demonstrated that long-term persistence is associated with the downregulation of seminal exosome miRNA eca-mir-128 and enhanced expression of its putative target, CXCL16, in the reproductive tract. For the first time, this study suggests complex interactions between eca-mir-128 and cellular elements at the site of EAV persistence and implicates this miRNA in the regulation of the CXCL16/CXCR6 axis in the reproductive tract during long-term persistence.

  • Lactic Acid Downregulates Viral MicroRNA To Promote Epstein-Barr Virus-Immortalized B Lymphoblastic Cell Adhesion and Growth [Virus-Cell Interactions]

  • High plasma lactate is associated with poor prognosis of many malignancies, but its role in virally mediated cancer progression and underlying molecular mechanisms are unclear. Epstein-Barr virus (EBV), the first human oncogenic virus, causes several cancers, including B-cell lymphoma. Here, we report that lactate dehydrogenase A (LDH-A) expression and lactate production are elevated in EBV-immortalized B lymphoblastic cells, and lactic acid (LA; acidic lactate) at low concentration triggers EBV-infected B-cell adhesion, morphological changes, and proliferation in vitro and in vivo. Moreover, LA-induced responses of EBV-infected B cells uniquely occurs in viral latency type III, and it is dramatically associated with the inhibition of global viral microRNAs, particularly the miR-BHRF1 cluster, and the high expression of SMAD3, JUN, and COL1A genes. The introduction of miR-BHRF1-1 blocks the LA-induced effects of EBV-infected B cells. Thus, this may be a novel mechanism to explain EBV-immortalized B lymphoblastic cell malignancy in an LA microenvironment.

    IMPORTANCE The tumor microenvironment is complicated, and lactate, which is created by cell metabolism, contributes to an acidic microenvironment that facilitates cancer progression. However, how LA operates in virus-associated cancers is unclear. Thus, we studied how EBV (the first tumor virus identified in humans; it is associated with many cancers) upregulates the expression of LDH-A and lactate production in B lymphoma cells. Elevated LA induces adhesion and the growth of EBV-infected B cells by inhibiting viral microRNA transcription. Thus, we offer a novel understanding of how EBV utilizes an acidic microenvironment to promote cancer development.

  • Vaccinia Virus C9 Ankyrin Repeat/F-Box Protein Is a Newly Identified Antagonist of the Type I Interferon-Induced Antiviral State [Virus-Cell Interactions]

  • Type I interferons (IFNs) induce expression of more than 300 cellular genes that provide protection against viruses and other pathogens. For survival, viruses evolved defenses to prevent the IFN response or counteract the IFN-induced antiviral state. However, because viruses and cells coevolved, the dynamic relationship between virus and host is difficult to discern. In the present study, we demonstrated that vaccinia virus with a large deletion near the left end of the genome had a diminished ability to replicate in cells that had been pretreated with beta interferon (IFN-bbeta;), suggesting that one or more of the missing 17 open reading frames (ORFs) encode an antagonist of the IFN-induced antiviral state. By systematically deleting groups of ORFs and then individual ORFs, the C9L gene was shown to be required for IFN resistance. Replication of the C9L deletion mutant (vC9) was impaired in human cells that had been pretreated with IFN-bbeta;. Expression of viral early genes occurred, but subsequent events, including genome uncoating, genome replication, and postreplicative gene expression, were inhibited. Expression of the C9 protein occurred prior to genome replication, consistent with an early role in counteracting the IFN-induced antiviral state. C9 contains six ankyrin repeat motifs and a near C-terminal F-box. Mass spectrometry and immunoblotting identified host proteins that copurified with a functional epitope-tagged C9. The most abundant proteins were components of the SCF (CUL1, SKP1, F-box) and signalosome/deneddylation complexes, which interact with each other, suggesting a possible role in proteolysis of one or more interferon-induced proteins.

    IMPORTANCE Poxviruses comprise a family of large DNA viruses that replicate in the cytoplasm of vertebrate and insect hosts and cause human and zoonotic diseases. In most cases the primary infection is moderated by innate immune defenses. Vertebrates, including fish, amphibians, reptiles, birds, and mammals, all produce type I interferon homologs. In humans, interferon stimulates the synthesis of more than 300 proteins thought to have roles in host defense. Conversely, viruses have evolved means to thwart the host defenses. We are attempting to deconstruct the established virus-host relationship in order to better understand the molecular mechanisms involved. In the present study, we identified a vaccinia virus gene that prevents interferon-mediated inhibition of very early stages of viral replication and is conserved in orthopoxviruses. The viral protein was shown to interact with host proteins involved in proteolysis, suggesting that vaccinia virus may subvert the cellular apparatus for its own defense.

  • Unique Phenotypic Characteristics of Recently Transmitted HIV-1 Subtype C Envelope Glycoprotein gp120: Use of CXCR6 Coreceptor by Transmitted Founder Viruses [Vaccines and Antiviral Agents]

  • Adequate information on the precise molecular and biological composition of the viral strains that establish HIV infection in the human host will provide effective means of immunization against HIV infection. In an attempt to identify the transmitted founder (TF) virus and differentiate the biological properties and infectious potential of the TF virus from those of the population of the early transmitted viruses, 250 patient-derived gp120 envelope glycoproteins were cloned in pMN-K7-Luc-IRESs-Nefgp120 to obtain chimeric viruses. Samples were obtained from eight infants who had recently become infected with HIV through mother-to-child transmission (MTCT) and two adults who acquired infection through the heterosexual route and were in the chronic stage of infection. Among the 250 clones tested, 65 chimeric viruses were infectious, and all belonged to HIV-1 subtype C. The 65 clones were analyzed for molecular features of the envelope, per-infectious-particle infectivity, coreceptor tropism, drug sensitivity, and sensitivity to broadly neutralizing antibodies. Based on genotypic and phenotypic analysis of the viral clones, we identified 10 TF viruses from the eight infants. The TF viruses were characterized by shorter V1V2 regions, a reduced number of potential N-linked glycosylation sites, and a higher infectivity titer compared to the virus variants from the adults in the chronic stage of infection. CXCR6 coreceptor usage, in addition to that of the CCR5 coreceptor, which was used by all 65 chimeric viruses, was identified in 13 viruses. The sensitivity of the TF variants to maraviroc and a standard panel of neutralizing monoclonal antibodies (VRC01, PG09, PG16, and PGT121) was found to be much lower than that of the virus variants from the adults in the chronic stage of infection.

    IMPORTANCE Tremendous progress has been made during the last three and half decades of HIV research, but some significant gaps continue to exist. One of the frontier areas of HIV research which has not seen a breakthrough yet is vaccine research, which is because of the enormous genetic diversity of HIV-1 and the unique infectious fitness of the virus. Among the repertoire of viral variants, the virus that establishes successful infection (transmitted founder [TF] virus) has not been well characterized yet. An insight into the salient features of the TF virus would go a long way toward helping with the design of an effective vaccine against HIV. Here we studied the biological properties of recently transmitted viruses isolated from infants who acquired infection from the mother and have come up with unique characterizations for the TF virus that establishes infection in the human host.

  • Karyopherin Alpha 6 Is Required for Replication of Porcine Reproductive and Respiratory Syndrome Virus and Zika Virus [Virus-Cell Interactions]

  • Movement of macromolecules between the cytoplasm and the nucleus occurs through the nuclear pore complex (NPC). Karyopherins comprise a family of soluble transport factors facilitating the nucleocytoplasmic translocation of proteins through the NPC. In this study, we found that karyopherin aalpha;6 (KPNA6; also known as importin aalpha;7) was required for the optimal replication of porcine reproductive and respiratory syndrome virus (PRRSV) and Zika virus (ZIKV), which are positive-sense, single-stranded RNA viruses replicating in the cytoplasm. The KPNA6 protein level in virus-infected cells was much higher than that in mock-infected controls, whereas the KPNA6 transcript remains stable. Viral infection blocked the ubiquitin-proteasomal degradation of KPNA6, which led to an extension of the KPNA6 half-life and the elevation of the KPNA6 level in comparison to mock-infected cells. PRRSV nsp12 protein induced KPNA6 stabilization. KPNA6 silencing was detrimental to the replication of PRRSV, and KPNA6 knockout impaired ZIKV replication. Moreover, KPNA6 knockout blocked the nuclear translocation of PRRSV nsp1bbeta; but had a minimal effect on two other PRRSV proteins with nuclear localization. Exogenous restitution of KPNA6 expression in the KPNA6-knockout cells results in restoration of the nuclear translocation of PRRSV nsp1bbeta; and the replication of ZIKV. These results indicate that KPNA6 is an important cellular factor for the replication of PRRSV and ZIKV.

    IMPORTANCE Positive-sense, single-stranded RNA (+ssRNA) viruses replicate in the cytoplasm of infected cells. The roles of transport factors in the nucleocytoplasmic trafficking system for the replication of +ssRNA viruses are not known. In this study, we discovered that PRRSV and ZIKV viruses needed karyopherin aalpha;6 (KPNA6), one of the transport factors, to enhance the virus replication. Our data showed that viral infection induced an elevation of the KPNA6 protein level due to an extension of the KPNA6 half-life via viral interference of the ubiquitin-proteasomal degradation of KPNA6. Notably, KPNA6 silencing or knockout dramatically reduced the replication of PRRSV and ZIKV. PRRSV nsp1bbeta; depended on KPNA6 to translocate into the nucleus. In addition, exogenous restitution of KPNA6 expression in KPNA6-knockout cells led to the restoration of nsp1bbeta; nuclear translocation and ZIKV replication. These results reveal a new aspect in the virus-cell interaction and may facilitate the development of novel antiviral therapeutics.

  • Functional Role of N-Linked Glycosylation in Pseudorabies Virus Glycoprotein gH [Virus-Cell Interactions]

  • Many viral envelope proteins are modified by asparagine (N)-linked glycosylation, which can influence their structure, physicochemical properties, intracellular transport, and function. Here, we systematically analyzed the functional relevance of N-linked glycans in the alphaherpesvirus pseudorabies virus (PrV) glycoprotein H (gH), which is an essential component of the conserved core herpesvirus fusion machinery. Upon gD-mediated receptor binding, the heterodimeric complex of gH and gL activates gB to mediate fusion of the viral envelope with the host cell membrane for viral entry. gH contains five potential N-linked glycosylation sites at positions 77, 162, 542, 604, and 627, which were inactivated by conservative mutations (asparagine to glutamine) singly or in combination. The mutated proteins were tested for correct expression and fusion activity. Additionally, the mutated gH genes were inserted into the PrV genome for analysis of function during virus infection. Our results demonstrate that all five sites are glycosylated. Inactivation of the PrV-specific N77 or the conserved N627 resulted in significantly reduced in vitro fusion activity, delayed penetration kinetics, and smaller virus plaques. Moreover, substitution of N627 greatly affected transport of gH in transfected cells, resulting in endoplasmic reticulum (ER) retention and reduced surface expression. In contrast, mutation of N604, which is conserved in the Varicellovirus genus, resulted in enhanced in vitro fusion activity and viral cell-to-cell spread. These results demonstrate a role of the N-glycans in proper localization and function of PrV gH. However, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles.

    IMPORTANCE Herpesvirus infection requires fusion of the viral envelope with cellular membranes, which involves the conserved fusion machinery consisting of gB and the heterodimeric gH/gL complex. The bona fide fusion protein gB depends on the presence of the gH/gL complex for activation. Viral envelope glycoproteins, such as gH, usually contain N-glycans, which can have a strong impact on their folding, transport, and functions. Here, we systematically analyzed the functional relevance of all five predicted N-linked glycosylation sites in the alphaherpesvirus pseudorabies virus (PrV) gH. Despite the fact that mutation of specific sites affected gH transport, in vitro fusion activity, and cell-to-cell spread and resulted in delayed penetration kinetics, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles. Thus, our results demonstrate a modulatory but nonessential role of N-glycans for gH function.

  • The Superimposed Deubiquitination Effect of OTULIN and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Nsp11 Promotes Multiplication of PRRSV [Virus-Cell Interactions]

  • Linear ubiquitination plays an important role in the regulation of the immune response by regulating nuclear factor B (NF-B). The linear ubiquitination-specific deubiquitinase ovarian tumor domain deubiquitinase with linear linkage specificity (OTULIN) can control the immune signaling transduction pathway by restricting the Met1-linked ubiquitination process. In our study, the porcine OTLLIN gene was cloned and deubiquitin functions were detected in a porcine reproductive and respiratory syndrome virus (PRRSV)-infected-cell model. PRRSV infection promotes the expression of the OTULIN gene; in turn, overexpression of OTULIN contributes to PRRSV proliferation. There is negative regulation of innate immunity with OTULIN during viral infection. The cooperative effects of swine OTULIN and PRRSV Nsp11 potentiate the ability to reduce levels of cellular protein ubiquitin associated with innate immunity. Importantly, PRRSV Nsp11 recruits OTULIN through a nonenzymatic combination to enhance its ability to remove linear ubiquitination targeting NEMO, resulting in a superimposed effect that inhibits the production of type I interferons (IFNs). Our report presents a new model of virus utilization of the ubiquitin-protease system in vivo from the perspective of the viral proteins that interact with cell deubiquitination enzymes, providing new ideas for prevention and control of PRRSV.

    IMPORTANCE Deubiquitination effects of swine OTULIN were identified. The interaction between porcine OTULIN and PRRSV Nsp11 is dependent on the OTU domain. PRRSV Nsp11 recruits OTULIN through a nonenzymatic combination to promote removal of linear ubiquitination targeting NEMO, resulting in a superimposed effect that inhibits the production of type I IFNs.

  • Single-Cell Analysis of the Impact of Host Cell Heterogeneity on Infection with Foot-and-Mouth Disease Virus [Virus-Cell Interactions]

  • Viral infection and replication are affected by host cell heterogeneity, but the mechanisms underlying the effects remain unclear. Using single-cell analysis, we investigated the effects of host cell heterogeneity, including cell size, inclusion, and cell cycle, on foot-and-mouth disease virus (FMDV) infection (acute and persistent infections) and replication. We detected various viral genome replication levels in FMDV-infected cells. Large cells and cells with a high number of inclusions generated more viral RNA copies and viral protein and a higher proportion of infectious cells than other cells. Additionally, we found that the viral titer was 10- to 100-fold higher in cells in G2/M than those in other cell cycle phases and identified a strong correlation between cell size, inclusion, and cell cycle heterogeneity, which all affected the infection and replication of FMDV. Furthermore, we demonstrated that host cell heterogeneity influenced the adsorption of FMDV due to differences in the levels of FMDV integrin receptors expression. Collectively, these results further our understanding of the evolution of a virus in a single host cell.

    IMPORTANCE It is important to understand how host cell heterogeneity affects viral infection and replication. Using single-cell analysis, we found that viral genome replication levels exhibited dramatic variability in foot-and-mouth disease virus (FMDV)-infected cells. We also found a strong correlation between heterogeneity in cell size, inclusion number, and cell cycle status and that all of these characteristics affect the infection and replication of FMDV. Moreover, we found that host cell heterogeneity influenced the viral adsorption as differences in the levels of FMDV integrin receptors' expression. This study provided new ideas for the studies of correlation between FMDV infection mechanisms and host cells.

  • WDR5 Facilitates Human Cytomegalovirus Replication by Promoting Capsid Nuclear Egress [Virus-Cell Interactions]

  • WD repeat-containing protein 5 (WDR5) is essential for assembling the VISA-associated complex to induce a type I interferon antiviral response to Sendai virus infection. However, the roles of WDR5 in DNA virus infections are not well described. Here, we report that human cytomegalovirus exploits WDR5 to facilitate capsid nuclear egress. Overexpression of WDR5 in fibroblasts slightly enhanced the infectious virus yield. However, WDR5 knockdown dramatically reduced infectious virus titers with only a small decrease in viral genome replication or gene expression. Further investigation of late steps of viral replication found that WDR5 knockdown significantly impaired formation of the viral nuclear egress complex and induced substantially fewer infoldings of the inner nuclear membrane. In addition, fewer capsids were associated with these infoldings, and there were fewer capsids in the cytoplasm. Restoration of WDR5 partially reversed these effects. These results suggest that WDR5 knockdown impairs the nuclear egress of capsids, which in turn decreases virus titers. These findings reveal an important role for a host factor whose function(s) is usurped by a viral pathogen to promote efficient replication. Thus, WDR5 represents an interesting regulatory mechanism and a potential antiviral target.

    IMPORTANCE Human cytomegalovirus (HCMV) has a large (~235-kb) genome with over 170 open reading frames and exploits numerous cellular factors to facilitate its replication. HCMV infection increases protein levels of WD repeat-containing protein 5 (WDR5) during infection, overexpression of WDR5 enhances viral replication, and knockdown of WDR5 dramatically attenuates viral replication. Our results indicate that WDR5 promotes the nuclear egress of viral capsids, the depletion of WDR5 resulting in a significant decrease in production of infectious virions. This is the first report that WDR5 favors HCMV, a DNA virus, replication and highlights a novel target for antiviral therapy.

  • Correction for Anthony et al., "Cooperation between Strain-Specific and Broadly Neutralizing Responses Limited Viral Escape and Prolonged the Exposure of the Broadly Neutralizing Epitope" [Author Correction]

  • Adenovirus Infection of Human Enteroids Reveals Interferon Sensitivity and Preferential Infection of Goblet Cells [Virus-Cell Interactions]

  • Human adenoviruses (HAdV) are significant human pathogens. Although only a subset of HAdV serotypes commonly cause gastroenteritis in humans, most HAdV species replicate in the gastrointestinal tract. Knowledge of the complex interaction between HAdVs and the human intestinal epithelium has been limited by the lack of a suitable cell culture system containing relevant cell types. Recently, this need has been met by the stable and prolonged cultivation of primary intestinal epithelial cells as enteroids. Human enteroids have been used to reveal novel and interesting aspects of rotavirus, norovirus, and enterovirus replication, prompting us to explore their suitability for HAdV culture. We found that both prototype strains and clinical isolates of enteric and nonenteric HAdVs productively replicate in human enteroids. HAdV-5p, a respiratory pathogen, and HAdV-41p, an enteric pathogen, are both sensitive to type I and III interferons in human enteroid monolayers but not A549 cells. Interestingly, HAdV-5p, but not HAdV-41p, preferentially infected goblet cells. And, HAdV-5p but not HAdV-41p was potently neutralized by the enteric human alpha-defensin HD5. These studies highlight new facets of HAdV biology that are uniquely revealed by primary intestinal epithelial cell culture.

    IMPORTANCE Enteric adenoviruses are a significant cause of childhood gastroenteritis worldwide, yet our understanding of their unique biology is limited. Here we report robust replication of both prototype and clinical isolates of enteric and respiratory human adenoviruses in enteroids, a primary intestinal cell culture system. Recent studies have shown that other fastidious enteric viruses replicate in human enteroids. Therefore, human enteroids may provide a unified platform for culturing enteric viruses, potentially enabling isolation of a greater diversity of viruses from patients. Moreover, both the ability of interferon to restrict respiratory and enteric adenoviruses and a surprising preference of a respiratory serotype for goblet cells demonstrate the power of this culture system to uncover aspects of adenovirus biology that were previously unattainable with standard cell lines.

  • Correction for Chaumorcel et al., "The Human Cytomegalovirus Protein TRS1 Inhibits Autophagy via Its Interaction with Beclin 1" [Author Correction]

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

  • Differential Contribution of RNA Interference Components in Response to Distinct Fusarium graminearum Virus Infections [Virus-Cell Interactions]

  • The mechanisms of RNA interference (RNAi) as a defense response against viruses remain unclear in many plant-pathogenic fungi. In this study, we used reverse genetics and virus-derived small RNA profiling to investigate the contributions of RNAi components to the antiviral response against Fusarium graminearum viruses 1 to 3 (FgV1, -2, and -3). Real-time reverse transcription-quantitative PCR (qRT-PCR) indicated that infection of Fusarium graminearum by FgV1, -2, or -3 differentially induces the gene expression of RNAi components in F. graminearum. Transcripts of the DICER-2 and AGO-1 genes of F. graminearum (FgDICER-2 and FgAGO-1) accumulated at lower levels following FgV1 infection than following FgV2 or FgV3 infection. We constructed gene disruption and overexpression mutants for each of the Argonaute and dicer genes and for two RNA-dependent RNA polymerase (RdRP) genes and generated virus-infected strains of each mutant. Interestingly, mycelial growth was significantly faster for the FgV1-infected FgAGO-1 overexpression mutant than for the FgV1-infected wild type, while neither FgV2 nor FgV3 infection altered the colony morphology of the gene deletion and overexpression mutants. FgV1 RNA accumulation was significantly decreased in the FgAGO-1 overexpression mutant. Furthermore, the levels of induction of FgAGO-1, FgDICER-2, and some of the FgRdRP genes caused by FgV2 and FgV3 infection were similar to those caused by hairpin RNA-induced gene silencing. Using small RNA sequencing analysis, we documented different patterns of virus-derived small interfering RNA (vsiRNA) production in strains infected with FgV1, -2, and -3. Our results suggest that the Argonaute protein encoded by FgAGO-1 is required for RNAi in F. graminearum, that FgAGO-1 induction differs in response to FgV1, -2, and -3, and that FgAGO-1 might contribute to the accumulation of vsiRNAs in FgV1-infected F. graminearum.

    IMPORTANCE To increase our understanding of how RNAi components in Fusarium graminearum react to mycovirus infections, we characterized the role(s) of RNAi components involved in the antiviral defense response against Fusarium graminearum viruses (FgVs). We observed differences in the levels of induction of RNA silencing-related genes, including FgDICER-2 and FgAGO-1, in response to infection by three different FgVs. FgAGO-1 can efficiently induce a robust RNAi response against FgV1 infection, but FgDICER genes might be relatively redundant to FgAGO-1 with respect to antiviral defense. However, the contribution of this gene in the response to the other FgV infections might be small. Compared to previous studies of Cryphonectria parasitica, which showed dicer-like protein 2 and Argonaute-like protein 2 to be important in antiviral RNA silencing, our results showed that F. graminearum developed a more complex and robust RNA silencing system against mycoviruses and that FgDICER-1 and FgDICER-2 and FgAGO-1 and FgAGO-2 had redundant roles in antiviral RNA silencing.

  • Reduced Chronic Lymphocyte Activation following Interferon Alpha Blockade during the Acute Phase of Simian Immunodeficiency Virus Infection in Rhesus Macaques [Pathogenesis and Immunity]

  • Pathogenic human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection of humans and rhesus macaques (RMs) induces persistently high production of type I interferon (IFN-I), which is thought to contribute to disease progression. To elucidate the specific role of interferon alpha (IFN-aalpha;) in SIV pathogenesis, 12 RMs were treated prior to intravenous (i.v.) SIVmac239 infection with a high or a low dose of an antibody (AGS-009) that neutralizes most IFN-aalpha; subtypes and were compared with six mock-infused, SIV-infected controls. Plasma viremia was measured postinfection to assess the effect of IFN-aalpha; blockade on virus replication, and peripheral blood and lymphoid tissue samples were analyzed by immunophenotypic staining. Consistent with the known antiviral effect of IFN-I, high-dose AGS-009 treatment induced a modest increase in acute-phase viral loads versus controls. Four out of 6 RMs receiving a high dose of AGS-009 also experienced an early decline in CD4+ T cell counts that was associated with progression to AIDS. Interestingly, 50% of the animals treated with AGS-009 (6/12) developed AIDS within 1 year of infection compared with 17% (1/6) of untreated controls. Finally, blockade of IFN-aalpha; decreased the levels of activated CD4+ and CD8+ T cells, as well as B cells, as measured by PD-1 and/or Ki67 expression. The lower levels of activated lymphocytes in IFN-aalpha;-blockaded animals supports the hypothesis that IFN-aalpha; signaling contributes to lymphocyte activation during SIV infection and suggests that this signaling pathway is involved in controlling virus replication during acute infection. The potential anti-inflammatory effect of IFN-aalpha; blockade should be explored as a strategy to reduce immune activation in HIV-infected individuals.

    IMPORTANCE Interferon alpha (IFN-aalpha;) is a member of a family of molecules (type I interferons) that prevent or limit virus infections in mammals. However, IFN-aalpha; production may contribute to the chronic immune activation that is thought to be the primary cause of immune decline and AIDS in HIV-infected patients. The study presented here attempts to understand the contribution of IFN-aalpha; to the natural history and progression of SIV infection of rhesus macaques, the primary nonhuman primate model system for testing hypotheses about HIV infection in humans. Here, we show that blockade of IFN-aalpha; action promotes lower chronic immune activation but higher early viral loads, with a trend toward faster disease progression. This study has significant implications for new treatments designed to impact the type I interferon system.

  • Formation of RNA Granule-Derived Capsid Assembly Intermediates Appears To Be Conserved between Human Immunodeficiency Virus Type 1 and the Nonprimate Lentivirus Feline Immunodeficiency Virus [Structure and Assembly]

  • During immature capsid assembly in cells, human immunodeficiency virus type 1 (HIV-1) Gag co-opts a host RNA granule, forming a pathway of intracellular assembly intermediates containing host components, including two cellular facilitators of assembly, ABCE1 and DDX6. A similar assembly pathway has been observed for other primate lentiviruses. Here we asked whether feline immunodeficiency virus (FIV), a nonprimate lentivirus, also forms RNA granule-derived capsid assembly intermediates. First, we showed that the released FIV immature capsid and a large FIV Gag-containing intracellular complex are unstable during analysis, unlike for HIV-1. We identified harvest conditions, including in situ cross-linking, that overcame this problem, revealing a series of FIV Gag-containing complexes corresponding in size to HIV-1 assembly intermediates. Previously, we showed that assembly-defective HIV-1 Gag mutants are arrested at specific assembly intermediates; here we identified four assembly-defective FIV Gag mutants, including three not previously studied, and demonstrated that they appear to be arrested at the same intermediate as the cognate HIV-1 mutants. Further evidence that these FIV Gag-containing complexes correspond to assembly intermediates came from coimmunoprecipitations demonstrating that endogenous ABCE1 and the RNA granule protein DDX6 are associated with FIV Gag, as shown previously for HIV-1 Gag, but are not associated with a ribosomal protein, at steady state. Additionally, we showed that FIV Gag associates with another RNA granule protein, DCP2. Finally, we validated the FIV Gag-ABCE1 and FIV Gag-DCP2 interactions with proximity ligation assays demonstrating colocalization in situ. Together, these data support a model in which primate and nonprimate lentiviruses form intracellular capsid assembly intermediates derived from nontranslating host RNA granules.

    IMPORTANCE Like HIV-1 Gag, FIV Gag assembles into immature capsids; however, it is not known whether FIV Gag progresses through a pathway of immature capsid assembly intermediates derived from host RNA granules, as shown for HIV-1 Gag. Here we showed that FIV Gag forms complexes that resemble HIV-1 capsid assembly intermediates in size and in their association with ABCE1 and DDX6, two host facilitators of HIV-1 immature capsid assembly that are found in HIV-1 assembly intermediates. Our studies also showed that known and novel assembly-defective FIV Gag mutants fail to progress past putative intermediates in a pattern resembling that observed for HIV-1 Gag mutants. Finally, we used imaging to demonstrate colocalization of FIV Gag with ABCE1 and with the RNA granule protein DCP2. Thus, we conclude that formation of assembly intermediates derived from host RNA granules is likely conserved between primate and nonprimate lentiviruses and could provide targets for future antiviral strategies.

  • Long Noncoding RNA uc002yug.2 Activates HIV-1 Latency through Regulation of mRNA Levels of Various RUNX1 Isoforms and Increased Tat Expression [Virus-Cell Interactions]

  • The HIV-1 reservoir is a major obstacle to complete eradication of the virus. Although many proteins and RNAs have been characterized as regulators in HIV-1/AIDS pathogenesis and latency, only a few long noncoding RNAs (lncRNAs) have been shown to be closely associated with HIV-1 replication and latency. In this study, we demonstrated that lncRNA uc002yug.2 plays a key role in HIV-1 replication and latency. uc002yug.2 potentially enhances HIV-1 replication, long terminal repeat (LTR) activity, and the activation of latent HIV-1 in both cell lines and CD4+ T cells from patients. Further investigation revealed that uc002yug.2 activates latent HIV-1 through downregulating RUNX1b and -1c and upregulating Tat protein expression. The accumulated evidence supports our model that the Tat protein has the key role in the uc002yug.2-mediated regulatory effect on HIV-1 reactivation. Moreover, uc002yug.2 showed an ability to activate HIV-1 similar to that of suberoylanilide hydroxamic acid or phorbol 12-myristate 13-acetate using latently infected cell models. These findings improve our understanding of lncRNA regulation of HIV-1 replication and latency, providing new insights into potential targeted therapeutic interventions.

    IMPORTANCE The latent viral reservoir is the primary obstacle to curing HIV-1 disease. To date, only a few lncRNAs, which play major roles in various biological processes, including viral infection, have been identified as regulators in HIV-1 latency. In this study, we demonstrated that lncRNA uc002yug.2 is important for both HIV-1 replication and activation of latent viruses. Moreover, uc002yug.2 was shown to activate latent HIV-1 through regulating alternative splicing of RUNX1 and increasing the expression of Tat protein. These findings highlight the potential merit of targeting lncRNA uc002yug.2 as an activating agent for latent HIV-1.

  • Maraviroc Is Associated with Latent HIV-1 Reactivation through NF-{kappa}B Activation in Resting CD4+ T Cells from HIV-Infected Individuals on Suppressive Antiretroviral Therapy [Pathogenesis and Immunity]

  • Maraviroc is a CCR5 antagonist used in the treatment of HIV-1 infection. We and others have suggested that maraviroc could reactivate latent HIV-1. To test the latency-reversing potential of maraviroc and the mechanisms involved, we performed a phase II, single-center, open-label study in which maraviroc was administered for 10 days to 20 HIV-1-infected individuals on suppressive antiretroviral therapy (EudraCT registration no. 2012-003215-66). All patients completed full maraviroc dosing and follow-up. The primary endpoint was to study whether maraviroc may reactivate HIV-1 latency, eliciting signaling pathways involved in the viral reactivation. An increase in HIV-1 transcription in resting CD4+ T cells, estimated by levels of HIV-1 unspliced RNA, was observed. Moreover, activation of the NF-B transcription factor was observed in these cells. To elucidate the mechanism of NF-B activation by maraviroc, we have evaluated in HeLa P4 C5 cells, which stably express CCR5, whether maraviroc could be acting as a partial CCR5 agonist, with no other mechanisms or pathways involved. Our results show that maraviroc can induce NF-B activity and that NF-B targets gene expression by CCR5 binding, since the use of TAK779, a CCR5 inhibitor, blocked NF-B activation and functionality. Taking the results together, we show that maraviroc may have a role in the activation of latent virus transcription through the activation of NF-B as a result of binding CCR5. Our results strongly support a novel use of maraviroc as a potential latency reversal agent in HIV-1-infected patients.

    IMPORTANCE HIV-1 persistence in a small pool of long-lived latently infected resting CD4+ T cells is a major barrier to viral eradication in HIV-1-infected patients on antiretroviral therapy. A potential strategy to cure HIV-1-infection is the use of latency-reversing agents to eliminate the reservoirs established in resting CD4+ T cells. As no drug has been shown to be completely effective so far, the search for new drugs and combinations remains a priority for HIV cure. We examined the ability of maraviroc, a CCR5 antagonist used as an antiretroviral drug, to activate latent HIV-1 in infected individuals on antiretroviral therapy. The study showed that maraviroc can activate NF-B and, subsequently, induce latent HIV-1-transcription in resting CD4+ T cells from HIV-1-infected individuals on suppressive antiretroviral therapy. Additional interventions will be needed to eliminate latent HIV-1 infection. Our results suggest that maraviroc may be a new latency-reversing agent to interfere with HIV-1 persistence during antiretroviral therapy.

  • Differential Delivery of Genomic Double-Stranded RNA Causes Reovirus Strain-Specific Differences in Interferon Regulatory Factor 3 Activation [Cellular Response to Infection]

  • Serotype 3 (T3) reoviruses induce substantially more type 1 interferon (IFN-I) secretion than serotype 1 (T1) strains. However, the mechanisms underlying differences in IFN-I production between T1 and T3 reoviruses remain undefined. Here, we found that differences in IFN-I production between T1 and T3 reoviruses correlate with activation of interferon regulatory factor 3 (IRF3), a key transcription factor for the production of IFN-I. T3 strain rsT3D activated IRF3 more rapidly and to a greater extent than the T1 strain rsT1L, in simian virus 40 (SV40) immortalized endothelial cells (SVECs). Differences in IRF3 activation between rsT1L and rsT3D were observed in the first hours of infection and were independent of de novo viral RNA and protein synthesis. NF-B activation mirrored IRF3 activation, with rsT3D inducing more NF-B activity than rsT1L. We also found that IRF3 and NF-B are activated in a mitochondrial antiviral-signaling protein (MAVS)-dependent manner. rsT1L does not suppress IRF3 activation, as IRF3 phosphorylation could be induced in rsT1L-infected cells. Transfected rsT1L and rsT3D RNA induced IRF3 phosphorylation, indicating that genomic RNA from both strains has the capacity to activate IRF3. Finally, bypassing the normal route of reovirus entry by transfecting in vitro-generated viral cores revealed that rsT1L and rsT3D core particles induced equivalent IRF3 activation. Taken together, our findings indicate that entry-related events that occur after outer capsid disassembly, but prior to deposition of viral cores into the cytoplasm, influence the efficiency of IFN-I responses to reovirus. This work provides further insight into mechanisms by which nonenveloped viruses activate innate immune responses.

    IMPORTANCE Detection of viral nucleic acids by the host cell triggers type 1 interferon (IFN-I) responses, which are critical for containing and clearing viral infections. Viral RNA is sensed in the cytoplasm by cellular receptors that initiate signaling pathways, leading to the activation of interferon regulatory factor 3 (IRF3) and NF-B, key transcription factors required for IFN-I induction. Serotype 3 (T3) reoviruses induce significantly more IFN-I than serotype 1 (T1) strains. In this work, we found that differences in IFN-I production by T1 and T3 reoviruses correlate with differential IRF3 activation. Differences in IRF3 activation are not caused by a blockade of the IRF3 activation by a T1 strain. Rather, differences in events during the late stages of viral entry determine the capacity of reovirus to activate host IFN-I responses. Together, our work provides insight into mechanisms of IFN-I induction by nonenveloped viruses.

  • Dynamics of Evolution of Poliovirus Neutralizing Antigenic Sites and Other Capsid Functional Domains during a Large and Prolonged Outbreak [Genetic Diversity and Evolution]

  • We followed the dynamics of capsid amino acid replacement among 403 Nigerian outbreak isolates of type 2 circulating vaccine-derived poliovirus (cVDPV2) from 2005 through 2011. Four different functional domains were analyzed: (i) neutralizing antigenic (NAg) sites, (ii) residues binding the poliovirus receptor (PVR), (iii) VP1 residues 1 to 32, and (iv) the capsid structural core. Amino acid replacements mapped to 37 of 43 positions across all 4 NAg sites; the most variable and polymorphic residues were in NAg sites 2 and 3b. The most divergent of the 120 NAg variants had no more than 5 replacements in all NAg sites and were still neutralized at titers similar to those of Sabin 2. PVR-binding residues were less variable (25 different variants; 0 to 2 replacements per isolate; 30/44 invariant positions), with the most variable residues also forming parts of NAg sites 2 and 3a. Residues 1 to 32 of VP1 were highly variable (133 different variants; 0 to 6 replacements per isolate; 5/32 invariant positions), with residues 1 to 18 predicted to form a well-conserved amphipathic helix. Replacement events were dated by mapping them onto the branches of time-scaled phylogenies. Rates of amino acid replacement varied widely across positions and followed no simple substitution model. Replacements in the structural core were the most conservative and were fixed at an overall rate ~20-fold lower than the rates for the NAg sites and VP1 1 to 32 and ~5-fold lower than the rate for the PVR-binding sites. Only VP1 143-Ile, a non-NAg site surface residue and known attenuation site, appeared to be under strong negative selection.

    IMPORTANCE The high rate of poliovirus evolution is offset by strong selection against amino acid replacement at most positions of the capsid. Consequently, poliovirus vaccines developed from strains isolated decades ago have been used worldwide to bring wild polioviruses almost to extinction. The apparent antigenic stability of poliovirus obscures a dynamic of continuous change within the neutralizing antigenic (NAg) sites. During 7 years of a large outbreak in Nigeria, the circulating type 2 vaccine-derived polioviruses generated 120 different NAg site variants via multiple independent pathways. Nonetheless, overall antigenic evolution was constrained, as no isolate had fixed more than 5 amino acid differences from the Sabin 2 NAg sites, and the most divergent isolates were efficiently neutralized by human immune sera. Evolution elsewhere in the capsid was also constrained. Amino acids binding the poliovirus receptor were strongly conserved, and extensive variation in the VP1 amino terminus still conserved a predicted amphipathic helix.

  • Intranasal Live Influenza Vaccine Priming Elicits Localized B Cell Responses in Mediastinal Lymph Nodes [Vaccines and Antiviral Agents]

  • Pandemic live attenuated influenza vaccines (pLAIV) prime subjects for a robust neutralizing antibody response upon subsequent administration of a pandemic inactivated subunit vaccine (pISV). However, a difference was not detected in H5-specific memory B cells in the peripheral blood between pLAIV-primed and unprimed subjects prior to pISV boost. To investigate the mechanism underlying pLAIV priming, we vaccinated groups of 12 African green monkeys (AGMs) with H5N1 pISV or pLAIV alone or H5N1 pLAIV followed by pISV and examined immunity systemically and in local draining lymph nodes (LN). The AGM model recapitulated the serologic observations from clinical studies. Interestingly, H5N1 pLAIV induced robust germinal center B cell responses in the mediastinal LN (MLN). Subsequent boosting with H5N1 pISV drove increases in H5-specific B cells in the axillary LN, spleen, and circulation in H5N1 pLAIV-primed animals. Thus, H5N1 pLAIV primes localized B cell responses in the MLN that are recalled systemically following pISV boost. These data provide mechanistic insights for the generation of robust humoral responses via prime-boost vaccination.

    IMPORTANCE We have previously shown that pandemic live attenuated influenza vaccines (pLAIV) prime for a rapid and robust antibody response on subsequent administration of inactivated subunit vaccine (pISV). This is observed even in individuals who had undetectable antibody (Ab) responses following the initial vaccination. To define the mechanistic basis of pLAIV priming, we turned to a nonhuman primate model and performed a detailed analysis of B cell responses in systemic and local lymphoid tissues following prime-boost vaccination with pLAIV and pISV. We show that the nonhuman primate model recapitulates the serologic observations from clinical studies. Further, we found that pLAIVs induced robust germinal center B cell responses in the mediastinal lymph node. Subsequent boosting with pISV in pLAIV-primed animals resulted in detection of B cells in the axillary lymph nodes, spleen, and peripheral blood. We demonstrate that intranasally administered pLAIV elicits a highly localized germinal center B cell response in the mediastinal lymph node that is rapidly recalled following pISV boost into germinal center reactions at numerous distant immune sites.

  • The 38K-Mediated Specific Dephosphorylation of the Viral Core Protein P6.9 Plays an Important Role in the Nucleocapsid Assembly of Autographa californica Multiple Nucleopolyhedrovirus [Structure and Assembly]

  • Encapsidation of the viral genomes, leading to the assembly of the nucleocapsids to form infectious progeny virions, is a key step in many virus life cycles. Baculovirus nucleocapsid assembly is a complex process that involves many proteins. Our previous studies showed that the deletion of the core gene 38K (ac98) interrupted the nucleocapsid assembly by producing capsid sheaths devoid of viral genomes by an unknown mechanism. All homologs of 38K contain conserved motifs of the haloacid dehalogenase superfamily, which are involved in phosphoryl transfer. The requirements of these motifs for nucleocapsid assembly, confirmed in the present study, suggest that 38K may be a functioning haloacid dehalogenase. P6.9 is also encoded by a core gene (ac100) and is required for viral genome encapsidation. It has been reported that multiple phosphorylated species of P6.9 are present in virus-infected cells, while only an unphosphorylated species is detected in the budded virus. Therefore, whether 38K mediates the dephosphorylation of P6.9 was investigated. An additional phosphorylated species of P6.9 in 38K-deleted or -mutated virus-transfected cells was detected, and the dephosphorylated sites mediated by 38K were determined by mass spectrometry. To assess the effects of dephosphorylation of P6.9 mediated by 38K on virus replication, these sites were mutated to glutamic acids (phosphorylation-mimic mutant) or to alanines (phosphorylation-deficient mutant). Studies showed that the nucleocapsid assembly was interrupted in phosphorylation-mimic mutant virus-transfected cells. Taken together, our findings demonstrate that 38K mediates the dephosphorylation of specific sites at the C terminus of P6.9, which is essential for viral genome encapsidation.

    IMPORTANCE Genome packaging is a fundamental process in the virus life cycle, and viruses have different strategies to perform this step. For several double-stranded DNA (dsDNA) viruses, the procapsid is formed before genome encapsidation, which may require basic proteins that help to neutralize the nucleic acid charge repulsion to facilitate the compaction of the genome within the confined capsid space. Baculovirus encodes a small basic protein, P6.9, which is required for a variety of processes in the virus infection cycle. The phosphorylation of P6.9 is thought to result in nucleocapsid uncoating, while the dephosphorylation of P6.9 is involved in viral DNA encapsidation during nucleocapsid assembly. Here, we demonstrate that a haloacid dehalogenase homolog encoded by baculovirus core gene 38K is involved in nucleocapsid assembly by mediating the dephosphorylation of 5 specific sites at the C terminus of P6.9. This finding contributes to the understanding of the mechanisms of virus nucleocapsid assembly.

  • Hepatitis C Virus core+1/ARF Protein Modulates the Cyclin D1/pRb Pathway and Promotes Carcinogenesis [Virus-Cell Interactions]

  • Viruses often encompass overlapping reading frames and unconventional translation mechanisms in order to maximize the output from a minimum genome and to orchestrate their timely gene expression. Hepatitis C virus (HCV) possesses such an unconventional open reading frame (ORF) within the core-coding region, encoding an additional protein, initially designated ARFP, F, or core+1. Two predominant isoforms of core+1/ARFP have been reported, core+1/L, initiating from codon 26, and core+1/S, initiating from codons 85/87 of the polyprotein coding region. The biological significance of core+1/ARFP expression remains elusive. The aim of the present study was to gain insight into the functional and pathological properties of core+1/ARFP through its interaction with the host cell, combining in vitro and in vivo approaches. Our data provide strong evidence that the core+1/ARFP of HCV-1a stimulates cell proliferation in Huh7-based cell lines expressing either core+1/S or core+1/L isoforms and in transgenic liver disease mouse models expressing core+1/S protein in a liver-specific manner. Both isoforms of core+1/ARFP increase the levels of cyclin D1 and phosphorylated Rb, thus promoting the cell cycle. In addition, core+1/S was found to enhance liver regeneration and oncogenesis in transgenic mice. The induction of the cell cycle together with increased mRNA levels of cell proliferation-related oncogenes in cells expressing the core+1/ARFP proteins argue for an oncogenic potential of these proteins and an important role in HCV-associated pathogenesis.

    IMPORTANCE This study sheds light on the biological importance of a unique HCV protein. We show here that core+1/ARFP of HCV-1a interacts with the host machinery, leading to acceleration of the cell cycle and enhancement of liver carcinogenesis. This pathological mechanism(s) may complement the action of other viral proteins with oncogenic properties, leading to the development of hepatocellular carcinoma. In addition, given that immunological responses to core+1/ARFP have been correlated with liver disease severity in chronic HCV patients, we expect that the present work will assist in clarifying the pathophysiological relevance of this protein as a biomarker of disease progression.

  • HAVCR1 (CD365) and Its Mouse Ortholog Are Functional Hepatitis A Virus (HAV) Cellular Receptors That Mediate HAV Infection [Virus-Cell Interactions]

  • The hepatitis A virus (HAV) cellular receptor 1 (HAVCR1), classified as CD365, was initially discovered as an HAV cellular receptor using an expression cloning strategy. Due to the lack of HAV receptor-negative replication-competent cells, it was not possible to fully prove that HAVCR1 was a functional HAV receptor. However, biochemistry, classical virology, and epidemiology studies further supported the functional role of HAVCR1 as an HAV receptor. Here, we show that an anti-HAVCR1 monoclonal antibody that protected African green monkey kidney (AGMK) cells against HAV infection only partially protected monkey Vero E6 cells and human hepatoma Huh7 cells, indicating that these two cell lines express alternative yet unidentified HAV receptors. Therefore, we focused our work on AGMK cells to further characterize the function of HAVCR1 as an HAV receptor. Advances in clustered regularly interspaced short palindromic repeat/Cas9 technology allowed us to knock out the monkey ortholog of HAVCR1 in AGMK cells. The resulting AGMK HAVCR1 knockout (KO) cells lost susceptibility to HAV infection, including HAV-free viral particles (vpHAV) and exosomes purified from HAV-infected cells (exo-HAV). Transfection of HAVCR1 cDNA into AGMK HAVCR1 KO cells restored susceptibility to vpHAV and exo-HAV infection. Furthermore, transfection of the mouse ortholog of HAVCR1, mHavcr1, also restored the susceptibility of AGMK HAVCR1 KO cells to HAV infection. Taken together, our data clearly show that HAVCR1 and mHavcr1 are functional HAV receptors that mediate HAV infection. This work paves the way for the identification of alternative HAV receptors to gain a complete understanding of their interplay with HAVCR1 in the cell entry and pathogenic processes of HAV.

    IMPORTANCE HAVCR1, an HAV receptor, is expressed in different cell types, including regulatory immune cells and antigen-presenting cells. How HAV evades the immune response during a long incubation period of up to 4 weeks and the mechanism by which the subsequent necroinflammatory process clears the infection remain a puzzle that most likely involves the HAV-HAVCR1 interaction. Based on negative data, a recent paper from the S. M. Lemon and W. Maury laboratories (A. Das, A. Hirai-Yuki, O. Gonzalez-Lopez, B. Rhein, S. Moller-Tank, R. Brouillette, L. Hensley, I. Misumi, W. Lovell, J. M. Cullen, J. K. Whitmire, W. Maury, and S. M. Lemon, mBio 8:e00969-17, 2017, https://doi.org/10.1128/mBio.00969-17) suggested that HAVCR1 is not a functional HAV receptor, nor it is it required for HAV infection. However, our data, based on regain of the HAV receptor function in HAVCR1 knockout cells transfected with HAVCR1 cDNA, disagree with their findings. Our positive data show conclusively that HAVCR1 is indeed a functional HAV receptor and lays the ground for the identification of alternative HAV receptors and how they interact with HAVCR1 in cell entry and the pathogenesis of HAV.

  • Escape of Hepatitis C Virus from Epitope I Neutralization Increases Sensitivity of Other Neutralization Epitopes [Pathogenesis and Immunity]

  • The hepatitis C virus (HCV) E2 glycoprotein is a major target of the neutralizing antibody (nAb) response, with multiple type-specific and broadly neutralizing antibody (bnAb) epitopes identified. The 412-to-423 region can generate bnAbs that block interaction with the cell surface receptor CD81, with activity toward multiple HCV genotypes. In this study, we reveal the structure of rodent monoclonal antibody 24 (MAb24) with an extensive contact area toward a peptide spanning the 412-to-423 region. The crystal structure of the MAb24nndash;peptide 412-to-423 complex reveals the paratope bound to a peptide hairpin highly similar to that observed with human MAb HCV1 and rodent MAb AP33, but with a different angle of approach. In viral outgrowth experiments, we demonstrated three distinct genotype 2a viral populations that acquired resistance to MAb24 via N415D, N417S, and N415D/H386R mutations. Importantly, the MAb24-resistant viruses exhibited significant increases in sensitivity to the majority of bnAbs directed to epitopes within the 412-to-423 region and in additional antigenic determinants located within E2 and the E1E2 complex. This study suggests that modification of N415 causes a global change in glycoprotein structure that increases its vulnerability to neutralization by other antibodies. This finding suggests that in the context of an antibody response to viral infection, acquisition of escape mutations in the 412-to-423 region renders the virus more susceptible to neutralization by other specificities of nAbs, effectively reducing the immunological fitness of the virus. A vaccine for HCV that generates polyspecific humoral immunity with specificity for the 412-to-423 region and at least one other region of E2 is desirable.

    IMPORTANCE Understanding how antibodies neutralize hepatitis C virus (HCV) is essential for vaccine development. This study reveals for the first time that when HCV develops resistance to a major class of bnAbs targeting the 412-to-423 region of E2, this results in a concomitant increase in sensitivity to neutralization by a majority of other bnAb specificities. Vaccines for the prevention of HCV infection should therefore generate bnAbs directed toward the 412-to-423 region of E2 and additional bnAb epitopes within the viral glycoproteins.

  • Epstein-Barr Virus Nuclear Antigen Leader Protein Coactivates EP300 [Virus-Cell Interactions]

  • Epstein-Barr virus nuclear antigen (EBNA) leader protein (EBNALP) is one of the first viral genes expressed upon B-cell infection. EBNALP is essential for EBV-mediated B-cell immortalization. EBNALP is thought to function primarily by coactivating EBNA2-mediated transcription. Chromatin immune precipitation followed by deep sequencing (ChIP-seq) studies highlight that EBNALP frequently cooccupies DNA sites with host cell transcription factors (TFs), in particular, EP300, implicating a broader role in transcription regulation. In this study, we investigated the mechanisms of EBNALP transcription coactivation through EP300. EBNALP greatly enhanced EP300 transcription activation when EP300 was tethered to a promoter. EBNALP coimmunoprecipitated endogenous EP300 from lymphoblastoid cell lines (LCLs). EBNALP W repeat serine residues 34, 36, and 63 were required for EP300 association and coactivation. Deletion of the EP300 histone acetyltransferase (HAT) domain greatly reduced EBNALP coactivation and abolished the EBNALP association. An EP300 bromodomain inhibitor also abolished EBNALP coactivation and blocked the EP300 association with EBNALP. EBNALP sites cooccupied by EP300 had significantly higher ChIP-seq signals for sequence-specific TFs, including SPI1, RelA, EBF1, IRF4, BATF, and PAX5. EBNALP- and EP300-cooccurring sites also had much higher H3K4me1 and H3K27ac signals, indicative of activated enhancers. EBNALP-only sites had much higher signals for DNA looping factors, including CTCF and RAD21. EBNALP coactivated reporters under the control of NF-B or SPI1. EP300 inhibition abolished EBNALP coactivation of these reporters. Clustered regularly interspaced short palindromic repeat interference targeting of EBNALP enhancer sites significantly reduced target gene expression, including that of EP300 itself. These data suggest a previously unrecognized mechanism by which EBNALP coactivates transcription through subverting of EP300 and thus affects the expression of LCL genes regulated by a broad range of host TFs.

    IMPORTANCE Epstein-Barr virus was the first human DNA tumor virus discovered over 50 years ago. EBV is causally linked to ~200,000 human malignancies annually. These cancers include endemic Burkitt lymphoma, Hodgkin lymphoma, lymphoma/lymphoproliferative disease in transplant recipients or HIV-infected people, nasopharyngeal carcinoma, and ~10% of gastric carcinoma cases. EBV-immortalized human B cells faithfully model key aspects of EBV lymphoproliferative diseases and are useful models of EBV oncogenesis. EBNALP is essential for EBV to transform B cells and transcriptionally coactivates EBNA2 by removing repressors from EBNA2-bound DNA sites. Here, we found that EBNALP can also modulate the activity of the key transcription activator EP300, an acetyltransferase that activates a broad range of transcription factors. Our data suggest that EBNALP regulates a much broader range of host genes than was previously appreciated. A small-molecule inhibitor of EP300 abolished EBNALP coactivation of multiple target genes. These findings suggest novel therapeutic approaches to control EBV-associated lymphoproliferative diseases.

  • Bovine Nebovirus Interacts with a Wide Spectrum of Histo-Blood Group Antigens [Virus-Cell Interactions]

  • Some viruses within the Caliciviridae family initiate their replication cycle by attachment to cell surface carbohydrate moieties, histo-blood group antigens (HBGAs), and/or terminal sialic acids (SAs). Although bovine nebovirus (BNeV), one of the enteric caliciviruses, is an important causative agent of acute gastroenteritis in cattle, its attachment factors and possibly other cellular receptors remain unknown. Using a comprehensive series of protein-ligand biochemical assays, we sought to determine whether BNeV recognizes cell surface HBGAs and/or SAs as attachment factors. It was found that BNeV virus-like particles (VLPs) bound to A type/H type 2/Ley HBGAs expressed in the bovine digestive tract and are related to HBGAs expressed in humans and other host species, suggesting a wide spectrum of HBGA recognition by BNeV. BNeV VLPs also bound to a large variety of different bovine and human saliva samples of all ABH and Lewis types, supporting previously obtained results and suggesting a zoonotic potential of BNeV transmission. Removal of aalpha;1,2-linked fucose and aalpha;1,3/4-linked fucose epitopes of target HBGAs by confirmation-specific enzymes reduced the binding of BNeV VLPs to synthetic HBGAs, bovine and human saliva, cultured cell lines, and bovine small intestine mucosa, further supporting a wide HBGA binding spectrum of BNeV through recognition of aalpha;1,2-linked fucose and aalpha;1,3/4-linked fucose epitopes of targeted HBGAs. However, removal of terminal aalpha;2,3- and aalpha;2,6-linked SAs by their specific enzyme had no inhibitory effects on binding of BNeV VLPs, indicating that BNeV does not use terminal SAs as attachment factors. Further details of the binding specificity of BNeV remain to be explored.

    IMPORTANCE Enteric caliciviruses such as noroviruses, sapoviruses, and recoviruses are the most important etiological agents of severe acute gastroenteritis in humans and many other mammalian host species. They initiate infection by attachment to cell surface carbohydrate moieties, HBGAs, and/or terminal SAs. However, the attachment factor(s) for BNeV, a recently classified enteric calicivirus genus/type species, remains unexplored. Here, we demonstrate that BNeV VLPs have a wide spectrum of binding to synthetic HBGAs, bovine and human saliva samples, and bovine duodenal sections. We further discovered that aalpha;1,2-linked fucose and aalpha;1,3/4-linked fucose epitopes are essential for binding of BNeV VLPs. However, BNeV VLPs do not bind to terminal SAs on cell carbohydrates. Continued investigation regarding the proteinaceous receptor(s) will be necessary for better understanding of the tropism, pathogenesis, and host range of this important viral genus.

  • Plasmid Partitioning by Human Tumor Viruses [Minireviews]

  • The human tumor viruses that replicate as plasmids (we use the term plasmid to avoid any confusion in the term episome, which was coined to mean DNA elements that occur both extrachromosomally and as integrated forms during their life cycles, as does phage lambda) share many features in their DNA synthesis. We know less about their mechanisms of maintenance in proliferating cells, but these mechanisms must underlie their partitioning to daughter cells. One amazing implication of how these viruses are thought to maintain themselves is that while host chromosomes commit themselves to partitioning in mitosis, these tumor viruses would commit themselves to partitioning before mitosis and probably in S phase shortly after their synthesis.

  • A Functional Link between RNA Replication and Virion Assembly in the Potyvirus Plum Pox Virus [Genome Replication and Regulation of Viral Gene Expression]

  • Accurate assembly of viral particles in the potyvirus Plum pox virus (PPV) has been shown to depend on the contribution of the multifunctional viral protein HCPro. In this study, we show that other viral factors, in addition to the capsid protein (CP) and HCPro, are necessary for the formation of stable PPV virions. The CP produced in Nicotiana benthamiana leaves from a subviral RNA termed LONG, which expresses a truncated polyprotein that lacks P1 and HCPro, together with HCPro supplied in trans, was assembled into virus-like particles and remained stable after in vitro incubation. In contrast, deletions in multiple regions of the LONG coding sequence prevented the CP stabilization mediated by HCPro. In particular, we demonstrated that the first 178 amino acids of P3, but not a specific nucleotide sequence coding for them, are required for CP stability and proper assembly of PPV particles. Using a sequential coagroinfiltration assay, we observed that the subviral LONG RNA replicates and locally spreads in N. benthamiana leaves expressing an RNA silencing suppressor. The analysis of the effect of both point and deletion mutations affecting RNA replication in LONG and full-length PPV demonstrated that this process is essential for the assembly of stable viral particles. Interestingly, in spite of this requirement, the CP produced by a nonreplicating viral RNA can be stably assembled into virions as long as it is coexpressed with a replication-proficient RNA. Altogether, these results highlight the importance of coupling encapsidation to other viral processes to secure a successful infection.

    IMPORTANCE Viruses of the family Potyviridae are among the most dangerous threats for basically every important crop, and such socioeconomical relevance has made them a subject of many research studies. In spite of this, very little is currently known about proteins and processes controlling viral genome encapsidation by the coat protein. In the case of Plum pox virus (genus Potyvirus), for instance, we have previously shown that the multitasking viral factor HCPro plays a role in the production of stable virions. Here, by using this potyvirus as a model, we move further to show that additional factors are also necessary for the efficient production of potyviral particles. More importantly, a comprehensive screening for such factors led us to the identification of a functional link between virus replication and packaging, unraveling a previously unknown connection of these two key events of the potyviral infection cycle.

  • A Prominent Role of the Human Cytomegalovirus UL8 Glycoprotein in Restraining Proinflammatory Cytokine Production by Myeloid Cells at Late Times during Infection [Virus-Cell Interactions]

  • Human cytomegalovirus (HCMV) persistence in infected individuals relies on a plethora of mechanisms to efficiently reduce host immune responses. To that end, HCMV uses a variety of gene products, some of which have not been identified yet. Here we characterized the UL8 gene, which consists of two exons, sharing the first with the HCMV RL11 family member UL7. UL8 is a transmembrane protein with an N-terminal immunoglobulin (Ig)-like domain in common with UL7 but with an extended stalk and a distinctive cytoplasmic tail. The UL8 open reading frame gives rise to a heavily glycosylated protein predominantly expressed on the cell surface, from where it can be partially endocytosed and subsequently degraded. Infections with UL8-tagged viruses indicated that UL8 was synthesized with late-phase kinetics. By virtue of its highly conserved Ig-like domain, this viral protein interacted with a surface molecule present on activated neutrophils. Notably, when ectopically expressed in THP-1 myeloid cells, UL8 was able to significantly reduce the production of a variety of proinflammatory cytokines. Mutations in UL8 indicated that this functional effect was mediated by the cell surface expression of its Ig-like domain. To investigate the impact of the viral protein in the infection context, we engineered HCMVs lacking the UL8 gene and demonstrated that UL8 decreases the release of a large number of proinflammatory factors at late times after infection of THP-1 cells. Our data indicate that UL8 may exert an immunosuppressive role key for HCMV survival in the host.

    IMPORTANCE HCMV is a major pathogen that causes life-threatening diseases and disabilities in infected newborns and immunocompromised individuals. Containing one of the largest genomes among all reported human viruses, HCMV encodes an impressive repertoire of gene products. However, the functions of a large proportion of them still remain unknown, a fact that complicates the design of new therapeutic approaches to prevent or treat HCMV-associated diseases. In this report, we have conducted an extensive study of UL8, one of the previously uncharacterized HCMV open reading frames. We found that the UL8 protein is expressed at late times postinfection and utilized by HCMV to reduce the production of proinflammatory factors by infected myeloid cells. Thus, the work presented here points to a key role of UL8 as a novel HCMV immune modulator capable of restraining host antiviral defenses.

  • Fibronectin Facilitates Enterovirus 71 Infection by Mediating Viral Entry [Virus-Cell Interactions]

  • Fibronectin (FN) is a high-molecular-weight extracellular matrix protein that contains the RGDS motif, which is required to bind to integrins. Synthetic RGDS peptides have been reported to compete with FN to bind to the cell surface and inhibit the function of FN. Here, we identified that synthetic RGDS peptides significantly inhibit human enterovirus 71 (EV71) infection in cell cultures. In addition, mice treated with RGDS peptides and infected with EV71 had a significantly higher survival rate and a lower viral load than the control group. Because RGDS peptides affect the function of FN, we questioned whether FN may play a role in virus infection. Our study indicates that overexpression of FN enhanced EV71 infection. In contrast, knockout of FN significantly reduced viral yield and decreased the viral binding to host cells. Furthermore, EV71 entry, rather than intracellular viral replication, was blocked by FN inhibitor pretreatment. Next, we found that FN could interact with the EV71 capsid protein VP1, and further truncated-mutation assays indicated that the D2 domain of FN could interact with the N-terminal fragment of VP1. Taken together, our results demonstrate that the host factor FN binds to EV71 particles and facilitates EV71 entry, providing a potential therapy target for EV71 infection.

    IMPORTANCE Hand, foot, and mouth disease outbreaks have occurred frequently in recent years, sometimes causing severe neurological complications and even death in infants and young children worldwide. Unfortunately, no effective antiviral drugs are available for human enterovirus 71 (EV71), one of the viruses that cause hand, foot, and mouth disease. The infection process and the host factors involved remain unknown, although several receptors have been identified. In this study, we found that the host factor fibronectin (FN) facilitated EV71 replication by interacting with EV71 particles and further mediated their entry. The RGDS peptide, an FN inhibitor, significantly inhibited EV71 replication in both RD cells and mice. In conclusion, our research identified a new host factor involved in EV71 infection, providing a new potential antiviral target for EV71 treatment.

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    JVI Accepts: Articles Published Ahead of Print

  • Hepatitis B virus core protein dephosphorylation occurs during pregenomic RNA encapsidation [PublishAheadOfPrint]

  • Hepatitis B virus (HBV) core protein consists of N-terminal assembly domain and C-terminal domain (CTD) with seven conserved serines or threonine that are dynamically phosphorylated/dephosphorylated during the viral replication cycle. Sulfamoylbenzaminde derivatives are small molecular core protein allosteric modulators (CpAMs) that bind to the HAP pocket between the core protein dimer-dimer interfaces. CpAM binding alters the kinetics and pathway of capsid assembly and can result in the formation of morphologically "normal" capsids devoid of viral pregenomic (pg) RNA and DNA polymerase. In order to investigate the mechanism underlying CpAM inhibition of pgRNA encapsidation, we developed an immunobloting assay that can resolve core protein based on its phosphorylation status and demonstrated, for the first time, that core protein is hyperphosphorylated in free dimers and empty capsids from both mock-treated and CpAM-treated cells, but is hypophosphorylated in pgRNA- and DNA-containing nucleocapsids. Interestingly, inhibition of pgRNA encapsidation by a heat shock protein (hsp) 90 inhibitor prevented core protein dephosphorylation. Moreover, core proteins with point mutations at wall of the HAP pocket, V124A or V124W, assembled empty capsids and nucleocapsids with altered phosphorylation status. The results thus suggest that core protein dephosphorylation occurs in the assembly of pgRNA and interference of the interaction between core protein subunits at dimer-dimer interfaces during nucleocapsid assembly not only alters capsid structure, but also core protein dephosphorylation. Hence, inhibition of pgRNA encapsidation by CpAMs might be due to disruption of core protein dephosphorylation during nucleocapsid assembly.

    IMPORTANCE Dynamic phosphorylation of HBV core protein regulates multiple steps of viral replication. However, the regulatory function was mainly investigated by phosphomimetic mutagenesis that disrupts the natural dynamics of core protein phosphorylation/dephosphorylation. Development of an immunobloting assay capable of resolving hyper- and hypo-phosphorylated core proteins allows to track the phosphorylation status of core proteins existing as free dimers and in the variety of intracellular capsids and investigate the role of core protein phosphorylation/dephosphorylation in viral replication. Herein, we found that disruption of core protein interaction at dimer-dimer interfaces during nucleocapsid assembly (by CpAMs or mutagenesis) inhibited core protein dephosphorylation and pgRNA packaging. Our work has thus revealed a novel function of core protein dephosphorylation in HBV replication and mechanism by which CpAMs, a class of compounds that are currently in clinical trials for treatment of chronic hepatitis B, induce the assembly of empty capsids.

  • Osteopontin regulates HCV replication and assembly via interaction with HCV proteins and lipid droplets and by binding to receptors, {alpha}V{beta}3 and CD44 [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) replication and assembly occur at the specialized site of endoplasmic reticulum (ER) membranes and lipid droplets (LDs), respectively. Recently, several host proteins have been shown to be involved in HCV replication and assembly. In the present study, we demonstrate the important relationship among osteopontin (OPN), ER and LDs. OPN is a secreted phosphoprotein and overexpression of OPN in hepatocellular carcinoma (HCC) tissue can lead to invasion and metastasis. OPN expression is also enhanced in HCV-associated HCC. Our recent studies have demonstrated the induction, proteolytic cleavage and secretion of OPN in response to HCV infection. We also defined the critical role of secreted OPN in human hepatoma cell migration and invasion through binding to receptors, integrin aalpha;Vbbeta;3 and CD44. However, the role of HCV-induced OPN in HCV life cycle has not been elucidated. In this study, we show a significant reduction in HCV replication, assembly and infectivity in HCV-infected cells transfected with siRNA against OPN, bbeta;3 and CD44. We also observed the association of endogenous OPN with HCV proteins (NS3, NS5A, NS4A/B, NS5B and core). Confocal microscopy revealed the colocalization of OPN with HCV NS5A and core in the ER and LDs, indicating the possible role of OPN in HCV replication and assembly. Interestingly, the secreted OPN activated HCV replication, infectivity and assembly through binding to aalpha;Vbbeta;3 and CD44. Collectively, these observations provide evidence that HCV-induced OPN is critical for HCV replication and assembly.

    IMPORTANCE

    Recently, our studies uncovered the critical role of HCV-induced endogenous and secreted OPN in migration and invasion of hepatocytes. However, the role of OPN in HCV life cycle has not been elucidated. In this study, we investigated the importance of OPN in HCV replication and assembly. We demonstrated whether endogenous OPN associates with HCV NS3, NS5A, NS5B and core proteins which are in the close proximity to ER and LDs. Moreover, we showed that the interactions of secreted OPN with cell surface receptors, aalpha;Vbbeta;3 and CD44 are critical for HCV replication and assembly. These observations provide evidence that HCV-induced endogenous and secreted OPN play pivotal role in HCV replication and assembly in HCV-infected cells. Taken together, our findings clearly demonstrate that targeting OPN may provide opportunities for therapeutic intervention of HCV pathogenesis.

  • Amino acid substitutions within HLA-B*27-restricted T cell epitopes prevent recognition by hepatitis delta virus-specific CD8+ T cells [PublishAheadOfPrint]

  • Virus-specific CD8 T-cell response seems to play a significant role in the outcome of hepatitis delta virus (HDV) infection. However, the HDV-specific T-cell epitope repertoire and mechanisms of CD8 T-cell failure in HDV infection have merely been characterized. We therefore aimed to characterize HDV-specific CD8 T cell epitopes and the impact of viral mutations on immune escape. In this study, we predicted peptide epitopes binding the most frequent HLA types and assessed their HLA binding capacity. These epitopes were characterized in HDV-infected patients by intracellular IFN- staining. Sequence analysis of L-HDAg and HLA typing were performed in 104 patients. The impact of substitutions within epitopes on CD8 T cell response was evaluated experimentally and by in silico studies. We identified two HLA-B*27-restricted CD8 T-cell epitopes within L-HDAg. These novel epitopes are located in a relatively conserved region of L-HDAg. However, we detected molecular footprints within these epitopes in HLA-B*27-positive patients with chronic HDV infection. The variant peptides were not cross-recognized in HLA-B*27-positive patients with resolved HDV infection, indicating these substitutions represent viral escape mutations. Molecular modeling of HLA-B*27 complexes with the L-HDAg epitope and its potential viral escape mutations indicates that the structural and electrostatic properties of the bound peptides differ considerably at the T-cell receptor interface, which provides a possible molecular explanation for the escape mechanism. This viral escape from the HLA-B*27-restricted CD8 T-cell response correlates to chronic outcome of hepatitis D infection. T-cell failure resulting from immune escape may contribute to the high chronicity rate in HDV infection.

    Importance Hepatitis D virus (HDV) causes severe chronic hepatitis which affects 20 million people worldwide. Only a small number of patients is able to clear the virus, possibly mediated by virus-specific T cell response. Here we performed a systematic screen to define CD8 epitopes and investigated the role of CD8 T cells in outcome of hepatitis D and how they fail in eliminating HDV. Overall the number of epitopes identified was very low as compared to other hepatotropic viruses. We identified, two HLA-B*27-restricted epitopes in patients with resolved infection. In HLA-B*27 positive patients with chronic HDV infection, however, we detected escape mutations within these identified epitopes which could lead to viral evasion from immune responses. These findings support the evidence that HLA-B*27 is important for viral-specific CD8 T cell responses, similar to other viral infections. These results have implications for the clinical prognosis of HDV infection and for vaccine development.

  • Comparative Analysis of UL16 Mutants Derived from Multiple Strains of HSV-2 and HSV-1 Reveals Species-Specific Requirements for the UL16 Protein [PublishAheadOfPrint]

  • Orthologs of the herpes simplex virus (HSV) UL16 gene are conserved throughout the Herpesviridae. Because of this conservation, one might expect that these proteins perform similar functions for all herpesviruses. Previous studies on a UL16 null mutant derived from HSV-2 strain 186 revealed a roughly 100-fold replication defect and a critical role for UL16 in the nuclear egress of capsids. These findings were in stark contrast to what has been observed with UL16 mutants of HSV-1 and pseudorabies virus where roughly 10-fold replication deficiencies were reported that were accompanied by defects in the secondary envelopment of cytoplasmic capsids. One possible explanation for this discrepancy is that the HSV-2 186 strain is not representative of the HSV-2 species. To address this possibility, multiple UL16 null mutants were constructed in multiple HSV-2 and HSV-1 strains by CRISPR/Cas9 mutagenesis and their phenotypes characterized side-by-side. This analysis showed that all the HSV-2 UL16 mutants had 50 to 100-fold replication deficiencies that were accompanied by defects in the nuclear egress of capsids as well as defects in the secondary envelopment of cytoplasmic capsids. By contrast, most HSV-1 UL16 mutants had 10-fold replication deficiencies that were accompanied by defects in secondary envelopment of cytoplasmic capsids. These findings indicated that UL16 has HSV species-specific functions. Interestingly, HSV-1 UL16 could promote the nuclear egress of HSV-2 UL16 null strains, suggesting that, unlike HSV-1, HSV-2 lacks an activity that can promote nuclear egress in the absence of UL16.

    IMPORTANCE HSV-2 and HSV-1 are important human pathogens that cause distinct diseases in their hosts. A complete understanding of the morphogenesis of these viruses is expected to reveal vulnerabilities that can be exploited in the treatment of HSV disease. UL16 is a virion structural component that is conserved throughout the Herpesviridae and functions in virus morphogenesis, however, previous studies have suggested different roles for UL16 in the morphogenesis of HSV-2 and HSV-1. This study sought to resolve this apparent discrepancy by analyzing multiple UL16 mutant viruses derived from multiple strains of HSV-2 and HSV-1. The data indicate that UL16 has HSV species-specific functions insofar as HSV-2 has a requirement for UL16 in the escape of capsids from the nucleus whereas both HSV-2 and HSV-1 require UL16 for final envelopment of capsids at cytoplasmic membranes.

  • Generation of recombinant rotaviruses expressing fluorescent proteins using an optimized reverse genetics system [PublishAheadOfPrint]

  • An entirely plasmid-based reverse genetics system for rotaviruses was established very recently. We improved the reverse genetics system to generate recombinant rotavirus by transfecting only 11 cDNA plasmids for its 11 gene segments under the condition of increasing the ratio of the cDNA plasmids for NSP2 and NSP5 genes. Utilizing this highly efficient system, we then engineered infectious recombinant rotaviruses expressing bioluminescent (NanoLuc luciferase) and fluorescent (EGFP and mCherry) reporters. These recombinant rotaviruses expressing reporters remained genetically stable during serial passages. Our reverse genetics approach and recombinant rotaviruses carrying reporter genes will be great additions to the tool kit for studying the molecular virology of rotavirus, and for developing future next-generation vaccines and expression vectors.

    IMPORTANCE Rotavirus is one of the most important pathogens causing severe gastroenteritis in young children worldwide. In this paper, we describe a robust and simple reverse genetics system based on only rotavirus cDNAs, and its application for engineering infectious recombinant rotaviruses harboring bioluminescent (NanoLuc) and fluorescent (EGFP and mCherry) protein genes. This highly efficient reverse genetics system and recombinant RVAs expressing reporters could be powerful tools for the study of different aspects of rotavirus replication. Furthermore, they may be useful for next-generation vaccine production for this medically important virus.

  • Evolution-guided structural and functional analyses of the HERC family reveals an ancient marine origin and determinants of antiviral activity. [PublishAheadOfPrint]

  • In humans, llsquo;homologous to the E6-AP carboxyl terminus (HECT) and regulator of chromosome condensation 1 (RCC1)-like domain-containing protein 5rrsquo; (HERC5) is an interferon-induced protein that inhibits replication of evolutionarily diverse viruses including human immunodeficiency virus type 1 (HIV-1). To better understand the origin, evolution and function of HERC5, we performed phylogenetic, structural and functional analyses of the entire human small HERC family, which includes HERC3, HERC4, HERC5 and HERC6. We demonstrated that the HERC family emerged ggt;595 million years ago and has undergone gene duplication and gene loss events throughout its evolution. The structural topology of the RCC1-like domain and HECT domains from all HERC paralogs are highly conserved among evolutionarily diverse vertebrates despite low sequence homology. Functional analyses showed that the human small HERCs exhibit different degrees of antiviral activity towards HIV-1 and that HERC5 provided the strongest level of inhibition. Notably, coelacanth HERC5 inhibited SIV, but not HIV-1, particle production suggesting that the antiviral activity of HERC5 emerged over 413 million years ago and exhibits species- and virus-specific restriction. In addition, we showed that both HERC5 and HERC6 are evolving under strong positive selection; particularly blade 1 of the RCC1-like domain, which we showed is a key determinant of antiviral activity. These studies provide insight into the origin, evolution and biological importance of the human restriction factor HERC5 and the other HERC family members.

    IMPORTANCE Intrinsic immunity plays an important role as the first line of defense against viruses. Studying the origin, evolution and function of proteins responsible for effecting this defense will provide key information about virus-host relationships that can be exploited for future drug development. We showed that HERC5 is one such antiviral protein that belongs to an evolutionarily conserved family of HERCs with an ancient marine origin. Not all vertebrates possess all HERC members, suggesting that different HERCs emerged at different times during evolution to provide the host with a survival advantage. Consistent with this, some of the more recently emerged HERC members, HERC5 and HERC6, displayed strong signatures of having been involved in an ancient evolutionary battle with viruses. Our findings provide new insights into the evolutionary origin and function of the HERC family in vertebrate evolution, identifying HERC5 and possibly HERC6 as important effectors of intrinsic immunity in vertebrates.

  • Incomplete downregulation of CD4 expression affects HIV-1 Env conformation and ADCC responses [PublishAheadOfPrint]

  • HIV-1 infected cells expressing envelope glycoproteins (Env) in the CD4-bound conformation on their surface are targeted by antibody-dependent cellular cytotoxicity (ADCC) mediated by CD4-induced (CD4i) antibodies and HIV+ sera. By downregulating the surface expression of CD4, Nef prevents Env-CD4 interaction thus protecting HIV-1-infected cells from ADCC. HIV-1 infectious molecular clones (IMC) are widely used to measure ADCC. In order to facilitate the identification of infected cells and high-throughput ADCC analysis, reporter genes (e.g. Renilla luciferase, LucR) are often introduced into IMC constructs. We evaluated the susceptibility of HIV-1 infected CD4+ T lymphocytes to ADCC using a panel of parental IMC, and derivatives that expressed the LucR reporter gene utilizing different molecular strategies, including that specifically designed to retain Nef expression. We found that in some of these constructs, Nef expression in CD4+ T cells was suboptimal and consequently CD4 downregulation was incomplete. CD4 molecules remaining on the cell surface resulted in the exposure of ADCC-mediating CD4i epitopes on Env and a dramatic increase in the susceptibility of the infected cells to ADCC. Strikingly, protection from ADCC was observed when cells were infected with the parental IMC that exhibited strong CD4 downregulation. This discrepancy between the parental and Nef-impaired viruses was independent of the strains of Env expressed, but rather correlated with levels of CD4 surface expression. Overall, our results indicate that caution should be taken when selecting IMC for ADCC measurements, and that CD4 downregulation needs to be carefully monitored when drawing conclusions about the nature and magnitude of ADCC.

    IMPORTANCE In-depth understanding of the susceptibility of HIV-1-infected cells to ADCC might help establish correlates of vaccine protection and guide the development of HIV-1 vaccine strategies. Different ADCC assays have been developed, including those using infectious molecular clones (IMC) coding for a LucR reporter gene that greatly facilitates large-scale quantitative analysis. We previously reported different molecular strategies for introducing LucR while maintaining Nef expression and function, and consequently, CD4 surface downregulation. Here we demonstrate that utilizing IMC that exhibit impaired Nef expression can have undesirable consequences due to incomplete CD4 downregulation. CD4 molecules remaining on the cell surface resulted in the exposure of ADCC-mediating CD4i epitopes on Env and a dramatic increase in the susceptibility of the infected cells to ADCC. Overall, our results indicate that CD4 downregulation needs to be carefully monitored when drawing conclusions about the nature and magnitude of ADCC.

  • Arenaviral Nucleoproteins Suppress PACT-Induced Augmentation of RIG-I Function To Inhibit Type I Interferon Production [PublishAheadOfPrint]

  • RIG-I is a major cytoplasmic sensor of viral pathogen-associated molecular pattern (PAMP) RNA and induces the production of type I interferon (IFN) upon viral infection. A dsRNA-binding protein PACT plays an important role in potentiating RIG-I function. We have previously shown that arenaviral nucleoproteins (NPs) suppress type I IFN production via its exoribonuclease (RNase) activity to degrade PAMP RNA. We report here that NPs of arenaviruses block PACT-induced enhancement of RIG-I function to mediate type I IFN production and that the inhibition is dependent on the NP's RNase function, which is different than a known mechanism of other viral proteins to disrupt PACT and RIG-I interaction. To understand the biological roles of PACT and RIG-I in authentic arenavirus infection, we analyze growth kinetics of recombinant Pichinde virus (PICV), a prototypic arenavirus, in RIG-I knock-out (KO) and PACT KO mouse embryonic (MEF) cells. WT PICV grew at higher titers in both KO MEF lines than in normal MEFs, suggesting the important roles of these cellular proteins in restricting virus replication. PICV carrying the NP RNase catalytically inactive mutation could not grow in normal MEFs but could replicate to some extent in both KO MEF lines. The level of viral growth inversely correlated with the amount of type I IFNs produced. These results suggest that PACT plays an important role in potentiating RIG-I function to induce type I IFNs in order to restrict arenavirus replication, and that viral NP RNase activity is essential for optimal viral replication by suppressing PACT-induced RIG-I activation.

    Importance: We report here a new role of the nucleoproteins of arenaviruses that can block type I IFN production via their specific inhibition of the cellular protein sensors of virus infection (RIG-I and PACT). Our results suggest that PACT plays an important role in potentiating RIG-I function to induce type I IFNs in order to restrict arenavirus replication. This new knowledge can be exploited for the development of novel antiviral treatments and/or vaccines against some arenaviruses that can cause severe and lethal hemorrhagic fever diseases in humans.

  • A GXXXA motif in the transmembrane domain of the Ebola virus glycoprotein is required for tetherin antagonism [PublishAheadOfPrint]

  • The interferon-induced antiviral host cell protein tetherin can inhibit the release of several enveloped viruses from infected cells. The Ebola virus (EBOV) glycoprotein (GP) antagonizes tetherin but the domains and amino acids in GP that are required for tetherin antagonism have not been fully defined. A GXXXA motif within the transmembrane domain (TMD) of EBOV-GP was previously shown to be important for GP-mediated cellular detachment. Here, we investigated whether this motif also contributes to tetherin antagonism. Mutation of the GXXXA motif did not impact GP expression or particle incorporation and only modestly reduced EBOV-GP-driven entry. In contrast, the GXXXA motif was required for tetherin antagonism in transfected cells. Moreover, alteration of the GXXXA motif increased tetherin-sensitivity of a replication-competent vesicular stomatitis virus (VSV) chimera encoding EBOV-GP. Although these results await confirmation with authentic EBOV, they indicate that a GXXXA motif in the TMD of EBOV-GP is important for tetherin antagonism. Moreover, they provide the first evidence that GP can antagonize tetherin in the context of an infectious EBOV surrogate.

    IMPORTANCE The glycoprotein (GP) of Ebola virus (EBOV) inhibits the antiviral host cell protein tetherin and may promote viral spread in tetherin-positive cells. However, tetherin antagonism by GP has so far only been demonstrated using virus-like particles and it is unknown whether GP can block tetherin in infected cells. Moreover, a mutation in GP that selectively abrogates tetherin antagonism is unknown. Here, we show that a GXXXA motif in the transmembrane domain of EBOV-GP, which was previously reported to be required for GP-mediated cell rounding, is also important for tetherin counteraction. Moreover, analysis of this mutation in the context of vesicular stomatitis virus chimeras encoding EBOV-GP revealed that GP-mediated tetherin counteraction is operative in infected cells. To our knowledge, these findings demonstrate for the first time that GP can antagonize tetherin in infected cells and provide a tool to study the impact of GP-dependent tetherin counteraction on EBOV spread.

  • Effects of adjuvants on HIV-1 envelope glycoprotein SOSIP trimers in vitro [PublishAheadOfPrint]

  • Native-like, soluble, recombinant SOSIP trimers of various designs and based on several env genes of human immunodeficiency virus type 1 (HIV-1) are being tested as immunogens in different animal models. These experiments almost always involve co-formulating the trimers with an adjuvant to boost the magnitude of the immune responses. One factor relevant to the choice of an adjuvant is that it should not physically damage the immunogen or impede its ability to present relevant epitopes. As examples, an adjuvant formulation that includes harsh detergents could disrupt the structural integrity of a trimer, and any charged compounds in the formulation could bind to counter-charged regions of the trimer and physically occlude nearby epitopes. While a few adjuvants have been tested for their potential effects on SOSIP trimers in vitro, there has been no systematic study. Here, we have assessed how nine different adjuvants of various compositions affect SOSIP trimers of the BG505 and B41 genotypes. We used negative-stain electron microscopy, thermal denaturation, and gel-electrophoresis to evaluate effects on trimer integrity, and immunoassays to measure effects on the presentation of various epitopes. We conclude that most of the tested adjuvants are benign from these perspectives, but some raise grounds for concern. An acidified alum formulation is highly disruptive to trimer integrity, and a DNA-based polyanionic CpG oligodeoxynucleotide adjuvant binds to trimers and occludes the trimer apex epitope for the PGT145 neutralizing antibody. The methods described here should be generalizable to protein subunit vaccines targeting various pathogens.

    IMPORTANCE Adjuvant formulations increase the magnitude of immune responses to vaccine antigens. They are critically important for formulation of HIV-1 envelope glycoprotein (Env) vaccines intended to induce antibody production, as Env proteins are otherwise only very weakly immunogenic. The HIV-1 vaccine field now uses the well-defined structures of trimeric Env glycoproteins, like SOSIPs, to present multiple known epitopes for broad and potent neutralizing human antibodies in a native-like conformation. Successful adjuvant formulations must not disrupt how the trimers are folded, as that could adversely affect their performance as immunogens. We studied whether the various adjuvants most commonly used in animal experiments affect the integrity of two different SOSIP trimers in vitro. Most adjuvant classes are not problematic, but an aluminum sulfate formulation is highly damaging as it exposes trimers to acidic pH and a nucleic acid-based adjuvant can bind to the trimer and block access to a key neutralizing epitope.

  • CD4 T cell epitope specificity and cytokine potential are preserved as cells transition from the lung vasculature to lung tissue following influenza infection [PublishAheadOfPrint]

  • Pulmonary CD4 T cells are critical in respiratory virus control, both by delivering direct effector function and through coordinating responses of other immune cells. Recent studies have shown that following influenza infection, virus-specific CD4 T cells are partitioned between pulmonary vasculature and lung tissue. However, very little is known about the peptide specificity or functional differences of CD4 T cells within these two compartments. Using a mouse model of influenza infection in conjunction with intravascular labeling in vivo, the cell surface phenotype, epitope specificity, and functional potential of the endogenous polyclonal CD4 T cell response was examined by tracking nine independent CD4 T cell epitope specificities. These studies revealed that tissue-localized CD4 cells were globally distinct from vascular cells in expression of markers associated with transendothelial migration, residency and micropositioning. Despite these differences, there was little evidence for remodeling of the viral epitope specificity or cytokine potential as cells transition from vasculature to the highly inflamed lung tissue. Our studies also distinguished cells in the pulmonary vasculature from peripheral circulating CD4 T cells, providing support for the concept that the pulmonary vasculature does not simply reflect circulating cells that are trapped within the narrow confines of capillary vessels, but rather is enriched in transitional cells primed in the draining lymph node that have specialized potential to enter the lung tissue.

    Importance: CD4 T cells convey a multitude of functions in immunity to influenza, including those delivered in the lymph node and others conveyed by CD4 T cells that leave the lymph node, enter the blood and extravasate into the lung tissue. Here, we show that the transition of recently primed CD4 cells detected in the lung vasculature undergo profound changes in expression of markers associated with tissue localization as they establish residence in the lung. However, this transition does not edit CD4 T cell epitope specificity or the cytokine potential of the CD4 T cells. Thus, CD4 T cells that enter the infected lung can convey diverse functions and have a sufficiently broad viral antigen specificity to detect the complex array of infected cells within the infected tissue, offering the potential for more effective protective function.

  • Characterization of Three Novel Linear Neutralizing B-Cell Epitopes in the Capsid Protein of Swine Hepatitis E Virus [PublishAheadOfPrint]

  • Hepatitis E virus (HEV) causes liver disease in humans and is thought to be a zoonotic infection with domestic animals being a reservoir including swine and rabbits. One of the proteins encoded by the virus is the capsid protein. This is likely the major immune-dominant protein and a target for vaccination. Four monoclonal antibodies (MAbs); three novel; 1E4, 2C7, 2G9, and one previously characterized (1B5), were evaluated for binding to the capsid protein from genotype 4 (swine) hepatitis E virus (HEV). The results indicated that 625DFCP628, 458PSRPF462, and 407EPTV410 peptides on the capsid protein comprised minimal amino acid sequence motifs recognized by 1E4, 2C7, and 2G9, respectively. The data suggested that 2C7 and 2G9 epitopes were partially exposed on the surface of the capsid protein. Truncated genotype 4 swine HEV capsid protein (sp239, amino acids 368-606), can exist in multimeric forms. Pre-incubation of swine HEV with 2C7, 2G9, or 1B5 before addition to HepG2 cells partially blocked sp239 cell binding and inhibited swine HEV infection. The study indicated that 2C7, 2G9, and 1B5 partially blocked swine HEV infection of rabbits better than 1E4 or normal mouse IgG. The cross reactivity of antibodies suggested that capsid epitopes recognized by 2C7 and 2G9 are common to HEV strains infecting most host species. Collectively, MAbs 2C7, 2G9, and 1B5 were shown to recognize three novel linear neutralizing B-cell epitopes of genotype 4 HEV capsid protein. These results enhance understanding of HEV capsid protein structure to guide vaccine and anti-viral design.

    IMPORTANCE Genotype 3 and 4 HEVs are zoonotic viruses. Here, genotype 4 HEV was studied due to its prevalence in human populations and pig herds in China. To improve HEV disease diagnosis and prevention, a better understanding of antigenic structure and neutralizing epitopes of HEV capsid protein are needed. In this study, the locations of three novel linear B-cell recognition epitopes within genotype 4 swine HEV capsid protein were characterized. Moreover, the neutralizing abilities of three MAbs specific for this protein, 2C7, 2G9, and 1B5, were studied in vitro and in vivo. Collectively, these findings reveal structural details of genotype 4 HEV capsid protein and should facilitate development of applications for design of vaccines and antiviral drugs for broader prevention, detection, and treatment of HEV infection of diverse human and animal hosts.

  • BHV-1 UL49.5 interacts with gM and VP22 to ensure virus cell-to-cell spread and virion incorporation: A novel role for VP22 in gM independent UL49.5 virion incorporation [PublishAheadOfPrint]

  • Alphaherpesvirus envelope glycoprotein N (gN) and gM form a covalently linked complex. Bovine herpesvirus type 1 (BHV-1) UL49.5 (a gN homolog) contains two predicted cysteine residues C42 and C78. The C42 is highly conserved among the alphaherpesvirus gN homologs (e.g. herpes simplex virus 1, pseudorabies virus). To identify which cysteine residue is required for the formation of the UL49.5/gM complex and to characterize the functional significance of the UL49.5/gM complex, we constructed and analyzed C42S and C78S substitution mutants either in BHV-1 wt or in BHV-1 UL49.5 cytoplasmic tail-null (CT-null) virus backgrounds. The results demonstrated that BHV-1 UL49.5 residue C42 but not C78 was essential for the formation of the covalently linked functional UL49.5/gM complex, gM maturation in the Golgi, and efficient cell-to-cell spread of the virus. Interestingly, the C42S and CT-null mutations separately did not affect mutant UL49.5 virion incorporation. However, when both the mutations were introduced simultaneously, the UL49.5 C42S/CT-null protein virion incorporation was severely reduced. Incidentally, the anti-VP22 antibody coimmunoprecipitated the UL49.5 C42S/CT-null mutant protein at a noticeably reduced level when compared to that of the individual UL49.5 C42S and CT-null mutant proteins. As expected, in a dual UL49.5 C42S/VP22 virus, the UL49.5 C42S virion incorporation was also severely reduced while in a gM virus, UL49.5 virion incorporation was affected only slightly. Together, these results suggested that UL49.5 virion incorporation is mediated redundantly, by both UL49.5/gM functional complex and VP22, through a putative gM independent novel UL49.5 and VP22 interaction.

    Importance

    Bovine herpesvirus 1 (BHV-1) envelope protein UL49.5 is an important virulence determinant because it down regulates MHC-I. UL49.5 also forms a covalently linked complex with gM. The results of this study demonstrate that UL49.5 regulates gM maturation and virus cell-to-cell spread since gM maturation in the Golgi depends on covalently linked UL49.5/gM complex. The results also show that the UL49.5 residue cysteine 42 (C42) mediates the formation of the covalently linked UL49.5/gM interaction. Furthermore, a C42S mutant virus in which UL49.5 cannot interact with gM has defective cell-to-cell spread. Interestingly, UL49.5 also interacts with the tegument protein VP22 via its cytoplasmic tail (CT). The putative UL49.5 CT-VP22 interaction is essential for a gM independent UL49.5 virion incorporation and is revealed when UL49.5 and gM are not linked. Therefore, UL49.5 virion incorporation is mediated by UL49.5/gM complex interaction and through a gM independent interaction between UL49.5 and VP22.

  • Subclinical cytomegalovirus infection associates with altered host immunity, gut microbiota and vaccine responses. [PublishAheadOfPrint]

  • Subclinical viral infections (SVI) including cytomegalovirus (CMV) are highly prevalent in humans, resulting in life-long persistence. However, the impact of SVI on the interplay between the host immunity and gut microbiota in the context of environmental exposures is not well defined. We utilized the preclinical nonhuman primate (NHP) model consisting of SVI-free (SPF) rhesus macaques and compared them to the animals with SVI (non-SPF) acquired through natural exposure and investigated the impact of SVI on immune cell distribution and function as well as on gut microbiota. These changes were examined in animals housed in the outdoor environment as compared to the controlled indoor environment. We report that SVI are associated with altered immune cell subsets and gut microbiota composition in animals housed in the outdoor environment. Non-SPF animals harbored a higher proportion of potential butyrate-producing Firmicutes and higher numbers of lymphocytes, effector T cells and cytokine-producing T cells. Surprisingly, these differences diminished following their transfer to the controlled indoor environment, suggesting that non-SPFs had increased responsiveness to environmental exposures. An experimental infection of indoor SPF animals with CMV resulted in an increased abundance of butyrate-producing bacteria, validating that CMV enhanced colonization of butyrate-producing commensals. Finally, non-SPF animals displayed lower antibody responses to influenza vaccination as compared to SPF animals. Our data show that subclinical CMV infection heightens host immunity and gut microbiota changes in response to environmental exposures. This may contribute to the heterogeneity in host immune response to vaccines and environmental stimuli at the population level.

    IMPORTANCE Humans harbor several latent viruses that modulate host immunity and commensal microbiota, thus introducing heterogeneity in their responses to pathogens, vaccines and environmental exposures. Most of our understanding of the effect of CMV on the immune system is based on studies of children acquiring CMV or of immune-compromised humans with acute or reactivated CMV infection or in ageing individuals. The experimental mouse models are genetically inbred and are completely adapted to the indoor laboratory environment. In contrast, nonhuman primates are genetically outbred and are raised in the outdoor environment. Our study is the first to report the impact of long-term subclinical CMV infection on host immunity and gut microbiota, which is evident only in the outdoor environment but not in the indoor environment. The significance of this study is in highlighting the impact of SVI on enhancing host immune susceptibility to environmental exposures and immune heterogeneity.

  • Discovery of novel bat coronaviruses in south China that use the same receptor as MERS coronavirus [PublishAheadOfPrint]

  • Middle East respiratory syndrome coronavirus (MERS-CoV) has represented a human health threat since 2012. Although several MERS-related CoVs, which belong to the same species as MERS-CoV, have been identified from bats, they do not use the MERS-CoV receptor, dipeptidyl peptidase 4 (DPP4). Here, we screened 1059 bat samples from at least 30 bat species collected in different regions in south China and identified 89 strains of lineage C betacoronaviruses, including Tylonycteris pachypus HKU4, Pipistrellus pipistrellus HKU5, and MERS-related CoVs. We sequenced the full-length genomes of two positive samples collected from the great evening bat, Ia io, from Guangdong Province. The two genomes were highly similar and exhibited genomic structures identical to those of other lineage C betacoronaviruses. While they exhibited genome-wide nucleotide identities of only 75.3 to 81.2% with other MERS-related CoVs, their gene-coding regions were highly similar to their counterparts, except in the case of the spike proteins. Further protein--protein interaction assays demonstrated that the spike proteins of these MERS-related CoVs bind to the receptor DPP4. Recombination analysis suggested that the newly discovered MERS-related CoVs might have acquired their spike genes from a DPP4-recognizing bat HKU4. Our study provides further evidence that bats represent the evolutionary origins of MERS-CoV.

    IMPORTANCE Previous studies suggested that the Middle East respiratory syndrome coronavirus (MERS-CoV) may have originated in bats. However, its evolutionary path from bats to humans remains unclear. In this study, we discovered 89 novel lineage C betacoronaviruses (BetaCoVs) in eight bat species. We provide the evidence of a MERS-related CoV derived from the great evening bat that uses the same host receptor as human MERS-CoV. This virus also provides evidence for a natural recombination event between the bat MERS-related CoV and another bat coronavirus HKU4. Our study expands the host ranges of MERS-related CoV and represents an important step toward establishing bats as the natural reservoir of MERS-CoV. These findings may lead to improved epidemiological surveillance of MERS-CoV and the prevention and control of the spread of MERS-CoV to humans.

  • RLRs and TLRs Signaling Pathways Cause Aberrant Production of Inflammatory Cytokines/Chemokines in an SFTSV Infection Mouse Model [PublishAheadOfPrint]

  • Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by a tick-borne phlebovirus of the family Bunyaviridae, the SFTS virus (SFTSV). Wild type and type I interferon (IFN-I) receptor 1-deficient (IFNAR1mmdash;/mmdash;) mice have been established as non-lethal and lethal models of SFTSV infection, respectively. However, the mechanisms of IFN-I production in vivo and the factors causing the lethal disease are not well understood. Using bone marrow chimeric mice, we found that IFN-I signaling in hematopoietic cells was essential for survival from lethal SFTSV infection. The disruption of IFN-I signaling in hematopoietic cells allowed an increase in viral loads in serum and produced an excess of multiple inflammatory cytokines and chemokines. The production of IFN-I and inflammatory cytokines was abolished by the deletion of signaling molecules IPS-1 and MyD88, essential for RLRs and TLRs signaling, respectively. However, IPS-1mmdash;/mmdash; MyD88mmdash;/mmdash; mice exhibited resistance to lethal SFTS with a moderate viral load in serum. Taken together, these results indicate that adequate activation of RLRs and TLRs signaling pathways under low to moderate levels of viremia contributed to survival through the IFN-I-dependent antiviral response during SFTSV infection, whereas overactivation of these signaling pathways under high levels of viremia resulted in abnormal induction of multiple inflammatory cytokines and chemokines causing the lethal disease.

    IMPORTANCE SFTSV causes a severe infectious disease in humans, with a high fatality rate of 12mmdash;30%. To know the pathogenesis of the virus, we need to clarify the innate immune response as a front line of defense against viral infection. Here, we report that the lethal animal model showed abnormal induction of multiple inflammatory cytokines and chemokines by an uncontrolled innate immune response, which triggered the lethal SFTS. Our findings suggest a new strategy to target inflammatory humoral factors to treat severe SFTS patients. Furthermore, this study may help the investigation of other tick-borne viruses.

  • Visualization of arenavirus RNA species in individual cells by single-molecule fluorescence in situ hybridization (smFISH) suggests a model of cyclical infection and clearance during persistence [PublishAheadOfPrint]

  • Lymphocytic choriomeningitis mammarenvirus (LCMV) is an enveloped, negative-strand RNA virus that causes serious disease in humans but establishes an asymptomatic, lifelong infection in reservoir rodents. Different models have been proposed to describe how arenaviruses regulate the replication and transcription of their bisegmented, single-stranded RNA genomes, particularly during persistent infection. However, these models were largely based on viral RNA profiling data derived from entire populations of cells. To better understand LCMV replication and transcription at the single-cell level, we established a high-throughput, single-molecule (sm)FISH image acquisition and analysis pipeline and examined viral RNA species at discrete time points from viral entry through the late stages of persistent infection in vitro. We observed transcription of viral nucleoprotein and polymerase mRNAs from the incoming S and L segment genomic RNAs, respectively, within 1 hr of infection, whereas transcription of glycoprotein mRNA from the S segment antigenome required ~4-6 hr. This confirms the temporal separation of viral gene expression expected due to the ambisense coding strategy of arenaviruses and also suggests that antigenomic RNA contained in virions is not transcriptionally active upon entry. Viral replication and transcription peaked at 36 hours post-infection, followed by a progressive loss of viral RNAs over the next several days. During persistence, the majority of cells showed repeating cyclical waves of viral transcription and replication followed by clearance of viral RNA. Thus, our data support a model of LCMV persistence whereby infected cells can spontaneously clear infection and become reinfected by viral reservoir cells that remain in the population.

    IMPORTANCE Arenaviruses are human pathogens that can establish asymptomatic, life-long infections in their rodent reservoirs. Several models have been proposed to explain how arenavirus spread is restricted within host rodents, including the periodic accumulation and loss of replication competent, but transcriptionally incompetent, viral genomes. A limitation of previous studies was the inability to enumerate viral RNA species at the single cell level. We developed a high throughput, single-molecule RNA (sm)FISH assay and used it to quantitate lymphocytic choriomeningitis mammarenavirus (LCMV) replicative and transcriptional RNA species in individual cells at distinct time points following infection. Our findings support a model whereby productively infected cells can clear infection, including viral RNAs and antigen, and later be reinfected. This information improves our understanding of the timing and possible regulation of LCMV genome replication and transcription during infection. Importantly, the smFISH assay and data analysis pipeline developed here is easily adaptable to other RNA viruses.

  • The pathway to establishing HIV latency is critical to how latency is maintained and reversed [PublishAheadOfPrint]

  • HIV infection requires lifelong antiretroviral therapy because of the persistence of latently infected CD4+ T cells. Induction of viral expression from latently infected cells occurs following T cell receptor (TCR) activation but not all latently infected cells respond to TCR stimulation. We compared two models of latently infected cells using an enhanced green fluorescent protein (EGFP) reporter virus to infect CCL19 treated resting CD4+ (rCD4) T cells (pre-activation latency) or activated CD4+ T cells that returned to a resting state (post-activation latency). We isolated latently infected cells by sorting for EGFP- cells after infection. These cells were cultured with antivirals and stimulated with anti-CD3/anti-CD28, mitogens, latency reversing agents (LRA) as well as co-cultured with monocytes and anti-CD3. Spontaneous EGFP expression was more frequent in post-activation than pre-activation latency. Stimulation of latently infected cells with monocytes/anti-CD3 resulted in an increase in EGFP expression compared to unstimulated controls using the pre-activation latency model but led to a reduction in EGFP expression in the post-activation latency model. The reduced EGFP expression was not associated with a reduction in viral DNA or the levels of T cell proliferation but depended on direct contact between monocytes and T cells. Monocytes added to the post-activation latency model during the establishment of latency reduced spontaneous viral expression suggesting that monocyte-T cell interactions at an early time point post infection can maintain HIV latency. This direct comparison of pre- and post- activation latency suggests that effective strategies needed to reverse latency will depend on how latency is established.

    IMPORTANCE One strategy being evaluated to eliminate latently infected cells that persist in HIV-infected individuals on antiretroviral therapy (ART) is to activate HIV expression or production with the goal of inducing virus mediated cytolysis or immune mediated clearance of the infected cells. The gold standard for activation of latent virus is T cell receptor stimulation with anti-CD3/anti-CD28. However, this stimulus only activates a small proportion of latently infected cells. We show clear differences in the response of latently infected cells to activating stimuli based on how latent infection is established, an observation that may potentially explain the persistence of non-induced intact proviruses in HIV-infected individuals on ART.

  • Developing and evaluating inhibitors against the RNase H active site of HIV-1 RT [PublishAheadOfPrint]

  • We tested three compounds for their ability to inhibit the RNase H (RH) and polymerase activities of HIV-1 reverse transcriptase (RT). A high-resolution crystal structure (2.2 AAring;) of one of the compounds showed that it chelates the two magnesium ions at the RH active site; this prevents the RH active site from interacting with, and cleaving, the RNA strand of an RNA/DNA heteroduplex. The compounds were tested using a variety of substrates: All three compounds inhibited the polymerase-independent RH activity of HIV-1 RT. Time of addition experiments showed that the compounds were more potent if they were bound to RT before the nucleic acid substrate was added. The compounds significantly inhibited the site-specific cleavage required to generate the polypurine tract (PPT) RNA primer that initiates the second strand of viral DNA synthesis. The compounds also reduced the polymerase activity of RT; this ability was a result of the compounds binding to the RH active site. These compounds appear to be relatively specific; they do not inhibit either E. coli RNase HI or human RNase H2. The compounds inhibit the replication of a HIV-1 based vector in a one-round assay, and their potencies were only modestly decreased by mutations that confer resistance to integrase strand transfer inhibitors (INSTIs), nucleoside analogs, or non-nucleoside RT inhibitors (NNRTIs), suggesting that their ability to block HIV replication is related to their ability to block RH cleavage. These compounds appear to be useful leads that can be used to develop more potent and specific compounds.

    IMPORTANCE Despite advances in HIV-1 treatment, drug resistance is still a problem. Of the four enzymatic activities found in HIV-1 proteins (protease, RT polymerase, RT RNase H, and integrase), only RNase H has no approved therapeutics directed against it. This new target could be used to design and develop new classes of inhibitors that would suppress the replication of the drug resistant variants that have been selected by the current therapeutics.

  • The dynamics of SIV 2-LTR Circles in the Presence and Absence of CD8+ Cells [PublishAheadOfPrint]

  • CD8+ cells play a key role in HIV/SIV infection, but their specific mechanism(s) of action in controlling the virus are unclear. 2-LTR circles are extrachromosomal products generated upon failed integration of HIV/SIV. To understand the specific effects of CD8+ cells on infected cells, we analyzed the dynamics of 2-LTR circles in SIVmac251-infected rhesus macaques (RM) treated with an integrase inhibitor (INT). Twenty RMs underwent CD8+ cell depletion, received RAL monotherapy or a combination of both. Blood, lymph nodes (LNs) and gut biopsies were routinely sampled. Plasma viral loads (pVLs) and 2-LTR circles from PBMCs and LN lymphocytes were measured with qRT-PCR. In the CD8 depletion group, an ~1 log increase in pVLs and a slow increase in PBMC 2-LTRs occurred following depletion. In the INT group, a strong decline in pVLs upon treatment initiation and no change in 2-LTR levels were observed. In the INT and CD8+ cell depletion group, a similar increase in pVLs following CD8 depletion was observed, with a modest decline following INT initiation, and 2-LTR circles significantly increased in PBMCs and LNs. Analyzing the 2-LTR data across all treatment groups with a mathematical model indicates that the data best supports an effect of CD8+ cells in killing cells prior to viral integration. Sensitivity analyses of these results confirm that effect, but also allow for additional effects, which the data does not discriminate well. Overall, we show that INT does not significantly increase the levels of 2-LTR circles. However, CD8+ cell depletion increases the 2-LTR levels, which are enhanced in the presence of an INT.

    IMPORTANCE

    CD8+ T cells play an essential role in controlling HIV and simian immunodeficiency virus (SIV) infection, but the specific mechanisms involved remain poorly understood. Due to failed viral infection, HIV and SIV can form 2-LTR extrachromosomal circles that can be quantified. We present novel data on the dynamics of these 2-LTR forms in a SIV-infected macaque model under three different treatment conditions: depletion of CD8+ cells; administration of the integrase inhibitor in a monotherapy, which favors the formation of 2-LTR circles; and combination of the two treatments. We used a new mathematical model to help interpret the data, and the results suggest that CD8+ cells exert a killing effect on infected cells prior to virus integration. These results provide new insights into the mechanisms of action of CD8+ cells in SIV infection. Confirmation of our results would be an important step in understanding immune control of HIV.

  • The functional oligomeric state of tegument protein GP41 is essential for baculovirus BV and ODV assembly [PublishAheadOfPrint]

  • gp41, one of the baculovirus core genes, encodes the only recognized tegument (O-glyco) protein of the occlusion-derived virion (ODV) phenotype so far. A previous study using a temperature-sensitive Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) mutant showed that GP41 plays a crucial role in budded virion (BV) formation. However, the precise function of GP41 in the baculovirus replication cycle remains unclear. In this study, AcMNPV GP41 was found to accumulate around the ring zone region (RZ) within the infected nucleus and finally assembled into both BV and ODV. Deletion of gp41 from the AcMNPV genome showed that BVs were no longer formed and ODVs were no longer assembled, suggesting its essential role in baculovirus virion morphogenesis. In infected cells, besides the 42 kDa-monomers, dimers and trimers were detected under non-reducing conditions, whereas only trimeric GP41 forms were selectively incorporated into BVs or ODVs. Mutations of all five cysteines in GP41 individually had minor effect on GP41 oligomer formation, albeit certain mutations impaired infectious BV production, suggesting flexibility in the intermolecular disulfide bonding. Single mutations of key leucines within two predicted leucine zipper-like motifs did not interfere GP41 oligomerization, nor BV and ODV formation, but double leucine mutations completely blocked oligomerization of GP41 and progeny BV production. In the latter case, the usual subcellular localization, especially RZ accumulation of GP41, was abolished. The above findings clearly pointed out a close correlation between GP41 oligomerization and its function, therefore highlighted the oligomeric state as the functional form of GP41 in baculovrius replication cycle.

    IMPORTANCE The tegument, which is sandwiched between nucleocapsid and virion envelope, is an important substructure of many enveloped viruses. It is composed of one or more proteins that have important functions during virus entry, replication, assembly and egress. Unlike another large DNA virus, herpesvirus that encodes an extensive set of tegument components, baculoviruses very likely exploit the major tegument protein, GP41, to execute functions in baculovirus virion morphogenesis and assembly. However, the function of this O-glycosylated baculovirus tegument protein remains largely unknown. In this study, we identified trimers as the functional structure of GP41 in baculovirus virion morphogenesis and that both disulfide bridging and protein-protein interactions via the two leucine zipper-like domains are involved in the formation of different oligomeric states. This study advances our understanding of the unique viral tegument protein GP41 participating in the life cycle of baculoviruses.

  • JC Polyomavirus: Let's Please Respect Privacy [PublishAheadOfPrint]

  • JC polyomavirus was discovered in 1971, and its name was derived from the initials of the individual from whose brain tissue it was isolated. While most scientists refer to the virus properly, i.e., calling it JCV or JCPyV, there is a small but palpable number of scientists who refer to the virus by the full name of the patient from whom it was isolated. This practice should stop.

  • Introduction of viral haemorrhagic septicaemia virus (VHSV) to freshwater cultured rainbow trout is followed by bursts of adaptive evolution [PublishAheadOfPrint]

  • Viral haemorrhagic septicaemia virus (VHSV), a rhabdovirus infecting teleost fish, has repeatedly crossed the boundary from marine fish species to freshwater cultured rainbow trout. These naturally replicated cross-species transmission events permit the study of general and repeatable evolutionary events occurring in connection with viral emergence into a novel host species.

    The purpose of this study was to investigate adaptive molecular evolution of the VHSV glycoprotein, one of the key virus proteins involved in viral emergence, following emergence from marine species into freshwater cultured rainbow trout. A comprehensive phylogenetic reconstruction of the complete coding region of the VHSV glycoprotein was conducted and adaptive molecular evolution was investigated using a maximum likelihood approach to compare different codon substitution models allowing for heterogeneous substitution rate ratios among amino acid sites.

    Evidence of positive selection was detected at six amino acid sites within the signal peptide, the confirmation-dependent major neutralizing epitope, and the intracellular tail of the VHSV G protein. Evidence of positive selection was exclusively found in rainbow trout-adapted virus isolates and amino acid combinations found at the six sites under positive selection differentiated rainbow trout- from non-rainbow trout-adapted isolates. Furthermore, four adaptive sites revealed signs of recurring identical changes across phylogenetic groups of rainbow trout-adapted isolates, suggesting that repeated VHSV emergence into freshwater cultured rainbow trout was established through convergent routes of evolution that are associated with immune escape.

    Importance This study is the first to demonstrate that VHSV emergence from marine species into freshwater cultured rainbow trout have been accompanied by bursts of adaptive evolution in the VHSV glycoprotein. Repeated detection of the same adaptive amino acid sites across phylogenetic groups of rainbow trout-adapted isolates furthermore indicates that adaptation to rainbow trout was established through parallel evolution. In addition, signals of convergent evolution towards the maintenance of genetic variation were detected in the conformation-dependent neutralizing epitope or in close proximity to disulfide-bonds involved in the structural conformation of the neutralizing epitope, indicating adaptation to immune response-related genetic variation across freshwater cultured rainbow trout.

  • Neuronal subtype determines HSV-1 Latency-Associated-Transcript (LAT) promoter activity during latency. [PublishAheadOfPrint]

  • Herpes simplex virus (HSV) latency in neurons remains poorly understood, and the heterogeneity of the sensory nervous system complicates mechanistic studies. In this study, we used primary culture of adult trigeminal ganglion (TG) mouse neurons in microfluidic devices, and an in vivo model, to examine the subtypes of sensory neurons involved in HSV latency. HSV-infected neurofilament heavy-positive neurons (NefH+) were more likely to express Latency-Associated Transcripts (LATs) relative to infected neurofilament heavy-negative neurons (NefH-). This differential expression of the LAT promoter correlated with differences in HSV-1 early infection that manifested as differences in the efficiency with which HSV particles reached the cell body following infection at the distal axon. In vivo, we further identified a specific subset of NefH+ neurons which co-expressed Calcitonin Gene Related Peptide aalpha; (NefH+ CGRP+) as the sensory neuron subpopulation with the highest LAT promoter activity following HSV-1 infection. Finally, an early-phase reactivation assay showed HSV-1 reactivating in NefH+ CGRP+ neurons, although other sensory neuron subpopulations were also involved. Together, these results show that sensory neurons expressing neurofilaments exhibit enhanced LAT promoter activity. We hypothesize that reduced efficiency of HSV-1 invasion at an early phase of infection may promote efficient establishment of latency in NefH+ neurons due to initiation of the antiviral state preceding arrival of the virus at the neuronal cell body. While the outcome of HSV-1 infection of neurons is determined by a broad variety of factors in vivo, neuronal subtypes are likely to play differential roles in modulating the establishment of latent infection.

    Importance: Two pivotal properties of HSV-1 make it a successful pathogen. Firstly, it infects neurons which are immune-privileged. Secondly, it establishes latency in these neurons. Together, these properties allow HSV to persist for the lifetime of its host. Neurons are diverse and highly organized cells, with specific anatomical, physiological and molecular characteristics. Previous work has shown that establishment of latency by HSV-1 does not occur equally in all types of neurons. Our results show that the kinetics of HSV infection and levels of latency-related gene expression differ in certain types of neurons. The neuronal subtype infected by HSV is therefore a critical determinant of the outcome of infection and latency.

  • Neutralizing Antibody Responses following Long-Term Vaccination with HIV-1 Env gp140 in Guinea Pigs [PublishAheadOfPrint]

  • A vaccination regimen capable of eliciting potent and broadly neutralizing antibodies (bNAbs) remains an unachieved goal of the HIV-1 vaccine field. Here we report the immunogenicity of longitudinal prime/boost vaccination regimens over a period of 200 weeks in guinea pigs with a panel of HIV-1 envelope (Env) gp140 protein immunogens. We assessed vaccine regimens that included a monovalent clade C gp140 regimen (C97), a tetravalent regimen consisting of four clade C gp140s (4C), and a tetravalent regimen consisting of a clade A, B, C, and mosaic gp140 (ABCM). We found that the 4C and ABCM prime/boost regimens were capable of eliciting a greater magnitude and breadth of binding antibodies targeting variable loop 2 (V2) over time, compared to the monovalent C97 only regimen. The longitudinal boosting regimen conducted over more than two years increased the magnitude of certain tier 1 NAbs, but did not increase the magnitude or breadth of heterologous tier 2 NAbs. These data suggest that additional immunogen design strategies are needed to induce broad, high titer tier 2 NAbs.

    IMPORTANCE The elicitation of potent, broadly neutralizing antibodies (bNAbs) remains an elusive goal for the HIV-1 vaccine field. In this study we explored the use of a long-term vaccination regimen with differing immunogens to determine if we could elicit bNAbs in guinea pigs. We found that longitudinal boosting over more than two years increased tier 1 NAbs but did not increase the magnitude and breadth of tier 2 NAbs. These data suggest that additional immunogen designs and vaccination strategies will be necessary to induce broad tier 2 NAb responses.

  • Truncated CPSF6 forms higher order complexes that bind and disrupt HIV-1 capsid [PublishAheadOfPrint]

  • Cleavage and polyadenylation specificity factor 6 (CPSF6) is a human protein that binds HIV-1 capsid and mediates nuclear transport and integration targeting of HIV-1 pre-integration complexes. Truncation of the protein at its C-terminal nuclear-targeting arginine/serine-rich (RS-) domain produces a protein, CPSF6-358, that potently inhibits HIV-1 infection by targeting the capsid and inhibiting nuclear entry. To understand the molecular mechanism behind this restriction, the interaction between CPSF6-358 and HIV-1 capsid was characterized using in vitro and in vivo assays. Purified CPSF6-358 protein formed oligomers and bound in vitro assembled wild-type (WT) capsid protein (CA) tubes but not CA tubes containing a mutation in the putative binding site of CPSF6. Intriguingly, binding of CPSF6-358 oligomers to WT CA tubes physically disrupted the tubular assemblies into small fragments. Further, fixed and live-cell imaging showed that stably expressed CPSF6-358 forms cytoplasmic punctae upon WT HIV-1 infection and leads to capsid permeabilization. These events did not occur when the HIV-1 capsid contained a mutation known to prevent CPSF6-binding nor did they occur in the presence of a small molecule inhibitor of capsid binding to CPSF6-358. Together, our in vitro biochemical and transmission electron microscopy data and in vivo intracellular imaging results provide the first direct evidence for an oligomeric nature of CPSF6-358 and suggest a plausible mechanism for restriction of HIV-1 infection by CPSF6-358.

    IMPORTANCE After entry into cells, the HIV-1 capsid, which contains the viral genome, interacts with numerous host cell factors to facilitate crucial events required for replication, including uncoating. One such host cell factor is called CPSF6, which is predominantly located in the cell nucleus and interacts with HIV-1 capsid. The interaction between CA and CPSF6 is critical during HIV-1 replication in vivo. Truncation of CPSF6 leads to its localization to the cell cytoplasm and inhibition of HIV-1 infection. Here, we determined that truncated CPSF6 protein forms large higher order complexes that binds directly to HIV-1 capsid, leading to its disruption. Truncated CPSF6 expression in cell leads to premature capsid uncoating that is detrimental to HIV-1 infection. Our study provides the first direct evidence for an oligomeric nature of truncated CPSF6 and insights into the highly regulated process of HIV-1 capsid uncoating.

  • A Single Amino Acid at the Polymerase Acidic Protein Determines the Pathogenicity of Influenza B Viruses [PublishAheadOfPrint]

  • Influenza B virus (IBV) is one of the human respiratory viruses and the targets of seasonal vaccination. However, the bifurcation of two antigenically distinct lineages of IBVs makes it difficult to arrange proper medical countermeasures. Moreover, compared with pathogenicity-related molecular markers known for influenza A virus, little has been known for IBVs. To understand pathogenicity caused by IBVs, we investigated the molecular determinants of IBV pathogenicity in animal models. After serial lung-to-lung passages of Victoria lineage B/Brisbane/60/2008 (Vc_BR60) and Yamagata lineage B/Wisconsin/01/2010 (Ym_WI01) viruses in BALB/c mice, we identified the mouse-adapted Vc_BR60 (maVc_BR60) and Ym_WI01 (maYm_WI01) viruses, respectively. To find out molecular clue(s) to the increased viral pathogenicity of maVc_BR60 and maYm_WI01, we determined their genetic sequences. Several amino acid mutations were identified in PB2, PB1, PA, BM2, and/or NS1 protein coding regions, and one concurrent lysine (K)-to-arginine (R) mutation in PA residue 338 (PA K338R) was found in both maVc_BR60 and maYm_WI01 viruses. When analyzed using reverse-genetically rescued viruses, it was shown that PA K338R alone could increase the pathogenicity of both IBVs in mice and viral replication property in the respiratory tracts of ferrets. In a subsequent mini-replicon assay, the effect of PA K338R was highlighted by the enhancement of viral polymerase complex activity of both Vc_BR60 and Ym_WI01 viruses. These results suggest that the PA K338R mutation may be a molecular determinant of IBV pathogenicity via modulating the viral polymerase function of IBVs.

    IMPORTANCE To investigate molecular pathogenic determinants of IBVs, which is one of the targets of seasonal influenza vaccines, we adapted both Victoria and Yamagata lineage IBVs independently in mice. The recovered mouse-adapted viruses exhibited increased virulence, and, of the various mutations identified from both mouse-adapted viruses, a concurrent amino acid mutation was found in the PA protein coding region. When analyzed using the reverse-genetically rescued viruses, the PA mutation appeared to exclusively contribute to viral pathogenicity in mice within the compatible genetic constellation between the IBV lineages and the replication property of IBVs in ferrets. Regarding the potential mechanism of increased viral pathogenicity, it was shown that the PA mutation could upregulate viral polymerase complex activity pf both IBV lineages. These indicate that the PA mutation can be a newly defined, molecular pathogenic determinant of IBVs that substantiates our understanding of viral pathogenicity and public health risks of IBVs.

  • SUN1 regulates HIV-1 nuclear import in a manner dependent on the interaction between the viral capsid and cellular cyclophilin A. [PublishAheadOfPrint]

  • HIV-1 can infect non-dividing cells via passing through the nuclear pore complex. The nuclear membrane imbedded protein SUN2 was recently reported to be involved in the nuclear import of HIV-1. Whether SUN1, which shares many functional similarities with SUN2, is involved in this process remained to be explored. Here, we report that overexpression of SUN1 specifically inhibited the infection of HIV-1, but not simian immunodeficiency virus (SIV) or murine leukemia virus (MLV). Overexpression of SUN1 did not affect reverse transcription, but led to reduced accumulation of the 2-LTR circular DNA and integrated viral DNA, suggesting a block in the process of nuclear import. HIV-1 CA was mapped as a determinant for the viral sensitivity to SUN1. Treatment of SUN1-expressing cells with cyclosporine A (CsA) significantly reduced the sensitivity of the virus to SUN1 and an HIV-1 mutant containing CA-G89A that does not interact with cyclophilin A (CypA) was resistant to SUN1 overexpression. Downregulation of endogenous SUN1 inhibited the nuclear entry of the wildtype virus, but not the G89A mutant. These results indicate that SUN1 participates in the HIV-1 nuclear entry process in a manner dependent on the interaction of CA with CypA.

    Importance

    HIV-1 infects both dividing and non-dividing cells. The viral pre-integration complex (PIC) can enter the nucleus through the nuclear pore complex. It has been well known that the viral protein CA plays an important role in determining the pathways by which the PIC enters the nucleus. In addition, the interaction between CA and the cellular protein CypA has been reported to be important in the selection of the nuclear entry pathways, though the underlying mechanisms are not very clear. Here we show that both SUN1 overexpression and downregulation inhibited HIV-1 nuclear entry. CA played an important role in determining the sensitivity of the virus to SUN1; the regulatory activity of SUN1 towards HIV-1 relied on the interaction between CA and CypA. These results help to explain how SUN1 is involved in the HIV-1 nuclear entry process.

  • KSHV mRNA accumulation in nuclear foci is influenced by viral DNA replication and the viral noncoding PAN RNA [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV), like other herpesviruses, replicates within the nuclei of its human cell host and hijacks host machinery for expression of its genes. The activities that culminate in viral DNA synthesis and assembly of viral proteins into capsids physically concentrate in nuclear areas termed viral replication compartments. We sought to better understand the spatiotemporal regulation of viral RNAs during the KSHV lytic phase by examining and quantifying the subcellular localization of select viral transcripts. We find that viral mRNAs, as expected, localize to the cytoplasm throughout the lytic phase. However, dependent on active viral DNA replication, viral transcripts also accumulate in the nucleus, often in foci in and around replication compartments, independent of the host shutoff effect. Our data point to involvement of the viral long noncoding PAN RNA in the localization of an early, intronless viral mRNA encoding ORF59-58 to nuclear foci that are associated with replication compartments.

    IMPORTANCE Late in the lytic phase, mRNAs from Kaposi's sarcoma-associated herpesvirus accumulate in the host-cell nucleus near viral replication compartments, centers of viral DNA synthesis and virion production. This work contributes spatiotemporal data on herpesviral mRNAs within the lytic host cell and suggests a mechanism for viral RNA accumulation. Our findings indicate that the mechanism is independent of the host shutoff effect and splicing, but dependent on active viral DNA synthesis and in part on the viral noncoding RNA, polyadenylated nuclear (PAN) RNA. PAN RNA is essential for the viral life cycle and its contribution to the nuclear accumulation of viral messages may facilitate propagation of the virus.

  • Inefficient HIV-1 trans infection of CD4+ T cells by macrophages from HIV-1 nonprogressors is associated with altered membrane cholesterol and DC-SIGN [PublishAheadOfPrint]

  • Professional antigen presenting cells (APC: myeloid dendritic cells (DC) and macrophages (M); B lymphocytes) mediate highly efficient HIV-1 infection of CD4+ T cells, termed trans infection, that could contribute to HIV-1 pathogenesis. We have previously shown that lower cholesterol content in DC and B lymphocytes is associated with a lack of HIV-1 trans infection in HIV-1 infected nonprogressors (NP). Here we assessed whether HIV-1 trans infection mediated by another major APC, M, is deficient in NP due to altered cholesterol metabolism. When comparing healthy HIV-1 seronegatives (SN), rapid progressors (PR), and NP, we found that monocyte-derived M from NP did not mediate HIV-1 trans infection of autologous CD4+ T cells, in contrast to efficient trans infection mediated by SN and PR M. M trans infection efficiency was directly associated with the number of DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN)-expressing M. Significantly fewer NP M expressed DC-SIGN. Unesterified (free) cholesterol in M cell membranes and lipid rafting was significantly lower in NP than PR, as well as virus internalization in early endosomes. Furthermore, simvastatin (SIMV), decreased the subpopulation of DC-SIGN+ M, as well as M cis and trans infection. Notably, SIMV decreased cell membrane cholesterol and led to lipid raft dissociation, effectively mimicking the incompetent APC trans infection environment characteristic of NP. Our data support that DC-SIGN and membrane cholesterol are central to M trans infection, and a lack of these limits HIV-1 disease progression. Targeting the ability of M to drive HIV-1 dissemination in trans could enhance HIV-1 therapeutic strategies.

    IMPORTANCE Despite the success of combination anti-retroviral therapy, neither a vaccine nor a cure for HIV infection has been developed, demonstrating a need for novel prophylactic and therapeutic strategies. Here we show that efficiency of macrophage (M) -mediated HIV trans infection of CD4+ T cells is a unique characteristic associated with control of disease progression, and it is impaired in HIV-infected nonprogressors (NP). In vitro treatment of M from healthy donors with simvastatin (SIMV) lowers their cholesterol content which results in a strongly reduced trans infection ability, similar to the levels of M from NP. Taken together, our data support the hypothesis that M-mediated HIV-1 trans infection plays a role in HIV infection and disease progression and demonstrate that the use of SIMV to decrease this mechanism of virus transfer should be considered for future HIV therapeutic development.

  • The proapoptotic, HIV protease generated Casp8p41 when bound and inactivated by Bcl2, is degraded by the proteasome [PublishAheadOfPrint]

  • HIV protease is known to cause cell death which is dependent upon cleavage of procaspase 8. HIV protease cleavage of procaspase 8 generates Casp8p41 which directly binds Bak with nanomolar affinity causing Bak activation and consequent cell death. Casp8p41 can also bind Bcl2 with nanomolar affinity in which case cell death is averted. Central memory CD4 T cells express high levels of Bcl2 possibly explaining why these cells do not die when they reactivate HIV. Here we determine that the Casp8p41:Bcl2 complex is polyubiquitinated and degraded by the proteasome. Ixazomib, a proteasome inhibitor in clinical use, blocks this pathway mmdash; increasing the abundance of Casp8p41 and causing more cells to die in a Casp8p41 dependent manner.

    Importance The Casp8p41 pathway of cell death is unique to HIV infected cells, yet is blocked by Bcl2. Once bound by Bcl2, Casp8p41 is polyubiquitinated and degraded by the proteasome. Proteasome inhibition blocks degradation of Casp8p41, increasing Casp8p41 levels and causing more HIV infected cell to die.

  • Recombinant porcine reproductive and respiratory syndrome virus expressing membrane-bound IL-15 as immunomodulatory adjuvant enhances NK and {gamma}{delta} T cell responses and confers heterologous protection [PublishAheadOfPrint]

  • Cytokines are often used as adjuvants to improve vaccine immunogenicity, since they are important in initiating and shaping the immune response. The available commercial modified live-attenuated vaccines (MLVs) against porcine reproductive and respiratory syndrome virus (PRRSV) are unable to mount sufficient heterologous protection, as they typically induce weak innate and inadequate T cell responses. In this study, we investigated the immunogenicity and vaccine efficacy of recombinant PRRSV MLVs incorporated with porcine cytokine IL-15 or IL-18 gene fusing to a glycophosphatidylinositol (GPI) modification signal that can anchor the cytokines to cell membrane. We demonstrated that both cytokines were successfully expressed on the cell membrane of porcine alveolar macrophage after infection with recombinant MLVs. Pigs vaccinated with recombinant MLVs or parental Suvaxyn MLV had significantly reduced lung lesions and viral RNA loads in the lung after heterologous challenge with PRRSV NADC20 strain. The recombinant MLVs SUV-IL-15 and SUV-IL-18 recovered the inhibition on NK cell response seen in Suvaxyn MLV. The recombinant MLV SUV-IL-15 significantly increased the IFN--producing cells in circulation at 49 days post-vaccination (dpv), especially for the IFN--producing CD4-CD8+ T cells and T cells when compared to the Suvaxyn MLV and SUV-IL-18. Additionally, the MLV SUV-IL-15-vaccinated pigs also had an elevated level of T cell response observed at 7 dpv, 49 dpv and 7 days post-challenge. These data demonstrate that the recombinant MLV expressing membrane-bound IL-15 enhances NK and T cell immune responses after vaccination, and confers an improved heterologous protection, although not statistically significant than parental MLV.

    Importance

    Porcine reproductive and respiratory syndrome (PRRS) has arguably been the most economically-important global swine disease, causing immense economic losses worldwide. The available commercial modified live-attenuated vaccines (MLVs) against PRRS virus (PRRSV) are generally effective only against homologous or closely-related virus strains but ineffective against heterologous strains, partially due to the insufficient immune response induced by the vaccine virus. To improve the immunogenicity of MLVs, we in this study present a novel approach of using porcine IL-15 or IL-18 as adjuvants by directly incorporating their coding genes into a PRRSV MLV and express them as adjuvants. Importantly, we directed the expression of the incorporated cytokines to the cell membrane surface by fusing the genes with a membrane-targeting signal from CD59. The recombinant MLV vaccine virus expressing membrane-bound IL-15 cytokine greatly enhanced NK cell and T cell responses, and also confers an improved protection against heterologous challenge with PRRSV NADC20 strain.

  • Molecular basis of unusually high neutralization resistance in tier 3 HIV-1 strain 253-11 [PublishAheadOfPrint]

  • Understanding the mechanisms used by HIV-1 to evade antibody neutralization may contribute to the design of a high-coverage vaccine. The tier 3 virus 253-11, is poorly neutralized by subtype-matched and subtype C sera, even when compared to other tier 3 viruses, and is also recognized poorly by V3/glycan targeting monoclonal antibodies. We found that sequence polymorphism in the V3 loop and N-linked glycosylation sites only minimally contribute to the high neutralization resistance of 253-11. Interestingly, the 253-11 membrane proximal external region (MPER) is rarely recognized by sera in the context of the wild-type virus, but is commonly recognized in the context of an HIV-2 chimeric virus, suggesting steric or kinetic hindrance of binding to MPER in the native Env. Mutations in the 253-11 MPER mmdash; which were previously reported to increase the lifetime of the pre-fusion Envelope (Env) conformation mmdash; affected the resistance of 253-11 to antibodies targeting various epitopes on HIV-1 Env, presumably destabilizing its otherwise stable, closed trimer structure. To gain insight into the structure of 253-11, we constructed and crystallized a recombinant 253-11 SOSIP trimer. The resulting structure revealed that the heptad repeat helices in gp41 are drawn in close proximity to the trimer axis and that gp120 protomers also showed a relatively compact disposition around the trimer axis. These observations give substantial insight into the molecular features of an envelope spike from a tier 3 virus and into possible mechanisms that may contribute to its unusually high neutralization resistance.

    IMPORTANCE HIV-1 isolates that are highly resistant to broadly neutralizing antibodies could limit the efficacy of an antibody-based vaccine. We studied 253-11, which is highly resistant to commonly-elicited neutralizing antibodies. To further understand its resistance, we made mutations that are known to delay fusion and thus increase the time the virus spends in the open conformation following CD4-binding. Interestingly, we found that these mutations affect the 253-11 Envelope (Env) spike before CD4 binding, presumably by destabilizing the trimer structure. To gain further information about the structure of the 253-11 Env trimer, we generated a recombinant 253-11 SOSIP trimer. The crystal structure of the SOSIP trimer revealed that the gp41 helices and the gp120 protomers were drawn in towards the center of the molecule compared to most solved HIV-1 Env structures. These observations provide insight into the distinct molecular features of a Tier 3 envelope spike.

  • A non-envelopment role for the ESCRT-III complex during HCMV infection [PublishAheadOfPrint]

  • Secondary envelopment of human cytomegalovirus (HCMV) occurs through a mechanism that is poorly understood. Many enveloped viruses utilize the endosomal sorting complexes required for transport (ESCRTs) for viral budding and envelopment. Although there are conflicting reports on the role of the ESCRT AAA-ATPase protein VPS4 in an HCMV infection, VPS4 may act in an envelopment role similar to its function during other viral infections. Because VPS4 is normally recruited by the ESCRT-III complex, we hypothesized that ESCRT-III subunits would also be required for HCMV infection. We investigated the role of ESCRT-III, the core ESCRT scission complex, during the late stages of infection. We show that inducible expression of dominant-negative ESCRT-III subunits during infection blocks endogenous ESCRT function, but does not inhibit virus production. We also show that HCMV forms enveloped intracellular and extracellular virions in the presence of dominant-negative ESCRT-III subunits, suggesting that ESCRT-III is not involved in the envelopment of HCMV. We also found that similar to ESCRT-III, inducible expression of a dominant-negative form of VPS4A did not inhibit the envelopment of virions or reduce virus titers. Thus, HCMV does not require the ESCRTs for secondary envelopment. However, we found that ESCRT-III subunits are required for efficient virus spread. This suggests a role for ESCRT-III during the spread of HCMV that is independent of viral envelopment.

    IMPORTANCE Human cytomegalovirus (HCMV) is a prevalent opportunistic pathogen in the human population. HCMV infection in neonatal and immunocompromised patients can cause severe and possibly life-threatening complications in these at-risk patients. It is important to define mechanisms of the viral replication cycle to identify potential targets for new therapies. Secondary envelopment, or acquisition of the membrane envelope, of HCMV is a mechanism that needs further study. Using an inducible fibroblast system to carefully control for the toxicity associated with blocking ESCRT-III function, this study determines that the ESCRT proteins are not required for viral envelopment. However, the study does discover a non-envelopment role for the ESCRT-III complex in the efficient spread of the virus. Thus, this study advances our understanding of an important process essential for the replication of HCMV.

  • Ortervirales: A new viral order unifying five families of reverse-transcribing viruses [PublishAheadOfPrint]

  • Reverse-transcribing viruses, which synthesize a copy of genomic DNA from an RNA template, are widespread in animals, plants, algae and fungi (1, 2)....

  • SIVmac239 Vif and Human APOBEC3B Interactions Resemble Those Between HIV-1 Vif and Human APOBEC3G [PublishAheadOfPrint]

  • Several members of the APOBEC3 DNA cytosine deaminase family can potently inhibit Vif-deficient HIV-1 by catalyzing cytosine deamination in viral cDNA and impeding reverse transcription. HIV-1 counteracts restriction with the virally encoded Vif protein, which targets relevant APOBEC3 proteins for proteasomal degradation. HIV-1 Vif is optimized for degrading the restrictive human APOBEC3 repertoire and, in general, lentiviral Vif proteins specifically target the restricting APOBEC3 enzymes of each host species. However, SIVmac239 Vif elicits a curiously wide range of APOBEC3 degradation capabilities that include several human APOBEC3s and even human APOBEC3B, a non-HIV-1 restricting APOBEC3 enzyme. To better understand the molecular determinants of the interaction between SIVmac239 Vif and human APOBEC3B, we analyzed an extensive series of mutants. We found that SIVmac239 Vif interacts with the N-terminal domain of human APOBEC3B and, interestingly, that this occurs within a structural region homologous to the HIV-1 Vif interaction surface of human APOBEC3G. An alanine scan of SIVmac239 Vif revealed several residues required for human APOBEC3B degradation activity. These residues overlap with HIV-1 Vif surface residues that interact with human APOBEC3G, and are distinct from those that engage APOBEC3F or APOBEC3H. Overall, these studies indicate that the molecular determinants of the functional interaction between human APOBEC3B and SIVmac239 Vif resemble those between human APOBEC3G and HIV-1 Vif. These studies contribute to growing knowledge of the APOBEC-Vif interaction and may help guide future efforts to disrupt this interaction as an antiviral therapy or exploit the interaction as a novel strategy to inhibit APOBEC3B-dependent tumor evolution.

    IMPORTANCE Primate APOBEC3 proteins provide innate immunity against retroviruses such as HIV and SIV. HIV-1, the primary cause of AIDS, utilizes its Vif protein to specifically counteract restrictive human APOBEC3 enzymes. SIVmac239 Vif exhibits a much wider range of anti-APOBEC3 activities that includes several rhesus macaque enzymes and extends to multiple proteins in the human APOBEC3 repertoire including APOBEC3B. Understanding the molecular determinants of the interaction between SIVmac239 Vif and human APOBEC3B adds to existing knowledge on the APOBEC3-Vif interaction and has potential to shed light on what processes may have shaped Vif functionality over evolutionary time. An intimate understanding of this interaction may also lead to a novel cancer therapy because, for instance, creating a derivative of SIVmac239 Vif that specifically targets human APOBEC3B could be used to suppress tumor genomic DNA mutagenesis by this enzyme, slow ongoing tumor evolution, and help prevent poor clinical outcomes.

  • Functional relevance of the transmembrane domain and cytoplasmic tail of the pseudorabies virus glycoprotein H for membrane fusion [PublishAheadOfPrint]

  • Herpesvirus membrane fusion depends on the core fusion machinery comprising glycoproteins (g)B and gH/gL. Although gB structurally resembles autonomous class III fusion proteins, it strictly depends on gH/gL to drive membrane fusion. Whether the gH/gL complex needs to be membrane-anchored to fulfill its function and which role the gH cytoplasmic (CD) and transmembrane domains (TMD) play in fusion is unclear. While the gH CD and TMD play an important role during infection, soluble gH/gL of herpes simplex virus 1 (HSV-1) seems to be sufficient to mediate cell-cell fusion in transient assays arguing against an essential contribution of the CD and TMD. To shed more light on this apparent discrepancy we investigated the role of the CD and TMD of the related alphaherpesvirus pseudorabies virus (PrV) gH. For this purpose, we expressed C-terminally truncated and soluble gH, and substituted the TMD by a glycosylphosphatidylinositol (gpi)-anchor. We also generated chimeras containing the TMD and/or CD of PrV gD or HSV-1 gH. Proteins were characterized in cell-based fusion assays and during virus infection. Although truncation of the CD resulted in decreased membrane fusion activity, the mutant proteins still supported replication of gH-negative PrV, indicating that the PrV gH CD is dispensable for viral replication. In contrast, PrV gH lacking the TMD, membrane-anchored via a lipid-linker, or comprising the PrV gD TMD were non-functional highlighting the essential role of the gH TMD for function. Interestingly, despite low sequence identity, the HSV-1 gH TMD could substitute for the PrV gH TMD pointing to functional conservation.

    IMPORTANCE Enveloped viruses depend on membrane fusion for virus entry. While this process can be mediated by only one or two proteins herpesviruses depend on the concerted action of at least three different glycoproteins. Although gB has features of bona-fide fusion proteins, it depends on gH and its complex partner gL for fusion. Whether gH/gL prevents premature fusion or actively triggers gB-mediated fusion is unclear and there are contradictory results on whether gH/gL function requires stable membrane anchorage or whether the ectodomains alone are sufficient. Our results show that in pseudorabies virus gH the transmembrane anchor plays an essential role for gB-mediated fusion while the cytoplasmic tail is not strictly required.

  • Porcine deltacoronavirus engages the transmissible gastroenteritis virus functional receptor porcine aminopeptidase N for infectious cellular entry [PublishAheadOfPrint]

  • Identification of cellular receptors used by coronavirus (CoV) entry into the host cells is critical to understand pathogenesis and to develop intervention strategies. The fourth CoV genus, Deltacoronavirus, evolutionally related to the Gammacoronavirus, has just been defined recently. In the current study, we demonstrate that porcine aminopeptidase N (pAPN) acts as a cross-genus CoV functional receptor for both enteropathogenic porcine DeltaCoV (PDCoV) and AlphaCoV (transmissible gastroenteritis virus, TGEV) based upon three lines of evidences. First, the soluble S1 protein of PDCoV efficiently bound to surface of target porcine cell lines known to express pAPN as TGEV-S1 did, which could be blocked by soluble pAPN pre-treatment. Second, either PDCoV-S1 or TGEV-S1 physically recognized and interacted with pAPN by co-immunoprecipitation in pAPN-cDNA-transfected cells and by dot blot hybridization assay. Finally, exogenous expression of pAPN in refractory cells conferred susceptibility to PDCoV-S1 binding and for PDCoV entry and productive infection. PDCoV-S1 appeared to have a lower pAPN-binding affinity and likely consequent lower infection efficiency in pAPN-expressing refractory cells as compared to TGEV-S1, suggesting that there may be difference in virus-binding regions in pAPN between these two viruses. This study paves the way for dissecting the molecular mechanisms of PDCoV-host interactions and pathogenesis as well as facilitates future vaccine development and intervention strategies against PDCoV infection.

    IMPORTANCE The emergence of new human and animal coronaviruses is believed to have occurred through interspecies transmission that is mainly mediated by species-specific receptor of the host. Among the four genera of the Coronavirinae, a couple functional receptors for the representative members in the genera Alphacoronavirus and Betacoronavirus have been identified, whereas receptors for Gammacoronavirus and Deltacoronavirus, which are believed to originate from birds, are still unknown. Porcine coronaviruses including the newly discovered porcine deltacoronavirus (PDCoV) associated with diarrhea in newborn piglets have posed a serious threat to the pork industry in Asia and North America. Here we report that PDCoV employs alphacoronavirus TGEV functional receptor porcine aminopeptidase N (pAPN) for cellular entry, demonstrating the usage of pAPN as a cross-genus CoV functional receptor. The identification of PDCoV receptor provides another example of the expanded host range of CoV, and paves the way for further investigation of PDCoV-host interaction and pathogenesis.

  • Comparison of Uncleaved and Mature Human Immunodeficiency Virus (HIV-1) Membrane Envelope Glycoprotein Trimers [PublishAheadOfPrint]

  • The mature envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus (HIV-1) infected cells and virions is derived by proteolytic cleavage of a trimeric gp160 glycoprotein precursor. In these studies, we compared the conformations of cleaved and uncleaved membrane Envs with truncated cytoplasmic tails to those of stabilized soluble gp140 SOSIP.664 Env trimers. Deletion of the gp41 cytoplasmic tail did not significantly affect the sensitivity of viruses with the HIV-1AD8 Env to inhibition by antibodies or a CD4-mimetic compound. After glutaraldehyde fixation and purification from membranes, a cleaved Env exhibited a hydrodynamic radius of ~10 nm and an antibody-binding profile largely consistent with that expected based on virus neutralization sensitivity. The purified cleaved Env trimers exhibited a hollow architecture with a central void near the trimer axis. Uncleaved Env, crosslinked and purified in parallel, exhibited a hydrodynamic radius similar to that of the cleaved Env. However, the uncleaved Env was recognized by poorly neutralizing antibodies and appeared to sample multiple conformations by negative-stain electron microscopy. Compared with membrane Envs, stabilized soluble gp140 SOSIP.664 Env trimers appear to be more compact, as reflected in their smaller hydrodynamic radii and negative-stain electron microscopy structures. The antigenic features of the soluble gp140 SOSIP.664 Env trimers differed from that of the cleaved membrane Env, particularly in gp120 V3 and some CD4-binding site epitopes. Thus, proteolytic maturation allows the membrane-anchored Env to achieve a conformation that retains functional metastability but masks epitopes for poorly neutralizing antibodies.

    IMPORTANCE The entry of human immunodeficiency virus (HIV-1) into host cells is mediated by the envelope glycoprotein (Env) spike on the surface of the virus. Host antibodies elicited during natural HIV-1 infection or by vaccination can potentially recognize the Env spike and block HIV-1 infection. However, the changing shape of the HIV-1 Env spike protects the virus from antibody binding. Understanding the shape of natural and man-made preparations of HIV-1 Envs will assist the development of effective vaccines against this virus. Here, we evaluate the effect of several Env modifications commonly used to produce Env preparations for vaccine studies and the determination of structure. We found that the cleavage of the HIV-1 Env precursor helps Env to assume its natural shape, which resists the binding of many commonly elicited antibodies. Stabilized soluble Envs exhibit more compact shapes, but expose some Env elements differently than the natural Env.

  • Identification of the RNA Pseudoknot within the 3 End of PRRSV Genome as Pathogen Associated Molecular Pattern to Activate Antiviral Signaling via RIG-I and TLR3 [PublishAheadOfPrint]

  • Once infected by viruses, cells can detect pathogen associated molecular patterns (PAMPs) on viral nucleic acid by host pattern recognition receptors (PRRs) to initiate the antiviral response. Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of porcine reproductive and respiratory syndrome (PRRS), characterized by reproductive failure in sows and respiratory diseases in pigs of different ages. To date, the sensing mechanism of PRRSV has not been elucidated. Here, we reported that the pseudoknot region residing in the 3rrsquo; untranslated regions (UTR) of PRRSV genome, which has been proposed to regulate RNA synthesis and virus replication, was sensed as non-self by retinoic acid-inducible gene I (RIG-I) and Toll like receptor (TLR3) and strongly induced type I IFNs and interferon-stimulated genes (ISGs) in porcine alveolar macrophages (PAMs). The interaction between the two stem-loops inside the pseudoknot structure was sufficient for IFN induction, since disruption of the pseudoknot interaction powerfully dampened the IFN induction. Furthermore, transfection of the 3rrsquo; UTR pseudoknot transcripts in PAMs inhibited PRRSV replication in vitro. Importantly, the predicted similar structures of other arterivirus members, including equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV) also displayed strong IFN induction activities. Together, in this work we identified an innate recognition mechanism by which PRRSV 3rrsquo; UTR pseudoknot region served as PAMPs of arteriviruses and activated innate immune signaling to produce IFNs that inhibit virus replication. All these results provide novel insights into innate immune recognition during virus infection.

    IMPORTANCE PRRS is the most common viral disease in pork industry. It is caused by PRRSV, a positive single-stranded RNA, whose infection often leads to persistent infection. To date, it is not yet clarified that how PRRSV is recognized by host and what is the exact mechanism of IFN induction. Here, we investigated the nature of PAMPs on PRRSV and the associated PRRs. We found that, the 3rrsquo; UTR pseudoknot region of PRRSV, which has been proposed to regulate viral RNA synthesis, could act as PAMPs recognized by RIG-I and TLR3 to induce type I IFN production to suppress PRRSV infection. This report is the first detailed description of pattern recognition for PRRSV, which is important in understanding the antiviral response of arteriviruses, especially PRRSV, and extends our knowledge on virus recognition.

  • Brain large artery lymphocytic inflammation and Human Immunodeficiency Virus-related brain arterial remodeling [PublishAheadOfPrint]

  • The pathogenesis of increased stroke risk in Human Immunodeficiency Virus (HIV) remains unclear. Our study investigated the relationship between adventitial and intimal CD3+ T cells and brain arterial remodeling that potentially contributes to HIV-related vasculopathy and stroke. Large brain arteries from 84 HIV+ cases and 78 HIV- cases were analyzed to determine interadventitial and luminal diameters, intimal and wall thickness, percentage stenosis, and presence of atherosclerosis. Immunohistochemical analysis was performed to detect and visually score CD3, a pan T cell marker, in the intima and adventitia. Our study showed that adventitial CD3+ T cells are lower among persons with HIV, especially if CD4llt;200, though intimal CD3+ T cells did not differ by HIV status. Among those with HIV but CD4llt;200 at time of death, intimal CD3+ T cells were associated with hypertrophic outward remodeling, while among those with HIV and CD4ggt;200 or HIV- controls, intimal CD3+ T cells were associated with hypertrophic inward remodeling. We conclude that intimal lymphocytic inflammation is involved in brain arterial remodeling that may contribute to HIV-related cerebrovascular pathology.

    IMPORTANCE Although mortality from Human Immunodeficiency Virus (HIV) has decreased with the use of combination antiretroviral therapies, there is now an increased risk of cardiovascular and cerebrovascular disease associated with HIV. Thus, there is a need to understand the pathogenesis of stroke in HIV infection. Our study examines how lymphocytic inflammation in brain arteries may contribute to increased cerebral vasculopathy. With this understanding, our study can potentially help direct future therapies to target and prevent brain arterial remodeling processes associated with HIV.

  • Large scale complete genome sequencing and phylodynamic analysis of eastern equine encephalitis virus reveal source-sink transmission dynamics in the United States [PublishAheadOfPrint]

  • Eastern equine encephalitis virus (EEEV) has a high case-fatality rate in horses and humans, and Florida has been hypothesized to be the source of EEEV epidemics for the northeastern U.S. To test this hypothesis, we sequenced complete genomes of 433 EEEV strains collected within the U.S. from 1934 to 2014. Phylogenetic analysis suggested EEEV evolves relatively slowly and that transmission is enzootic in Florida, characterized by higher genetic diversity and long-term local persistence. In contrast, EEEV in New York and Massachusetts were characterized by lower genetic diversity, multiple introductions, and shorter local persistence. Our phylogeographic analysis supported a source-sink model in which Florida is the major source of EEEV compared to the other localities sampled. In sum, this study revealed the complex epidemiological dynamics of EEEV in different geographic regions in the U.S., and provided general insights into the evolution and transmission of other avian mosquito-borne viruses in this region.

    IMPORTANCE Eastern equine encephalitis virus (EEEV) infections are severe in horses and humans on the east coast of the United States with over 90% mortality rate in horses, approximately 33% mortality rate in humans, and significant brain damage in most human survivors. However, little is known about the evolutionary characteristics of EEEV due to the lack of genome sequences. By generating large collection of publicly-available complete genome sequences, this study comprehensively determined the evolution of the virus, described the epidemiological dynamics of EEEV in different states in the U.S., and identified Florida as one of the major sources. These results may have important implications for the control and prevention of other mosquito-borne viruses in the Americas.

  • Feline herpesvirus 1 (FHV-1) US3 blocks type I IFN signal pathway by targeting IRF3 dimerization in a kinase-independent manner [PublishAheadOfPrint]

  • As a prevalent agent in cats, feline herpesvirus 1 (FHV-1) infection contributes to feline respiratory disease and acute and chronic conjunctivitis. FHV-1 can successfully evade the host innate immune response and persist for the lifetime of the cat. Several mechanisms of immune evasion by human herpesviruses have been elucidated, but the mechanism by FHV-1 remains unknown. In this study, we screened for FHV-1 ORFs responsible for inhibiting type I interferon (IFN) pathway with an IFN-bbeta; promoter reporter and an analysis of IFN-bbeta; mRNA levels in HEK 293T cells and CRFK feline cell line, and we identified the Ser/Thr kinase US3 as the most powerful inhibitor. Furthermore, we found that the anti-IFN activity of US3 depended on its N-terminus (1-75 aa) and was independent of its kinase activity. Mechanistically, the ectopic expression of US3 selectively inhibited IFN regulatory factor 3 (IRF3) promoter activation. Furthermore, US3 bound to the IRF association domain (IAD) of IRF3 and prevented IRF3 dimerization. Finally, US3-deleted and US3-repaired recombinant FHV-1 (rFHV-dUS3 and rFHV-rUS3, respectively) were constructed. Compared with wild-type FHV-1 and rFHV-rUS3, infection with rFHV-dUS3 induced large amounts of IFN-bbeta; in vitro and in vivo. More importantly, US3 deletion significantly attenuated virulence, reduced virus shedding and blocked the invasion of trigeminal ganglia. These results indicate that FHV-1 US3 efficiently inhibits IFN induction using a novel immune evasion mechanism and that FHV-1 US3 is a potential regulator of neurovirulence.

    IMPORTANCE Despite widespread vaccination, the prevalence of FHV-1 remains high, suggesting that it can successfully evade the host innate immune response and infect cats. In this study, we screened viral proteins for inhibiting IFN pathway and identified the Ser/Thr kinase US3 as the most powerful inhibitor. In contrast to other members of alphaherpesvirus, FHV-1 US3 blocked host type I IFN pathway in a kinase-independent manner and via binding to the IRF3 IAD domain and preventing IRF3 dimerization. More importantly, depletion of US3 attenuated the anti-IFN activity of FHV-1 and prevented efficient viral replication in vitro and in vivo. Also, US3 deletion significantly attenuated virulence and blocked the invasion of trigeminal ganglia. We believe that these findings will not only help us to better understand the mechanism about how FHV-1 manipulates host IFN response, but also highlight the potential role of US3 in the establishment of latent infection in vivo.

  • Ac102 Participates in Nuclear Actin Polymerization by Modulating BV/ODV-C42 Ubiquitination during Autographa californica Multiple Nucleopolyhedrovirus Infection [PublishAheadOfPrint]

  • As a virus-encoded actin nucleation promoting factor (NPF), P78/83 induces actin polymerization to assist in Autographa californica multiple nucleopolyhedrovirus (AcMNPV) propagation. According to our previous study, although P78/83 actively undergoes ubiquitin-independent proteasomal degradation, AcMNPV encodes BV/ODV-C42 (C42), which allows P78/83 to function as a stable NPF by inhibiting its degradation during viral infection. However, whether there are other viral proteins involved in regulating P78/83-induced actin polymerization has yet to be determined. In this study, we found that Ac102, an essential viral gene product previously reported to play a key role in mediating the nuclear accumulation of actin during AcMNPV infection, is a novel regulator of P78/83-induced actin polymerization. By characterizing an ac102 knockout bacmid, we demonstrated that Ac102 participates in regulating nuclear actin polymerization as well as the morphogenesis and distribution of capsid structures in the nucleus. These regulatory effects are heavily dependent on an interaction between Ac102 and C42. Further investigation revealed that Ac102 binds to C42 to suppress K48-linked ubiquitination of C42, which decreases C42 proteasomal degradation and consequently allows P78/83 to function as a stable NPF to induce actin polymerization. Thus, Ac102 and C42 form a regulatory cascade to control viral NPF activity, representing a sophisticated mechanism for AcMNPV to orchestrate actin polymerization in both a ubiquitin-dependent and ubiquitin-independent manner.

    IMPORTANCE Actin is one of the most functionally important proteins in eukaryotic cells. Morphologically, actin can be found in two forms: a monomeric form called globular actin (G-actin) and a polymeric form called filamentous actin (F-actin). G-actin can polymerize to form F-actin, and nucleation promoting factor (NPF) is the initiator of this process. Many viral pathogens harness the host actin polymerization machinery to assist in virus propagation. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) induces actin polymerization in host cells. P78/83, a viral NPF, is responsible for this process. Previously, we identified that BV/ODV-C42 (C42) binds to P78/83 and protects it from degradation. In this report, we determined that another viral protein, Ac102, is involved in modulating C42 ubiquitination and consequently ensures P78/83 activity as an NPF to initiate actin polymerization. This regulatory cascade represents a novel mechanism by which a virus can harness the cellular actin cytoskeleton to assist in viral propagation.

  • A Highly Conserved Leucine In Mammarenavirus Matrix Z Protein Is Required For Z Interaction With The Virus L Polymerase And Z Stability In Cells Harboring An Active Viral Ribonucleoprotein [PublishAheadOfPrint]

  • Mammarenaviruses cause chronic infections in their natural rodent hosts. Infected rodents shed infectious virus into excreta. Humans are infected through mucosal exposure to aerosols or direct contact of abraded skin with fomites, resulting in a wide-range of manifestations from asymptomatic or mild febrile illness to severe life-threatening hemorrhagic fever. The mammarenavirus matrix Z protein has been shown to be a main driving force of virus budding and to act as a negative regulator of viral RNA synthesis. To gain a better understanding of how the Z protein exerts its several different functions, we investigated the interaction between Z and viral polymerase L protein using the prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV). We found that in the presence of an active viral ribonucleoprotein (vRNP), the Z protein translocated from non-ionic detergent-resistant, membrane-rich structures to a subcellular compartment with a different membrane composition susceptible to disruption by non-ionic detergents. Alanine (A) substitution of a highly conserved leucine (L) at position 72 in LCMV Z protein abrogated Z-L interaction. The L72A mutation did not affect the stability or budding activity of Z when expressed alone, but in the presence of an active vRNP, mutation L72A promoted rapid degradation of Z via a proteasome and lysosome independent pathway. Accordingly, L72A mutation in the Z protein resulted in non-viable LCMV. Our findings have uncovered novel aspects of the dynamics of the Z protein for which a highly conserved L residue was strictly required.

    IMPORTANCE Several mammarenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever disease in humans and pose important public health concerns in their endemic regions. Moreover, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), is a neglected human pathogen of clinical significance. The mammarenavirus matrix Z protein plays critical roles in different steps of the viral life cycle by interacting with viral and host cellular components. Here we report that alanine substitution of a highly conserved leucine residue, located at position 72 in LCMV Z protein, abrogated Z-L interaction. The L72A mutation did not affect Z budding activity, but promoted its rapid degradation in the presence of an active viral ribonucleoprotein (vRNP). Our findings have uncovered novel aspects of the dynamics of the Z protein for which a highly conserved L residue was strictly required.

  • A vaccine platform against arenaviruses based on a recombinant hyper-attenuated Mopeia virus expressing heterologous glycoproteins [PublishAheadOfPrint]

  • Several Old and New-World arenaviruses are responsible for severe endemic and epidemic hemorrhagic fevers, whereas other members of the Arenaviridae family are nonpathogenic. To date, no approved vaccines, antiviral or specific treatments are available, except for Juniiacute;n virus. Protection in non-human primates against Lassa fever virus (LASV) is however possible through the inoculation of the closely related but nonpathogenic Mopeia virus (MOPV) before challenge with LASV. We reasoned that this virus, modified using reverse genetics, would represent the basis for the generation of a vaccine platform against LASV and other pathogenic arenaviruses. After showing evidence of exoribonuclease (ExoN) activity in the NP of MOPV, we found that this activity was essential for multiplication in antigen presenting cells. The introduction of multiple mutations in the ExoN site of MOPV NP generated a hyper-attenuated strain (MOPVExoN6b) which is (i) genetically stable over passages, (ii) has increased immunogenic properties compared to MOPV and (iii) still promotes a strong type I IFN response. The MOPVExoN6b virus was further modified to harbor the envelope glycoproteins of heterologous pathogenic arenaviruses, such as LASV, Lujo, Machupo, Guanarito, Chapare, or Sabia viruses in order to broaden specific antigenicity while preserving the hyper-attenuated characteristics of the parental strain. Our MOPV based vaccine candidate for LASV, MOPEVACLASV, was used in a one-shot immunization assay in non-human primates and fully protected them from a lethal challenge with LASV. Thus, our hyper-attenuated strain of MOPV constitutes a promising new live-attenuated vaccine platform to immunize against several, if not all, pathogenic arenaviruses.

    Importance

    Arenaviruses are emerging pathogens transmitted to humans by rodents and responsible for endemic and epidemic hemorrhagic fevers of global concern. Nonspecific symptoms associated with the onset of infection make these viruses difficult to distinguish from other endemic pathogens. Moreover, the unavailability of rapid diagnosis in the field delays the identification of the virus, early care for treatment, and favors spreading. The vaccination of exposed populations would be of great help to decrease morbidity and human-to-human transmission. Using reverse genetics, we generated a vaccine platform for pathogenic arenaviruses based on a modified and hyper-attenuated strain of the nonpathogenic Mopeia virus and showed that the Lassa virus candidate fully protected non-human primates from a lethal challenge. These results showed that a rationally designed recombinant MOPV-based vaccine is safe, immunogenic and efficacious in non-human primates.

  • IL-27R signalling mediates early viral containment and impacts innate and adaptive immunity after chronic lymphocytic choriomeningitis virus infection [PublishAheadOfPrint]

  • Chronic viral infections represent a major challenge to host's immune response and a unique network of immunological elements, including cytokines, are required for their containment. By using a model persistent infection with the natural murine pathogen lymphocytic choriomeningitis virus clone 13 (LCMV Cl13) we investigated the role of one such cytokine, interleukin 27 (IL-27), in the control of chronic infection. We found that IL-27R signalling promoted control of LCMV Cl13 as early as day 1 and 5 after infection and that il27p28 transcripts were rapidly elevated in multiple subsets of dendritic cells (DCs) and myeloid cells. In particular, plasmacytoid DCs (pDCs), the most potent type-1-interferon (IFN-I) producing cells, significantly increased il27p28 in a TLR7 dependent fashion. Notably, mice deficient in IL-27 specific receptor (R), WSX-1, exhibited a pleiotropy of innate and adaptive immune alterations after chronic LCMV infection, including compromised NK cell cytotoxicity and antibody responses. While, the majority of these immune alterations appeared cell-extrinsic, cell-intrinsic IL-27R was necessary to maintain early pDC numbers, which, alongside lower IFN-I transcription in CD11b+ DCs and myeloid cells, may explain the compromised IFN-I elevation that we observed early after LCMV Cl13 infection in IL-27R-deficient mice. Together these data highlight the critical role of IL-27 in enabling optimal anti-viral immunity early and late after infection with a systemic persistent virus and suggest that a previously unrecognized positive feedback-loop mediated by IL-27 in pDCs might be involved in this process.

    IMPORTANCE Persistently replicating pathogens such as Human Immunodeficiency virus, Hepatitis B virus and Hepatitis C virus represent a major health problem worldwide. These infections impose a long-term challenge on the host immune system, which must be heavily and continuously regulated to keep the pathogen replication in check without causing fatal immunopathology. Using a persistently replicating rodent pathogen, lymphocytic choriomeningitis virus (LCMV), in its natural host we identified the cellular sources and effects of one important regulatory pathway, the interleukin-27 receptor WSX-1 signalling, that is required for both very early and late restriction of chronic (but not acute) infection. We found that WSX-1 was necessary to promote innate immunity and the development of aberrant adaptive immune responses. This not only highlights the role of IL-27 receptor signalling in regulating distinct host responses that are known to be necessary to control chronic infections but also positions IL-27 as a potential therapeutic target for their modulation.

  • Extracellular conformational changes in the capsid of human papillomaviruses contribute to asynchronous uptake into host cells [PublishAheadOfPrint]

  • The human papillomavirus type 16 (HPV16) is the leading cause of cervical cancer. For initial infection, HPV16 utilizes a novel endocytic pathway for host cell entry. Unique amongst viruses, uptake occurs asynchronously over a protracted period of time with half-times between 9-12 h. To trigger endocytic uptake, the virus particles need to undergo a series of structural modifications after initial binding to heparan sulfate proteoglycans (HSPG). These changes involve proteolytic cleavage of the major capsid protein L1 by kallikrein-8 (KLK8), exposure of the N-terminus of the minor capsid protein L2 by cyclophilins, and cleavage of this N-terminus by furin. Overall, the structural changes are thought to facilitate the engagement of an elusive secondary receptor for internalization. Here, we addressed whether structural changes are the rate-limiting steps during infectious internalization of HPV16 by using structurally-primed HPV16 particles. Our findings indicate that the structural modifications mediated by cyclophilins and furin, which lead to exposure and cleavage of the L2 N-terminus, respectively, contribute to the slow and asynchronous internalization kinetics, whereas conformational changes elicited by HSPG binding and KLK8 cleavage did not. However, these structural modifications only accounted for 30-50% of the delay in internalization. Therefore, we propose that limited internalization receptor availability for engagement of HPV16 causes slow and asynchronous internalization in addition to rate-limiting structural changes in the viral capsid.

    IMPORTANCE HPVs are the main cause for anogenital cancers. Their unique biology is linked to the differentiation program of skin or mucosa. Here, we analyzed another unique aspect of HPV infections using the prototype HPV16. After initial cell binding, HPVs display an unusually protracted residence time on the plasma membrane prior to asynchronous uptake. As viruses typically do not expose themselves to host immune sensing, we analyzed the underlying reasons for this unusual behavior. This study provides evidence that both extracellular structural modifications and possibly a limited availability of the internalization receptor contribute to the slow internalization process of the virus. These findings indicated that perhaps a unique niche for initial infection exists that could allow for rapid infection. In addition, our results may help to develop novel, preventive antiviral measures.

  • Degradation of a novel DNA damage response protein, tankyrase 1 binding protein 1 (Tab182), following adenovirus infection. [PublishAheadOfPrint]

  • Infection by most DNA viruses activates a cellular DNA damage response (DDR), which may be to the detriment or advantage of the virus. In the case of adenoviruses, they neutralise anti-viral effects of DDR activation by targeting a number of proteins for rapid proteasome-mediated degradation. We have now identified a novel DDR protein, tankyrase 1 binding protein 1 (TNKS1BP1 also known as Tab182), which is degraded during infection by adenovirus 5 and adenovirus 12. In both cases, degradation requires the action of E1B55K and E4orf6 viral proteins and is mediated through the proteasome by the action of cullin-based cellular E3 ligases. The degradation of Tab182 appears to be serotype specific as the protein remains relatively stable following infection with adenoviruses 4, 7, 9 and 11. We have gone on to confirm that Tab182 is an integral component of the CNOT complex, which has transcriptional regulatory, deadenylation and E3 ligase activity. At least 2 other members of the complex (CNOT3 and CNOT7) are also reduced in level during adenovirus infection whereas levels of CNOT4 and CNOT1 remain stable. Depletion of Tab182 with siRNA enhances expression of E1As to a limited extent during adenovirus infection but depletion of CNOT1 is particularly advantageous to the virus and results in a marked increase in expression of adenovirus early proteins. In addition, depletion of Tab182 and CNOT1 results in a limited increase in viral DNA during infection. We conclude that the cellular CNOT complex is a previously unidentified major target for adenoviruses during infection.

    Importance

    Adenoviruses target a number of cellular proteins involved in the DNA damage response for rapid degradation. We have now shown that Tab182, which we have confirmed to be an integral component of the mammalian CNOT complex, is degraded following infection by adenovirus serotypes 5 and 12. This requires the viral E1B55K and E4orf6 proteins and is mediated by cullin-based E3 ligases and the proteasome. In addition to Tab182, other CNOT proteins are also reduced during adenovirus infection. Thus, CNOT3 and CNOT7, for example, are degraded whereas CNOT4 and CNOT1 are not. siRNA-mediated depletion of components of the complex enhances the expression of adenovirus early proteins and increases the concentration of viral DNA produced during infection. This study highlights a novel protein complex, CNOT, which is targeted for adenovirus-mediated protein degradation. To our knowledge this is the first time that the CNOT complex has been identified as an adenoviral target.

  • eCD4-Ig variants that more potently neutralize HIV-1 [PublishAheadOfPrint]

  • The HIV-1 entry inhibitor eCD4-Ig is a fusion of CD4-Ig and a coreceptor-mimetic peptide. eCD4-Ig is markedly more potent than CD4-Ig, with neutralization efficiencies approaching those of HIV-1 broadly neutralizing antibodies (bNAbs). However, unlike bNAbs, eCD4-Ig neutralizes all HIV-1, HIV-2 and SIV isolates that it has been tested against, suggesting that it may be useful in clinical settings where antibody escape is a concern. Here we characterize three new eCD4-Ig variants, each with different architectures and each utilizing D1.22, a stabilized form of CD4 domain 1. These variants were 10- to 20-fold more potent than our original eCD4-Ig variant, with a construct bearing four D1.22 domains (eD1.22-HL-Ig) exhibiting the greatest potency. However, this variant mediated less efficient antibody-dependent cell-mediated cytotoxicity (ADCC) activity than eCD4-Ig itself or several other eCD4-Ig variants, including the smallest variant (eD1.22-Ig). A variant with the same architecture as original eCD4-Ig (eD1.22-D2-Ig) showed modestly higher thermal stability and best prevented promotion of infection of CCR5-positive, CD4-negative cells. All three variants, and eCD4-Ig itself, mediated more efficient shedding of the HIV-1 envelope glycoprotein gp120 than did CD4-Ig. Finally, we show that only three D1.22 mutations contributed to the potency of eD1.22-D2-Ig, and that introduction of these changes into eCD4-Ig resulted in a variant 9-fold more potent than eCD4-Ig and 2-fold more potent than eD1.22-D2-Ig. These studies will assist in developing eCD4-Ig variants with properties optimized for prophylaxis, therapy, and cure applications.

    IMPORTANCE HIV-1 bNAbs have properties different from antiretroviral compounds. Specifically, antibodies can enlist immune effector cells to eliminate infected cells, whereas antiretroviral compounds simply interfere with various steps in the viral lifecycle. Unfortunately, HIV-1 is adept at evading antibody recognition, limiting the utility of antibodies as a treatment for HIV-1 infection or part of an effort to eradicate latently infected cells. eCD4-Ig is an antibody-like entry inhibitor that closely mimics HIV-1's obligate receptors. eCD4-Ig appears to be qualitatively different from antibodies since it neutralizes all HIV-1, HIV-2, and SIV isolates. Here we characterize three new structurally distinct eCD4-Ig variants, and show that each excels in a key property useful to prevent, treat or cure an HIV-1 infection. For example, one variant neutralized HIV-1 most efficiently, while others best enlisted natural killer cells to eliminate infected cells. These observations will help generate eCD4-Ig variants optimized for different clinical applications.

  • Mosquito saliva re-shapes alphavirus infection and immunopathogenesis [PublishAheadOfPrint]

  • Alphaviruses are transmitted to humans via bites of infected mosquitoes. Although alphaviruses have caused a wide magnitude of outbreaks and crippling disease, licensed vaccines or antiviral therapies remain limited. Mosquito vectors such as Aedes and Culex are the main culprits in the transmission of alphaviruses. This review explores how mosquito saliva may promote alphavirus infection. Identifying the roles of mosquito-derived factors in alphavirus pathogenesis will generate novel tools to circumvent and control mosquito-borne alphavirus infections in humans.

  • Human group C rotavirus VP8*s recognize type A histo-blood group antigens as ligands [PublishAheadOfPrint]

  • Group/species C rotaviruses (RVCs) have been identified as important pathogens of acute gastroenteritis (AGE) in children, family-based outbreaks, as well as animal infections. However, little is known regarding their host-specific interaction, infection, and pathogenesis. In this study, we performed serial studies to characterize the function and structural features of a human G4P[lsqb]2[rsqb] RVC VP8* that is responsible for host receptor interaction. Glycan microarrays demonstrated that the human RVC VP8* recognizes type A histo-blood antigens (HBGAs), which was confirmed by synthetic glycan-/saliva-based binding assays, and hemagglutination of red blood cells, establishing a paradigm of RVC VP8*-glycan interactions. Furthermore, high resolution crystal structure of the human RVC VP8* was solved, showing a typical galectin-like structure consisting of two bbeta;-sheets but with significant differences to cogent proteins of RVAs. The VP8* in complex with a type A trisaccharide displays a novel ligand binding site that consists of a particular set of amino acid (aa) residues of the C-D, G-H, and K-L loops. RVC VP8* interacts with type A HBGAs through a unique mechanism compared with RVAs. Our findings shed light on the host-virus interaction and co-evolution of RVCs and will facilitate the development of specific antivirals and vaccines.

    IMPORTANCE Group/species C rotaviruses (RVCs), members of Reoviridae family, infect both humans and animals, but our knowledge about the host factors that control host susceptibility and specificity is rudimentary. In this work, we characterized the glycan binding specificity and structural basis of a human RVC that recognizes type A HBGAs. We found that human RVC VP8*, the RV host ligand binding domain that shares only ~15% homology to those of RVAs, recognizes type A HBGA at an as-yet-unknown glycan binding site through a distinct mechanism compared with RVAs. Our new advancements provide insights into RVC-cell attachment, the critical step of virus infection, which will in turn help the development of control and prevention strategies against RVs.

  • Central Role of the NF{kappa}B Pathway in the Scgb1a1-Expressing Epithelium in Mediating Respiratory Syncytial Virus-induced Airway Inflammation [PublishAheadOfPrint]

  • Lower respiratory tract infection with respiratory syncytial virus (RSV) produces profound inflammation. Despite an understanding of the role of adaptive immunity in RSV infection, the identity of the major sentinel cells initially triggering inflammation is controversial. Here we evaluate the role of nonciliated secretoglobin (Scgb1a1)-expressing bronchiolar epithelial cells in RSV infection. Knock-in mice expressing a tamoxifen (TMX)-inducible Cre recombinase (CreERTM) from the Scgb1a1 locus were crossed with mice that harbor a RelA conditional allele (RelAfl), with loxP sites flanking exons 5-8 of the Rel homology domain. The Scgb1a1CreERTM/+ X RelAfl/fl mouse is a RelA conditional knockout (RelACKO) of a nonciliated epithelial cell population enriched in the small bronchioles. TMX-treated RelACKO mice have reduced pulmonary neutrophilic infiltration and impaired expression and secretion of NFB-dependent cytokines in response to RSV. In addition, RelACKO mice had reduced expression of IRF1/7 and RIG-I, components of the mucosal IFN positive feedback loop. We demonstrate that RSV replication induces RelA to complex with bromodomain-containing protein 4 (BRD4), a cofactor required for RNA Pol II phosphorylation, activating the atypical histone acetyltransferase (HAT) activity of BRD4 required for phospho-Ser 2 Pol II formation, histone H3K122 acetylation and cytokine secretion in vitro and in vivo. TMX-treated RelACKO mice have less weight loss, reduced airway obstruction/hyperreactivity, yet similar levels of IFN production despite higher viral production. These data indicate that the nonciliated Scgb1a1-expressing epithelial is a major innate sensor for restricting RSV infection by mediating neutrophilic inflammation, chemokine and mucosal IFN production via the RelA-BRD4 pathway.

    IMPORTANCE RSV infection is the most common cause of infant hospitalization in the US, resulting in 2.1 M children annually requiring medical attention. RSV primarily infects nasal epithelial cells, spreading distally to produce severe lower respiratory tract infections. Our study examines the role of a nonciliated respiratory epithelial cell population in RSV infection. We genetically engineered a mouse that can be selectively depleted of the NFB/RelA transcription factor in this subset of epithelial cells. These mice show impaired activation of the bromodomain-containing protein 4 (BRD4) coactivator, resulting in reduced cytokine expression and neutrophilic inflammation. During the course of RSV infection, epithelial RelA-depleted mice have reduced disease scores, airway hyperreactivity yet increased levels of virus replication. We conclude RelAssdot;BRD4 signaling in nonciliated bronchiolar epithelial cells mediates neutrophilic airway inflammation and disease severity. This complex is an attractive target to reduce the severity of infection.

  • Improvement of Dengue Non-Human Primate Model via a Reverse Translational Approach Based on Dengue Vaccine Clinical Efficacy Data against Dengue 2 and Dengue 4 [PublishAheadOfPrint]

  • Recent data obtained with the live-attenuated tetravalent dengue CYD-TDV vaccine showed higher protective efficacy against DENV-4 compared with DENV-2. In contrast, results from earlier studies in non-human primates predicted comparable high levels of protection against each serotype. Maximum viral loads achieved in macaques by subcutaneous inoculation of DENV are generally much lower than those observed in naturally dengue-infected humans. This may contribute to overestimation of vaccine efficacy. Using more stringent DENV infection conditions consisting of intravenous inoculation of a 107 CCID50 dose in CYD-TDV-vaccinated macaques, complete protection (i.e. undetectable viral RNA), was achieved in all 6 monkeys challenged with DENV-4, and in 6/18 of those challenged with DENV-2, including transiently positive animals. All other infected macaques (12/18) developed sustained DENV-2 RNAemia, although 1 to 3 log10 below the levels achieved in control animals. Similar results were obtained in macaques immunized with either CYD-TDV or MV CYD-2. This suggests that the partial protection against DENV-2 was mainly mediated by CYD-2 and not by the other CYDs. Post-challenge induction of strong anamnestic responses, suggesting efficient vaccine priming, likely contributed to the reduction of DENV-2 RNAemia. Finally, an inverse correlation was found between DENV RNA titers post-challenge and vaccine-induced homotypic neutralizing antibody titers pre-challenge, emphasizing the key role of these antibodies in controlling DENV infection. Collectively, these data show better agreement with CYD-TDV clinical vaccine efficacy data reported against DENV-2 and DENV-4. Despite inherent limitations of the non-human primate model, these results reinforce its value in assessing the efficacy of dengue vaccines.

    IMPORTANCE The NHP model is the most widely recognized tool for assessing the protective activity of dengue vaccine candidates, based on prevention of post-infection DENV viremia. However, its use has been questioned after the recent CYD vaccine Phase III trials in which moderate protective efficacy against DENV-2 was reported, despite full protection against DENV-2 viremia previously being demonstrated in CYD-vaccinated monkeys. Using a reverse translational approach, we show here that the NHP model can be improved to achieve DENV-2 protection levels which show better agreement with clinical efficacy. With this new model, we demonstrate that injection of the CYD-2 component of the vaccine, either in monovalent or tetravalent formulation, is able to reduce DENV-2 viremia in all immunized animals, and we provide clear statistical evidence that DENV-2 neutralizing antibodies are able to reduce viremia in a dose-dependent manner.

  • Dynein engages and disassembles cytosol-localized SV40 to promote infection [PublishAheadOfPrint]

  • During entry, polyomavirus (PyV) is endocytosed and sorts to the endoplasmic reticulum (ER) where it penetrates the ER membrane to reach the cytosol. From the cytosol, the virus moves to the nucleus to cause infection. How PyV is transported from the cytosol into the nucleus, a crucial infection step, is unclear. We found that upon reaching the cytosol, the archetype PyV SV40 recruits the cytoplasmic dynein motor, which disassembles the viral particle. This reaction enables the resulting disassembled virus to enter the nucleus to promote infection. Our findings reveal how a cytosolic motor can be hijacked to impart conformational changes to a viral particle, which is essential for successful infection.

    IMPORTANCE How a non-enveloped virus successfully traffics from the cell surface to the nucleus to cause infection remains enigmatic in many instances. In the case of the non-enveloped PyV, the viral particle is sorted from the plasma membrane to the ER and then the cytosol from where it enters the nucleus to promote infection. The molecular mechanism by which PyV reaches the nucleus from the cytosol is not entirely clear. Here we demonstrate that the prototype PyV SV40 recruits dynein upon reaching the cytosol. Importantly, this cellular motor disassembles the viral particle during cytosol-to-nuclear transport to cause infection.

  • Control of HIV-1 pathogenesis in viremic nonprogressors is independent of Gag-specific CTL responses [PublishAheadOfPrint]

  • Viremic nonprogressors (VNP) constitute a very scarce group of untreated HIV-1-infected individuals who maintain stable CD4+ T cell-counts despite high levels of HIV-1 replication. The specific factors associated to this atypical control of the HIV infection are poorly described. Since specific T-cell responses seem to be one of the main causes of HIV-1 control in elite controllers, herein we study if HIV-1 Gag-specific cytotoxic T lymphocyte (CTL) responses could also modulate disease control in VNP.

    We characterized immune responses from four VNPs compared to five subjects with standard progression (SP) during the first years of HIV-1 infection. We observed no differences in the breadth and frequency of Gag-specific cellular responses. Furthermore, we obtained 217 HIV-1Gag clonal sequences in which viral variability of Gag increased over 3 years of infection for synonymous and non-synonymous mutations in both VNP and SP. VNPs evolution rates in gag were comparable to SP. This observation is in line with a similar accumulation of CTL putative escape mutations in Gag epitopes targeted by CTL responses.

    Altogether, the absence of viral pathogenesis in VNP individuals seems to be independent of HIV-Gag-specific CTL-cell responses. This novel information guides to the study of alternative mechanism of HIV-1 pathogenesis control.

    IMPORTANCE Control of the HIV infection has been widely studied in elite controllers or long-term nonprogressors models. However, there is a less known group of individuals, termed viremic nonprogressors (VNP), who maintain stable CD4+ T-cell counts despite high plasma viremia. The mechanisms involved in this remarkable control of HIV-1 pathogenesis clearly have implications for the development of new drugs and vaccines.

    We show for the first time that VNPs have similar immune responses and HIV-gag evolution than standard progressors. Remarkably we demonstrate that the mechanism of pathogenesis control in these individuals differs from some elite controllers that are reported to have improved immune control. This is noteworthy as opens the door to, yet unknown, new mechanisms of HIV control.

    Our novel results advance the understanding of mechanisms involved in the viremic nonprogression and suggest that there are alternative mechanisms to the adaptive immune responses to an effective control of viral pathogenesis.

  • VZV ORF63 protects human neuronal and keratinocyte cell lines from apoptosis and changes its localization upon apoptosis induction [PublishAheadOfPrint]

  • There are many facets of varicella zoster virus (VZV) pathogenesis that are not fully understood, such as the mechanisms involved in the establishment of lifelong latency, reactivation and development of serious conditions such as post herpetic neuralgia (PHN). Virus-encoded modulation of apoptosis has been suggested to play an important role in these processes. VZV open reading frame (ORF) 63 has been shown to modulate apoptosis in a cell type specific manner, but the impact of ORF63 on cell death pathways has not been examined in isolation, in the context of human cells. We sought to elucidate the effect of VZV ORF63 on apoptosis induction in human neuron and keratinocyte cell lines. VZV ORF63 was shown to protect differentiated SH-SY5Y neuronal cells against staurosporine-induced apoptosis. In addition, VZV infection did not induce high levels of apoptosis in the human keratinocyte cell line, HaCaTs, highlighting a delay in apoptosis induction. VZV ORF63 was shown to protect HaCaTs against both staurosporine and Fas ligand induced apoptosis. Confocal microscopy was utlilised to examine VZV ORF63 localisation during apoptosis induction. In VZV infection and ORF63 expression alone, VZV ORF63 became more cytoplasmic with aggregate formation during apoptosis induction. Together this suggests VZV ORF63 protects both differentiated SH-SY5Ys and HaCaTs from apoptosis induction and may mediate this effect through its localization change during apoptosis. VZV ORF63 is a prominent VZV gene product in both productive and latent infection and thus may play a critical role in VZV pathogenesis through aiding neuron and keratinocyte survival.

    IMPORTANCE VZV, a human specific alphaherpesvirus causes chicken pox during primary infection and establishes lifelong latency in the dorsal root ganglia (DRG). Reactivation of VZV causes shingles, which is often followed by a prolonged pain syndrome called post herpetic neuralgia. It has been suggested the ability of the virus to modulate cell death pathways is linked to its ability to establish latency and reactivate. The significance of our research is in investigating the ability of ORF63, a VZV gene product, to inhibit apoptosis in novel cell types crucial for VZV pathogenesis. This will allow for an increased understanding of critical enigmatic components of VZV pathogenesis.

  • Stem Cell Properties of Normal Human Keratinocytes Determine Transformation Responses to HPV16 DNA [PublishAheadOfPrint]

  • Human papillomavirus (HPV) infection of the genital tract is common, however only about 10-15% of infections persist, and approximately 10-15% of these persistent infections result in cancer. Basal epidermal stem cells are the presumed target cells for HPV infection, providing a reservoir of latently infected cells that persist over time and initiate lesions. Yet, it is not known whether stem cell density has any influence on transformation of human keratinocytes by HPV. We explored the relationship between stem cell properties of normal human keratinocytes and their susceptibility to transformation by HPV16 DNA. Normal human keratinocyte isolates (NHKc) derived from different donors were cultured in 3-D anchorage-free suspension to assess their spheroid forming ability. NHKc spheroids were then plated back into plastic monolayer culture and transfected with full-length HPV16 DNA, which we have previously shown to integrate into the host cell genome upon transfection. Spheroid-derived NHKc (SD-NHKc) and FACS-purified populations of basal stem-like keratinocytes -expressing low levels of Epidermal Growth Factor Receptor and high levels of Integrin alpha 6 high (EGFRlo/ITGaalpha;6hi)- responded to transfection with HPV16 DNA with more vigorous proliferation, greater immortalization efficiency, and faster progression to differentiation resistance (DR) than autologous mass cultured cells. Conversely, cells committed to terminal differentiation (EGFRhi/ITGaalpha;6lo) grew slowly after transfection with HPV16 and failed to generate immortalized or DR clones. HPV16 DNA induced stem cell properties in mass cultured NHKc. We conclude that HPV16 preferentially immortalizes basal keratinocytes with stem cell properties and that these cells readily achieve a differentiation resistant phenotype, upon immortalization by HPV16.

    IMPORTANCE This paper explores the relationship between the stem cell properties of normal human epidermal cells in culture and these cells' susceptibility to transformation by HPV16 DNA, the HPV type present in about 50% of cervical cancers. We report variable susceptibilities to HPV16-mediated transformation among different keratinocyte isolates derived from neonatal foreskin. Our findings provide strong experimental evidence that HPV16 preferentially transforms basal keratinocytes with stem cell properties. Insights gained from these studies increase our understanding of the host cell-specific factors influencing individual susceptibility to HPV-driven transformation and the contributing factors leading to pre-neoplastic and neoplastic progression of HPV-positive lesions.

  • EFFECT OF SHORT-TERM ART INTERRUPTION ON LEVELS OF INTEGRATED HIV DNA [PublishAheadOfPrint]

  • Analytic treatment interruption (ATI) studies are required to evaluate strategies aimed at achieving ART-free HIV remission, but the impact of ATI on the viral reservoir remains unclear. We validated a DNA size selection-based assay for measuring levels of integrated HIV DNA and applied it to assess the effects of short-term ATI on the HIV reservoir. Samples from participants from four AIDS Clinical Trials Group (ACTG) ATI studies were assayed for integrated HIV DNA levels. Cryopreserved PBMCs were obtained for 12 participants with available samples pre-ATI and approximately 6 months after ART resumption. Four participants also had samples available during the ATI. The median duration of ATI was 12 weeks. Validation of the HIV Integrated DNA size-Exclusion (HIDE) assay was performed using samples spiked with unintegrated HIV DNA, HIV-infected cell lines, and participant PBMCs. The HIDE assay eliminated 99% of unintegrated HIV DNA species and strongly correlated with the established Alu-gag assay. For the majority of individuals, integrated DNA levels increased during ATI and subsequently declined upon ART resumption. There was no significant difference in levels of integrated HIV DNA between the pre- and post-ATI time points, with the median ratio of post:pre-ATI HIV DNA levels of 0.95. Using a new integrated HIV DNA assay, we found minimal change in the levels of integrated HIV DNA in participants who underwent an ATI followed by 6 months of ART. This suggests that short-term ATI can be conducted without a significant impact on levels of integrated proviral DNA in the peripheral blood.

    IMPORTANCE Interventions aimed at achieving sustained antiretroviral therapy (ART)-free HIV remission require treatment interruption trials to assess their efficacy. However, these trials are accompanied by safety concerns related to the expansion of the viral reservoir. We validated an assay that uses an automated DNA size-selection platform for quantifying levels of integrated HIV DNA and is less sample- and labor-intensive than current assays. Using stored samples from AIDS Clinical Trials Group studies, we found that short-term ART discontinuation had minimal impact on integrated HIV DNA levels after ART resumption, providing reassurance about the reservoir effects of short-term treatment interruption trials.

  • Conformational stability of the hemagglutinin of H5N1 influenza A viruses influences susceptibility to broadly neutralizing stem antibodies [PublishAheadOfPrint]

  • Vaccines that elicit broadly neutralizing antibodies to the conserved stem of hemagglutinin (HA) are being developed as universal influenza vaccines that protect against influenza across multiple years. However, different influenza strains, even those in the same subtype with identical stem sequences, can vary in susceptibility to broadly neutralizing stem antibodies, and the reasons are not understood. Here we studied potential mechanisms underlying different sensitivities of a panel of H5N1 HA-pseudoviruses to broadly neutralizing stem antibodies. We found that greater HA conformational stability, as measured by thermal inactivation and pH triggering of conformational changes, correlates with reduced neutralization sensitivity and antibody binding to HA under neutral and low pH conditions. Our data indicate that conformational stability of HA is an important attribute of susceptibility to broadly neutralizing stem antibodies and is influenced by residues outside the stem antibody epitopes.

    IMPORTANCE The influenza surface glycoprotein hemagglutinin (HA) mediates virus attachment and membrane fusion between virus and host cells, allowing the viral core to enter the host cell cytoplasm for replication. Fusion occurs when HA undergoes low-pH-induced-conformational changes during endocytosis. Broadly neutralizing antibodies to the conserved stem region of HA interfere with conformational changes required for fusion. Vaccines that elicit such antibodies are being developed as novel universal influenza vaccines for multiyear protection. We investigated why H5N1 HA from different strains vary in sensitivity to broadly neutralizing stem antibodies despite having conserved epitopes. We report that HA conformational stability due to residues outside the antibody binding site accounted for much of the variation in susceptibility to neutralization by stem antibodies. These findings highlight the importance of non-epitope residues in influencing neutralization sensitivity to stem antibodies and the complexities in developing universal vaccines targeting conserved epitopes in the HA stem.

  • IL-15 STIMULATED NATURAL KILLER CELLS CLEAR HIV-1 INFECTED CELLS FOLLOWING LATENCY REVERSAL EX VIVO [PublishAheadOfPrint]

  • Current efforts towards HIV eradication include approaches to augment immune recognition and elimination of persistently infected cells following latency reversal. Natural killer (NK) cells, the main effectors of the innate immune system, recognize and clear targets using different mechanisms than CD8+T cells, offering an alternative or complementary approach for HIV clearance strategies. We assessed the impact of IL-15 treatment on NK cell function and the potential of stimulated NK cells to clear the HIV reservoir. We measured NK cell receptor expression, antibody-dependent cell-dependent cytotoxicity (ADCC), cytotoxicity, IFN- production and antiviral activity in autologous HIV replication systems. All NK cell functions were uniformly improved by IL-15, and more importantly, IL-15-treated NK cells were able to clear latently HIV infected cells after exposure to vorinostat, a clinically relevant latency reversing agent. We also demonstrate that NK cells from HIV infected individuals aviremic on antiretroviral therapy can be efficiently stimulated with IL-15. Our work opens a promising line of investigation towards future immunotherapies to clear persistent HIV infection using NK cells.

    IMPORTANCE In the search for an HIV cure, strategies to enhance immune function to allow recognition and clearance of HIV infected cells following latency reversal are being evaluated. Natural killer (NK) cells possess characteristics that can be exploited for immunotherapy against persistent HIV infection. We demonstrate that NK cells from HIV-positive donors can be strongly stimulated with IL-15, improving their antiviral and cytotoxic potential, and more importantly, clearing HIV infected cells after latency reversal with a clinically relevant drug. Our results encourage further investigation to design NK cell-based immunotherapies to achieve HIV eradication.

  • Variation at extra-epitopic amino acid residues influences suppression of influenza virus replication by M158-66 epitope-specific CD8+ T lymphocytes [PublishAheadOfPrint]

  • Influenza virus-specific CD8+ T lymphocytes (CTL) contribute to clearance of influenza virus infections and reduce disease severity. Variation at amino acid residues located in- or outside CTL epitopes has been shown to affect viral recognition by virus-specific CD8+ T lymphocytes. Here we investigated the effect of naturally occurring variation at residues outside the conserved immuno-dominant and HLA*0201 restricted M158-66 epitope located in the influenza virus M1 protein on the extent of virus replication in the presence of CTLs specific for the epitope. To this end, isogenic viruses with an M1 gene segment derived from either an avian or a human influenza virus, HLA-transgenic human epithelial cells, human T cell clones specific for the M158-66 epitope or a control epitope and a novel purposely developed, in vitro system to co-culture influenza virus-infected cells with T cells were used. It was found that the M gene segment of a human influenza A/H3N2 virus afforded the virus the capacity to replicate better in the presence of M158-66-specific CTLs than the M gene segment of avian viruses. These findings are in concordance with differential CTL activation observed previously caused by variation at extra-epitopic residues, and may reflect an immune adaptation strategy of human influenza viruses that allows them to cope with potent CTL immunity to the M158-66 epitope in HLA-A*0201 positive individuals, resulting in increased virus replication and shedding and possibly increasing disease severity.

    IMPORTANCE Influenza viruses are among the leading causes of acute respiratory tract infections. CD8+ T lymphocytes display a high degree of cross-reactivity with influenza A viruses of various subtypes and are considered an important correlate of protection. Unraveling immune viral evasion strategies and identifying signs of immune adaptation is important to define the role of CD8+ T lymphocytes in affording protection more accurately. Improving our insight of the interaction between influenza viruses on the one hand and virus-specific CD8+ T lymphocyte immunity on the other, may help advance our understanding of influenza virus epidemiology, may aid risk assessment of potentially pandemic influenza virus strains and may benefit design of vaccines that induce more broadly protective immunity.

  • Host factor SPCS1 regulates the replication of Japanese encephalitis virus through interactions with transmembrane domains of NS2B [PublishAheadOfPrint]

  • Signal peptidase complex subunit 1 (SPCS1) is a newly identified host factor that regulates flavivirus replication, but the molecular mechanism is not fully understood. Herein, using Japanese encephalitis virus (JEV) as a model, we investigated the mechanism through which host factor SPCS1 regulates the replication of flaviviruses. We first validated the regulatory function of SPCS1 in JEV propagation by knocking down and knocking out endogenous SPCS1. Loss of SPCS1 function markedly reduced intracellular virion assembly and production of infectious JEV particles, but did not affect virus cell entry, RNA replication, or translation. SPCS1 was found to interact with NS2B, which is involved in post-translational protein processing and viral assembly. Serial deletion mutation of the JEV NS2B protein revealed that two transmembrane domains, NS2B (1-49) and NS2B (84-131), interact with SPCS1. Further mutagenesis analysis of conserved flavivirus residues in two SPCS1 interaction domains of NS2B demonstrated that G12A, G37A, and G47A in NS2B (1-49), and P112A in NS2B (84-131), weakened the interaction with SPCS1. Deletion mutation of SPCS1 revealed that SPCS1 (91-169) which containing two transmembrane domains was involved in the interaction with both NS2B (1-49) and NS2B (84-131). Taken together, the results demonstrate that SPCS1 affects viral replication by interacting with NS2B, thereby influencing post-translational processing of JEV proteins and the assembly of virions.

    IMPORTANCE

    Understanding viral-host interactions is important for elucidating the molecular mechanisms of viral propagation, and identifying potential anti-viral targets. Previous reports demonstrated that SPCS1 is involved in the flavivirus life cycle, but the mechanism remains unknown. In this study, we confirmed that SPCS1 participates in the post-translational protein processing and viral assembly stages of the JEV lifecycle, but not in the cell entry, genome RNA replication, or translation stages. Furthermore, we found that SPCS1 interacts with two independent transmembrane domains of the Flavivirus NS2B protein. NS2B also interacts with NS2A, which is proposed to mediate viral assembly. Therefore, we propose a protein-protein interaction model showing how SPCS1 participates in the assembly of JEV particles. The findings expand our understanding of how host factors participate in the flavivirus replication lifecycle, and identify potential anti-viral targets for combatting flavivirus infection.

  • Deaminase-Dead Mouse APOBEC3 is an In Vivo Retroviral Restriction Factor [PublishAheadOfPrint]

  • The apolipoprotein B editing complex 3 (APOBEC3) proteins are potent retroviral restriction factors that are under strong positive selection, both in terms of gene copy number and sequence diversity. A common feature of all the members of the APOBEC3 family is the presence of one or two cytidine deamination domains, essential for cytidine deamination of retroviral reverse transcripts as well as packaging into virions. Several studies have indicated that human and mouse APOBEC3 restrict retrovirus infection via cytidine deaminase (CD)-dependent and mmdash;independent means. To understand the relative contribution of CD-independent restriction in vivo, we created strains of transgenic mice on an APOBEC3 knockout background that express a deaminase-dead mouse APOBEC3 due to point mutations in both CD domains (E73Q/E253Q). Here we show that the CD-dead APOBEC3 can restrict murine retroviruses in vivo. Moreover, unlike the wild type protein, the mutant APOBEC3 is not packaged into virions but acts only as a cell-intrinsic restriction factor that blocks reverse transcription by incoming viruses. Finally, we show that wild type- and CD-dead mouse APOBEC3 can bind to MLV reverse transcriptase. Our findings suggest that the mouse APOBEC3 cytidine deaminase activity is not required for retrovirus restriction.

    IMPORTANCE: APOBEC3 proteins are important host cellular restriction factors essential for restricting retrovirus infection, by causing mutations in the virus genome and by blocking reverse transcription. While both methods of restriction function in vitro, little is known about their role during in vivo infection. By developing transgenic mice with mutations in the cytidine deamination domains needed for enzymatic activity and interaction with viral RNA, we show that APOBEC3 proteins can still restrict in vivo infection by interacting with reverse transcriptase and blocking its activity. These studies demonstrate that APOBEC3 proteins have evolved multiple means for blocking retrovirus infection, all of which function in vivo.

  • Within-Host Variations of Human Papillomavirus Reveal APOBEC-Signature Mutagenesis in the Viral Genome [PublishAheadOfPrint]

  • Persistent infection with oncogenic human papillomaviruses (HPVs) causes cervical cancer, accompanied with the accumulation of somatic mutations into the host genome. There are concomitant genetic changes in the HPV genome during viral infection; however, their relevance to cervical carcinogenesis is poorly understood. Here we explored within-host genetic diversity of HPV by performing deep sequencing analyses of viral whole-genome sequences in clinical specimens. The whole genomes of HPV types 16, 52 and 58 were amplified by type-specific PCR from total cellular DNA of cervical exfoliated cells collected from patients with cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC), and were deep-sequenced. After constructing a reference vial genome sequence for each specimen, nucleotide positions showing changes with ggt; 0.5% frequencies compared to the reference sequence were determined for individual samples. In total, 1,052 positions of nucleotide variations were detected in HPV genomes from 151 samples (CIN1, n = 56; CIN2/3, n = 68; ICC, n = 27), with varying numbers per sample. Overall, C-to-T and C-to-A substitutions were the dominant changes observed across all histological grades. While C-to-T transitions were predominantly detected in CIN1, their prevalence was decreased in CIN2/3 and fell below that of C-to-A transversions in ICC. Analysis of the tri-nucleotides context encompassing substituted bases revealed that TpCpN, a preferred target sequence for cellular APOBEC cytosine deaminases, was a primary site for C-to-T substitutions in the HPV genome. These results strongly imply that the APOBEC proteins are drivers of HPV genome mutation, particularly in CIN1 lesions.

    IMPORTANCE HPVs exhibit surprisingly high levels of genetic diversity, including a large repertoire of minor genomic variants in each viral genotype. Here, by conducting deep sequencing analyses, we show for the first time a comprehensive snapshot of the "within-host" genetic diversity of high-risk HPVs during cervical carcinogenesis. Quasi-species harboring minor nucleotide variations in viral whole-genome sequences were extensively observed across different grades of CIN and cervical cancer. Among the within-host variations, C-to-T transitions, a characteristic change mediated by cellular APOBEC cytosine deaminases, were predominantly detected throughout the whole viral genome, most strikingly in low-grade CIN lesions. The results strongly suggest that within-host variations of the HPV genome are primarily generated through the interaction with host cell DNA-editing enzymes, and that such within-host variability might be an evolutionary source of the genetic diversity of HPVs.

  • Intracellular hepatitis C modeling predicts infection dynamics and viral protein mechanisms [PublishAheadOfPrint]

  • Hepatitis C virus infection is a global health problem, with nearly 2 million new infections occurring every year and up to 85% of these becoming chronic infections that pose serious long-term health risks. To effectively reduce the prevalence of HCV infection and associated diseases, it is important to understand the intracellular dynamics of the viral lifecycle. Here, we present a detailed mathematical model that represents the full hepatitis C lifecycle. It is the first full HCV model to be fit to acute intracellular infection data and the first to explore the functions of distinct viral proteins, probing multiple hypotheses of cis- and trans-acting mechanisms to provide insights for drug targeting. Model parameters were derived from the literature, experiments, and fitting to experimental intracellular viral RNA, extracellular viral titer, and HCV core and NS3 protein kinetic data from viral inoculation to steady-state. Our model predicts faster rates for protein translation and polyprotein cleavage than previous replicon models and demonstrates that the processes of translation and synthesis of viral RNA have the most influence on the levels of the species we tracked in experiments. Overall, our experimental data and the resulting mathematical infection model reveal information about the regulation of core protein during infection, produce specific insights into the roles of the viral core, NS5A, and NS5B proteins, and demonstrate the sensitivities of viral proteins and RNA to distinct reactions within the lifecycle.

    IMPORTANCE We have designed a model for the full lifecycle of hepatitis C virus. Past efforts have largely focused on modeling hepatitis C replicon systems, in which transfected subgenomic HCV RNA maintains autonomous replication in the absence of virion production or spread. We started with the general structure of these previous replicon models and expanded to create a model that incorporates the full virus lifecycle as well as additional intracellular mechanistic detail. We compared several different hypotheses that have been proposed for different parts of the lifecycle and applied the corresponding model variations to infection data to determine which hypotheses are most consistent with the empirical kinetic data. Because the infection data we have collected for this study is a more physiologically relevant representation of a viral lifecycle than data obtained from a replicon system, our model can make more accurate predictions about clinical hepatitis C infections.

  • Characterization of H5N1 influenza virus quasispecies with adaptive hemagglutinin mutations from single-virus infections of human airway cells [PublishAheadOfPrint]

  • Transmission of avian influenza (AI) viruses to mammals involves phylogenetic bottlenecks that select small numbers of variants for transmission to new host species. However, little is known about the AI virus quasispecies diversity that produces variants for virus adaptation to humans. Here, we analyzed the hemagglutinin (HA) genetic diversity produced during AI H5N1 single-virus infection of primary human airway cells and characterized the phenotypes of these variants. During single-virus infection, HA variants emerged with increased fitness to infect human cells. These variants generally had decreased HA thermostability, an indicator of decreased transmissibility, that appeared to compensate for their increase in aalpha;2,6 Sia binding specificity and/or membrane fusion pH threshold, which are advantageous mutational changes for viral infection of human airway epithelia. An HA variant with increased HA thermostability also emerged, but could not outcompete variants with less HA thermostability. These results provided data on HA quasispecies diversity in human airway cells.

    IMPORTANCE The diversity of the influenza virus quasispecies that emerges from a single infection is the starting point for viral adaptation to new hosts. A few studies have investigated AI virus quasispecies diversity during human adaptation using clinical samples. However, those studies could be appreciably affected by individual variability and multifactorial respiratory factors, which complicate identification of quasispecies diversity produced by selective pressure for increased adaptation to infect human airway cells. Here, we found that detectable HA genetic diversity was produced by H5N1 single-virus infection of human airway cells. Most of the HA variants had increased fitness to infect human airway cells, but incurred a fitness cost of less HA stability. To our knowledge, this is the first report to characterize the adaptive changes of AI virus quasispecies produced by infection of human airway cells. These results provide a better perspective on AI virus adaptation to infect humans.

  • H5N1 influenza A virus PB1-F2 relieves HAX-1-mediated restriction of avian virus polymerase PA in human lung cells [PublishAheadOfPrint]

  • Highly pathogenic IAV from avian hosts were first reported to directly infect humans 20 years ago. However, these are rare events and our understanding of factors promoting or restricting zoonotic transmission is still limited. One accessory protein of IAV, PB1-F2, was associated with pathogenicity of pandemic and zoonotic IAV. This 90-amino-acid-short peptide does not harbor an enzymatic function. We thus identified host factors interacting with H5N1 PB1-F2, which could explain its importance for virulence. PB1-F2 binds to HCLS1 associated protein X1 (HAX-1), a recently identified host restriction factor of the PA subunit of IAV polymerase complexes. We demonstrate that the PA of a mammalian adapted H1N1 IAV is resistant to HAX-1 imposed restriction while the PA of an avian origin H5N1 IAV remains sensitive. We also showed HAX-1 sensitivity for PAs of A/Brevig Mission/1/1918 (H1N1) and A/Shanghai/1/2013 (H7N9), two avian origin zoonotic IAV. Inhibition of H5N1 polymerase by HAX-1 can be alleviated by its PB1-F2 through direct competition. Accordingly, replication of PB1-F2 deficient H5N1 IAV is attenuated in presence of high amounts of HAX-1. Mammalian adapted H1N1 and H3N2 viruses do not display this dependence on PB1-F2 for efficient replication in presence of HAX-1. We propose that PB1-F2 plays a key role in zoonotic transmission of avian H5N1 IAV into humans.

    IMPORTANCE Aquatic and shore birds are the natural reservoir of influenza A viruses from which the virus can jump into a variety of bird and mammal host species including humans. H5N1 influenza viruses are a good model for this process. They pose an ongoing threat to human and animal health due to their high mortality rates. However, it is currently unclear, what restricts these inter-species-jumps on the host side or what promotes them on the virus side. Here, we show that a short viral peptide, PB1-F2, helps H5N1 bird influenza viruses to overcome a human restriction factor of the viral polymerase complex, HAX-1. Interestingly, we find that human influenza A virus polymerase complexes are already adapted to HAX-1 and do not require this function of PB1-F2. We thus propose that a functional full length PB1-F2 supports direct transmission of bird viruses into humans.

  • Atomic structure of the murine norovirus protruding domain and sCD300lf receptor complex [PublishAheadOfPrint]

  • Human noroviruses are the leading cause of acute gastroenteritis in human. Noroviruses also infect animals such as cow, mice, cat, and dog. How noroviruses bind and enter host cells is still incompletely understood. Recently, the type I transmembrane protein CD300lf was recently identified as the murine norovirus receptor, yet it is unclear how the virus capsid and receptor interact at the molecular level. In this study, we determined the X-ray crystal structure of the soluble CD300lf (sCD300lf) and murine norovirus capsid-protruding domain complex at 2.05 AAring; resolution. We found that the sCD300lf binding site is located on the topside of the protruding domain and involves a network of hydrophilic and hydrophobic interactions. The sCD300lf locked nicely into a complementary cavity on the protruding domain that is additionally coordinated with a positive surface charge on the sCD300lf and a negative surface charge on the protruding domain. Five of six protruding domain residues interacting with sCD300lf were maintained between different murine norovirus strains, suggesting that the sCD300lf was capable of binding to a highly conserved pocket. Moreover, a sequence alignment with other CD300 paralogs showed that the sCD300lf interacting residues were partially conserved in CD300ld, but variable in other CD300 family members, consistent with previously reported infection selectivity. Overall, these data provide insights into how a norovirus engages a protein receptor and will be important for a better understanding of selective recognition and norovirus attachment and entry mechanisms.

    IMPORTANCE Noroviruses exhibit exquisite host-range specificity due to species-specific interactions between the norovirus capsid protein and host molecules. Given this strict host-range restriction it has been unclear how the viruses are maintained within a species between relatively sporadic epidemics. While much data demonstrates that noroviruses can interact with carbohydrates, recent work has shown that expression of the protein CD300lf is both necessary and sufficient for murine norovirus infection of mice and binding of the virus to permissive cells. Importantly, the expression of this murine protein by human cells renders them fully permissive for murine norovirus infection, indicating that at least in this case host-range restriction is determined by molecular events that control receptor binding and entry. Defining the atomic-resolution interactions between the norovirus capsid protein and its cognate receptor is essential for a molecular understanding of host-range restriction and norovirus tropism.

  • The N terminus of Autographa californica Multiple Nucleopolyhedrovirus DNA Polymerase is Required for Efficient Viral DNA Replication and Virus and Occlusion Body Production [PublishAheadOfPrint]

  • Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA polymerase (DNApol) plays a crucial role in viral DNA synthesis and the N terminus (residues 1 to 186) is highly conserved in the baculovirus DNApol family. However, the functional role of the N terminus of DNApol has not yet been characterized. Here we report a functional analysis of the AcMNPV DNApol N terminus. We truncated the DNApol N-terminus and constructed corresponding truncation mutants: Bac-GFP-Pol64, Bac-GFP-Pol110 and Bac-GFP-Pol186 that lack N-terminal 64, 110 and 186 residues, respectively. Although the truncation mutants rescued viral DNA synthesis and infectious viral production, the level of DNA replication decreased, and Bac-GFP-Pol64, Bac-GFP-Pol110 and Bac-GFP-Pol186 showed 10-fold, 89-fold and 891-fold reduction in infectious viral yield compared to wild-type repair virus, respectively. Production of occlusion bodies was compromised for all truncation mutants. Further bioinformatic analysis showed that the first 64 amino acids at the extreme N terminus contains a conserved aalpha;(-helix)-bbeta;(-sheet)-bbeta;-bbeta; secondary structure region, and further downstream sequence from aa 67 to 186 is comprised of four conserved sequence motifs. Multiple alanine point substitutions in the aalpha;bbeta;bbeta;bbeta; structure region or the four sequence motifs in the N terminus impaired viral DNA replication and resulted in reduction of virus yield and occlusion body production. Together our results suggested that the secondary structure and four conserved motifs within the N terminus of AcMNPV DNApol are important for viral DNA synthesis, infectious virus yield and production of occlusion bodies.

    IMPORTANCE DNA polymerase (DNApol) is highly conserved in all baculoviruses and is required for viral DNA replication. The N terminus is one of the highly conserved regions of baculovirus DNApols. Our results showed that the N terminus of baculovirus DNA polymerase plays an important role in efficient viral DNA synthesis and infectious virus yield and production of occlusion bodies. We identified five features, including a highly conserved secondary structure and four conserved amino acid motifs in the AcMNPV DNApol N terminus, all of which are important for efficient viral DNA synthesis, infectious virus yield and production of occlusion bodies.

  • Deep-Sequencing Analysis of the Dynamics of HIV-1 Quasiespecies in Naive Patients during a Short Exposure to Maraviroc [PublishAheadOfPrint]

  • In this study we have characterized quasispecies dynamics and the evolution of viral tropism in naive HIV-1-infected patients treated with a short-course of maraviroc monotherapy (NCT01060618) independently of their tropism. We randomly selected 20 patients displaying different basal tropism -10 R5 and 10 DMX4- at recruitment as determined by phenotypic tests (Trofilerreg;). Evolution of viral quasiespecies at the end of treatment was determined by ultra-deep sequencing of the V3 region using a 454 Life Sciences Platform and geno2pheno (g2p) algorithm for viral tropism prediction. False Positive Rate (FPR) that defines the probability of classifying an R5-virus falsely as X4 was set at 10% .Viral load (VL) X4 was calculated from sequences with FPR llt; 3.75.

    Virological response as defined as ggt;1 log10 reduction in VL was detected in 70% of patients independently of their basal tropism. Viral tropism remained stable and non-significant differences in FPR values before and after treatment was found for the majority of patients in both tropism groups. Only 3 patients (1 R5 and 2 DM/X4) showed an increased (ggt;1log) VL-X4 and one patient with DM/X4 tropism displayed a significant reduction in FPR values at the end of treatment. Fast changes in the composition of viral populations were observed in all patients after 10-day of MVC monotherapy treatment and a complete replacement of viral quasiespecies was found in 3/10 patients carrying R5-using viruses and 4/10 DM/X4-using viruses.

    IMPORTANCE Initiation of treatment with maraviroc requires previous determination of tropism by genotypic or phenotypic methods because the risk of treatment failure and selection of DM/X4-tropic variants. In this study we confirm previous work showing that virologic response to maraviroc is independent of basal tropism. By deep-sequencing analysis we determined that fast changes in viral populations were due to the emergence of minority variants in some patients whereas in others generation of new strains were detected. The risk of DM/X4 selection was very low as FPR values remained stable and only one patient showed a detrimental switch to DM/X4 variants. Our data show that some DM/X4 viruses are sensitive to maraviroc treatment probably because only a low proportion of DM/X4 uses preferentially the X4 receptor and contain authentically maraviroc-resistant viruses that are not accurately detected by current assays.

  • Divergent Pathogenic Properties of Circulating Coxsackievirus A6 Associated with Emerging HFMD [PublishAheadOfPrint]

  • Coxsackievirus A6 (CV-A6) is an emerging pathogen associated with hand, foot, and mouth disease (HFMD). Its genetic characterization and pathogenic properties are largely unknown. Here, we report 39 circulating CV-A6 strains isolated in 2013 from HFMD patients in Northeast China. Three major clusters of CV-A6 were identified and related to CV-A6 mostly from Shanghai, indicating that domestic CV-A6 strains were responsible for HFMD emerging in Northeast China. Four full-length CV-A6 genomes representing each cluster were sequenced and analyzed further. Bootscanning tests indicated that all four CV-A6-Changchun strains were most likely recombinants between CV-A6 prototype Gdula and prototype CV-A4 or CV-A4-related viruses, while the recombination pattern was related to yet distinct from those strains isolated from other regions of China. Furthermore, different CV-A6 viruses showed different capabilities of viral replication, release, and pathogenesis in a mouse model. Further analyses indicated that viral protein 2C contributed to the diverse pathogenic abilities of CV-A6 by causing autophagy and inducing cell death. To our knowledge, this study is the first to report lethal and non-lethal strains of CV-A6 associated with HFMD. The 2C protein region may play a key role in the pathogenicity of CV-A6 viruses.

    IMPORTANCE Hand, foot, and mouth disease (HFMD) is a major and persistent threat to infants and children. Besides the most common pathogens such as enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16), other enteroviruses are increasingly contributing to HFMD. The present study focused on the recently emerging CV-A6 strain. We found that CV-A6 strains isolated in Changchun city in Northeast China were associated with domestic origins. These Changchun viruses were novel recombinants of the CV-A6 prototype Gdula and CV-A4. Our results imply that measures to control CV-A6 transmission are urgently needed. Further analyses revealed differing pathogenicities in strains isolated in a neonatal mouse model. One of the possible causes has been narrowed down to the viral protein 2C, using phylogenetic studies, viral sequences, and direct tests on cultured human cells. Thus, the viral 2C protein is a promising target for anti-viral drugs to prevent CV-A6-induced tissue damaging.

  • New threats of H7N9 influenza virus: the spread and evolution of highly and low pathogenic variants with high genomic diversity in Wave Five [PublishAheadOfPrint]

  • H7N9 virus has caused five infection waves since it emerged in 2013. The highest number of human cases was seen in Wave Five; however, the underlying reasons have not been thoroughly elucidated. In this study, the geographical distribution, phylogeny and genetic evolution of 240 H7N9 viruses in Wave Five, including 35 new isolates from patients and poultry in nine provinces, were comprehensively analyzed together with strains from first four waves. Geographical distribution analysis displayed the newly-emerging highly pathogenic (HP) and low pathogenic (LP) H7N9 viruses were co-circulating, causing human and poultry infections across China. Genetic analysis indicated that dynamic reassortment of the internal genes among LP-H7N9/H9N2/H6Ny and HP-H7N9, as well as the surface genes between Yangtze and Pearl River Delta lineages resulted in at least 36 genotypes, with three major genotypes (G1, A/chicken/Jiangsu/SC537/2013-like, G3, A/Chicken/Zhongshan/ZS/2017-like and G11, A/Anhui/40094/2015-like). The HP-H7N9 likely evolved from G1 LP-H7N9 by the insertion of a "KRTA" motif at the cleavage site (CS), then evolved into fifteen genotypes with four different CS motifs including PKGKRTAR/G, PKGKRIAR/G, PKRKRAAR/G and PKRKRTAR/G. Approximately 46% (28/61) of HP strains belonged to G3. Importantly, neuraminidase (NA) inhibitor resistance (R292K in NA) and mammalian adaptation (eg. E627K and A588V in PB2) mutations were found in a few non-human-derived HP-H7N9 strains. In summary, the enhanced prevalence and diverse genetic characteristics with mammalian-adapted and NAI-resistant mutations may have contributed towards increased numbers of human infections in Wave Five.

    IMPORTANCE The highest numbers of human H7N9 infections were observed during Wave Five from October 2016 to September 2017. Our results showed that HP-H7N9 and LP-H7N9 has spread virtually throughout China and underwent dynamic reassortment with different subtypes (H7N9/H9N2 and H6Ny) and lineages (Yangtze and Pearl River Delta lineages), resulting in a total of 36 and three major genotypes. Notably, the NAI drug-resistant (R292K in NA) and mammalian-adapted (eg. E627K in PB2) mutations were found in HP-H7N9 not only from humans, but also from poultry and environmental isolates, indicating increased risks for human infections. The broad dissemination of LP- and HP-H7N9 with high genetic diversity, host adaptation and drug-resistant mutations likely accounted for the sharp increases in the number of human infections during Wave Five. Therefore, more strategies are needed against the further spread and damage of H7N9 in the world.

  • Central Nervous System Inflammation and Infection During Early, Non-Accelerated Simian-Human Immunodeficiency Virus Infection in Rhesus Macaques [PublishAheadOfPrint]

  • Studies utilizing highly pathogenic simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV) have largely focused on the immunopathology of the central nervous system (CNS) during end-stage neuro AIDS and SIV encephalitis. However, this may not model pathophysiology in earlier stages of infection. In this non-accelerated SHIV model, plasma SHIV RNA levels and peripheral blood and colonic CD4 T+ cell counts mirrored early HIV infection in humans. At 12 weeks post infection, cerebrospinal fluid (CSF) detection of SHIV RNA and elevations in IP-10 and MCP-1 reflected a discrete neurovirologic process. Immunohistochemical staining revealed a diffuse, low-level CD3+, CD4- cellular infiltrate in the brain parenchyma, without a concomitant increase in CD68/CD163+ monocytes, macrophages and activated microglial cells. Rare SHIV-infected cells in the brain parenchyma and meninges were identified by RNAscoperreg; in situ hybridization. In the meninges, there was also a trend toward increased CD4+ infiltration in SHIV-infected animals, but no differences in CD68/CD163+ cells between SHIV-infected and uninfected control animals. These data suggest that in a model that closely recapitulates human disease, CNS inflammation and SHIV in CSF may be predominantly mediated by T-cell mediated processes during early infection in both brain parenchyma and meninges. Because SHIV expresses an HIV rather than SIV envelope, this model could inform studies to understand potential HIV cure strategies targeting the HIV envelope.

    IMPORTANCE Animal models of the neurologic effects of HIV are needed because brain pathology is difficult to assess in humans. Many current models focus on the effects of late stage disease utilizing simian immunodeficiency virus (SIV). In the era of antiretroviral therapy, manifestations of late stage HIV are less common. Furthermore, new interventions such as monoclonal antibodies and therapeutic vaccinations target HIV envelope. We therefore describe a new model of central nervous system involvement in rhesus macaques infected with simian-human immunodeficiency virus (SHIV) expressing HIV envelope in earlier, less aggressive stages of disease. Here, we demonstrate that SHIV mimics the early clinical course in humans, and that early neurologic inflammation is characterized by predominantly T cell mediated inflammation, accompanied by SHIV infection in the brain and meninges. This model can be utilized to assess the effect of novel therapies targeted to HIV envelope on reducing brain inflammation before end stage disease.

  • Nonstructural 5A impairs DNA damage repair: Implication of hepatitis C virus-mediated hepatocarcinogenesis [PublishAheadOfPrint]

  • RAD51-associated protein 1 (RAD51AP1) is a member of the multiprotein complexes postulated to carry out RAD51-mediated homologous recombination and DNA repair in mammalian cells. In the present study, we showed that hepatitis C virus (HCV) NS5A directly bound RAD51AP1 and increased protein level of RAD51AP1 through modulation of the ubiquitin-proteasome pathway. We also demonstrated that RAD51AP1 protein levels were increased in the liver tissues of HCV-infected patients and NS5A transgenic mice. Importantly, NS5A impaired DNA repair by disrupting RAD51/RAD51AP1/UAF1 complex and rendered HCV-infected cells more sensitive to DNA damage. Silencing of RAD51AP1 expression resulted in a decrease of viral propagation. We further demonstrated that RAD51AP1 was involved in the assembly step of the HCV life cycle by protecting viral RNA. These data suggest that HCV exploits RAD51AP1 to promote viral propagation and thus host DNA repair is compromised in HCV-infected cells. Overall, our findings provide mechanistic insight into the pathogenesis of HCV infection

    IMPORTANCE Chronic infection with HCV is the leading cause of hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying HCV-induced HCC are not fully understood. Here, we demonstrated that HCV NS5A protein physically interacted with RAD51AP1 and increased RAD51AP1 protein level through modulation of the ubiquitin-proteasome pathway. HCV coopts host RAD51AP1 to protect viral RNA at an assembly step of the HCV life cycle. Of note, RAD51 protein was accumulated in the cytoplasm of the HCV-infected cells and thus RAD51/RAD51AP1/UAF1-mediated DNA damage repair system in the nucleus is compromised in HCV-infected cells. Our data may provide new insight into the molecular mechanisms of HCV-induced pathogenesis.

  • Immunogenicity and Cross-reactivity of Rhesus Adenoviral Vectors [PublishAheadOfPrint]

  • Adenovirus (Ad) vectors are being investigated as vaccine candidates, but baseline anti-vector immunity exists in human populations to both human Ad (HuAd) and chimpanzee Ad (ChAd) vectors. In this study, we investigated the immunogenicity and cross-reactivity of a panel of recently described rhesus adenoviral (RhAd) vectors. RhAd vectors elicited T cells with low exhaustion markers and robust anamnestic potential. Moreover, RhAd vector immunogenicity was unaffected by high levels of pre-existing anti-HuAd immunity. Both HuAd/RhAd and RhAd/RhAd prime-boost vaccine regimens were highly immunogenic, despite a degree of cross-reactive neutralizing antibodies (NAbs) between phylogenetically related RhAd vectors. We observed extensive vector-specific cross-reactive CD4 T cell responses and more limited CD8 T cell responses between RhAd and HuAd vectors, but the impact of vector-specific cellular responses was far less than vector-specific NAbs. These data suggest the potential utility of RhAd vectors and define novel heterologous prime-boost strategies for vaccine development.

    IMPORTANCE To date, most adenoviral vectors developed for vaccination have been HuAds from species B, C, D, and E and display moderate to high levels of pre-existing immunity in human populations. There is a clinical need for new adenoviral vectors that are not hindered by pre-existing immunity. Moreover, the development of RhAd vector vaccines expands our ability to vaccinate against multiple pathogens in a population that may have received other HuAd or ChAd vectors. We evaluated the immunogenicity and cross-reactivity of RhAd vectors, which belong to the poorly described adenovirus species G. These vectors induce robust cellular and humoral immune responses and were not hampered by pre-existing anti-HuAd vector immunity. Such properties make RhAd vectors attractive as potential vaccine vectors.

  • A Naturally Occurring Deletion in the Effector Domain of H5N1 Swine Influenza Virus Nonstructural Protein 1 Regulates Viral Fitness and Host Innate Immunity [PublishAheadOfPrint]

  • Nonstructural protein 1 (NS1) of influenza A virus regulates innate immune responses via various mechanisms. We previously showed that a naturally occurring deletion (the EALQR motif) in the NS1 effector domain of an H5N1 swine-origin avian influenza virus impairs the inhibition of type I interferon (IFN) in chicken fibroblasts and attenuates virulence in chickens. Here, we found that the virus bearing this deletion in its NS1 effector domain showed diminished inhibition of IFN-related cytokine expression and attenuated virulence in mice. We further show that deletion of the EALQR motif disrupts NS1 dimerization, impairing double-stranded RNA (dsRNA) sequestration and competitive binding with RIG-I. In addition, the EALQR-deleted NS1 could not bind to TRIM25, unlike full-length NS1, and was less able to block TRIM25 oligomerization and self-ubiquitination, further impairing the inhibition of TRIM25-mediated RIG-I ubiquitination compared with full-length NS1. Our data demonstrate that the EALQR deletion prevents NS1 from blocking RIG-I-mediated IFN induction via a novel mechanism to attenuate the viral replication and virulence in mammalian cells and animals.

    IMPORTANCE H5 highly pathogenic avian influenza viruses have infected more than 800 individuals with an overall 53% case fatality rate across 16 countries. Among viral proteins, nonstructural protein 1 (NS1) of influenza virus is considered a key determinant for type I interferon (IFN) antagonism, pathogenicity, and host range. However, that how NS1 precisely modulates virus-host interaction facilitating virus survival is not fully understood. Here, we report that a naturally occurring deletion (the EALQR motif) in the NS1 effector domain of an H5N1 swine-origin avian influenza virus disrupted the NS1 dimerization, which diminished the blockade of IFN induction via the RIG-I signaling pathway, thereby impairing virus replication and virulence in the host. Our study demonstrates the EALQR motif of NS1 regulates virus fitness to attain a virus-host compromise state in animals, and identifies this critical motif as a potential target for the future development of small molecular drugs and attenuated vaccines.

  • Activation and induction of antigen-specific T follicular helper cells (TFH) play a critical role in LAIV-induced human mucosal anti-influenza antibody response [PublishAheadOfPrint]

  • There is increasing interest recently in developing intranasal vaccines against respiratory tract infections. Antibody response is critical in vaccine-induced protection and TFH is considered important in mediating antibody response. Most data supporting the role for TFH in antibody response are from animal studies, and direct evidence from humans is limited, apart from TFH-like cells in blood. We studied activation and induction of TFH and its role on anti-influenza antibody response by live-attenuated influenza vaccine(LAIV) in human nasopharynx-associated lymphoid tissue(NALT). TFH activation in adenotonsillar tissues were analysed by flow-cytometry, and anti-hemagglutinin(HA) antibodies examined following LAIV stimulation of tonsillar mononuclear cells(MNC). Induction of antigen-specific TFH by LAIV was studied by flow-cytometry for induced TFH and CD154 expression. LAIV induced TFH proliferation which correlated with anti-HA antibody production, and TFH was shown critical for antibody response. Induction of TFH from naïve T cells by LAIV was shown in newly induced TFH expressing BCL6 and CD21, which was followed by the detection of anti-HA antibodies. Antigen specificity of LAIV-induced TFH was demonstrated by the expression of antigen-specific T cell activation marker CD154 upon challenge by H1N1 virus antigen or HA. LAIV-induced TFH differentiation was inhibited by BCL6, IL21, ICOS and CD40 signalling blocking respectively, and that diminished anti-HA antibody production. Conclusion: We demonstrate for the first time the induction of antigen-specific TFH by LAIV in human NALT that provide critical support for anti-influenza antibody response. Promoting antigen-specific TFH in NALT by intranasal vaccines may provide an effective vaccination strategy against respiratory infections in humans.

    IMPORTANCE. Airway infection such as influenza is common in humans. Intranasal vaccination has been considered a more biologically relevant and effective way of immunization against airway infection. Vaccine-induced antibody response is crucial for protection against infection. Recent data from animal studies suggest one type of T cells, named TFH is important for the antibody response. However, data on whether this TFH-mediated help for antibody production operates in humans is limited, due to the lack of access to human immune tissue containing the TFH. In this study, we demonstrated the induction of TFH cells by an intranasal influenza vaccine in human immune tissue that provide critical support for anti-influenza antibody response. Our findings provide direct evidence that TFH cells play a critical role in vaccine-induced immunity in humans, and suggest a novel strategy to promote such cells by intranasal vaccines against respiratory infections.

  • Age-associated differences in infection of human skin in the SCID mouse model of varicella-zoster virus pathogenesis [PublishAheadOfPrint]

  • Varicella-zoster virus (VZV) is the skin-tropic human alphaherpesvirus responsible for both varicella and herpes zoster. Varicella and herpes zoster skin lesions have similar morphology but herpes zoster occurs disproportionally in older individuals and is often associated with a more extensive local rash and severe zoster-related neuralgia. We hypothesized that skin aging could also influence the outcome of the anterograde axonal transport of VZV to skin. We utilized human skin xenografts maintained in immunodeficient (SCID) mice to study VZV-induced skin pathology in vivo in fetal and adult skin xenografts. Here we find that VZV replication is enhanced in skin from older compared to younger adults, correlating with clinical observations. In addition to measures of VZV infection, we examined the expression of type I interferon (IFN) pathway components in adult skin and investigated elements of the cutaneous proliferative and inflammatory response to VZV infection in vivo. Our results demonstrated that VZV infection of adult skin triggers intrinsic IFN-mediated responses as we have described in VZV-infected fetal skin xenografts, including MxA as well as PML, in skin cells surrounding lesions. Further, we observed that VZV elicited altered cell-signaling, proliferative and inflammatory responses that are involved in wound healing, driven by follicular stem cells. These cellular changes are consistent with VZV-induced activation of STAT3 and suggest that VZV exploits the wound healing process to ensure efficient delivery of the virus to keratinocytes. Adult skin xenografts offer an approach to further investigate VZV-induced skin pathologies in vivo.

    IMPORTANCE Varicella-zoster virus (VZV) is the agent responsible for both varicella and herpes zoster. Herpes zoster occurs disproportionally in older individuals and is often associated with a more extensive local rash and severe zoster-related neuralgia. To examine the effect of skin aging on VZV skin lesions, we utilized fetal and adult human skin xenografts maintained in immunodeficient (SCID) mice. We measured VZV-induced skin pathology, examined the expression of type I interferon (IFN) pathway components in adult skin, and investigated elements of the cutaneous proliferative and inflammatory response to VZV infection in vivo. Our results demonstrate that characteristics of aging skin are preserved in xenografts, that VZV replication is enhanced in skin from older compared to younger adults, correlating with clinical observations, and that VZV infection elicits altered cell-signaling and inflammatory responses. Adult skin xenografts offer an approach to further investigate VZV-induced skin pathologies in vivo.

  • Domains of the hepatitis B virus small surface protein S mediating oligomerization [PublishAheadOfPrint]

  • During hepatitis B virus infections subviral particles (SVP) consisting only of viral envelope proteins and lipids are secreted. Heterologous expression of the small envelope protein S in mammalian cells is sufficient for SVP generation. S is synthesized as a transmembrane protein with N-terminal (TM1), central (TM2), and C-terminal (HCR) transmembrane domains. The loops between TM1 and TM2 (CL) and between TM2 and HCR (LL) are located in the cytosol and in the ER lumen, respectively. To define domains of S mediating oligomerization during SVP morphogenesis S mutants were characterized by expression in transiently transfected cells. Substitution of 12 out of 15 amino acids of TM1 to alanines as well as the deletion of HCR still allowed SVP formation demonstrating that these two domains were not essential for contacts between S proteins. Furthermore, the oligomerization of S was measured with a FACS-based FRET (Foerster resonance energy transfer) assay. This approach demonstrated that CL, TM2, and LL independently contributed to S oligomerization while TM1 and HCR played a minor role. Apparently, intermolecular homooligomerization of CL, TM2, and LL drive S protein aggregation. Detailed analyses revealed that the point mutation C65S in CL, the exchange of 13 out of 19 amino acids of TM2 to alanine residues, and the simultaneous substitution of all eight cysteine residues in LL by serine residues blocked the ability of these domains to support S protein interactions. Altogether, specific domains and residues were defined in the HBV S protein required for oligomerization and SVP generation.

    Importance The small hepatitis B virus envelope protein S has the intrinsic capability to direct the morphogenesis of spherical 20 nm subviral lipoprotein particles. Such particles expressed in yeast or mammalian cells represent the antigenic component of current hepatitis B vaccines. Our knowledge about the steps leading from the initial, monomeric, transmembrane translation product of S to SVP is very limited as is our information on the structure of the complex main epitope of SVP that induces the formation of protective antibodies after vaccination. This study contributes to our understanding of the oligomerization process of S chains during SVP formation and shows that the cytoplasmic, one membrane embedded, and the luminal domain of S independently drive S-S oligomerization.

  • Vaccinia virus phospholipase protein F13 promotes the rapid entry of extracellular virions into cells [PublishAheadOfPrint]

  • The vaccinia virus protein F13, encoded by the F13L gene, is conserved across the subfamily Chordopoxvirinae and is critical among orthopoxviruses to produce the wrapped form of virus that is required for cell-to-cell spread. F13 is the major envelope protein on the membrane of extracellular forms of virus, however it is not known if F13 is required in steps post-wrapping. In this report, we utilize two temperature-sensitive vaccinia virus mutants from the Condit collection of temperature-sensitive viruses whose small plaque phenotypes have been mapped to the F13L gene. Despite the drastic reduction in plaque size, the temperature-sensitive viruses were found to produce similar levels of extracellular virions to the parental strain, Western Reserve (WR), at the permissive and non-permissive temperature, suggesting that they are not defective in extracellular virion formation. Analyses of extracellular virions produced by one temperature-sensitive mutant found that those produced at the non-permissive temperature had undetectable levels of F13 and bound cells with similar efficiency to WR, but displayed delayed cell entry kinetics. Additionally, low-pH treatment of cells bound by extracellular virions produced at the non-permissive temperature by the temperature-sensitive reporter virus was unable to overcome a block in infection by bafilomycin A1, suggesting that these virions display increased resistance to dissolution of the extracellular virion envelope. Taken together, our results suggest F13 plays a role in both the formation of extracellular virions, and promotes their rapid entry into cells by enhancing the sensitivity of the membrane to acid-induced dissolution.

    IMPORTANCE Vaccinia virus (VACV) is an orthopoxvirus, and produces two infectious forms, mature virions (MV) and extracellular virions (EV). EV are derived from MV and contain an additional membrane that must first be removed prior to cell entry. F13 is critical for the formation of EV, but a post envelopment role has not been described. Here, two temperature-sensitive VACV mutants whose deficiencies were previously mapped to the F13L locus are characterized. Both viruses produced EV at the non-permissive temperature at levels similar to a virus that has F13L, yet had a small plaque phenotype and rate of spread similar to an F13L-deletion virus. F13 was undetectable on the EV membrane at the non-permissive temperature, and these EV exhibited delayed cell entry kinetics compared to EV containing F13. This study is the first to conclusively demonstrate a novel role for F13 in cell entry of the EV form of the virus.

  • Role of the E2 hypervariable region (HVR1) in the immunogenicity of a recombinant HCV vaccine [PublishAheadOfPrint]

  • Current evidence supports a protective role for virus neutralizing antibodies in immunity against HCV infection. Many cross-neutralizing monoclonal antibodies have been identified. These antibodies have been shown to protect or clear infection in animal models. Previous clinical trials have shown a gpE1/gpE2 vaccine can induce antibodies that neutralize the in vitro infectivity of all the major HCVcc genotypes around the world. However, cross-neutralization appeared to favour certain genotypes with significant but lower neutralization against others. HCV may employ epitope masking to avoid antibody-mediated neutralization. The hypervariable region-1 (HVR1) at the amino-terminus of glycoprotein E2 has been shown to restrict access to many neutralizing antibodies. Consistent with this, other groups have reported that recombinant viruses lacking the HVR1 are hypersensitive to neutralization. It has been proposed that gpE1/gpE2 lacking this domain could be a better vaccine antigen to induce broadly neutralizing antibodies. In this study, we examined the immunogenicity of recombinant gpE1/gpE2 lacking the HVR1 (HVR1). Our results indicate that WT and HVR1 gpE1/gpE2 induced antibodies targeting many well-characterized cross-genotype neutralizing epitopes. However, while WT gpE1/gpE2 vaccine can induce cross-genotype protection against various genotypes of HCVcc and/or HCVpp, antisera from HVR1 gpE1/gpE2 immunized animals exhibited either reduced homologous neutralization activity or similar heterologous neutralization activity compared to WT. This data suggests HVR1 gpE1/gpE2 is not a superior vaccine antigen. Based on chimpanzee protection data reported previously using WT gpE1/gpE2 and our current findings, we are preparing a combination vaccine including a wild type recombinant gpE1/gpE2 for clinical testing in the future.

    IMPORTANCE An HCV vaccine is an unmet medical need. Current evidence suggests neutralizing antibodies play an important role in viral clearance along with cellular immune responses. Previous clinical data showed gpE1/gpE2 can effectively induce cross neutralizing antibodies although they favour certain genotypes. HCV employs the hypervariable HVR1 region within gpE2 to evade host immune control. It has been hypothesized that removal of this domain would improve the production of cross-neutralizing antibodies. In this study, we compared the immunogenicity of WT and HVR1 gpE1/gpE2 antigens as vaccine candidates. Our results indicate that HVR1 gpE1/gpE2 antigen confers no advantages in the neutralization of HCV when compared with WT antigen. Previously we have shown that this WT antigen remains the only vaccine candidate to protect chimpanzees from chronic infection, contains multiple cross-neutralizing epitopes, and is well-tolerated and immunogenic in humans. The current data supports the further clinical development of this vaccine antigen component.

  • Herpesvirus evasion of Natural Killer cells [PublishAheadOfPrint]

  • Natural killer (NK) cells play an important role in the host response against viral infections and cancer development. They are able to kill virus-infected and tumor cells, and produce different important cytokines that stimulate the antiviral and antitumor adaptive immune response, particularly interferon gamma. NK cells are of particular importance in herpesvirus infections, which is illustrated by systemic and life-threatening herpesvirus disease symptoms in patients with deficiencies in NK cell activity, and by the myriad of reports describing herpesvirus NK cell evasion strategies. The latter is particularly obvious for cytomegaloviruses but increasing evidence indicates that most, if not all, members of the herpesvirus family suppress NK cell activity to some extent. The current review discusses the different NK cell evasion strategies described for herpesviruses and how this knowledge may translate to clinical applications.

  • Positive regulation of hepatitis E virus replication by microRNA-122 [PublishAheadOfPrint]

  • Molecular mechanisms of liver pathology and clinical disease in HEV infection remain unclear. MicroRNAs are known to modulate viral pathogenesis either by directly altering viral gene expression or by enhancing cellular antiviral responses. Given the importance of microRNA-122 (miR-122) in liver pathobiology, we investigated possible role of miR-122 in HEV infection. In silico predictions using genotype 1, 2, 3 and 4 HEV sequences showed that majority of genomes (203/222) harbor at least one miR-122/miR-122* target site. Interestingly, HEV-1 genomes showed a highly conserved (97%) miR-122 target site in RdRp encoding region (RdRpc). We analyzed significance of miR-122 target sites in HEV-1/3 genomes by using replicon based cell culture system. HEV infection did not change basal levels of miR-122 in hepatoma cells. However, transfection of these cells with miR-122 mimics enhanced HEV-1/3 replication and depletion of miR-122 with inhibitors lead to suppression of HEV-1/3 replication. Mutant HEV-1 replicons with altered target RdRpc sequence (CACTCC) showed a drastic decrease in virus replication. While, introduction of alternative miR-122 target sites in mutant replicons rescued viral replication. There was enrichment of HEV-1 RNA and miR-122 molecules in RNA-induced silencing complexes in HEV infected cells. Furthermore, pulldown of miR-122 molecules from HEV-infected cells resulted in pulldown of HEV genomic RNA along with miR-122 molecules. These observations indicate that miR-122 facilitates HEV-1 replication probably via direct interaction with target site in viral genome. The positive role of miR-122 in viral replication presents novel opportunities for antiviral therapy and management of Hepatitis E.

    IMPORTANCE Hepatitis E is a problem of both developing and developed countries. HEV infection in most patients follows a self-limited course; however, 20-30% mortality is seen in infected pregnant women. HEV super-infections in patients with chronic hepatitis B or hepatitis C virus infections are associated with adverse clinical outcomes and both these conditions warrant therapy. Chronic HEV infections in immunocompromised transplant recipients are known to rapidly progress into cirrhosis. Currently, off-label use of ribavirin (RBV) and PEG-IFN as antiviral therapy have shown promising results in both acute and chronic hepatitis E patients, however teratogenecity of RBV limits its use during pregnancy, while, IFN-aalpha; increases risk of transplant rejections. Experimental data with genotype 1 virus in the current study shows that miR-122 facilitates HEV replication. These observations present novel opportunities for antiviral therapy and management of Hepatitis E.

  • Infectious Bursal Disease Virus hijacks endosomal membranes as the scaffolding structure for viral replication [PublishAheadOfPrint]

  • Birnaviruses are unconventional members of the double-stranded RNA (dsRNA) viruses group that are characterized by the lack of a transcriptionally active inner core. Instead, the birnaviral particles organize their genome in ribonucleoprotein complexes (RNPs) composed by dsRNA segments, the dsRNA-binding VP3 protein, and the viral encoded RNA-dependent RNA-polymerase (RdRp). This and other structural features suggests that birnaviruses may follow a completely different replication program from that followed by members of the Reoviridae family, supporting the hypothesis that birnaviruses are the evolutionary link between single-stranded positive RNA (+ssRNA) and dsRNA viruses. Here, we demonstrated that the Infectious Bursal Disease Virus (IBDV), a prototypical member of the Birnaviridae family, hijacks endosomal membranes of infected cells through the interaction of viral protein, VP3, with the phospholipids on the cytosolic leaflet of these compartments for replication. Employing a mutagenesis approach, we demonstrated that VP3 domain PATCH 2 (P2) mediates the association of VP3 with the endosomal membranes. To determine the role of VP3 P2 in the context of virus replication cycle, we used avian cells stably overexpressing VP3 P2 for IBDV infection. Importantly, the intra- and extra-cellular virus yields, as well as the intracellular levels of VP2 viral capsid protein, significantly diminished in VP3 P2 stably overexpressing cells. Altogether, our results indicate that the association of VP3 with endosomes has a relevant role in IBDV replication cycle. This report provides direct experimental evidence for membranous compartments such as endosomes being required by a dsRNA virus for its replication. The results also support the previously proposed role of birnaviruses as an evolutionary link between +ssRNA and dsRNA viruses.

    IMPORTANCE Infectious Bursal Disease (IBD, also called Gumboro disease) is an acute, highly contagious immunosuppressive disease that affects young chickens and spreads worldwide. The etiological agent of IBD is the Infectious Bursal Disease Virus (IBDV). This virus destroys the central immune organ (bursa of Fabricius), resulting in immunosuppression and reduced responses of chickens to vaccines, which increases their susceptibility to other pathogens.

    IBDV is a member of Birnaviridae family, which comprises unconventional members of dsRNA viruses, whose replication strategy has been scarcely studied. In this report we show that IBDV hijacks the endosomes of the infected cells for establishing viral replication complexes via the association of the ribonucleoprotein complex component VP3,with the phospholipids in the cytosolic leaflet of endosomal membranes. We show that this interaction is mediated by VP3 PATCH2 domain and demonstrated its relevant role in the context of viral infection.

  • Exacerbated apoptosis of cells infected with infectious bursal disease virus (IBDV) upon exposure to Interferon alpha (IFN-{alpha}) [PublishAheadOfPrint]

  • Infectious bursal disease virus (IBDV) belongs to the Birnaviridae family and is the etiological agent of a highly contagious and immunosuppressive disease (IBD) that affects domestic chickens (Gallus gallus). IBD or Gumboro disease leads to high morbidity and mortality of infected animals, and is responsible for major economic losses to the poultry industry world-wide. IBD is characterized by a massive loss of IgM-bearing B lymphocytes and the destruction of the bursa of Fabricius. The molecular bases of the IBDV pathogenicity are still poorly understood, nonetheless, an exacerbated cytokine immune response and B cell depletion due to apoptosis are considered main factors contributing to the severity of the disease. Here we have studied the role of type I interferon (IFN) on IBDV infection. While IFN pre-treatment confers protection against subsequent IBDV infection, IFN addition to infected cell cultures early after infection drives to a massive apoptotic cell death. Downregulation of PKR, TNF-aalpha; or nuclear factor-B (NF-B) expression drastically reduces the extent of apoptosis, indicating that these are critical proteins in the apoptotic response induced by IBDV upon treatment with IFN-aalpha;. Our results indicate that IBDV genomic dsRNA is a major viral factor contributing to apoptosis triggering. These findings provide novel insights into the potential mechanisms of IBDV-induced immunosuppression and pathogenesis in chickens.

    IMPORTANCE IBDV infection represents an important threat to the poultry industry world-wide. IBDV-infected chickens develop a severe immunosuppression, which render them highly susceptible to secondary infections, and unresponsive to vaccination against other pathogens. The early dysregulation of the innate immune response lead by IBDV infection, and the exacerbated apoptosis of B cells, have been proposed as the main factors contributing to virus-induced immunopathogenesis. Our work contributes for the first time to elucidating a potential mechanism driving apoptotic death of IBDV infected cells upon exposure to type I IFN. We provide solid evidence about the critical importance of PKR, TNF-aalpha; and NF-B in this phenomenon. The described mechanism could facilitate an early clearance of infected cells, thereby aiding to amelioration of IBDV-induced pathogenesis, but it could also contribute to B-cell depletion and immunosuppression. The balance between these two opposing effects might be dramatically affected by the genetic backgrounds of both the host and the infecting virus strain.

  • Baculovirus AC102 is a nucleocapsid protein that is crucial for nuclear actin polymerization and nucleocapsid morphogenesis [PublishAheadOfPrint]

  • The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type species of alphabaculoviruses, is an enveloped DNA virus that infects lepidopteran insects and is commonly known as a vector for protein expression and cell transduction. AcMNPV belongs to a diverse group of viral and bacterial pathogens that target the host cell actin cytoskeleton during infection. AcMNPV is unusual, however, in that it absolutely requires actin translocation into the nucleus early in infection, and actin polymerization within the nucleus late in infection coincident with viral replication. Of the six viral factors that are sufficient, when coexpressed, to induce the nuclear localization of actin, only AC102 is essential for viral replication and the nuclear accumulation of actin. We therefore sought to better understand the role of AC102 in actin mobilization in the nucleus early and late in infection. Although AC102 was proposed to function early in infection, we found that AC102 is predominantly expressed as a late protein. In addition, we observed that AC102 is required for F-actin assembly in the nucleus during late infection, as well as for proper formation of viral replication structures and nucleocapsid morphogenesis. Finally, we found that AC102 is a nucleocapsid protein and a newly recognized member of a complex consisting of the viral proteins EC27, C42, and the actin polymerization protein P78/83. Taken together, our findings suggest that AC102 is necessary for nucleocapsid morphogenesis and actin assembly during late infection through its role as a component of the P78/83-C42-EC27-AC102 protein complex.

    IMPORTANCE The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an important biotechnological tool for protein expression and cell transduction, and related nucleopolyhedroviruses are also employed as environmentally benign insecticides. One impact of our work is to better understand the fundamental mechanisms through which AcMNPV exploits the cellular machinery of the host for replication, which may aid in the development of improved baculovirus-based research and industrial tools. Moreover, AcMNPV's ability to mobilize the host actin cytoskeleton within the cell's nucleus during infection makes it a powerful cell biological tool. It is becoming increasingly clear that actin plays important roles in the cell's nucleus, yet the regulation and function of nuclear actin is poorly understood. Our work to better understand how AcMNPV relocalizes and polymerizes actin within the nucleus may reveal fundamental mechanisms that govern nuclear actin regulation and function, even in the absence of viral infection.

  • Antigenically diverse swine-origin H1N1 variant influenza viruses exhibit differential ferret pathogenesis and transmission phenotypes [PublishAheadOfPrint]

  • Influenza A(H1) viruses circulating in swine represent an emerging virus threat as zoonotic infections occur sporadically following exposure to swine. A fatal infection caused by an H1N1 variant (H1N1v) virus was detected in a patient with reported exposure to swine and who presented with pneumonia, respiratory failure, and cardiac arrest. To understand the genetic and phenotypic characteristics of the virus, genome sequence analysis, antigenic characterization, and ferret pathogenesis and transmissibility experiments were performed. Antigenic analysis of the virus isolated from the fatal case, A/Ohio/09/2015, demonstrated significant antigenic drift away from classical swine H1N1 variant viruses and H1N1 pandemic 2009 viruses. A substitution in the H1 hemagglutinin (G155E) was identified that likely impacted antigenicity, and reverse genetics was employed to understand the molecular mechanism of antibody escape. Reversion of the substitution to 155G, in a reverse genetics A/Ohio/09/2015 virus, showed that this residue was central to the loss of hemagglutination inhibition by ferret antisera raised against a prototypical H1N1 pandemic 2009 virus (A/California/07/2009), as well as gamma lineage classical swine H1N1 viruses, demonstrating the importance of this residue for antibody recognition of this H1 lineage. When analyzed in the ferret model, A/Ohio/09/2015 and another H1N1v virus (A/Iowa/39/2015), as well as A/California/07/2009, replicated efficiently in the respiratory tract of ferrets. The two H1N1v viruses transmitted efficiently among cohoused ferrets, but respiratory droplet transmission studies showed that A/California/07/2009 transmitted through the air more efficiently. Pre-existing immunity to A/California/07/2009 did not fully protect ferrets from challenge with A/Ohio/09/2015.

    IMPORTANCE Human infections with classical swine influenza A(H1N1) viruses that circulate in pigs continue to occur in the United States following exposure to swine. To understand the genetic and virologic characteristics of a virus (A/Ohio/09/2015) associated with a fatal infection and a virus associated with a non-fatal infection (A/Iowa/39/2015), we performed genome sequence analysis, antigenic testing, and pathogenicity and transmission studies in a ferret model. Reverse genetics was employed to identify a single antigenic site substitution (HA G155E) responsible for antigenic variation of A/Ohio/09/2015 compared to related classical swine influenza A(H1N1) viruses. Ferrets with pre-existing immunity to the pandemic A(H1N1) virus were challenged with A/Ohio/09/2015 demonstrating decreased protection. This data illustrates the potential for currently circulating swine influenza viruses to infect and cause illness in humans with pre-existing immunity to H1N1 pandemic 2009 viruses and a need for ongoing risk assessment and development of candidate vaccine viruses for improved pandemic preparedness.

  • Development of a Broadly Accessible VEE Replicon Particle Vaccine Platform [PublishAheadOfPrint]

  • Zoonotic viruses circulate as swarms in animal reservoirs and can emerge into human populations causing epidemics that adversely affect public health. Portable, safe, and effective vaccine platforms are needed in the context of these outbreak and emergence situations. In this manuscript, we report the generation and characterization of an alphavirus replicon vaccine platform based on a non-select agent, attenuated Venezuelan equine encephalitis (VEE) virus vaccine strain 3526 (VRP 3526). Using both noroviruses and coronaviruses as model systems, we demonstrate utility of the VRP 3526 platform in generation of recombinant proteins, production of virus like particles, and in vivo efficacy as a vaccine against emergent viruses. Importantly, packaging under BSL2 conditions distinguishes VRP 3526 from previously reported alphavirus platforms and makes this approach accessible to the majority of labs around the world. In addition, improved outcomes in the vulnerable aged models as well as against heterologous challenge suggest improved efficacy compared to previous attenuated VRP approaches. Taken together, the VRP 3526 platform represents a safe and highly portable system that can be rapidly deployed under BSL2 conditions for generation of candidate vaccines against emerging microbial pathogens.

    IMPORTANCE While VEE replicon particles provide a robust, established platform for antigen expression and vaccination, its utility has been limited by the requirement for high containment facilities for production and packaging. In this manuscript, we utilize an attenuated vaccine strain capable of use at lower biocontainment, but retaining the capacity of the wild type replicon particle. Importantly, the new replicon platform provides equal protection for aged mice and following heterologous challenge, which distinguishes it from other attenuated replicon platforms. Together, the new system represents a highly portable, safe system for use in the context of disease emergence.

  • Reduced cell-associated DNA and improved viral control in macaques following passive transfer of a single anti-V2 monoclonal antibody and repeated SHIV challenges [PublishAheadOfPrint]

  • A high level of V1V2-specific IgG antibodies in vaccinees' sera was the only independent variable that correlated with a reduced risk of HIV acquisition in the RV144 clinical trial. In contrast, IgG avidity, antibody neutralization, and antibody-dependent cellular cytotoxicity each failed as independent correlates of infection. Extended analyses of RV144 samples demonstrated antiviral activity of V1V2-specific vaccine-induced antibodies. V2-specific antibodies have also been associated with protection from SIV, and the V2i-specific subset of human monoclonal antibodies (mAbs), while poor neutralizers, mediate Fc-dependent antiviral functions in vitro. The objective of this study was to determine the protective efficacy of a V2i-specific human mAb 830A against mucosal SHIV challenge. V2i mAb binding sites overlap the integrin binding site in the V2 region and are similar to the epitopes bound by antibodies associated with reduced HIV infection rate in RV144. Because the IgG3 subclass was a correlate of reduced infection rate in RV144, we compared passive protection by both IgG1 and IgG3 subclasses of V2i mAb 830A. This experiment represents the first in vivo test of the hypothesis emanating from RV144 and SIV studies that V2i Abs can reduce infection risk. The results show that passive transfer with a single V2i mAb, IgG1 830A, reduced plasma and PBMC virus levels and decreased viral DNA in lymphoid tissues compared to controls, but too few animals remained uninfected to achieve significance in reducing infection risk. Based on these findings, we conclude that V2i antibodies can impede viral seeding following mucosal challenge, resulting in improved viral control.

    IMPORTANCE Since the results of the HIV RV144 clinical trial were reported, there has been significant interest in understanding how protection was mediated. Antibodies directed to a subregion of the Envelope protein called V1V2 were directly correlated with reduced risk, and surprisingly low virus neutralization was observed. To determine whether these antibodies alone could mediate protection, we used a human monoclonal antibody directed to V2 with properties similar to those elicited in the vaccine trial for passive infusions in rhesus macaques and challenge with SHIV. The single V2 antibody at the dose given did not significantly reduce the number of infections, but there was a significant reduction in seeding of virus to the lymph nodes and a decrease in plasma viremia in the HIV antibody-infused macaques compared with the control antibody-infused animals. This finding shows that V2 antibodies mediate antiviral activities in vivo that could contribute to a protective HIV vaccine.

  • Human T lymphocytes are permissive for dengue virus replication [PublishAheadOfPrint]

  • Dengue virus (DV) infection can cause either a self-limiting flu-like disease or a threatening hemorrhage that may evolve to shock and death. A variety of cell types, such as dendritic cells, monocytes and B cells, can be infected by DV. However, despite the role of T lymphocytes in the control of DV replication, there remains a paucity of information on possible DV-T cell interactions during the disease course. In the present study, we have demonstrated that primary human naïve CD4+ and CD8+ T cells are permissive for DV infection. Importantly, both T cell subtypes support viral replication and secrete viable virus particles. DV infection triggers the activation of both CD4+ and CD8+ T lymphocytes, but the pre-activation of T cells reduces the susceptibility of T cells to DV infection. Interestingly, the cytotoxicity-inducing protein granzyme A is highly secreted by human CD4+ but not CD8+ T cells after exposure to DV in vitro. Additionally, using Annexin V and poly-caspase assays, we demonstrate that T lymphocytes, in contrast to monocytes, are resistant to DV-induced apoptosis. Strikingly, both CD4+ and CD8+ T cells were found to be infected with DV in acutely infected dengue patients. Together, these results show that T cells are permissive for DV infection in vitro and in vivo, suggesting that this cell population may be a viral reservoir during acute phase of the disease.

    IMPORTANCE Infection by dengue virus (DV) causes a flu-like disease that can evolve to severe hemorrhaging and death. T lymphocytes are important cells that regulate antibody secretion by B cells and trigger the death of infected cells. However, little is known about the direct interaction between DV and T lymphocytes. Here, we showed that T lymphocytes from healthy donors are susceptible to infection by DV, leading to cell activation. Additionally, T cells seem to be resistant to DV-induced apoptosis, suggesting a potential role as a viral reservoir in humans. Finally, we showed that both CD4+ and CD8+ T lymphocytes from acutely infected DV patients are infected by DV. Our results raise new questions about DV pathogenesis and vaccine development.

  • Contribution of NK cell education to both direct and anti-HIV-1 antibody-dependent NK cell functions [PublishAheadOfPrint]

  • Antibody Fc-dependent functions are linked to prevention and control of HIV-1 infection. Basic NK cell biology is likely key to understanding the contributions anti-HIV-1 antibody-dependent NK cell activation and cytolysis make to HIV-1 susceptibility and disease progression. The importance of NK cell education through inhibitory receptors specific for self-HLA-I in determining the potency of anti-HIV-1 antibody mediated NK cell activation and cytolysis is controversial. To address this issue more definitively we utilized HLA-I genotyping, flow cytometry staining panels and cytolysis assays to assess the functionality of educated and non-educated peripheral blood NK cells. We now demonstrate that educated NK cells are superior in terms of their capacity to become activated and/or mediate cytolysis following anti-HIV-1 antibody-dependent stimulation. The profiles of activation observed were similar to those observed upon direct stimulation of NK cells with HLA-I devoid target cells. Non-educated NK cells make significantly lower contributions to total NK cell activation than would be expected from their frequency within the total NK cell population (i.e., are hypofunctional) and educated NK cells make similar or higher contributions as their frequency in the total NK cell population. Finally, NK cells educated through at least one killer immunoglobulin-like receptor and NKG2A exhibited the most significant difference between actual and expected contribution to the total NK cell response, based on their frequency within the total NK cell population, suggesting summation of NK cell education through inhibitory receptors determines overall NK cell functionality. These observations have potential implications for understanding HIV-1 vaccine efficacy and disease progression.

    IMPORTANCE NK cells are major mediators of anti-HIV-1 antibody-dependent functions, including cytokine production and cytolysis. The mechanisms controlling the capacity of individual NK cells to mediate antibody-dependent functions remain poorly defined. We now show that NK cell education determines the capacity of NK cells to exhibit anti-HIV-1 antibody-dependent activation and mediate antibody-dependent cellular cytotoxicity. These observations suggest the process of NK cell education could be of importance for understanding HIV-1 pathogenesis and designing immune-based prophylactics or therapeutics.

  • Adenovirus vector vaccination impacts NK cell rheostat function following LCMV infection [PublishAheadOfPrint]

  • Natural killer (NK) cells respond rapidly as a first line of defense against infectious pathogens. In addition, NK cells may provide a "rheostat" function and have been shown to reduce the magnitude of antigen-specific T cell responses following infection to avoid immunopathology. However, it remains unknown whether NK cells similarly modulate vaccine-elicited T cell responses following viral challenge. We used the LCMV clone 13 infection model to address whether NK cells regulate T cell responses in adenovirus vector vaccinated mice following challenge. As expected, NK cell depletion in unvaccinated mice resulted in increased virus-specific CD4+ and CD8+ T cell responses and immunopathology following LCMV challenge. In contrast, NK cell depletion had minimal to no impact on antigen-specific T cell responses in mice that were vaccinated with an Ad5-GP vector prior to LCMV challenge. Moreover, NK cell depletion in vaccinated mice prior to challenge did not result in immunopathology and did not compromise protective efficacy. These data suggest that adenovirus vaccine-elicited T cells may be less sensitive to NK cell rheostat regulation than T cells primed by LCMV infection.

    IMPORTANCE Recent data has shown that NK cell depletion leads to enhanced virus-elicited T cell responses that can result in severe immunopathology following LCMV infection in mice. In this study, we observed that NK cells exerted minimal to no impact on vaccine-elicited T cells following LCMV challenge, suggesting that adenovirus vaccine-elicited T cells may be less subject to NK cell regulation. These data contribute to our understanding of NK cell regulatory functions and T cell-based vaccines.

  • Reovirus-Induced Apoptosis in the Intestine Limits Establishment of Enteric Infection [PublishAheadOfPrint]

  • Several viruses induce intestinal epithelial cell death during enteric infection. However, it is unclear whether pro-apoptotic capacity promotes or inhibits replication in this tissue. We infected mice with two reovirus strains that infect the intestine but differ in the capacity to alter immunological tolerance to new food antigen. Infection with reovirus strain T1L, which induces an inflammatory immune response to fed antigen, is prolonged in the intestine, whereas T3D-RV, which does not induce this response, is rapidly cleared from the intestine. Compared with T1L, T3D-RV infection triggered apoptosis of intestinal epithelial cells and subsequent sloughing of dead cells into the intestinal lumen. We conclude that the infection advantage of T1L derives from its capacity to subvert host restriction by epithelial cell apoptosis, providing a possible mechanism by which T1L enhances inflammatory signals during antigen feeding. Using a panel of T1L x T3D-RV reassortant viruses, we identified the viral M1 and M2 gene segments as determinants of reovirus-induced apoptosis in the intestine. Expression of the T1L M1 and M2 genes in a T3D-RV background was sufficient to limit epithelial cell apoptosis and enhance viral infection to levels displayed by T1L. These findings define additional reovirus gene segments required for enteric infection of mice and illuminate the antiviral effect of intestinal epithelial cell apoptosis in limiting enteric viral infection. Viral strain-specific differences in the capacity to infect the intestine may be useful in identifying viruses capable of ameliorating tolerance to fed antigen in autoimmune conditions like celiac disease.

    IMPORTANCE Acute viral infections are thought to be cleared by the host with few lasting consequences. However, there may be much broader and long-lasting effects of viruses on immune homeostasis. Infection with reovirus, a common, nonpathogenic virus, triggers inflammation against innocuous food antigens, implicating this virus in the development of celiac disease, an autoimmune intestinal disorder triggered by exposure to dietary gluten. Using two reovirus strains that differ in the capacity to abrogate oral tolerance, we found that strain-specific differences in the capacity to replicate in the intestine inversely correlate with the capacity to induce apoptotic death of intestinal epithelial cells, providing a host-mediated process to restrict intestinal infection. This work contributes new knowledge about virus-host interactions in the intestine and establishes a foundation for future studies to define mechanisms by which viruses break oral tolerance in celiac disease.

  • Importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on MERS-CoV Spike to avoid neutralization escape [PublishAheadOfPrint]

  • Middle East Respiratory Syndrome coronavirus (MERS-CoV) causes a highly lethal pulmonary infection with ~35% mortality. The potential for a future pandemic originating from animal reservoirs or healthcare-associated events is a major public health concern. There are no vaccines or therapeutic agents currently available for MERS-CoV. Using a probe-based single B cell-cloning strategy, we have identified and characterized multiple neutralizing mAbs specifically binding to the receptor binding domain (RBD) or S1 (non-RBD) regions from a convalescent MERS-CoV-infected patient and from immunized rhesus macaques. RBD-specific mAbs tended to have greater neutralizing potency than non-RBD S1-specific mAbs. Six RBD-specific and five S1-specific mAbs could be sorted into four RBD and three non-RBD distinct binding patterns, based on competition assays, mapping neutralization escape variants, and structural analysis. We determined co-crystal structures for two mAbs targeting RBD from different angles and show they can only bind RBD in the "out" position. We then showed that selected RBD-specific, non-RBD S1, and S2-specific mAbs given prophylactically prevented MERS-CoV replication in lungs and protected mice from lethal challenge. Importantly, combining RBD- and non-RBD mAbs delayed the emergence of escape mutations in a cell-based virus-escape assay. These studies identify mAbs targeting different antigenic sites on S that will be useful for defining mechanisms of MERS-CoV neutralization, and for developing more effective interventions to prevent or treat MERS-CoV infections.

    IMPORTANCE: MERS-CoV causes a highly lethal respiratory infection for which no vaccines or antiviral therapeutic options are currently available. Based on continuing exposure from established reservoirs in dromedary camels and bats, transmission of MERS-CoV into humans and future outbreaks are expected. Using structurally-defined probes for the MERS-CoV Spike (S) glycoprotein, the target for neutralizing antibodies, single B cells were sorted from a convalescent human and immunized non-human primates (NHPs). mAbs produced from paired immunoglobulin gene sequences were mapped to multiple epitopes within and outside the receptor-binding domain (RBD) and protected against lethal MERS infection in a murine model following passive immunization. Importantly, combining mAbs targeting distinct epitopes prevented viral neutralization escape from RBD-directed mAbs. These data suggest that antibody responses to multiple domains on CoV Spike may improve immunity and will guide future vaccine and therapeutic development efforts.

  • A stem-loop structure in potato leafroll virus ORF5 that is essential for readthrough translation of the coat protein ORF stop codon 700 bases upstream [PublishAheadOfPrint]

  • Translational readthrough of the stop codon of the capsid protein (CP) open reading frame (ORF) is used by members of the Luteoviridae to produce their minor capsid protein as a readthrough protein (RTP). The elements regulating RTP expression are not well understood, but involve long-distance interactions between RNA domains. Using high-resolution mass spectrometry, glutamine and tyrosine were identified as the primary amino acids inserted at the stop codon of Potato leafroll virus (PLRV) CP ORF. We characterized the contributions of a cytidine-rich domain immediately downstream, and a branched stem-loop structure 600-700 nucleotides downstream of the CP stop codon. Mutations predicted to disrupt and restore the base of the distal stem-loop structure prevented and restored stop codon readthrough. Motifs in the downstream readthrough element (DRTE) are predicted to base pair to a site within 27 nt of the CP ORF stop codon. Consistent with a requirement for this base pairing, the DRTE of Cereal yellow dwarf virus was not compatible with the stop codon-proximal element of PLRV in facilitating readthrough. Moreover, deletion of the complementary tract of bases from the stop codon-proximal region or the DRTE of PLRV prevented readthrough. In contrast, the distance and sequence composition between the two domains was flexible. Mutants deficient in RTP translation moved long distance in plants, but fewer infection foci developed in systemically infected leaves. SHAPE probing to determine the secondary structure of the mutant DRTEs revealed that the functional mutants were more likely to have bases accessible for long-distance base pairing than in the nonfunctional mutants. This study reveals a heretofore unknown combination of RNA structure and sequence that reduces stop codon efficiency, allowing translation of a key viral protein.

    IMPORTANCE Programmed stop codon readthrough is used by many animal and plant viruses to produce key viral proteins. Moreover, such "leaky" stop codons are used in host mRNAs, or can arise from mutations that cause genetic disease. Thus it is important to understand the mechanism(s) of stop codon readthrough. Here, we shed light on the mechanism of readthrough of the stop codon of the coat protein ORF of viruses in the Luteoviridae, by identifying the amino acids inserted at the stop codon and RNA structures that facilitate this "leakiness" of the stop codon. Members of the Luteoviridae encode a C-terminal extension to the capsid protein known as the readthrough protein (RTP). We characterize two RNA domains in Potato leafroll virus (PLRV), located 600 to 700 nucleotides apart, that are essential for efficient RTP translation. We further determine PLRV readthrough process involves both local structures and long-range RNA-RNA interactions. Genetic manipulation of the RNA structure altered the ability of PLRV to translate RTP and systemically infect the plant. This demonstrates plant virus RNA contains multiple layers of information beyond the primary sequence and extends our understanding of stop codon readthrough. Strategic targets were revealed that can be exploited to disrupt the virus lifecycle and reduce its ability to move within and between plant hosts.

  • Distinct Immunogenicity and Efficacy of Poxvirus-based Vaccine Candidates against Ebola Virus expressing GP and VP40 Proteins [PublishAheadOfPrint]

  • Zaire and Sudan ebolavirus species cause a severe disease in humans and non-human primates (NHPs) characterized by high mortality rate. There are no licensed therapies or vaccines against Ebola virus disease (EVD), and the recent 2013-2016 outbreak in West Africa highlighted the need of EVD-specific medical countermeasures. Here, we have generated and characterized head-to-head the immunogenicity and efficacy of five vaccine candidates against Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV) based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA), expressing either the virus glycoprotein (GP) or GP together with the virus protein 40 (VP40) forming virus-like particles (VLPs). In a human monocytic cell line the different MVA vectors (termed MVA-EBOVs and MVA-SUDVs) triggered robust innate immune responses, with production of beta interferon (IFN-bbeta;), pro-inflammatory cytokines, and chemokines. Additionally, several innate immune cells, such as dendritic cells, neutrophils, and natural killer cells were differentially recruited in the peritoneal cavity of mice inoculated with MVA-EBOVs. After immunization of mice with a homologous prime/boost protocol (MVA/MVA) total IgG antibodies against GP or VP40 from Zaire and Sudan were differentially induced by these vectors, which were mainly of the IgG1 and IgG3 isotypes. Remarkably, an MVA-EBOV construct co-expressing GP and VP40 protected chimeric mice challenged with EBOV to higher extend than a vector expressing GP alone. These results support the consideration of MVA-EBOVs and MVA-SUDVs expressing GP and VP40 and producing VLPs as best-in-class potential vaccine candidates against EBOV and SUDV.

    IMPORTANCE EBOV and SUDV cause a severe hemorrhagic fever affecting humans and NHPs. Since their discovery in 1976, they have caused several sporadic epidemics, being the recent outbreak in West Africa in 2013-2016 the largest and most severe with more than 11,000 deaths reported. Although some vaccines are in advanced clinical phases, cheaper, safer and more effective licensed vaccines are desirable. We have generated and characterized head-to-head the immunogenicity and efficacy of five novel vaccines against EBOV and SUDV based on the poxvirus MVA expressing GP or GP and VP40. The expression of GP and VP40 leads to the formation of VLPs. These MVA-EBOV and MVA-SUDV recombinants triggered robust innate and humoral immune responses in mice. Furthermore, MVA-EBOV recombinants expressing GP and VP40 induced high protection against EBOV in a mouse challenge model. Thus, MVA expressing GP and VP40 and producing VLPs are promising vaccine candidates against EBOV and SUDV.

  • The experimental analysis of mimivirus translation initiation factor 4a reveals its importance for viral proteins translation during infection of Acanthamoeba polyphaga [PublishAheadOfPrint]

  • Acanthamoeba polyphaga mimivirus is the first giant virus ever described, with a 1.2-Mb genome which encodes 979 proteins including central components of the translation apparatus. One of these proteins, R458, was predicted to initiate translation, although its specific role remains unknown.

    We silenced the R458 gene using siRNA and compared viral fitness and protein expression in silenced versus wild-type mimivirus. Silencing decreased growth rate but viral particle production at the end of the viral cycle was unaffected. A comparative proteomic approach using 2D-DIGE revealed deregulation of the expression of 32 proteins in silenced mimivirus, defined as up- or down-regulated. Besides revealing proteins with unknown functions, silencing R458 also revealed deregulation in proteins associated with viral particle structures, transcriptional machinery, oxidative pathways, modification of proteins/lipids, and DNA topology/repair. Most of these proteins belong to genes transcribed at the end of the viral cycle.

    Overall, our data suggest that the R458 protein regulates the expression of mimivirus proteins and, thus, mimivirus translational proteins may not be strictly redundant in relation to those from the amoeba host. As is the case for the eIF4a eukaryotic factor, the R458 protein is the prototypical member of the ATP-dependent DEAD-box RNA helicase mechanism. We suggest that the R458 protein is required to unwind the secondary structures at the 5rrsquo; ends of mRNAs and to bind the mRNA to the ribosome, making it possible to scan for the start codon. These data are the first experimental evidence of mimivirus translation-related genes, predicted to initiate protein biosynthesis.

    IMPORTANCE The presence in the genome of Mimivirus of genes coding for many translational process, with the exception of ribosome constituents, has been the subject of debate since its discovery in 2003. In this work, we focused on the R458 mimivirus gene, predicted to initiate proteins biosynthesis. After silencing, we observed that it has no major effect on mimivirus multiplication, but affects protein expression and fitness. This suggests that it is effectively used by mimivirus during its developmental cycle. Until large scale genetic manipulation of giant viruses becomes possible, the silencing strategy used here on mimivirus translation related-factors opens the way to understanding the functions of these translational genes.

  • Identification of H209 essential for pH 8-triggered receptor-independent syncytium formation by S protein of MHV-A59 [PublishAheadOfPrint]

  • The spike glycoprotein (S) of murine coronavirus mouse hepatitis virus (MHV) strain A59 uses murine carcinoembryonic antigen-related cell adhesion molecule 1a as its receptor for cell entry, but S protein can also be triggered in the absence of receptor by pH 8.0 alone at 37ddeg;C. The mechanism by which conformational changes of this S glycoprotein can be triggered by pH 8.0 has not yet been determined. Here we show that MHV-A59 S protein is triggered by pH 8.0 at 37ddeg;C to induce receptor-independent syncytium (RIS) formation on 293T cells, and that the conformational changes in S proteins triggered by pH 8.0 are very similar to those triggered by receptor binding. We systemically mutated each of 15 histidine residues in S protein, and found that H209 is essential for pH 8.0-triggered RIS formation while H179, H441, H643 and H759 also play important roles in this process. Substitution of H209 with Ala had no effect on receptor binding, but in murine 17Cl.1 cells mutant H209A MHV-A59 showed delayed growth kinetics and was readily outcompeted by wild type virus when mixed together, indicating that the H209A mutation caused a defect on virus fitness. Finally, the H209A mutation significantly increased the thermostability of S protein in its prefusion conformation, which may raise the energy barrier for conformational change of S protein required for membrane fusion and lead to decrease in virus fitness in cell culture. Thus, MHV-A59 may have evolved to lower the stability of its S protein in order to increase virus fitness.

    IMPORTANCE Enveloped viruses enter cells through fusion of viral and cellular membranes, and the process is mediated by interactions between viral envelope proteins and their host receptors. In the prefusion conformation, viral envelope proteins are metastable, and activation to the fusion conformation is tightly regulated since premature activation would lead to loss of viral infectivity. The stability of viral envelope proteins greatly influences their activation and virus fitness. Here, we report that, similar to the A82V mutation in Ebola glycoprotein, in the S glycoprotein of murine coronavirus MHV-A59, the histidine residue at position of 209 significantly affects the thermal stability of the S protein, determines whether S protein can be activated at 37ddeg;C by either pH 8.0 alone or by receptor binding, and affects viral fitness in cell culture. Thus the spike glycoprotein of MHV-A59 has evolved to retain histidine at position of 209 to optimize virus fitness.

  • HIV-1 Nef antagonizes SERINC5 restriction by downregulation of SERINC5 via the endosome/lysosome system [PublishAheadOfPrint]

  • The primate lentiviral accessory protein Nef downregulates CD4 and MHC-I from the cell surface via independent endosomal trafficking pathways to promote viral pathogenesis. In addition, Nef antagonizes a novel restriction factor, SERINC5 (Ser5), to increase viral infectivity. To explore the molecular mechanism of Ser5 antagonism by Nef, we determined how Nef affects Ser5 expression and intracellular trafficking in comparison with CD4 and MHC-I. We confirm that Nef excludes Ser5 from HIV-1 virions by downregulating its cell surface expression via similar functional motifs required for CD4-downregulation. We find that Nef decreases both Ser5 and CD4 expression at steady-state levels, which are rescued by NH4Cl or bafilomycin A1 treatment. Nef binding to Ser5 was detected in living cells using a bimolecular fluorescence complementation (BiFC) assay, where Nef membrane association is required for interaction. In addition, Nef triggers rapid Ser5 internalization via receptor-mediated endocytosis, and re-localizes Ser5 to Rab5+ early, Rab7+ late, and Rab11+ recycling endosomes. Manipulation of AP-2, Rab5, Rab7, and Rab11 expression levels affects the Nef-dependent Ser5 and CD4 downregulation. Moreover, although Nef does not promote Ser5 polyubiquitination, Ser5 downregulation relies on the ubiquitination pathway, and both K48- and K63-specific ubiquitin linkages are required for the downregulation. Finally, Nef promotes Ser5 co-localization with LAMP1, which is enhanced by bafilomycin A1 treatment, suggesting that Ser5 is targeted to lysosomes for destruction. We conclude that Nef employs a similar mechanism to downregulate Ser5 and CD4, which sorts Ser5 into a point-of-no-return degradative pathway to counteract its restriction.

    IMPORTANCE HIV and SIV express an accessory protein called Nef to promote viral pathogenesis. Nef drives immune escape in vivo through downregulation of CD4 and MHC-I from the host cell surface. Recently, Nef was reported to counteract a novel host restriction factor, Ser5, to increase viral infectivity. Nef downregulates cell surface Ser5, thus preventing its incorporation into virus particles, resulting in disruption of its antiviral activity. Here, we report mechanistic studies of Nef-mediated Ser5 downregulation in comparison with CD4 and MHC-I. We demonstrate that Nef binds directly to Ser5 in living cells and that Nef-Ser5 interaction requires Nef-association with the plasma membrane. Subsequently, Nef internalizes Ser5 from the plasma membrane via receptor-mediated endocytosis, and targets ubiquitinated Ser5 to endosomes and lysosomes for destruction. Collectively, these results provide new insights into our ongoing understanding of the Nef-Ser5 arms race in HIV-1 infection.

  • Depletion of the insulator protein CTCF results in HSV-1 reactivation in vivo [PublishAheadOfPrint]

  • Herpes Simplex Virus 1 (HSV-1) establishes a lifelong latent infection in host peripheral neurons including the neurons of the trigeminal ganglia (TG). HSV-1 can reactivate from neurons to cause recurrent infection. During latency, the insulator protein CTCF occupies DNA binding sites on the HSV-1 genome and these sites have been previously characterized as functional enhancer-blocking insulators. Previously, CTCF was found to be dissociated from wild type virus post-reactivation but not in mutants that do not reactivate, indicating that CTCF eviction may also be an important component of reactivation. To further elucidate the role of CTCF in reactivation of HSV-1, we used recombinant adeno-associated virus (rAAV) vectors to deliver an siRNA targeting CTCF to peripheral neurons latent with HSV-1 in rabbit TG. Our data show that CTCF depletion resulted in long-term and persistent shedding of infectious virus in the cornea and increased ICP0 expression in the ganglia, indicating that CTCF depletion facilitates HSV-1 reactivation.

    IMPORTANCE Increasing evidence has shown that the insulator protein CTCF regulates gene expression of DNA viruses, including the gammaherpesviruses. While CTCF occupation and insulator function control gene expression in DNA viruses, CTCF eviction has been correlated to increased lytic gene expression and the dissolution of transcriptional domains. Our previous data have shown that in the alphaherpesvirus HSV-1, CTCF was found to be dissociated from the HSV-1 genome post-reactivation, further indicating a global role for CTCF eviction in the transition from latency to reactivation in HSV-1 genomes. Using an rAAV8, we targeted HSV-1 infected peripheral neurons for CTCF depletion to show that CTCF depletion precedes the shedding of infectious virus and increased lytic gene expression in vivo, providing the first evidence that CTCF depletion facilitates HSV-1 reactivation.

  • A DNA vaccine expressing consensus hemagglutinin-esterase fusion protein protected guinea pigs from infection by two lineages of influenza D virus [PublishAheadOfPrint]

  • Two lineages of Influenza D virus (IDV) have been found to infect cattle and promote bovine respiratory disease complex (BRDC), one of the most commonly diagnosed causes of morbidity and mortality within the cattle industry. Furthermore, IDV can infect other economically important domestic livestock including pigs and has the potential to infect humans, which necessitates the need for an efficacious vaccine. In this study, we designed a DNA vaccine expressing consensus hemagglutinin-esterase-fusion (HEF) protein (FluD-Vax) and tested its protective efficacy against two lineages of IDV (D/OK and D/660) in guinea pigs. Animals that received FluD-Vax (n=12) developed appreciable titers of neutralizing antibodies against IDV lineage representatives, D/OK and D/660. Importantly, vaccinated animals were protected against intranasal challenge with IDV (3E5 TCID50) D/OK (n=6) or D/600 (n=6) based on the absence of viral RNA in necropsied tissues (5 and 7 days post challenge) using qRT-PCR and in situ hybridization (ISH). In contrast, animals that received a sham DNA vaccine (n=12) had no detectable neutralizing antibodies against IDV and viral RNA was readily detectable in respiratory tract tissues after intranasal challenge with IDV (3E5 TCID50) D/OK (n=6) or D/660 (n=6). Using a TUNEL assay, we found that IDV D/OK and D/600 infections induced apoptosis in epithelial cells lining alveoli and bronchioles as well as non-epithelial cells in lung tissues. Our results have demonstrated for the first time that the consensus IDV HEF DNA vaccine can elicit complete protection against infection from two lineages of IDV in the guinea pig model.

    IMPORTANCE IDV infection has been associated with BRDC, one of the most devastating diseases of the cattle population. Moreover, with broad host range and high environmental stability, IDV has the potential to further gain virulence, or even infect humans. An efficacious vaccine is needed to prevent infection and stop potential cross-species transmission. In this study, we designed a DNA vaccine encoding the consensus HEF of two lineages of IDV (D/OK and D/660) and tested its efficacy in a guinea pig model. Our results showed that the consensus DNA vaccine elicited high-titer neutralizing antibodies and achieved sterilizing protection against two lineage-representative IDV intra-nasal infections. To our knowledge, this is the first study showing a DNA vaccine-expressing consensus HEF is efficacious in preventing different lineages of influenza D virus infections.

  • Porcine MKRN1 modulates the replication and pathogenesis of PCV2 by inducing capsid protein ubiquitination and degradation [PublishAheadOfPrint]

  • Porcine circovirus type 2 (PCV2) capsid protein (Cap) is a unique structure protein that plays pivotal roles in the process of viral replication and pathogenesis. Herein, we characterized putative porcine Makorin ring finger protein 1 variant (pMKRN1), a N-terminal-truncated variant of putative full-size porcine MKRN1, which is a unique expression pattern resulting from the porcine mkrn1 gene and interacts with PCV2 Cap. Domain mapping assay showed that the C terminus of pMKRN1 and the fragments (108-198 aa) of Cap are required for this interaction. PCV2 up-regulated pMKRN1 transiently in PK-15 cells, but persistent viral infection downregulated pMKRN1 in major pathological tissues of PCV2-infected piglets. Over-expression of pMKRN1 significantly inhibited the generation of progeny PCV2 via ubiquitination and degradation of Cap, whereas knockout of pMKRN1 blocked Cap degradation and promoted progeny virus replication. pMKRN1 specifically targeted PCV2 Cap lysine residues 164, 179, and 191 to induce polyubiquitination and subsequent degradation. Mutation of either of the three lysine residues in Cap protein, or mutation of histidine at residue 243 within the ring finger domain of pMKRN1 abrogated the E3 ligase activity of pMKRN, rendered cells incapable of inducing Cap ubiquitination and degradation. Consistent with this finding, a Cap ubiquitination-deficient PCV2 strain showed enhanced virus replication and severe histological lesions in the lung and lymph node tissues compared with wild-type PCV2. Taken together, the results presented here suggested that PCV2 downregulates pMKRN1 variant to avoid pMKRN1-mediated Cap ubiquitination and degradation, thus promoting viral replication and pathogenesis in its targeted tissues.

    IMPORTANCE Porcine circovirus type 2 is the most susceptible pathogen of pigs causing immense economic losses in the global swine industry, but whether host cells have developed some strategies to prevent viral replication is still unclear. Here, we found that porcine MKRN1(pMKRN1) was up-regulation in early stage of PCV2 infection and mediated polyubiquitination and degradation of Cap protein to block PCV2 replication. Yet persistant PCV2 infection down-regulated pMKRN1 levels to avoid degradation, promoting viral replication and pathogenesis in its targeted tissues. These data present a new insight for molecular mechanisms underlying the antiviral effects of pMKRN1 E3 ligase during PCV2 infection and also suggest potential new control measures for PCV2 outbreaks.

  • Role of microvesicles in the spread of Herpes simplex virus type 1 in oligodendrocytic cells [PublishAheadOfPrint]

  • Herpes simplex virus type 1 (HSV-1) is a neurotropic pathogen that can infect many types of cells and establishes latent infections in neurons of sensory ganglia. In some cases, the virus spreads into the CNS causing encephalitis or meningitis. Cells infected with several different types of viruses may secrete microvesicles containing viral proteins and RNAs. In some instances, extracellular microvesicles harboring infectious virus have been found. Here we describe the features of shedding microvesicles released by the human oligodendroglial HOG cell line infected with HSV-1 and their participation in the viral cycle. Using transmission electron microscopy, we detected for the first time microvesicles containing HSV-1 virions. Interestingly, Chinese hamster ovary (CHO) cell line, which is resistant to infection by free HSV-1 virions, was susceptible to HSV-1 infection after being exposed to virus-containing microvesicles. Therefore, our results indicate for the first time that MVs released by infected cells contain virions, are endocytosed by naïve cells and lead to a productive infection. Furthermore, infection of CHO cells was not completely neutralized when virus-containing microvesicles were pre-incubated with neutralizing anti-HSV-1 antibodies. The lack of complete neutralization and the ability of MVs to infect nectin-1/HVEM negative CHO-K1 cells suggest a novel way for HSV-1 to spread to and enter target cells. Taken together, our results suggest that HSV-1 could spread through microvesicles to expand its tropism and shield the virus from neutralizing antibodies as a possible mechanism to escape the host immune response.

    Importance Herpes simplex virus type 1 (HSV-1) is a neurotropic pathogen that can infect many types of cells and establishes latent infections in neurons. Extracellular vesicles are a heterogeneous group of membrane vesicles secreted by most cell types. Microvesicles -extracellular vesicles which derive from the shedding of the plasma membrane- isolated from the supernatant of HSV-1-infected HOG cells were analysed to find out whether they were involved in the viral cycle. The importance of our investigation lies in the detection, for the first time, of microvesicles containing HSV-1 virions. In addition, virus-containing microvesicles were endocytosed into CHO-K1 cells and were able to actively infect these otherwise non-permissive cells. Finally, the infection of CHO cells with these virus-containing microvesicles was not completely neutralized by anti-HSV-1 antibodies, suggesting that these extracellular vesicles might shield the virus from neutralizing antibodies as a possible mechanism of immune evasion.

  • Caspase-dependent suppression of type I interferon signaling promotes KSHV lytic replication [PublishAheadOfPrint]

  • An important component of lytic infection by Kaposi's sarcoma-associated herpesvirus (KSHV) is the ability of the virus to evade the innate immune response, specifically type I interferon (IFN) responses that are triggered by recognition of viral nucleic acids. Inhibition of type I IFN responses by the virus promotes viral replication. Here we report that KSHV uses a caspase-dependent mechanism to block type I IFN, in particular IFNbbeta;, responses during lytic infection. Inhibition of caspases during KSHV reactivation resulted in increased TBK1/IKK-dependent phosphorylation of IRF3 as well as elevated levels of IFNbbeta; transcription and secretion. The increased secretion of IFNbbeta; upon caspase inhibition reduced viral gene expression, viral DNA replication and virus production. Blocking IFNbbeta; production or signaling restored viral replication. Overall, our results show that caspase-mediated regulation of pathogen sensing machinery is an important mechanism exploited by KSHV to evade innate immune responses.

    IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), an AIDS-defining tumor that is one of the most common causes of cancer death in sub-Saharan Africa. In this study, we examined the role of a set of cellular proteases called caspases in the regulation of immune responses during KSHV infection. We demonstrate that caspases prevent the induction and secretion of the anti-viral factor interferon bbeta; during replicative KSHV infection. The reduced interferon bbeta; production allows for high viral gene expression and viral replication. Therefore, caspases are important for maintaining KSHV replication. Overall, our results suggest that KSHV utilizes caspases to evade innate immune responses, and that inhibiting caspases could boost the innate immune response to this pathogen and potentially be a new antiviral strategy.

  • Dynamics of tissue-specific CD8+ T cell responses during West Nile virus infection [PublishAheadOfPrint]

  • The mouse model of West Nile virus, which is a leading cause of mosquito-borne encephalitis worldwide, has provided fundamental insights into the host and viral factors that regulate viral pathogenesis and infection outcome. In particular, CD8+ T cells are critical for controlling WNV replication and promoting protection against infection. Here, we present the characterization of a T cell receptor (TCR) transgenic mouse with specificity to the immunodominant epitope in the WNV NS4B protein (herein referred to as transgenic WNV-I mice). Using an adoptive transfer model, we found that WNV-I CD8+ T cells behave similar to endogenous CD8+ T cell responses, with an expansion phase in the periphery beginning around day 7 post-infection (pi) followed by a contraction phase through day 15 pi. Through the use of in vivo intravascular immune cell staining, we determined the kinetics, expansion and differentiation into effector and memory subsets of WNV-I CD8+ T cells within the spleen and brain. We found that red pulp WNV-I CD8+ T cells were more effector-like as compared to white pulp WNV-I CD8+ T cells, which displayed increased differentiation into memory precursor cells. Within the central nervous system (CNS), we found that WNV-I CD8+ T cells were polyfunctional (IFN-, TNF-aalpha;), displayed tissue-resident characteristics (CD69+ and CD103+), persisted in the brain through day 15 pi and reduced viral burden within the brain. The use of these TCR transgenic WNV-I mouse provide a new resource to dissect the immunological mechanisms of CD8+ T cell-mediated protection during WNV infection.

    Author summary West Nile Virus (WNV) is the leading cause of mosquito-borne encephalitis worldwide. There are currently no approved therapeutics or vaccines for use in humans to treat or prevent WNV infection. CD8+ T cells are critical for controlling WNV replication and protecting against infection. Here, we present the generation and comprehensive characterization of a novel TCR transgenic mouse with specificity to the immunodominant epitope in the WNV NS4B protein. In this study, we determine the kinetics, proliferation, differentiation into effector and memory subsets, homing, and clearance of WNV in the CNS. Our findings provide a new resource to dissect the immunological mechanisms of CD8+ T cell-mediated protection during WNV infection.

  • Relative abundance of the integrant-derived viral RNAs in infected tissues harvested from chronic HBV carriers [PublishAheadOfPrint]

  • Five matching sets of non-malignant liver tissues and HCCs from individuals chronically infected with hepatitis B virus (HBV) were examined. The HBV genomic sequences were determined using overlapping PCR amplicons covering the entire viral genome. Four pairs of tissues were infected with HBV of genotype C, while one pair - with genotype B. HBV replication markers were found in all tissues. In majority of HCC samples, the levels of pre-genomic/pre-core RNA (pgRNA) and covalently closed circular DNA (cccDNA) were lower than those of liver tissue counterparts. Regardless of the presence of HBV replication markers, (i) integrant-derived HBV RNAs (id-RNAs) were found using RT-PCR analysis in all tissues, and were considerably abundant or predominant in 6/10 tissue samples (2 livers and 4 HCCs); (ii) the RNAs that were polyadenylated using cryptic HBV polyadenylation signal and therefore could be produced by HBV replication or derived from integrated HBV DNA were found in 5/10 samples (3 livers and 2 HCCs), and were considerably abundant species in 3/10 tissues (2 livers and 1 HCC); and (iii) cccDNA-transcribed RNAs polyadenylated near position 1931 were not abundant in 7/10 tissues (2 livers and 5 HCCs), and were predominant only in two livers. Subsequent RNA sequencing analysis of selected liver/HCC samples also showed relative abundance of id-RNAs in most of examined tissues. Our findings suggesting that id-RNAs could represent a significant source of HBV envelope proteins, which is independent of viral replication, are discussed in the context of possible contribution of id-RNAs to the HBV life cycle.

    IMPORTANCE The relative abundance of integrant-derived HBV RNAs (id-RNAs) in chronically infected tissues suggests that id-RNAs coding for the envelope proteins may facilitate production of considerable fraction of surface antigens (HBsAg) in infected cells bearing HBV integrants. If the same cells support HBV replication, then a significant fraction of assembled HBV virions could bear id-RNA-derived HBsAg as a major component of their envelopes. Therefore, infectivity of these HBV virions, and their ability to facilitate virus cell-to-cell spread could be determined mainly by the properties of id-RNA-derived envelope proteins, and not by the properties of replication-derived HBsAg. These interpretations suggest that id-RNAs may play a role in maintenance of chronic HBV infection, and therefore contribute to HBV life cycle. Furthermore, the production of HBsAg from id-RNAs independently of viral replication may at least in part explain why treatment with interferon or nucleos(t)ides in most cases failed to achieve loss of serum HBsAg.

  • Asymmetric modification of HBV genomes by an endogenous cytidine deaminase inside HBV cores informs a model of reverse transcription [PublishAheadOfPrint]

  • Cytidine deaminases inhibit replication of broad range of DNA viruses by deaminating cytidines on single stranded DNA to generate uracil. While several lines of evidence have revealed HBV genome editing by deamination, it is still unclear which nucleic acid intermediate of HBV is modified. Hepatitis B virus has a relaxed circular double-stranded DNA (rcDNA) genome that is reverse transcribed within virus cores from a RNA template. The HBV genome also persists as covalently closed circular DNA (cccDNA) in the nucleus of an infected cell. In the present study, we find that in HBV-producing HepAD38 and Hep2.2.15 cell lines, endogenous cytidine deaminases edited 10-25% of HBV rcDNA genomes, asymmetrically with almost all mutations on the 5rrsquo; half of the minus strand. This region corresponds to the last half of the minus strand to be protected by plus strand synthesis. Within this half of the genome, the number of mutations peaks in the middle. Over-expressed APOBEC3A and APOBEC3G could be packaged in HBV capsids but did not change the amount or distribution of mutations. We found no deamination on pgRNA indicating that an intact genome is encapsidated and deaminated during or after reverse transcription. The deamination pattern suggests a model of rcDNA synthesis where pgRNA and then newly synthesized minus-sense single stranded DNA are protected from deaminase by interaction with the virus capsid; during plus strand synthesis, when enough dsDNA has been synthesized to displace the remaining minus strand from the capsid surface that single stranded DNA becomes deaminase-sensitive.

    IMPORTANCE Host-induced mutation of the HBV genome, as by APOBEC proteins, may be a path to clearing the virus. We examined Cytidine to Thymidine mutations in the genomes of HBV particles grown in the presence or absence of overexpressed APOBEC proteins. We found that genomes were subjected to deamination activity during reverse transcription, which takes place within the virus capsid. These observations provide a direct insight into the mechanics of reverse transcription, suggesting that newly synthesized dsDNA displaces ssDNA from the capsid walls making the ssDNA accessible to deaminase activity.

  • Virulent poxviruses inhibit DNA sensing by preventing STING activation [PublishAheadOfPrint]

  • Cytosolic recognition of DNA has emerged as a critical cellular mechanism of host immune activation upon pathogen invasion. The central cytosolic DNA sensor cGAS activates STING, which is phosphorylated, dimerises and translocates from the ER to a perinuclear region to mediate IRF-3 activation. Poxviruses are dsDNA viruses replicating in the cytosol and hence likely to trigger cytosolic DNA sensing. Here we investigated the activation of innate immune signalling by 4 different strains of the prototypic poxvirus vaccinia virus (VACV) in a cell line proficient in DNA sensing. Infection with the attenuated VACV strain MVA activated IRF-3 via cGAS and STING, and accordingly STING dimerised and was phosphorylated during MVA infection. Conversely, VACV strains Copenhagen and Western Reserve inhibited STING dimerisation and phosphorylation during infection and in response to transfected DNA and cGAMP, thus efficiently suppressing DNA sensing and IRF-3 activation. A VACV deletion mutant lacking protein C16, thought to be the only viral DNA sensing inhibitor acting upstream of STING, retained the ability to block STING activation. Similar inhibition of DNA-induced STING activation was also observed for cowpox and ectromelia viruses. Our data demonstrate that virulent poxviruses possess mechanisms for targeting DNA sensing at the level of the cGAS-STING axis and that these mechanisms do not operate in replication-defective strains such as MVA. These findings shed light on the role of cellular DNA sensing in poxvirus-host interactions and will open new avenues to determine its impact on VACV immunogenicity and virulence.

    IMPORTANCE Poxviruses are dsDNA viruses infecting a wide range of vertebrates and include the causative agent of smallpox (variola virus) and its vaccine vaccinia virus (VACV). Despite smallpox eradication VACV remains of interest as a therapeutic. Attenuated strains are popular vaccine candidates, whereas replication-competent strains are emerging as efficient oncolytics in virotherapy. The successful therapeutic use of VACV depends on a detailed understanding of its ability to modulate host innate immune responses. DNA sensing is a critical cellular mechanism for pathogen detection and activation of innate immunity that is centrally coordinated by the ER-resident protein STING. Here STING is shown to mediate immune activation in response to MVA, but not to virulent VACV strains or other virulent poxviruses, which prevent STING activation and DNA sensing during infection and after DNA transfection. These results provide new insights into poxvirus immune evasion and have implications in the rational design of VACV-based therapeutics.

  • Euphyllophyte paleoviruses illuminate hidden diversity and macroevolutionary mode of Caulimoviridae [PublishAheadOfPrint]

  • Endogenous viral elements (paleoviruses) provide llsquo;molecular fossilsrrsquo; for studying the deep history and macroevolution of viruses. Endogenous plant pararetroviruses (EPRVs) are widespread in angiosperms, but little is known about EPRVs in earlier branching plants. Here we use a large-scale phylogenomic approach to investigate the diversity and macroevolution of plant pararetroviruses (formally known as Caulimoviridae). We uncover an unprecedented and unappreciated diversity of EPRVs within the genomes of gymnosperms and ferns. The known angiosperm viruses only constitute a minor part of the Caulimoviridae diversity. By characterizing the distribution of EPRVs, we show that no major euphyllophyte lineages escape the activity of Caulimoviridae, raising the possibility that many exogenous Caulimoviridae remain to be discovered in euphyllophytes. We find that the copy numbers of EPRVs are generally high, suggesting that EPRVs might define a unique group of repetitive elements and represent important components of euphyllophyte genomes. Evolutionary analyses suggest an ancient origin of Caulimoviridae and at least three independent origins of Caulimoviridae in angiosperms. Our findings reveal the remarkable diversity of Caulimoviridae and have important implications in understanding the origin and macroevolution of plant pararetroviruses.

    IMPORTANCE Few viruses have been documented in plants outside angiosperms. Viruses can occasionally integrate into host genomes, forming endogenous viral elements (EVEs). Endogenous plant pararetroviruses (EPRVs) are widespread in angiosperms. In this study, we performed comprehensive comparative and phylogenetic analyses of EPRVs and found EPRVs are present in the genomes of gymnosperms and ferns. We identified numerous EPRVs in gymnosperm and fern genomes, revealing an unprecedented depth in the diversity of plant pararetroviruses. Plant pararetroviruses mainly underwent cross-species transmission and angiosperm pararetroviruses arose at least three times. Our study provides novel insights into the diversity and macroevolution of plant pararetroviruses.

  • Intragastric administration of Lactobacillus plantarum and AT-2-inactivated SIV does not protect Indian rhesus macaques from intra-rectal SIV challenge nor reduce virus replication after transmission. [PublishAheadOfPrint]

  • A major obstacle to development of an effective AIDS vaccine is that along with intended beneficial responses, immunization regimen may activate CD4+ T cells that can facilitate acquisition of HIV by serving as target cells for the virus. Lu et al. reported that intra-gastric administration of chemically inactivated SIVmac239 (iSIV) and Lactobacillus plantarum (LP) (iSIV+LP) protected 15/16 Chinese-origin rhesus macaques (RMs) from high-dose intra-rectal SIVmac239 challenge at three months post-immunization. They attributed the observed protection to induction of immune tolerance, mediated by "MHC-Ib/E-restricted CD8+ regulatory T cells that suppressed SIV-harboring CD4+ T cell activation and ex vivo SIV replication in 15/16 animals without inducing SIV-specific antibodies or cytotoxic T". Andrieu et al subsequently reported protection from infection in 23/24 RM immunized intragastrically or intravaginally with iSIV and BCG, LP or Lactobacillus rhamnosus, which they ascribed to the same tolerogenic mechanism. Using vaccine materials obtained from our co-authors, we conducted an immunization and challenge experiment in 54 Indian RMs, and included control groups receiving iSIV only or LP only, as well as unvaccinated animals. Intra-rectal challenge with SIVmac239 resulted in rapid infection in all groups of vaccinated RMs as well as unvaccinated controls. iSIV+LP vaccinated animals that became SIV infected showed viral loads similar to those observed in animals receiving iSIV only, LP only, and unvaccinated controls. The protection from SIV transmission conferred by intra-gastric iSIV+LP administration reported previously for Chinese origin RMs was not observed when the same experiment was conducted in a larger cohort of Indian-origin animals.

    IMPORTANCE: Despite increased understanding in immune responses against HIV, a safe and effective AIDS vaccine is not yet available. One obstacle is that immunization may activate CD4+ T cells that could act as target cells for acquisition of HIV. An alternative strategy could involve induction of a tolerizing response that limits the availability of activated CD4+ T cells, thus limiting the ability of virus to establish infection. In this regard, exciting results were obtained in Chinese-origin rhesus macaques by using a "tolerogenic" vaccine consisting of intra-gastric administration of Lactobacillus plantarum and AT-2-inactivated SIV (iSIV+LP) which showed highly significant protection from virus transmission. Here, we administered iSIV+LP immunizations to Indian-origin rhesus macaques, and failed to observe any protective effect from virus acquisition in this experimental setting. This work is important as it contributes to the overall assessment of the clinical potential of a new candidate AIDS vaccine platform based on the iSIV+LP.

  • Enhanced human-type receptor binding by ferret transmissible H5N1 with a K193T mutation [PublishAheadOfPrint]

  • All human influenza pandemics have originated from avian influenza viruses. Although multiple changes are needed for an avian virus to be able to transmit between humans, binding to human-type receptors is essential. Several research groups have reported mutations in H5N1 viruses that exhibit specificity to human-type receptors and promote respiratory droplet transmission between ferrets. Upon detailed analysis we have found that these mutants exhibit significant differences in fine receptor specificity compared to human H1N1 and H3N2 and retain avian-type receptor binding. We have recently shown that human influenza viruses preferentially bind to aalpha;2-6 sialylated branched N-linked glycans, where the sialic acids on each branch can bind to receptor sites on two protomers of the same HA trimer. In this binding mode the glycan projects over the 190-helix at the top of the receptor-binding pocket, which in H5N1 would create stearic clash with lysine at 193. Thus we hypothesized that a K193T mutation, would improve binding to branched N-linked receptors. Indeed, adding the K193T mutation to the H5 HA of a respiratory droplet transmissible virus dramatically improves both binding to human trachea epithelial cells and specificity for extended aalpha;2-6 sialylated N-linked glycans recognized by human influenza viruses.

    IMPORTANCE Infections by avian H5N1 viruses are associated with a high mortality rate in several species including humans. Fortunately H5N1 viruses do not transmit between humans because they don't bind to human-type receptors. In 2012, three seminal papers have shown how these viruses can be engineered to transmit between ferrets, the human model for influenza virus infection. Receptor binding, amongst others, was changed, and now binds to human-type receptors. Receptor specificity was still markedly different compared to human influenza viruses. Here we report an additional mutation in ferret transmissible H5N1 that increases human-type receptor binding. K193T seems to be a common receptor specificity determinant as it increases human-type receptor binding in multiple subtypes. The K193T mutation can now be used as a marker during surveillance of emerging viruses to assess potential pandemic risk.

  • IDENTIFICATION OF PIPERAZINYLBENZENESULFONAMIDES AS NEW INHIBITORS OF CLAUDIN-1 TRAFFICKING AND HEPATITIS C VIRUS ENTRY [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) infection causes 500,000 deaths annually, associated with end-stage liver diseases. Investigations of the HCV life cycle widened the knowledge in virology, and here we discovered that two piperazinylbenzenesulfonamides inhibit HCV entry into liver cells. The entry process of HCV into host cells is a complex process, not fully understood, but characterized by multiple spatially and temporally regulated steps involving several known host factors. Through a high-content virus-infection screening analysis with a library of 1,120 biologically active chemical compounds, we identified SB258585, an antagonist of the serotonin receptor 6 (5-HT6), as a new inhibitor of HCV entry in liver-derived cell lines, as well as in primary hepatocytes. A functional characterization suggested a role for this compound, as well as for the compound SB399885 sharing a similar structure, as inhibitors of a late HCV entry step, modulating the localization of the co-receptor tight junction protein claudin 1 (CLDN1), in a 5-HT6 independent manner. Both chemical compounds induced an intracellular accumulation of CLDN1, reflecting export impairment. This regulation correlated with the modulation of protein kinase A (PKA) activity. The PKA inhibitor H89 fully reproduced these phenotypes. Furthermore, PKA activation resulted in increased CLDN1 accumulation at the cell surface. Interestingly, increase of CLDN1 recycling did not correlate with an increased interaction with CD81 or HCV entry. These findings reinforce the hypothesis of a common pathway shared by several viruses, which involves G-protein coupled receptor -dependent signaling in late steps of viral entry.

    IMPORTANCE The HCV entry process is highly complex and important details of this structured event are poorly understood. By screening a library of biologically active chemical compounds, we identified two piperazinylbenzenesulfonamides as inhibitors of HCV entry. The mechanism of inhibition was not through previously described activity of these inhibitors as antagonists of the serotonin receptor 6, but instead through modulation of PKA activity in a 5-HT6 independent manner, as proven by the lack of 5-HT6 in liver. We thus highlighted the involvement of PKA pathway in modulating HCV post-binding step entry and in the recycling of the tight junction protein claudin-1 (CLDN1) towards the cell surface. Our work underscores once more the complexity of HCV entry steps and suggests a role for PKA pathway as regulator of CLDN1 recycling, having an impact on both cell biology and virology.

  • IFN{alpha}/{beta} signaling in astrocytes mediates protection against viral encephalomyelitis and regulates IFN{gamma} dependent responses [PublishAheadOfPrint]

  • The contribution of distinct central nervous system (CNS) resident cells to protective IFNaalpha;/bbeta; function following viral infections is poorly understood. Based on numerous immune regulatory functions of astrocytes, we evaluated the contribution of astrocyte-IFNaalpha;/bbeta; signaling towards protection against a non-lethal glia- and neurono-tropic mouse hepatitis virus (MHV-A59). Analysis of gene expression associated with IFNaalpha;/bbeta; function, e.g. pattern recognition receptors (PRRs) and interferon-stimulated genes (ISGs) revealed lower basal mRNA levels in brain derived astrocytes compared to microglia. Although astrocytes poorly induced Ifnbbeta; mRNA following infection, they upregulated various mRNAs in the IFNaalpha;/bbeta; pathway to a higher extent than microglia, supporting effective IFNaalpha;/bbeta; responsiveness. Ablation of the IFNaalpha;/bbeta; receptor (IFNAR) in astrocytes using mGFAPcre IFNARfl/fl mice resulted in severe encephalomyelitis and mortality, coincident with uncontrolled virus replication. Further, virus spread was not restricted to astrocytes, but also affected microglia and neurons, despite increased and sustained Ifnaalpha;/bbeta; and ISG mRNA levels within the CNS. IFN, a crucial mediator for MHV control was not impaired in infected mGFAPcre IFNARfl/fl mice despite reduced T cell CNS infiltration. Unexpectedly however, poor induction of IFN dependent MHC class II expression on microglia supported that defective IFN signaling contributes to uncontrolled virus replication. A link between sustained elevated IFNaalpha;/bbeta; and impaired responsiveness to IFN supports the novel concept that temporally limited early IFNaalpha;/bbeta; responses are critical for effective antiviral IFN function. Overall our results imply that IFNaalpha;/bbeta; signaling in astrocytes is not only critical in limiting early CNS viral spread, but also promotes protective antiviral IFN function.

    IMPORTANCE An antiviral state established by IFNaalpha;/bbeta; contains initial viral spread as adaptive immunity develops. While it is apparent that the CNS lacks professional IFNaalpha;/bbeta; producers and resident cells have distinct abilities to elicit innate IFNaalpha;/bbeta; responses, protective interactions between inducer and responder cells require further investigation. Infection with a glia- and neurono-tropic coronavirus demonstrates that astrocytes mount a delayed but more robust response to infection than microglia, despite their lower basal mRNA levels of IFNaalpha;/bbeta; inducing components. Lethal, uncontrolled viral dissemination following ablation of astrocyte IFNaalpha;/bbeta; signaling revealed the importance of IFNaalpha;/bbeta; responses in a single cell type for protection. Sustained global IFNaalpha;/bbeta; expression associated with uncontrolled virus did not suffice to protect neurons and further impaired responsiveness to protective IFN. The results support astrocytes as critical contributors to innate immunity and the concept that limited IFNaalpha;/bbeta; responses are critical for effective subsequent antiviral IFN function.

  • Caspase-dependent apoptosis induction via viral protein ORF4 of porcine circovirus 2 binding to mitochondrial adenine nucleotide translocase 3 [PublishAheadOfPrint]

  • Apoptosis is an essential strategy of host defense responses and is used by viruses to maintain their life cycles. However, the apoptotic signals involved in virus replication are poorly known. In the present study, we report the molecular mechanism of apoptotic induction by viral protein ORF4, a newly identified viral protein of porcine circovirus type 2 (PCV2). Apoptosis detection revealed that not only is the activity of caspases 3 and 9 is increased in PCV2-infected and ORF4-transfected cells, but also cytochrome c release from the mitochondria to the cytosol is upregulated. Subsequently, ORF4 colocalization with adenine nucleotide translocase 3 (ANT3) was observed using structured illumination microscopy. Moreover, co-immunoprecipitation and pulldown analyses confirmed that ORF4 interacts directly with mitochondrial ANT3. Binding domain analysis further confirmed that N-terminal residues 1 to 30 of ORF4, comprising a mitochondrial targeting signal, are essential for the interaction with ANT3. Knockdown of ANT3 markedly inhibited the apoptotic induction of both ORF4 and PCV2, indicating that ANT3 plays an important role in ORF4-induced apoptosis during PCV2 infection. Taken together, these data indicated that ORF4 is a mitochondrial targeting protein that induces apoptosis by interacting with ANT3 through the mitochondrial pathway.

    IMPORTANCE Porcine circovirus type 2 (PCV2) encoded protein ORF4 is a newly identified viral protein; however, little is known about its functions. Apoptosis is an essential strategy of the host defense response and is used by viruses to maintain their life cycles. In the present study, we report the molecular mechanism of the apoptosis induced by ORF4. ORF4 contains a mitochondrial targeting signal and is an unstable protein that is degraded by the proteasome-dependent pathway. Viral protein ORF4 triggers caspases-3 and -9-dependent cellular apoptosis in mitochondria by directly binding to ANT3. We concluded that ORF4 is a mitochondrial targeting protein and revealed a mechanism whereby circovirus recruits ANT3 to induce apoptosis.

  • Annexin A2 Mediates the Localization of Measles Virus Matrix Protein at the Plasma Membrane [PublishAheadOfPrint]

  • Annexins are a family of structurally related proteins that bind negatively charged membrane phospholipids in a Ca2+-dependent manner. Annexin A2 (AnxA2), a member of the family, has been implicated in a variety of cellular functions including the organization of membrane domains, vesicular trafficking and cell-cell adhesion. AnxA2 generally forms the heterotetrameric complex with a small Ca2+-binding protein S100A10. Measles virus (MV), a member of the family Paramyxoviridae, is an enveloped virus with a nonsegmented negative strand RNA genome. Knockdown of AnxA2 greatly reduced MV growth in cells, without affecting its entry and viral RNA production. In MV-infected, AnxA2-knockdown cells, the expression level of the matrix (M) protein, but not other viral proteins, was reduced compared with that in control cells, and the distribution of the M protein at the plasma membrane was decreased. The M protein lines the inner surface of the envelope and plays an important role in virus assembly by connecting the nucleocapsid to the envelope proteins. The M protein bound to AnxA2 independently of AnxA2's phosphorylation or its association with S100A10, and was co-localized with AnxA2 within cells. Truncation of the N-terminal 10 amino acid residues, but not the N-terminal 5 residues, compromised the ability of the M protein to interact with AnxA2 and localize at the plasma membrane. These results indicate that AnxA2 mediates the localization of the MV M protein at the plasma membrane by interacting with its N-terminal region (especially residues at positions 6-10), thereby aiding in MV assembly.

    IMPORTANCE Measles virus (MV) is an important human pathogen, still claiming ~ 100,000 lives per year despite the presence of effective vaccines, and causes occasional outbreaks even in developed countries. Replication of viruses largely relies on the functions of host cells. Our study revealed that the reduction of the host protein annexin A2 compromises the replication of MV within the cell. Further studies demonstrated that annexin A2 interacts with the MV matrix (M) protein and mediates the localization of the M protein at the plasma membrane where MV particles are formed. The M protein lines the inner surface of the MV envelope membrane and plays a role in MV particle formation. Our results provide useful information for the understanding of the MV replication process and potential development of anti-viral agents.

  • Copy number heterogeneity, large origin tandem repeats, and interspecies recombination in HHV-6A and HHV-6B reference strains [PublishAheadOfPrint]

  • Quantitative PCR is the diagnostic pillar for clinical virology testing, and reference materials are necessary for accurate, comparable quantitation between clinical laboratories. Accurate quantitation of HHV-6A/B is important for detection of viral reactivation and inherited chromosomally integrated HHV-6A/B in immunocompromised patients. Reference materials in clinical virology commonly consist of laboratory-adapted viral strains that may be affected by the culture process. We performed next-generation sequencing to make relative copy number measurements at single nucleotide resolution of eight candidate HHV-6A and seven HHV-6B reference strains and DNA materials from the HHV-6 Foundation and Advanced Biotechnologies. 11 of 17 (65%) HHV-6A/B candidate reference materials showed multiple copies of the origin of replication upstream of the U41 gene by next-generation sequencing. These large tandem repeats arose independently in culture-adapted HHV-6A and HHV-6B strains, measuring 1254 bp and 983 bp, respectively. Average copy number measured between 5-10X copies relative to the rest of the genome. We also report the first interspecies recombinant HHV-6A/B strain with a HHV-6A backbone and ggt;5.5kb region from HHV-6B from U41-U43 that covered the origin tandem repeat. Specific HHV-6A reference strains demonstrated duplication of regions at U1/U2, U87, and U89, as well as deletion in the U12-U24 region and U94/95 genes. HHV-6A/B strains derived from cord blood mononuclear cells from different labs on different continents with fewer passages revealed no copy number differences throughout the viral genome. These data indicate large origin tandem duplications are an adaptation of both HHV-6A and HHV-6B in culture and show interspecies recombination is possible within the Betaherpesvirinae.

    IMPORTANCE: Anything in science that needs to be quantitated requires a standard unit of measurement. This includes viruses, for which quantitation increasingly determines definitions of pathology and guidelines for treatment. However, the act of making standard or reference material in virology can alter its very accuracy through genomic duplications, insertions, and rearrangements. We used deep sequencing to examine candidate reference strains for HHV-6, a ubiquitous human virus that can reactivate in the immunocompromised population and is integrated into the human genome in every cell of the body for 1% of people worldwide. We found large tandem repeats in the origin of replication for both HHV-6A and HHV-6B that are selected for in culture. We also found the first interspecies recombinant between HHV-6A and HHV-6B, a phenomenon that is well-known in alphaherpesviruses but to date has not been seen in betaherpesviruses. These data critically inform HHV-6A/B biology and the standard selection process.

  • Mutations in the basic region of the Mason-Pfizer monkey virus nucleocapsid protein affect reverse transcription, gRNA packaging and the site of viral assembly [PublishAheadOfPrint]

  • In addition to specific RNA-binding zinc finger domains, retroviral Gag polyprotein contains clusters of basic amino acid residues thought to support Gag-viral genomic RNA (gRNA) interactions. One of these clusters is the basic K16NK18EK20 region, located upstream of the first zinc finger of the Mason-Pfizer monkey virus (M-PMV) nucleocapsid protein (NC). To investigate the role of this basic region in the M-PMV life cycle, we used a combination of in vivo and in vitro methods to study a series of mutants in which the overall charge of this region was more positive (RNRER), more negative (AEAEA), or neutral (AAAAA). The mutations markedly affected gRNA incorporation and the onset of reverse transcription. Introducing a more negative charge (AEAEA) significantly reduced the incorporation of M-PMV gRNA into nascent particles. Moreover, the assembly of immature particles of the AEAEA Gag mutant was relocated from the perinuclear region to the plasma membrane. In contrast, enhancing the basicity of this region of M-PMV NC (RNRER) caused substantially more efficient incorporation of gRNA, subsequently resulting in an increase in M-PMV RNRER infectivity. Nevertheless, despite the higher amount of gRNA packaged by the RNRER mutant, the onset of reverse transcription was delayed in comparison to that of the wild type. Our data clearly show the requirement for certain positively charged amino acid residues upstream of the first zinc finger for proper gRNA incorporation, assembly of immature particles and proceeding of reverse transcription.

    Importance: We identified a short sequence within Gag polyprotein that, together with the zinc finger domains and previously identified RKK motif, contributes to packaging of genomic RNA (gRNA) of Mason-Pfizer monkey virus (M-PMV). Importantly, in addition to gRNA incorporation this basic region (KNKEK) at the N-terminus of nucleocapsid protein is crucial for an onset of reverse transcription. The mutations changing the positive charge of the region to a negative one significantly reduced specific gRNA packaging. The assembly of immature particles of this mutant was reoriented from the perinuclear region to the plasma membrane. On the contrary, enhancing the basic character of this region increased the efficiency of both gRNA packaging and infectivity of the virus. However, the onset of reverse transcription was delayed even in this mutant. Summing up, the basic region in M-PMV Gag plays a key role in packaging of genomic RNA and consequently in the assembly and reverse transcription.

  • Tunneling nanotubes (TNT) as a novel route of cell-to-cell spread of herpesviruses [PublishAheadOfPrint]

  • Various types of intercellular connections that are essential for communication between cells are often utilized by pathogens. Recently, a new type of cellular connection, long, thin, actin-rich membrane extensions named tunneling nanotubes (TNTs), have been shown to play an important role in cell-to-cell spread of HIV and influenza virus. In the present report, we show that TNTs are frequently formed by cells infected by an alpha-herpesvirus BoHV-1 (bovine herpesvirus 1). Viral proteins, such as envelope glycoprotein gE, capsid protein VP26 and tegument protein Us3, as well as cellular organelles (mitochondria) were detected by immunofluorescence and live cell imaging of nanotubes formed by bovine primary fibroblasts and oropharynx cells (KOP). Time-lapse confocal studies of live cells infected with fluorescently labeled viruses showed that viral particles were transmitted via TNTs. This transfer also occurred in the presence of neutralizing antibodies, which prevented free entry of BoHV-1. We conclude that TNT formation contributes to successful cell-to-cell spread of BoHV-1 and demonstrate for the first time the participation of membrane nanotubes in intercellular transfer of a herpesvirus in live cells.

    IMPORTANCE Efficient transmission of viral particles between cells is an important factor in successful infection by herpesviruses. Herpesviruses can spread by the free entry mode or direct cell-to-cell transfer via cell junctions and long extensions of neuronal cells. In this report, we show for the first time that an alpha-herpesvirus can also spread between various types of cells using tunneling nanotubes, intercellular connections that are utilized by HIV and other viruses. Live cells monitoring revealed that viral transmission occurs between the cells of the same type as well as between epithelial cells and fibroblasts.This newly discovered route of herpesviruses spread may contribute to efficient transmission despite the presence of host immune responses, especially after reactivation from latency that developed after primary infection. Long-range communication provided by TNTs may facilitate the spread of herpesviruses between many tissues and organs of an infected organism.

  • Mechanisms of Host IFI16, PML and Daxx Protein Restriction of Herpes Simplex Virus 1 Replication [PublishAheadOfPrint]

  • The initial events after DNA virus infection involve a race between epigenetic silencing of the incoming viral DNA by host cell factors and expression of viral genes. Several host gene products, including the nuclear domain 10 (ND10) components PML (promyelocytic leukemia) and Daxx (death domain-associated protein 6), as well as IFI16 (interferon-inducible protein 16), have been shown to restrict herpes simplex virus 1 (HSV-1) replication. Whether IFI16 and ND10 components work together or separately to restrict HSV-1 replication is not known. To determine the combinatorial effects of IFI16 and ND10 proteins on viral infection, we depleted Daxx or PML in primary human fibroblasts in the presence or absence of IFI16. Daxx or IFI16 depletion resulted in higher ICP0 mutant viral yields, and their effects were additive. Surprisingly, siRNA depletion of PML in the HFF cells led to decreased ICP0-null virus replication, while shRNA depletion led to increased ICP0-null virus replication, arguing that different PML isoforms or PML-related proteins may have restrictive or pro-viral functions. In normal human cells, viral DNA replication increases expression of all classes of HSV-1 genes. We observed that IFI16 repressed transcription from both parental and progeny DNA genomes. Taken together, our results show that the mechanisms of action of IFI16 and ND10 proteins are independent, at least in part, and that IFI16 exerts restrictive effects on both input and replicated viral genomes. These results raise the potential for distinct mechanisms of action of IFI16 on parental and progeny viral DNA molecules.

    IMPORTANCE Many human DNA viruses transcribe their genome and replicate in the nucleus of a host cell, where they exploit the host cell nuclear machinery for their own replication. Host factors attempt to restrict viral replication by blocking those events, and viruses have evolved mechanisms to neutralize the host restriction factors. In this study, we provide information about the mechanism of action of three host cell factors that restrict replication of herpes simplex virus (HSV), and we found that these factors function independently and one acts to restrict viral transcription from parental and progeny viral DNA genomes. These results provide new information how cells counter DNA virus replication in the nucleus and provide possible approaches to enhance the ability of human cells to resist HSV infection.

  • An M2 rather than TH2 response contributes to better protection against latency-reactivation following ocular infection of naive mice with a recombinant HSV-1 expressing murine IL-4 [PublishAheadOfPrint]

  • We found previously that altering macrophage polarization toward M2 responses by injection of colony stimulating factor-1 (CSF-1) was more effective in reducing both primary and latent infection in HSV-1 ocularly infected mice than M1 polarization by IFN- injection. Cytokines can coordinately regulate macrophage and TH responses, with IL-4 inducing TH2 as well as M2 responses and IFN- inducing TH1 as well as M1 responses. We have now differentiated the contributions of these immune compartments on protection against latency/reactivation and corneal scarring by comparing the effects of infection with recombinant HSV-1 in which the latency associated transcript (LAT) gene was replaced with either the IL-4 (HSV-IL-4) or IFN- (HSV-IFN-) genes using infection with the parental (LAT-negative) virus as a control. Analysis of peritoneal macrophages in vitro established that the replacement of LAT with the IL-4 or IFN- genes did not affect virus infectivity and promoted polarization appropriately. Protection against corneal scarring was significantly higher in mice ocularly infected with HSV-IL-4 than those infected with HSV-IFN- or parental virus. Primary virus replication in the eyes and trigeminal ganglia (TG) was similar in the three groups of mice but the numbers of gC+ cells were lower on day 5 post infection in the eyes of HSV-IL-4-infected mice than those infected with HSV-IFN- or parental virus. Latency and explant reactivation were lower in both HSV-IL-4- or HSV-IFN--infected mice than those infected with parental virus with the lowest level of latency being associated with HSV-IL-4 infection. Higher latency correlated with higher levels of CD8, PD-1 and IFN- mRNA, while reduced latency and T-cell exhaustion correlated with lower gC+ expression in the TG. Depletion of macrophages increased the levels of latency in all ocularly infected mice compared with their undepleted counterparts, with macrophage depletion increasing latency in the HSV-IL-4 group by greater than 3000-fold. Our results suggest that shifting the innate macrophage immune responses toward M2, rather than M1 responses, in HSV-1 infection would improve protection against establishment of latency, reactivation and eye disease.

    IMPORTANCE Ocular HSV-1 infections are among the most frequent serious viral eye infections in the U.S. and a major cause of viral-induced blindness. As establishment of a latent infection in the trigeminal ganglia results in recurrent infection and is associated with corneal scarring, prevention of latency/reactivation is a major therapeutic goal. It is well-established that absence of latency associated transcripts (LAT) reduces latency/reactivation. Here, we demonstrate that recombinant HSV-1 expressing IL-4 (an inducer of TH2/M2 responses) or IFN- (an inducer of TH1/M1 responses) in place of LAT further reduced latency with HSV-IL-4 showing the highest overall protective efficacy. In naive mice, this higher protective efficacy was mediated by innate rather than adaptive immune responses. Although both M1 and M2 macrophage responses were protective, shifting macrophages towards an M2 response through expression of IL-4 was more effective in curtailing ocular HSV-1 latency-reactivation.

  • Hepatitis B virus DNA integration occurs early in the viral life cycle in an in vitro infection model via NTCP-dependent uptake of enveloped virus particles [PublishAheadOfPrint]

  • Chronic infection by the Hepatitis B Virus (HBV) is the major contributor to liver disease worldwide. Though HBV replicates via a nuclear episomal DNA (cccDNA), integration of HBV DNA into the host cell genome is regularly observed in the liver of infected patients. While reported as a pro-oncogenic alteration, the mechanism(s) and timing of HBV DNA integration are not well-understood, chiefly due to the lack of in vitro infection models that have detectable integration events. Here, we have established an in vitro system in which integration can be reliably detected following HBV infection. We measured HBV DNA integration using inverse nested PCR in primary human hepatocytes, HepaRG-NTCP, HepG2-NTCP, and Huh7-NTCP cells after HBV infection. Integration was detected in all cell types at a rate of ggt;1 per 10000 cells, with the most consistent detection in Huh7-NTCP cells. Integration rate remained stable between 3 and 9 days post-infection. HBV DNA integration was efficiently blocked by treatment with 200nM of the HBV entry inhibitor Myrcludex B, but not with 10mmu;M Tenofovir, 100U Interferon alpha, or 1mmu;M of the capsid assembly inhibitor GLS4. This suggests integration of HBV DNA occurs immediately after infection of hepatocytes and is likely independent of de novo HBV replication in this model. Site analysis revealed that HBV DNA integrations were distributed over the entire human genome. Further, integrated HBV DNA sequences were consistent with double-stranded linear HBV DNA being the major precursor. Thus, we have established an in vitro system to interrogate the mechanisms of HBV DNA integration.

    Importance

    Hepatitis B Virus (HBV) is a common blood-borne pathogen and, following a chronic infection, can cause liver cancer and liver cirrhosis. Integration of HBV DNA into the host genome occurs in all known members of the hepadnaviridae family, despite this form not being necessary for viral replication. HBV DNA integration has been reported to drive liver cancer formation and persistence of virus infection. However, when and the mechanism(s) by which HBV DNA integration occurs is not clear. Here, we have developed and characterized an in vitro system to reliably detect HBV DNA integrations that result from a true HBV infection event and that closely resemble those found in patient tissues. Using this model, we show that integration already occurs when the infection is first established. Importantly, we provide here a system to analyze molecular factors involved in HBV integration, which can be used to develop strategies to halt its formation.

  • A new quinoline BRD4 inhibitor targets a distinct latent HIV-1 reservoir for re-activation from other 'shock drugs [PublishAheadOfPrint]

  • Upon HIV-1 infection, a reservoir of latently infected resting T cells prevents the eradication of the virus from patients. To achieve complete depletion, the existing virus-suppressing antiretroviral therapy must be combined with drugs that reactivate the dormant viruses. We previously described a novel chemical scaffold compound, MMQO (8-methoxy-6-methylquinolin-4-ol) that is able to reactivate viral transcription in several models of HIV latency including J-Lat cells through an unknown mechanism. MMQO potentiates the activity of known latency-reversing agents (LRAs) or llsquo;shockrrsquo; drugs such as PKC agonists or HDAC inhibitors. Here we demonstrate that MMQO activates HIV-1 independently of the Tat transactivator. Gene expression microarrays in Jurkat cells indicated that MMQO treatment results in robust immunosuppression, diminishes expression of c-Myc, and causes the dysregulation of acetylation sensitive genes. These hallmarks indicated that MMQO mimics acetylated lysines of core histones and might function as a bromodomain and extraterminal domain protein family inhibitor (BETi). MMQO functionally mimics the effects of JQ1, a well-known BETi. We confirmed that MMQO interacts with the BET family protein BRD4. Utilizing MMQO and JQ1, we demonstrate how the inhibition of BRD4 targets a distinct subset of latently integrated barcoded proviruses from those targeted by HDAC inhibitors or PKC pathway agonists. Thus, the quinoline-based compound MMQO represents a new class of the BET bromodomain inhibitors, which due to its minimalistic structure holds promise for further optimization for increased affinity and specificity for distinct bromodomain family members and could potentially be of use against a variety of diseases, including HIV.

    IMPORTANCE The tentative "shock and kill" therapy aims to eradicate the latent functional proportion of HIV-1 proviruses in a patient. Yet to this day, clinical studies investigating the "shocking" element of this strategy have proven it to be considerably more difficult than anticipated. While the proportion of intracellular viral RNA production and general plasma viral load have been shown to increase upon a "shock" regimen, the global viral reservoir remains unaffected, highlighting both the inefficiency of the treatments used and the gap in our understanding of viral reactivation in vivo.

    Utilizing a new BRD4 inhibitor and barcoded HIV-1 minigenomes, we demonstrate that PKC pathway activators, HDAC and bromodomain inhibitors all target different subsets of proviral integrations. Considering the fundamental differences of these compounds and the synergies displayed between them, we propose that the field should concentrate on investigating the development of combinatory "shock" cocktail therapies for an improved reservoir reactivation.

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