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Jean-Yves Sgro
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Table of Contents for this page:

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

    JVI Current Issue

  • Editorial Board [Masthead]

  • Herpes Simplex Virus 1 UL24 Abrogates the DNA Sensing Signal Pathway by Inhibiting NF-{kappa}B Activation [Virus-Cell Interactions]

  • Cyclic GMP-AMP synthase (cGAS) is a newly identified DNA sensor that recognizes foreign DNA, including the genome of herpes simplex virus 1 (HSV-1). Upon binding of viral DNA, cGAS produces cyclic GMP-AMP, which interacts with and activates stimulator of interferon genes (STING) to trigger the transcription of antiviral genes such as type I interferons (IFNs), and the production of inflammatory cytokines. HSV-1 UL24 is widely conserved among members of the herpesviruses family and is essential for efficient viral replication. In this study, we found that ectopically expressed UL24 could inhibit cGAS-STING-mediated promoter activation of IFN-bbeta; and interleukin-6 (IL-6), and UL24 also inhibited interferon-stimulatory DNA-mediated IFN-bbeta; and IL-6 production during HSV-1 infection. Furthermore, UL24 selectively blocked nuclear factor B (NF-B) but not IFN-regulatory factor 3 promoter activation. Coimmunoprecipitation analysis demonstrated that UL24 bound to the endogenous NF-B subunits p65 and p50 in HSV-1-infected cells, and UL24 was also found to bind the Rel homology domains (RHDs) of these subunits. Furthermore, UL24 reduced the tumor necrosis factor alpha (TNF-aalpha;)-mediated nuclear translocation of p65 and p50. Finally, mutational analysis revealed that the region spanning amino acids (aa) 74 to 134 of UL24 [UL24(74nndash;134)] is responsible for inhibiting cGAS-STING-mediated NF-B promoter activity. For the first time, UL24 was shown to play an important role in immune evasion during HSV-1 infection.

    IMPORTANCE NF-B is a critical component of the innate immune response and is strongly induced downstream of most pattern recognition receptors (PRRs), leading to the production of IFN-bbeta; as well as a number of inflammatory chemokines and interleukins. To establish persistent infection, viruses have evolved various mechanisms to counteract the host NF-B pathway. In the present study, for the first time, HSV-1 UL24 was demonstrated to inhibit the activation of NF-B in the DNA sensing signal pathway via binding to the RHDs of the NF-B subunits p65 and p50 and abolishing their nuclear translocation.

  • Classic Spotlight, 1980 and 1981: Articles of Significant Interest Selected from the Journal of Virology Archives by the Editors [Editorial]

  • The PTPN14 Tumor Suppressor Is a Degradation Target of Human Papillomavirus E7 [Transformation and Oncogenesis]

  • Activation of signaling pathways ensuring cell growth is essential for the proliferative competence of human papillomavirus (HPV)-infected cells. Tyrosine kinases and phosphatases are key regulators of cellular growth control pathways. A recently identified potential cellular target of HPV E7 is the cytoplasmic protein tyrosine phosphatase PTPN14, which is a potential tumor suppressor and is linked to the control of the Hippo and Wnt/beta-catenin signaling pathways. In this study, we show that the E7 proteins of both high-risk and low-risk mucosal HPV types can interact with PTPN14. This interaction is independent of retinoblastoma protein (pRb) and involves residues in the carboxy-terminal region of E7. We also show that high-risk E7 induces proteasome-mediated degradation of PTPN14 in cells derived from cervical tumors. This degradation appears to be independent of cullin-1 or cullin-2 but most likely involves the UBR4/p600 ubiquitin ligase. The degree to which E7 downregulates PTPN14 would suggest that this interaction is important for the viral life cycle and potentially also for the development of malignancy. In support of this we find that overexpression of PTPN14 decreases the ability of HPV-16 E7 to cooperate with activated EJ-ras in primary cell transformation assays.

    IMPORTANCE This study links HPV E7 to the deregulation of protein tyrosine phosphatase signaling pathways. PTPN14 is classified as a potential tumor suppressor protein, and here we show that it is very susceptible to HPV E7-induced proteasome-mediated degradation. Intriguingly, this appears to use a mechanism that is different from that employed by E7 to target pRb. Therefore, this study has important implications for our understanding of the molecular basis for E7 function and also sheds important light on the potential role of PTPN14 as a tumor suppressor.

  • A Temperature-Sensitive Lesion in the N-Terminal Domain of the Rotavirus Polymerase Affects Its Intracellular Localization and Enzymatic Activity [Genome Replication and Regulation of Viral Gene Expression]

  • Temperature-sensitive (ts) mutants of simian rotavirus (RV) strain SA11 have been previously created to investigate the functions of viral proteins during replication. One mutant, SA11-tsC, has a mutation that maps to the gene encoding the VP1 polymerase and shows diminished growth and RNA synthesis at 39ddeg;C compared to that at 31ddeg;C. In the present study, we sequenced all 11 genes of SA11-tsC, confirming the presence of an L138P mutation in the VP1 N-terminal domain and identifying 52 additional mutations in four other viral proteins (VP4, VP7, NSP1, and NSP2). To investigate whether the L138P mutation induces a ts phenotype in VP1 outside the SA11-tsC genetic context, we employed ectopic expression systems. Specifically, we tested whether the L138P mutation affects the ability of VP1 to localize to viroplasms, which are the sites of RV RNA synthesis, by expressing the mutant form as a green fluorescent protein (GFP) fusion protein (VP1L138P-GFP) (i) in wild-type SA11-infected cells or (ii) in uninfected cells along with viroplasm-forming proteins NSP2 and NSP5. We found that VP1L138P-GFP localized to viroplasms and interacted with NSP2 and/or NSP5 at 31ddeg;C but not at 39ddeg;C. Next, we tested the enzymatic activity of a recombinant mutant polymerase (rVP1L138P) in vitro and found that it synthesized less RNA at 39ddeg;C than at 31ddeg;C, as well as less RNA than the control at all temperatures. Together, these results provide a mechanistic basis for the ts phenotype of SA11-tsC and raise important questions about the role of leucine 138 in supporting key protein interactions and the catalytic function of the VP1 polymerase.

    IMPORTANCE RVs cause diarrhea in the young of many animal species, including humans. Despite their medical and economic importance, gaps in knowledge exist about how these viruses replicate inside host cells. Previously, a mutant simian RV (SA11-tsC) that replicates worse at higher temperatures was identified. This virus has an amino acid mutation in VP1, which is the enzyme responsible for copying the viral RNA genome. The mutation is located in a poorly understood region of the polymerase called the N-terminal domain. In this study, we determined that the mutation reduces the ability of VP1 to properly localize within infected cells at high temperatures, as well as reduced the ability of the enzyme to copy viral RNA in a test tube. The results of this study explain the temperature sensitivity of SA11-tsC and shed new light on functional protein-protein interaction sites of VP1.

  • Conserved Interaction of Lentiviral Vif Molecules with HIV-1 Gag and Differential Effects of Species-Specific Vif on Virus Production [Virus-Cell Interactions]

  • The virion infectivity factor (Vif) open reading frame is conserved among most lentiviruses. Vif molecules contribute to viral replication by inactivating host antiviral factors, the APOBEC3 cytidine deaminases. However, various species of lentiviral Vif proteins have evolved different strategies for overcoming host APOBEC3. Whether different species of lentiviral Vif proteins still preserve certain common features has not been reported. Here, we show for the first time that diverse lentiviral Vif molecules maintain the ability to interact with the human immunodeficiency virus type 1 (HIV-1) Gag precursor (Pr55Gag) polyprotein. Surprisingly, bovine immunodeficiency virus (BIV) Vif, but not HIV-1 Vif, interfered with HIV-1 production and viral infectivity even in the absence of APOBEC3. Further analysis revealed that BIV Vif demonstrated an enhanced interaction with Pr55Gag compared to that of HIV-1 Vif, and BIV Vif defective for the Pr55Gag interaction lost its ability to inhibit HIV-1. The C-terminal region of capsid (CA) and the p2 region of Pr55Gag, which are important for virus assembly and maturation, were involved in the interaction. Transduction of CD4+ T cells with BIV Vif blocked HIV-1 replication. Thus, the conserved Vif-Pr55Gag interaction provides a potential target for the future development of antiviral strategies.

    IMPORTANCE The conserved Vif accessory proteins of primate lentiviruses HIV-1, simian immunodeficiency virus (SIV), and BIV all form ubiquitin ligase complexes to target host antiviral APOBEC3 proteins for degradation, with different cellular requirements and using different molecular mechanisms. Here, we demonstrate that BIV Vif can interfere with HIV-1 Gag maturation and suppress HIV-1 replication through interaction with the precursor of the Gag (Pr55Gag) of HIV-1 in virus-producing cells. Moreover, the HIV-1 and SIV Vif proteins are conserved in terms of their interactions with HIV-1 Pr55Gag although HIV-1 Vif proteins bind Pr55Gag less efficiently than those of BIV Vif. Our research not only sheds new light on this feature of these conserved lentiviral Vif proteins but also provides a formerly unrecognized target for the development of antiviral strategies. Since increasing the Vif-Pr55Gag interaction could potentially suppress virus proliferation, this approach could offer a new strategy for the development of HIV inhibitors.

  • Rhinovirus C, Asthma, and Cell Surface Expression of Virus Receptor CDHR3 [Gem]

  • Human rhinoviruses (RVs) of the A, B, and C species are defined agents of the common cold. But more than that, RV-A and RV-C are the dominant causes of hospitalization category infections in young children, especially those with asthma. The use of cadherin-related family member 3 (CDHR3) by RV-C as its cellular receptor creates a direct phenotypic link between human genetics (G versus A alleles cause Cys529 versus Tyr529 protein variants) and the efficiency with which RV-C can infect cells. With a lower cell surface display density, the human-specific Cys529 variant apparently confers partial protection from the severest virus-induced asthma episodes. Selective pressure favoring the Cys529 codon may have coemerged with the evolution of RV-C and helped shape modern human genomes against the virus-susceptible, albeit ancestral Tyr529.

  • Correction for Baig et al., Determinants of Efficient Degradation of APOBEC3 Restriction Factors by HIV-1 Vif [Author Correction]

  • The FACT Complex Promotes Avian Leukosis Virus DNA Integration [Virus-Cell Interactions]

  • All retroviruses need to integrate a DNA copy of their genome into the host chromatin. Cellular proteins regulating and targeting lentiviral and gammaretroviral integration in infected cells have been discovered, but the factors that mediate alpharetroviral avian leukosis virus (ALV) integration are unknown. In this study, we have identified the FACT protein complex, which consists of SSRP1 and Spt16, as a principal cellular binding partner of ALV integrase (IN). Biochemical experiments with purified recombinant proteins show that SSRP1 and Spt16 are able to individually bind ALV IN, but only the FACT complex effectively stimulates ALV integration activity in vitro. Likewise, in infected cells, the FACT complex promotes ALV integration activity, with proviral integration frequency varying directly with cellular expression levels of the FACT complex. An increase in 2-long-terminal-repeat (2-LTR) circles in the depleted FACT complex cell line indicates that this complex regulates the ALV life cycle at the level of integration. This regulation is shown to be specific to ALV, as disruption of the FACT complex did not inhibit either lentiviral or gammaretroviral integration in infected cells.

    IMPORTANCE The majority of human gene therapy approaches utilize HIV-1- or murine leukemia virus (MLV)-based vectors, which preferentially integrate near genes and regulatory regions; thus, insertional mutagenesis is a substantial risk. In contrast, ALV integrates more randomly throughout the genome, which decreases the risks of deleterious integration. Understanding how ALV integration is regulated could facilitate the development of ALV-based vectors for use in human gene therapy. Here we show that the FACT complex directly binds and regulates ALV integration efficiency in vitro and in infected cells.

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

  • Differences in Glycoprotein Complex Receptor Binding Site Accessibility Prompt Poor Cross-Reactivity of Neutralizing Antibodies between Closely Related Arenaviruses [Virus-Cell Interactions]

  • The glycoprotein complex (GPC) of arenaviruses, composed of stable signal peptide, GP1, and GP2, is the only antigen correlated with antibody-mediated neutralization. However, despite strong cross-reactivity of convalescent antisera between related arenavirus species, weak or no cross-neutralization occurs. Two closely related clade B viruses, Machupo virus (MACV) and Juniiacute;n virus (JUNV), have nearly identical overall GPC architecture and share a host receptor, transferrin receptor 1 (TfR1). Given structural and functional similarities of the GP1 receptor binding site (RBS) of these viruses and the recent demonstration that the RBS is an important target for neutralizing antibodies, it is not clear how these viruses avoid cross-neutralization. To address this, MACV/JUNV chimeric GPCs were assessed for interaction with a group of aalpha;-JUNV GPC monoclonal antibodies (MAbs) and mouse antisera against JUNV or MACV GPC. All six MAbs targeted GP1, with those that neutralized JUNV GPC-pseudovirions competing with each other for RBS binding. However, these MAbs were unable to bind to a chimeric GPC composed of JUNV GP1 containing a small disulfide bonded loop (loop 10) unique to MACV GPC, suggesting that this loop may block MAbs interaction with the GP1 RBS. Consistent with this loop causing interference, mouse anti-JUNV GPC antisera that solely neutralized pseudovirions bearing autologous GP1 provided enhanced neutralization of MACV GPC when this loop was removed. Our studies provide evidence that loop 10, which is unique to MACV GP1, is an important impediment to binding of neutralizing antibodies and contributes to the poor cross-neutralization of aalpha;-JUNV antisera against MACV.

    IMPORTANCE Multiple New World arenaviruses can cause severe disease in humans, and some geographic overlap exists among these viruses. A vaccine that protects against a broad range of New World arenaviruses is desirable for purposes of simplicity, cost, and broad protection against multiple National Institute of Allergy and Infectious Disease-assigned category A priority pathogens. In this study, we sought to better understand how closely related arenaviruses elude cross-species neutralization by investigating the structural bases of antibody binding and avoidance. In our studies, we found that neutralizing antibodies against two New World arenaviruses, Machupo virus (MACV) and Juniiacute;n virus (JUNV), bound to the envelope glycoprotein 1 (GP1) with JUNV monoclonal antibodies targeting the receptor binding site (RBS). We further show that altered structures surrounding the RBS pocket in MACV GP1 impede access of JUNV-elicited antibodies.

  • Human Rhinovirus Diversity and Evolution: How Strange the Change from Major to Minor [Genetic Diversity and Evolution]

  • Rhinoviruses are the most common causes of the common cold. Their many distinct lineages fall into "major" and "minor" groups that use different cell surface receptors to enter host cells. Minor-group rhinoviruses are more immunogenic in laboratory studies, although their patterns of transmission and their cold symptoms are broadly similar to those of the major group. Here we present evolutionary evidence that minor-group viruses are also more immunogenic in humans. A key finding is that rates of amino acid substitutions at exposed sites in the capsid proteins VP2, VP3, and VP1 tend to be elevated in minor-group relative to major-group viruses, while rates at buried sites show no consistent differences. A reanalysis of historical virus watch data also indicates a higher immunogenicity of minor-group viruses, consistent with our findings about evolutionary rates at amino acid positions most directly exposed to immune surveillance. The increased immunogenicity and speed of evolution in minor-group lineages may contribute to the very large numbers of rhinovirus serotypes that coexist while differing in virulence.

    IMPORTANCE Most colds are caused by rhinoviruses (RVs). Those caused by a subset known as the minor-group members of rhinovirus species A (RV-A) are correlated with the inception and aggravation of asthma in at-risk populations. Genetically, minor-group viruses are similar to major-group RV-A, from which they were derived, although they tend to elicit stronger immune responses. Differences in their rates and patterns of molecular evolution should be highly relevant to their epidemiology. All RV-A strains show high rates of amino acid substitutions in the capsid proteins at exposed sites not previously identified as being immunogenic, and this increase is significantly greater in minor-group viruses. These findings will inform future studies of the recently discovered RV-C, which also appears to exacerbate asthma in adults and children. In addition, these findings draw attention to the difficult problem of explaining the long-term coexistence of many serotypes of major- and minor-group RVs.

  • Latent Membrane Protein 1 (LMP1) and LMP2A Collaborate To Promote Epstein-Barr Virus-Induced B Cell Lymphomas in a Cord Blood-Humanized Mouse Model but Are Not Essential [Transformation and Oncogenesis]

  • Epstein-Barr virus (EBV) infection is associated with B cell lymphomas in humans. The ability of EBV to convert human B cells into long-lived lymphoblastoid cell lines (LCLs) in vitro requires the collaborative effects of EBNA2 (which hijacks Notch signaling), latent membrane protein 1 (LMP1) (which mimics CD40 signaling), and EBV-encoded nuclear antigen 3A (EBNA3A) and EBNA3C (which inhibit oncogene-induced senescence and apoptosis). However, we recently showed that an LMP1-deleted EBV mutant induces B cell lymphomas in a newly developed cord blood-humanized mouse model that allows EBV-infected B cells to interact with CD4 T cells (the major source of CD40 ligand). Here we examined whether the EBV LMP2A protein, which mimics constitutively active B cell receptor signaling, is required for EBV-induced lymphomas in this model. We find that the deletion of LMP2A delays the onset of EBV-induced lymphomas but does not affect the tumor phenotype or the number of tumors. The simultaneous deletion of both LMP1 and LMP2A results in fewer tumors and a further delay in tumor onset. Nevertheless, the LMP1/LMP2A double mutant induces lymphomas in approximately half of the infected animals. These results indicate that neither LMP1 nor LMP2A is absolutely essential for the ability of EBV to induce B cell lymphomas in the cord blood-humanized mouse model, although the simultaneous loss of both LMP1 and LMP2A decreases the proportion of animals developing tumors and increases the time to tumor onset. Thus, the expression of either LMP1 or LMP2A may be sufficient to promote early-onset EBV-induced tumors in this model.

    IMPORTANCE EBV causes human lymphomas, but few models are available for dissecting how EBV causes lymphomas in vivo in the context of a host immune response. We recently used a newly developed cord blood-humanized mouse model to show that EBV can cooperate with human CD4 T cells to cause B cell lymphomas even when a major viral transforming protein, LMP1, is deleted. Here we examined whether the EBV protein LMP2A, which mimics B cell receptor signaling, is required for EBV-induced lymphomas in this model. We find that the deletion of LMP2A alone has little effect on the ability of EBV to cause lymphomas but delays tumor onset. The deletion of both LMP1 and LMP2A results in a smaller number of lymphomas in infected animals, with an even more delayed time to tumor onset. These results suggest that LMP1 and LMP2A collaborate to promote early-onset lymphomas in this model, but neither protein is absolutely essential.

  • Sterol Binding by the Tombusviral Replication Proteins Is Essential for Replication in Yeast and Plants [Genome Replication and Regulation of Viral Gene Expression]

  • Membranous structures derived from various organelles are important for replication of plus-stranded RNA viruses. Although the important roles of co-opted host proteins in RNA virus replication have been appreciated for a decade, the equally important functions of cellular lipids in virus replication have been gaining full attention only recently. Previous work with Tomato bushy stunt tombusvirus (TBSV) in model host yeast has revealed essential roles for phosphatidylethanolamine and sterols in viral replication. To further our understanding of the role of sterols in tombusvirus replication, in this work we showed that the TBSV p33 and p92 replication proteins could bind to sterols in vitro. The sterol binding by p33 is supported by cholesterol recognition/interaction amino acid consensus (CRAC) and CARC-like sequences within the two transmembrane domains of p33. Mutagenesis of the critical Y amino acids within the CRAC and CARC sequences blocked TBSV replication in yeast and plant cells. We also showed the enrichment of sterols in the detergent-resistant membrane (DRM) fractions obtained from yeast and plant cells replicating TBSV. The DRMs could support viral RNA synthesis on both the endogenous and exogenous templates. A lipidomic approach showed the lack of enhancement of sterol levels in yeast and plant cells replicating TBSV. The data support the notion that the TBSV replication proteins are associated with sterol-rich detergent-resistant membranes in yeast and plant cells. Together, the results obtained in this study and the previously published results support the local enrichment of sterols around the viral replication proteins that is critical for TBSV replication.

    IMPORTANCE One intriguing aspect of viral infections is their dependence on efficient subcellular assembly platforms serving replication, virion assembly, or virus egress via budding out of infected cells. These assembly platforms might involve sterol-rich membrane microdomains, which are heterogeneous and highly dynamic nanoscale structures usurped by various viruses. Here, we demonstrate that TBSV p33 and p92 replication proteins can bind to sterol in vitro. Mutagenesis analysis of p33 within the CRAC and CARC sequences involved in sterol binding shows the important connection between the abilities of p33 to bind to sterol and to support TBSV replication in yeast and plant cells. Together, the results further strengthen the model that cellular sterols are essential as proviral lipids during viral replication.

  • Neutralization of Diverse Human Cytomegalovirus Strains Conferred by Antibodies Targeting Viral gH/gL/pUL128-131 Pentameric Complex [Vaccines and Antiviral Agents]

  • Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection, and developing a prophylactic vaccine is of high priority to public health. We recently reported a replication-defective human cytomegalovirus with restored pentameric complex glycoprotein H (gH)/gL/pUL128-131 for prevention of congenital HCMV infection. While the quantity of vaccine-induced antibody responses can be measured in a viral neutralization assay, assessing the quality of such responses, including the ability of vaccine-induced antibodies to cross-neutralize the field strains of HCMV, remains a challenge. In this study, with a panel of neutralizing antibodies from three healthy human donors with natural HCMV infection or a vaccinated animal, we mapped eight sites on the dominant virus-neutralizing antigenmmdash;the pentameric complex of glycoprotein H (gH), gL, and pUL128, pUL130, and pUL131. By evaluating the site-specific antibodies in vaccine immune sera, we demonstrated that vaccination elicited functional antiviral antibodies to multiple neutralizing sites in rhesus macaques, with quality attributes comparable to those of CMV hyperimmune globulin. Furthermore, these immune sera showed antiviral activities against a panel of genetically distinct HCMV clinical isolates. These results highlighted the importance of understanding the quality of vaccine-induced antibody responses, which includes not only the neutralizing potency in key cell types but also the ability to protect against the genetically diverse field strains.

    IMPORTANCE HCMV is the leading cause of congenital viral infection, and development of a preventive vaccine is a high public health priority. To understand the strain coverage of vaccine-induced immune responses in comparison with natural immunity, we used a panel of broadly neutralizing antibodies to identify the immunogenic sites of a dominant viral antigenmmdash;the pentameric complex. We further demonstrated that following vaccination of a replication-defective virus with the restored pentameric complex, rhesus macaques can develop broadly neutralizing antibodies targeting multiple immunogenic sites of the pentameric complex. Such analyses of site-specific antibody responses are imperative to our assessment of the quality of vaccine-induced immunity in clinical studies.

  • Expression of Ifnlr1 on Intestinal Epithelial Cells Is Critical to the Antiviral Effects of Interferon Lambda against Norovirus and Reovirus [Pathogenesis and Immunity]

  • Lambda interferon (IFN-) has potent antiviral effects against multiple enteric viral pathogens, including norovirus and rotavirus, in both preventing and curing infection. Because the intestine includes a diverse array of cell types, however, the cell(s) upon which IFN- acts to exert its antiviral effects is unclear. Here, we sought to identify IFN--responsive cells by generation of mice with lineage-specific deletion of the receptor for IFN-, Ifnlr1. We found that expression of IFNLR1 on intestinal epithelial cells (IECs) in the small intestine and colon is required for enteric IFN- antiviral activity. IEC Ifnlr1 expression also determines the efficacy of IFN- in resolving persistent murine norovirus (MNoV) infection and regulates fecal shedding and viral titers in tissue. Thus, the expression of Ifnlr1 by IECs is necessary for the response to both endogenous and exogenous IFN-. We further demonstrate that IEC Ifnlr1 expression is required for the sterilizing innate immune effects of IFN- by extending these findings in Rag1-deficient mice. Finally, we assessed whether our findings pertained to multiple viral pathogens by infecting mice specifically lacking IEC Ifnlr1 expression with reovirus. These mice phenocopied Ifnlr1-null animals, exhibiting increased intestinal tissue titers and enhanced reovirus fecal shedding. Thus, IECs are the critical cell type responding to IFN- to control multiple enteric viruses. This is the first genetic evidence that supports an essential role for IECs in IFN--mediated control of enteric viral infection, and these findings provide insight into the mechanism of IFN--mediated antiviral activity.

    IMPORTANCE Human noroviruses (HNoVs) are the leading cause of epidemic gastroenteritis worldwide. Type III interferons (IFN-) control enteric viral infections in the gut and have been shown to cure mouse norovirus, a small-animal model for HNoVs. Using a genetic approach with conditional knockout mice, we identified IECs as the dominant IFN--responsive cells in control of enteric virus infection in vivo. Upon murine norovirus or reovirus infection, Ifnlr1 depletion in IECs largely recapitulated the phenotype seen in Ifnlr1nndash;/nndash; mice of higher intestinal tissue viral titers and increased viral shedding in the stool. Moreover, IFN--mediated sterilizing immunity against murine norovirus requires the capacity of IECs to respond to IFN-. These findings clarify the mechanism of action of this cytokine and emphasize the therapeutic potential of IFN- for treating mucosal viral infections.

  • Sex-Dependent Intestinal Replication of an Enteric Virus [Pathogenesis and Immunity]

  • Coxsackievirus is an enteric virus that initiates infection in the gastrointestinal tract before disseminating to peripheral tissues to cause disease, but intestinal factors that influence viral replication are understudied. Furthermore, a sex bias for severe sequelae from coxsackievirus infections has been observed in humans. While mouse models mimicking human pathogenesis have been well characterized, many of these experiments use intraperitoneal injection of coxsackievirus to infect mice, bypassing the intestine. In light of recent studies identifying intestinal factors, such as the microbiota, that alter enteric viral replication, we sought to investigate coxsackievirus replication within the intestine. Here, we orally infected mice with coxsackievirus B3 (CVB3) and found that CVB3 replication in the intestine is sex dependent. CVB3 replicated efficiently in the intestine of male mice but not female mice. Additionally, we found that the type I interferon response and sex hormones can alter both viral replication and lethality. Overall, these data suggest that sex and the immune response play a vital role in CVB3 replication in the intestine and should be considered in light of the sex bias observed in human disease.

    IMPORTANCE Sex bias in severe sequelae from enteric viral infections has been observed. Since viruses have evolved to achieve optimal levels of fitness in their environmental niches, it is imperative to study viruses at the site of initial replication. Here, we used an oral inoculation system for CVB3, which follows the natural route of infection in the gastrointestinal tract. We found that sex can influence the replication of CVB3 in the intestine. Additionally, the type I interferon response and sex hormones alter both CVB3 intestinal replication and lethality. Overall this work highlights the fact that sex should be considered in investigations of enteric viral replication and pathogenesis.

  • Dynamic Modulation of Expression of Lentiviral Restriction Factors in Primary CD4+ T Cells following Simian Immunodeficiency Virus Infection [Pathogenesis and Immunity]

  • Although multiple restriction factors have been shown to inhibit HIV/SIV replication, little is known about their expression in vivo. Expression of 45 confirmed and putative HIV/SIV restriction factors was analyzed in CD4+ T cells from peripheral blood and the jejunum in rhesus macaques, revealing distinct expression patterns in naive and memory subsets. In both peripheral blood and the jejunum, memory CD4+ T cells expressed higher levels of multiple restriction factors compared to naive cells. However, relative to their expression in peripheral blood CD4+ T cells, jejunal CCR5+ CD4+ T cells exhibited significantly lower expression of multiple restriction factors, including APOBEC3G, MX2, and TRIM25, which may contribute to the exquisite susceptibility of these cells to SIV infection. In vitro stimulation with anti-CD3/CD28 antibodies or type I interferon resulted in upregulation of distinct subsets of multiple restriction factors. After infection of rhesus macaques with SIVmac239, the expression of most confirmed and putative restriction factors substantially increased in all CD4+ T cell memory subsets at the peak of acute infection. Jejunal CCR5+ CD4+ T cells exhibited the highest levels of SIV RNA, corresponding to the lower restriction factor expression in this subset relative to peripheral blood prior to infection. These results illustrate the dynamic modulation of confirmed and putative restriction factor expression by memory differentiation, stimulation, tissue microenvironment and SIV infection and suggest that differential expression of restriction factors may play a key role in modulating the susceptibility of different populations of CD4+ T cells to lentiviral infection.

    IMPORTANCE Restriction factors are genes that have evolved to provide intrinsic defense against viruses. HIV and simian immunodeficiency virus (SIV) target CD4+ T cells. The baseline level of expression in vivo and degree to which expression of restriction factors is modulated by conditions such as CD4+ T cell differentiation, stimulation, tissue location, or SIV infection are currently poorly understood. We measured the expression of 45 confirmed and putative restriction factors in primary CD4+ T cells from rhesus macaques under various conditions, finding dynamic changes in each state. Most dramatically, in acute SIV infection, the expression of almost all target genes analyzed increased. These are the first measurements of many of these confirmed and putative restriction factors in primary cells or during the early events after SIV infection and suggest that the level of expression of restriction factors may contribute to the differential susceptibility of CD4+ T cells to SIV infection.

  • Glycosylation of the Hemagglutinin Protein of H5N1 Influenza Virus Increases Its Virulence in Mice by Exacerbating the Host Immune Response [Pathogenesis and Immunity]

  • The highly pathogenic avian influenza (HPAI) H5N1 viruses continue to circulate in nature and threaten public health. Although several viral determinants and host factors that influence the virulence of HPAI H5N1 viruses in mammals have been identified, the detailed molecular mechanism remains poorly defined and requires further clarification. In our previous studies, we characterized two naturally isolated HPAI H5N1 viruses that had similar viral genomes but differed substantially in their lethality in mice. In this study, we explored the molecular determinants and potential mechanism for this difference in virulence. By using reverse genetics, we found that a single amino acid at position 158 of the hemagglutinin (HA) protein substantially affected the systemic replication and pathogenicity of these H5N1 influenza viruses in mice. We further found that the G158N mutation introduced an N-linked glycosylation at positions 158 to 160 of the HA protein and that this N-linked glycosylation enhanced viral productivity in infected mammalian cells and induced stronger host immune and inflammatory responses to viral infection. These findings further our understanding of the determinants of pathogenicity of H5N1 viruses in mammals.

    IMPORTANCE Highly pathogenic avian influenza (HPAI) H5N1 viruses continue to evolve in nature and threaten human health. Key mutations in the virus hemagglutinin (HA) protein or reassortment with other pandemic viruses endow HPAI H5N1 viruses with the potential for aerosol transmissibility in mammals. A thorough understanding of the pathogenic mechanisms of these viruses will help us to develop more effective control strategies; however, such mechanisms and virulent determinants for H5N1 influenza viruses have not been fully elucidated. In this study, we identified glycosylation at positions 158 to 160 of the HA protein of two naturally occurring H5N1 viruses as an important virulence determinant. This glycosylation event enhanced viral productivity, exacerbated the host response, and thereby contributed to the high pathogenicity of H5N1 virus in mice.

  • The Envelope Gene of Transmitted HIV-1 Resists a Late Interferon Gamma-Induced Block [Virus-Cell Interactions]

  • Type I interferon (IFN) signaling engenders an antiviral state that likely plays an important role in constraining HIV-1 transmission and contributes to defining subsequent AIDS pathogenesis. Type II IFN (IFN-) also induces an antiviral state but is often primarily considered to be an immunomodulatory cytokine. We report that IFN- stimulation can induce an antiviral state that can be both distinct from that of type I interferon and can potently inhibit HIV-1 in primary CD4+ T cells and a number of human cell lines. Strikingly, we find that transmitted/founder (TF) HIV-1 viruses can resist a late block that is induced by type II IFN, and the use of chimeric IFN--sensitive/resistant viruses indicates that interferon resistance maps to the env gene. Simultaneously, in vitro evolution also revealed that just a single amino acid substitution in the envelope can confer substantial resistance to IFN-mediated inhibition. Thus, the env gene of transmitted HIV-1 confers resistance to a late block that is phenotypically distinct from blocks previously described to be resisted by env and is therefore mediated by unknown IFN--stimulated factor(s) in human CD4+ T cells and cell lines. This important unidentified block could play a key role in constraining HIV-1 transmission.

    IMPORTANCE The human immune system can hinder invading pathogens through interferon (IFN) signaling. One consequence of this signaling is that cells enter an antiviral state, increasing the levels of hundreds of defenses that can inhibit the replication and spread of viruses. The majority of HIV-1 infections result from a single virus particle (the transmitted/founder) that makes it past these defenses and colonizes the host. Thus, the founder virus is hypothesized to be a relatively interferon-resistant entity. Here, we show that certain HIV-1 envelope genes have the unanticipated ability to resist specific human defenses mediated by different types of interferons. Strikingly, the envelope gene from a founder HIV-1 virus is far better at evading these defenses than the corresponding gene from a common HIV-1 lab strain. Thus, these defenses could play a role in constraining the transmission of HIV-1 and may select for transmitted viruses that are resistant to this IFN-mediated inhibition.

  • The Measles Virus Receptor SLAMF1 Can Mediate Particle Endocytosis [Virus-Cell Interactions]

  • The signaling lymphocyte activation molecule F1 (SLAMF1) is both a microbial sensor and entry receptor for measles virus (MeV). Herein, we describe a new role for SLAMF1 to mediate MeV endocytosis that is in contrast with the alternative, and generally accepted, model that MeV genome enters cells only after fusion at the cell surface. We demonstrated that MeV engagement of SLAMF1 induces dramatic but transient morphological changes, most prominently in the formation of membrane blebs, which were shown to colocalize with incoming viral particles, and rearrangement of the actin cytoskeleton in infected cells. MeV infection was dependent on these dynamic cytoskeletal changes as well as fluid uptake through a macropinocytosis-like pathway as chemical inhibition of these processes inhibited entry. Moreover, we identified a role for the RhoA-ROCK-myosin II signaling axis in this MeV internalization process, highlighting a novel role for this recently characterized pathway in virus entry. Our study shows that MeV can hijack a microbial sensor normally involved in bacterial phagocytosis to drive endocytosis using a complex pathway that shares features with canonical viral macropinocytosis, phagocytosis, and mechanotransduction. This uptake pathway is specific to SLAMF1-positive cells and occurs within 60 min of viral attachment. Measles virus remains a significant cause of mortality in human populations, and this research sheds new light on the very first steps of infection of this important pathogen.

    IMPORTANCE Measles is a significant disease in humans and is estimated to have killed over 200 million people since records began. According to current World Health Organization statistics, it still kills over 100,000 people a year, mostly children in the developing world. The causative agent, measles virus, is a small enveloped RNA virus that infects a broad range of cells during infection. In particular, immune cells are infected via interactions between glycoproteins found on the surface of the virus and SLAMF1, the immune cell receptor. In this study, we have investigated the steps governing entry of measles virus into SLAMF1-positive cells and identified endocytic uptake of viral particles. This research will impact our understanding of morbillivirus-related immunosuppression as well as the application of measles virus as an oncolytic therapeutic.

  • The Pseudorabies Virus Glycoprotein gE/gI Complex Suppresses Type I Interferon Production by Plasmacytoid Dendritic Cells [Pathogenesis and Immunity]

  • Plasmacytoid dendritic cells (pDC) play a central role in the antiviral immune response, both in the innate response and in shaping the adaptive response, mainly because of their ability to produce massive amounts of type I interferon (TI-IFN). Here, we report that cells infected with the live attenuated Bartha vaccine strain of porcine alphaherpesvirus pseudorabies virus (PRV) trigger a dramatically increased TI-IFN response by porcine primary pDC compared to cells infected with wild-type PRV strains (Becker and Kaplan). Since Bartha is one of the relatively few examples of a highly successful alphaherpesvirus vaccine, identification of factors that may contribute to its efficacy may provide insights for the rational design of other alphaherpesvirus vaccines. The Bartha vaccine genome displays several mutations compared to the genome of wild-type PRV strains, including a large deletion in the unique short (US) region, encompassing the glycoprotein E (gE), gI, US9, and US2 genes. Using recombinant PRV Becker strains harboring the entire Bartha US deletion or single mutations in the four affected US genes, we demonstrate that the absence of the viral gE/gI complex contributes to the observed increased IFN-aalpha; response. Furthermore, we show that the absence of gE leads to an enhanced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in pDC, which correlates with a higher TI-IFN production by pDC. In conclusion, the PRV Bartha vaccine strain triggers strongly increased TI-IFN production by porcine pDC. Our data further indicate that the gE/gI glycoprotein complex suppresses TI-IFN production by pDC, which represents the first alphaherpesvirus factor that suppresses pDC activity.

    IMPORTANCE Several alphaherpesviruses, including herpes simpex virus, still lack effective vaccines. However, the highly successful Bartha vaccine has contributed substantially to eradication of the porcine alphaherpesvirus pseudorabies virus (PRV) in several countries. The impact of Bartha on the immune response is still poorly understood. Type I interferon (TI-IFN)-producing plasmacytoid dendritic cells (pDC) may play an important role in vaccine development. Here, we show that Bartha elicits a dramatically increased type I interferon (TI-IFN) response in primary porcine pDC compared to wild-type strains. In addition, we found that the gE/gI complex, which is absent in Bartha, inhibits the pDC TI-IFN response. This is the first description of an immune cell type that is differentially affected by Bartha versus wild-type PRV and is the first report describing an alphaherpesvirus protein that inhibits the TI-IFN response by pDC. These data may therefore contribute to the rational design of other alphaherpesvirus vaccines.

  • Overexpression of Interleukin-7 Extends the Humoral Immune Response Induced by Rabies Vaccination [Vaccines and Antiviral Agents]

  • Rabies continues to present a public health threat in most countries of the world. The most efficient way to prevent and control rabies is to implement vaccination programs for domestic animals. However, traditional inactivated vaccines used in animals are costly and have relatively low efficiency, which impedes their extensive use in developing countries. There is, therefore, an urgent need to develop single-dose and long-lasting rabies vaccines. However, little information is available regarding the mechanisms underlying immunological memory, which can broaden humoral responses following rabies vaccination. In this study, a recombinant rabies virus (RABV) that expressed murine interleukin-7 (IL-7), referred to here as rLBNSE-IL-7, was constructed, and its effectiveness was evaluated in a mouse model. rLBNSE-IL-7 induced higher rates of T follicular helper (Tfh) cells and germinal center (GC) B cells from draining lymph nodes (LNs) than the parent virus rLBNSE. Interestingly, rLBNSE-IL-7 improved the percentages of long-lived memory B cells (Bmem) in the draining LNs and plasma cells (PCs) in the bone marrow (BM) for up to 360 days postimmunization (dpi). As a result of the presence of the long-lived PCs, it also generated prolonged virus-neutralizing antibodies (VNAs), resulting in better protection against a lethal challenge than that seen with rLBNSE. Moreover, consistent with the increased numbers of Bmem and PCs after a boost with rLBNSE, rLBNSE-IL-7-immunized mice promptly produced a more potent secondary anti-RABV neutralizing antibody response than rLBNSE-immunized mice. Overall, our data suggest that overexpressing IL-7 improved the induction of long-lasting primary and secondary antibody responses post-RABV immunization.

    IMPORTANCE Extending humoral immune responses using adjuvants is an important method to develop long-lasting and efficient vaccines against rabies. However, little information is currently available regarding prolonged immunological memory post-RABV vaccination. In this study, a novel rabies vaccine that expressed murine IL-7 was developed. This vaccine enhanced the numbers of Tfh cells and the GC responses, resulting in upregulated quantities of Bmem and PCs. Moreover, we found that the long-lived PCs that were elicited by the IL-7-expressing recombinant virus (rLBNSE-IL-7) were able to sustain VNA levels much longer than those elicited by the parent rLBNSE virus. Upon reexposure to the pathogen, the longevous Bmem, which maintained higher numbers for up to 360 dpi with rLBNSE-IL-7 compared to rLBNSE, could differentiate into antibody-secreting cells, resulting in rapid and potent secondary production of VNAs. These results suggest that the expression of IL-7 is beneficial for induction of potent and long-lasting humoral immune responses.

  • Impact of Type I Interferon on the Safety and Immunogenicity of an Experimental Live-Attenuated Herpes Simplex Virus 1 Vaccine in Mice [Vaccines and Antiviral Agents]

  • Viral fitness dictates virulence and capacity to evade host immune defenses. Understanding the biological underpinnings of such features is essential for rational vaccine development. We have previously shown that the live-attenuated herpes simplex virus 1 (HSV-1) mutant lacking the nuclear localization signal (NLS) on the ICP0 gene (0NLS) is sensitive to inhibition by interferon beta (IFN-bbeta;) in vitro and functions as a highly efficacious experimental vaccine. Here, we characterize the host immune response and in vivo pathogenesis of HSV-1 0NLS relative to its fully virulent parental strain in C57BL/6 mice. Additionally, we explore the role of type 1 interferon (IFN-aalpha;/bbeta;) signaling on virulence and immunogenicity of HSV-1 0NLS and uncover a probable sex bias in the induction of IFN-aalpha;/bbeta; in the cornea during HSV-1 infection. Our data show that HSV-1 0NLS lacks neurovirulence even in highly immunocompromised mice lacking the IFN-aalpha;/bbeta; receptor. These studies support the translational viability of the HSV-1 0NLS vaccine strain by demonstrating that, while it is comparable to a virulent parental strain in terms of immunogenicity, HSV-1 0NLS does not induce significant tissue pathology.

    IMPORTANCE HSV-1 is a common human pathogen associated with a variety of clinical presentations ranging in severity from periodic "cold sores" to lethal encephalitis. Despite the consistent failures of HSV subunit vaccines in clinical trials spanning the past 28 years, opposition to live-attenuated HSV vaccines predicated on unfounded safety concerns currently limits their widespread acceptance. Here, we demonstrate that a live-attenuated HSV-1 vaccine has great translational potential.

  • Azacytidine Treatment Inhibits the Progression of Herpes Stromal Keratitis by Enhancing Regulatory T Cell Function [Pathogenesis and Immunity]

  • Ocular infection with herpes simplex virus 1 (HSV-1) sets off an inflammatory reaction in the cornea which leads to both virus clearance and chronic lesions that are orchestrated by CD4 T cells. Approaches that enhance the function of regulatory T cells (Treg) and dampen effector T cells can be effective to limit stromal keratitis (SK) lesion severity. In this report, we explore the novel approach of inhibiting DNA methyltransferase activity using 5-azacytidine (Aza; a cytosine analog) to limit HSV-1-induced ocular lesions. We show that therapy begun after infection when virus was no longer actively replicating resulted in a pronounced reduction in lesion severity, with markedly diminished numbers of T cells and nonlymphoid inflammatory cells, along with reduced cytokine mediators. The remaining inflammatory reactions had a change in the ratio of CD4 Foxp3+ Treg to effector Th1 CD4 T cells in ocular lesions and lymphoid tissues, with Treg becoming predominant over the effectors. In addition, compared to those from control mice, Treg from Aza-treated mice showed more suppressor activity in vitro and expressed higher levels of activation molecules. Additionally, cells induced in vitro in the presence of Aza showed epigenetic differences in the Treg-specific demethylated region (TSDR) of Foxp3 and were more stable when exposed to inflammatory cytokines. Our results show that therapy with Aza is an effective means of controlling a virus-induced inflammatory reaction and may act mainly by the effects on Treg.

    IMPORTANCE HSV-1 infection has been shown to initiate an inflammatory reaction in the cornea that leads to tissue damage and loss of vision. The inflammatory reaction is orchestrated by gamma interferon (IFN-)-secreting Th1 cells, and regulatory T cells play a protective role. Hence, novel therapeutics that can rebalance the ratio of regulatory T cells to effectors are a relevant issue. This study opens up a new avenue in treating HSV-induced SK lesions by increasing the stability and function of regulatory T cells using the DNA methyltransferase inhibitor 5-azacytidine (Aza). Aza increased the function of regulatory T cells, leading to enhanced suppressive activity and diminished lesions. Hence, therapy with Aza, which acts mainly by its effects on Treg, can be an effective means to control virus-induced inflammatory lesions.

  • The V3 Loop of HIV-1 Env Determines Viral Susceptibility to IFITM3 Impairment of Viral Infectivity [Virus-Cell Interactions]

  • Interferon-inducible transmembrane proteins (IFITMs) inhibit a broad spectrum of viruses, including HIV-1. IFITM proteins deter HIV-1 entry when expressed in target cells and also impair HIV-1 infectivity when expressed in virus producer cells. However, little is known about how viruses resist IFITM inhibition. In this study, we have investigated the susceptibilities of different primary isolates of HIV-1 to the inhibition of viral infectivity by IFITMs. Our results demonstrate that the infectivity of different HIV-1 primary isolates, including transmitted founder viruses, is diminished by IFITM3 to various levels, with strain AD8-1 exhibiting strong resistance. Further mutagenesis studies revealed that HIV-1 Env, and the V3 loop sequence in particular, determines the extent of inhibition of viral infectivity by IFITM3. IFITM3-sensitive Env proteins are also more susceptible to neutralization by soluble CD4 or the 17b antibody than are IFITM3-resistant Env proteins. Together, data from our study suggest that the propensity of HIV-1 Env to sample CD4-bound-like conformations modulates viral sensitivity to IFITM3 inhibition.

    IMPORTANCE Results of our study have revealed the key features of the HIV-1 envelope protein that are associated with viral resistance to the IFITM3 protein. IFITM proteins are important effectors in interferon-mediated antiviral defense. A variety of viruses are inhibited by IFITMs at the virus entry step. Although it is known that envelope proteins of several different viruses resist IFITM inhibition, the detailed mechanisms are not fully understood. Taking advantage of the fact that envelope proteins of different HIV-1 strains exhibit different degrees of resistance to IFITM3 and that these HIV-1 envelope proteins share the same domain structure and similar sequences, we performed mutagenesis studies and determined the key role of the V3 loop in this viral resistance phenotype. We were also able to associate viral resistance to IFITM3 inhibition with the susceptibility of HIV-1 to inhibition by soluble CD4 and the 17b antibody that recognizes CD4-binding-induced epitopes.

  • Influence of the Envelope gp120 Phe 43 Cavity on HIV-1 Sensitivity to Antibody-Dependent Cell-Mediated Cytotoxicity Responses [Pathogenesis and Immunity]

  • HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cellular-mediated cytotoxicity (ADCC). HIV-1 has evolved sophisticated mechanisms to avoid the exposure of Env ADCC epitopes by downregulating CD4 and by limiting the overall amount of Env on the cell surface. In HIV-1, substitution of large residues such as histidine or tryptophan for serine 375 (S375H/W) in the gp120 Phe 43 cavity, where Phe 43 of CD4 contacts gp120, results in the spontaneous sampling of an Env conformation closer to the CD4-bound state. While residue S375 is well conserved in the majority of group M HIV-1 isolates, CRF01_AE strains have a naturally occurring histidine at this position (H375). Interestingly, CRF01_AE is the predominant circulating strain in Thailand, where the RV144 trial took place. In this trial, which resulted in a modest degree of protection, ADCC responses were identified as being part of the correlate of protection. Here we investigate the influence of the Phe 43 cavity on ADCC responses. Filling this cavity with a histidine or tryptophan residue in Env with a natural serine residue at this position (S375H/W) increased the susceptibility of HIV-1-infected cells to ADCC. Conversely, the replacement of His 375 by a serine residue (H375S) within HIV-1 CRF01_AE decreased the efficiency of the ADCC response. Our results raise the intriguing possibility that the presence of His 375 in the circulating strain where the RV144 trial was held contributed to the observed vaccine efficacy.

    IMPORTANCE HIV-1-infected cells presenting Env in the CD4-bound conformation on their surface are preferentially targeted by ADCC mediated by HIV-positive (HIV+) sera. Here we show that the gp120 Phe 43 cavity modulates the propensity of Env to sample this conformation and therefore affects the susceptibility of infected cells to ADCC. CRF01_AE HIV-1 strains have an unusual Phe 43 cavity-filling His 375 residue, which increases the propensity of Env to sample the CD4-bound conformation, thereby increasing susceptibility to ADCC.

  • Recombinant Adenoviruses Displaying Matrix 2 Ectodomain Epitopes on Their Fiber Proteins as Universal Influenza Vaccines [Vaccines and Antiviral Agents]

  • Influenza is a zoonotic disease that poses severe threats to public health and the global economy. Reemerging influenza pandemics highlight the demand for universal influenza vaccines. We developed a novel virus platform using extracellular domain IV of the matrix 2 protein (M2e), AdC68-F3M2e, by introducing three conserved M2e epitopes into the HI loop of the chimpanzee adenovirus (AdV) fiber protein. The M2e epitopes were expressed sufficiently on the AdV virion surface without affecting fiber trimerization. Additionally, one recombinant adenovirus, AdC68-F3M2e(H1-H5-H7), induced robust M2e-specific antibody responses in BALB/c mice after two sequential vaccinations and conferred efficient protection against homologous and heterologous influenza virus (IV) challenges. We found that the use of AdV with tandem M2e epitopes in fiber is a potential strategy for influenza prevention.

    IMPORTANCE Influenza epidemics and pandemics severely threaten public health. Universal influenza vaccines have increasingly attracted interest in recent years. Here, we describe a new strategy that incorporates triple M2e epitopes into the fiber protein of chimpanzee adenovirus 68. We optimized the process of inserting foreign genes into the AdC68 structural protein by one-step isothermal assembly and demonstrated that this 225-bp HI loop insertion could be well tolerated. Furthermore, two doses of adjuvant-free fiber-modified AdC68 could confer sufficient protection against homologous and heterologous influenza virus infections in mice. Our results show that AdC68-F3M2e could be pursued as a novel universal influenza vaccine.

  • Role of the PB2 627 Domain in Influenza A Virus Polymerase Function [Genome Replication and Regulation of Viral Gene Expression]

  • The RNA genome of influenza A viruses is transcribed and replicated by the viral RNA-dependent RNA polymerase, composed of the subunits PA, PB1, and PB2. High-resolution structural data revealed that the polymerase assembles into a central polymerase core and several auxiliary highly flexible, protruding domains. The auxiliary PB2 cap-binding and the PA endonuclease domains are both involved in cap snatching, but the role of the auxiliary PB2 627 domain, implicated in host range restriction of influenza A viruses, is still poorly understood. In this study, we used structure-guided truncations of the PB2 subunit to show that a PB2 subunit lacking the 627 domain accumulates in the cell nucleus and assembles into a heterotrimeric polymerase with PB1 and PA. Furthermore, we showed that a recombinant viral polymerase lacking the PB2 627 domain is able to carry out cap snatching, cap-dependent transcription initiation, and cap-independent ApG dinucleotide extension in vitro, indicating that the PB2 627 domain of the influenza virus RNA polymerase is not involved in core catalytic functions of the polymerase. However, in a cellular context, the 627 domain is essential for both transcription and replication. In particular, we showed that the PB2 627 domain is essential for the accumulation of the cRNA replicative intermediate in infected cells. Together, these results further our understanding of the role of the PB2 627 domain in transcription and replication of the influenza virus RNA genome.

    IMPORTANCE Influenza A viruses are a major global health threat, not only causing disease in both humans and birds but also placing significant strains on economies worldwide. Avian influenza A virus polymerases typically do not function efficiently in mammalian hosts and require adaptive mutations to restore polymerase activity. These adaptations include mutations in the 627 domain of the PB2 subunit of the viral polymerase, but it still remains to be established how these mutations enable host adaptation on a molecular level. In this report, we characterize the role of the 627 domain in polymerase function and offer insights into the replication mechanism of influenza A viruses.

  • Functional Analysis of Hepatitis C Virus (HCV) Envelope Protein E1 Using a trans-Complementation System Reveals a Dual Role of a Putative Fusion Peptide of E1 in both HCV Entry and Morphogenesis [Structure and Assembly]

  • Hepatitis C virus (HCV) is an enveloped RNA virus belonging to the Flaviviridae family. It infects mainly human hepatocytes and causes chronic liver diseases, including cirrhosis and cancer. HCV encodes two envelope proteins, E1 and E2, that form a heterodimer and mediate virus entry. While E2 has been extensively studied, less has been done so for E1, and its role in the HCV life cycle still needs to be elucidated. Here we developed a new cell culture model for HCV infection based on the trans-complementation of E1. Virus production of the HCV genome lacking the E1-encoding sequence can be efficiently rescued by the ectopic expression of E1 in trans. The resulting virus, designated HCVE1, can propagate in packaging cells expressing E1 but results in only single-cycle infection in naive cells. By using the HCVE1 system, we explored the role of a putative fusion peptide (FP) of E1 in HCV infection. Interestingly, we found that the FP not only contributes to HCV entry, as previously reported, but also may be involved in virus morphogenesis. Finally, we identified amino acid residues in FP that are critical for biological functions of E1. In summary, our work not only provides a new cell culture model for studying HCV but also provides some insights into understanding the role of E1 in the HCV life cycle.

    IMPORTANCE Hepatitis C virus (HCV), an enveloped RNA virus, encodes two envelope proteins, E1 and E2, that form a heterodimeric complex to mediate virus entry. Compared to E2, the biological functions of E1 in the virus life cycle are not adequately investigated. Here we developed a new cell culture model for single-cycle HCV infection based on the trans-complementation of E1. The HCV genome lacking the E1-encoding sequence can be efficiently rescued for virus production by the ectopic expression of E1 in trans. This new model renders a unique system to dissect functional domains and motifs in E1. Using this system, we found that a putative fusion peptide in E1 is a multifunctional structural element contributing to both HCV entry and morphogenesis. Our work has provided a new cell culture model to study HCV and provides insights into understanding the biological roles of E1 in the HCV life cycle.

  • HIV Controllers Exhibit Enhanced Frequencies of Major Histocompatibility Complex Class II Tetramer+ Gag-Specific CD4+ T Cells in Chronic Clade C HIV-1 Infection [Pathogenesis and Immunity]

  • Immune control of viral infections is heavily dependent on helper CD4+ T cell function. However, the understanding of the contribution of HIV-specific CD4+ T cell responses to immune protection against HIV-1, particularly in clade C infection, remains incomplete. Recently, major histocompatibility complex (MHC) class II tetramers have emerged as a powerful tool for interrogating antigen-specific CD4+ T cells without relying on effector functions. Here, we defined the MHC class II alleles for immunodominant Gag CD4+ T cell epitopes in clade C virus infection, constructed MHC class II tetramers, and then used these to define the magnitude, function, and relation to the viral load of HIV-specific CD4+ T cell responses in a cohort of untreated HIV clade C-infected persons. We observed significantly higher frequencies of MHC class II tetramer-positive CD4+ T cells in HIV controllers than progressors (P = 0.0001), and these expanded Gag-specific CD4+ T cells in HIV controllers showed higher levels of expression of the cytolytic proteins granzymes A and B. Importantly, targeting of the immunodominant Gag41 peptide in the context of HLA class II DRB1*1101 was associated with HIV control (r = nndash;0.5, P = 0.02). These data identify an association between HIV-specific CD4+ T cell targeting of immunodominant Gag epitopes and immune control, particularly the contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infection. Furthermore, these results highlight the advantage of the use of class II tetramers in evaluating HIV-specific CD4+ T cell responses in natural infections.

    IMPORTANCE Increasing evidence suggests that virus-specific CD4+ T cells contribute to the immune-mediated control of clade B HIV-1 infection, yet there remains a relative paucity of data regarding the role of HIV-specific CD4+ T cells in shaping adaptive immune responses in individuals infected with clade C, which is responsible for the majority of HIV infections worldwide. Understanding the contribution of HIV-specific CD4+ T cell responses in clade C infection is particularly important for developing vaccines that would be efficacious in sub-Saharan Africa, where clade C infection is dominant. Here, we employed MHC class II tetramers designed to immunodominant Gag epitopes and used them to characterize CD4+ T cell responses in HIV-1 clade C infection. Our results demonstrate an association between the frequency of HIV-specific CD4+ T cell responses targeting an immunodominant DRB1*11-Gag41 complex and HIV control, highlighting the important contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infections.

  • A Cytoplasmic RNA Virus Alters the Function of the Cell Splicing Protein SRSF2 [Virus-Cell Interactions]

  • To replicate efficiently, viruses must create favorable cell conditions and overcome cell antiviral responses. We previously reported that the reovirus protein mmu;2 from strain T1L, but not strain T3D, represses one antiviral response: alpha/beta interferon signaling. We report here that T1L, but not T3D, mmu;2 localizes to nuclear speckles, where it forms a complex with the mRNA splicing factor SRSF2 and alters its subnuclear localization. Reovirus replicates in cytoplasmic viral factories, and there is no evidence that reovirus genomic or messenger RNAs are spliced, suggesting that T1L mmu;2 might target splicing of cell RNAs. Indeed, RNA sequencing revealed that reovirus T1L, but not T3D, infection alters the splicing of transcripts for host genes involved in mRNA posttranscriptional modifications. Moreover, depletion of SRSF2 enhanced reovirus replication and cytopathic effect, suggesting that T1L mmu;2 modulation of splicing benefits the virus. This provides the first report of viral antagonism of the splicing factor SRSF2 and identifies the viral protein that determines strain-specific differences in cell RNA splicing.

    IMPORTANCE Efficient viral replication requires that the virus create favorable cell conditions. Many viruses accomplish this by repressing specific antiviral responses. We demonstrate here that some mammalian reoviruses, RNA viruses that replicate strictly in the cytoplasm, express a protein variant that localizes to nuclear speckles, where it targets a cell mRNA splicing factor. Infection with a reovirus strain that targets this splicing factor alters splicing of cell mRNAs involved in the maturation of many other cell mRNAs. Depletion of this cell splicing factor enhances reovirus replication and cytopathic effect. Our results provide the first evidence of viral antagonism of this splicing factor and suggest that downstream consequences to the cell are global and benefit the virus.

  • Hierarchical and Redundant Roles of Activating Fc{gamma}Rs in Protection against Influenza Disease by M2e-Specific IgG1 and IgG2a Antibodies [Pathogenesis and Immunity]

  • The ectodomain of matrix protein 2 is a universal influenza A virus vaccine candidate that provides protection through antibody-dependent effector mechanisms. Here we compared the functional engagement of Fc receptor (FcR) family members by two M2e-specific monoclonal antibodies (MAbs), MAb 37 (IgG1) and MAb 65 (IgG2a), which recognize a similar epitope in M2e with similar affinities. The binding of MAb 65 to influenza A virus-infected cells triggered all three activating mouse Fc receptors in vitro, whereas MAb 37 activated only FcRIII. The passive transfer of MAb 37 or MAb 65 in wild-type, Fcer1gnndash;/nndash;, Fcgr3nndash;/nndash;, and Fcgr1nndash;/nndash; Fcgr3nndash;/nndash; BALB/c mice revealed the importance of these receptors for protection against influenza A virus challenge, with a clear requirement of FcRIII for IgG1 MAb 37 being found. We also report that FcRIV contributes to protection by M2e-specific IgG2a antibodies.

    IMPORTANCE There is increased awareness that protection by antibodies directed against viral antigens is also mediated by the Fc domain of these antibodies. These Fc-mediated effector functions are often missed in clinical assays, which are used, for example, to define correlates of protection induced by vaccines. The use of antibodies to prevent and treat infectious diseases is on the rise and has proven to be a promising approach in our battle against newly emerging viral infections. It is now also realized that Fc receptors significantly enhance the in vivo protective effect of broadly neutralizing antibodies directed against the conserved parts of the influenza virus hemagglutinin. We show here that two M2e-specific monoclonal antibodies with close to identical antigen-binding specificities and affinities have a very different in vivo protective potential that is controlled by their capacity to interact with activating Fc receptors.

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    JVI Accepts: Articles Published Ahead of Print

  • Structural characterization of HCoV-NL63 N-protein. [PublishAheadOfPrint]

  • Coronaviruses are responsible for upper and lower respiratory tract infections in humans. It is estimated that 1mmdash;10% of the population suffers annually from cold-like symptoms related to infection with HCoV-NL63, an alphacoronavirus. The nucleocapsid (N) protein, the major structural component of the capsid, facilitates RNA packing, links the capsid to the envelope and is also involved in multiple other processes including viral replication and evasion of the immune system. Although the role of N protein in viral replication is relatively well described, no structural data are currently available regarding the N proteins of alphacoronaviruses. Moreover, our understanding of the mechanisms of RNA binding and nucleocapsid formation remains incomplete. In this study, we solved the crystal structures of the N- and C-terminal domains (NTD, residues 10mmdash;140, and CTD, residues 221mmdash;340, respectively) of the N protein of HCoV-NL63, both at 1.5 AAring; resolution. Based on our structure of NTD solved here, we proposed and experimentally evaluated a model of RNA binding. The structure of the CTD reveals the mode of N protein dimerization. Overall, this study expands our understanding of the initial steps of N protein/nucleic acid interaction, and may facilitate future efforts to control the associated infections.

    IMPORTANCE Coronaviruses are responsible for the common cold and other respiratory tract infections in humans. According to multiple studies, 1mmdash;10% of the population is infected each year with HCoV-NL63.

    Viruses are relatively simple organisms composed of a few proteins and the nucleic acids that carry the information determining their composition. The nucleocapsid (N) protein studied here protects the nucleic acid from the environmental factors during virus transmission. This study investigated the structural arrangement of N protein, explaining the first steps of its interaction with nucleic acid at the initial stages of virus structure assembly. The results expand our understanding of coronavirus physiology and may facilitate future efforts to control the associated infections.

  • Kaposi's Sarcoma-associated Herpesvirus Hijacks RNA Polymerase II to Create a Viral Transcriptional Factory [PublishAheadOfPrint]

  • Locally concentrated nuclear factors ensure efficient binding to the DNA templates, facilitating RNA polymerase II recruitment and frequent reutilization of stable pre-initiation complexes. Here we have uncovered a mechanism for effective viral transcription by focal assembly of RNA polymerase II around KSHV genomes in the host cell nucleus. Using immunofluorescent labeling of latent nuclear antigen (LANA) protein, together with fluorescence in situ RNA hybridization (RNA-FISH) of the intron region of immediate-early transcripts, we visualized active transcription of viral genomes in naturally infected cells. At single cell level, we found that not all episomes were uniformly transcribed following stimuli. However, those episomes that were being transcribed, would spontaneously aggregate to form transcriptional "factories", which recruited a significant fraction of cellular RNA polymerase II. Focal assembly of "viral transcriptional factories" decreased the pool of cellular RNA polymerase II available for cellular genes transcription, which consequently impaired cellular gene expression globally, with the exception of selected ones. The viral transcriptional factories localized with replicating viral genomic DNAs. The observed colocalization of viral transcriptional factories with replicating viral genomic DNA suggests that KSHV assembles an "all-in-one" factory for both gene transcription and DNA replication. We propose that the assembly of RNA polymerase II around viral episomes in the nucleus may be a previously unexplored aspect of KSHV gene regulation by confiscation of a limited supply of RNA polymerase II in infected cells.

    IMPORTANCE B-cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) harbor multiple copies of the KSHV genome in the form of episomes. 3D imaging of viral gene expression in the nucleus allows us to study interactions and changes in the physical distribution of these episomes following stimulation. The results showed heterogeneity in the responses of individual KSHV episomes to stimuli within a single reactivating cell -- and those episomes that did respond to stimulation, aggregated within large domains that appear to function as viral transcription factories. A significant portion of cellular RNA polymerase II was trapped in these factories and was serving to transcribe viral genomes, which coincided with an overall decrease in cellular gene expression. Our findings uncover a strategy of KSHV gene regulation through focal assembly of KSHV episomes and a molecular mechanism of late gene expression.

  • Nectin-4 Interactions Govern Measles Virus Virulence in a New Model of Pathogenesis, Squirrel Monkeys (Simia sciureus) [PublishAheadOfPrint]

  • In addition to humans, only certain non-human primates are naturally susceptible to measles virus (MeV) infection. Disease severity is species-dependent, ranging from mild to moderate for macaques to severe and even lethal for certain New World monkey species. To investigate if squirrel monkeys (Simia sciureus), which are reported to develop a course of disease similar to humans, may be better suited than macaques for the identification of virulence determinants or the evaluation of therapeutics, we infected them with a green fluorescent protein-expressing MeV. Compared to cynomolgus macaques (Macaca fascicularis) infected with the same virus, the squirrel monkeys developed more severe immunosuppression, higher viral load, and a broader range of clinical signs typical for measles. In contrast, infection with a MeV unable to interact with the epithelial receptor nectin-4, while causing immunosuppression, resulted in only a mild and transient rash and a short-lived elevation of the body temperature. Similar titers of the wild type and nectin-4 blind MeV were detected in peripheral blood mononuclear cells and lymph node homogenates, but only the wild type virus was found in tracheal lavages and urine. Thus, our study demonstrates the importance of MeV interactions with nectin-4 for clinical disease in the new and more performing Simia sciureus model of measles pathogenesis.

    IMPORTANCE The characterization of mechanisms underlying measles virus clinical disease has been hampered by the lack of an animal model that reproduces the course of disease seen in human patients. Here we report that infection of squirrel monkeys (Simia sciureus) fulfills these requirements. Comparative infection with wild type and epithelial cell receptor-blind viruses demonstrated the importance of epithelial cell infection for clinical disease, highlighting the spread to epithelia as an attractive target for therapeutic strategies.

  • Evaluation of the immune responses to and cross-protective efficacy of Eurasian H7 avian influenza viruses [PublishAheadOfPrint]

  • Due to increasing concerns of human infection by various H7 viruses, including recent H7N9 viruses, we evaluated the genetic relationships and the cross-protective efficacies of three different Eurasian H7 avian influenza viruses. Phylogenic and molecular analysis revealed that recent Eurasian H7 viruses can be separated into two different lineages with relatively high amino acid identity within groups (94.8 to 98.8%), and low amino acid identity (90.3 to 92.6 %) between the groups. In vivo immunization with representatives of each group revealed that while group-specific cross-reactivity was induced, cross-reactive HI titers were approximately fourfold lower against heterologous group viruses compared to homologous group viruses. Moreover, the group I (RgW109/06) vaccine could protect 100% of immunized mice from various group I viruses, while only 20 to 40% of immunized mice survived lethal challenge with heterologous group II viruses and exhibited high viral titers in the lung. Moreover, while the group II (RgW478/14) vaccine could also protect from lethal challenge with group II viruses, it failed to elicit cross-protection against group I viruses in mice. However, it is noteworthy that vaccination with RgAnhui1/13, which is a sublineage of group I, cross-protected immunized mice against lethal challenge with both group I and II viruses and significantly attenuated lung viral titers. Interestingly, immune sera from RgAnhui1/13 vaccinated mice showed a broad neutralizing spectrum rather than the group-specific pattern observed with the other viruses. These results suggest that the recent, human infectious H7N9 strain could be a candidate, broad cross-protective vaccine for Eurasian H7 viruses.

    IMPORTANCE Genetic and phylogenic analyses have demonstrated that the Eurasian H7 viruses can be separated into at least two different lineages, both of which contain human infectious, fatal H7 viruses including the recent, novel H7N9 viruses isolated in China since 2013. Due to the increasing concerns regarding the global public health risk posed by H7 viruses, we evaluated the genetic relationships between Eurasian H7 avian influenza viruses and the cross-protective efficacy of three different H7 viruses: W109/06 (group I), W478/14 (group II), and Anhui1/13 (a sublineage of group I). While each vaccine induced group-specific antibody responses and cross-protective efficacy, only Anhui1/13 was able to cross-protect immunized hosts against lethal challenge across groups. In fact, the Anhui1/13 virus not only induced cross-protection, but also broad serum-neutralizing antibody responses against both groups of viruses. This suggests that Anhui1/13-like H7N9 viruses may be viable vaccine candidates for broad protection against Eurasian H7 viruses.

  • Divergent requirement of Fc-Fc{gamma}R interactions for in vivo protection against influenza viruses by two pan-H5 hemagglutinin antibodies [PublishAheadOfPrint]

  • Recent studies have shown that Fc-FcR interactions are required for in vivo protection against influenza viruses by broadly reactive anti-HA stem, but not virus strain-specific, anti-receptor binding site (RBS), antibodies (Abs). Since only a few Abs recognizing epitopes in the head region but outside the RBS have been tested against single challenge virus strains, it remains unknown whether Fc-FcR interactions are required for in vivo protection by Abs recognizing epitopes outside the RBS, and whether the requirement is virus strain-specific or epitope-specific. In the present study, we therefore investigated the requirements for in vivo protection using two pan-H5 Abs, 65C6 and 100F4. We generated chimeric Abs, 65C6/IgG2a and 100F4/IgG2a, which preferentially engage activating FcRs, and isogenic forms, 65C6/D265A and 100F4/D265A, which do not bind FcR. Virus neutralizing activity, binding, ADCC, and in vivo protection of these Abs were compared using three H5 strains: A/Shenzhen/406H/2006 (SZ06), A/chicken/Shanxi/2/2006 (SX06), and A/chicken/Netherlands/14015526/2014 (NE14). We found that all four chimeric Abs bound and neutralized the SZ06 and NE14 strains, but poorly inhibited the SX06 strain. 65C6/IgG2a and 100F4/IgG2a, but not 65C6/D265A and 100F4/D265A, mediated ADCC against target cells expressing HA derived from all three virus strains. Interestingly, both 65C6/IgG2a and 65C6/D265A demonstrated comparable protection against all three virus strains in vivo; however, 100F4/IgG2a, but not 100F4/D265A, showed in vivo protection. Thus, we conclude that Fc-FcR interactions are required for in vivo protection by 100F4, but not by 65C6; and therefore, protection is not virus strain-specific, but epitope-specific.

    IMPORTANCE Abs play an important role in immune protection against influenza virus infection. Fc-FcR interactions are required for in vivo protection by broadly-neutralizing anti-stem, but not by virus strain-specific, anti-receptor binding site (RBS), Abs. Whether such interactions are necessary for protection by Abs that recognize epitopes outside RBS is not fully understood. In the present study, we investigated in vivo protection mechanisms against three H5 strains by two pan-H5 Abs 65C6 and 100F4. We show that although these two Abs have similar neutralizing, binding, and ADCC activities agsint all three H5 strains in vitro, they have divergent requirements for Fc-FcR interactions to protect against the three H5 strains in vivo. The Fc-FcR interactions are required for in vivo protection by 100F4, but not by 65C6. Thus, we conclude that Fc-FcR interactions for in vivo protection by pan-H5 Abs is not strain-specific, but epitope-specific.

  • Interferon-Inducible Oligoadenylate Synthetase-Like Protein Acts as an Antiviral Effector against Classical Swine Fever Virus via the MDA5-Mediated Type I Interferon Signaling Pathway [PublishAheadOfPrint]

  • Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), which poses a serious threat to the global pig industry. Interferons (IFNs) and IFN-stimulated genes (ISGs) play a key role in host antiviral defense. We have previously screened the porcine 2rrsquo; -5rrsquo; -oligoadenylate synthase-like protein (pOASL) as a potential anti-CSFV ISG using a reporter CSFV. This study aimed to clarify the underlying antiviral mechanism of pOASL against CSFV. We confirmed that CSFV replication was significantly suppressed in lentivirus-delivered, pOASL-overexpressing PK-15 cells, whereas silencing the expression of endogenous pOASL by small interfering RNAs markedly enhanced CSFV growth. In addition, the transcriptional level of pOASL was upregulated both in vitro and in vivo upon CSFV infection. Interestingly, the anti-CSFV effects of pOASL are independent of canonical RNase L pathway but depend on the activation of the type I IFN response. Glutathione S-transferase pulldown and coimmunoprecipitation assays revealed that pOASL interacts with MDA5, a double-stranded RNA sensor, and further enhances the MDA5-mediated type I IFN signaling. Moreover, we showed that pOASL exerts anti-CSFV effects in an MDA5-dependent manner. In conclusion, pOASL suppresses CSFV replication via the MDA5-mediated type I IFN signaling pathway.

    IMPORTANCE The host innate immune response plays an important role in mounting the initial resistance to viral infection. Here, we identify the porcine 2rrsquo; -5rrsquo; -oligoadenylate synthase-like protein (pOASL) as an interferon (IFN)-stimulated gene (ISG) against classical swine fever virus (CSFV). We demonstrate that the anti-CSFV effects of pOASL depend on the activation of type I IFN response. In addition, we show that pOASL, as an MDA5-interacting protein, is a coactivator of MDA5-mediated IFN induction to exert anti-CSFV actions. This work will be beneficial to the development of novel anti-CSFV strategies by targeting pOASL.

  • Recombinant modified vaccinia virus Ankara generating Ebola virus-like particles [PublishAheadOfPrint]

  • There are currently no approved therapeutics or vaccines to treat or protect against the severe hemorrhagic fever and death caused by Ebola virus (EBOV). Ebola virus-like particles (EBOV-VLPs) consisting of the matrix protein VP40, the glycoprotein (GP) and the nucleoprotein (NP) are highly immunogenic and protective in non-human primates against Ebola virus disease (EVD). We have constructed a modified vaccinia virus Ankara-Bavarian Nordicrreg; (MVA-BNrreg;) recombinant co-expressing VP40 and glycoprotein (GP) of EBOV Mayinga and the nucleoprotein (NP) of Taï Forest virus (TAFV) (MVA-BN-EBOV-VLP) to launch non-infectious EBOV-VLPs as a second vaccine modality in the MVA-BN-EBOV-VLP-vaccinated organism. Human cells infected with either MVA-BN-EBOV-VLP or MVA-BN-EBOV-GP showed comparable GP expression levels and transport of complex N-glycosylated GP to the cell surface. Human cells infected with MVA-BN-EBOV-VLP produced large amounts of EBOV-VLPs that were decorated with GP spikes but excluded the poxviral membrane protein B5, thus resembling authentic EBOV particles. The heterologous TAFV-NP enhanced EBOV-VP40-driven VLP formation with comparable efficiency as the homologous EBOV-NP in a transient expression assay, and both NPs were incorporated into EBOV-VLPs. EBOV-GP-specific CD8 T cell responses were comparably efficient between MVA-BN-EBOV-VLP and MVA-BN-EBOV-GP immunized mice. The levels of EBOV-GP-specific neutralizing and binding antibodies as well as GP-specific IgG1/IgG2a ratios induced by the two constructs in mice were also similar, raising the question whether the quality rather than the quantity of the GP-specific antibody response might be altered by an EBOV-VLP-generating MVA recombinant.

    IMPORTANCE The recent outbreak of Ebola virus (EBOV), claiming more than 11,000 lives, has underscored the need to advance the development of safe and effective filovirus vaccines. Virus-like particles (VLPs) as well as recombinant viral vectors have proved to be promising vaccine candidates. Modified vaccinia virus Ankara-Bavarian Nordicrreg; (MVA-BNrreg;) is a safe and immunogenic vaccine vector with a large capacity to accommodate multiple foreign genes. In this study, we combined the advantages of VLPs and the MVA platform by generating a recombinant MVA-BN-EBOV-VLP that would produce non-infectious EBOV-VLPs in the vaccinated individual. Our results show that human cells infected with MVA-BN-EBOV-VLP indeed formed and released EBOV-VLPs, thus producing a highly authentic viral immunogen. MVA-BN-EBOV-VLP efficiently induced EBOV-specific humoral and cellular immune responses in vaccinated mice. These results are the basis for future advancements, e.g. by including antigens from various filoviral species to develop multivalent VLP-producing MVA based filovirus vaccines.

  • Structural insights into emerging pediatric pathogens human bocaparvoviruses [PublishAheadOfPrint]

  • Bocaparvoviruses are emerging pathogens of the Parvoviridae family. Human bocaviruses 1 (HBoV1) causes severe respiratory infections and HBoV2-4 gastrointestinal infections in young children. Recent reports of life threatening cases, lack of direct treatment or vaccination, and a limited understanding of their disease mechanisms demand the need to study these pathogens on a molecular and structural level for the development of therapeutics. Towards this end, the capsid structures of HBoV1, HBoV3, and HBoV4 were determined to 2.8 mmdash; 3.0 AAring; resolution using cryo-electron microscopy and 3D image reconstruction. The bocaparvovirus capsids, that display different tissue tropisms, share common features with other parvoviruses, such as depressions at the icosahedral 2-fold and surrounding the 5-fold symmetry axes, protrusions surrounding the 3-fold, and a channel at the 5-fold axis. However, unlike other parvoviruses densities extending the 5-fold channel into the capsid interior are conserved among the bocaparvoviruses and are suggestive of genus specific function. Additionally, their major viral protein 3 contains loops with variable regions at their apexes conferring unique capsid surface topologies relative to other parvoviruses. Structural comparisons at strain (HBoVs) and genus (bovine parvovirus and HBoVs) levels identified differences in surface loops functionally important in host/tissue tropism, pathogenicity, and antigenicity in other parvoviruses, and likely play similar roles for these viruses. This study thus provides a structural framework to characterize determinants of host/tissue tropism, pathogenicity, and antigenicity for the development of anti-viral strategies to control human bocavirus infections.

    Importance Human bocaviruses are one of only a few members of the Parvoviridae family pathogenic to humans, especially young children and immunocompromised adults. There are currently no treatments or vaccines for these viruses or the related enteric bocaviruses. This study reports the first high-resolution structures of three human bocaparvoviruses determined by cryo-reconstruction. HBoV1 infects the respiratory tract, HBoV3 and HBoV4 the gastrointestinal tract, tissues that are likely targeted by the capsid. Comparison of these viruses provides information on conserved bocaparvovirus-specific features, and variable regions resulting in unique surface topologies that can serve as guides to characterize HBoV determinants of tissue tropism and antigenicity in future experiments. When compared to other existing parvovirus capsid structures this study suggests capsid regions that likely control successful infection, including determinants for receptor attachment, host cell trafficking, and antigenic reactivity. Overall these observations could impact efforts to design anti-viral strategies and vaccines for the HBoVs.

  • Feline immunodeficiency virus evolutionarily acquires two proteins, Vif and protease, capable of antagonizing feline APOBEC3 [PublishAheadOfPrint]

  • The interplay between viral and host proteins has been well studied to elucidate virus-host interactions and their relevance to virulence. Mammalian genes encode apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins, which act as intrinsic restriction factors against lentiviruses. To overcome APOBEC3-mediated anti-viral actions, lentiviruses have evolutionarily acquired an accessory protein, viral infectivity factor (Vif), and Vif degrades host APOBEC3 proteins via a ubiquitin/proteasome-dependent pathway. Although Vif-APOBEC3 interaction and its evolutionary significance, particularly those of primate lentiviruses (including HIV) and primates (including humans), have been well investigated, those of non-primate lentiviruses and non-primates are poorly understood. Moreover, the factors that determine lentiviral pathogenicity remain unclear. Here, we focus on feline immunodeficiency virus (FIV), a pathogenic lentivirus in domestic cats, and the interaction between FIV Vif and feline APOBEC3 in terms of viral virulence and evolution. We reveal the significantly reduced diversity of FIV subtype B compared to that of other subtypes, which may associate with the low pathogenicity of this subtype. We also demonstrate that FIV subtype B Vif is less active with regards to feline APOBEC3 degradation. More intriguingly, we further reveal that FIV protease cleaves feline APOBEC3 in released virions. Taken together, our findings provide evidence that a lentivirus encodes two types of anti-APOBEC3 factors, Vif and viral protease.

    IMPORTANCE During the history of mammalian evolution, mammals co-evolved with retroviruses, including lentiviruses. All pathogenic lentiviruses, excluding equine infectious anemia virus, have acquired the vif gene via evolution to combat APOBEC3 proteins, which are intrinsic restriction factors against exogenous lentiviruses. Here we demonstrate that FIV, a pathogenic lentivirus in domestic cats, antagonizes feline APOBEC3 proteins by both Vif and a viral protease. Furthermore, the Vif proteins of an FIV subtype (subtype B) have attenuated their anti-APOBEC3 activity through evolution. Our findings can be a clue to elucidate the complicated evolutionary processes by which lentiviruses adapt to mammals.

  • Comprehensive transcriptome analyses reveal that potato spindle tuber viroid triggers genome-wide changes in alternative splicing, inducible trans-acting activity of phasiRNAs and immune responses [PublishAheadOfPrint]

  • Many pathogens express non-coding RNAs (ncRNAs) during infection processes. In the most extreme case, pathogenic ncRNAs alone (such as viroids) can infect eukaryotic organisms leading to diseases. While a few of pathogenic ncRNAs have been implicated in regulating gene expression, functions of most pathogenic ncRNAs in host-pathogen interactions remain unclear. Here we employ potato spindle tuber viroid (PSTVd)-infecting tomato as a system to dissect host interactions with pathogenic ncRNAs, using comprehensive transcriptome analyses. We uncover various new activities in regulating gene expression during PSTVd infection, such as genome-wide alteration in alternative splicing of host protein-coding genes, enhanced guided-cleavage activities of a host microRNA and the induction of trans-acting function of phased secondary short interfering RNAs. Furthermore, we reveal that PSTVd infection massively activates genes involved in plant immune responses, mainly those in the calcium-dependent protein kinase and mitogen-activated protein kinase cascades as well as prominent genes involved in hypersensitive responses, cell wall fortification and various hormone signaling. Intriguingly, our data supports a notion that plant immune systems can respond to pathogenic ncRNAs, which has broad implications in providing new opportunities to understand the complex of immune systems in differentiating "self" and "non-self" as well as lays the foundation to resolve the long-standing question regarding the pathogenesis mechanisms of viroids and perhaps other infectious RNAs.

    IMPORTANCE Numerous pathogens, including viruses, express pathogenic non-coding transcripts during infection. In the most extreme case, pathogenic non-coding RNAs alone (i.e. viroids) can cause disease in plants. While some work has demonstrated that pathogenic non-coding RNAs interact with host factors for function, it remains largely unclear regarding the biological significance of pathogenic non-coding RNAs in host-pathogen interactions. Here we apply comprehensive genome-wide analyses on plant-viroid interactions and discover several novel molecular activities underlying nuclear-replicating viroid infection processes in plants, including effects on the expression and function of host non-coding transcripts as well as the alternative splicing of host protein-coding genes. Importantly, we show that plant immunity is activated upon the infection of a neclear-replicating viroid, which is a new concept that helps to understand viroid-based pathogenesis. Our finding has broad implications for understanding the complexity of host immune system and the diverse functions of non-coding RNAs.

  • Hili inhibits HIV replication in activated T cells [PublishAheadOfPrint]

  • Piwil proteins restrict the replication of mobile genetic elements in the germline. They are also expressed in many transformed cell lines. In this report, we discovered that the human piwil 2 (hili) can also inhibit HIV replication, especially in activated CD4+ T cells that are the preferred target cells for this virus in the infected host. Although resting cells did not express hili, it was rapidly induced following T cell activation. In these cells and transformed cell lines, depletion of hili increased levels of viral proteins and new viral particles. Further studies revealed that hili binds to tRNA. Some of them represent rare tRNA species, whose codons are over-represented in the viral genome. Targeting tRNAArg(UCU) with an antisense oligonucleotide replicated effects of hili and also inhibited HIV replication. Finally, hili also inhibited the retrotransposition of the endogenous intracysternal A particle (IAP) by a similar mechanism. Thus, hili joins a list of host proteins that inhibit the replication of HIV and other mobile genetic elements.

    IMPORTANCE Piwil proteins inhibit the movement of mobile genetic elements in the germline. In their absence, sperm does not form and male mice are sterile. This inhibition is thought to occur via small piRNAs. However, in some species and in human somatic cells, piwil proteins bind primarily to tRNA. In this report, we demonstrate that human piwil proteins, especially hili, not only bind to select tRNA species that include rare tRNAs, but also inhibit HIV replication. Importantly, T cell activation induces the expression of hili in CD4+ T cells. Since hili also inhibited the movement of an endogenous retrovirus (IAP), our finding shed new light on this intracellular resistance to exogenous and endogenous retroviruses as well as other mobile genetic elements.

  • BST-2 Expression Modulates Small CD4 Mimetic Sensitization of HIV-1-infected cells to ADCC [PublishAheadOfPrint]

  • Antibodies recognizing conserved CD4-induced (CD4i) epitopes on HIV-1 Env and able to mediate antibody-dependent cellular cytotoxicity (ADCC) have been shown to be present in sera from most HIV-1-infected individuals. These antibodies preferentially recognize Env in its CD4-bound conformation. CD4 downregulation by Nef and Vpu dramatically reduces exposure of CD4i HIV-1 Env epitopes and therefore reduce the susceptibility of HIV-1-infected cells to ADCC mediated by HIV+ sera. Importantly, this mechanism of immune evasion can be circumvented with small-molecule CD4-mimetics (CD4mc) which are able to transition Env into the CD4-bound conformation and sensitize HIV-1-infected cells to ADCC mediated by HIV+ sera. However, HIV-1 developed additional mechanisms to avoid ADCC including Vpu-mediated BST-2 antagonism, which decreases the overall amount of Env present at the cell surface. Accordingly, BST-2 up-regulation in response to IFN-aalpha; was shown to increase the susceptibility of HIV-1-infected cells to ADCC despite the activity of Vpu. Here we show that BST-2 upregulation by IFN-bbeta; and IL-27 also increases the surface expression of Env and thus boosts the ability of CD4mc to sensitize HIV-1-infected cells to ADCC by sera from HIV-1-infected individuals.

    IMPORTANCE HIV-1 evolved sophisticated strategies to conceal Env epitopes from ADCC-mediating antibodies present in HIV+ sera. Vpu-mediated BST-2 downregulation was shown to decrease ADCC responses by limiting the amount of Env present at the cell surface. This effect of Vpu was shown to be attenuated by IFN-aalpha; treatment. Here we show that in addition to IFN-aalpha;, IFN-bbeta; and IL-27 also affect Vpu-mediated BST-2 downregulation and greatly enhance ADCC responses against HIV-1-infected cells in the presence of CD4mc. These findings may inform strategies aimed at HIV prevention and eradication.

  • INVESTIGATION OF THE ROLE OF PROTEIN KINASE D IN HUMAN RHINOVIRUS REPLICATION [PublishAheadOfPrint]

  • Picornavirus replication is known to cause extensive remodelling of Golgi and endoplasmic reticulum membranes and a number of the host proteins involved in the viral replication complex have been identified, including oxysterol binding protein (OSBP) and phosphatidylinositol 4-kinase III beta (PI4KB). Since both OSBP and PI4KB are substrates for protein kinase D (PKD) and PKD is known to be involved in the control of Golgi vesicular and lipid transport, we hypothesised that PKD played a role in viral replication. We present multiple lines of evidence in support of that hypothesis. Firstly, infection of HeLa cells with human rhinovirus (HRV) induced phosphorylation of PKD. Secondly, PKD inhibitors reduced HRV viral genome replication, protein expression and viral titres in a concentration dependent fashion and also blocked the replication of poliovirus (PV) and foot-and-mouth disease virus (FMDV) in a variety of cells. Thirdly, HRV replication was significantly reduced in HeLa cells overexpressing wild type and mutant forms of PKD1 and fourthly HRV genome replication was reduced in HAP1 cells in which the PKD1 gene was knocked out by CRISPR/Cas9. Although we have not identified the molecular mechanism through which PKD regulates viral replication our data suggests this is not due to enhanced interferon signalling nor inhibition of clathrin-mediated endocytosis and PKD inhibitors do not need to be present during viral uptake. Our data shows for the first time that targeting PKD with small molecules can inhibit replication of HRV, PV and FMDV and therefore, PKD may represent a novel anti-viral target for drug discovery.

    IMPORTANCE Picornaviruses remain an important family of human and animal pathogens for which we have a very limited arsenal of anti-viral agents. HRV is the causative agent of the common cold, which in itself is a relatively trivial infection, however in asthma and chronic obstructive pulmonary disease (COPD) patients this virus is a major cause of exacerbations resulting in increased use of medication, worsening symptoms and frequently hospital admission. Thus, HRV represents a substantial healthcare and economic burden for which there are no approved therapies. We sought to identify a novel host target as a potential anti-HRV therapy. HRV infection induces phosphorylation of PKD and inhibitors of this kinase effectively block HRV replication at an early stage in the viral life cycle. Moreover, PKD inhibitors also block PV and FMDV replication. This is the first description that PKD may represent a target for anti-viral drug discovery.

  • Loss of the Human Cytomegalovirus US16 Protein Abrogates Virus Entry into Endothelial and Epithelial Cells by Reducing the Virion Content of the Pentamer [PublishAheadOfPrint]

  • The Human Cytomegalovirus (HCMV) US12 gene family encodes a group of predicted seven-transmembrane proteins whose functions have yet to be established. While inactivation of individual US12 members in laboratory strains of HCMV does not affect viral replication in fibroblasts, disruption of the US16 gene in the low-passage TR strain prevents viral growth in endothelial and epithelial cells. In these cells, the US16-null viruses fail to express IE, E, and L viral proteins due to a defect which occurs prior to IE gene expression. Here, we show that this defective phenotype is a direct consequence of deficiencies in the entry of US16-null viruses in these cell types due to an impact on the gH/gL/UL128/UL130/UL131A (Pentamer) complex. Indeed, viral particles released from fibroblasts infected with US16-null viruses were defective for the Pentamer, thus preventing entry during infections of endothelial and epithelial cells.

    A link between pUS16 and the Pentamer was further supported by the colocalization of pUS16 and Pentamer proteins within the cytoplasmic viral assembly compartment (cVAC) of infected fibroblasts. Deletion of the C-terminal tail of pUS16 reproduced the defective growth phenotype and alteration of virion composition as US16-null viruses. However, the Pentamer assembly and trafficking to the cVAC were not affected by the lack of the C-terminus of pUS16. Coimmunoprecipitation results then indicated that US16 interacts with pUL130, but not with the mature Pentamer or gH/gL/gO. Together, these results suggest that pUS16 contributes to the tropism of HCMV by influencing the content of the Pentamer into virions.

    IMPORTANCE Human Cytomegalovirus (HCMV) is major pathogen in newborns and immunocompromised individuals. A hallmark of HCMV pathogenesis is its ability to productively replicate in an exceptionally broad range of target cells. The virus infects a variety of cell types by exploiting different forms of the envelope glycoprotein gH/gL heteroligomers, which allow entry into many cell types through different pathways. For example, incorporation of the pentameric gH/gL/UL128/UL130/UL131A complex into virions is a prerequisite for infection of endothelial and epithelial cells. Here, we show that the absence of US16, a thus far uncharacterized HCMV multi transmembrane protein, abrogates virus entry into endothelial and epithelial cells, and that this defect is due to the lack of adequate amounts of the pentameric complex into extracellular viral particles. Our study suggests pUS16 as a novel viral regulatory protein important for shaping virion composition in a manner that influences HCMV cell tropism.

  • Heterologous packaging signals on HA, but not NA or NS, limit influenza A virus reassortment [PublishAheadOfPrint]

  • Influenza A virus (IAV) RNA packaging signals serve to direct the incorporation of IAV gene segments into virus particles, and this process is thought to be mediated by segment-segment interactions. These packaging signals are segment- and strain-specific, and as such, they have the potential to impact reassortment outcomes between different IAV strains. Our study aimed to quantify the impact of packaging signal mismatch on IAV reassortment using the human seasonal influenza A/Panama/2007/99 (H3N2) and pandemic influenza A/Netherlands/602/2009 (H1N1) viruses. Focusing on the three most divergent segments, we constructed pairs of viruses that encoded identical proteins but differed in the packaging signal regions on a single segment. We then evaluated the frequency with which segments carrying homologous vs. heterologous packaging signals were incorporated into reassortant progeny viruses. We found that, when the HA segments of co-infecting parental viruses were modified, there was a significant preference for the segment containing matched packaging signals relative to the background of the virus. This preference was apparent even when the homologous HA constituted a minority of the HA segment population available in the cell for packaging. Conversely, when NA or NS segments carried modified packaging signals, there was no significant preference for homologous packaging signals. These data suggest that movement of NA and NS segments between the human H3N2 and H1N1 lineages is unlikely to be restricted by packaging signal mismatch, while movement of the HA segment would be more constrained. Our results indicate that the importance of packaging signals in IAV reassortment is segment-dependent.

    IMPORTANCE Influenza A viruses (IAVs) can exchange genes through reassortment. This process contributes to both the highly diverse population of IAVs found in nature and the formation of novel epidemic and pandemic IAV strains. Our study sought to determine the extent to which IAV packaging signal divergence impacts reassortment between seasonal IAVs. Our knowledge in this area is lacking, and insight into the factors that influence IAV reassortment will inform and strengthen ongoing public health efforts to anticipate the emergence of new viruses. We found that the packaging signals on the HA segment, but not the NA or NS segments, restricted IAV reassortment. Thus, the packaging signals of the HA segment could be an important factor in determining the likelihood that two IAV strains of public health interest will undergo reassortment.

  • Ebolaviruses associated with differential pathogenicity induce distinct host responses in human macrophages [PublishAheadOfPrint]

  • Ebola virus (EBOV) and Reston virus (RESTV) are members of the Ebolavirus genus which greatly differ in their pathogenicity. While EBOV causes a severe disease in humans characterized by a dysregulated inflammatory response and elevated cytokine and chemokine production, there are no reported disease-associated human cases of RESTV infection, suggesting that RESTV is non-pathogenic for humans. The underlying mechanisms determining the pathogenicity of different ebolavirus species are not yet known. In this study we dissect the host response to EBOV and RESTV infection in primary human monocyte-derived macrophages (MDMs). As expected, EBOV infection led to a profound proinflammatory response, including strong induction of type I and type III IFNs. In contrast, RESTV-infected macrophages remained surprisingly silent. Early activation of IRF3 and NFB was observed in EBOV-infected, but not in RESTV-infected MDMs. In concordance with previous results, MDMs treated with inactivated EBOV and Ebola virus-like particles (VLPs) induced NFB activation mediated by TLR4 in a glycoprotein (GP)-dependent manner. This was not the case in cells exposed to live RESTV, inactivated RESTV, or VLPs containing RESTV GP, indicating that RESTV GP does not trigger TLR4 signaling. Our results suggest that the lack of immune activation in RESTV-infected MDMs contributes to lower pathogenicity by preventing the cytokine storm observed in EBOV infection. We further demonstrate that inhibition of TLR4 signaling abolishes EBOV GP-mediated NFB activation. This finding indicates that limiting the excessive TLR4-mediated proinflammatory response in EBOV infection should be considered as a potential supportive treatment option for EBOV disease.

    Importance Emerging infectious diseases are a major public health concern as exemplified by the recent devastating Ebola virus (EBOV) outbreak. Different ebolavirus species are associated with widely varying pathogenicity in humans ranging from asymptomatic infections for Reston virus (RESTV) to severe disease with fatal outcomes for EBOV. In this comparative study of EBOV- or RESTV-infected human macrophages, we identified key differences in host cell responses. Consistent with previous data, EBOV infection is associated with a proinflammatory signature triggered by the surface glycoprotein (GP) which can be inhibited by blocking TLR4 signaling. In contrast, infection with RESTV failed to stimulate a strong host response in infected macrophages due to the inability of RESTV GP to stimulate TLR4. We propose that disparate proinflammatory host signatures contribute to the differences in pathogenicity reported for ebolavirus species and suggest that proinflammatory pathways represent an intriguing target for the development of novel therapeutics.

  • Diversity, distribution, and evolution of tomato viruses in China uncovered by small RNA sequencing [PublishAheadOfPrint]

  • Tomato is a major vegetable crop that has tremendous popularity. However, viral disease is still a major factor limiting tomato production. Here we report the tomato virome identified through sequencing small RNAs of 170 field-grown samples collected in China. A total of 22 viruses were identified including both well-documented and newly detected viruses. The tomato viral community is dominated by a few species, and they exhibit polymorphisms and recombination in the genomes with coldspots and hotspots. Most samples were co-infected by multiple viruses and the majority of identified viruses are positive-sense single-stranded RNA viruses. Evolutionary analysis of one of the most dominant tomato viruses, Tomato yellow leaf curl virus (TYLCV), predicts its origin and the time back to its most recent common ancestor. The broadly sampled data has enabled us to identify several unreported viruses in tomato including a completely new virus, which has a genome of ~13.4 kb and groups with aphid-transmitted viruses in genus Cytorhabdovirus. Although both DNA and RNA viruses can trigger the biogenesis of virus-derived small interfering RNAs (vsiRNAs), we show that features such as length distribution, paired distance and base selection bias of vsiRNA sequences reflect different plant Dicer-like proteins and Argonautes involved in vsiRNA biogenesis. Collectively, this study offers insights into host-virus interaction in tomato and provides valuable information to facilitate the management of viral diseases.

    IMPORTANCE Tomato is an important source of micronutrient in human diet and is extensively consumed in the world. Virus is among the major constrains to tomato production. Categorizing virus species that are capable of infecting tomato and understanding their diversity and evolution are challenging due to difficulties in detecting such fast evolving biological entities. Here we report the landscape of tomato virome in China, the leading country of tomato production. We identified dozens of viruses present in tomato including both well-documented and completely new viruses. Some newly-emerged viruses in tomato were found to spread fast and therefore prompt attention is needed to control them. Moreover, we show that the virus genomes exhibit considerable degree of polymorphisms and recombination, and the virus-derived small interfering RNA (vsiRNA) sequences indicate distinct vsiRNA biogenesis mechanisms for different viruses. The Chinese tomato virome we developed provides valuable information to facilitate the management of tomato viral diseases.

  • Multiple sources of genetic diversity of influenza A viruses during the Hajj [PublishAheadOfPrint]

  • Outbreaks of respiratory virus infection at mass gatherings pose significant health risks to attendants, host communities and ultimately the global population if they help facilitate viral emergence. However, little is known about the genetic diversity, evolution and patterns of viral transmission during mass gatherings, particularly how much diversity is generated by in situ transmission compared to that imported from other locations. Here we describe the genomic-scale evolution of influenza A viruses sampled from the Hajj at Makkah during 2013-2015. Phylogenetic analysis revealed that the diversity of influenza virus at the Hajj was shaped by multiple introduction events, comprising multiple co-circulating lineages in each year, including those that have circulated in the Middle East and those whose origins likely lie on different continents. At the scale of individual hosts, the majority of minor variants resulted from de novo mutation with only limited evidence of minor variant transmission or minor variants circulating at sub-consensus level despite the likely identification of multiple transmission clusters. Together, these data highlight the complexity of influenza infection at the Hajj, reflecting a mix of global genetic diversity drawn from multiple sources combined with local transmission, and reemphasize the need for vigilant surveillance at mass gatherings.

    IMPORTANCE Large population sizes and densities at mass gatherings such as the Hajj (Makkah, Saudi Arabia) can contribute to outbreaks of respiratory virus infection by providing local hot-spots for transmission followed by spread to other localities. Using a genome-scale analysis we show that the genetic diversity of influenza A viruses at the Hajj during 2013-2015 was largely shaped by the introduction of multiple viruses from diverse geographic regions, including the Middle East, with only little evidence of inter-host virus transmission at the Hajj and seemingly limited spread of sub-consensus mutational variants. The diversity of viruses at the Hajj highlights the potential for lineage co-circulation during mass gatherings, in turn fuelling segment reassortment and the emergence of novel variants, such that the continued surveillance of respiratory pathogens at mass gatherings should be a public health priority.

  • Herpes simplex virus gE/gI and US9 promote both envelopment and sorting of virus particles in the cytoplasm of neurons, two processes that precede anterograde transport in axons. [PublishAheadOfPrint]

  • Herpes simplex virus (HSV) anterograde transport in neuronal axons is vital, allowing spread from latently-infected ganglia to epithelial tissues where viral progeny are produced in numbers allowing spread to other hosts. HSV membrane proteins gE/gI and US9 initiate the process of anterograde axonal transport ensuring that virus particles are transported from the cytoplasm into the most proximal segments of axons. These proteins do not appear to be important once HSV is inside axons. Previously we described HSV double mutants lacking both gE and US9 that failed to transport virus particles into axons. Here, we show that gE-/US9- double mutants accumulate large quantities of unenveloped and partially enveloped capsids in neuronal cytoplasm. These defects in envelopment can explain defects in axonal transport of enveloped virions. In addition, the unenveloped capsids that accumulated were frequently bound onto cytoplasmic membranes, apparently immobilized in intermediate stages of envelopment. A gE-null mutant produced enveloped virions, but these accumulated in large numbers in the neuronal cytoplasm, rather than reaching cell surfaces like wild type HSV virions. Thus, in addition to the defects in envelopment there was missorting of capsids and enveloped particles in the neuronal cytoplasm which can explain reduced anterograde transport of unenveloped capsids and enveloped virions. These mechanisms differ substantially from existing models suggesting that gE/gI and US9 function by tethering HSV particles onto kinesin microtubule motors. The defects in assembly of virus particles with gE-/US9- mutants were novel as they were neuron-specific, in keeping with observations that US9 is neuron-specific.

    IMPORTANCE Herpes simplex virus (HSV) and other alpha-herpesviruses such as varicella zoster virus depend upon the capacity to navigate in neuronal axons. To do this virus particles tether onto dyneins and kinesins that motor along microtubules from axon tips to neuronal cell bodies (retrograde) or from cell bodies to axon tips (anterograde). This transit in axons is essential in order for alpha-herpesviruses to establish latency in ganglia then reactivate and move back to peripheral tissues for spread to other hosts. Anterograde transport of HSV requires two membrane proteins, gE/gI and US9. Our studies describe new mechanisms for how gE/gI and US9 initiate anterograde axonal transport. HSV mutants lacking both gE and US9 fail to properly assemble enveloped virus particles in the cytoplasm which blocks anterograde transport of enveloped particles. As well, there were defects in the sorting of virus particles so that particles when formed do not enter proximal axons.

  • Maintenance of the HIV reservoir is antagonized by selective BCL2 inhibition [PublishAheadOfPrint]

  • Decay of the HIV reservoir is slowed over time in part by expansion of the pool of HIV infected cells. This expansion reflects homeostatic proliferation of infected cells by IL-7 or antigenic stimulation, as well as new rounds of infection of susceptible target cells. As novel therapies are being developed to accelerate the decay of the latent HIV reservoir, it will be important to identify interventions that prevent expansion and/or repopulation of the latent HIV reservoir. Our previous studies showed that HIV protease cleaves the host protein procaspase 8 to generate Casp8p41, which can bind and activate BAK to induce apoptosis of infected cells. In circumstances where expression of the anti-apoptotic protein BCL2 is high, Casp8p41 instead binds BCL2, and cell death does not occur. This effect can be overcome by treating cells with the clinically approved BCL2 antagonist venetoclax, which prevents Casp8p41 from binding BCL2, thereby allowing Casp8p41 to bind BAK and kill the infected cell. Herein we assess whether the events that maintain the HIV reservoir are also antagonized by venetoclax. Using the J-Lat 10.6 model of persistent infection, we demonstrate that proliferation and HIV expression is countered by venetoclax, which causes preferential killing of the HIV expressing cells. Similarly, during new rounds of infection of primary CD4 T cells, venetoclax causes selective killing of HIV infected cells, resulting in decreased number of HIV DNA containing cells.

    IMPORTANCE Cure of HIV infection requires an intervention that reduces HIV reservoir size. A variety of approaches are being tested for their ability to impact HIV reservoir size. Even if successful, however, these approaches will need to be combined with additional complementary approaches that prevent replenishment or repopulation of the HIV reservoir. Our previous studies have shown that the FDA approved BCL2 antagonist venetoclax has a beneficial effect on the HIV reservoir size following HIV reactivation. Herein we demonstrate that venetoclax also has a beneficial effect on HIV reservoir size in a model of homeostatic proliferation of HIV as well as in acute spreading infection of HIV in primary CD4 T cells. These results suggest that venetoclax is a compound worthy of further study for its effects on HIV reservoir size, either alone, or in combination with other approaches to reducing HIV reservoir size.

  • Monkeypox virus host factor screen in haploid cells identifies essential role of GARP complex in extracellular virus formation [PublishAheadOfPrint]

  • Monkeypox virus (MPXV) is a human pathogen that is a member of the Orthopoxvirus genus, which includes Vaccinia virus and Variola virus (the causative agent of smallpox). Human monkeypox is considered an emerging zoonotic infectious disease. To identify host factors required for MPXV infection, we performed a genome-wide insertional mutagenesis screen in human haploid cells. The screen revealed several candidate genes, including those involved in Golgi trafficking, glycosaminoglycan biosynthesis and glycosylphosphatidylinositol (GPI) mmdash; anchor biosynthesis. We validated the role of a set of vacuolar protein sorting (VPS) genes during infection, VPS51-54, which comprise the Golgi-associated retrograde protein (GARP) complex. The GARP complex is a tethering complex involved in retrograde transport of endosomes to the trans-Golgi apparatus. Our data demonstrate that VPS52 and VPS54 were dispensable for mature virus (MV) production but were required for extracellular virus (EV) formation. For comparison, a known antiviral compound, ST-246, was used in our experiments demonstrating that EV titers in VPS52 and VPS54 knockout (KO) cells were comparable to levels exhibited by ST-246 treated wildtype cells. Confocal microscopy was used to examine actin tail formation, one of the viral egress mechanisms for cell-to-cell dissemination, and revealed an absence of actin tails in VPS52KO or VPS54KO infected cells. Further evaluation of these cells by electron microscopy demonstrated a decrease in wrapped viruses (WV) compared to wild type control. Collectively, our data demonstrate the role of GARP complex genes in double-membrane wrapping of MV necessary for EV formation, implicating the host endosomal trafficking pathway in orthopoxvirus infection.

    IMPORTANCE Human monkeypox is an emerging zoonotic infectious disease caused by Monkeypox virus (MPXV). Of the two MPXV clades, the Congo Basin strain is associated with severe disease, higher mortality, and increased human-to-human transmission relative to the West African strain. Monkeypox is endemic to regions of western and central Africa but was introduced into the United States in 2003 from the importation of infected animals. The threat of MPXV and other orthopoxviruses is increasing due to the absence of routine smallpox vaccination leading to a higher proportion of naïve populations. In this study, we have identified and validated candidate genes that are required for MPXV infection, specifically the Golgi-associated retrograde protein (GARP) complex. Identifying host targets required for infection that prevents extracellular virus formation such as the GARP complex or the retrograde pathway can provide a potential target for anti-viral therapy.

  • CXCR5 Dependent Entry of CD8 T Cells into Rhesus Macaque B-Cell Follicles Achieved Through T-Cell Engineering [PublishAheadOfPrint]

  • Follicular helper CD4 T cells, TFH, residing in B-cell follicles within secondary lymphoid tissues, are readily infected by AIDS viruses and are a major source of persistent virus despite relative control of viral replication. This persistence is due at least in part to a relative exclusion of effective antiviral CD8 T cells from B-cell follicles. To determine whether CD8 T cells could be engineered to enter B-cell follicles, we genetically modified unselected CD8 T cells to express CXCR5, the chemokine receptor implicated in cellular entry into B-cell follicles. Engineered CD8 T cells expressing CXCR5 (CD8hCXCR5) exhibited ligand specific signaling and chemotaxis in vitro. Six infected rhesus macaques were infused with differentially fluorescent dye-labeled autologous CD8hCXCR5 and untransduced CD8 T cells and necropsied 48h later. Flow cytometry of both spleen and lymph node samples revealed higher frequencies of CD8hCXCR5 than untransduced cells, consistent with preferential trafficking to B-cell follicle-containing tissues. Confocal fluorescence microscopy of thin-sectioned lymphoid tissues demonstrated strong preferential localization of CD8hCXCR5 T cells within B-cell follicles with only rare cells in extrafollicular locations. CD8hCXCR5 T cells were present throughout the follicles with some observed near infected TFH. In contrast, untransduced CD8 T cells were found in the extrafollicular T-cell zone. Our ability to direct localization of unselected CD8 T cells into B-cell follicles using CXCR5 expression provides a strategy to place highly effective virus-specific CD8 T cells into these AIDS virus sanctuaries and potentially suppress residual viral replication.

    IMPORTANCE AIDS virus persistence in individuals under effective drug therapy or those who spontaneously control viremia remains an obstacle to definitive treatment. Infected follicular helper CD4 T cells, TFH, present inside B-cell follicles represent a major source of this residual virus. While effective CD8 T-cell responses can control viral replication in conjunction with drug therapy or in rare cases spontaneously, most antiviral CD8 T cells do not enter B-cell follicles and those that do fail to robustly control viral replication in the TFH population. Thus, these sites are a sanctuary and a reservoir for replicating AIDS viruses. Here, we demonstrate that engineering unselected CD8 T cells to express CXCR5, a chemokine receptor on TFH associated with B-cell follicle localization, redirects them into B-cell follicles. These proof of principle results open a pathway for directing engineered antiviral T cells into these viral sanctuaries to help eliminate this source of persistent virus.

  • Bushmeat hunting and zoonotic transmission of Simian T-lymphotropic virus 1 in tropical West and Central Africa [PublishAheadOfPrint]

  • Simian T-lymphotropic virus 1 (STLV-1) enters human populations through contact with non-human primate (NHP) bushmeat. We tested whether differences in the extent of contact to STLV-1 infected NHP bushmeat foster regional differences in prevalence of human HTLV-1. Using serological and PCR assays, we screened humans and NHP at two sub-Saharan African sites where subsistence hunting was expected to be less (Taï region, Coocirc;te d'Ivoire, CIV) or more developed (Bandundu region, Democratic Republic of the Congo, DRC). Only 0.7% of human participants were infected with HTLV-1 in CIV (N=574), and 1.3% of humans in DRC (N=302). Two of the Ivorian human virus sequences were closely related to simian counterparts, indicating ongoing zoonotic transmission. Multivariate analysis of human demographic parameters and behavior confirmed that participants from CIV were less often exposed to NHP than participants from DRC through direct contact, e.g. butchering. At the same time, numbers of STLV-1 infected NHP were higher at CIV (39%, N=111) than at DRC (23%, N=39). We conclude that a similar ultimate risk of zoonotic STLV-1 transmission - defined as the product of prevalence in local NHP and human rates of contact to fresh NHP carcasses - contributes to the observed comparable rates of HTLV-1 infection in humans in CIV and DRC. We find that young adult men and mature women are most likely exposed to NHP at both sites. In view of the continued difficulties to control zoonotic disease outbreaks, the identification of such high risk groups of NHP exposition may guide future prevention efforts.

    Importance Multiple studies report a high risk for zoonotic transmission of blood-borne pathogens like retroviruses through contact to NHP, and this risk seems to be particularly high in tropical Africa. Here, we reveal high levels of exposure to NHP bushmeat in two regions of Western and Central tropical Africa. We provide evidence for continued zoonotic origin of HTLV-1 in humans at CIV, and we find that young men and mature women represent risk groups for zoonotic transmission of pathogens from NHP. Identifying such risk groups can contribute to mitigate not only zoonotic STLV-1 transmission, but transmission of any blood-borne pathogen onto humans in sub-Saharan Africa.

  • African swine fever virus NP868R capping enzyme promotes reovirus rescue during reverse genetics by promoting reovirus protein expression, virion assembly, and RNA incorporation into infectious virions [PublishAheadOfPrint]

  • Reoviruses, like many eukaryotic viruses, contain an inverted 7-methylguanosine (m7G) cap linked to the 5rrsquo; nucleotide of mRNA. Traditional functions of capping are to promote mRNA stability, protein translation, and concealment from cellular proteins that recognize foreign RNA. To address the role of mRNA capping during reovirus replication, we assessed the benefits of adding the African swine fever virus NP868R capping enzyme. C3P3, a fusion protein containing T7 RNA polymerase and NP868R, was found to increase protein expression 5 to 10-fold, as compared to T7 RNA polymerase alone, while enhancing reovirus rescue from the current reverse genetics system by 100-fold. Surprisingly, RNA stability was not increased by C3P3, suggesting a direct effect on protein translation. A time course analysis revealed that C3P3 increased protein synthesis within the first 2 days of a reverse genetics transfection. This analysis also revealed that C3P3 enhanced processing of outer capsid mmu;1 protein to mmu;1C, a previously described hallmark of reovirus assembly. Finally, to determine the rate of infectious RNA incorporation into new virions, we developed a new recombinant reovirus S1 gene that expressed the fluorescent protein UnaG. Following transfection of cells with UnaG and infection with wild-type virus, passage of UnaG through progeny was significantly enhanced by C3P3. This data suggests that capping provides non-traditional functions to reovirus, such as promoting assembly and infectious RNA incorporation.

    IMPORTANCE The findings in this manuscript expand our understanding of how viruses utilize capping, suggesting that capping provides non-traditional functions to reovirus such as promoting assembly and infectious RNA incorporation, in addition to enhancing protein translation. Beyond providing mechanistic insight into reovirus replication, our findings also show that reovirus reverse genetics rescue is enhanced 100-fold by the NP868R capping enzyme. Since reovirus shows promise as a cancer therapy, efficient reovirus reverse genetics rescue will accelerate production of recombinant reoviruses as candidates to enhance therapeutic potency. NP868R-assisted reovirus rescue will also expedite production of recombinant reovirus for mechanistic insights on reovirus protein function and structure.

  • PRION STRAIN CHARACTERIZATION OF A NOVEL SUBTYPE OF CREUTZFELDT-JAKOB DISEASE [PublishAheadOfPrint]

  • In 2007, we reported a patient with an atypical form of Creutzfeldt-Jakob disease (CJD), heterozygous for methionine-valine (MV) at codon 129, who showed a novel pathological prion protein (PrPTSE) conformation with an atypical glycoform (AG) profile and an intraneuronal PrP deposition. In the present study, we further characterized the conformational properties of this pathological prion protein (PrPTSE MVAG) showing that PrPTSE MVAG is composed by multiple conformers with biochemical properties distinct from PrPTSE type 1 and type 2 of MV sporadic CJD (sCJD). Experimental transmission of CJD-MVAG to bank voles and gene-targeted transgenic mice carrying the human prion protein gene (TgHu) showed unique transmission rates, survival times, neuropathological changes, PrPTSE deposition patterns and PrPTSE glycotypes that are distinct from sCJD-MV1 and sCJD-MV2. These biochemical and experimental data suggest the presence of a novel prion strain in CJD-MVAG.

    IMPORTANCE Sporadic Creutzfeldt-Jakob disease is caused by the misfolding of the cellular prion protein, which assumes two major different conformations (type 1 and type 2) and, together with the methionine/valine polymorphic codon 129 of the prion protein gene, contribute to the occurrence of distinct clinical-pathological phenotypes. Inoculation in laboratory rodents of brain tissues from the six possible combination of pathological prion protein type with codon 129 genotypes results in the identification of 3 to 4 strains of prions. We report on the identification of a novel strain of Creutzfeldt-Jakob disease isolated from a patient who carried an abnormally glycosylated pathological prion protein. This novel strain has unique biochemical characteristics, does not transmit to humanized transgenic mice, and shows exclusive transmission properties in bank voles. The identification of a novel human prion strain improves our understanding on the pathogenesis of the disease and on possible mechanisms of prion transmission.

  • SAT2 foot-and-mouth disease virus (FMDV) structurally modified for increased thermostability [PublishAheadOfPrint]

  • Foot-and-mouth disease virus (FMDV) is notoriously unstable, particularly the O and SAT serotypes. Consequently, vaccines derived from heat-labile SAT viruses have been linked to the induction of poor duration immunity and hence require more frequent vaccinations to ensure protection. In-silico calculations predicted residue substitutions that would increase interactions at the inter-pentameric interface supporting increased stability. We assessed the stability of the 18 recombinant mutant viruses for their growth kinetics; antigenicity; plaque morphology; genetic stability; temperature, ionic and pH stability using the thermofluor and inactivation assays, in order to evaluate potential SAT2 vaccine candidates with improved stability. The most stable mutation was the single mutant S2093Y for temperature and pH stability, whilst other promising single mutants were E3198A, L2094V,S2093H and the triple mutant F2062Y-H2087M-H3143V. Although the S2093Y mutant had the greatest stability it exhibited smaller plaques; a reduced growth rate; a change in a monoclonal antibody footprint, and poor genetic stability properties compared to the wild-type virus. However, these factors affecting production can be overcome. The addition of 1M NaCl salt was found to further increase the stability of the SAT2 panel of viruses. The S2093Y and S2093H mutants were selected for future use in stabilising SAT2 vaccines.

    IMPORTANCE Foot-and-mouth disease virus (FMDV) causes a highly contagious acute vesicular disease in cloven-hoofed livestock and wildlife. The control of the disease by vaccination is essential, especially at livestock-wildlife interfaces. The instability of serotypes such as SAT2 affects the quality of the vaccine and therefore the duration of immunity. We have shown that by mutating residues at the capsid interface through predictive modelling we can improve the stability of SAT2 viruses. This is an important finding for the potential use of such mutants in improving the stability of SAT2 vaccines in endemic countries that rely heavily on the maintenance of the cold-chain, with potential improvement to the duration of immune responses.

  • Human herpesvirus 6B induces hypomethylation on chromosome 17p13.3 correlating with increased gene expression and virus integration [PublishAheadOfPrint]

  • Human herpesvirus 6B (HHV-6B) is a neurotropic beta-herpesvirus that achieves latency by integrating its genome into host cell chromosomes. Several viruses can induce epigenetic modifications in their host cells, but no study has investigated the epigenetic modifications induced by HHV-6B. This study analyzed methylation with Illumina 450K array comparing HHV-6B infected and uninfected Molt-3 T cells three days post infection. Bisulfite pyrosequencing was used to validate Illumina results and investigate methylation over time in vitro. Expression of genes was investigated using qPCR, and virus integration was investigated with PCR. A total of 406 CpG sites showed significant HHV-6B induced change in methylation in vitro. Remarkably, 86% (351/406) of these CpGs were located llt;1Mb from chromosomal ends and were all hypomethylated in virus-infected cells. This was most evident at chromosome 17p13.3 where HHV-6B induced CpG hypomethylation already after two days infection, possibly through TET2, which was found upregulated together with an identified cytosine hydroxymethylation. Genes located in the hypomethylated region at 17p13.3 showed significantly upregulated expression in HHV-6B infected cells. A temporal experiment showed HHV-6B integration in Molt-3 cell DNA three days after infection. The telomere at 17p has repeatedly been described as an integration site for HHV-6B and we show for the first time that HHV-6B induces hypomethylation in this region during acute infection, which may play a role in the integration process, possibly by making the DNA more accessible.

    IMPORTANCE The ability to establish latency in the host is a hallmark of herpesviruses, but mechanisms vary. Human herpesvirus 6B (HHV-6B) is known to establish latency through integration of its genome into the telomeric regions of host cells, with ability to reactivate. Our study is the first to show that HHV-6B specifically induces hypomethylated regions close to the telomeres, and that integrating viruses may use the host methylation machinery to facilitate their integration process. The results from this study contribute to knowledge of HHV-6B biology and virus-host- interaction. This in turn will further progress our understanding of the underlying mechanisms by which HHV-6B contributes to pathological processes and may have important implications in both disease prevention and treatment.

  • Dengue virus activates the AMP kinase-mTOR axis to stimulate a proviral lipophagy. [PublishAheadOfPrint]

  • Robust dengue virus (DENV) replication requires lipophagy, a selective autophagy that targets lipid droplets. The autophagic mobilization of lipids leads to increased bbeta;-oxidation in DENV-infected cells. The mechanism by which DENV induces lipophagy is unknown. Here, we show that infection with DENV activates the metabolic regulator 5rrsquo; adenosine-monophosphate activated kinase (AMPK), and that the silencing or pharmacological inhibition of AMPK activity decreases DENV replication and the induction of lipophagy. The activity of mechanistic target of rapamycin complex 1 (mTORC1), decreases in DENV-infected cells and is inversely correlated to lipophagy induction. Constitutive activation of mTORC1 by depletion of tuberous sclerosis complex 2 (TSC2) inhibits lipophagy induction in DENV-infected cells, and decreases viral replication. While AMPK normally stimulates TSC2-dependent inactivation of mTORC1 signaling, mTORC1 inactivation is independent of AMPK activation during DENV infection. Thus, DENV stimulates and requires AMPK signaling, as well as AMPK-independent suppression of mTORC1 activity for proviral lipophagy.

    IMPORTANCE Dengue virus alters host cell lipid metabolism to promote its infection. One mechanism for altered metabolism is the induction of a selective autophagy that targets lipid droplets, termed lipophagy. Lipophagy mobilizes lipid stores resulting in enhanced bbeta;-oxidation and viral replication. We show here that DENV infection activates and requires the central metabolic regulator AMPK for its replication and the induction of lipophagy. This is required for the induction of lipophagy, but not basal autophagy, in DENV-infected cells.

  • PROTEOLYTIC CLEAVAGE OF BOVINE ADENOVIRUS-3 ENCODED pVIII [PublishAheadOfPrint]

  • Proteolytic maturation involving cleavage of one non-structural and six structural precursor proteins including pVIII by adenovirus protease is an important aspect of adenovirus life cycle. The pVIII encoded by BAdV-3 is a protein of 216 amino acids, which contains two potential protease cleavage sites. Here, we report that BAdV-3 pVIII is cleaved by adenovirus protease at both potential consensus protease cleavage sites. Usage of at least one cleavage site appears essential for the production of progeny BAdV-3 virions as glycine to alanine mutation of both protease cleavage sites appears lethal for the production of progeny virions. However, mutation of a single protease cleavage site of BAdV-3 pVIII significantly affects the efficient production of infectious progeny virions. Further analysis revealed no significant defect in endosome escape, genome replication, capsid formation and virus assembly. Interestingly, cleavage of pVIII at both potential cleavage sites appears essential for the production of stable BAdV-3 virions as BAdV-3 expressing pVIII containing glycine to alanine mutation of either of the potential cleavage site are thermolabile and lead to the production of non- infectious virions.

    IMPORTANCE Here, we demonstrated that the BAdV-3 adenovirus protease cleaves BAdV-3 pVIII at both potential protease cleavage sites. Although cleavage of pVIII at one of the two adenoviral protease cleavage site is required for the production of progeny virions, the mutation of a single cleavage site of pVIII affects the efficient production of infectious progeny virions. Further analysis indicated that the mutation of single protease cleavage site (glycine to alanine) of pVIII produce thermolabile virions, which lead to the production of non-infectious virions with disrupted capsids. We thus provide evidence about the requirement of proteolytic cleavage of pVIII for production of infectious progeny virions. We feel our study has significantly advanced the understanding of requirement of adenovirus protease cleavage of pVIII.

  • Simultaneous single cell in situ analysis of human adenovirus type 5 DNA and mRNA expression patterns in lytic and persistent infection [PublishAheadOfPrint]

  • An efficient adenovirus infection results in high-level accumulation of viral DNA and mRNAs in the infected cell population. However, the average viral DNA and mRNA content in a heterogeneous cell population do not necessarily reflect the same abundance in individual cells. Here we describe a novel padlock probe-based rolling circle amplification technique that enables simultaneous detection and analysis of human adenovirus type 5 (HAdV-5) genomic DNA and virus-encoded mRNAs in individual infected cells. We demonstrate that the method is applicable for detection and quantification of HAdV-5 DNA and mRNAs in short-term infections in human epithelial cells and in long-term infections in human B lymphocytes. Single cell evaluation of these infections revealed high heterogeneity and unique cell subpopulations defined by differential viral DNA content and mRNA expression. Further, our single cell analysis shows that the specific expression pattern of viral E1A 13S and 12S mRNA splice variants is linked to HAdV-5 DNA content in the individual cells. Furthermore, we show that expression of mature form of the HAdV-5 histone-like protein VII affects virus genome detection in HAdV-5 infected cells. Collectively, padlock probes combined with rolling circle amplification should be a welcome addition to the method repertoire to characterize the molecular details of the HAdV life cycle in individual infected cells.

    IMPORTANCE Human adenoviruses (HAdVs) have been extensively used as model systems to study various aspects of eukaryotic gene expression and genome organization. The vast majority of the HAdV studies are based on standard experimental procedures carried out using heterogeneous cell populations, where data averaging often masks biological differences. As every cell is unique, characteristics and efficiency of a HAdV infection can vary from cell to cell. Therefore, the analysis of HAdV gene expression and genome organization would benefit from a method that permits analysis of individual infected cells in the heterogeneous cell population. Here, we show that the padlock-probe based rolling circle amplification method can be used to study concurrent viral DNA accumulation and mRNA expression patterns in individual HAdV-5-infected cells. Hence, this versatile method can be applied to detect extent of infection and virus gene expression changes in different HAdV-5 infections.

  • A Single Dose Recombinant PIV5 Expressing Respiratory Syncytial Virus F and G Proteins Protected Cotton Rats and African Green Monkeys from RSV Challenge [PublishAheadOfPrint]

  • Human respiratory syncytial virus (RSV) is a common cause of severe respiratory disease among infants, immunocompromised individuals and the elderly. No licensed vaccine is currently available. In this study, we evaluated two parainfluenza virus 5 (PIV5)-vectored vaccines expressing RSV F (PIV5/F) or G (PIV5/G) proteins in the cotton rat and African green monkey models for their replication, immunogenicity and efficacy of protection against RSV challenge. Following a single intranasal inoculation, both animal species shed the vaccine viruses for a limited time but without noticeable clinical symptoms. In cotton rats, the vaccines elicited RSV F or G specific serum antibodies, and conferred complete lung protection against RSV challenge at doses as low as 103 plaque forming units (PFU). Neither vaccine produced the enhanced lung pathology as observed in animals immunized with formalin inactivated RSV. In African green monkeys, vaccine induced serum and mucosal antibody responses were readily detected as well. PIV5/F provided near complete protection against RSV infection in the upper and lower respiratory tracts at a dose of 106 PFU of vaccine. At the same dose levels, PIV5/G was less efficacious. Both PIV5/F and PIV5/G were also able to boost neutralization titers in RSV pre-exposed African green monkeys. Overall, our data indicated that PIV5/F is a promising RSV vaccine candidate.

    IMPORTANCE A safe and efficacious respiratory syncytial virus (RSV) vaccine remains elusive. We tested the recombinant parainfluenza virus 5 (PIV5) vectors expressing RSV glycoproteins, for their immunogenicity and protective efficacy, in cotton rats and African green monkeys which are among the best available animal models to study RSV infection. In both species, a single dose of intranasal immunization with PIV5 vectored vaccines was able to produce systemic and local immunities, and protect animals from RSV challenge. The vaccines could also boost RSV neutralization antibody titers in African green monkeys that had been infected previously. Our data suggested that PIV5 vectored vaccines could potentially protect both the pediatric and elderly populations, and supported continued development of the vector platform.

  • Dengue Virus NS2B/NS3 Protease Inhibitors Exploiting the Prime Side [PublishAheadOfPrint]

  • The mosquito-transmitted dengue virus (DENV) infects millions of people in tropical and sub-tropical regions. Maturation of DENV particles requires proper cleavage of the viral polyprotein, including processing 8 of the 13 substrate cleavage sites by dengue NS2Bnndash;S3 protease. With no available direct-acting antiviral targeting DENV, NS2nndash;S3 protease is a promising target for inhibitor design. Current design efforts focus on the nonprime side of the DENV protease active site, resulting in highly hydrophilic and nonspecific scaffolds. However, the prime side also significantly modulates DENV protease binding affinity, as revealed by engineering the binding loop of aprotinin, a small protein with high affinity to DENV protease. In this study, we designed a series of cyclic peptides interacting with both sides of the active site as inhibitors of dengue protease. The design was based on two aprotinin loops, and aimed to leverage both key specific interactions of substrate sequences and the entropic advantage driving aprotinin's high affinity. By optimizing the cyclization linker, length, and amino acid sequence, the tightest cyclic peptide achieved a Ki value of 2.9 mmu;M against DENV3 WT protease. These inhibitors provide proof of concept that both sides of DENV protease active site can be exploited to potentially achieve specificity and lower hydrophilicity in the design of inhibitors targeting DENV.

    IMPORTANCE Viruses of the flaviviral family, including DENV and Zika viruses transmitted by A. aegypti, continue to be a threat to global health by causing major outbreaks in tropical and subtropical regions, with no available direct-acting antivirals for treatment. A better understanding of the molecular requirements for the design of potent and specific inhibitors against flaviviral proteins will contribute to the development of targeted therapies for infections by these viruses. The cyclic peptides reported here as DENV protease inhibitors provide novel scaffolds that enable exploiting the prime side of the protease active site, toward achieving better specificity and lower hydrophilicity compared to current scaffolds in the design of anti-flaviviral inhibitors.

  • Internal disequilibria and phenotypic diversification during replication of hepatitis C virus in a non-coevolving cellular environment [PublishAheadOfPrint]

  • Viral quasispecies evolution upon long-term virus replication in a non-coevolving cellular environment raises relevant general issues such as the attainment of population equilibrium, compliance with the molecular clock hypothesis or stability of the phenotypic profile. Here we evaluate adaptation, mutant spectrum dynamics, and phenotypic diversification of hepatitis C virus (HCV) in the course of two hundred passages in human hepatoma cells in an experimental design that precluded coevolution of the cells with the virus. Adaptation to the cells was evidenced by increase in progeny production. The rate of accumulation of mutations in the genomic consensus sequence deviated slightly from linearity, and mutant spectrum analyses revealed a complex dynamics of mutational waves, which was sustained beyond passage 100. The virus underwent several phenotypic changes some of which impacted the virus-host relationship, such as enhanced cell killing, shift towards higher virion density, and increased shut-off of host cell protein synthesis. Fluctuations in progeny production and failure to reach population equilibrium at the genomic level suggest internal instabilities that anticipate an unpredictable HCV evolution in the complex liver environment.

    IMPORTANCE Long-term virus evolution in an unperturbed cellular environment can reveal features of virus evolution that cannot be answered by comparing natural viral isolates. In the present study we investigate genetic and phenotypic changes that occur upon prolonged passage of hepatitis C virus (HCV) in human hepatoma cells in an experimental design in which host cell evolutionary change is prevented. Despite replication in a non-coevolving cellular environment, the virus exhibited internal population disequilibria that did not decline with increased adaptation to the host cells. The diversification of phenotypic traits suggests that disequilibria inherent to viral populations may provide a selective advantage to viruses that can be fully exploited in changing environments.

  • Vaccinia virus A6 is a two-domain protein requiring a cognate N-terminal domain for full viral membrane assembly activity [PublishAheadOfPrint]

  • Poxvirus virion biogenesis is a complex, multistep process, starting with the formation of crescent-shaped viral membranes, followed by their enclosure of viral core to form the spherical immature virions. Crescent formation requires a group of proteins that are highly conserved among poxviruses, including A6 and A11 of vaccinia virus (VACV). To gain a better understanding of the molecular function of A6, we established a HeLa cell line that inducibly expressed VACV-A6, which allowed us to construct VACV mutants with A6 deletion or mutation. As expected, A6 deletion VACV mutant failed to replicate in non-complementing cell lines with defects in crescent formation and A11 localization. Surprisingly, a VACV mutant that had A6 substituted with a close ortholog from Yaba-like disease virus, YLDV-97, also failed to replicate. This mutant, however, developed crescents and had normal A11 localization despite failing to form immature virions. A limited proteolysis of the recombinant A6 protein identified an N- and a C-domain of approximately 121 and 251 residues, respectively. Various chimeras of VACV-A6 and YLDV-97 were constructed, but only one that precisely combined the N-domain of VACV-A6 and the C-domain of YLDV-97 supported VACV replication, albeit at reduced efficiency. Our results show that VACV A6 has a two-domain architecture and functions in both crescent formation and its enclosure to form immature virions. While a cognate N-domain is not required for crescent formation, it is required for virion formation, suggesting that interactions of N-domain with cognate viral proteins may be critical for virion assembly.

    IMPORTANCE Poxviruses are unique among enveloped viruses in that they acquire their primary envelope not through budding from cellular membranes but by forming and extending crescent membranes. The crescents are highly unusual, open-ended membranes, and their origin and biogenesis have perplexed virologists for decades. A group of five viral proteins have been recently identified as essential for crescent formation, including A6 protein of vaccinia virus. It is thus important to understand the structure and function of A6 in order to solve the long-standing mystery of poxvirus membrane biogenesis. Here, we established an experimental system that allowed the genetic manipulation of the essential A6L gene. By studying A6 mutant viruses, we found that A6 plays an essential role not only in crescent formation but also in its subsequent enclosure to form immature virions. We defined A6 domain architecture and suggested that one of its two domains cooperates with cognate viral proteins.

  • Broadly reactive anti-RSV G antibodies from exposed individuals effectively inhibit infection of primary airway epithelial cells [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) causes severe respiratory disease in young children. Antibodies specific for the RSV prefusion F protein have guided RSV vaccine research and in human serum these antibodies attribute to ggt;90% of the neutralization response, however detailed insight in the composition of the human B cell repertoire against RSV is still largely unknown. In order to study the B cell repertoire of 3 healthy donors for specificity against RSV, CD27+ memory B cells were isolated and immortalized using BCL6 and Bcl-xL. Of the circulating memory B cells 0.35% recognized RSV-A2 infected cells, of which 59% were IgA and 41% were IgG expressing cells. When we generated monoclonal B cells selected for high binding to RSV infected cells, 44.5% of IgGs and 56% of IgAs reacted to the F protein, while unexpectedly 41.5% of IgG and 44% of IgA expressing B cells reacted to the G protein. Analysis of the G-specific antibodies revealed that 4 different domains on the G protein were recognized. These epitopes predict cross-reactivity between RSV-A and RSV-B and matched with their potency to neutralize RSV in HEp-2 cells and in primary epithelial cell cultures. G-specific antibodies were also able to induce antibody dependent cellular cytotoxicity and antibody dependent cellular phagocytosis of RSV-A2 infected cells. However, these processes did not seem to dependent on a specific epitope. In conclusion, healthy adults harbor a diverse repertoire of RSV glycoprotein specific antibodies with a broad range of effector functions that likely play an important role in anti-viral immunity.

    Importance Human RSV remains the most common cause of severe lower respiratory tract disease in premature babies, young infants, the elderly and immunocompromised patients, and plays an important role in asthma exacerbations. In developing countries RSV lower respiratory tract disease is a disease with a high mortality. Without an effective vaccine, only passive immunization with palivizumab is approved for prophylactic treatment. However, highly potent RSV specific monoclonal antibodies could potentially serve as a therapeutic treatment and contribute to disease control and mortality reduction. In addition, these antibodies could guide further vaccine development. In this study, we isolated and characterized several novel antibodies directed at the RSV G protein, which can add to our understanding and treatment of RSV disease.

  • Nucleic and amino acid sequences support structure-based viral classification [PublishAheadOfPrint]

  • Viral capsids ensure viral genome integrity by protecting the enclosed nucleic acids. Interactions between the genome, capsid and between individual capsid proteins (i.e. "capsid architecture") are intimate and expected to be characterized by strong evolutionary conservation. For this reason, a capsid structure-based viral classification has been proposed as a way to bring order to the viral universe. The seeming lack of sufficient sequence similarity to reproduce this classification has made it difficult to reject structural convergence as the basis for the classification. We reinvestigate whether the structure-based classification, for viral coat proteins making icosahedral virus capsids, is in fact supported by previously undetected sequence similarity. Since codon choices can influence nascent protein folding cotranslationally, we have searched for both amino acid and nucleotide sequence similarity. To demonstrate the sensitivity of the approach, we identify a candidate gene for the Pandoravirus capsid protein. We show that the structure-based classification is strongly supported by amino acid and also nucleotide sequence similarity, suggesting that similarities are due to common descent. The correspondence between structure-based and sequence-based analyses of the same proteins shown here allow them to be used in future analyses of the relationship between linear sequence information and macromolecular function, as well as between linear sequence and protein folds.

    IMPORTANCE Viral capsids protect nucleic acid genomes which in turn encode capsid proteins. This tight coupling of protein shell and nucleic acids, together with strong functional constraints on capsid protein folding and architecture, leads to the hypothesis that capsid protein coding nucleotide sequences may retain signatures of ancient viral evolution. We have been able to show that this is indeed the case, using the major capsid proteins of viruses forming icosahedral capsids. Importantly, we detect similarity at nucleotide level between capsid protein coding regions from viruses infecting cells belonging to all three domains of life, reproducing a previously established structure-based classification of icosahedral viral capsids.

  • Structure-Based Design of a Soluble Prefusion-Closed HIV-1-Env Trimer with Reduced CD4 Affinity and Improved Immunogenicity [PublishAheadOfPrint]

  • The HIV-1-envelope (Env) trimer is a target for vaccine design as well as a conformational machine that facilitates virus entry by transitioning between prefusion-closed, CD4-bound, and co-receptor-bound conformations before rearranging into a postfusion state. Vaccine designers have sought to restrict the conformation of the HIV-1-Env trimer to its prefusion-closed state, as this state is recognized by most broadly neutralizing nndash;but not by non-neutralizingnndash; antibodies. We previously identified a disulfide bond, I201C-A433C (DS), which stabilizes Env in the vaccine-desired prefusion-closed state. When placed into the context of BG505 SOSIP.664, a soluble Env-trimer mimic developed by Sanders, Moore and colleagues, the engineered DS-SOSIP trimer showed reduced conformational triggering by CD4. Here, we further stabilize DS-SOSIP through a combination of structure-based design and 96-well-based expression and antigenic assessment. From 103 designs, we identified one, named DS-SOSIP.4mut, with four additional mutations at the interface of potentially mobile domains of the prefusion-closed structure. We also determined the crystal structures of DS-SOSIP.4mut at 4.1-AAring; resolution nndash; and of an additional DS-SOSIP.6mut variant at 4.3-AAring; resolution nndash; and these confirmed the formation of engineered disulfide bonds. Notably, DS-SOSIP.4mut elicited a higher ratio of tier-2 autologous titers versus tier-1 V3-sensitive titers as compared to BG505 SOSIP.664. DS-SOSIP.4mut also showed reduced recognition of CD4 and increased thermostability. The improved antigenicity, thermostability, and immunogenicity of DS-SOSIP.4mut suggests utility as an immunogen or a serologic probe; moreover, the specific 4mut alterations identified here, M154, M300, M302 and L320, can also be transferred to other HIV-1 Env trimers of interest to improve their properties.

    IMPORTANCE One approach to elicit broadly neutralizing antibodies against HIV-1 is to stabilize the structurally flexible HIV-1-envelope (Env) trimer in a conformation that displays predominantly broadly neutralizing epitopes, and few to no non-neutralizing epitopes. The prefusion-closed conformation of HIV-1 Env has been identified as one such preferred conformation, and a current leading vaccine candidate is the "BG505 DS-SOSIP" variant, comprising two disulfides and an Ile to Pro mutation of strain BG505. Here, we introduced additional mutations to further stabilize BG505 DS-SOSIP in the vaccine-preferred prefusion-closed conformation. In guinea pigs, our best mutant, DS-SOSIP.4mut, elicited a significantly higher ratio of autologous versus V3-directed neutralizing antibody responses than the SOSIP-stabilized form. We also observed an improvement in thermostability and a reduction in CD4 affinity. With improved antigenicity, stability, and immunogenicity, DS-SOSIP.4mut-stabilized trimers may have utility as HIV-1 immunogens, or in other antigen-specific contexts, such as with B-cell probes.

  • Glycolysis, Glutaminolysis and Fatty Acid Synthesis are Required for Distinct Stages of KSHV Lytic Replication [PublishAheadOfPrint]

  • Kaposi's Sarcoma-associated Herpesvirus (KSHV) is the etiologic agent of Kaposi's Sarcoma (KS). KSHV infection induces and requires multiple metabolic pathways, including glycolysis, glutaminolysis and fatty acid synthesis (FAS) for the survival of latently infected endothelial cells. To determine the metabolic requirements for productive KSHV infection, we induced lytic replication in the presence of inhibitors of different metabolic pathways. We found that glycolysis, glutaminolysis and FAS are all required for maximal KSHV virus production and that these pathways appear to participate in virus production at different stages of the viral life cycle. Glycolysis and glutaminolysis, but not FAS, inhibit viral genome replication and interestingly, are required for different early steps of lytic gene expression. Glycolysis is necessary for early gene transcription while glutaminolysis is necessary for early gene translation, but not transcription. Inhibition of FAS resulted in decreased production of extracellular virions, but did not reduce intracellular genome levels or block intracellular virion production. However, in the presence of FAS inhibitors, the intracellular virions are non-infectious indicating that FAS is required for virion assembly or maturation. KS tumors support both latent and lytic KSHV replication. Previous work has shown that multiple cellular metabolic pathways are required for latency, and we now show that these metabolic pathways are required for efficient lytic replication providing novel therapeutic avenues for KS tumors.

    IMPORTANCE KSHV is the etiologic agent of Kaposi's Sarcoma, the most common tumor of AIDS patients. KS spindle cells, the main tumor cells, all contain KSHV, mostly in the latent state where there is limited viral gene expression. However, a percentage of spindle cells support lytic replication and production of virus and these cells are thought to contribute to overall tumor formation. Our previous findings showed that latently infected cells are sensitive to inhibitors of cellular metabolic pathways including glycolysis, glutaminolysis and fatty acid synthesis. Here we find that these same inhibitors block the production of infectious virus from lytically infected cells, each at a different stage of viral replication. Therefore, inhibition of specific cellular metabolic pathways can both eliminate latently infected cells, as well as block lytic replication thereby inhibiting infection of new cells. Inhibition of metabolic pathways provides novel therapeutic approaches for KS tumors.

  • Modulation of HCV-specific CD8 effector T-cell function with antiviral effect in infectious hepatitis C virus co-culture model [PublishAheadOfPrint]

  • Antiviral effects of hepatitis C virus (HCV)-specific CD8 T-cells have been shown in HCV replicon, but not in authentic infectious cell culture system (HCVcc). Here, we developed tools to examine the antigenicity of HCV-infected HLA A2+ Huh7.5 hepatoma cells (Huh7.5A2) in activating HCV-specific CD8 T-cells with downstream antiviral effects. Infectious HCV epitope mutants encoding well-defined genotype 1a-derived HLA A2-restricted HCV NS3 1073 or NS5 2594 epitope were generated from genotype 2a-derived HCV clone (Jc1Gluc2A) by site-directed mutagenesis. CD8 T-cell lines specific for NS3 1073 and NS5 2594 were expanded from HCV-seropositive persons by peptide stimulation in-vitro or engineered from HCV-seronegative donor T-cells by lentiviral transduction of HCV-specific T-cell receptors. HCV-specific CD8 T-cells were co-cultured with Huh7.5 cells that were pulsed with titrating doses of HCV epitope peptides or infected with HCV epitope mutants. HCV-specific CD8 T-cell activation (CD107, IFN, MIP-1bbeta;, TNFaalpha;) was dose-dependent on peptide concentrations and relative percentages of HCV-infected Huh7.5A2 cells. HCV-infected Huh7.5A2 cells activated HCV-specific CD8 T-cells at levels comparable to 0.1-2mmu;M pulsed peptides, providing a novel estimate at which endogenously processed HCV epitopes are presented in HCV-infected cells. While HCV-specific CD8 T-cell activation with cytolytic and antiviral effects was blunted by PD-L1 expression on HCV-infected Huh7.5A2 cells with improved viability of Huh7.5A2 cells, PD-1 blockade reversed this effect with enhanced cytolytic elimination of HCV-infected Huh7.5A2 cells. Our findings using infectious HCVcc system show that HCV-specific CD8 T-cell function is modulated by antigen expression levels, percentage of HCV-infected cells and PD-1/PD-L1 pathways, with antiviral and cytotoxic effects.

    IMPORTANCE We developed several novel molecular and immunological tools to study interactions between HCV, HCV-infected hepatocytes and HCV-specific CD8 T-cells. Using these tools, we show the level at which HCV-infected hepatoma cells present endogenously processed HCV epitopes to HCV-specific CD8 T-cells with antiviral and cytotoxic effects. We also show marked protective effect of PD-L1 expression in HCV-infected hepatoma cells against HCV-specific CD8 T-cells.

  • CpG in combination with an inhibitor of Notch signaling suppresses FI-RSV-enhanced airway hyperresponsiveness and inflammation through inhibiting Th17 memory responses and promoting tissue resident memory cells in lungs [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) is the leading cause of childhood hospitalizations. The formalin-inactivated RSV (FI-RSV) vaccine enhanced respiratory disease (ERD) has been an obstacle to the development of a safe and effective killed RSV vaccine. Agonists of Toll-like receptor (TLR) have been shown to regulate immune responses induced by FI-RSV. Notch signaling plays critical roles during the differentiation and effector function phases of innate and adaptive immune responses. Cross-talk between TLR and Notch signaling pathways results in fine tuning of TLR-triggered innate inflammatory responses. We evaluated the impact of TLR and Notch signaling on ERD in a murine model by administering CpG, an agonist of TLR9, in combination with L685,458, an inhibitor of Notch signaling during FI-RSV immunization. Activation with CpG or deficiency of MyD88-dependent TLR signaling did not alleviate airway inflammation in FI-RSV-immunized mice. Activation or inhibition of Notch signaling with Dll4 or L685,458 did not suppress FI-RSV enhanced airway inflammation either. However, the CpG together with L685,458 markedly inhibited FI-RSV-enhanced airway hyperresponsiveness, weight loss, and lung inflammation. Interestingly, CpG+L685,458 completely inhibited FI-RSV associated Th17, and Th17-associated proinflammatory chemokine responses in lungs following RSV challenge, but not Th1 or Th2, memory responses. In addition, FI-RSV+CpG+L685,458 promoted protective CD8+ lung tissue-resident memory cells (TRM). These results indicate that activation of TLR signaling combined with inhibition of Notch signaling prevent FI-RSV ERD, and the mechanism appears to involve suppressing proinflammatory Th17 memory responses and promoting protective TRM in lungs.

    IMPORTANCE RSV is the most important cause of lower respiratory tract infections in infants. The FI-RSV enhanced respiratory disease (ERD) is a major impediment to the development of a safe and effective killed RSV vaccine. Using adjuvants to regulate innate and adaptive immune responses could be an effective method to prevent ERD. We evaluated the impact of TLR and Notch signaling on ERD by administering CpG, an agonist of TLR9, in combination with L685,458, an inhibitor of Notch signaling, during FI-RSV immunization. The data showed that treatment of TLR or Notch signaling alone did not suppress FI-RSV enhanced airway inflammation, while CpG+L685,458 markedly inhibited ERD. The mechanism appears to involve suppressing Th17 memory responses and promoting tissue resident memory cells. Moreover, these results suggest that regulation of lung immune memory with adjuvant compounds containing more than one immune-stimulatory molecule may be a good strategy to prevent FI-RSV ERD.

  • Mutating the CX3C motif in the G protein should make a live respiratory syncytial virus vaccine safer and more effective [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae and is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. Through a CX3C chemokine motif (182CWAIC186) in the G protein, RSV binds to the corresponding chemokine receptor, CX3CR1. Since RSV binding to CX3CR1 contributes to disease pathogenesis, we investigated whether a mutation in the CX3C motif by insertion of an alanine A186 within the CX3C motif to CX4C (182CWAIAC187), known to block binding to CX3CR1, might decrease disease. We studied the effect of the CX4C mutation in two strains of RSV (A2 and r19F) in a mouse challenge model. We included the RSV r19F because it induces mucous production and airway resistance, two manifestations of RSV infection in humans, in mice. Compared to wildtype virus (wt), mice infected with the CX4C had a 0.7 to 1.2 log10-fold lower virus titer in the lung at 5 days p.i. and had markedly reduced weight loss, pulmonary inflammatory cell infiltration, mucous production, and airway resistance after challenge. This decrease in disease was not dependent on decrease in virus replication but did correspond to a decrease in pulmonary Th2 and inflammatory cytokines. Mice infected with CX4C viruses also had higher antibody titers and a Th1 biased T cell memory response at 75 days pi. These results suggest that the CX4C mutation in the G protein could improve the safety and efficacy of a live attenuated RSV vaccine.

    Importance RSV binds to the corresponding chemokine receptor, CX3CR1, through a CX3C chemokine motif (182CWAIC186) in the G protein. RSV binding to CX3CR1 contributes to disease pathogenesis, therefore, we investigated whether a mutation in the CX3C motif by insertion of an alanine A186 within the CX3C motif to CX4C (182CWAIAC187), known to block binding to CX3CR1, might decrease disease. The effect of this mutation and treatment with the F(ab')2 form of the anti-RSV G 131-2G mAb show that mutating the CX3C motif to CX4C blocks much of the disease and immune modulation associated with the G protein and should improve the safety and efficacy of a live attenuated RSV vaccine.

  • RNA-associated Early-stage Anti-viral Factor (REAF) is a major component of LV2 restriction [PublishAheadOfPrint]

  • HIV and SIV replication in human cells is restricted at early post-entry steps by host inhibitory factors. We previously described and characterised an early phase restriction of HIV-1 and 2 replication in human cell lines, primary macrophages and PBMCs. The restriction was termed Lentiviral restriction 2 (Lv2). The viral determinants of Lv2 susceptibility mapped to the HIV-2 Env and CA. We subsequently reported a whole genome siRNA screen for factors involved in to HIV which identified RNA-associated Early-stage Anti-viral Factor (REAF). Using HIV-2 chimeras of susceptible and non-susceptible viruses we show here that REAF is a major component of the previously described Lv2. Further studies of the viral CA demonstrate that the CA mutation I73V (previously called I207V), a potent determinant for HIV-2, is a weak determinant of susceptibility for HIV-1. More potent CA determinants for HIV-1 REAF restriction were identified at P38A, N74D, G89V and G94D. These results firmly establish that in HIV-1 CA is a strong determinant of susceptibility to LV2/REAF. Similar to HIV-2 the HIV-1 Env can rescue sensitive CAs from restriction. We conclude that REAF is a major component of the previously described Lv2 restriction.

    Importance Measures taken by the host cell to combat infection drive the evolution of pathogens to counteract or side step them. The study of such virus-host conflicts can point to possible weaknesses in the arsenal of viruses and may lead to the rational design of anti-viral agents. Here we describe our discovery that the host restriction factor REAF fulfils the same criteria previously used to describe Lentiviral restriction (Lv2). We show that, like HIV-1 CA, the CA of HIV-1 is a strong determinant of LV2/REAF susceptibility. We illustrate how HIV counteracts LV2/REAF by using an envelope with alternative routes of entry into cells.

  • The Product of the Herpes Simplex Virus Type 2 UL16 Gene is Critical for the Egress of Capsids from the Nuclei of Infected Cells [PublishAheadOfPrint]

  • The herpes simplex virus (HSV) UL16 gene is conserved throughout the Herpesviridae and encodes a poorly understood tegument protein. The HSV-1 UL16 protein forms complexes with several viral proteins including UL11, gE, VP22 and UL21. We previously demonstrated that HSV-2 UL21 was essential for virus propagation due to the failure of DNA-containing capsids to exit the nucleus. We hypothesized that if a UL16/UL21 complex were required for nuclear egress then HSV-2 lacking UL16 would have a similar phenotype as HSV-2 lacking UL21. Deletion of HSV-2 UL16 (16) resulted in a 950-fold reduction in virus propagation in mouse L cell fibroblasts and a 200-fold reduction in virus propagation in Vero cells that was fully reversed upon repair of 16 (16R), and partially reversed by infecting UL16 expressing cells with 16. The kinetics of viral gene expression in cells infected with 16 was indistinguishable from cells infected with 16R or parental virus. Additionally, similar numbers of capsids were isolated from the nuclei of cells infected with 16 and parental virus. However, transmission electron microscopy, fluorescence in situ hybridization experiments and fluorescent capsid localization assays all indicated a reduction in the ability of 16 C-capsids to exit the nucleus of infected cells. Taken together, these data indicate that, like UL21, UL16 is critical for HSV-2 propagation and suggest that UL16 and UL21 proteins may function together to facilitate the nuclear egress of capsids.

    IMPORTANCE HSV-2 is a highly prevalent sexually transmitted human pathogen that is the main cause of genital herpes infections and is fueling the epidemic transmission of HIV in sub-Saharan Africa. Despite important differences in the pathological features of HSV-1 and HSV-2 infections, HSV-2 is understudied compared to HSV-1. Here we demonstrate that deletion of the HSV-2 UL16 gene results in a substantial inhibition of virus replication due to a reduction in the ability of DNA-containing capsids to exit the nucleus of infected cells. The phenotype of this UL16 mutant resembles that of an HSV-2 UL21 mutant described previously by our laboratory. Because UL16 and UL21 interact these findings suggest that a complex containing both proteins may function together in nuclear egress.

  • Quantitative evaluation of Protein Heterogeneity within Herpes simplex type I Viral Particles [PublishAheadOfPrint]

  • Several virulence genes have thus far been identified in the herpes simplex virus type 1 genome. It is also generally accepted that protein heterogeneity among virions further impacts viral fitness. However, linking this variability directly with infectivity has been challenging at the individual viral particle level. To address this issue, we resorted to flow cytometry (flow virometry), a powerful approach we recently employed to analyze individual viral particles, to identify which tegument proteins vary and directly address if such variability is biologically relevant. We found that the stoichiometry of the UL37, ICP0 and VP11/12 tegument proteins in virions is more stable than the VP16 and VP22 tegument proteins, which varied significantly among viral particles. Most interestingly, viruses sorted for their high VP16 or VP22 content yielded modest but reproducible increases in infectivity when compared to their corresponding low containing VP16 or VP22 counterparts. These findings were corroborated for VP16 in siRNA experiments but proved intriguingly more complex for VP22. An analysis by quantitative Western blotting revealed substantial alterations of virion composition upon manipulation of individual tegument proteins and suggests that VP22 protein levels acted indirectly on viral fitness. These findings reaffirm the interdependence of the virion components and corroborate that viral fitness is not only influenced by the genome of viruses but also by the stoichiometry of proteins within each virion.

    IMPORTANCE The ability of viruses to spread in animals has been mapped to several viral genes but other factors are clearly involved, including virion heterogeneity. To directly probe whether the latter influences viral fitness, we analyzed the protein content of individual herpes simplex virus type 1 particles using an innovative flow cytometry approach. The data confirm that some viral proteins are incorporated in more controlled amounts, while others vary substantially. Interestingly, this correlates with the VP16 trans-activating viral protein and indirectly to VP22, a second virion component whose modulation profoundly alters virion composition. This reaffirms that not only the presence but also the amount of specific tegument proteins is an important determinant of viral fitness.

  • Tight junction protein occludin is a porcine epidemic diarrhea virus entry factor [PublishAheadOfPrint]

  • Porcine epidemic diarrhea virus (PEDV), the causative agent of porcine epidemic diarrhea, has caused huge economic losses in pig-producing countries. Although it was long believed to replicate in the intestinal epithelium using aminopeptidase N as a receptor, the mechanisms of PEDV infection are not fully characterized. In this study, we found that PEDV infection of epithelial cells resulted in the disruption of the tight junctional distribution of occludin to the intracellular localization. Overexpression of occludin in target cells makes them more susceptible for PEDV infection, whereas ablation of occludin expression using siRNA in target cells significantly reduces their susceptibility to virus infection. However, the results observed with occludin siRNA indicate that occludin is not required for virus attachment. We conclude that occludin plays an essential role in PEDV infection at the post-binding stages. Furthermore, we observed that macropinocytosis inhibitors blocked occludin internalization and virus entry, indicating that virus entry and occludin internalization appear to be closely coupled. But the macropinocytosis inhibitors cannot impede virus replication once the virus has entered into host cells. These suggest that occludin internalization by macropinocytosis or a macropinocytosis-like process is involved in the virus entry events. Immunofluorescence confocal microscopy showed that PEDV was trapped at cellular junctional regions upon macropinocytosis inhibitor treatment, indicating that occludin may serve as a scaffold in the vicinity of virus entry. Collectively, these data show that occludin plays an essential role in PEDV infection at late entry events. Our observation may provide novel insights into PEDV infection and related pathogenesis.

    IMPORTANCE Tight junctions are highly specialized membrane domains whose main function is to attach adjacent cells together thereby forming intercellular seals. Here, we investigate for the first time the role of tight junction protein occludin in PEDV infection. We observed that PEDV infection induced the internalization of occludin. By using genetic modification methods, we demonstrate that occludin plays an essential role in PEDV infection. Moreover, PEDV entry and occludin internalization seem to be closely coupled. Our findings reveal a new mechanism of PEDV infection.

  • Identification of SERINC5-001 as the Predominant Spliced Isoform for HIV-1 Restriction [PublishAheadOfPrint]

  • Among the five serine incorporator (SERINC) family members, SERINC5 (Ser5) was reported to strongly inhibit HIV-1 replication, which is counteracted by Nef. Ser5 produces 5 alternatively spliced isoforms: Ser5-001 has 10 putative transmembrane domains, whereas Ser5-004, -005, -008a, and -008b do not have the last one. Here, we confirmed the strong Ser5 anti-HIV-1 activity and investigated its isoforms' expression and antiviral activity. It was found that Ser5-001 transcripts were detected at least 10-fold more than the other isoforms by real-time quantitative PCR. When Ser5-001 and its two isoforms Ser5-005, and Ser5-008a were expressed from the same mammalian expression vector, only Ser5-001 was stably expressed, whereas the others were poorly expressed due to rapid degradation. In addition, unlike the other isoforms that are located mainly in the cytoplasm, Ser5-001 is localized primarily to the plasma membrane. To map the critical determinant, Ser5 mutants bearing C-terminal deletions were created. It was found that the 10th transmembrane domain is required for Ser5 stable expression and plasma membrane localization. As expected, only Ser5-001 strongly inhibits HIV-1 infectivity, whereas the other Ser5 isoforms and mutants that do not have the 10th transmembrane show a very poor activity. It was also observed that the Nef counteractive activity could be easily saturated by Ser5 overexpression. Thus, we conclude that Ser5-001 is the predominant antiviral isoform that restricts HIV-1, and the 10th transmembrane domain plays a critical role in this process by regulating its protein stability and plasma membrane targeting.

    IMPORTANCE HIV and SIV express a small protein Nef to enhance viral pathogenesis in vivo. Nef has an important in vitro function, which is to make virus particles more infectious, but the mechanism has been unclear. Recently, Nef was reported to counteract a novel anti-HIV host protein SERINC5 (Ser5). Ser5 has five alternatively spliced isoforms including Ser5-001, 004, 005, 008a, and 008b, and only Ser5-001 has an extra C-terminal transmembrane domain. We now show that the Ser5-001 transcripts are produced at least 10-fold more than the others, and only Ser5-001 produces stable proteins that are targeted to plasma membrane. Importantly, only Ser5-001 shows a strong anti-HIV-1 activity. We further demonstrate that the extra transmembrane domain is required for Ser5 stable expression and plasma membrane localization. These results suggest that plasma membrane localization is required for Ser5 antiviral activity, and Ser5-001 is the predominant isoform that contributes to the activity.

  • Oral prion disease pathogenesis is impeded in the specific absence of CXCR5-expressing dendritic cells [PublishAheadOfPrint]

  • After oral exposure the early replication of certain prion strains upon stromal-derived follicular dendritic cells (FDC) in the Peyer's patches in the small intestine is essential for the efficient spread of disease to the brain. However, little is known of how prions are initially conveyed from the gut lumen to establish infection on FDC. Our previous data suggest that mononuclear phagocytes such as CD11c+ conventional dendritic cells play an important role in the initial propagation of prions from the gut lumen into Peyer's patches. But whether these cells conveyed orally-acquired prions towards FDC within Peyer's patches was not known. The chemokine CXCL13 is expressed by FDC and follicular stromal cells and modulates the homing of CXCR5-expressing cells towards the FDC-containing B cell follicles. Here, novel compound transgenic mice were created in which CXCR5-deficiency was specifically restricted to CD11c+ cells. These mice were used to determine whether CXCR5-expressing conventional dendritic cells propagate prions towards FDC after oral exposure. Our data show that in the specific absence of CXCR5-expressing conventional dendritic cells the early accumulation of prions upon FDC in Peyer's patches and the spleen was impaired, and disease susceptibility significantly reduced. These data suggest that CXCR5-expressing conventional dendritic cells play an important role in the efficient propagation of orally-administered prions towards FDC within Peyer's patches in order to establish host infection.

    IMPORTANCE Many natural prion diseases are acquired by oral consumption of contaminated food or pasture. Once the prions reach the brain they cause extensive neurodegeneration which ultimately leads to death. In order for the prions to efficiently spread from the gut to the brain, they first replicate upon follicular dendritic cells within intestinal Peyer's patches. How the prions are first delivered to follicular dendritic cells to establish infection was unknown. Understanding this process is important since treatments which prevent prions from infecting follicular dendritic cells can block their spread to the brain. We created mice in which mobile conventional dendritic cells were unable to migrate towards follicular dendritic cells. In these mice the early accumulation of prions on follicular dendritic cells was impaired and oral prion disease susceptibility reduced. This suggests that prions exploit conventional dendritic cells to facilitate their initial delivery towards follicular dendritic cells to establish host infection.

  • The deacetylase SIRT1 regulates the replication properties of human papillomavirus 16 E1 and E2 [PublishAheadOfPrint]

  • Human papillomaviruses (HPV) replicate their genomes in differentiating epithelium using the viral proteins E1 and E2 in association with host proteins. While the roles of E1 and E2 in this process are understood, the host factors involved and how they interact with and regulate E1-E2 are not. Our previous work identified the host replication and repair factor TopBP1 as an E2 partner protein essential for optimal E1-E2 replication and for the viral life cycle. The role of TopBP1 in host DNA replication is regulated by the class III deacetylase SIRT1; activation of the DNA damage response prevents SIRT1 deacetylation of TopBP1 resulting in a switch from DNA replication to repair functions for this protein and cell cycle arrest. Others have demonstrated an essential role for SIRT1 in regulation of the HPV31 life cycle; here we report that SIRT1 can directly regulate HPV16 E1-E2 mediated DNA replication. SIRT1 is part of the E1-E2 DNA replication complex and is recruited to the viral origin of replication in an E1-E2 dependent manner. CRISPR/Cas9 was used to generate C33a clones with undetectable SIRT1 expression and lack of SIRT1 elevated E1-E2 DNA replication, in part due to an increased acetylation and stabilization of the E2 protein in the absence of SIRT1. The results demonstrate that SIRT1 is a member of, and can regulate, the HPV16 replication complex. We discuss the potential role of this protein in the viral life cycle.

    Importance Human papillomaviruses (HPV) are causative agents in a number of human diseases and currently only the symptoms of these diseases are treated. To identify novel therapeutic approaches for combating these diseases the viral life cycle must be understood in more detail. This report demonstrates that a cellular enzyme, SIRT1, is part of the HPV16 DNA replication complex and is brought to the viral genome by the viral proteins E1 and E2. Using gene editing technology (CRISPR/Cas9) the SIRT1 gene was removed from cervical cancer cells and the consequence of this was that viral replication was elevated, probably due to a stabilization of the viral replication factor E2. The overall results demonstrate that an enzyme with known inhibitors, SIRT1, plays an important role in controlling how HPV16 makes copies of itself. Targeting this enzyme could be a new therapeutic approach for combating HPV spread and disease.

  • A strategy for cultivation of retargeted oncolytic HSVs in non-cancer cells [PublishAheadOfPrint]

  • The oncolytic herpes simplex virus (HSV) approved for clinical practice, and those in clinical trials are attenuated viruses, often deleted in the neurovirulence gene 134.5, and in additional genes. A strategy to engineer non-attenuated oncolytic HSVs consists in retargeting the virus tropism to a cancer-specific receptor of choice - exemplified by HER2 (human epidermal growth factor receptor 2) present in breast, ovary and other cancers - and in detargeting from the natural receptors. Because the HER2-retargeted HSVs strictly depend on this receptor for infection, the viruses employed in preclinical studies were cultivated in HER2-positive cancer cells. The production of clinical grade viruses destined to humans should avoid the use of cancer cells. Here, we engineered the R-213 recombinant, by insertion of a 20-aa short peptide (named GCN4) in the gH of R-LM113, a recombinant retargeted to HER2 through insertion in gD of a single chain antibody (scFv) to HER2. Next, we generated a Vero cell line expressing an artificial receptor (GCN4R), whose N-terminus consists of a scFv to GCN4, and therefore is capable to interact with GCN4 present in gH of R-213. R-213 replicated as well as R-LM113 in SK-OV-3 cells, implying that addition of the GCN4 peptide was not detrimental to gH. R-213 grew to relatively high titers in Vero-GCN4R, efficiently spread from cell to cell, and killed both Vero-GCN4R and SK-OV-3 cells, as expected of an oncolytic virus. Altogether, Vero-GCN4R cells represent an efficient system for cultivation of retargeted oncolytic HSV in non-cancer cells.

    IMPORTANCE There is a growing interest in viruses as oncolytic agents, which can be administered in combination with immunotherapeutic compounds, including immunocheckpoint inhibitors. The oncolytic HSV approved for clinical practice, and those in clinical trials are attenuated viruses. An alternative to attenuation is a higher cancer-specificity, as can be attained by tropism retargeting to selected cancer receptors. However, the retargeted oncolytic HSVs strictly depend on cancer receptors for infection. Here, we devised a strategy for in vitro cultivation of retargeted HSVs in non-cancer cells. The strategy envisions a double retargeting approach: one retargeting is via gD to the cancer receptor; the second retargeting is via gH to an artificial receptor expressed in Vero cells. The double-retargeted HSV uses alternatively the two receptors to infect cancer cells or producer cells. A universal non-cancer cell line for growth of clinical grade retargeted HSVs represents a step forward in the translational phase.

  • Attenuated Human Parainfluenza Virus type 1 expressing Ebola Virus Glycoprotein GP Administered Intranasally is Immunogenic in African Green Monkeys [PublishAheadOfPrint]

  • The recent 2014-2016 Ebola virus (EBOV) outbreak prompted increased efforts to develop vaccines against EBOV disease. We describe the development and preclinical evaluation of an attenuated recombinant human parainfluenza virus type 1 (rHPIV1) expressing the membrane-anchored form of EBOV glycoprotein GP, as an intranasal (IN) EBOV vaccine. GP was codon optimized and expressed either as a full-length protein or an engineered chimeric form in which its transmembrane and cytoplasmic tail (TMCT) domains were substituted with those of the HPIV1 F protein in an effort to enhance packaging into the vector particle and immunogenicity. GP was inserted either preceding the N gene (pre-N) or between the N and P genes (N-P) of rHPIV1 bearing a stabilized attenuating mutation in the P/C gene (C170). The constructs grew to high titers and efficiently and stably expressed GP. Viruses were attenuated, replicating at low titers over several days, in the respiratory tract of African green monkeys (AGMs). Two doses of candidates expressing GP from the pre-N position elicited higher GP neutralizing serum antibody titers than the N-P viruses, and unmodified GP induced higher levels than its TMCT counterpart. Unmodified EBOV GP was packaged into the HPIV1 particle, and the TMCT modification did not increase packaging or immunogenicity but rather reduced the stability of GP expression during in vivo replication. In conclusion, we identified an attenuated and immunogenic IN vaccine candidate expressing GP from the pre-N position. It is expected to be well-tolerated in humans and is available for clinical evaluation.

    IMPORTANCE. EBOV hemorrhagic fever is one of the most lethal viral infections and lacks a licensed vaccine. Contact of fluids from infected individuals, including droplets or aerosols, with mucosal surfaces is an important route of EBOV spread during a natural outbreak, and aerosols also might be exploited for intentional virus spread. Therefore, vaccines that protect against mucosal as well as systemic inoculation are needed. We evaluated a version of human parainfluenza virus type 1 (HPIV1) bearing a stabilized attenuating mutation in the P/C gene (C170) as an intranasal vaccine vector to express the EBOV glycoprotein GP. We evaluated expression from two different genome positions (pre-N and N-P), and investigated the use of vector packaging signals. African green monkeys immunized with two doses of the vector expressing GP from the pre-N position developed high titers of GP neutralizing serum antibodies. The attenuated vaccine candidate is expected to be safe and immunogenic and is available for clinical development.

  • A Neonatal Murine Model of Coxsackievirus A6 Infection for Evaluation of Antiviral and Vaccine Efficacy [PublishAheadOfPrint]

  • Hand, foot and mouth disease (HFMD) is a global health concern. Family Picornaviridae members, particularly enterovirus 71 (EVA71) and Coxsackievirus A16 (CVA16), are the primary etiological agents of HFMD, however, a third enterovirus A species, CoxsackievirusA6 (CVA6), has been recently associated with epidemic outbreaks. Study of the pathogenesis of CVA6 infection and development of antivirals and vaccines are hindered due to the lack of appropriate animal models. We have developed and characterized a murine model of CVA6 infection which was employed to evaluate the antiviral activities of different drugs and the protective efficacies of CVA6-inactivated vaccines. Neonatal mice were susceptible to CVA6 infection via intramuscular inoculation and the susceptibility of mice to CVA6 infection was age- and dose-dependent. Five day-old mice infected with 105.5 TCID50 of the CVA6 WF057R strain consistently exhibited clinical signs, including reduced mobility, lower weight gain and quadriplegia with significant pathology in the brain, hindlimb skeletal muscles and lungs of the infected mice in the moribund state. Immunohistochemistry and qRT-PCR analyses showed high viral loads (11 log10) in skeletal muscle and elevated levels of IL-6 (ggt;2000pg/ml) were associated with severe viral pneumonia and encephalitis. Ribavirin and IFN- administered prophylactically diminished CVA6-associated pathology in vivo and treatment with IL-6 accelerated the death of neonatal mice. Both specific anti-CVA6 serum and maternal antibody play an important role in controlling CVA6 infection and viral replication. Collectively, these findings indicate that this neonatal murine model will be invaluable in future studies to develop CVA6-specific antivirals and vaccines.

    IMPORTANCE Although Coxsackievirus A6 (CVA6) infections are commonly mild and self-limiting, a small proportion of children may have serious complications, such as encephalitis, acute flaccid paralysis, neuro-respiratory syndrome, leading to fatalities. We have established a mouse model of CVA6 infection by inoculation of neonatal mice with a CVA6 clinical isolate, which produced consistent pathological outcomes. Here, using this model of CVA6 infection we found that high levels of IL-6 was associated with severe viral pneumonia and encephalitis, as in evaluation of antiviral efficacy in vivo, IL-6 failed to confer any protective effect and instead accelerated mortality in neonatal mice. We demonstrated that, as antiviral drugs, both IFN- and ribavirin played important protective roles in the early stages of infection with increased survival in treated neonatal mice challenged with CVA6. Moreover, active and passive immunization with the inactivated vaccines and anti-CVA6 serum also conferred protection against homologous infections in mice.

  • The ND10 component promyelocytic leukemia protein acts as an E3 ligase for SUMOylation of the major immediate-early protein IE1 of human cytomegalovirus [PublishAheadOfPrint]

  • Previous studies identified the nuclear domain 10 (ND10) components PML, hDaxx, and Sp100 as factors of an intrinsic immune response against human cytomegalovirus (HCMV). This antiviral function of ND10, however, is antagonized by viral effector proteins like IE1p72, which induces a dispersal of ND10. Furthermore, we have shown that both major immediate-early proteins of HCMV, IE1p72 and IE2p86, transiently co-localize with ND10 subnuclear structures and undergo modification by the covalent attachment of SUMO. Since recent reports indicate that PML acts as a SUMO E3 ligase we asked whether the SUMOylation of IE1p72 and IE2p86 is regulated by PML. To address this, PML-depleted fibroblasts as well as cells overexpressing individual PML isoforms were infected with HCMV. Western blot experiments revealed a clear correlation between the degree of IE1p72 SUMO conjugation and the abundance of PML. On the contrary, the SUMOylation of IE2p86 was not affected by PML. By performing in vitro SUMOylation assays we were able to provide direct evidence that IE1p72 is a substrate for PML-mediated SUMOylation. Interestingly, disruption of the RING finger domain of PML, which is proposed to confer SUMO E3 ligase activity, abolished PML-induced SUMOylation of IE1p72. In contrast, IE1p72 was still efficiently SUMO-modified by a SUMOylation-defective PML mutant, indicating that intact ND10 bodies are not necessary for this effect. Thus, this is the first report that the E3 ligase PML is capable of stimulating the SUMOylation of a viral protein which is supposed to serve as a cellular mechanism to compromise specific functions of IE1p72.

    IMPORTANCE The major immediate-early proteins of human cytomegalovirus, termed IE1p72 and IE2p86, have previously been shown to undergo posttranslational modification by covalent coupling to SUMO moieties at specific lysine residues. However, the enzymatic activities that are responsible for this modification have not been identified, yet. Here, we demonstrate that the PML protein which mediates an intrinsic immune response against HCMV, specifically serves as an E3 ligase for SUMO modification of IE1p72. Since SUMO modification of IE1p72 has previously been shown to interfere with STAT factor binding, thus compromising the interferon-antagonistic function of this viral effector protein, our finding highlights an additional mechanism how PML is able to restrict viral infections.

  • Respiratory syncytial virus (RSV) vaccine vectored by a stable chimeric and replication-deficient Sendai virus protects mice without inducing enhanced disease. [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) is a major cause of severe respiratory infections in children and elderly people and no marketed vaccine exists. In this study, we generated and analyzed a subunit vaccine against RSV based on a novel genome replication-deficient Sendai virus (SeV) vector. We inserted the RSV F protein, known as a genetically stable antigen, into our vector in a specific way to optimize the vaccine features. Exchanging the ectodomain of the SeV F protein with its counterpart from RSV, we created a chimeric vectored vaccine that contains the RSV F protein as an essential structural component. In this way, the antigen is actively expressed, on the surface of vaccine particles, in its prefusion conformation and, as recently reported for other vectored vaccines, the occurrence of silencing mutations of the transgene in the vaccine genome can be prevented. In addition, its active gene expression contributes to a further stimulation of the immune response. In order to understand the best route of immunization, we compared vaccine efficacy after intranasal (i.n.) or intramuscular (i.m.) immunization of BALB/c mice. Via both routes, substantial RSV-specific immune responses were induced, consisting in serum IgG and neutralizing antibodies, as well as cytotoxic T-cells. Moreover, i.n. immunization was also able to stimulate specific mucosal IgA in the upper and lower respiratory tract. In virus challenge experiments, animals were protected against RSV infection after both i.n. and i.m. immunization, without inducing vaccine-enhanced disease. Above all, the administration of the replication-deficient SeV appeared to be safe and well tolerated.

    IMPORTANCE Respiratory syncytial virus (RSV) is a major cause of respiratory diseases in young children and elderly people worldwide. There is a great demand for a licensed vaccine. Promising existing vaccine approaches based on live-attenuated vaccines or viral vectors have suffered from unforeseen drawbacks related to immunogenicity and attenuation. We provide a novel RSV vaccine concept based on a genome-replication deficient Sendai vector that has many favorable vaccine characteristics. Specific vaccine design guarantees genetic stability of the transgene, furthermore, it supports a favorable presentation of the antigen, activating the adaptive response mmdash; features that other vectored vaccine approaches often had often difficulties with. Wide immunological and pathological analyses in mice confirmed the validity and efficacy of this approach after both parenteral and mucosal administration. Above all, this concept is suitable for initiating clinical studies and it could also be applied to other infectious diseases.

  • Viral Epitranscriptomics [PublishAheadOfPrint]

  • Although it has been known for over 40 years that eukaryotic mRNAs bear internal base modifications, it is only in the last five years that the importance of these modifications has begun to come into focus. The most common mRNA modification, the addition of a methyl group to the N6 position of adenosine (m6A), has been shown to affect splicing, translation and stability, and m6A is also essential for embryonic development in organisms ranging from plants to mice. While all viral transcripts examined so far have been found to be extensively m6A modified, the role, if any, of m6A in regulating viral gene expression and replication was previously unknown. However, recent data generated using HIV-1 as a model system strongly suggest that sites of m6A addition are not only evolutionarily conserved but also enhance virus replication. It is therefore likely that the field of viral epitranscriptomics, which can be defined as the study of functionally relevant post-transcriptional modifications of viral RNA transcripts that do not change the nucleotide sequence of that RNA, is poised for a major expansion in scientific interest and may well fundamentally change our understanding of how viral replication is regulated.

  • Measles virus enters breast and colon cancer cell lines through a PVRL4-mediated macropinocytosis pathway [PublishAheadOfPrint]

  • Measles virus (MeV) is a member of the family Paramixoviridae that causes a highly contagious respiratory disease, but has emerged as a promising oncolytic platform. Previous studies of MeV entry have focused on the identification of cellular receptors. However, the endocytic and trafficking pathways utilized during MeV entry remain poorly described. The contribution of each endocytic pathway has been examined in cells that express MeV receptors, SLAM (Signaling Lymphocyte-Activating Molecule) and PVRL4 (Poliovirus Receptor-Like-4, nectin-4). Recombinant MeV expressing either firefly luciferase or the green fluorescence protein together with a variety of inhibitors, were used in. Results showed that MeV uptake was dynamin-independent in the Vero.hPVRL4, Vero.hSLAM, and PVRL4-positive MCF7 breast cancer cell lines. However, MeV infection was blocked by EIPA, the hallmark inhibitor of macropinocytosis, as well as inhibitors of actin polymerization. Using phalloidin staining, MeV entry was shown to induce actin rearrangements and formation of membrane ruffles accompanied by a transient elevated fluid uptake. The siRNA knockdown of PAK1 demonstrated that MeV enters both Vero.hPVRL4 and Vero.hSLAM cells in a PAK1-independent manner using a macropinocytosis-like pathway. In contrast, MeV entry into MCF7 human breast cancer cells relied upon Rac1 and its effector PAK1, through a PVRL4-mediated macropinocytosis pathway. MeV entry into DLD-1 colon and HTB-20 breast cancer cells also appeared to use the same pathway. Overall, these findings provide new insight into the lifecycle of MeV, which could lead to therapies that block virus entry, or methods that improve the uptake of MeV by cancer cells during oncolytic therapy.

    IMPORTANCE In the past decades, Measles virus (MeV) has emerged as a promising oncolytic platform. Previous studies concerning MeV entry have mainly focused on the identification of putative receptors for MeV. Nectin-4 (PVRL4) was recently identified as the epithelial cell receptor for MeV. However, the specific endocytic and trafficking pathways utilized during MeV infections are poorly documented. In this study, we demonstrated that MeV enters host cells via a dynamin-independent and actin-dependent endocytic pathway. Moreover, we show that MeV gains entry into the MCF7, DLD-1, HTB-20 cancer cells through a PVRL4-mediated macropinocytosis pathway, and identified the typical cellular GTPase and kinase involved. Our findings provide new insight into the lifecycle of MeV which may lead to the development of therapies that block entry of the virus into the host cell, or alternatively promote the uptake of oncolytic MeV into cancer cells.

  • Cytoplasmic Motifs in the Nipah Virus Fusion Protein Modulate Virus Particle Assembly and Egress [PublishAheadOfPrint]

  • Nipah virus (NiV), a paramyxovirus in the genus Henipavirus, has a mortality rate in humans of approximately 75%. While several studies have begun our understanding of NiV particle formation, the mechanism of this process remains to be fully elucidated. For many paramyxoviruses, M proteins drive viral assembly and egress; however, some paramyxoviral glycoproteins have been reported as important or essential in budding. For NiV the matrix protein (M), the fusion glycoprotein (F), and to a much lesser extent the attachment glycoprotein (G), autonomously induce the formation of virus-like particles (VLPs). However, functional interactions between these proteins during assembly and egress remain to be fully understood. Moreover, if the F-driven formation of VLPs occurs through interactions with host cell machinery, the cytoplasmic tail (CT) of F is a likely interactive domain. Therefore, we analyzed NiV F CT deletion and alanine mutants and report that several but not all regions of the F CT are necessary for efficient VLP formation. Two of these regions contain YXXOOslash; or di-tyrosine motifs previously shown to interact with cellular machinery involved in F endocytosis and transport. Importantly, our results showed that F-driven, M-driven, and M/F-driven viral particle formation enhanced the recruitment of G into VLPs. By identifying key motifs, specific residues, and functional viral protein interactions important for VLP formation, we improve our understanding of the viral assembly/egress process and point to potential interactions with host cell machinery.

    IMPORTANCE Henipaviruses can cause deadly infections of medical, veterinary, and agricultural importance. With recent discoveries of new henipa-like viruses, understanding the mechanisms by which these viruses reproduce is paramount. We have focused this study on identifying the functional interactions of three Nipah virus proteins during viral assembly, and particularly on the role of one of these proteins, the fusion glycoprotein, in incorporation of other viral proteins into viral particles. By identifying several regions in the fusion glycoprotein that drive viral assembly, we further our understanding of how these viruses assemble and egress from infected cells. The results presented will likely be useful towards designing treatments targeting this aspect of the viral life cycle and for the production of new viral particle-based vaccines.

  • Immunization with DNA Plasmids Coding for Crimean-Congo Hemorrhagic Fever Virus Capsid and Envelope Proteins and/or Virus-Like Particles Induces Protection and Survival in Challenged Mice [PublishAheadOfPrint]

  • Crimean Congo hemorrhagic fever virus (CCHFV) is a bunyavirus causing severe hemorrhagic fever disease in humans, with high mortality rates. The requirement of high-containment laboratory and the lack of animal model hampered the study of the immune response and protection of vaccine candidates.

    Using the recently developed interferon-alpha-receptor knock-out (IFNAR -/-) mouse model, which replicates human disease, we investigated the immunogenicity and protection of two novel CCHFV vaccine candidates: a DNA vaccine encoding an ubiquitin-linked version of CCHFV Gc, Gn, and N, and transcriptionally-competent virus-like particles (tc-VLPs). Contrary to most studies that focus on neutralizing antibodies, we measured both humoral and cellular immune responses. We demonstrated a clear and 100% efficient preventive immunity against lethal CCHFV challenge with the DNA vaccine. Interestingly, there was no correlation with the neutralizing antibody titers alone, which were higher in the tc-VLP vaccinated mice. However, the animals with a lower neutralizing titer, but a dominant cell-mediated Th1 response and a balanced Th2 response, resisted the CCHFV challenge. Moreover, we found that challenged mice with a Th1 response (immunized by DNA/DNA and boosted by tc-VLPs) changed the immune response to Th2 at day 9 post challenge. In addition, we were able to identify new linear B-cell epitopes regions that are highly conserved between CCHFV strains. Altogether, our results suggest that a predominantly Th1-type immune response, provides the most efficient protective immunity against CCHFV challenge. However, we can not exclude the importance of the neutralizing antibodies, as the surviving immunized mice exhibited substantial amounts of them.

    IMPORTANCE Crimean Congo Hemorrhagic fever virus (CCHFV) is responsible for hemorrhagic diseases in humans, with a high mortality rate. There is no FDA-approved vaccine, and there are still gaps in our knowledge of the immune responses to infection. The recently developed mouse models mimic the human CCHF disease, and are useful to study the immunogenicity and protection by vaccine candidates. Our study shows the full protection of mice vaccinated with a specific DNA vaccine. Importantly, we show that neutralising antibodies are not sufficient for protection against CCHFV challenge, but that an extra Th1-specific cellular response is required. Moreover, we describe the identification of five conserved B-cell epitopes, of which only one was previously known, that could be of great importance for the development of diagnostics tools and the improvement of vaccines candidates.

  • MiR-126-5p enhances the inflammatory responses of monocytes to LPS stimulation by suppressing CYLD in chronic HIV-1 infection [PublishAheadOfPrint]

  • Persistent immune activation during chronic human immunodeficiency virus-1 (HIV-1) infection facilitates immune dysfunction and thereby fuels disease progression. The translocation of bacterial derivatives into blood and the hyper-inflammatory responsiveness of monocytes have been considered important causative factors for persistent immune activation. Whether microRNAs (miRNAs) involve in regulating monocyte-mediated inflammatory responses during chronic HIV-1 infection remains elusive. In this study, we show that miR-126-5p functions as a positive regulator of monocyte-mediated inflammatory responses. Significantly increased miR-126-5p and decreased cylindromatosis (CYLD) were observed in primary monocytes from chronic HIV-1 patients. Inhibition of miR-126-5p in monocytes from chronic HIV-1 patients attenuated the responsiveness of these cells to lipopolysaccharide (LPS) stimulation. Gain-of-function assays confirmed that miR-126-5p could down-regulate CYLD, which in turn caused an up-regulation of phosphorylation of JNK protein (pJNK) and enhanced inflammatory responses of monocytes to LPS stimulation. Overall, miR-126-5p up-regulates the responsiveness of monocytes to LPS stimulation in chronic HIV-1 infection, and the suppression of miR-126-5p and the promotion of CYLD expression in primary monocytes may represent a practical immune intervention strategy to contain persistent inflammation in chronic HIV-1 infection.

    IMPORTANCE Monocyte-mediated hyper-inflammatory responses during chronic HIV-1 infection are important causative factors driving acquired immune deficiency syndrome (AIDS) progression; however, the underlying mechanism has not been fully addressed. We demonstrated that miR-126-5p, one of the most up-regulated miRNAs during chronic HIV-1 infection, could enhance the inflammatory responses of monocytes to LPS by suppressing the inhibitory protein CYLD and thereby unleashing the expression of pJNK in the LPS/toll-like receptor 4 (TLR4)/mitogen-activated protein kinase (MAPK) pathway. This observation reveals a new mechanism for HIV-1 pathogenesis, which could be targeted by immune intervention.

  • mosGCTL-7, a C-type lectin protein, mediates Japanese encephalitis virus infection in mosquitoes [PublishAheadOfPrint]

  • Japanese encephalitis virus (JEV) is an arthropod-borne flavivirus prevalent in Asia and the Western Pacific, and is the leading cause of viral encephalitis. JEV is maintained in a transmission cycle between mosquitoes and vertebrate hosts, but the molecular mechanisms by which the mosquito vector participates in transmission are unclear. We investigated the expression of all C-type lectins during JEV infection in Aedes aegypti. C-type lectin mosGCTL-7 (AAEL002524) was significantly up-regulated by JEV infection and facilitated infection in vivo and in vitro. mosGCTL-7 bound to the N-glycan at N154 on JEV envelope protein. This recognition of viral N-glycan by mosGCTL-7 is required for JEV infection, and we found that this interaction was Ca2+ dependent. After mosGCTL-7 bound to the glycan, mosPTP-1 bound to mosGCTL-7, promoting JEV entry. The viral burden in vivo and in vitro was significantly decreased by mosPTP-1 dsRNA treatment, and infection was abolished by anti-mosGCTL-7 antibodies. Our results indicate that the mosGCTL-7/mosPTP-1 pathway plays a key role in JEV infection in mosquitoes. An improved understanding of the mechanisms underlying flavivirus infection in mosquitoes will provide further opportunities for developing new strategies to control viral dissemination in nature.

    IMPORTANCE Japanese encephalitis virus is a mosquito-borne flavivirus, and is the primary cause of viral encephalitis in the Asianndash;Pacific region. Twenty-four countries in the WHO South-East Asia and Western Pacific Regions have endemic JEV transmission, which exposes ggt;3 billion people to the risks of infection, although JEV primarily affects children. C-type lectins are host factors that play a role in flavivirus infection in humans, swine, and other mammals. In this study, we investigated C-type lectins functions in JEV-infected Aedes aegypti, Culex pipiens pallens and cultured cells. JEV infection changed the expression of almost all C-type lectins in vivo and in vitro, and mosGCTL-7 bound to the JEV envelope protein via an N-glycan at N154. Cell surface mosPTP-1 interacted with the mosGCTL-7nndash;JEV complex to facilitate virus infection in vivo and in vitro. Our findings provide further opportunities for developing new strategies to control arbovirus dissemination in nature.

  • Pathology in Permissive Syrian Hamsters after Infection with Human Adenovirus Is the Result of Virus Replication; HAdV-C6 Replicates More and Causes More Pathology than HAdV-C5 [PublishAheadOfPrint]

  • Syrian hamsters are permissive for the replication of species C human adenoviruses. The virus replicates to high titers in the liver of these animals after intravenous infection, while respiratory infection results in virus replication in the lung. Here we show that two types belonging to species C, HAdV-C5 and HAdV-C6, replicate to a significantly different extent and cause pathology with significantly different severity, with HAdV-C6 replicating better and inducing more severe and more widespread lesions. The virus burden in the liver of HAdV-C6-infected hamsters is higher than in HAdV-C5-infected ones because more of the permissive hepatocytes get infected. Further, when hamsters are infected intravenously with HAdV-C6, live, infectious virus can be isolated from the lung and the kidney, which was not seen with HAdV-C5. Similarly to mouse models, in hamsters HAdV-C6 is sequestered by macrophages to a lesser degree than HAdV-C5. Depletion of Kupffer cells from the liver greatly increases the replication of HAdV-C5 in the liver, while it has only a modest effect on the replication of HAdV-C6. Elimination of Kupffer cells also dramatically increases the pathology induced by HAdV-C5. These findings indicate that in hamsters, pathology resulting from intravenous infection with adenoviruses is caused mostly by replication in hepatocytes and not by the abortive infection of Kupffer cells and the following cytokine storm.

    IMPORTANCE Immunocompromised human patients can develop severe, often lethal adenovirus infections. Respiratory adenovirus infection among military recruits is a serious problem, in some cases requiring hospitalization of the patient. Further, adenovirus-based vectors are frequently used as experimental viral therapeutic agents. Thus, it is imperative that we investigate the pathogenesis of adenoviruses in a permissive animal model. Syrian hamsters are susceptible to infection with certain human adenoviruses, and the pathology accompanying these infections is similar to what is observed with adenovirus-infected human patients. We demonstrate that replication in permissive cells in a susceptible host animal is a major part of the mechanism by which systemic adenovirus infection induces pathology, as opposed to a chiefly immune-mediated pathology observed in non-susceptible hosts. These findings support the use of compounds inhibiting adenovirus replication as a means to block adenovirus-induced pathology.

  • Influenza A virus virulence depends on two amino acids in the N-terminal domain of its NS1 protein facilitating inhibition of PKR [PublishAheadOfPrint]

  • The RNA-dependent protein kinase (PKR) has broad antiviral activity inducing translational shut-down of viral and cellular genes and is therefore targeted by various viral proteins to facilitate pathogen propagation. The pleiotropic NS1 protein of influenza A virus acts as silencer of PKR activation and ascertains high level viral replication and virulence. However, the exact way of this inhibition remains controversial. To elucidate the structural requirements within the NS1 protein for PKR inhibition, we generated a set of mutant viruses identifying highly conserved arginine residues 35 and 46 within the NS1 N-terminus as being most critical not only for binding to and blocking activation of PKR, but also for efficient virus propagation. Biochemical and FRET-based interaction studies showed that mutation of each of R35 or R46 allowed formation of NS1 dimers, but eliminated any detectable binding to PKR as well as to dsRNA. Using in vitro and in vivo approaches of phenotypic restoration we demonstrate the essential role of the NS1 N-terminus for blocking PKR. The strong attenuation conferred by NS1 mutations R35A or R46A was substantially alleviated by stable knock-down of PKR in human cells. Intriguingly, both NS1 mutant viruses did not trigger any signs of disease in PKR+/+ mice, but replicated to high titers in lungs of PKR-/- mice and caused lethal infections. These data not only establish the NS1 N-terminus as highly critical for neutralization of PKR's antiviral activity, but also identify this blockade as an indispensable contribution of NS1 to the viral life cycle.

    IMPORTANCE Influenza A virus inhibits activation of the RNA-dependent protein kinase PKR by means of its non-structural NS1 protein, but the underlying mode of inhibition is debated. Using mutational analysis, we identify arginine residues 35 and 46 within the N-terminal NS1 domain as highly critical for binding to and functional silencing of PKR. In addition, our data show that this is a main activity of the amino acids 35 and 46 as the strong attenuation of corresponding mutant viruses in human cells was rescued to a large extent by lowering PKR expression levels. Significantly, this corresponded with restoration of viral virulence for NS1 R35A and R46A mutant viruses in PKR-/- mice. Therefore, our data establish a model in which the NS1 N-terminal domain engages in a binding interaction to inhibit activation of PKR and ensure efficient viral propagation and virulence.

  • Conformational states of a soluble, uncleaved HIV-1 envelope trimer [PublishAheadOfPrint]

  • HIV-1 envelope spike [Env; trimeric (gp160)3, cleaved to (gp120/gp41)3] induces membrane fusion, leading to viral entry. It is also the viral component targeted by neutralizing antibodies. Vaccine development requires production, in quantities suitable for clinical studies, of a recombinant form that resembles functional Env. HIV-1 gp140 trimers mmdash; the uncleaved ectodomains of (gp160)3 mmdash; from a few selected viral isolates adopt a compact conformation with many antigenic properties of native Env spikes. One is currently being evaluated in a clinical trial. We report here low-resolution (20AAring;) cryoEM (electron cryomicroscopy) structures of this gp140 trimer, which adopts two principal conformations, one closed and the other slightly open. The former is indistinguishable at this resolution from those adopted by a stabilized, cleaved trimer (SOSIP) or by a membrane-bound Env trimer with truncated cytoplasmic tail (EnvCT). The latter conformation is closer to a partially open Env trimer than to the fully open conformation induced by CD4. These results show that a stable, uncleaved HIV-1 gp140 trimer has a compact structure close to that of native Env.

    IMPORTANCE Development of any HIV vaccine with a protein component (either prime or boost) requires production of a recombinant form to mimic the trimeric, functional HIV-1 envelope spike, in quantities suitable for clinical studies. Our understanding of the envelope structure has depended in part on a cleaved, soluble trimer, known as SOSIP.664, stabilized by several modifications including an engineered disulfide. This construct, difficult to produce in large quantities, has yet to induce better antibody responses than other envelope-based immunogens, even in animal models. The uncleaved ectodomain of the envelope protein, called gp140, has also been made as a soluble form to mimic the native Env present on the virion surface. Most HIV-1 gp140 preparations are not stable, however, and of inhomogeneous conformation. The results presented here show that gp140 preparations from suitable isolates can adopt a compact, native-like structure, supporting its use as a vaccine candidate.

  • Interferon gamma prevents infectious entry of HPV16 via an L2-dependent mechanism [PublishAheadOfPrint]

  • In this study, we report that IFN-, but not IFN aalpha;, bbeta; or treatment, dramatically decreased infection of HPV16 pseudovirus (PsV). In a survey of 20 additional HPV and animal papillomavirus type, we found that many, but not all, PsV types were also inhibited by IFN-. Microscopic and biochemical analyses of HPV16 PsV determined that the antiviral effect was exerted at the level of endosomal processing of the incoming capsid and depended on the JAK2/STAT1 pathway. In contrast to infection in the absence of IFN-, where L1 proteolytic products are produced during endosomal capsid processing and L2/DNA complexes segregate from L1 in the late endosome and travel to the nucleus, IFN- treatment led to decreased L1 proteolysis and retention of L2 and the viral genome in the late endosome/lysosome. PsV sensitivity or resistance to IFN- treatment was mapped to the L2 protein, as determined with infectious hybrid PsV in which the L1 protein was derived from an IFN--sensitive HPV type and the L2 protein from an IFN--insensitive type, or vice versa.

    Importance: A subset of human papillomaviruses (HPV) are the causative agents of many human cancers, most notably cervical cancer. This manuscript describes the inhibition of infection of multiple HPV types, including oncogenic types, by treatment with interferon-, an antiviral cytokine that is released from stimulated immune cells. Exposure of cells to IFN- has been shown to trigger the expression of proteins with broad antiviral effector functions, most of which act to prevent viral transcription or translation. Interestingly, in this study, we show that infection is blocked at the early step of virus entry into the host cell by retention of the minor capsid protein, L2, and the viral genome, instead of trafficking into the nucleus. Thus, a novel antiviral mechanism for interferon- has been revealed.

  • New connections: Cell to cell HIV-1 transmission, resistance to broadly neutralizing antibodies, and an envelope sorting motif [PublishAheadOfPrint]

  • HIV-1 infection from cell to cell may provide an efficient mode of viral spread in vivo and could therefore present a significant challenge for preventative or therapeutic strategies based on broadly neutralizing antibodies. Indeed, Li et al show that the potency and magnitude of multiple HIV-1 broadly neutralizing antibody classes are decreased during cell to cell infection in a context dependent manner. A functional motif in gp41 appears to contribute to this differential susceptibility by modulating exposure of neutralization epitopes.

  • Functional evidence of the involvement of the dynein light chain DYNLRB2 in murine leukemia virus infection. [PublishAheadOfPrint]

  • How murine leukemia virus (MLV) travels from the cell membrane to the nucleus, and the mechanism for nuclear entry of MLV viral DNA in dividing cells, still remain unclear. It seems likely that the MLV preintegration complex (PIC) interacts with cellular proteins to perform those tasks. We have recently published that microtubule motor cytoplasmic dynein complex and its regulator proteins interact with MLV PIC at early times of infection, suggesting a functional interaction between the incoming viral particles, the dynein complex and dynein regulators. To understand better the role of the dynein complex on MLV infection, we performed shRNA screening of the dynein light chains on MLV infection. We found that silencing of a specific light chain of the cytoplasmic dynein complex, DYNLRB2, reduced the efficiency of infection by MLV reporter viruses, without affecting HIV-1 infection. Furthermore, the over expression of DYNLRB2 increases the infection of MLV. We conclude that the light chain of the cytoplasmic dynein complex DYNLRB2 is an important and specific piece of the host machinery needed for MLV infection.

    IMPORTANCE Retroviruses must reach the chromatin of their host to integrate their viral DNA, but first they must get into the nucleus. The cytoplasm is a crowded environment where simple diffusion is slow, thus viruses utilize retrograde transport along the microtubule network mediated by the dynein complex. Different viruses use different components of this multisubunit complex. We have found that murine leukemia virus (MLV) associates functionally and specifically with the dynein light chain DYNLRB2, which is required for infection. Our study provides more insight into the molecular requirements for retrograde transport of MLV preintegration complex, and demonstrates a role for DYNLRB2 in viral infection for the first time.

  • C-terminal HIV-1 transframe p6* tetra-peptide blocks enhanced Gag cleavage incurred by leucine zipper replacement of a deleted p6* domain [PublishAheadOfPrint]

  • HIV-1 protease (PR) functions as a homodimer mediating virus maturation following virus budding. Gag-Pol dimerization is believed to trigger embedded PR activation by promoting PR dimer formation. Early PR activation can lead to markedly reduced virus yields due to premature Gag cleavage. The p6* peptide, located between Gag and PR, is believed to ensure virus production by preventing early PR maturation. Studies aimed at finding supporting evidence for this proposal are limited due to a reading frame overlap between p6* and the p6gag budding domain. To determine if p6* affects virus production via the modulation of PR activation, we engineered multiple constructs derived from Dp6*PR (an assembly- and processing-competent construct with Pol fused at the inactivated PR C-terminus). The data indicate that a p6* deletion adjacent to active PR significantly impaired virus processing. We also observed that the insertion of a leucine zipper (LZ) dimerization motif in the deleted region eliminated virus production in a PR activity-dependent manner, suggesting that the LZ insertion triggered premature PR activation by facilitating PR dimer formation. As few as four C-terminal p6* residues remaining at the p6*/PR junction were sufficient to restore virus yields, with a Gag processing profile similar to that of the wild type. Our study provides supporting evidence in a virus assembly context that the C-terminal p6* tetra-peptide plays a role in preventing premature PR maturation.

    IMPORTANCE Supporting evidence is lacking for the assumption that p6* retards PR maturation in the context of virus assembly. We found that replacing p6* with a leucine-zipper peptide abolished virus assembly due to the significant enhancement of Gag cleavage. However, as few as four C-terminal p6* residues remaining in the deleted region were sufficient for significant PR release, as well as for counteracting leucine zipper-incurred premature Gag cleavage. Our data provide evidence that (a) p6* ensures virus assembly by preventing early PR activation, and (b) four C-terminal p6* residues are critical for modulating PR activation. Current PR inhibitor development efforts are largely aimed at mature PR, but there is a tendency for HIV-1 variants to emerge that are resistant to multiple protease inhibitors. Our data support the idea of modulating PR activation by targeting PR precursors as an alternative approach to controlling HIV-1/AIDS.

  • Pathways of Prion Spread during Early Chronic Wasting Disease in Deer [PublishAheadOfPrint]

  • Among prion infections, two scenarios of prion spread are generally observed: (a) early lymphoid tissue replication or (b) direct neuroinvasion without substantial antecedent lymphoid amplification. In nature, cervids are infected with chronic wasting disease (CWD) prions by oral and nasal mucosal exposure, and studies of early CWD pathogenesis have implicated pharyngeal lymphoid tissue as the earliest sites of prion accumulation. However, knowledge of chronological events in prion spread during early infection remains incomplete. To investigate this knowledge gap in early CWD pathogenesis, we exposed white-tailed deer to CWD prions by mucosal routes and performed serial necropsies to assess PrPCWD tissue distribution by real-time quaking-induced conversion (RT-QuIC) and tyramide signal amplification immunohistochemistry (TSA-IHC). Although PrPCWD was not detected by either method in the initial days (1 and 3) post-exposure, we observed PrPCWD seeding activity and follicular immunoreactivity in oropharyngeal lymphoid tissues at 1 and 2 months post-exposure (MPE). At 3 MPE, PrPCWD replication had expanded to all systemic lymphoid tissues. By 4 MPE, the PrPCWD burden in all lymphoid tissues had increased, and approached levels observed in terminal disease, yet there was no evidence of nervous system invasion. These results indicate the first site of CWD prion entry is in the oropharynx and the initial phase of prion amplification occurs in the oropharyngeal lymphoid tissues followed by rapid dissemination to systemic lymphoid tissues. This lymphoid replication phase appears to precede neuroinvasion.

    IMPORTANCE Chronic wasting disease (CWD) is a universally fatal transmissible spongiform encephalopathy affecting cervids and natural infection occurs through oral and nasal mucosal exposure to infectious prions. Terminal disease is characterized by PrPCWD accumulation in the brain and lymphoid tissues of affected animals. However, the initial sites of prion accumulation and pathways of prion spread during early CWD infection remain unknown. To investigate the chronological events of early prion pathogenesis, we exposed deer to CWD prions and monitored the tissue distribution of PrPCWD over the first 4 months of infection. We show CWD uptake occurs in the oropharynx with initial prion replication in the draining oropharyngeal lymphoid tissues, rapidly followed by dissemination to systemic lymphoid tissues without evidence of neuroinvasion. This data highlights the two phases of CWD infection: a robust prion amplification in systemic lymphoid tissues prior to neuroinvasion or establishment of a carrier state.

  • Imaging HIV-1 Genomic DNA from Entry through Productive Infection [PublishAheadOfPrint]

  • In order to track the fate of HIV-1 particles from early entry events through productive infection, we developed a method to visualize HIV-1 DNA reverse transcription complexes by incorporation and fluorescent labeling of the thymidine analog 5-ethynyl-2rrsquo; -deoxyuridine (EdU) into nascent viral DNA during cellular entry. Monocyte-derived macrophages were chosen as natural targets of HIV-1 which do not divide and therefore do not incorporate the EdU into chromosomal DNA, which would obscure detection of intranuclear HIV-1 genomes. Using this approach, we observed distinct EdU puncta in the cytoplasm of infected cells within 12 hours post-infection and subsequent accumulation of puncta in the nucleus that remained stable through 5 days. Depletion of the restriction factor SAMHD1 resulted in a markedly increased number of EdU puncta, allowing for efficient quantification of HIV-1 reverse transcription events. Analysis of HIV-1 bearing defined mutations in the capsid protein revealed differences in their cytoplasmic and nuclear accumulation, and quantitative PCR analysis closely recapitulated the EdU results. RNA fluorescence in situ hybridization identified actively transcribing, EdU-labeled HIV-1 genomes in productively infected cells, and immunofluorescence analysis confirmed that CDK9, phosphorylated at Serine 175, was recruited to the RNA-positive HIV-1 DNA, providing a means to directly observe transcriptionally active HIV-1 genomes in productively infected cells. Overall, this system allows for stable labeling and monitoring of HIV genomic DNA within infected cells during cytoplasmic transit, nuclear import, and mRNA synthesis.

    IMPORTANCE The fates of HIV-1 reverse transcription products within infected cells are not well understood. Although previous imaging approaches identified HIV-1 intermediates during early stages of infection, few have connected these events with the later stages that ultimately lead to proviral transcription and production of progeny virus. Here we developed a technique to label HIV-1 genomes using modified nucleosides, allowing subsequent imaging of cytoplasmic and nuclear HIV-1 DNA in infected monocyte-derived macrophages. We used the technique to track the efficiency of nuclear entry as well as the fates of HIV-1 genomes in productively and non-productively infected macrophages. We visualized transcriptionally active HIV-1 DNA; revealing that transcription occurs in a subset of HIV-1 genomes in productively infected cells. Collectively, the approach provides new insights into the nature of transcribing HIV-1 genomes and allows us to track the entire course of infection in macrophages, a key target of HIV-1 in infected individuals.

  • Porcine Deltacoronavirus Nsp5 Antagonizes Type I Interferon Signaling by Cleaving STAT2 [PublishAheadOfPrint]

  • Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus. The first outbreak of PDCoV was announced from the United States in 2014, followed by reports in Asia. The nonstructural protein nsp5 is a 3C-like protease of coronavirus and our previous study showed that PDCoV nsp5 inhibits type I interferon (IFN) production. In this study, we found that PDCoV nsp5 significantly inhibited IFN-stimulated response element (ISRE) promoter activity and transcription of IFN-stimulated genes (ISGs), suggesting that PDCoV nsp5 also suppresses IFN signaling. Detailed analysis showed that nsp5 cleaved signal transducer and activator of transcription 2 (STAT2), but not Janus kinase 1 (JAK1), tyrosine kinase 2 (TYK2), STAT1 and interferon regulatory factor 9 (IRF9), key molecules of the JAK-STAT pathway. STAT2 cleavage was dependent on the protease activity of nsp5. Interestingly, nsp5 cleaved STAT2 at two sites, glutamine (Q) 685 and Q758, and similar cleavage was observed in PDCoV-infected cells. As expected, cleaved STAT2 impaired the ability to induce ISGs, demonstrating that STAT2 cleavage is an important mechanism utilized by PDCoV nsp5 to antagonize IFN signaling. We also discussed the substrate selection and binding mode of PDCoV nsp5 by homologous modeling of PDCoV nsp5 with the two cleaved peptide substrates. Taken together, our study demonstrates that PDCoV nsp5 antagonizes type I IFN signaling by cleaving STAT2 and provides structural insights to comprehend the cleavage mechanism of PDCoV nsp5, revealing a potential new function for PDCoV nsp5 in type I IFN signaling.

    IMPORTANCE The 3C-like protease encoded by nsp5 is a major protease of coronaviruses; thus it is an attractive target for development of anti-coronavirus drugs. Previous studies have revealed that the 3C-like protease of coronaviruses, including PDCoV and porcine epidemic diarrhea virus (PEDV), antagonizes type I IFN production by targeting NF-B essential modulator (NEMO). Here, for the first time, we demonstrate that overexpression of PDCoV nsp5 also antagonizes IFN signaling by cleaving STAT2, an essential component of transcription factor complex ISGF3, and that PDCoV infection reduces the levels of STAT2, which may affect the innate immune response.

  • Prophylactic Herpes Simplex Virus 2 (HSV-2) Vaccines Adjuvanted with Stable Emulsion and Toll-Like Receptor 9 Agonist Induce a Robust HSV-2-Specific Cell-Mediated Immune Response, Protect against Symptomatic Disease, and Reduce the Latent Viral Reservoir [PublishAheadOfPrint]

  • Several prophylactic vaccines targeting HSV-2 have failed in the clinic to demonstrate a sustained depression in viral shedding or protection from recurrences. Although these vaccines have generated high titers of neutralizing antibodies, their induction of robust CD8 T cells has largely been unreported, even though evidence for the importance of HSV-2 antigen-specific CD8 T cells is mounting in animal models and in translational studies involving subjects with active HSV-2-specific immune responses. We developed a subunit vaccine composed of the neutralizing antibody (nAb) targets gD and gB, the novel T cell antigen and tegument protein UL40, and we compared this to a whole-inactivated virus vaccine (FI-HSV-2). We evaluated different formulations in combination with several Th1-inducing TLR agonists in vivo. In mice, the TLR9 agonist cytosine-phosphate-guanine (CpG) oligodeoxynucleotide formulated in a squalene-based oil-in-water emulsion promoted the most robust, functional HSV-2 antigen-specific CD8 T cell responses and high neutralizing antibodies, demonstrating superiority to vaccines adjuvanted by monophosphoryl lipid A (MPL)/alum. We further established that FI-HSV-2 alone or in combination with adjuvants as well as adjuvanted subunit vaccines were successful in the induction of nAbs and T cell responses in guinea pigs. These immunological responses were coincident with a suppression of vaginal HSV-2 shedding, low lesion scores, and a reduction in latent HSV-2 DNA in dorsal root ganglia to undetectable levels. These data support the further preclinical and clinical development of prophylactic HSV-2 vaccines that contain appropriate antigen and adjuvant components responsible for programming elevated CD8 T cell responses.

    IMPORTANCE Millions of people worldwide are infected with herpes simplex virus type 2 (HSV-2), and to date, an efficacious prophylactic vaccine has not met the rigors of clinical trials. Attempts to develop a vaccine have focused primarily on glycoproteins necessary for HSV-2 viral entry as target antigens, and to which the dominant neutralizing antibody response is directed during natural infection. Individuals with asymptomatic infection have exhibited T-cell responses against specific HSV-2 antigens not observed in symptomatic individuals. We describe for the first time the immunogenicity profile in animal models of UL40, a novel HSV-2 T-cell antigen that has been correlated with asymptomatic HSV-2 disease. Additionally, vaccine candidates adjuvanted by a robust formulation of CpG oligonucleotide delivered in emulsion were superior to unadjuvanted or MPL/alum-adjuvanted formulations at eliciting a robust cell mediated immune response and blocking establishment of a latent viral reservoir in the guinea pig challenge model of HSV-2 infection.

  • ER protein SCAP inhibits Dengue virus NS2B3 protease by suppressing its K27-linked polyubiquitylation [PublishAheadOfPrint]

  • Dengue viruses (DENVs) are an emerging threat to global public health. The NS2B3 protease complex of DENV has recently been shown to cleave antiviral protein STING and thereby subverting the innate immune signaling to facilitate virus replication. Whether host cells have mechanism to counteract this virus-mediated immunosuppression is unclear. We discovered that the K27-linked poly-ubiquitination of NS3 protein facilitates its recruitment of NS2B and the formation of NS2B3, and consequently the enhanced cleavage of STING. However, an ER protein SCAP, through binding to NS2B protein, inhibits the ubiquitination of NS3, rendering NS2B3 protease incapable of binding and cleavage of STING. Importantly, ectopic-expression of SCAP impaired DENV infection, whereas silencing of SCAP potentiated DENV infection. Collectively, this study uncovered a novel function of SCAP to counteract the inhibitory action of DENV NS2B3 protease on the STING signaling, suggesting that modulation of SCAP levels may have therapeutic implications.

    IMPORTANCE This study reports the first ubiquitylation target protein in DENV: the NS3 protein and the unique role of the K27-linked poly ubiquitylation on NS3rrsquo; ability to recruit NS2B and formation of the NS2B3 protease complex. Additionally, this study identified novel functions of the ER protein SCAP: one is to compete with NS2B for binding to STING; another is to inhibit the ubiquitination of NS3. Both of these functions protect STING from being cleaved by the NS2B3 protease, and thus contribute to host antiviral response.

  • The B cell response to foot-and-mouth disease virus in cattle following sequential vaccination with multiple serotypes. [PublishAheadOfPrint]

  • Foot-and mouth disease virus is a highly contagious viral disease. Antibodies are pivotal in providing protection against FMDV infection. Serological protection against one FMDV serotype doesn't confer inter-serotypic protection. However, some historical data have shown inter-serotypic protection can be induced following sequential FMDV challenge with multiple FMDV serotypes. In this study we have investigated the kinetics of the FMDV-specific antibody secreting cell response following homologous and heterologous inactivated FMDV vaccination regimes. We have demonstrated that the kinetics of the B cell response are similar between all four FMDV serotypes tested following a homologous FMDV vaccination regime. When a heterologous vaccination regime was used with the sequential inoculation of three different inactivated FMDV serotypes (O-, A- and Asia1-serotypes) a B cell response to FMDV SAT1 and C-serotype was induced. The studies also revealed that the local lymphoid tissue had detectable FMDV-specific antibody secreting cells (ASCs) in the absence of circulating FMDV-specific ASCs indicating the presence of short-lived ASCs; a hallmark of a T-independent 2 (TI-2) antigenic response to inactivated FMDV capsid.

    Importance

    bbull; Development of intra-serotypic response following a sequential vaccination regime of four different FMDV serotypes.

    bbull; Indication of short-lived ASCs in the local lymphoid tissue mmdash; further evidence of a TI-2 type response to FMDV.

  • The SWI/SNF Chromatin Regulator, BRG1, Modulates the Transcriptional Regulatory Activity of the EBV DNA Polymerase Processivity Factor BMRF1 [PublishAheadOfPrint]

  • During lytic phase of Epstein-Barr virus (EBV), binding of the transactivator Zta to the origin of lytic replication (oriLyt) and the BHLF1 transcript, forming a stable RNA-DNA hybrid, are required to initiate viral DNA replication. EBV-encoded viral DNA replication proteins form complexes to amplify viral DNA. BMRF1, the viral DNA polymerase accessory factor, is essential for lytic DNA replication and also known as a transcriptional regulator of the expression of BHLF1 and BALF2 (ssDNA-binding protein). In order to determine systematically how BMRF1 regulates viral transcription, a BMRF1 knockout bacmid was generated to analyze viral gene expression using a viral DNA microarray. We found a subset of Rta-responsive late genes, including BcLF1, BLLF1, BLLF2 and BDLF3, were down regulated in cells harboring p2089BMRF1. In reporter assays, BMRF1 appears to transactivate a subset of viral late promoters through distinct pathways. BMRF1 activates the BDLF3 promoter in an SP1-dependent manner. Notably, BMRF1 associates with the transcriptional regulator BRG1 in EBV-reactivated cells. BMRF1-mediated transactivation activities on the BcLF1 and BLLF1 promoters were attenuated by knockdown of BRG1. In BRG1-depleted EBV-reactivated cells, BcLF1 and BLLF1 transcripts were reduced in number, resulting in reduced virion secretion. BMRF1 and BRG1 bound to the adjacent upstream regions of the BcLF1 and BLLF1 promoters and depletion of BRG1 attenuated the recruitment of BMRF1 onto both promoters, suggesting BRG1 is involved in BMRF1-mediated regulation of these two genes. Overall, we reveal a novel pathway by which BMRF1 can regulate viral promoters through interaction with BRG1.

    Importance The cascade of viral gene expression during Epstein-Barr virus (EBV) replication is exquisitely regulated by the coordination of the viral DNA replication machinery and cellular factors. Upon lytic replication, the EBV immediate early proteins Zta and Rta turn on the expression of early proteins that assemble into viral DNA replication complexes. The DNA polymerase accessory factor, BMRF1, also is known to transactivate early gene expression through its interaction with SP1 or Zta on specific promoters. Through a global analysis, we demonstrate that BMRF1 also turns on a subset of Rta-regulated, late structural gene promoters. Searching for BMRF1-interacting cellular partners revealed that the SWI/SNF chromatin modifier, BRG1, contributes to BMRF1-mediated transactivation of a subset of late promoters through protein-protein interaction and viral chromatin binding. Our findings indicate that BMRF1 regulates the expression of more viral genes than thought previously, through distinct viral DNA replication-independent mechanisms.

  • A polymorphism within the internal fusion loop of the Ebola virus glycoprotein modulates host cell entry [PublishAheadOfPrint]

  • The large scale of the Ebola virus disease (EVD) outbreak in West Africa in 2013-2016 raised the question whether the host cell interactions of the responsible Ebola virus (EBOV) strain differed from those of other ebolaviruses. We previously reported that the glycoprotein (GP) of the virus circulating in West Africa in 2014 (EBOV2014) exhibited reduced ability to mediate entry into two non-human primate (NHP)-derived cell lines relative to the GP of EBOV1976. Here, we investigated the molecular determinants underlying the differential entry efficiency. We found that EBOV2014-GP-driven entry into diverse NHP-derived cell lines as well as human monocyte-derived macrophages and dendritic cells was reduced as compared to EBOV1976-GP, although entry into most human- and all bat-derived cell lines tested was comparable. Moreover, EBOV2014 replication in NHP but not human cells was diminished relative to EBOV1976, suggesting that reduced cell entry translated into reduced viral spread. Mutagenic analysis of EBOV2014-GP and EBOV1976-GP revealed that an amino acid polymorphism in the receptor-binding domain, A82V, modulated entry efficiency in a cell line-independent manner and did not account for the reduced EBOV2014-GP-driven entry into NHP cells. In contrast, polymorphism T544I, located in the internal fusion loop in the GP2 subunit, was found to be responsible for the entry phenotype. These results suggest that position 544 is an important determinant of EBOV infectivity for NHP- and certain human target cells.

    IMPORTANCE The Ebola virus disease outbreak in West Africa in 2013 entailed more than 10,000 deaths. The scale of the outbreak and its dramatic impact on human health raised the question whether the responsible virus was particularly adept at infecting human cells. Our study shows that an amino acid exchange, A82V, that the virus acquired during the epidemic and that was not observed in previously circulating viruses, increases viral entry into diverse target cells. In contrast, the epidemic virus showed a reduced ability to enter cells of non-human primates as compared to the virus circulating in 1976 and a single amino acid exchange in the internal fusion loop of the viral glycoprotein was found to account for this phenotype.

  • Capsid Phosphorylation State and Hepadnavirus Virion Secretion [PublishAheadOfPrint]

  • The C-terminal domain (CTD) of hepadnavirus core protein is involved in multiple steps of viral replication. In particular, CTD is initially phosphorylated at multiple sites to facilitate viral RNA packaging into immature nucleocapsids (NCs) and the early stage of viral DNA synthesis. For the avian hepadnavirus, the duck hepatitis B virus (DHBV), CTD is dephosphorylated subsequently to facilitate the late stage of viral DNA synthesis and to stabilize NCs containing mature viral DNA. The role of CTD phosphorylation in virion secretion, if any, has remained unclear. Here, the CTD from the human hepatitis B virus (HBV) was found to be dephosphorylated in association with NC maturation and secretion of DNA-containing virions, as in DHBV. In contrast, the CTD in empty HBV virions (i.e., enveloped capsids with no RNA or DNA) was found to be phosphorylated. The potential role of CTD dephosphorylation in virion secretion was analyzed through mutagenesis. For secretion of empty HBV virions, which is independent of either viral RNA packaging or DNA synthesis, multiple substitutions in the CTD to mimic either phosphorylation or dephosphorylation showed little detrimental effect. Similarly, phosphor-mimetic substitutions in the DHBV CTD did not block the secretion of DNA-containing virions. These results indicate that CTD dephosphorylation, though associated with NC maturation in both HBV and DHBV, is not essential for the subsequent NC-envelope interaction to secrete DNA-containing virions, and the CTD state of phosphorylation also does not play an essential role in the interaction between empty capsids and the envelope for secretion of empty virions.

    IMPORTANCE The phosphorylation state of the C-terminal domain (CTD) of hepatitis B virus (HBV) core or capsid protein is highly dynamic and plays multiple roles in the viral life cycle. To study the potential role of CTD state of phosphorylation in virion secretion, we have analyzed the CTD phosphorylation state in complete (containing the genomic DNA) vs. empty (genome-free) HBV virions. Whereas CTD is unphosphorylated in complete virions, it is phosphorylated in empty virions. Mutational analyses indicate that neither phosphorylation nor dephosphorylation of CTD is required for virion secretion. These results demonstrate that while CTD dephosphorylation is associated with HBV DNA synthesis, the CTD state of phosphorylation may not regulate virion secretion.

  • Functional relevance of the N-terminal domain of pseudorabies virus envelope glycoprotein H and its interaction with glycoprotein L [PublishAheadOfPrint]

  • Several envelope glycoproteins are involved in herpesvirus entry into cells, direct cell-to-cell spread, and induction of cell fusion. The membrane fusion protein gB and the presumably gB-activating heterodimer gH/gL are essential for these processes and conserved throughout the Herpesviridae. However, after extended cell culture passage of gL-negative mutants of the alphaherpesvirus pseudorabies virus (PrV) phenotypic revertants could be isolated which had acquired spontaneous mutations affecting the gL-interacting N-terminal part of the gH ectodomain (gDH, gHB4.1) (B. G. Klupp and T. C. Mettenleiter, J Virol 73:3014 nndash;3022, 1999; C. Schröter, M. Vallbracht, J. Altenschmidt, S. Kargoll, W. Fuchs, B. G. Klupp and T. C. Mettenleiter, J Virol 90:2264-2272, 2016). To investigate the functional relevance of this part of gH in more detail, we introduced an in-frame deletion of 66 codons at the 5rrsquo; end of the plasmid-cloned gH gene (gH32/98). The N-terminal signal peptide was retained and the deletion did not affect expression or processing of gH, but abrogated its function in in vitro fusion assays. Insertion of the engineered gH gene into the PrV genome resulted in a defective mutant (pPrV-gH32/98K), which was incapable of entry and spread. Interestingly, in vitro activity of mutated gH32/98 was restored when co-expressed with hyperfusogenic gBB4.1, obtained from a passaged gL-deletion mutant of PrV. Moreover, the entry and spread defects of pPrV-gH32/98K were compensated by the mutations in gBB4.1 in cis, as well as in trans independent of gL. Thus, PrV gL and the gL-interacting domain of gH are not strictly required for function.

    Importance Membrane fusion is crucial for infectious entry and spread of enveloped viruses. While many enveloped viruses require only one or two proteins for receptor binding and membrane fusion, herpesvirus infection depends on several envelope glycoproteins. Besides subfamily-specific receptor binding proteins, the core fusion machinery consists of the conserved fusion protein gB and the gH/gL complex. The role of the latter is unclear but it is hypothesized to interact with gB for fusion activation. Using isogenic virus recombinants we demonstrate here that gL and the gL-binding domain of PrV gH are not strictly required for membrane fusion during virus entry and spread when concomitantly mutations in gB are present which increase its fusogenicity. Thus, our results strongly support the notion of a functional gB-gH interaction during the fusion process.

  • Hepatitis C Virus-Specific T Cell Receptor mRNA-engineered Human T Cells: Impact of Antigen-Specificity on Functional Properties [PublishAheadOfPrint]

  • Therapy with genetically modified autologous T cells has shown great promise in cancer therapy. For an efficient control of hepatitis C virus (HCV) infection, cytotoxic T cells (CTL) are pivotal but persistence of activated T cells may lead to liver toxicity. Here, anti-HCV T cell receptors (TCRs) recognising the HCV non-structural (NS) NS3 or NS5 viral peptide target were examined by mRNA transfection of human peripheral blood lymphocytes (PBL) derived from healthy donors as well as chronically infected HCV patients. Immunological analysis show that whilst the CTLs expressing the NS5-specific TCR reduced HCV RNA replication by a non-cytotoxic mechanism, the NS3-specific TCR-redirected CTLs were polyfunctional and inhibited HCV RNA replication through antigen-specific cytotoxicity. Transcriptome signatures from these two types of CTL responses revealed uniquely expressed gene clusters upon encountering hepatoma target cells presenting endogenously expressed HCV proteins. The NS3 TCR induced a rapid expression of apoptotic signalling pathways and formation of embryonic gene clusters, whereas the NS5A TCR activation induced extended proliferative and metabolic pathways as the HCV target cells survived. Our results provide detailed insights into basic HCV T cell immunology and have clinical relevance for redirecting T cells to target virally infected hepatoma cells.

    Importance Due to the protective ability of HCV-specific T cells and the hepatotoxic potential they possess, there is a great need for the understanding the functional aspects of HCV-specific T cells. To circumvent the low level of precursor frequency in patients we engineered primary CD8+ T cells by mRNA TCR vectors to confer HCV-specificity to new T cells. HCV TCRs that differ in antigen specificity and polyfunctionality were examined. mRNA TCR-engineering of peripheral blood lymphocyte from healthy donors or chronically infected HCV patients resulted in strikingly high levels of HCV TCR expression and HCV-specific responses. Whilst a cytotoxicity response from a polyfunctional T cell activation caused hepatoxicity and rapid induction of apoptotic signalling pathways, the non-cytotoxic T cell activation showed extended proliferative, metabolic pathways and persistence of HCV target cells. Our results provide detailed insights into basic HCV T cell immunology and have clinical relevance for immune protection of HCV-associated diseases.

  • Restriction of HCMV replication by ISG15, a host effector regulated by cGAS-STING dsDNA sensing [PublishAheadOfPrint]

  • Accumulation of the interferon-stimulated gene (ISG) 15 protein product, which is reversibly conjugated to numerous polypeptide targets, impacts the proteome and physiology of uninfected and infected cells. While many viruses, including human cytomegalovirus (HCMV) blunt host antiviral defenses by limiting ISG expression, the overall abundance of ISG15 monomer and protein conjugates rises in HCMV-infected cells. However, the molecular signals underlying ISG15 accumulation and whether the ISG15 polypeptide itself influences HCMV infection biology remain unknown. Here, we establish that the ISG15 gene product itself directly regulates HCMV replication and its accumulation restricts productive virus growth. Although ISG15 monomer and protein conjugate accumulation was induced in cells infected with UV-inactivated HCMV, they were subsequently reduced, but not eliminated, by an immediate-early (IE) or early (E) virus-encoded function(s). Instead, HCMV-induced ISG15 monomer and protein conjugate accumulation was dependent upon the double-stranded DNA (dsDNA) sensor cGAS, the innate immune adaptor STING, and interferon signaling. Significantly, dsDNA itself was sufficient to induce cGAS-, STING-, and interferon signaling-dependent ISG15 monomer and conjugate protein accumulation in uninfected cells. Accumulation of ISGylated proteins in uninfected cells treated with dsDNA was prevented by expressing the HCMV multifunctional IE1 transactivator. This demonstrates that expression of a single host interferon-stimulated gene, ISG15, restricts HCMV replication, and that IE1 is sufficient to blunt ISGylation in response to dsDNA sensing in uninfected cells. Moreover, it establishes that ISGylation modifies the proteomes of virus-infected and uninfected normal cells in response to cell intrinsic dsDNA sensing dependent upon cGAS-STING.

    IMPORTANCE By antagonizing type I interferon production and action, many viruses, including human cytomegalovirus (HCMV), evade host defenses. However, levels of the interferon-induced ISG15 protein, which is covalently conjugated to host and viral proteins, increases in HCMV-infected cells. How ISG15 accumulation is regulated and whether the ISG15 polypeptide influences HCMV replication remains unknown. This study establishes that ISG15 itself restricts HCMV replication and HCMV-induced ISG15 accumulation is triggered by host defenses that detect cytoplasmic double strand (ds) DNA. Remarkably, dsDNA triggered ISG15 accumulation even in uninfected cells and this was reduced by HCMV IE1 expression. This shows that ISG15 itself controls replication of HCMV, which causes life-threatening disease among the immunocompromised and is a significant source of congenital morbidity and mortality among newborns. Moreover, it demonstrates that ISG15 modifies the uninfected cell proteome in response to dsDNA, potentially impacting responses to DNA vaccines, gene therapy and autoimmune disease pathogenesis.

  • A glycosylation benchmark profile for HIV-1 envelope glycoprotein production based on eleven Env trimers. [PublishAheadOfPrint]

  • HIV-1 envelope glycoprotein (Env) glycosylation is important because individual glycans are components of multiple broadly neutralizing antibody epitopes, while shielding other sites that might otherwise be immunogenic. The glycosylation on Env is influenced by a variety of factors, including the genotype of the protein, the cell line used for its expression, and the details of the construct design. Here, we used a mass spectrometry-based approach to map the complete glycosylation profile at every site in multiple HIV-1 Env trimers, accomplishing two goals: 1) We determined which glycosylation sites contain conserved glycan profiles across many trimeric Envs. 2) We identified the variables that impact Env's glycosylation profile at sites with divergent glycosylation. Over half of the gp120 glycosylation sites on eleven different trimeric Env proteins have a conserved glycan profile, indicating that a native consensus glycosylation profile does indeed exist among trimers. We showed that some soluble gp120s and gp140s exhibit highly divergent glycosylation profiles compared to trimeric Env. We also assessed the impact of several variables on Env glycosylation: truncating the full-length Env; producing Env in CHO cells, instead of more virologically relevant T lymphocytes; and purifying Env with different chromatographic platforms: Ni-NTA, 2G12, or PGT151 affinity. This study provides the first consensus glycosylation profile of Env trimers, which should serve as a useful benchmark for HIV-1 vaccine developers. This study also defines the sites where glycosylation may be impacted when Env trimers are truncated or produced in CHO cells.

    IMPORTANCE: A protective HIV-1 vaccine will likely include a recombinant version of the viral envelope glycoprotein (Env). Env is highly glycosylated, yet vaccine developers have lacked guidance on how to assess whether their immunogens have optimal glycosylation. Important questions are still unanswered: 1) What is the "target" glycosylation profile, when the goal is to generate a natively glycosylated protein? 2) What variables exert the greatest influence on Env glycosylation? We identified numerous sites on Env where the glycosylation profile does not deviate in eleven different Env trimers, and we investigated the impact on the divergent glycosylation profiles of changing the genotype of the Env sequence, the construct design, the purification method, and the producer cell type. The data herein give vaccine developers a "glycosylation target" for their immunogens, and they show how protein production variables can impact Env glycosylation.

  • Maternal binding and neutralizing IgG responses targeting the C terminal region of the V3 loop are predictive of reduced peripartum HIV-1 transmission risk [PublishAheadOfPrint]

  • The development of an effective maternal HIV-1 vaccine that could synergize with antiretroviral therapy (ART) to eliminate pediatric HIV-1 infection will require the characterization of maternal immune responses capable of blocking transmission of autologous HIV viruses to the infant. We previously identified that maternal plasma antibody binding to linear epitopes within the variable loop 3 (V3) region of HIV envelope (Env) and neutralizing responses against easy to neutralize tier 1 viruses were associated with reduced risk of peripartum HIV infection in the historic U.S. Woman and Infant Transmission Study (WITS) cohort. Here, we defined the fine-specificity and function of the potentially-protective maternal V3-specific IgG antibodies associated with reduced peripartum HIV transmission risk in this cohort. The V3-specific IgG binding that predicted low risk of mother-to-child-transmission (MTCT) was dependent on the C-terminal flank of the V3 crown and particularly on amino acid position 317, a residue that has also been associated with breakthrough transmission in the RV144 vaccine trial. Remarkably, the fine-specificity of potentially-protective maternal plasma V3-specific tier 1 virus neutralizing responses was dependent on the same region in the V3 loop. Our findings suggest that MTCT risk is associated with neutralizing maternal IgG that target amino acid residues in the C-terminal region of the V3 loop crown, suggesting the importance of this region in immunogen design for maternal vaccines to prevent MTCT.

    IMPORTANCE Efforts to curb HIV-1 transmission in pediatric populations by antiretroviral therapy (ART) have been highly successful in both developed and developing countries. However, more than 150,000 infants continue to be infected each year likely due to a combination of late maternal HIV diagnosis, lack of ART access or adherence, and drug-resistant viral strains. Defining the fine-specificity of maternal humoral responses that partially protect against MTCT of HIV is required to inform the development of a maternal HIV vaccine that will enhance these responses during pregnancy. In this study, we identified amino acid residues targeted by potentially-protective maternal V3-specific IgG binding and neutralizing responses, localizing the potentially-protective response in the C terminal region of the V3 loop crown. Our findings have important implications in the design of maternal vaccination strategies that could synergize with ART during pregnancy to achieve the elimination of pediatric HIV infections.

  • The Long Noncoding RNA NEAT1 Exerts Anti-Hantaviral Effects by Acting as a Positive Feedback for RIG-I Signaling [PublishAheadOfPrint]

  • Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs (lncRNAs) play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA, NEAT1, as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus (HTNV) infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced interferon (IFN) bbeta; production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I-IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline-glutamine rich (SFPQ) by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections.

    IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs (lncRNAs) as key regulators of innate immunity; however, their roles in Hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon (IFN) responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I-IRF7 pathway in a time and dose-dependent manner, and promoted HTNV-induced IFN production by facilitating RIG-I and DDX60 expression. Intriguingly, NEAT1 relocated SFPQ and formed paraspeckles after HTNV infection, which might reverse inhibitive effects of SFPQ on the transcription of RIG-I and DDX60. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections.

  • Genotype-specific evolution of hepatitis E virus [PublishAheadOfPrint]

  • Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis globally. HEV comprises four genotypes with different geographic distributions and host ranges. We utilise this natural case-control study for investigating the evolution of zoonotic viruses compared to single host viruses, using 244 near full length HEV genomes. Genome wide estimates of dN/dS located a region of overlapping reading frames, which is subject to positive selection in genotypes 3 and 4. The open reading frames (ORFs) involved have functions related to host-pathogen interaction, so genotype specific evolution of these regions may reflect their fitness. Bayesian inference of evolutionary rates shows genotypes 3 and 4 have significantly elevated rates relative to genotypes 1 across all ORFs. Reconstructing phylogenies of zoonotic genotypes demonstrates significant intermingling of isolates between hosts. We speculate that the genotype specific differences may result from cyclical adaptation to different hosts in genotypes 3 and 4.

    Importance: Hepatitis E virus (HEV) is increasingly recognised as a pathogen which affects both the developing, and the developed world. While most often clinically mild, HEV can be severe or fatal in certain demographics, such as expectant mothers. Like many other viral pathogens, HEV has been grouped into several distinct genotypes. We show that most of the HEV genome is evolutionarily constrained. One locus of positive selection is unusual as it encodes two distinct protein products. We are the first to detect positive selection in this overlap region. Genotype 1, which only infects humans, appears to be evolving differently to genotypes 3 and 4, which infect multiple species, possibly because genotypes 3 and 4 are unable to achieve the same fitness due to repeated host jumps.

  • Low Pathogenic Influenza A Viruses in North American Diving Ducks Contribute to the Emergence of a Novel Highly Pathogenic Influenza A(H7N8) Virus [PublishAheadOfPrint]

  • Introductions of low pathogenic avian influenza (LPAI) viruses of subtypes H5 and H7 into poultry from wild birds have the potential to mutate to highly pathogenic avian influenza (HPAI), but such viruses' origins are often unclear. In January 2016, a novel H7N8 HPAI virus caused an outbreak in turkeys in Indiana, USA. To determine the virus's origin, we sequenced genomes of 441 wild birdmmdash;origin influenza A viruses (IAVs) from North America and subjected them to evolutionary analyses. Results showed that the H7N8 LPAI virus most likely circulated among diving ducks in the Mississippi flyway during autumn 2015 and was subsequently introduced to Indiana turkeys, in which it evolved high pathogenicity. Preceding the outbreak, an isolate with six gene segments (PB2, PB1, PA, HA, NA, and NS) sharing ggt;99% sequence identity with those of H7N8 turkey isolates was recovered from a diving duck sampled in Kentucky, USA. H4N8 IAVs from other diving ducks possessed five H7N8mmdash;like gene segments (PB2, PB1, NA, MP, and NS, ggt;98% sequence identity). Our findings suggest that viral gene constellations circulating among diving ducks can contribute towards the emergence of IAVs that affect poultry. Therefore, diving ducks may serve an important and understudied role in the maintenance, diversification, and transmission of IAVs in the wild bird reservoir.

    IMPORTANCE In January 2016, a novel H7N8 HPAI virus caused a disease outbreak in turkeys in Indiana, USA. Toward determining the origin of this virus, we sequenced and analyzed 441 wild birdmmdash;origin influenza strains isolated from wild birds inhabiting North America. We found that H7N8 LPAI virus most likely circulated among diving ducks in the Mississippi flyway during autumn 2015 and was subsequently introduced to Indiana turkeys, in which it evolved high pathogenicity. Our results suggest that viral gene constellations circulating among diving ducks can contribute towards the emergence of IAVs that affect poultry. Therefore diving ducks may serve an important and understudied role in the maintenance, diversification, and transmission of IAVs in the wild bird reservoir. Our study also highlights the importance of a coordinated, systematic, and collaborative surveillance for IAVs in both poultry and wild bird populations.

  • Endogenous brain lipids inhibit prion amyloid formation in vitro [PublishAheadOfPrint]

  • The normal cellular prion protein (PrPC) resides in detergent-resistant outer membrane lipid rafts in which conversion to the pathogenic misfolded form is believed to occur. Once misfolding occurs, the pathogenic isoform polymerizes into highly stable amyloid fibrils. In vitro assays have demonstrated an intimate association between prion conversion and lipids, specifically phosphatidylethanolamine, which is a critical cofactor in the formation of synthetic infectious prions. In the current work, we demonstrate an alternative inhibitory function of lipids in the prion conversion process as assessed in vitro by real-time, quaking induced conversion (RT-QuIC). Using an alcohol-based extraction technique, we removed the lipid content from chronic wasting disease (CWD)-infected white tailed deer brain homogenates and found lipid extraction enabled RT-QuIC detection of CWD prions in two logs greater concentration of brain sample. Conversely, addition of brain-derived lipid extracts to CWD-prion brain or lymph node samples inhibited amyloid formation in a dose-dependent manner. Subsequent lipid analysis demonstrated this inhibitory function was restricted to the polar lipid fraction in brain. We further investigated three phospholipids commonly found in lipid membranes, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol, and found all three similarly inhibited RT-QuIC. These results demonstrating polar-lipid, and specifically phospholipid, inhibition of prion-seeded amyloid formation highlight the diverse roles lipid constituents may play in the prion conversion process.

    IMPORTANCE Prion conversion is likely influenced by lipid interactions given the location of normal prion protein (PrPC) in lipid rafts and lipid cofactors generating infectious prions in in vitro models. Here, we use real-time quaking induced conversion (RT-QuIC) to demonstrate that endogenous brain polar lipids can inhibit prion seeded amyloid formation, suggesting, prion conversion is guided by an environment of pro-conversion and anti-conversion lipids. These experiments also highlight the applicability of RT-QuIC to identify potential therapeutic inhibitors of prion conversion.

  • Inhibition of HIV-1 Maturation via Small Molecule Targeting of the Amino-Terminal Domain in the Viral Capsid Protein [PublishAheadOfPrint]

  • The HIV-1 capsid protein is an attractive therapeutic target owing to its multifunctionality in virus replication and the high fitness cost of amino acid substitutions in capsid to HIV-1 infectivity. To date, small molecule inhibitors have been identified that inhibit HIV-1 capsid assembly and/or impair its function in target cells. Here we describe the mechanism of action of the previously reported capsid-targeting HIV-1 inhibitor, BI compound 1 (C1). We show that C1 acts during HIV-1 maturation to prevent assembly of a mature viral capsid. However, unlike the maturation inhibitor Bevirimat, C1 did not significantly affect the kinetics or fidelity of Gag processing. HIV-1 particles produced in the presence of C1 contained unstable capsids that lacked associated electron density and exhibited impairments in early postentry stages of infection, most notably reverse transcription. C1 inhibited assembly of recombinant HIV-1 CA in vitro and induced aberrant crosslinks in mutant HIV-1 particles capable of spontaneous intersubunit disulfide bonds at the interhexamer interface in the capsid lattice. Resistance to C1 was conferred by a single amino acid substitution within the compound-binding site in the N-terminal domain of the CA protein. Our results demonstrate that the binding site for C1 represents a new pharmacological vulnerability in the capsid assembly stage of the HIV-1 life cycle.

    IMPORTANCE The HIV-1 capsid protein is an attractive but unexploited target for clinical drug development. Prior studies have identified HIV-1 capsid-targeting compounds that display different mechanisms of action, which in part reflects the requirement for capsid function at both the efferent and afferent phases of viral replication. Here we show that one such compound, Compound 1, interferes with assembly of the conical viral capsid during virion maturation, and results in perturbations at a specific protein-protein interface in the capsid lattice. We also identify and characterize a mutation in the capsid protein that confers resistance to the inhibitor. This study reveals a novel mechanism by which a capsid-targeting small molecule can inhibit HIV-1 replication.

  • Human herpesvirus 6A (HHV-6A) exhibits restrictive propagation with limited activation of PKR-eIF2{alpha} stress pathway [PublishAheadOfPrint]

  • The eIF2aalpha; protein plays a critical role in the regulation of translation. The production of dsRNA during viral replication can activate protein kinase R (PKR) which phosphorylates the eIF2aalpha;, leading to inhibition of the initial step of translation. Many viruses have evolved gene products targeting PKR-eIF2a pathway, indicating its importance in antiviral defense. In the present study we focus on alternations of PKR-eIF2a pathway along HHV-6A infection while monitoring viral gene expression and infectious viral yields. We have found increased phosphorylated PKR as well as phosphorylated eIF2aalpha; coincidental with accumulation of the late gp82-105 viral protein. The level of total PKR was relatively constant, but it decreased by 144 hours post infection. The phosphorylation of eIF2a led to a moderate increase in ATF4 accumulation, indicating moderate inhibition of protein translation during HHV-6A infection. The overexpression of PKR led to decreased viral propagation coincidental with increased accumulation of phosphorylated PKR and phosphorylated eIF2a. Moreover, addition of dominant-negative PKR mutant resulted in a moderate increase in viral replication. HHV-6A exhibits relatively low propagation efficiency of progeny virus secreted into the culture media. This study suggests that the replicative strategy of HHV-6A involves a mild infection over lengthy life cycle in culture, while preventing severe activation of the PKR-eIF2aalpha; pathway.

    IMPORTANCE Human herpesvirus 6A (HHV-6A) and HHV-6B are common, wildly prevalent viruses, causing from mild to severe disease. Our study focuses on the PKR-eIF2aalpha; stress pathway that limits viral replication. The HHV-6 genome encodes multiple genes transcribed from the two strands predicting accumulation of dsRNAs which can activates PKR and inhibition of protein synthesis. We report that HHV-6A induced the accumulation of phosphorylated PKR, phosphorylated eIF2aalpha; and a moderate increase of the activating transcription factor 4 (ATF4), known to transcribe stress genes. Overexpression of PKR led to increased eIF2aalpha; phosphorylation and decreased viral replication whereas overexpression of dominant-negative PKR mutant resulted in a moderate increase in viral replication. These results suggest that HHV-6A replication strategy involves restricted activation of the PKR-eIF2aalpha; pathway, partial translation inhibition and lower yields of infectious virus. In essence, HHV-6A limits its own replication due to the inability to bypass the eIF2aalpha; phosphorylation.

  • The lateral organization of influenza virus proteins in the budozone region of the plasma membrane [PublishAheadOfPrint]

  • Influenza virus assembles and buds at the plasma membrane of virus-infected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some, like HA, NA, and M2 are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with the viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immuno-gold staining. The distribution of these proteins was examined individually and pair-wise using the Ripley K function, a type of nearest neighbor analysis. Individually HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly co-clustered in the plasma membrane; however, in the case of NA and M2 clustering depends upon the expression system used. Despite both being raft-resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly co-cluster but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the co-expression of other viral proteins. Similarly, M2 and NP occupy separate compartments but an association can be bridged by co-expression of M1.

    Importance The complement of influenza proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft like domains whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships between viral proteins in the plasma membrane. Some proteins such as HA and M2 inherently co-cluster within the membrane. Although M2 is mostly found on the periphery of regions of HA consistent with the proposed role of M2 in scission at the end of budding. The association between some pairs of influenza proteins, such as M2 and NP, appear to be brokered by additional influenza proteins, in this case M1. HA and NA, while both raft associated, reside in distinct domains, reflecting their distributions in the viral membrane.

  • BCA2/RABRING7 Interferes with HIV-1 Proviral Transcription by Enhancing the SUMOylation of I{kappa}B{alpha} [PublishAheadOfPrint]

  • BCA2/Rabring7 is a BST2 co-factor that promotes the lysosomal degradation of trapped HIV-1 virions, but also functions as a BST2-independent anti-HIV factor by targeting Gag for lysosomal degradation. Since many antiviral factors regulate the NF-B innate signaling pathway, we investigated whether BCA2 is also connected to this pro-inflammatory cascade. Here we show for the first time that BCA2 is induced by NF-B-activating pro-inflammatory cytokines, and that up-regulation of BCA2 provides a regulatory negative feedback on NF-B. Specifically, BCA2 serves as an E3 SUMO-ligase in the SUMOylation of IBaalpha;, which in turn enhances the sequestration of NF-B components in the cytoplasm. Since HIV-1 utilizes NF-B to promote proviral transcription, the BCA2-mediated inhibition of NF-B significantly decreases the transcriptional activity of HIV-1 (up to 4.4-fold in CD4+ T cells). Therefore, our findings indicate that BCA2 poses an additional barrier to HIV-1 infection: not only does BCA2 prevent assembly and release of nascent virions, but also significantly restricts HIV-1 transcription by inhibiting the NF-B pathway.

    IMPORTANCE Understanding the interactions between HIV-1 and its host cells is highly relevant for the design of new drugs aimed at eliminating HIV-1 from affected individuals. We have previously shown that BCA2, a co-factor of BST2 in the restriction of HIV-1, also prevents virion assembly in a BST2-independent manner. In this study we found that BCA2 negatively regulates the NF-B pathway nndash; a signaling cascade necessary for HIV-1 replication and infectivity nndash;, which in turn detrimentally affects proviral transcription and virus propagation. Thus, our results indicate that besides its already described functions as an antiviral factor, BCA2 poses an additional barrier to HIV-1 replication at the transcriptional level.

  • Relationship between measures of HIV reactivation and the decline of latent reservoir under latency-reversing agents [PublishAheadOfPrint]

  • Antiretroviral-free HIV remission requires substantial reduction of the number of latently infected cells and enhanced immune control of viremia. Latency-reversing agents (LRA) aim to eliminate latently infected cells by increasing the rate of reactivation of HIV transcription, which exposes these cells to killing by the immune system. As LRA are explored in clinical trials, it becomes increasingly important to assess the effect of increased HIV reactivation rate on the decline of latently infected cells, and estimate LRA efficacy in increasing virus reactivation. However, whether the extent of HIV reactivation is a good predictor of the rate of decline of the number of latently infected cells is dependent on a number of factors. Our modeling shows that mechanisms of maintenance and clearance of the reservoir, lifespan of cells with reactivated HIV and other factors may significantly impact the relationship between measures of HIV reactivation and the decline of the number of latently infected cells.

    The usual measures of HIV reactivation are the increase in cell-associated HIV RNA (CA RNA) and/or plasma HIV RNA soon after administration. We analyze two recent studies where CA RNA was used to estimate the impact of two novel LRAs, panobinostat and romidepsin. Both drugs increased the CA RNA level 3-4 fold in clinical trials. However, cells with panobinostat-reactivated HIV appeared long-lived (half-life ggt; month), suggesting that HIV reactivation rate increased approximately by 8%. With romidepsin, the lifespan of cells that reactivated HIV was short (2 days), suggesting that HIV reactivation rate may have doubled under treatment.

    Importance Long-lived latently infected cells that persist on antiretroviral treatment (ART) are thought to be the source of viral rebound soon after ART interruption. The elimination of latently infected cells is an important step to achieving antiretroviral-free HIV remission. Latency-reversing agents (LRA) aim to activate HIV expression in latently infected cells, which could lead to their death. Here we discuss the possible impact of the LRA on the reduction of the number of latently infected cells, depending on the mechanisms of their loss and self-renewal and on the lifespan of the cells that have activated HIV transcription by the LRAs.

  • Superiority in Rhesus Macaques of Targeting HIV-1 Env Gp140 to CD40 Versus LOX-1 in Combination with Replication Competent NYVAC-KC for Induction of Env-Specific Antibody and T Cell Responses [PublishAheadOfPrint]

  • We compared the HIV-1-specific immune responses generated by targeting HIV-1 envelope protein (Env gp140) to either CD40 or LOX-1, two endocytic receptors on dendritic cells (DCs), in Rhesus macaques primed with a poxvirus vector (NYVAC-KC) expressing Env gp140. The DC-targeting vaccines, humanized recombinant monoclonal antibodies fused to Env gp140, were administered as a boost with poly ICLC adjuvant either alone or co-administered with the NYVAC-KC vector. All the DC-targeting vaccine administrations with poly ICLC increased the low-level serum anti-Env IgG responses elicited by NYVAC-KC priming significantly more (up to P =0.01) than a group without poly ICLC. The responses were robust, cross-reactive, and contained antibodies specific to multiple epitopes within gp140 including the C1, C2, V1-3, C4, C5, and gp41 immuno-dominant regions. The DC-targeting vaccines also elicited modest serum Env-specific IgA responses. All groups gave serum neutralization activity limited to Tier 1 viruses and antibody dependent cytotoxicity responses (ADCC) after DC-targeting boosts. Furthermore, CD4+ and CD8+ T cell responses specific to multiple Env epitopes were strongly boosted by the DC-targeting vaccines + poly ICLC. Together, these results indicate that prime/boost immunization via NYVAC-KC and either aalpha;CD40.Env gp140/poly ICLC or aalpha;LOX-1.Env gp140/poly ICLC induced balanced antibody and T cell responses against HIV-1 Env. Co-administration of NYVAC-KC with the DC-targeting vaccines increased T cell responses, but had minimal effects on antibody responses except for suppressing serum IgA responses. Overall, compared to LOX-1, targeting Env to CD40 gave more robust T cell and serum antibody responses with broader epitope representation and greater durability.

    IMPORTANCE An effective vaccine to prevent HIV-1 infection does not yet exist. An approach to elicit strong protective antibody development is to direct virus protein antigens specifically to dendritic cells, which are now known to be the key cell type for controlling immunity. Here we have tested in non-human primates two prototype vaccines engineered to direct the HIV-1 coat protein Env to dendritic cells. These vaccines bind to either CD40 or LOX-1, two dendritic cell surface receptors with different functions and tissue distributions. We tested the vaccines described above in combination with attenuated virus vectors that express Env. Both vaccines, but especially that delivered via CD40, raised robust immunity against HIV-1 as measured by monitoring potentially protective antibody and T cell responses in the blood. The safety and efficacy of the CD40-targeted vaccine justifies further development for future human clinical trials.

  • Careers in Virology: Working at the Centers for Disease Control and Prevention [PublishAheadOfPrint]

  • As non-academic careers in science have become less and less "alternative," one field that has consistently attracted early-career virologists is public health research. The desire to make tangible contributions towards public health needs and better protect the public from infectious disease often motivate the transition. In this Career Gem, two academically-trained virologists offer insights into pursuing a research career in public health at the Centers for Disease Control and Prevention.

  • Inhibition of Vpx-mediated SAMHD1 and Vpr-mediated HLTF degradation by selective disruption of viral CRL4 (DCAF1) E3 ubiquitin ligase assembly [PublishAheadOfPrint]

  • The lentiviral accessory proteins Vpx and Vpr are known to utilize CRL4 (DCAF1) E3 ligase to induce the degradation of the host restriction factor SAMHD1 or transcriptional factor HLTF, respectively. Selective disruption of viral CRL4 (DCAF1) E3 ligase could be a promising antiviral strategy. Recently, we have determined that post-translational modification (neddylation) of Cullin-4 is required for the activation of Vpx-CRL4 (DCAF1) E3 ligase. However, the mechanism of Vpx/Vpr-CRL4 (DCAF1) E3 ligase assembly is still poorly understood. Here, we report that zinc coordination is an important regulator of Vpx-CRL4 E3 ligase assembly. Residues in a conserved zinc-binding motif of Vpx were essential for the recruitment of the CRL4 (DCAF1) E3 complex and Vpx-induced SAMHD1 degradation. Importantly, altering the intracellular zinc concentration by treatment with the zinc chelator, N,N,N,N-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN) potently blocked Vpx-mediated SAMHD1 degradation and inhibited wild-type SIVmac infection of myeloid cells, even in the presence of Vpx. TPEN selectively inhibited Vpx and DCAF1 binding, but not the Vpx-SAMHD1 interaction or Vpx virion packaging. Moreover, we have shown that zinc coordination is also important for the assembly of the HIV-1 Vpr-CRL4 E3 ligase. In particular, Vpr zinc-binding motif mutation or TPEN treatment efficiently inhibited Vpr-CRL4 (DCAF1) E3 ligase assembly and Vpr-mediated HTLF degradation or Vpr-induced G2 cell cycle arrest. Collectively, our study sheds light on a conserved strategy by the viral proteins Vpx and Vpr to recruit host CRL4 (DCAF1) E3 ligase, which represents a target for novel anti-HIV drug development.

    IMPORTANCE The Vpr and its paralog Vpx are accessory proteins encoded by different HIV/SIV lentiviruses. To facilitate viral replication, Vpx has evolved to induce SAMHD1 degradation and Vpr to mediate HLTF degradation. Both Vpx and Vpr perform their functions by recruiting CRL4 (DCAF1) E3 ligase. In this study, we demonstrate that the assembly of the Vpx- or Vpr-CRL4 E3 ligase requires a highly conserved zinc-binding motif. This motif is specifically required for the DCAF1 interaction but not for the interaction of Vpx or Vpr with their substrates. Selective disruption of Vpx- or Vpr-CRL4 E3 ligase function was achieved by zinc sequestration using N,N,N' -tetrakis-(2' -pyridylmethyl) ethylenediamine (TPEN). At the same time, zinc sequestration had no effect on zinc-dependent cellular protein functions. Therefore, information obtained from this study may be important for novel anti-HIV drug development.

  • The viral gene ORF79 encodes a repressor regulating induction of the lytic life cycle in the haloalkaliphilic virus {varphi}Ch1 [PublishAheadOfPrint]

  • In this study, we describe the construction of the first genetically modified mutant of a halovirus infecting haloalkaliphilic Archaea. By random choice we targeted ORF79, a so far uncharacterized viral gene of the haloalkaliphilic virus Ch1. We used a PEG-mediated transformation method to deliver a disruption cassette into a lysogenic strain of the haloalkaliphilic archaeon Natrialba magadii bearing Ch1 as provirus. This approach yielded mutant virus particles carrying a disrupted version of the ORF79. Disruption of ORF79 did not influence morphology of the mature virions. The mutant virus was able to infect cured strains of N. magadii resulting in a lysogenic, ORF79-disrupted strain. Analysis of this strain carrying the mutant virus revealed a repressor function of ORF79. In absence of gp79, onset of lysis as well as expression of viral proteins occurred prematurely compared to the wild-type strain. Constitutive expression of ORF79 in a cured strain of N. magadii reduced the plating efficiency of Ch1 by seven orders of magnitude. Overexpression of ORF79 in a lysogenic strain of N. magadii resulted in an inhibition of lysis and total absence of viral proteins as well as viral progeny. In further experiments, gp79 directly regulated the expression of the tail fiber protein ORF34 but did not influence the methyltransferase gene ORF94. Further, we describe the establishment of an inducible promoter for in vivo studies in N. magadii.

    IMPORTANCE Genetic analyses of haloalkaliphilic Archaea or haloviruses are only rarely reported. Therefore, only little insight into the in vivo roles of proteins and their functions has been gained so far. We used a reverse genetic approach to identify the function of a yet undescribed gene of Ch1. We provide evidence that gp79, a so far unknown protein of Ch1, acts as a repressor protein of the viral life cycle, affecting the transition from the lysogenic to the lytic state of the virus. Thus, repressor genes in other haloviruses could be identified by sequence homologies to gp79 in the future. Moreover, we describe the use of an inducible promoter of N. magadii. Our work provides valuable tools for the identification of other unknown viral genes by our approach as well as for functional studies of proteins by inducible expression.

  • TRIM25 Is Required for the Antiviral Activity of Zinc-finger Antiviral Protein (ZAP) [PublishAheadOfPrint]

  • Zinc-finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses by binding to viral mRNAs, and repressing the translation and/or promoting the degradation of target mRNA. In addition, ZAP regulates the expression of certain cellular genes. Here, we report that tripartite motif-containing protein 25 (TRIM25), a ubiquitin E3 ligase, is required for the antiviral activity of ZAP. Downregulation of endogenous TRIM25 abolished ZAP's antiviral activity. The E3 ligase activity of TRIM25 is required for this regulation. TRIM25 mediated ZAP ubiquitination, but the ubiquitination of ZAP itself did not seem to be required for its antiviral activity. Downregulation of endogenous ubiquitin or overexpression of deubiquitinase OTUB1 impaired ZAP's activity. We provide evidence indicating that TRIM25 modulates the target RNA binding activity of ZAP. These results uncover a mechanism by which the antiviral activity of ZAP is regulated.

    IMPORTANCE ZAP is a host antiviral factor that specifically inhibits the replication of certain viruses, including HIV-1, Sindbis virus and Ebola virus. ZAP binds directly to target mRNA, and represses the translation and promotes the degradation of target mRNA. While the mechanisms by which ZAP post-transcriptionally inhibits target RNA expression has been extensively studied, how its antiviral activity is regulated is not very clear. Here we report that TRIM25, a ubiquitin E3 ligase, is required for the antiviral activity of ZAP. Downregulation of endogenous TRIM25 remarkably abolished ZAP's activity. TRIM25 is required for ZAP optimal binding to target mRNA. These results help to better understand how the antiviral activity of ZAP is regulated.

  • JAK-STAT Signaling Pathways and Inhibitors Affect Reversion of Envelope Mutated HIV-1 [PublishAheadOfPrint]

  • HIV can spread by both cell-free and cell-to-cell transmission. Here, we show that numerous of the amino acid changes in Env that are close to the CD4 binding pocket can affect HIV replication. We generated a number of mutant viruses that were unable to infect T cells as cell-free viruses but were nevertheless able to infect certain T cell lines as cell-associated viruses, followed by reversion to wild type. However, the activation of JAK-STAT signaling pathways caused inhibition of such cell-to-cell infection as well as the reversion of multiple HIV Env mutants that displayed differences in ability to bind to the CD4 receptor. Specifically, two T cell activators, Interleukin-2 (IL2) and phorbol-12-myristate 13-acetate (PMA), both capable of activation of JAK-STAT pathways, were able to inhibit cell-to-cell viral transmission. In contrast, but consistent with the above result, a number of JAK-STAT-mTOR inhibitors actually promoted HIV-1 transmission and reversion. Hence, JAK-STAT signaling pathways may differentially affect the replication of a variety of HIV Env mutants in ways that differ from the role that these pathways play in the replication of wild type viruses.

    Importance: Specific alterations in HIV Env close to the CD4 binding site can differentially change the ability of HIV to mediate infection for cell-free and cell-associated viruses. However, such differences are dependent to some extent on the types of target cells used. JAK-STAT signaling pathways are able to play major roles in these processes. This work sheds new light on factors that can govern HIV infection of target cells.

  • Mutation of the 2nd Sialic Acid-Binding Site Resulting in Reduced Neuraminidase Activity Preceded Emergence of H7N9 Influenza A Virus [PublishAheadOfPrint]

  • The emergence of novel influenza A virus (IAV) H7N9 since 2013 has caused concerns about the ability of this virus to spread between humans. Analysis of the receptor-binding properties of the H7 protein of a human isolate revealed modestly increased binding to aalpha;2,6 sialosides and reduced, but still dominant, binding to aalpha;2,3-linked sialic acids (SIAs) compared to a closely related avian H7N9 virus from 2008. Here we show that the corresponding N9 neuraminidases display equal enzymatic activity on a soluble monovalent substrate and similar substrate specificity on a glycan array. In contrast, solid phase activity and binding assays demonstrated reduced specific activity and decreased binding of the novel N9 protein. Mutational analysis showed these differences to result from substitution T401A in the 2nd SIA-binding site, indicating that substrate binding via this site enhances NA catalytic activity. Substitution T401A in the novel N9 protein appears to functionally mimic the substitutions that are found in the 2nd SIA-binding site of NA proteins of avian-derived IAVs that became human pandemic viruses. Our phylogenetic analyses show that substitution T401A occurred prior to substitutions in HA causing the altered receptor-binding properties mentioned above. Hence, in contrast to the widespread assumption that such changes in NA are only obtained after acquisition of functional changes in HA, our data indicate that mutations in the 2nd SIA binding site may have enabled and even driven the acquisition of altered HA receptor-binding properties, and may have contributed to the spread of the novel H7N9 viruses.

    IMPORTANCE Novel H7N9 IAVs continue to cause human infections and pose an ongoing public health threat. Here, we show that their N9 protein displays reduced binding to and lower enzymatic activity against multivalent substrates, resulting from mutation of the 2nd sialic acid-binding site. This mutation preceded and may have driven the selection of substitutions in H7 that modify H7 receptor-binding properties. Of note, all animal IAVs that managed to cross the host species barrier and became human viruses carry mutated 2nd sialic acid-binding sites. Screening of animal IAVs to monitor their potential to cross the host species barrier should therefore not only focus on the HA protein, but also on the functional properties of NA.

  • Differential Disruption of Nucleocytoplasmic Trafficking Pathways by Rhinovirus 2A Proteases [PublishAheadOfPrint]

  • The RNA rhinoviruses (RV) encode 2A proteases (2Apro) that contribute essential polyprotein processing and host-cell shutoff functions during infection, including the cleavage of Phe/Gly-containing nucleoporin proteins (Nups) within nuclear pore complexes (NPC). Within the 3 RV species, multiple divergent genotypes encode diverse 2Apro sequences which act differentially on specific Nups. Since only subsets of Phe/Gly motifs, particularly those within Nup62, Nup98 and Nup153, are recognized by transport receptors (karyopherins) when trafficking large molecular cargos through the NPC, the processing preferences of individual 2Apro predict RV genotype-specific targeting of NPC pathways and cargos. To test this idea, transformed HeLa cells lines were created with fluorescent cargos (mCherry) for the importin aalpha;/bbeta;, transportin-1, and transportin-3 import pathways, and the Crm1-mediated export pathway. Live cell imaging of single cells expressing recombinant RV 2Apro (A16, A45, B04, B14, B52, C02, and C15) showed disruption of each pathway with measurably different efficiencies and reaction rates. The B04 and B52 proteases preferentially targeted Nups in the import pathways, while B04 and C15 proteases were more effective against the export pathway. Virus type-specific trends were also observed during infection of cells with A16, B04, B14, and B52 viruses or their chimeras, as measured by NF-KB (p65/Rel) translocation into the nucleus and the rates of virus-associated cytopathic effects. This study provides new tools for evaluating the host cell response to RV infections in real time, and suggests that differential 2Apro activities explain, in part, strain-dependent host responses and diverse RV disease phenotypes.

    IMPORTANCE Genetic variation among human rhinovirus types includes unexpected diversity in the viral protease genes (2Apro) which help these viruses achieve anti-host responses. When the enzyme activities of 7 different 2Apro were measured comparatively in transformed cells programed with fluorescent reporter systems and quantitative cell imaging, indeed, the cellular substrates, particularly in the nuclear pore complex, used by these proteases were attacked with different rates and affinities. The importance of this finding is that it provides a mechanistic explanation for how different types (strains) of rhinoviruses may elicit different cell responses which directly or indirectly lead to distinct disease phenotypes.

  • A murine herpesvirus closely related to ubiquitous human herpesviruses causes T-cell depletion [PublishAheadOfPrint]

  • Mouse models of human herpesvirus infections The human roseoloviruses HHV6A, HHV6B, and HHV7 comprise the Roseolovirus genus of the human Betaherpesvirinae subfamily. Infections with these viruses have been implicated in many diseases; however, it has been challenging to establish infections with Roseoloviruses as direct drivers of pathology because they are nearly ubiquitous and display species-specific tropism. Furthermore, controlled study of infection has been hampered by the lack of experimental models, and until now, a mouse roseolovirus has not been identified. Herein we describe a virus that causes severe thymic necrosis in neonatal mice, characterized by a loss of CD4+ T-cells. These phenotypes resemble those caused by the previously described mouse thymic virus (MTV), a putative herpesvirus that has not been molecularly characterized. By Next Generation sequencing of infected tissue homogenates, we assembled a contiguous 174Kb genome sequence encoding 128 unique predicted open reading frames (ORFs), many of which were most closely related to herpesvirus genes. Moreover, the structure of the virus genome and phylogenetic analysis of multiple genes strongly suggested that this virus is a betaherpesvirus more closely related to the roseoloviruses, HHV6A, HHV6B, and HHV7, than another murine betaherpesvirus, mouse cytomegalovirus (MCMV). As such, we have named this virus murine roseolovirus (MRV) because these data strongly suggest that MRV is a mouse homolog of HHV6A/HHV6B/HHV7.

    Importance: Herein we describe the complete genome sequence of a novel murine herpesvirus. By sequence and phylogenetic analyses, we show that it is a betaherpesvirus most closely related to the roseoloviruses, human herpesvirus 6A, 6B, and 7. These data combined with physiological similarities with human roseoloviruses collectively suggest that this virus is a murine roseolovirus (MRV), the first definitively described rodent roseolovirus, to our knowledge. Many biological and clinical ramifications of roseolovirus infection in humans have been hypothesized, but studies showing definitive causative relationships between infection and disease susceptibility are lacking. Here we show that MRV infects the thymus and causes T-cell depletion, suggesting that other roseoloviruses may have similar properties.

  • Differentiation-dependent LMP1 expression is required for efficient lytic EBV reactivation in epithelial cells [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV)-associated diseases of epithelial cells, including tumors such as nasopharyngeal carcinoma (NPC) that have latent infection, and oral hairy leukoplakia (OHL) lesions that have lytic infection, frequently express the viral latent membrane protein 1 (LMP1). In lytically-infected cells, LMP1 expression is activated by the BRLF1 (R) immediate-early (IE) protein. However, the mechanisms by which LMP1 expression is normally regulated in epithelial cells remain poorly understood, and its potential roles in regulating lytic reactivation in epithelial cells are as yet unexplored. We previously showed that the differentiation-dependent cellular transcription factors, KLF4 and BLIMP1, induce lytic EBV reactivation in epithelial cells by synergistically activating the two EBV immediate-early promoters (Zp and Rp). Here we show that epithelial cell differentiation also induces LMP1 expression. We demonstrate that KLF4 and BLIMP1 cooperatively induce the expression of LMP1, even in the absence of the EBV IE proteins BZLF1 (Z) and R, via activation of the two LMP1 promoters. Furthermore, we find that differentiation of NOKs-Akata cells by either methylcellulose suspension, or by organotypic culture, induces LMP1 expression prior to Z and R expression. We show that LMP1 enhances the lytic-inducing effects of epithelial cell differentiation, as well as TPA and sodium butyrate treatment, in EBV-infected epithelial cells by increasing expression of the Z and R proteins. Our results suggest that differentiation of epithelial cells activates a feed-forward loop in which KLF4 and BLIMP1 first activate LMP1 expression, and then cooperate with LMP1 to activate Z and R expression.

    IMPORTANCE The EBV protein LMP1 is expressed in EBV-associated epithelial cell diseases, regardless of whether these diseases are due to lytic infection (such as oral hairy leukoplakia), or latent infection (such as nasopharyngeal carcinoma). However, surprisingly little is known about how LMP1 expression is regulated in epithelial cells, and there are conflicting reports about whether it plays any role in regulating viral lytic reactivation. In this study, we show that epithelial cell differentiation induces LMP1 expression by increasing expression of two cellular transcription factors (KLF4 and BLIMP1) which cooperatively activate the two LMP1 promoters. We also demonstrate that LMP1 promotes efficient lytic reactivation in EBV-infected epithelial cells by enhancing expression of the Z and R proteins. Thus, in EBV-infected epithelial cells, LMP1 expression is promoted by differentiation and positively regulates lytic viral reactivation.

  • A Sequence-Independent, Unstructured IRES is Responsible for Internal Expression of the Coat Protein of Turnip Crinkle Virus [PublishAheadOfPrint]

  • To maximize the coding potential of viral genomes, internal ribosome entry sites (IRES) can be used to bypass the traditional requirement of a 5' cap and some/all of the associated translation initiation factors. Although viral IRES typically contain higher order RNA structure, an unstructured sequence of about 84-nt immediately upstream of the Turnip crinkle virus (TCV) coat protein (CP) ORF has been found to promote internal expression of the CP from the genomic (g)RNA both in vitro and in vivo. Absence of extensive RNA structure was predicted using RNA folding algorithms and confirmed by SHAPE structure probing. Analysis of the IRES region in vitro using both the TCV gRNA and reporter constructs did not reveal any sequence-specific elements but rather suggested that overall lack of structure was an important feature for IRES activity. The CP IRES is A-rich, independent of orientation, and is strongly conserved among viruses in the same genus. The IRES was dependent on eIF4G, but not eIF4E, for activity. Low levels of CP accumulated in vivo in the absence of detectable TCV subgenomic RNAs, strongly suggesting that the IRES was active in the gRNA in vivo. Since the TCV CP also serves as the viral silencing suppressor, early translation of the CP from the viral gRNA is likely important for countering host defenses. Cellular mRNA IRES also lack extensive RNA structures or sequence conservation suggesting that this viral IRES and cellular IRES may share a similar strategy for internal translation initiation.

    IMPORTANCE Cap-independent translation is a common strategy among positive-sense, single-stranded RNA viruses for bypassing the host cell requirement of a 5' cap structure. Viral IRES in general contain extensive secondary structure that is critical for activity. In contrast, we demonstrate that a region of viral RNA devoid of extensive secondary structure has IRES activity and produces low levels of viral coat protein in vitro and in vivo. Our findings may be applicable to cellular mRNA IRES that also have little or no sequences/structures in common.

  • Two white spot syndrome virus miRNAs target Dorsal to promote virus infection in shrimp Marsupenaeus japonicas [PublishAheadOfPrint]

  • In eukaryotes, microRNAs (miRNAs) serve as regulators of many biological processes, including virus infection. A miRNA can generally target diverse genes during virusmmdash;host interactions. However, regulation of gene expression by multiple miRNAs has not yet been extensively explored during virus infection. This study found that the Spz-Toll-Dorsal-ALF signaling pathway played a very important role in antiviral immunity in shrimp (Marsupenaeus japonicus) against invasion of white spot syndrome virus (WSSV). Dorsal, the central gene in Toll pathway, was targeted by two viral miRNAs (WSSV-miR-N13 and WSSV-miR-N23) during WSSV infection. Regulation of Dorsal expression by viral miRNAs suppressed the Spz-Toll-Dorsal-ALF signaling pathway in shrimp in vivo, leading to virus infection. Our study contributed novel insights into the viral miRNA-mediated Toll signaling pathway during virusmmdash;host interaction.

    Importance A miRNA can target diverse genes during virusmmdash;host interactions. However, regulation of gene expression by multiple miRNAs has not yet been extensively explored during virus infection. Results of this study indicated that shrimp Dorsal, the central gene in Toll pathway, was targeted by two viral miRNAs during infection with white spot syndrome virus. Regulation of Dorsal expression by viral miRNAs suppressed the Spz-Toll-Dorsal-ALF signaling pathway in shrimp in vivo, leading to virus infection. Our study provided a new insight into the viral miRNA-mediated Toll signaling pathway in virusmmdash;host interactions.

  • HIV-specific granzyme B, but not interferon-{gamma} secreting T Cells are associated with reduced viral reservoirs in early HIV infection [PublishAheadOfPrint]

  • A major barrier to an Human Immunodeficiency Virus Type 1 (HIV) infection cure is the establishment of a viral reservoir despite cART. It is unclear how HIV-specific CTLs influence the size of the reservoir in early HIV infection. 28 subjects with early HIV infection were recruited to receive cART and followed for 48 weeks. HIV reservoirs in peripheral CD4+ T cells measured by cell associated proviral DNA and viral outgrowth cultures were determined at baseline and 48 weeks of cART. At baseline, Granzyme B and IFN- ELISpot assays were performed with peptides spanning the HIV proteome. All subjects had detectable HIV-specific granzyme B and IFN- responses at baseline. The quantity and specificity of granzyme B responses did not correlate with IFN- responses. For Granzyme B, Tat/Rev was the most dominant whereas for IFN-, Gag predominated. HIV-specific granzyme B T cell responses negatively correlated with HIV proviral loads at baseline and 48 weeks, and with replication competent viral IUPM at baseline but not significantly at 48 weeks. Tat/Rev-, Env-, Gag- and Vif-specific granzyme B responses correlated most strongly with reservoir control. There was no correlation with HIV-specific IFN- responses with reservoir size at baseline or 48 weeks. The majority of granzyme B responses were contributed by CD8+ T cells. Thus, our findings suggest that the induction of potent granzyme B producing CTL to Tat, Rev, Env, Gag and Vif during early infection may be able to prevent the establishment of a large viral reservoir, thereby facilitating a reduced HIV burden.

    IMPORTANCE A major barrier to the cure of Human Immunodeficiency Virus Type 1 (HIV) infection is the establishment of a viral reservoir that must be significantly reduced or eradicated entirely to enable a cure. Combined antiretroviral therapy (cART) alone is unable to clear this viral reservoir. It has been shown that CD8+ cytotoxic T lymphocytes (CTLs) are important in controlling early HIV infection by reducing plasma viremia. However, it is not known if these HIV-specific CTLs influence the establishment of the viral reservoir in early HIV infection. We show that HIV-specific granzyme B targeting HIV Tat/rev, Env, Gag and Vif, but not IFN- responses are associated with reduced virus reservoirs at baseline and at 48 weeks of cART. These findings shed light on the nature of the effector CTL response that might limit reservoir size with implications on cure research and HIV vaccines.

  • A point mutation in the RNA-binding domain of human parainfluenza virus type 2 nucleoprotein elicits an abnormally enhanced polymerase activity [PublishAheadOfPrint]

  • The genome RNA of human parainfluenza virus type 2 (hPIV2) that acts as template for the polymerase complex is entirely encapsidated by the nucleoprotein (NP). Recently, the crystal structure of NP of PIV5, a virus closely related to hPIV2, was resolved in association with RNA. Ten amino acids that contact the bound RNA-binding were identified, and are strictly conserved between PIV5 and hPIV2 NP. Mutation of hPIV2 NP Q202 (that contacts a base rather than the RNA backbone) to some amino acids resulted in an over thirty-fold increase of polymerase activity as evidenced by a minireplicon assay, even though RNA-binding affinity was unaltered. Using various modified minireplicons, we found that enhanced reporter gene expression could be accounted for by increased minigenome replication, whereas mRNA synthesis itself was not affected by Q202 mutation. Moreover, the enhanced activities were still observed in minigenomes partially lacking the leader sequence and which were not of hexamer genome length. Unexpectedly, recombinant hPIV2 possessing the NP Q202A mutation could not be recovered from cDNA.

    IMPORTANCE We examined the importance of amino acids in the putative RNA-binding domain of hPIV2 NP for polymerase activity using minireplicons. Abnormally enhanced genome replication was observed by the substitution in NP Q202 position to various amino acids. Surprisingly, this mutation enabled polymerase to use minigenomes partially lacking the leader sequence and not of hexamer genome length. This mutation does not affect fundamental properties of NP, e.g., recognition of gene junctional and editing signals. However, the strongly enhanced polymerase activity may not be viable for infectious life-cycle. This report highlights the potential of the polymerase complex with point mutations in NP, and helps our detailed understanding of the molecular basis of gene expression.

  • HIV/AIDS vaccine candidates based on replication-competent recombinant poxvirus NYVAC-C-KC expressing trimeric gp140 and Gag-derived VLPs or lacking the viral molecule B19 that inhibits type I interferon activate relevant HIV-1-specific B and T cell immune functions in non-human primates [PublishAheadOfPrint]

  • The non-replicating attenuated poxvirus vector NYVAC expressing clade C(CN54) HIV-1 Env(gp120), Gag-Pol-Nef antigens (NYVAC-C) showed in phase I clinical trials limited immunogenicity. To enhance the capacity of the NYVAC vector to trigger broad humoral responses and a more balanced activation of CD4+ and CD8+ T cells, here we compared the HIV-1-specific immunogenicity elicited in non-human primates immunized with two replicating NYVAC vectors that have been modified by the insertion of K1L and C7L vaccinia viral host-range genes and express clade C(ZM96) trimeric HIV-1 gp140 protein or a Gag(ZM96)-Pol-Nef(CN54) polyprotein as Gag-derived virus-like particles (termed NYVAC-C-KC). Additionally, one NYVAC-C-KC vector was generated by deleting the viral gene B19R, an inhibitor of type I interferon response (NYVAC-C-KC-B19R). An immunization protocol mimicking the RV144 phase III clinical trial was used. Two groups of macaques received two doses of the corresponding NYVAC-C-KC vectors (weeks 0 and 4), and booster doses with NYVAC-C-KC vectors plus clade C HIV-1 gp120 protein (weeks 12 and 24). The two replicating NYVAC-C-KC vectors induced an enhanced and similar HIV-1-specific CD4+ and CD8+ T cell responses, similar levels of binding IgG antibodies, low levels of IgA antibodies, high levels of antibody-dependent cellular cytotoxicity responses and HIV-1-neutralizing antibodies. Small differences within the NYVAC-C-KC-B19R group were seen in the magnitude of CD4+ and CD8+ T cells, induction of some cytokines and in the neutralization of some HIV-1 isolates. Thus, replication-competent NYVAC-C-KC vectors acquired relevant immunological properties as vaccine candidates against HIV/AIDS, and the viral B19 molecule exerts some control of immune functions.

    IMPORTANCE It is of special importance to find a safe and effective HIV/AIDS vaccine that can induce strong and broad T cell and humoral immune responses correlating with HIV-1 protection. Here we developed novel replicating poxvirus NYVAC-based HIV/AIDS vaccine candidates expressing clade C HIV-1 antigens, with one of them lacking the vaccinia B19 protein, an inhibitor of type I interferon response. Immunization of non-human primates with these novel NYVAC-C-KC vectors and the protein component gp120 elicited a high level of T cell and humoral immune responses, with the vector containing the deletion in B19R inducing a trend to higher magnitude of CD4+ and CD8+ T cells and neutralization of some HIV-1 strains. These poxvirus vectors could be considered as HIV/AIDS vaccine candidates based on their activation of potential immune correlates of protection.

  • Toll-Like Receptor 7 Agonist GS-9620 Induces HIV Expression and HIV-Specific Immunity in Cells from HIV-Infected Individuals on Suppressive Antiretroviral Therapy [PublishAheadOfPrint]

  • Antiretroviral therapy can suppress HIV replication to undetectable levels but does not eliminate latent HIV, thus necessitating lifelong therapy. Recent efforts to target this persistent reservoir have focused on inducing the expression of latent HIV so that infected cells may be recognized and eliminated by the immune system. Toll like receptor (TLR) activation stimulates antiviral immunity and has been shown to induce HIV from latently infected cells. Activation of TLR7 leads to the production of several stimulatory cytokines, including type I interferons (IFNs). In this study, we show that the selective TLR7 agonist, GS-9620, induced HIV in peripheral blood mononuclear cells (PBMCs) from HIV-infected individuals on suppressive antiretroviral therapy. GS-9620 increased extracellular HIV RNA 1.5-2-fold through a mechanism that required type I IFN signaling. GS-9620 also activated HIV-specific T cells and enhanced antibody-mediated clearance of HIV-infected cells. Activation by GS-9620 in combination with HIV peptide stimulation increased CD8 T cell degranulation, production of intracellular cytokines, and cytolytic activity. T cell activation was again dependent on type I IFNs produced by plasmacytoid dendritic cells. GS-9620 induced phagocytic cell maturation and improved effector-mediated killing of HIV-infected CD4 T cells by the HIV envelope-specific broadly neutralizing antibody PGT121. Collectively, these data show that GS-9620 can activate HIV production and improve the effector functions that target latently infected cells. GS-9620 may effectively complement orthogonal therapies designed to stimulate antiviral immunity, such as therapeutic vaccines or broadly neutralizing antibodies. Clinical studies are underway to determine if GS-9620 can target HIV reservoirs.

    IMPORTANCE Though antiretroviral therapies effectively suppress viral replication, they do not eliminate integrated proviral DNA. This stable intermediate of viral infection is persistently maintained in reservoirs of latently infected cells. Consequently, lifelong therapy is required to maintain viral suppression. Ultimately, new therapies are needed that specifically target and eliminate the latent HIV reservoir. Toll-like receptor agonists are potent enhancers of innate antiviral immunity that can also improve the adaptive immune response. Here, we show that a highly selective TLR7 agonist, GS-9620, activated HIV from peripheral blood mononuclear cells isolated from HIV-infected individuals with suppressed infection. GS-9620 also improved immune effector functions that specifically targeted HIV-infected cells. Previously published studies on this compound in other chronic viral infections show that it can effectively induce immune activation at safe and tolerable clinical doses. Together, the results of these studies suggest that GS-9620 may be useful for treating HIV-infected individuals on suppressive antiretroviral therapy.

  • Progesterone-based contraceptives reduce adaptive immune responses and protection against sequential influenza A virus infections [PublishAheadOfPrint]

  • In addition to their intended use, progesterone (P4)-based contraceptives promote anti-inflammatory immune responses, yet their effects on the outcome of infectious diseases, including influenza A virus (IAV), are rarely evaluated. To evaluate their impact on immune responses to sequential IAV infections, adult female mice were treated with placebo or one of two progestins, P4 or levonorgestrel (LNG), and infected with mouse adapted (ma) H1N1 virus. Treatment with P4 or LNG reduced morbidity, but had no effect on pulmonary virus titers, during primary H1N1 infection as compared to placebo treatment. In serum and bronchoalveolar lavage fluid, total anti-IAV IgG and IgA titers and virus neutralizing antibody titers, but not hemagglutinin stalk antibody titers, were lower in progestin-treated mice as compared with placebo-treated mice. Females were challenged six weeks later with either a maH1N1 drift variant (maH1N1dv) or maH3N2 IAV. Protection following infection with the maH1N1dv was similar among all groups. In contrast, following challenge with maH3N2, progestin treatment reduced survival as well as numbers and activity of H1N1- and H3N2-specific memory CD8+ T cells, including tissue resident cells, compared with placebo treatment. In contrast to primary IAV infection, progestin treatment increased neutralizing and IgG antibody titers against both challenge viruses compared with placebo treatment. While the immunomodulatory properties of progestins protected naïve females against severe outcome from IAV infection, it made them more susceptible to secondary challenge with a heterologous IAV, despite improving their antibody responses against a secondary IAV infection. Taken together, the immunomodulatory effects of progestins differentially regulate the outcome of infection depending on exposure history.

    IMPORTANCE The impact of hormone-based contraceptives on the outcome of infectious diseases outside of the reproductive tract is rarely considered. Using a mouse model, we have made the novel observation that treatment with either progesterone or a synthetic analog found in hormonal contraceptives, levonorgestrel, impacts sequential influenza A virus infection, by modulating antibody responses and decreasing memory CD8+ T cells. Progestins reduced antibody responses during primary H1N1 virus infection, but increased antibody titers following a sequential infection with either a H1N1 drift variant or a H3N2 virus. Following challenge with a H3N2 virus, female mice treated with progestins experienced greater mortality with increased pulmonary inflammation and reduced numbers and activity of CD8+ T cell. This study suggests that progestins significantly affect adaptive immune responses to influenza A virus infection, with their effect on influence outcome depending on exposure history.

  • A bivalent heterologous DNA-virus-like particles prime-boost vaccine elicits broad protection against both groups 1 and 2 influenza A viruses [PublishAheadOfPrint]

  • Current seasonal influenza vaccines are efficacious when vaccine strains are matched with circulating strains. But they do not protect antigenic variants and newly emerging pandemic and outbreak strains. Thus there is a critical need for developing so-called "universal" vaccines that protect against all influenza viruses. In the present study we developed a bivalent heterologous DNA-virus-like particles (VLP) prime-boost vaccine strategy. We show that mice immunized with this vaccine were broadly protected against lethal challenge of group 1 (H1, H5 and H9) and group 2 (H3 and H7) viruses with 94% aggregate survival. To determine immune correlates of protection, we performed passive immunizations and in vitro assays. We show that this vaccine elicited antibody responses that bound HA from group 1 (H1, H2, H5, H6, H8, H9, H11 and H12) and group 2 (H3, H4, H7, H10, H14 and H15) and neutralized homologous and intrasubtypic H5 and H7 and heterosubtypic H1 viruses and HA-specific CD4 and CD8 T cell responses. As a result, passive immunization with immune sera fully protected mice from H5, H7 and H1 challenge; whereas with both immune sera and T cells completely survived from heterosubtypic H3 and H9 challenge. Thus it appears that neutralizing antibodies alone fully protect homologous and intrasubtypic H5 and H7; neutralizing and binding antibodies are sufficient to protect heterosubtypic H1; but against heterosubtypic H3 and H9 binding antibodies and T cells are required for complete survival. We believe that this vaccine regimen could potentially be a candidate for "universal" influenza vaccine.

    IMPORTANCE Influenza virus infection is global health problem. Current seasonal influenza vaccines are efficacious only when vaccine strains are matched with circulating strains. But they do not protect antigenic variants and newly emerging pandemic and outbreak strains. Because of this, there is an urgent need to develop so-called "universal" influenza vaccines that can protect against both current and future influenza strains. In the present study we developed a bivalent heterologous prime-boost vaccine strategy. We show that this bivalent vaccine regimen elicited broad binding and neutralizing antibody and T cell responses that conferred broad protection against diverse challenge viruses in mice, suggesting that this bivalent prime-boost strategy could practically be a candidate for "universal" influenza vaccine.

  • Identification of novel functions for hepatitis C virus envelope glycoprotein E1 in virus entry and assembly [PublishAheadOfPrint]

  • Hepatitis C Virus (HCV) envelope glycoprotein complex is composed of E1 and E2 subunits. E2 is the receptor-binding protein as well as the major target of neutralizing antibodies, whereas the functions of E1 remain poorly defined. Here, we took advantage of the recently published structure of the N-terminal region of E1 ectodomain to interrogate the functions of this glycoprotein by mutating residues within this 79 amino acid region in the context of an infectious clone. The phenotypes of the mutants were characterized to determine the effects of the mutations on virus entry, replication and assembly. Furthermore, biochemical approaches were also used to characterize the folding and assembly of E1E2 heterodimers. Thirteen out of nineteen mutations led to viral attenuation or inactivation. Interestingly, two attenuated mutants, T213A and I262A, were less dependent on claudin-1 for cellular entry in Huh-7 cells. Instead, these viruses relied on claudin-6, indicating a shift in receptor dependence for these two mutants in the target cell line. An unexpected phenotype was also observed for mutant D263A which was no longer infectious but still showed a good level of core protein secretion. Furthermore, genomic RNA was absent from these non-infectious viral particles, indicating that D263A mutation leads to the assembly and release of viral particles devoid of genomic RNA. Finally, a change in subcellular co-localization between HCV RNA and E1 was observed for D263A mutant. This unique observation highlights for the first time a crosstalk between HCV glycoprotein E1 and the genomic RNA during HCV morphogenesis.

    IMPORTANCE Hepatitis C virus (HCV) infection is a major public health problem worldwide. It encodes two envelope proteins, E1 and E2, which play a major role in the life cycle of this virus. E2 has been extensively characterized, whereas E1 remains poorly understood. Here, we investigated E1 functions by using site-directed mutagenesis in the context of the viral life cycle. Our results identify unique phenotypes. Unexpectedly, two mutants clearly showed a shift in receptor dependence for cell entry, highlighting a role for E1 in modulating HCV particle interaction with cellular receptor(s). More importantly, another mutant led to the assembly and release of viral particles devoid of genomic RNA. This unique phenotype was further characterized and we observed a change in subcellular co-localization between HCV RNA and E1. This unique observation highlights for the first time a crosstalk between a viral envelope protein and the genomic RNA during morphogenesis.

  • Japanese Encephalitis Virus NS5 Inhibits the Type I Interferon Production by Blocking the Nuclear Translocation of IRF3 and NF-{kappa}B [PublishAheadOfPrint]

  • The type I interferon (IFN) response is part of a first-line defense against viral infection. To initiate replication, viruses have developed powerful evasion strategies to counteract host IFN responses. In present study, we found that Japanese encephalitis virus (JEV) NS5 protein could inhibit double strand RNA (dsRNA)-induced IFN-bbeta; expression in a dose-dependent manner. Our data further demonstrated that JEV NS5 suppressed the activation of IFN transcriptional factors, IRF3 and NF-B. However, there was no defect in the phosphorylation of IRF3 and degradation of IB, an upstream inhibitor of NF-B, upon NS5 expression, indicating a direct inhibition of the nuclear localization of IRF3 and NF-B by NS5. Mechanically, NS5 was shown to interact with the nuclear transport proteins, KPNA2, KPNA3 and KPNA4, which competitively blocked the interaction of KPNA3 and KPNA4 with their cargo molecules, IRF3 and p65, a subunit of NF-B, and thus inhibited the nuclear translocation of IRF3 and NF-B. Furthermore, overexpression of KPNA3 and KPNA4 restored the activity of IRF3 and NF-B and increased the production of IFN-bbeta; in NS5-expressing or JEV-infected cells. Additionally, an up-regulated replication level of JEV was shown upon KPNA3 or KPNA4 overexpression. These results suggest that JEV NS5 inhibits the induction of type I IFN by targeting KPNA3 and KPNA4.

    IMPORTANCE JEV is the major cause of viral encephalitis in South and Southeast Asia with high mortality. However, the molecular mechanisms contributing to the severe pathogenesis are poorly understood. The ability of JEV to counteract the host innate immune response may be one of the potential mechanisms responsible for JEV virulence. Here, we demonstrate the ability of JEV NS5 to interfere with the dsRNA-induced nuclear translocation of IRF3 and NF-B by competitively inhibiting the interaction of IRF3 and NF-B with nuclear transport proteins. Via this mechanism, JEV NS5 suppresses the induction of type I IFN and antiviral response in host cells. These findings reveal a novel strategy for JEV to escape the host innate immune response and provide us new insight into the pathogenesis of JEV.

  • Impaired STING pathway in the human osteosarcoma U2OS cells contributes to the growth of ICP0-null mutant herpes simplex virus [PublishAheadOfPrint]

  • Human herpes simplex virus-1 (HSV-1) is a wide spread pathogen with 80% of the population being latently infected. To successfully evade the host the virus has evolved strategies to counteract antiviral responses, including the gene silencing and innate immunity machineries. The immediately early protein of the virus, Infected Cell Protein 0 (ICP0) plays central role in these processes. ICP0 blocks innate immunity and one mechanism is by degrading hostile factors with its intrinsic E3 ligase activity. ICP0 functions also as a promiscuous transactivator and it blocks repressor complexes to enable viral gene transcription. For these reasons the growth of a ICP0 virus is impaired in most cells except the human osteosarcoma cells U2OS and it is only partially impaired in the human osteosarcoma cells Saos-2. We found that the two human osteosarcoma cell lines that supported the growth of the ICP0 virus failed to activate innate immune responses upon treatment with 2rrsquo; 3rrsquo; -cGAMP, the natural agonist of STING (STimulator of INterferon Genes) or after infection with the ICP0 mutant virus. Innate immune responses were restored in these cells by transient expression of the STING protein but not after overexpression of the IFI16 protein. Restoration of STING expression resulted in suppression of ICP0 virus gene expression and a decrease in viral yields. Overexpression of IFI16 also suppressed ICP0 virus gene expression, albeit to a lesser extent than STING. These data suggest that the susceptibility of U2OS and Saos-2 cells to the ICP0 HSV-1 is in part due to an impaired STING pathway.

    IMPORTANCE The DNA sensor STING plays pivotal role in controlling HSV-1 infection both in cell culture and in mice. HSV-1 genome encodes for numerous proteins that are dedicated to combat host antiviral responses. The immediate-early protein of the virus ICP0 plays major role in this process as it targets hostile host proteins for degradation with its E3 ligase activity and it disrupts repressor complexes via protein-protein interaction to enable viral gene transcription. Therefore, the ICP0 HSV-1 virus is defective for growth in most cells, except the human osteosarcoma cell lines U2OS and Saos-2. We found that both cell lines that support ICP0 virus infection have defects in the STING DNA sensing pathway, which partially accounts for the rescue of the ICP0 virus growth. Restoration of STING expression in these cells rescued innate immunity and suppressed ICP0 virus infection. This study underscores the importance of STING in the control of HSV-1.

  • Atypical RNA Elements Modulate Translational Readthrough in Tobacco necrosis virus-D [PublishAheadOfPrint]

  • Tobacco necrosis virus-D (TNV-D) is a positive-strand RNA virus in the genus Betanecrovirus and family Tombusviridae. The production of its RNA-dependent RNA polymerase (RdRp), p82, is achieved by translational readthrough. This process is stimulated by an RNA structure that is positioned immediately downstream of the recoding site, termed the readthrough stem-loop (RTSL), and a sequence in the 3rrsquo; -untranslated region of the TNV-D genome, called the distal readthrough element (DRTE). Notably, a base pairing interaction between the RTSL and the DRTE, spanning ~3,000 nt, is required for enhancement of readthrough. Here, some of the structural features of the RTSL, as well as RNA sequences and structures that flank either the RTSL or DRTE, were investigated for their involvement in translational readthrough and virus infectivity. The results revealed that (i) the RTSL-DRTE interaction cannot be functionally replaced by stabilizing the RTSL structure, (ii) a novel tertiary RNA structure positioned just 3rrsquo; to the RTSL is required for optimal translational readthrough and virus infectivity, and (iii) these same activities also rely on an RNA stem-loop located immediately upstream of the DRTE. Functional counterparts for the RTSL-proximal structure may also be present in other tombusvirids. The identification of additional distinct RNA structures that modulate readthrough suggests that regulation of this process by genomic features may be more complex than previously appreciated. Possible roles for these novel RNA elements are discussed.

    IMPORTANCE The analysis of factors that affect recoding events in viruses is leading to an ever more complex picture of this important process. In this study, two new atypical RNA elements were shown to contribute to efficient translational readthrough of the TNV-D polymerase and to mediate robust viral genome accumulation in infections. One of the structures, located close to the recoding site, could have functional equivalents in related genera, while the other structure, positioned 3rrsquo; -proximally in the viral genome, is likely limited to betanecroviruses. Irrespective of their prevalence, the identification of these novel RNA elements adds to the current repertoire of viral genome-based modulators of translational readthrough and provides a notable example of the complexity of regulation of this process.

  • HIV-1 Vpu downmodulates ICAM-1 expression resulting in decreased killing of infected CD4+ T cells by NK cells [PublishAheadOfPrint]

  • HIV-1 Vpu is known to alter the expression of numerous cell surface molecules. Given the ever-increasing list of Vpu targets identified to date, we undertook a proteomic screen to discover novel cell membrane proteins modulated by this viral protein. Plasma membrane proteome isolates from Vpu-inducible T cells were subjected to SILAC-based mass spectrometry analysis and putative targets were validated by infection of primary CD4+ T cells. We report here that while Intercellular Adhesion Molecule (ICAM)-1 and ICAM-3 are upregulated by HIV-1 infection, expression of Vpu offsets this increase by downregulating these molecules from the cell surface. Specifically, we show that Vpu is sufficient to downregulate and deplete ICAM-1 in a manner requiring the Vpu transmembrane domain and a dual serine (S52,56) motif necessary for recruitment of the SCFbbeta;-TrCP E3 ubiquitin ligase. Vpu interacts with ICAM-1 to induce its proteasomal degradation. Interestingly, the E3 ubiquitin ligase component bbeta;-TrCP-1 is dispensable for ICAM-1 surface downregulation, yet is necessary for ICAM-1 degradation. Functionally, Vpu-mediated ICAM-1 downregulation lowers packaging of this adhesion molecule into virions, resulting in decreased infectivity. Importantly, while Vpu-mediated downregulation of ICAM-3 has a limited effect on the conjugation of NK cells to HIV-1-infected CD4+ T cells, downregulation of ICAM-1 by Vpu results in a reduced ability of NK cells to bind and kill infected T cells. Vpu-mediated ICAM-1 downregulation may therefore represent an evolutionary compromise in viral fitness by impeding the formation of cell-to-cell contacts between immune cells and infected T cells at the cost of decreased virion infectivity.

    IMPORTANCE The major barrier to eradicating HIV-1 infection is the establishment of treatment-resistant reservoirs early in infection. Vpu-mediated ICAM-1 downregulation may contribute to the evasion of cell-mediated immunity during acute infection to promote viral dissemination and the development of viral reservoirs. By aiding the immune system to clear infection prior to the development of reservoirs, novel treatments designed to disrupt Vpu-mediated ICAM-1 downregulation may be beneficial during acute infection or as a prophylactic treatment.

  • Targeting Vaccine-induced Extrafollicular Pathway of B cell Differentiation Improves Rabies Post-exposure Prophylaxis [PublishAheadOfPrint]

  • Vaccine-induced B cells differentiate along two pathways. The follicular pathway gives rise to germinal centers (GCs) that can take weeks to fully develop. The extrafollicular pathway gives rise to short-lived plasma cells (PCs) that can rapidly secrete protective antibodies within days of vaccination. Rabies virus (RABV) post-exposure prophylaxis (PEP) requires rapid vaccine-induced humoral immunity for protection. Therefore, we hypothesized that targeting extrafollicular B cell responses for activation would improve the speed and magnitude of RABV PEP. To test this hypothesis, we constructed, recovered, and characterized a recombinant RABV-based vaccine expressing murine B cell activating factor (BAFF) (rRABV-mBAFF). BAFF is an ideal molecule to improve early pathways of B cell activation as it links innate and adaptive immunity, promoting potent B cell responses. Indeed, rRABV-mBAFF induced a faster, higher antibody response in mice and enhanced survivorship in PEP settings compared to rRABV. Interestingly, rRABV-mBAFF and rRABV induced equivalent numbers of GC B cells, suggesting that rRABV-mBAFF augmented the extrafollicular B cell pathway. To confirm that rRABV-mBAFF modulated the extrafollicular pathway, we used a signaling lymphocytic activation molecule (SLAM)-associated protein (SAP)-deficient mouse model. In response to antigen, SAP-deficient mice form extrafollicular B cell responses but do not generate GCs. rRABV-mBAFF induced similar anti-RABV antibody responses in SAP-deficient and wild-type mice, demonstrating that BAFF modulated immunity through the extrafollicular and not the GC B cell pathway. Collectively, strategies that manipulate pathways of B cell activation may facilitate the development of a single-dose RABV vaccine that replaces current complicated and costly RABV PEP.

    IMPORTANCE Effective RABV PEP is currently resource- and cost-prohibitive in regions of the world where RABV is most prevalent. In order to diminish the requirements for rabies immunoglobulin (RIG) and multiple vaccinations for effective prevention of clinical rabies, a more rapidly protective vaccine is needed. This work presents a successful approach to rapidly generate antibody-secreting PCs in response to vaccination by targeting the extrafollicular B cell pathway. We demonstrate that the improved early antibody responses induced by rRABV-mBAFF confer improved protection against RABV in a PEP model. Significantly, activation of the early extrafollicular B cell pathway, such as that demonstrated herein, could improve the efficacy of vaccines targeting other pathogens against which rapid protection would decrease morbidity and mortality.

  • Reduced potency and incomplete neutralization of broadly neutralizing antibodies against cell-to-cell transmission of HIV-1 with transmitted founder Envs [PublishAheadOfPrint]

  • Broadly neutralizing antibodies (bNAbs) have been isolated from HIV-1 patients and can potently block infection of a wide spectrum of HIV-1 subtypes. These antibodies define common epitopes shared by many viral isolates. While bNAbs potently antagonize infection with cell-free virus, inhibition of HIV-1 transmission from infected to uninfected CD4+ T cells through virological synapses (VS), has been found to require greater amounts of antibody. In this study, we examined two well-studied molecular clones and two transmitted founder (T/F) viruses for their sensitivities to a panel of bNAbs in cell-free and cell-to-cell infection assays. We observed a relative resistance of cell-to-cell transmission to antibody neutralization that is reflected not only by reductions of antibody potency, but also by decreases in maximum neutralization capacity relative to cell-free infections. BNAbs targeting different epitopes exhibited incomplete neutralization against cell-associated virus with T/F Envs, which was not observed with cell-free form of the same virus. We further identified the membrane proximal internal tyrosine-based sorting motif as a determinant that can affect the incomplete neutralization of these T/F clones in cell-to-cell infection. These findings indicate that the signal that affects surface expression and/or internalization of Env from the plasma membrane can modulate the presentation of neutralizing epitopes on infected cells. These findings highlight that a fraction of virus can escape from high concentrations of antibody through cell-to-cell infection while maintaining sensitivity to neutralization in cell-free infection. The ability to fully inhibit cell-to-cell transmission may represent an important consideration in development of antibodies for treatment or prophylaxis.

    IMPORTANCE In recent years, isolation of new generation HIV-1 bNAbs has invigorated HIV vaccine research. These bNAbs display remarkable potency and breadth against cell-free virus, however, they exhibit diminished ability to block HIV-1 cell-to-cell transmission. The mechanism(s) by which HIV-1 resists neutralization when transmitting through VS remains uncertain. We examined a panel of bNAbs for their ability to neutralize HIV-1 T/F viruses in cell-to-cell infection assays. We find that some antibodies exhibit not only reduced potency but also decreased maximum neutralization capacity, or in vitro efficacy against cell-to-cell infection of HIV-1 with T/F Envs compared to cell-free infection of the same virus. We further identify the membrane proximal internal tyrosine-based sorting motif YXXL as a determinant that can affect the incomplete neutralization phenotype of these T/F clones. When the maximum neutralization capacity falls short of 100%, this can have a major impact on the ability of antibodies to halt viral replication.

  • The ATP-dependent RNA Helicase DDX3X modulates Herpes Simplex Virus Type 1 Gene Expression [PublishAheadOfPrint]

  • The human protein DDX3X is a DEAD-box ATP-dependent RNA helicase that regulates transcription, mRNA maturation, mRNA export and translation. DDX3X concomitantly modulates the replication of several RNA viruses and promotes innate immunity. We previously showed that herpes simplex virus type 1 (HSV-1), a human DNA virus, incorporates DDX3X into its mature particles and that DDX3X is required for optimal HSV-1 infectivity. Here, we show that viral gene expression, replication and propagation depend on optimal DDX3X protein levels. Surprisingly, DDX3X from incoming viral particles was not required for the early stages of the HSV-1 infection but the protein rather controlled the assembly of new viral particles. This was independent of the previously reported ability of DDX3X to stimulate interferon type I production. Instead, both the lack and overexpression of DDX3X disturbed viral gene transcription and thus subsequent genome replication. This suggests that in addition to its effect on RNA viruses, DDX3X impacts DNA viruses such as HSV-1 by an interferon-independent pathway.

    IMPORTANCE Viruses interact with a variety of cellular proteins to complete their life cycle. Among them is DDX3X, a RNA helicase that participates in most aspects of RNA biology, including transcription, splicing, nuclear export and translation. Several RNA viruses and a limited number of DNA viruses are known to manipulate DDX3X for their own benefit. In contrast, DDX3X is also known to promote interferon production to limit viral propagation. Here we show that DDX3X, which we previously identified in mature HSV-1 virions, stimulates HSV-1 gene expression and consequently virion assembly by a process that is independent of its ability to promote the interferon pathway.

  • Extreme mutation tolerance: Nearly half of the archaeal fusellovirus SSV1 genes are not required for virus function, including the minor capsid protein gene, vp3. [PublishAheadOfPrint]

  • Viruses infecting the Archaea harbor a tremendous amount of genetic diversity. This is especially true for the spindle-shaped viruses of the family Fuselloviridae, where ggt;90% of the viral genes do not have detectable homologs in public databases. This significantly limits our ability to elucidate the role of viral proteins in the infection cycle. To address this, we have developed genetic techniques to study the well-characterized fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1) which infects Sulfolobus solfataricus in volcanic hot springs at 80ddeg;C and pH 3.

    Here, we present a new comparative genome analysis and a thorough genetic analysis of SSV1 using both specific and random mutagenesis and thereby generate mutations in all ORFs. We demonstrate that almost half of the SSV1 genes are not essential for infectivity and the requirement for a particular gene correlates well with its degree of conservation within the Fuselloviridae. The major capsid gene vp1 is essential for SSV1 infectivity. However, the universally conserved minor capsid gene vp3 could be deleted without a loss in infectivity and results in virions with abnormal morphology.

    Importance: Most of the putative genes in the spindle-shaped archaeal hyperthermophile Fuselloviruses have no clearly similar sequences to characterized genes. In order to determine which of these SSV genes are important for function we disrupted all of the putative genes in the prototypical fusellovirus, SSV1. Surprisingly, about half of the genes could be disrupted without destroying virus function. Even deletions of one of the known structural protein genes that is present in all known Fuselloviruses, vp3, allow the production of infectious viruses. However, viruses lacking vp3 have abnormal shapes, indicating that the vp3 gene is important for virus structure. Identification of essential genes will allow focused research on minimal SSV genomes and further understanding of the structure of these unique, ubiquitous and extremely stable archaeal viruses.

  • A microRNA screen identifies the Wnt signaling pathway as a regulator of the interferon response during flavivirus infection. [PublishAheadOfPrint]

  • The impact of mosquito-borne flavivirus infections worldwide is significant, and many critical aspects of these viruses' biology, including virus-host interactions, host cell requirements for replication, and how virus-host interactions impact pathology, remain to be fully understood. The recent re-emergence and spread of flaviviruses, including dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV), highlights the importance of performing basic research on this important group of pathogens. MicroRNAs (miRNAs) are small, noncoding RNAs that modulate gene expression post-transcriptionally and have been demonstrated to regulate a broad range of cellular processes. Our research is focused on identifying pro- and anti-flaviviral miRNAs as a means of characterizing cellular pathways that support or limit viral replication. We have screened a library of known human miRNA mimics for their effect on replication of three flavivirusesmmdash;DENV, WNV, and Japanese encephalitis virus (JEV)mmdash;using a high content immunofluorescence screen. Several families of miRNAs were identified as inhibiting multiple flaviviruses, including the miR-34, miR-15, and miR-517 families. Members of the miR-34 family, which have been extensively characterized for their ability to repress Wnt/bbeta;-catenin signaling, demonstrated strong anti-flaviviral effects, and this inhibitory activity extended to other viruses, including ZIKV, alphaviruses, and herpesviruses. Previous research has suggested a possible link between the Wnt and type I interferon (IFN) signaling pathways. Therefore, we investigated the role of type I IFN induction in the antiviral effects of the miR-34 family and confirmed that these miRNAs potentiate IRF3 phosphorylation and translocation to the nucleus, induction of IFN-responsive genes, and release of type I IFN from transfected cells. We further demonstrate that the intersection between the Wnt and IFN signaling pathways occurs at the point of GSK3bbeta;-TBK1 binding, inducing TBK1 to phosphorylate IRF3 and initiate downstream IFN signaling. In this way, we have identified a novel cellular signaling network with a critical role in regulating replication of multiple virus families. These findings highlight the opportunities for using miRNAs as tools to discover and characterize unique cellular factors involved in supporting or limiting virus replication, opening up new avenues for antiviral research.

    Importance MicroRNAs are a class of small regulatory RNAs that modulate cellular processes through post-transcriptional repression of multiple transcripts. We hypothesized that individual miRNAs may be capable of inhibiting viral replication through their effects on host proteins or pathways. To test this, we performed a high content screen for miRNAs that inhibit replication of three medically relevant members of the flavivirus family: West Nile virus, Japanese encephalitis virus, and dengue virus-2. The results of this screen identify multiple miRNAs that inhibit one or more of these viruses. Extensive follow up on members of the miR-34 family of miRNAs, which are active against all three viruses as well as the closely related Zika virus, demonstrated that miR-34 functions through increasing the infected cell's ability to respond to infection through the interferon-based innate immune pathway. Our results not only add to the knowledge of how viruses interact with cellular pathways, but provide a basis for more extensive data mining by providing a comprehensive list of miRNAs capable of inhibiting flavivirus replication. Finally, the miRNAs themselves or cellular pathways identified as modulating virus infection may prove to be novel candidates for the development of therapeutic interventions.

  • Dinucleotide composition in animal RNA viruses is shaped more by virus family than host species [PublishAheadOfPrint]

  • Viruses use the cellular machinery of their hosts for replication. It has therefore been proposed that the nucleotide and dinucleotide composition of viruses should match that of their host species. If upheld, it may then be possible to use dinucleotide composition to predict the true host species of viruses sampled in metagenomic surveys. However, it is also clear that different taxonomic groups of viruses tend to have distinctive patterns of dinucleotide composition that may be independent of host species. To determine the relative strength of the effect of host versus virus family in shaping dinucleotide composition we performed a comparative analysis of 20 RNA virus families from 15 host groupings, spanning two animal phyla and more than 900 virus species. In particular, we determined the odds ratios for the 16 possible dinucleotides and performed a discriminant analysis to evaluate the capability of virus dinucleotide composition to predict the correct virus family or host taxon from which it was isolated. Notably, while 81% of the data analyzed here were predicted to the correct virus family, only 62% of these data were predicted to their correct subphylum/class host, and a mere 32% to their correct mammalian order. Similarly, dinucleotide composition has a weak predictive power for different hosts within individual virus families. We therefore conclude that dinucleotide composition is generally uniform within a virus family but less well reflects that of its host species. This has obvious implications for attempts to accurately predict host species from virus genome sequences alone.

    IMPORTANCE Determining the processes that shape virus genomes is central to understanding virus evolution and emergence. One question of particular importance is why nucleotide and dinucleotide frequencies differ so markedly between viruses? In particular, it is currently unclear whether host species or virus family has the biggest impact on dinucleotide frequencies, and whether dinucleotide composition can be used to accurately predict host species. Using a comparative analysis we show that dinucleotide composition has a strong phylogenetic association across different RNA virus families, such that dinucleotide composition can predict the family a virus sequence has been isolated from. Conversely, dinucleotide composition has a poorer predictive power for the different host species within a virus family and across different virus families, indicating that the host has a relatively small impact on the dinucleotide composition of a virus genome.

  • Computational prediction of the heterodimeric and higher order structure of gpE1/gpE2 envelope glycoproteins encoded by the hepatitis C virus (HCV). [PublishAheadOfPrint]

  • Despite the recent success of newly developed direct acting antivirals against Hepatitis C, the disease continues to be a global health threat due to lack of diagnosis of most carriers and the high cost of treatment. The heterodimer formed by the glycoproteins E1 and E2 within the hepatitis C virus (HCV) lipid envelope is a potential vaccine candidate and antiviral target. While the structure of E1/E2 has not yet been resolved, partial crystal structures of the E1 and E2 ectodomains have been determined. The unresolved parts of the structure are within the realm of what can be modeled with current computational modeling tools. Further, a variety of additional experimental data is available to support computational predictions of E1/E2 structure such as antibody binding studies, cryoEM, mutational analyses, peptide binding, linker-scanning mutagenesis and NMR studies. In accordance with these rich experimental data, we have built an in silico model of the full-length E1/E2 heterodimer. Our model supports that E1/E2 assembles into a trimer, which was previously suggested from a study by Falson and coworkers. Size exclusion chromatography and Western blotting data obtained using purified recombinant E1/E2 support our hypothesis. Our model suggests that during virus assembly the trimer of E1/E2 may further be assembled into a pentamer with 12 pentamers comprising a single HCV virion. We anticipate that this new model will provide a useful framework for HCV envelope structure and the development of antiviral strategies.

    Importance 150 million people have been estimated as infected with the Hepatitis C virus and many more are at risk for infection. A better understanding of the structure of the HCV viral envelope, which is responsible for attachment and fusion, could aid in the development of a vaccine and/or new treatments for this disease. We draw upon computational techniques to predict a full-length model of the E1/E2 heterodimer based on the partial crystal structures of the envelope glycoproteins E1 and E2. E1/E2 has been widely studied experimentally and this provides valuable data, which has assisted us in our modeling. Our proposed structure is used to suggest the organization of the HCV viral envelope. We also present new experimental data from size exclusion chromatography that supports our computational prediction of a trimeric oligomeric state of E1/E2.

  • Immunization with low doses of recombinant post-fusion or pre-fusion RSV F primes for vaccine-enhanced disease in the cotton rat model independent of the presence of a Th1-biasing (GLA-SE) or Th2-biasing (Alum) adjuvant [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) infection of children previously immunized with a non-live, formalin-inactivated (FI)-RSV vaccine was associated with serious enhanced respiratory disease (ERD). Consequently, detailed studies of potential ERD are a critical step in the development of non-live RSV vaccines targeting RSV-naïve children and infants. The fusion glycoprotein (F) of RSV in either its post- or pre-fusion conformation is a target for neutralizing antibodies and therefore an attractive antigen candidate for a pediatric RSV subunit vaccine. Here we report the evaluation of RSV post-F and pre-F in combination with GLA-SE and Alum adjuvants in the cotton rat model. Immunization with optimal doses of RSV F antigens in the presence of GLA-SE induced high titers of virus neutralizing antibodies, and conferred complete lung protection from virus challenge, with no ERD signs in the form of alveolitis. To mimic a waning immune response, and to assess priming for ERD under suboptimal conditions, an antigen dose de-escalation study was performed in the presence of either GLA-SE or Alum. At low RSV F doses, alveolitis-associated histopathology was unexpectedly observed with either adjuvant, at levels comparable to FI-RSV immunized controls. This occurred despite neutralizing antibody titers above the minimum levels required for protection, and with no/low virus replication in the lungs. These results emphasize the need to investigate a pediatric RSV vaccine candidate carefully for priming of ERD over a wide dose range, even in the presence of strong neutralizing activity, Th1 bias inducing adjuvant, and protection from virus replication in the lower respiratory tract.

    IMPORTANCE RSV disease is of great importance worldwide, with the highest burden of serious disease occurring upon primary infection in infants and children. FI-RSV-induced enhanced disease, observed in the 1960s, presented a major and ongoing obstacle for the development of non-live RSV vaccine candidates. The findings presented here underscore the need to evaluate a non-live RSV vaccine candidate during pre-clinical development over a wide dose range in the cotton rat RSV-enhanced disease model as suboptimal dosing of several RSV F subunit vaccine candidates led to the priming for ERD. These observations are relevant to the validity of the cotton rat model itself, and to safe development of non-live RSV vaccines for seronegative infants and children.

  • SARS Coronavirus Nucleocapsid Inhibits Type I Interferon Production by Interfering with TRIM25-Mediated RIG-I Ubiquitination [PublishAheadOfPrint]

  • Severe acute respiratory syndrome (SARS) is a respiratory disease caused by a coronavirus (SARS-CoV) that is characterized by atypical pneumonia. The nucleocapsid protein (N protein) of SARS-CoV plays an important role in inhibition of type I interferon (IFN) production via an unknown mechanism. In this study, the SARS-CoV N protein was found to bind to the SPRY domain of the tripartite motif protein 25 (TRIM25) E3 ubiquitin ligase, thereby interfering with the association between TRIM25 and retinoic acid-inducible gene I (RIG-I) and inhibiting TRIM25-mediated RIG-I ubiquitination and activation. Type I IFN production induced by poly I:C or Sendai virus (SeV) was suppressed by the SARS-CoV N protein. SARS-CoV replication was increased by over-expression of the full-length N protein but not N (1-361), which could not interact with TRIM25. These findings provide an insightful interpretation of the SARS-CoV-mediated host innate immune suppression caused by the N protein.

    Importance The SARS-CoV N protein is essential for the viral life cycle and plays a key role in the virus-host interaction. We demonstrated that the interaction between the C-terminus of the N protein and the SPRY domain of TRIM25 inhibited TRIM25-mediated RIG-I ubiquitination, which resulted in the inhibition of IFN production. We also found that the MERS-CoV N protein interacted with TRIM25 and inhibited RIG-I signaling. The outcomes of these findings indicate the function of the coronavirus N protein in modulating the host's initial innate immune response.

  • Patterns of cellular immunity associated with experimental infection with rDEN2{Delta}30 (Tonga/74) supports its suitability as a human DENV challenge strain [PublishAheadOfPrint]

  • A deletion variant of the DENV2 Tonga/74 strain lacking 30 nucleotides from its 3rrsquo; untranslated region (rDEN230) has previously been established for use in a controlled DENV human challenge model. To evaluate if this model is appropriate to derive correlates of protection for DENV vaccines based on cellular immunity, we wanted to compare how the cellular immune response to this challenge strain compares to the response induced by natural infection. To achieve this, we predicted HLA class I and class II restricted peptides from rDEN230 and used them, in an IFN-gamma ELISPOT assay, to interrogate CD8+ and CD4+ T cell responses in healthy volunteers infected with rDEN230. At the level of CD8 responses, vigorous ex vivo responses were detected in approximately 80% of donors. These responses were similar in terms of magnitude and numbers of epitopes recognized to previously reported responses observed in PBMC from donors from DENV hyper-endemic regions. The similarity extended to the immunodominance hierarchy of the DENV nonstructural proteins NS3, NS5, and NS1 being dominant in both donor cohorts. At the CD4 level, responses were less vigorous compared to natural DENV infection, and were more focused on nonstructural proteins. The epitopes recognized following rDEN230 infection and natural infection were largely overlapping for both CD8 (100%) and CD4 (85%) responses. Finally, rDEN230 induced stronger CD8 responses compared to other more attenuated DENV isolates.

    IMPORTANCE The lack of a known correlate of protection and the failure of a neutralizing antibody to correlate with protection against dengue have highlighted the need for human DENV challenge model to better evaluate the candidate live attenuated dengue vaccines. In this study we sought to characterize the immune profiling of rDEN230 infected subjects comparing them with subjects from areas where DENV is hyperendemic. Our data demonstrate that T cell responses to rDENV230 are largely similar to natural infection in terms of specificity, highlighting this virus as an appropriate human model for the T cell response to primary DENV2 infection.

  • Mutations during the adaptation of H9N2 avian influenza virus to the respiratory epithelium of pigs enhance the sialic acid binding activity and the virulence in mice [PublishAheadOfPrint]

  • The natural reservoir for influenza viruses is waterfowl from where they succeeded to cross the barrier to different mammalian species. We analyzed the adaptation of avian influenza viruses to a mammalian host by passaging an H9N2 strain three times in differentiated swine airway epithelial cells. Using precision-cut slices from the porcine lung to passage the parental virus, isolates from each of the three passages (P1-P3) were characterized by assessing growth curves and ciliostatic effects. The only difference noted was an increased growth kinetics of the P3 virus. Sequence analysis revealed four mutations: one each in the PB2 and NS1, and two in the HA protein. The HA mutations, A190V and T212I, were characterized by generating recombinant viruses containing either one or both amino acid exchanges. Whereas the parental virus recognized aalpha;2,3-linked sialic acids preferentially, the HA190 mutant bound to a broad spectrum of glycans with aalpha;2,6/8/9-linked sialic acids. The HA212 mutant alone differed only slightly from the parental virus; however, the combination of both mutations (HA190+HA212) increased the binding affinity to those glycans recognized by the HA190 mutant. Remarkably, only the HA double mutant showed a significantly increased pathogenicity in mice. By contrast, none of those mutations affected the ciliary activity of the epithelial cells which is characteristic for virulent swine influenza viruses. Taken together, our results indicate that shifts in the HA receptor affinity are just an early adaptation step of avian H9N2 strains; further mutational changes may be required to become virulent for pigs.

    IMPORTANCE Swine play an important role in the interspecies transmission of influenza viruses. Avian influenza A viruses (IAV) of the H9N2 subtype have successfully infected hosts from different species but not established a stable lineage. We have analyzed the adaptation of IAV-H9N2 virus to target cells of a new host by passaging the virus three times in differentiated porcine respiratory epithelial cells. Among the four mutations detected the two HA mutations were analyzed by generating recombinant viruses. Depending on the infection system used, the mutations differed in their phenotypic expression, e.g sialic acid binding activity, replication kinetics, plaque size, and pathogenicity in inbred mice. But none of the mutations affected the ciliary activity which serves as a virulence marker. Thus, early adaptative mutation enhance the replication kinetics but more mutations are required for IAV of the H9N2 subtype to become virulent.

  • Inactivated Recombinant Rabies Viruses Displaying the Canine Distemper Virus Glycoproteins Induce Protective Immunity Against Both Pathogens [PublishAheadOfPrint]

  • The development of multivalent vaccines is an attractive methodology for the simultaneous prevention of several infectious diseases in vulnerable populations. Canine distemper (CDV) and rabies (RABV) viruses both cause lethal disease in wild and domestic carnivores. While RABV vaccines are inactivated, the live-attenuated CDV vaccines retain residual virulence for highly susceptible wild life species. In the current study, we have developed recombinant bivalent vaccine candidates based on recombinant vaccine strain rabies virus particles, which concurrently display the protective CDV and RABV glycoprotein antigens. The recombinant viruses replicated to near wild type titers and the heterologous glycoproteins were efficiently expressed and incorporated in the viral particles. Immunization of ferrets with beta-propiolactone inactivated recombinant virus particles elicited protective RABV antibody titers, and animals immunized with a combination of CDV attachment and fusion protein-expressing recombinant viruses were protected from lethal CDV challenge. However, animals that were only immunized with a RABV expressing the attachment protein of the CDV vaccine strain Onderstepoort succumbed to the infection with a more recent wild type strain, indicating that immune responses to the more conserved fusion protein contribute to protection against heterologous CDV strains.

    IMPORTANCE Rabies and canine distemper virus (CDV) cause high mortality and death in many carnivores. While rabies vaccines are inactivated and thus have an excellent safety profile and high stability, live-attenuated CDV vaccines can retain residual virulence in highly susceptible species. Here we generated recombinant inactivated rabies viruses that carry one of the CDV glycoproteins on their surface. Ferrets immunized twice with a mix of recombinant rabies viruses carrying the CDV fusion and attachment glycoproteins resulted in protection from lethal CDV challenge, whereas all animals receiving recombinant rabies viruses carrying only the CDV attachment protein following the same immunization scheme died. Irrespective of the CDV antigens used, all animals developed protective titers against rabies virus, illustrating that a bivalent rabies-based vaccine against CDV induces protective immune responses against both pathogens.

  • M gene reassortment in H9N2 influenza virus promotes early infection and replication: contribution to rising virus prevalence in chickens in China [PublishAheadOfPrint]

  • Segment reassortment and base mutagenesis of influenza A viruses are the primary routes to the rapid evolution of high fitness virus genotypes. We recently described a predominant G57 genotype of avian H9N2 viruses that caused country-wide outbreaks in chickens in China during 2010-2013 which led to the zoonotic emergence of H7N9 viruses. One of the key features of the G57 genotype is the substitution of the earlier BJ/94-like M gene with the G1-like M gene of quail origin. We report here on the functional significance of the G1-like M gene in H9N2 viruses in conferring increased infection severity and infectivity in primary chicken embryonic fibroblasts and chickens. H9N2 virus housing the G1-like M gene, in place of BJ/94-like M gene, showed early surge in viral mRNA and vRNA transcription that were associated with enhanced viral protein production, and with early elevated release of progeny virus comprising largely spherical rather than filamentous virions. Importantly, H9N2 virus with G1-like M gene conferred extrapulmonary virus spread in chickens. Five highly represented signature amino acid residues (37A, 95K, 224N and 242N in M1 protein, and 21G in M2 protein) encoded by the prevalent G1-like M gene were demonstrated as prime contributors to enhanced infectivity. Therefore, the genetic evolution of M gene in H9N2 virus increases reproductive virus fitness, indicating its contribution to rising virus prevalence in chickens in China.

    Importance We recently described the circulation of a dominant genotype (G57) of H9N2 viruses in country-wide outbreaks in chickens in China, which was responsible through reassortment for the emergence of H7N9 viruses that cause severe human infections. A key feature of the G57 genotype H9N2 virus is the presence of quail origin G1-like M gene which had replaced the earlier BJ/94-like M gene. We found that H9N2 virus with G1-like M gene, but not BJ/94-like M gene, showed early surge in progeny virus production, more severe pathology and extrapulmonary virus spread in chickens. Five highly represented amino acid residues in M1 and M2 proteins derived from G1-like M gene were shown to mediate enhanced virus infectivity. These observations enhance what we currently know about the roles of reassortment and mutations on virus fitness and have implications for assessing the potential of variant influenza viruses that can cause rising prevalence in chickens.

  • Hepatitis B virus-encoded miRNA controls viral replication [PublishAheadOfPrint]

  • microRNAs (miRNAs) are a class of small single-stranded non-coding functional RNAs. Hepatitis B virus (HBV) is an enveloped DNA virus with virions and subviral forms of particles that lack a core. It was not known whether HBV encodes miRNAs. Here, we identified an HBV-encoded miRNA (called HBV-miR-3) by deep sequencing and northern blot. HBV-miR-3 is located at nts 373-393 of the HBV genome and was generated from 3.5Kb, 2,4Kb and 2.1Kb HBV in classic miRNA biogenesis (Drosha-Dicer dependent) manner. HBV-miR-3 was highly expressed in hepatoma cell lines with an integrated HBV genome and HBV (+) hepatoma tumors. In patients with HBV infection, HBV-miR-3 was released to circulation by exosome and HBV viron, and HBV-miR-3 expression was a positive correlation with HBV titers in the serum in the acute phase of patients with HBV infection. More interestingly, we found that HBV-miR-3 represses HBsAg, HBeAg and replication of HBV. HBV-miR-3 represses HBV replication by targeting the region of HBV 3.5 kb mRNA encoding HBV core protein (HBc) to reduce HBc protein and HBV pregenomic RNA (pgRNA), which in turn led to attenuate HBV replication. Overall, these results indicate that HBV encodes miRNA which may provide new potential biomarkers for clinical HBV infection and shed lights on a new mechanism of HBV replication regulation by which HBV-encoded miRNAs control self-replication via targeting viral transcripts.

    IMPORTANCE Hepatitis B is a liver infection caused by the hepatitis B virus (HBV), which can become a long-term, chronic infection and lead to cirrhosis or liver cancer. Hepatitis B virus (HBV) is a small DNA virus that belongs to the hepadnavirus family with virions and subviral forms of particles that lack a core. microRNA (miRNA), a small (~22 nt) non-coding RNA, is recently found to be an important regulator of gene expression. We found that HBV encodes miRNA (HBV-miR-3). More importantly, we revealed that HBV-miR-3 targets itself transcripts to attenuate HBV replication. This may contribute to explain that HBV infection leads to mild damage in live cells and the subsequent establishment/maintenance of its persistent infection. Our findings highlight a mechanism by which HBV-encoded miRNA controls the process of self-replication by regulating the virus itself during infection, and might provide new biomarkers for diagnostic and treatment of hepatitis B.

  • REPLICATION CAPACITY OF VIRUSES FROM ACUTE INFECTION DRIVES HIV-1 DISEASE PROGRESSION [PublishAheadOfPrint]

  • Viral genotype has been shown to play an important role in HIV pathogenesis following transmission. However, the viral phenotypic properties that contribute to disease progression remain unclear. Most studies have been limited to the evaluation of Gag function in the context of a recombinant virus backbone. Using this approach, important biological information may be lost making the evaluation of viruses obtained during acute infection, representing the transmitted virus, a more biologically relevant model. Here we evaluate the role of viral infectivity and replication capacity of viruses from acute infection on disease progression in women who seroconverted in the CAPRISA 004 tenofovir microbicide trial. We show that viral replication capacity but not viral infectivity correlates with set point viral load (Spearman r=0.346, p=0.045) and that replication capacity (HR=4.52, p=0.01) can predict CD4 decline independently of viral load (HR=2.9, p=0.004) or protective HLA alleles (HR=0.61, p=0.36). We further demonstrate that Gag-Pro is not the main driver of this association suggesting that additional properties of the transmitted virus play a role in disease progression. Finally, we find that although viruses from the tenofovir arm were two fold less infectious, they replicated at similar rates compared to viruses from the placebo arm. This indicates that the use of tenofovir gel did not select for viral variants with higher replication capacity. Overall, this study supports a strong influence of replication capacity in acute infection on disease progression, potentially driven by interaction of multiple genes rather than a dominant role of the major structural gene gag.

    IMPORTANCE HIV disease progression is known to differ between individuals and defining which fraction of this variation can be attributed to the virus is of importance both clinically and epidemiologically. In this study we show that the replication capacity of viruses isolated during acute infection predicts subsequent disease progression and drives CD4 decline independently of viral load. This provides further support for the hypothesis that the replication capacity of the transmitted virus determines the initial damage to the immune system, setting the pace for later disease progression. However, we did not find evidence that the major structural gene gag is driving this correlation, highlighting the importance of other genes in determining disease progression.

  • Recombinant Isfahan virus and vesicular stomatitis virus vaccine vectors provide durable, multivalent, single dose protection against lethal alphavirus challenge [PublishAheadOfPrint]

  • The demonstrated clinical efficacy of a recombinant vesicular stomatitis virus (rVSV) vaccine vector has stimulated the investigation of additional serologically distinct Vesiculovirus vectors as therapeutic and/or prophylactic vaccine vectors to combat emerging viral diseases. Amongst these viral threats are encephalitic alphaviruses, Venezuelan and eastern equine encephalitic viruses (VEEV, EEEV), which have demonstrated potential for natural disease outbreaks, yet no licensed vaccines are available in the event of an epidemic. Here we report rescue of recombinant Isfahan virus (rISFV) from genomic cDNA as a potential new vaccine vector platform. The rISFV genome was modified to attenuate virulence and engineered to express VEEV and EEEV E2/E1 surface glycoproteins as vaccine antigens. A single dose of the rISFV vaccine vectors elicited neutralizing antibody responses and protected mice from lethal VEEV and EEEV challenge at one month post-vaccination as well as lethal VEEV challenge at eight months post-vaccination. A mixture of rISFV vectors expressing VEEV and EEEV E2/E1 glycoproteins also provided durable, single-dose protection from lethal VEEV and EEEV challenge, demonstrating the potential for a multivalent vaccine formulation. These findings were paralleled in studies with an attenuated form of rVSV expressing VEEV E2/E1 glycoproteins. Both rVSV and rISFV vectors were attenuated using an approach which has demonstrated safety in human trials of an rVSV/HIV-1 vaccine. Vaccines based on either of these vaccine vector platforms may present a safe and effective approach to prevent alphavirus induced disease in humans.

    Importance: This work introduces rISFV as a novel vaccine vector platform that is serologically distinct and phylogenetically distant from VSV. The rISFV vector has been attenuated by an approach used for an rVSV vector that has demonstrated safety in clinical studies. The vaccine potential of the rISFV vector was investigated in a well-established alphavirus disease model. The findings indicate the feasibility of producing a safe, efficacious, multivalent vaccine against the encephalitic alphaviruses VEEV and EEEV, both of which can cause fatal disease. The work also demonstrates efficacy of an attenuated rVSV vector that has already demonstrated safety and immunogenicity in multiple HIV-1 Phase I clinical studies. The absence of serological cross-reactivity between rVSV and rISFV and their phylogenetic divergence within the Vesiculovirus genus indicates potential for two stand-alone vaccine vector platforms that could be used to target multiple bacterial and/or viral agents in successive immunization campaigns, or as heterologous prime-boost agents.

  • Neurovirulent murine coronavirus JHM.SD uses cellular zinc metalloproteases for virus entry and cell-cell fusion [PublishAheadOfPrint]

  • The coronavirus S protein requires cleavage by host cell proteases to mediate virus-cell and cell-cell fusion. Many strains of the murine coronavirus mouse hepatitis virus (MHV) have distinct, S-dependent organ and tissue tropisms despite using a common receptor, suggesting that they employ different cellular proteases for fusion. In support of this hypothesis, we found that inhibition of endosomal acidification only modestly decreased entry and overexpression of the cell surface protease TMPRSS2 greatly enhanced entry of the highly neurovirulent MHV strain JHM.SD relative to their effects on the reference strain A59. However, TMPRSS2 overexpression decreased MHV structural protein expression, release of infectious particles, and syncytia formation, and endogenous serine protease activity did not contribute greatly to infection. We therefore investigated the importance of other classes of cellular proteases and found that inhibition of MMP- and ADAM-family zinc metalloproteases markedly decreased both entry and cell-cell fusion. Suppression of virus by metalloprotease inhibition varied among tested cell lines and MHV S proteins, suggesting a role for metalloprotease use in strain-dependent tropism. We conclude that zinc metalloproteases must be considered potential contributors to coronavirus fusion.

    IMPORTANCE The family Coronaviridae includes viruses that cause two emerging diseases of humans, Severe Acute Respiratory Syndrome (SARS) Middle East Respiratory Syndrome (MERS), as well as a number of important animal pathogens. Because coronaviruses depend on host protease-mediated cleavage of their S proteins for entry, a number of protease inhibitors have been proposed as antiviral agents. However, it is unclear which proteases mediate in vivo infection: for example, SARS-CoV infection of cultured cells depends on endosomal acid pH-dependent proteases rather than on the cell-surface acid pH-independent serine protease TMPRSS2, but Zhou et al. (Antiviral Res 116:76-84, 2015, doi:10.1016/j.antiviral.2015.01.011) found that a serine protease inhibitor was more protective than a cathepsin inhibitor in SARS-CoV-infected mice. This paper explores the contributions of endosomal acidification and various proteases to coronavirus infection and identifies an unexpected class of proteases, the matrix metalloproteinase and A-Disintegrin-And-Metalloprotease (ADAM) families, as potential targets for anti-coronavirus therapy.

  • Different Expression of Interferon Stimulated Genes in Response to HIV-1 Infection in Dendritic Cells According to Their Maturation State. [PublishAheadOfPrint]

  • Dendritic cells (DCs) are professional antigen presenting cells whose functions are dependent on their degree of differentiation. In their immature state, DCs, capture pathogens and migrate to the lymph nodes. During this process DCs become resident mature cells specialized in antigen presentation. DCs are characterized by a highly limiting environment to HIV-1 replication due to the expression of restriction factors as SAMHD1 and APOBEC3G. However, uninfected DCs capture and transfer viral particles to CD4 lymphocytes through a trans-enhancement mechanism in which chemokines are involved. We analyzed changes in gene expression with whole-genome-microarray when immature (IDCs) or mature (MDCs) dendritic cells were productively infected using Vpx-loaded HIV-1 particles. Whereas productive HIV infection of IDCs induced expression of interferon stimulated genes (ISGs), such induction was not produced in MDCs in which a sharp decrease in ISG and CXCR3-binding chemokines was observed lessening trans-infection of CD4 lymphocytes. Similar patterns of gene expression were found when DCs were infected with HIV-2 that naturally express Vpx. Differences were also observed in conditions of restrictive HIV-1 infection, in the absence of Vpx. ISGs expression was not modified in IDCs whereas an increase of ISG and CXCR3-binding chemokines was observed in MDCs. Overall these results suggest that sensing and restriction of HIV-1 infection are different between IDCs and MDCs. We propose that restrictive infection results in increased virulence through different mechanisms. In IDC avoiding sensing and induction of ISGs whereas in MDC increased production of CXCR3-binding chemokines would result in lymphocyte attraction and enhanced infection at the immune synapse.

    IMPORTANCE In this work we describe for the first time the activation of a different genetic program during HIV-1 infection depending on the state of maturation of DCs. This represents a breakthrough in the understanding of the restriction to HIV-1 infection by DCs.

    The results show that infection of DCs by HIV-1, reprogram their gene expression pattern. In immature cells, productive HIV-1 infection activates IFN-related-genes involved in the control of viral replication thus inducing an antiviral state in surrounding cells. Paradoxically restriction of HIV-1 by SAMHD1 would result in lack of sensing and IFN activation thus favoring initial HIV-1 scape from innate immune response.

    In mature DCs restrictive infection results in HIV-1 sensing and induction of ISGs, in particular CXCR3-binding chemokines, that could favor the transmission of HIV to lymphocytes.

    Our data support the hypothesis that genetic DC reprograming by HIV-1 infection favors viral escape and dissemination thus increasing HIV-1 virulence.

  • Beta-propiolactone inactivation of coxsackievirus A16 induces structural alteration and surface modification of viral capsids [PublishAheadOfPrint]

  • Beta-propiolactone (BPL) is an inactivating agent widely used in the vaccine industry. However, its effects on vaccine protein antigens and its mechanisms of action remain poorly understood. Here we presented cryo-EM structures of BPL-treated coxsackievirus A16 (CVA16) mature virion and procapsid at resolutions of 3.9 AAring; and 6.5 AAring;, respectively. Notably, both particles were found to adopt an expanded conformation resembling the 135S-like uncoating intermediate, with characteristic features including an opened 2-fold channel, the externalization of the N-terminus of VP1 capsid protein as well as the absence of pocket factor. However, major neutralizing epitopes are very well preserved on these particles. Further biochemical analyses revealed that BPL treatment impairs the binding abilities of CVA16 particles to the attachment receptor heparan sulfate and to a conformation-dependent monoclonal antibody in a BPL dose-dependent manner, indicating that BPL is able to modify surface-exposed amino acid residues. Taken together, our results demonstrate that BPL treatment may induce alteration of the overall structure and surface properties of a non-enveloped viral capsid, thus revealing a novel mode of action of BPL.

    IMPORTANCE Beta-propiolactone (BPL) is commonly used as an inactivating reagent to produce viral vaccines. It is recognized that BPL inactivates viral infectivity through modification of viral nucleic acids. However, its effect on viral proteins remains largely unknown. Here, we present high-resolution cryo-EM structures of BPL-treated coxsackievirus A16 (CVA16) mature virion and procapsid, which reveals an expanded overall conformation and characteristic features that are typical for the 135S-like uncoating intermediate. We further show that BPL concentration affects the binding of inactivated CVA16 particles to its receptor/antibody. Thus, BPL treatment can alter overall structure and surface properties of viral capsids, which may lead to antigenic and immunogenic variations. Our findings provide important information for future development of BPL-inactivated vaccines.

  • Animal Models of Zika Virus Infection, Pathogenesis, and Immunity [PublishAheadOfPrint]

  • Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that now causes epidemics affecting millions of people on multiple continents. The virus has reached global attention because of some of its unusual epidemiological and clinical features including persistent infection in the male reproductive tract and sexual transmission, an ability to cross the placenta during pregnancy and infect the developing fetus to cause congenital malformations, and its association with Guillain-Barreeacute; syndrome in adults. This past year has witnessed an intensive effort by the global scientific community to understand the biology of ZIKV and to develop pathogenesis models for the rapid testing of possible countermeasures. Here, we review the recent advances, utility, and limitations of newly developed mouse and non-human primate models of ZIKV infection and pathogenesis.

  • Suppression of type I interferon signaling by E1A via RuvBL1/Pontin. [PublishAheadOfPrint]

  • Suppression of interferon signaling is of paramount importance to a virus. Interferon signaling will significantly reduce or halt the ability of a virus to replicate, therefore viruses have evolved sophisticated mechanisms that suppress activation of the interferon pathway or responsiveness of the infected cell to interferon. Adenovirus has multiple modes of inhibiting cellular response to interferon. Here we report that E1A, previously shown to regulate interferon signaling in multiple ways, inhibits interferon stimulated gene expression by modulating RuvBL1 function. RuvBL1 was previously shown to affect type I interferon signaling. E1A binds to RuvBL1 and is recruited to RuvBL1-regulated promoters in an interferon-dependent manner, preventing their activation. Depletion of RuvBL1 impairs adenovirus growth, but does not appear to significantly affect viral protein expression. Although RuvBL1 has been shown to play a role in cell growth, depletion of it had no effect on the ability of the virus to replicate its genome or drive cells into S-phase. E1A was found to bind to RuvBL1 via the C-terminus of E1A and this interaction was important for suppression of interferon stimulated gene transcriptional activation and recruitment of E1A to interferon-regulated promoters. Here we report the identification of RuvBL1 as a new target for adenovirus in its quest to suppress interferon response.

    IMPORTANCE For most viruses, suppression of the interferon signaling pathway is crucial to ensure a successful replicative cycle. Human adenovirus has evolved several different mechanisms that prevent activation of interferon or the ability of the cell to respond to interferon. The viral immediate early gene, E1A, was previously shown to affect interferon signaling in several different ways. Here we report a novel mechanism reliant on RuvBL1 that E1A uses to prevent activation of interferon stimulated gene expression following infection or interferon treatment. This adds to the growing knowledge of how viruses are able to inhibit interferon, and identifies a novel target used by adenovirus for modulation of cellular interferon pathway.

  • Innate Immune Response of Bat and Human Cells to Filoviruses: Commonalities and Distinctions [PublishAheadOfPrint]

  • Marburg (MARV) and Ebola (EBOV) viruses are zoonotic pathogens that cause severe hemorrhagic fever in humans. The natural reservoir of MARV is the Egyptian rousette bat (Rousettus aegyptiacus); that of EBOV is unknown but believed to be another bat species. The Egyptian rousette develops subclinical productive infection with MARV but is refractory to EBOV. Interaction of filoviruses with hosts is greatly affected by the viral interferon (IFN)-inhibiting domains (IID). Our study was aimed at characterization of innate immune responses to filoviruses and the role of filovirus IID in bat and human cells. The study demonstrated that EBOV and MARV replicate to similar levels in all tested cell lines, indicating that permissiveness for EBOV at cell and organism levels do not necessarily correlate. Filoviruses, particularly MARV, induced a potent innate immune response in rousette cells, which was generally stronger than in human cells. Both EBOV VP35 and VP24 IID were found to suppress the innate immune response in rousette cells, but only VP35 IID appeared to promote virus replication. Along with IFNaalpha; and IFNbbeta;, IFN was demonstrated to control filovirus infection in bat cells but not in human cells suggesting host species specificity of the antiviral effect. The antiviral effects of bat IFNs appeared not to correlate with induction of IFN-stimulated genes 54 and 56, which was detected in human cells ectopically expressing bat IFNaalpha; and IFNbbeta;. As bat IFN induced the type I IFN pathway, its antiviral effect is likely to be partially induced via llsquo;cross talk'.

    IMPORTANCE Bats serve as reservoirs for multiple emerging viruses including filoviruses, henipaviruses, lyssaviruses, and zoonotic coronaviruses. Although there is no evidence for symptomatic disease caused by either Marburg (MARV) or Ebola (EBOV) viruses in bats, spillover of these viruses into human populations causes deadly outbreaks. The reason for the lack of symptomatic disease in bats infected with filoviruses remains unknown. The outcome of a virus-host interaction depends on the ability of the host immune system to suppress viral replication and the ability of a virus to counteract the host defenses. Our study is a comparative analysis of the host innate immune response to either MARV or EBOV infection in bat and human cells and the role of viral interferon-inhibiting domains in the host innate immune responses. The data are useful for understanding the interactions of filoviruses with natural and accidental hosts and for identification of factors that influence filovirus evolution.

  • Transgenic CRISPR/Cas9-mediated viral gene targeting for antiviral therapy of Bombyx mori nucleopolyhedrovirus (BmNPV) [PublishAheadOfPrint]

  • We developed a novel anti-viral strategy by combining transposon-based transgenesis and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system for direct cleavage of the Bombyx mori nucleopolyhedrovirus (BmNPV) genome DNA to promote virus clearance in silkworms. We demonstrate that transgenic silkworms constitutively-expressing Cas9 and guide RNAs (gRNAs) targeting the BmNPV immediate early-1 (ie-1) gene and me53 gene effectively induce target-specific cleavage and subsequent mutagenesis, especially large segment deletions (~ 7 kilobase-pairs [kb]) in BmNPV genomes and thus exhibit a robust suppression of BmNPV proliferation. Transgenic animals exhibited higher and inheritable resistance to BmNPV infection when compared to wild-type animals. Our approach will not only contribute to modern sericulture but also shed light on future anti-viral therapy.

    Importance Pathogen genome targeting has shown its potential in anti-viral research. However, transgenic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system-mediated viral genome targeting has not been reported as an antiviral strategy in a natural animal host of a virus. Our data provide an effective approach against Bombyx mori nucleopolyhedrovirus (BmNPV) infection in a real-world biological system and demonstrate the potential of transgenic CRISPR/Cas9 systems in anti-viral research in other species.

  • Lack of ADCC breadth of human non-neutralizing anti-HIV-1 antibodies [PublishAheadOfPrint]

  • Anti-HIV-1 non-neutralizing antibodies (nnAbs) capable of antibody-dependent cellular cytotoxicity (ADCC) have been identified as a protective immune correlate in the RV144 vaccine efficacy trial. Broadly neutralizing antibodies (bNAbs) also mediate ADCC in cell culture and rely on their Fc region for optimal efficacy in animal models. Here, we selected 9 monoclonal nnAbs and 5 potent bNAbs, targeting various epitopes and conformations of the gp120/41 complex, and analyzed the potency of the two types of antibodies to bind and eliminate HIV-1-infected cells in culture. Regardless of their neutralizing activity, most of the selected antibodies recognized and killed cells infected with two laboratory-adapted HIV-1 strains. Some nnAbs also bound bystander cells that may have captured viral proteins. However, in contrast to the bNAbs, the nnAbs poorly bound to reactivated infected cells from 8 HIV-positive individuals and did not mediate effective ADCC against those cells. The nnAbs also inefficiently recognize cells infected with 8 different transmitted founder (T/F) isolates. Addition of a synthetic CD4 mimetic enhanced the binding and killing efficacy of some of the nnAbs in an epitope-dependent manner, without reaching the levels achieved by the most potent bNAbs. Overall, our data reveal important qualitative and quantitative differences between nnAbs and bNAbs in their ADCC capacity and strongly suggest that the breadth of recognition of HIV-1 by nnAbs is narrow.

    Importance Most of the anti-HIV antibodies generated by infected individuals do not display potent neutralizing activities. These non-neutralizing antibodies (nnAbs) with antibody-dependent cellular cytotoxicity (ADCC) have been identified as a protective immune correlate in the RV144 vaccine efficacy trial. However, in primate models, the nnAbs do not protect against SHIV acquisition. Thus, the role of nnAbs with ADCC activity in protecting from infection remains debatable. In contrast, broadly neutralizing antibodies (bNAbs) neutralize a large array of viral strains and mediate ADCC in cell culture. We analyzed the capacity of 9 nnAbs and 5 bNAbs to eliminate infected cells. We selected 18 HIV-1 strains, including virus reactivated from the reservoir of HIV+ individuals and transmitted-founder isolates. We report that the nnAbs poorly bind to cells infected with primary HIV-1 strains and do not mediate potent ADCC. Overall, our data show that the breadth of recognition of HIV-1 by nnAbs is narrow.

  • Parvovirus Expresses a Small Noncoding RNA that Plays an Essential Role in Virus Replication [PublishAheadOfPrint]

  • Human bocavirus 1 (HBoV1) belongs to species Primate bocaparvovirus of the genus Bocaparvovirus of the Parvoviridae family. HBoV1 causes acute respiratory tract infections in young children and has a selective tropism for the apical surface of well-differentiated human airway epithelia (HAE). In this study, we identify an additional HBoV1 gene, bocavirus-transcribed small non-coding RNA (BocaSR) within the 3rrsquo; non-coding region (nt 5199-5338) of the viral genome of positive sense. BocaSR is transcribed by RNA polymerase III (Pol III) from an intragenic promoter at similar levels to that of the capsid protein-coding mRNA and is essential for replication of the viral DNA in both transfected HEK293 and infected HAE cells. Mechanistically, we show that BocaSR regulates the expression of HBoV1-encoded non-structural proteins NS1, NS2, NS3, and NP1, but not NS4. BocaSR is similar to the adenovirus-associated type I (VAI) RNA in terms of both nucleotide sequence and secondary structure, but differs from it in that its regulation of viral protein expression is independent of RNA-activated protein kinase (PKR) regulation. Notably, BocaSR accumulates in the viral DNA replication centers within the nucleus and likely plays a direct role in replication of the viral DNA. Our findings reveal BocaSR to be a novel viral noncoding RNA that coordinates the expression of viral proteins and regulates replication of viral DNA within the nucleus. Thus, BocaSR may be a target for anti-viral therapies for HBoV and may also have utility in the production of recombinant HBoV vectors.

    Significance Human bocavirus 1 (HBoV1) is pathogenic to humans, causing acute respiratory tract infections in young children. In this study, we identify a novel HBoV1 gene that lies in the 3rrsquo; non-coding region of the viral positive sense genome and is transcribed by RNA polymerase III into a noncoding RNA of 140 nts. This bocavirus-transcribed small RNA (BocaSR) diverges from both adenovirus-associated (VA) RNAs and Epstein-Barr virus-encoded small RNAs (EBERs) with respect to RNA sequence, representing a third species of this kind of Pol III-dependent viral noncoding RNA and the first noncoding RNA identified in autonomous parvoviruses. Unlike the VA RNAs, BocaSR localizes to the viral DNA replication centers of the nucleus and is essential for expression of viral nonstructural proteins independent of RNA-activated protein kinase R and replication of HBoV1 genomes. The identification of BocaSR and its role in virus DNA replication reveals potential avenues for developing anti-viral therapies.

  • Monoclonal Antibodies to V2, V3, the CD4-binding site and gp41 HIV-1 Mediate Phagocytosis in a Dose-dependent Manner. [PublishAheadOfPrint]

  • In light of weak or absent neutralizing activity mediated by anti-V2 monoclonal Abs (mAbs), we tested whether they can mediate Ab-dependent cellular phagocytosis (ADCP) which is an important element of anti-HIV-1 immunity. We tested six anti-V2 mAbs and compared them with 21 mAbs specific for V3, the CD4-binding site (CD4bs), and gp41 derived from HIV-1 chronically infected individuals and produced by hybridoma cells. ADCP activity was measured by flow cytometry using uptake by THP-1 monocytic cells of fluorescent beads coated with gp120, gp41, BG505 SOSIP.664 or BG505 DS-SOSIP.664 complexed with mAbs. The ADCP activity measured by the area under the curve showed significantly higher activity of anti-gp41 mAbs compared to three other groups of mAbs tested using beads coated with monomeric gp41 or gp120; anti-V2 mAbs were dominant over anti-V3 and anti-CD4bs against clade C gp120ZM109. ADCP mediated by V2 and V3 mAbs was positive against stabilized DS-SOSIP.664 trimer but negligible against SOSIP.664 targets, suggesting a closed envelope conformation better exposes the variable loops. Two IgG3 mAbs against V2 and V3 regions displayed dominant ADCP activity over a panel of IgG1 mAbs. This superior ADCP activity was confirmed when two of three recombinant IgG3 anti-V2 mAbs were compared to IgG1 counterparts. The study demonstrated dominant ADCP activity of anti-gp41 against monomers but not trimers with some higher activity of anti-V2 mAbs over anti-V3 and anti-CD4bs mAbs. The ability to mediate ADCP suggests a mechanism by which anti-HIV-1 envelope Abs can contribute to protective efficacy.

    IMPORTANCE Anti-V2 antibodies (Abs) correlated with reduced risk of HIV-1 infection in recipients of the RV144 vaccine suggesting that they play a protective role, but a mechanism providing such protection remains to be determined. The rare and weak neutralizing activities of anti-V2 mAbs prompted us to study Fc-mediated activities. We compared anti-V2 mAbs with other mAbs specific for V3, CD4bs and gp41 for Ab-dependent cellular phagocytosis (ADCP) activity, implicated in protective immunity. The anti-V2 mAbs displayed stronger activity compared to other anti-gp120 mAbs when screened against one of two gp120s and against DS-SOSIP which mimics the native trimer. Anti-gp41 mAbs were superior when targeting monomeric gp41, but were comparable against trimers which may not adequately expose gp41 epitopes. While anti-envelope mAbs in general mediated ADCP, anti-V2 mAbs displayed some dominance compared to other mAbs. Our demonstration that anti-V2 mAbs mediate ADCP suggests a functional mechanism for their contribution to protective efficacy.

  • TNF{alpha} Induced Recruitment of Inflammatory Mononuclear Cells Leads to Inflammation and Altered Brain Development in MCMV Infected Newborn Mice [PublishAheadOfPrint]

  • Congenital human cytomegalovirus (HCMV) infection is a significant cause of abnormal neurodevelopment and long term neurological sequelae in infants and children. Resident cell populations of the developing brain have been suggested to be more susceptible to virus-induced cytopathology, a pathway thought to contribute to the clinical outcomes following intrauterine HCMV infection. However, recent findings in a newborn mouse model of this infection in the developing brain have indicated that elevated levels of proinflammatory mediators leading to mononuclear cell activation and recruitment could underlie the abnormal neurodevelopment. In this study, we demonstrated that treatment with TNFaalpha; neutralizing antibodies decreased the frequency of CD45+/Ly6C hi/CD11b+/CCR2+ activated myeloid mononuclear cells (MMC) and the levels of proinflammatory cytokines in the blood and the brains of murine CMV infected mice. This treatment also normalized neurodevelopment in infected mice without significantly impacting the level of virus replication. These results indicate that TNFaalpha; is a major component of the inflammatory response associated with altered neurodevelopment that follows murine CMV infection of the developing brain and that a subset of peripheral blood myeloid mononuclear cells represent a key effector cell population in this model of virus-induced inflammatory disease of the developing brain.

    IMPORTANCE Congenital human cytomegalovirus (HCMV) is the most common viral infection of the developing human fetus and can result in neurodevelopmental sequelae. Mechanisms of disease leading to neurodevelopmental deficits in infected infants remain undefined but postulated pathways include loss of neuronal progenitor cells, damage to the developing vascular system of the brain, and altered cellular positioning. Direct virus-mediated cytopathic effects cannot explain the phenotypes of brain damage in most infected infants. Using a mouse model that recapitulates characteristics of the brain infection described in human infants, we have shown that TNFaalpha; plays a key role in brain inflammation, including recruitment of inflammatory mononuclear cells. Neutralization of TNFaalpha; normalized neurodevelopmental abnormalities in infected mice providing evidence that virus-induced inflammation is a major component of disease in the developing brain. These results suggest that interventions limiting inflammation associated with this infection could potentially improve the neurologic outcome of infants infected in-utero with HCMV.

  • MMP-9 Facilitates Hepatitis B Virus Replication through Binding with IFNAR1 to Repress IFN/JAK/STAT Signaling [PublishAheadOfPrint]

  • Hepatitis B virus (HBV) infection may cause acute hepatitis B (AHB), chronic hepatitis B (CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). However, the mechanisms by which HBV evades host immunity and maintains chronic infection are largely unknown. Here, we revealed that matrix metalloproteinase-9 (MMP-9) is activated in peripheral blood mononuclear cells (PBMCs) of HBV-infected patients, and HBV stimulates MMP-9 expression in macrophages and PBMCs isolated from healthy individuals. MMP-9 plays important roles in the breakdown of extracellular matrix and in the facilitation of tumor progression, invasion, metastasis and angiogenesis. MMP-9 also regulates respiratory syncytial virus (RSV) replication, but the mechanism underlying such regulation is unknown. We further demonstrated that MMP-9 facilitates HBV replication by repressing interferon/Janus kinase/signal transducers and activators of transcription (IFN/JAK/STAT) pathway, IFN action, STAT1/2 phosphorylation and IFN-stimulated genes (ISGs) expression. Moreover, MMP-9 binds to type I IFN receptor 1 (IFNAR1) and facilitates IFNAR1 phosphorylation, ubiquitination, sub-cellular distribution and degradation to interfere the binding of IFANR1 with IFN-aalpha;. Thus, we identified a novel positive feedback regulation loop between HBV replication and MMP-9 production. On one hand, HBV activates MMP-9 in infected patients and leukocytes. On the other hand, MMP-9 facilitates HBV replication through repressing IFN/JAK/STAT signaling, IFNAR1 function, and IFN-aalpha; action. Therefore, HBV may take the advantage of MMP-9 function to establish or maintain chronic infection.

    Importance Hepatitis B virus (HBV) infection may cause chronic hepatitis B (CHB) and hepatocellular carcinoma (HCC). However, the mechanisms by which HBV maintains chronic infection are largely unknown. Matrix metalloproteinase-9 (MMP-9) plays important roles in the facilitation of tumor progression, invasion, metastasis and angiogenesis. However, the effects of MMP-9 on HBV replication and pathogenesis are not known. This study reveals that MMP-9 expression is activated in patients with CHB and HBV stimulates MMP-9 production in PBMCs and macrophages. More interestingly, MMP-9 in turn promotes HBV replication through suppressing IFN-aalpha; action. Moreover, MMP-9 interacts with type I interferon receptor 1 (IFNAR1) to disturb the binding of IFN-aalpha; with IFNAR1 and facilitate phosphorylation, ubiquitination, sub-cellular distribution and degradation of IFNAR1. Therefore, these results discover a novel role of MMP-9 in viral replication, and reveal a new mechanism by which HBV evades host immunity to maintain persistent infection.

  • Molecular analysis of Arthrobacter myovirus vB_ArtM-ArV1: we blame it on the tail [PublishAheadOfPrint]

  • This is the first report on a myophage that infects Arthrobacter. A novel virus vB_ArtM-ArV1 (ArV1) was isolated from soil using Arthrobacter sp. 68b strain for phage propagation. Transmission electron microscopy showed its resemblance to members of the family Myoviridae: ArV1 has an isometric head (~74 nm in diameter) and a contractile non-flexible tail (~192 nm). Phylogenetic and comparative sequence analyses, however, revealed that ArV1 has more genes in common with phages from the family Siphoviridae than it does with any myovirus characterized to date. The genome of ArV1 is a linear, circularly permuted, double-stranded DNA molecule (71,200 bp) with a GC content of 61.6 %. The genome codes for 101 ORFs, yet contains no tRNA genes. More than 50% of ArV1 genes encode unique proteins that either have no reliable identity to database entries, or have homologues only in Arthrobacter phages, both sipho- and myoviruses. Using bioinformatics approaches, 13 ArV1 structural genes were identified, including those coding for head, tail, tail fiber, and baseplate proteins. A further 6 ArV1 ORFs were annotated as putative structural proteins based on the results of proteomic analysis. Phylogenetic analysis based on the alignment of four conserved virion proteins revealed that Arthrobacter myophages form a discreet clade that seems to occupy a somewhat intermediate position between myo- and siphoviruses. Thus, the data presented here will help to advance our understanding of genetic diversity and evolution of phages that constitute the order Caudovirales.

    Importance Bacteriophages, which likely have originated in the early Precambrian Era, represent the most numerous population on the planet. Approximately 95% of known phages are tailed viruses that comprise three families: Podoviridae (short tail), Siphoviridae (long noncontractile tail), and Myoviridae (contractile tail). Based on the current hypothesis, myophages, which may have evolved from siphophages, are thought to have first emerged among Gram-negative bacteria, whereas only later - among Gram-positive bacteria. The results on the molecular characterization of a myophage vB_ArtM-ArV1 presented here conform to the aforementioned hypothesis since, at a glance, bacteriophage vB_ArtM-ArV1 appears to be a siphovirus that possesses a seemingly functional contractile tail. Our work demonstrates that such "chimeric" myophages are of cosmopolitan nature, and are likely characteristic to ecologically important soil bacterium Arthrobacter.

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