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Jean-Yves Sgro
Inst. for Mol.Virology
731B Bock Labs
1525 Linden Drive Madison, WI 53706

Table of Contents for this page:

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

    JVI Current Issue

  • Editorial Board [Masthead]

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

  • How Viruses Hijack the ERAD Tuning Machinery [Gems]

  • An essential step during the intracellular life cycle of many positive-strand RNA viruses is the rearrangement of host cell membranes to generate membrane-bound replication platforms. For example, Nidovirales and Flaviviridae subvert the membrane of the endoplasmic reticulum (ER) for their replication. However, the absence of conventional ER and secretory pathway markers in virus-induced ER-derived membranes has for a long time hampered a thorough understanding of their biogenesis. Recent reports highlight the analogies between mouse hepatitis virus-, equine arteritis virus-, and Japanese encephalitis virus-induced replication platforms and ER-associated degradation (ERAD) tuning vesicles (or EDEMosomes) that display nonlipidated LC3 at their cytosolic face and segregate the ERAD factors EDEM1, OS-9, and SEL1L from the ER lumen. In this Gem, we briefly summarize the current knowledge on ERAD tuning pathways and how they might be hijacked for viral genome replication. As ERAD tuning components, such as SEL1L and nonlipidated LC3, appear to contribute to viral infection, these cellular pathways represent novel candidate drug targets to combat positive-strand RNA viruses.

  • High-Resolution Structure of a Virally Encoded DNA-Translocating Conduit and the Mechanism of DNA Penetration [Gems]

  • Although X174 DNA pilot protein H is monomeric during procapsid assembly, it forms an oligomeric tube on the host cell surface. Reminiscent of a double-stranded DNA phage tail in form and function, the H tube transports the single-stranded X174 genome across the Escherichia coli cell wall. The 2.4-AAring; resolution H-tube crystal structure suggests functional and energetic mechanisms that may be common features of DNA transport through virally encoded conduits.

  • Unexpected Structural Features of the Hepatitis C Virus Envelope Protein 2 Ectodomain [Minireviews]

  • Hepatitis C virus (HCV), a member of the family Flaviviridae, is a leading cause of chronic liver disease and cancer. Recent advances in HCV therapeutics have resulted in improved cure rates, but an HCV vaccine is not available and is urgently needed to control the global pandemic. Vaccine development has been hampered by the lack of high-resolution structural information for the two HCV envelope glycoproteins, E1 and E2. Recently, Kong and coworkers (Science 342:1090nndash;1094, 2013, doi:10.1126/science.1243876) and Khan and coworkers (Nature 509[7500]:381nndash;384, 2014, doi:10.1038/nature13117) independently determined the structure of the HCV E2 ectodomain core with some unexpected and informative results. The HCV E2 ectodomain core features a globular architecture with antiparallel bbeta;-sheets forming a central bbeta; sandwich. The residues comprising the epitopes of several neutralizing and nonneutralizing human monoclonal antibodies were also determined, which is an essential step toward obtaining a fine map of the human humoral response to HCV. Also clarified were the regions of E2 that directly bind CD81, an important HCV cellular receptor. While it has been widely assumed that HCV E2 is a class II viral fusion protein (VFP), the newly determined structure suggests that the HCV E2 ectodomain shares structural and functional similarities only with domain III of class II VFPs. The new structural determinations suggest that the HCV glycoproteins use a different mechanism than that used by class II fusion proteins for cell fusion.

  • Contribution of Glutamine Residues in the Helix 4-5 Loop to Capsid-Capsid Interactions in Simian Immunodeficiency Virus of Macaques [Structure and Assembly]

  • Following retrovirus entry, the viral capsid (CA) disassembles into its component capsid proteins. The rate of this uncoating process, which is regulated by CA-CA interactions and by the association of the capsid with host cell factors like cyclophilin A (CypA), can influence the efficiency of reverse transcription. Inspection of the CA sequences of lentiviruses reveals that several species of simian immunodeficiency viruses (SIVs) have lost the glycine-proline motif in the helix 4-5 loop important for CypA binding; instead, the helix 4-5 loop in these SIVs exhibits an increase in the number of glutamine residues. In this study, we investigated the role of these glutamine residues in SIVmac239 replication. Changes in these residues, particularly glutamine 89 and glutamine 92, resulted in a decreased efficiency of core condensation, decreased stability of the capsids in infected cells, and blocks to reverse transcription. In some cases, coexpression of two different CA mutants produced chimeric virions that exhibited higher infectivity than either parental mutant virus. For this complementation of infectivity, glutamine 89 was apparently required on one of the complementing pair of mutants and glutamine 92 on the other. Modeling suggests that glutamines 89 and 92 are located on the distal face of hexameric capsid spokes and thus are well positioned to contribute to interhexamer interactions. Requirements to evade host restriction factors like TRIMCyp may drive some SIV lineages to evolve means other than CypA binding to stabilize the capsid. One solution used by several SIV strains consists of glutamine-based bonding.

    IMPORTANCE The retroviral capsid is an assembly of individual capsid proteins that surrounds the viral RNA. After a retrovirus enters a cell, the capsid must disassemble, or uncoat, at a proper rate. The interactions among capsid proteins contribute to this rate of uncoating. We found that some simian immunodeficiency viruses use arrays of glutamine residues, which can form hydrogen bonds efficiently, to keep their capsids stable. This strategy may allow these viruses to forego the use of capsid-stabilizing factors from the host cell, some of which have antiviral activity.

  • Interferon-Induced Protein Ifit2 Protects Mice from Infection of the Peripheral Nervous System by Vesicular Stomatitis Virus [Pathogenesis and Immunity]

  • The interferon system provides the first line of host defense against virus infection. Mouse pathogenesis studies have revealed the importance of specific interferon-induced proteins in providing protection against specific viruses. We have previously reported that one such protein, Ifit2, protects neurons of the central nervous system from intranasal infection by the neurotropic rhabdovirus, vesicular stomatitis virus (VSV). Here, we demonstrate that Ifit2 protects the peripheral nervous system from VSV infection as well. In Ifit2nndash;/nndash; mice, VSV, injected subcutaneously into the footpad, entered the proximal lymph node, where it replicated and infected the nodal nerve endings. The infection spread to the sciatic nerve, the spinal cord, and the brain, causing paralysis. In contrast, in the wild-type mice, although VSV replicated equally well in the lymph node, infection of the sciatic nerve and the rest of the nervous system was impaired, thus preventing paralysis. Ifit2 protected only the nervous system from VSV infection; other tissues were well protected even in Ifit2nndash;/nndash; mice. These results indicate that Ifit2 is the interferon-induced protein that prevents VSV infection of neurons of both the peripheral and the central nervous systems, thus inhibiting the consequent neuropathy, but it is dispensable for protecting the cells of other tissues from VSV infection.

    IMPORTANCE Although viral infection is quite common, the immune system effectively protects us from viral diseases. A major part of this protection is mediated by interferon, the antiviral cytokine secreted by virus-infected cells. To empower the neighboring uninfected cells in combating the oncoming infection, interferon induces the synthesis of more than 200 new proteins, many of which have antiviral activities. The virus studied here, vesicular stomatitis virus (VSV), like its relative, rabies virus, can cause neuropathy in mice if it enters the peripheral nervous system through skin lesions; however, interferon can protect neurons from VSV infection. We have identified a specific interferon-induced protein, Ifit2, as the protein that protects neurons from VSV infection. Surprisingly, Ifit2 was not needed to protect other cell types from VSV. Our results indicate that the effector antiviral proteins of the interferon system have highly specialized functions.

  • Development of a Gaussia Luciferase-Based Human Norovirus Protease Reporter System: Cell Type-Specific Profile of Norwalk Virus Protease Precursors and Evaluation of Inhibitors [Vaccines and Antiviral Agents]

  • Norwalk virus (NV) is the prototype strain of human noroviruses (HuNoVs), a group of positive-strand RNA viruses in the Caliciviridae family and the leading cause of epidemic gastroenteritis worldwide. Investigation of HuNoV replication and development of antiviral therapeutics in cell culture remain challenging tasks. Here, we present NoroGLuc, a HuNoV protease reporter system based on a fusion of NV p41 protein with a naturally secreted Gaussia luciferase (GLuc), linked by the p41/p22 cleavage site for NV protease (Pro). trans cleavage of NoroGLuc by NV Pro or Pro precursors results in release and secretion of an active GLuc. Using this system, we observed a cell type-specific activity profile of NV Pro and Pro precursors, suggesting that the activity of NV Pro is modulated by other viral proteins in the precursor forms and strongly influenced by cellular factors. NoroGLuc was also cleaved by Pro and Pro precursors generated from replication of NV stool RNA in transfected cells, resulting in a measurable increase of secreted GLuc. Truncation analysis revealed that the N-terminal membrane association domain of NV p41 is critical for NoroGLuc activity. Although designed for NV, a genogroup GI.1 norovirus, NoroGLuc also efficiently detects Pro activities from GII.3 and GII.4 noroviruses. At noncytotoxic concentrations, protease inhibitors ZnCl2 and Naalpha;-p-tosyl-l-lysine chloromethyl ketone (TLCK) exhibited dose-dependent inhibitory effects on a GII.4 Pro by NoroGLuc assay. These results establish NoroGLuc as a pan-genogroup HuNoV protease reporter system that can be used for the study of HuNoV proteases and precursors, monitoring of viral RNA replication, and evaluation of antiviral agents.

    IMPORTANCE Human noroviruses are the leading cause of epidemic gastroenteritis worldwide. Currently, there are no vaccines or antiviral drugs available to counter these highly contagious viruses. These viruses are currently noncultivatable in cell culture. Here, we report the development of a novel cell-based reporter system called NoroGLuc that can be used for studying norovirus replication and also for screening/evaluation of antiviral agents. This system is based on the fusion between viral protein p41 and a naturally secreted Gaussia luciferase (GLuc) with a cleavage site that can be recognized by the viral protease. Cleavage of this fusion protein by the viral protease results in the release and secretion of an active GLuc. Using NoroGLuc, we demonstrated a cell type-specific activity profile of the viral protease and its precursors and dose-dependent inhibitory effects of two protease inhibitors. This novel reporter system should be useful in probing norovirus replication and evaluating antiviral agents.

  • Three-Dimensional Imaging of HIV-1 Virological Synapses Reveals Membrane Architectures Involved in Virus Transmission [Virus-Cell Interactions]

  • HIV transmission efficiency is greatly increased when viruses are transmitted at virological synapses formed between infected and uninfected cells. We have previously shown that virological synapses formed between HIV-pulsed mature dendritic cells (DCs) and uninfected T cells contain interdigitated membrane surfaces, with T cell filopodia extending toward virions sequestered deep inside invaginations formed on the DC membrane. To explore membrane structural changes relevant to HIV transmission across other types of intercellular conjugates, we used a combination of light and focused ion beam scanning electron microscopy (FIB-SEM) to determine the three-dimensional (3D) architectures of contact regions between HIV-1-infected CD4+ T cells and either uninfected human CD4+ T cells or human fetal astrocytes. We present evidence that in each case, membrane extensions that originate from the uninfected cells, either as membrane sheets or filopodial bridges, are present and may be involved in HIV transmission from infected to uninfected cells. We show that individual virions are distributed along the length of astrocyte filopodia, suggesting that virus transfer to the astrocytes is mediated, at least in part, by processes originating from the astrocyte itself. Mechanisms that selectively disrupt the polarization and formation of such membrane extensions could thus represent a possible target for reducing viral spread.

    IMPORTANCE Our findings lead to new insights into unique aspects of HIV transmission in the brain and at T cell-T cell synapses, which are thought to be a predominant mode of rapid HIV transmission early in the infection process.

  • Host Factors That Interact with the Pestivirus N-Terminal Protease, Npro, Are Components of the Ribonucleoprotein Complex [Virus-Cell Interactions]

  • The viral N-terminal protease Npro of pestiviruses counteracts cellular antiviral defenses through inhibition of IRF3. Here we used mass spectrometry to identify a new role for Npro through its interaction with over 55 associated proteins, mainly ribosomal proteins and ribonucleoproteins, including RNA helicase A (DHX9), Y-box binding protein (YBX1), DDX3, DDX5, eIF3, IGF2BP1, multiple myeloma tumor protein 2, interleukin enhancer binding factor 3 (IEBP3), guanine nucleotide binding protein 3, and polyadenylate-binding protein 1 (PABP-1). These are components of the translation machinery, ribonucleoprotein particles (RNPs), and stress granules. Significantly, we found that stress granule formation was inhibited in MDBK cells infected with a noncytopathic bovine viral diarrhea virus (BVDV) strain, Kyle. However, ribonucleoproteins binding to Npro did not inhibit these proteins from aggregating into stress granules. Npro interacted with YBX1 though its TRASH domain, since the mutant C112R protein with an inactive TRASH domain no longer redistributed to stress granules. Interestingly, RNA helicase A and La autoantigen relocated from a nuclear location to form cytoplasmic granules with Npro. To address a proviral role for Npro in RNP granules, we investigated whether Npro affected RNA interference (RNAi), since interacting proteins are involved in RISC function during RNA silencing. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) silencing with small interfering RNAs (siRNAs) followed by Northern blotting of GAPDH, expression of Npro had no effect on RNAi silencing activity, contrasting with other viral suppressors of interferon. We propose that Npro is involved with virus RNA translation in the cytoplasm for virus particle production, and when translation is inhibited following stress, it redistributes to the replication complex.

    IMPORTANCE Although the pestivirus N-terminal protease, Npro, has been shown to have an important role in degrading IRF3 to prevent apoptosis and interferon production during infection, the function of this unique viral protease in the pestivirus life cycle remains to be elucidated. We used proteomic mass spectrometry to identify novel interacting proteins and have shown that Npro is present in ribosomal and ribonucleoprotein particles (RNPs), indicating a translational role in virus particle production. The virus itself can prevent stress granule assembly from these complexes, but this inhibition is not due to Npro. A proviral role to subvert RNA silencing through binding of these host RNP proteins was not identified for this viral suppressor of interferon.

  • Late Endosomal Trafficking of Alternative Serotype Adenovirus Vaccine Vectors Augments Antiviral Innate Immunity [Vaccines and Antiviral Agents]

  • Adenovirus (Ad) vaccine vectors have found widespread use as vaccine platforms against multiple infections and cancers, and multiple serotypes have been shown to differ significantly in their biological properties and immune phenotypes. Our laboratory and others have previously described differential innate immune stimulation elicited by various Ad serotypes. Here, we show that Ad serotype 5 (Ad5) traffics rapidly to the nucleus following infection, whereas Ad35 and Ad26 accumulate in late endosomes 2 to 8 h postinfection. Innate immune cytokine elicitation by all Ad serotypes was abrogated by blockade of endosomal acidification, cathepsin B, and caspase 1, suggesting that virus interactions with acid-dependent sensors, such as Toll-like receptor- and cathepsin-dependent inflammasome activation in late endosomes, may trigger innate immunity. These data suggest a mechanism by which Ad vectors from various serotypes differentially trigger innate antiviral pathways via distinct intracellular trafficking to late endosomes.

    IMPORTANCE Adenoviruses (Ad) are widely used for vaccination and gene therapy applications. Importantly, Ad vectors have been shown to differ significantly in their innate immune profiles both in vivo and in vitro. The molecular mechanism that underlies these observed differences has important implications for the development of improved vaccines. In this study, we propose a mechanism in which the degree of late endosomal trafficking of Ad vectors results in differential stimulation of late endosomal pattern recognition receptors.

  • Modulation of Stop Codon Read-Through Efficiency and Its Effect on the Replication of Murine Leukemia Virus [Genome Replication and Regulation of Viral Gene Expression]

  • Translational readthroughmmdash;suppression of termination at a stop codonmmdash;is exploited in the replication cycles of several viruses and represents a potential target for antiviral intervention. In the gammaretroviruses, typified by Moloney murine leukemia virus (MuLV), gag and pol are in the same reading frame, separated by a UAG stop codon, and termination codon readthrough is required for expression of the viral Gag-Pol fusion protein. Here, we investigated the effect on MuLV replication of modulating readthrough efficiency. We began by manipulating the readthrough signal in the context of an infectious viral clone to generate a series of MuLV variants in which readthrough was stimulated or reduced. In carefully controlled infectivity assays, it was found that reducing the MuLV readthrough efficiency only 4-fold led to a marked defect and that a 10-fold reduction essentially abolished replication. However, up to an ~8.5-fold stimulation of readthrough (up to 60% readthrough) was well tolerated by the virus. These high levels of readthrough were achieved using a two-plasmid system, with Gag and Gag-Pol expressed from separate infectious clones. We also modulated readthrough by silencing expression of eukaryotic release factors 1 and 3 (eRF1 and eRF3) or by introducing aminoglycosides into the cells. The data obtained indicate that gammaretroviruses tolerate a substantial excess of viral Gag-Pol synthesis but are very sensitive to a reduction in levels of this polyprotein. Thus, as is also the case for ribosomal frameshifting, antiviral therapies targeting readthrough with inhibitory agents are likely to be the most beneficial.

    IMPORTANCE Many pathogenic RNA viruses and retroviruses use ribosomal frameshifting or stop codon readthrough to regulate expression of their replicase enzymes. These translational "recoding" processes are potential targets for antiviral intervention, but we have only a limited understanding of the consequences to virus replication of modulating the efficiency of recoding, particularly for those viruses employing readthrough. In this paper, we describe the first systematic analysis of the effect of increasing or decreasing readthrough efficiency on virus replication using the gammaretrovirus MuLV as a model system. We find unexpectedly that MuLV replication is only slightly inhibited by substantial increases in readthrough frequency, but as with other viruses that use recoding strategies, replication is quite sensitive to even modest reductions. These studies provide insights into both the readthrough process and MuLV replication and have implications for the selection of antivirals against gammaretroviruses.

  • Genogroup IV and VI Canine Noroviruses Interact with Histo-Blood Group Antigens [Virus-Cell Interactions]

  • Human noroviruses (HuNV) are a significant cause of viral gastroenteritis in humans worldwide. HuNV attaches to cell surface carbohydrate structures known as histo-blood group antigens (HBGAs) prior to internalization, and HBGA polymorphism among human populations is closely linked to susceptibility to HuNV. Noroviruses are divided into 6 genogroups, with human strains grouped into genogroups I (GI), II, and IV. Canine norovirus (CNV) is a recently discovered pathogen in dogs, with strains classified into genogroups IV and VI. Whereas it is known that GI to GIII noroviruses bind to HBGAs and GV noroviruses recognize terminal sialic acid residues, the attachment factors for GIV and GVI noroviruses have not been reported. This study sought to determine the carbohydrate binding specificity of CNV and to compare it to the binding specificities of noroviruses from other genogroups. A panel of synthetic oligosaccharides were used to assess the binding specificity of CNV virus-like particles (VLPs) and identified aalpha;1,2-fucose as a key attachment factor. CNV VLP binding to canine saliva and tissue samples using enzyme-linked immunosorbent assays (ELISAs) and immunohistochemistry confirmed that aalpha;1,2-fucose-containing H and A antigens of the HBGA family were recognized by CNV. Phenotyping studies demonstrated expression of these antigens in a population of dogs. The virus-ligand interaction was further characterized using blockade studies, cell lines expressing HBGAs, and enzymatic removal of candidate carbohydrates from tissue sections. Recognition of HBGAs by CNV provides new insights into the evolution of noroviruses and raises concerns regarding the potential for zoonotic transmission of CNV to humans.

    IMPORTANCE Infections with human norovirus cause acute gastroenteritis in millions of people each year worldwide. Noroviruses can also affect nonhuman species and are divided into 6 different groups based on their capsid sequences. Human noroviruses in genogroups I and II interact with histo-blood group antigen carbohydrates, bovine noroviruses (genogroup III) interact with alpha-galactosidase (aalpha;-Gal) carbohydrates, and murine norovirus (genogroup V) recognizes sialic acids. The canine-specific strains of norovirus are grouped into genogroups IV and VI, and this study is the first to characterize which carbohydrate structures they can recognize. Using canine norovirus virus-like particles, this work shows that representative genogroup IV and VI viruses can interact with histo-blood group antigens. The binding specificity of canine noroviruses is therefore very similar to that of the human norovirus strains classified into genogroups I and II. This raises interesting questions about the evolution of noroviruses and suggests it may be possible for canine norovirus to infect humans.

  • Impact of HLA Selection Pressure on HIV Fitness at a Population Level in Mexico and Barbados [Pathogenesis and Immunity]

  • Previous studies have demonstrated that effective cytotoxic T lymphocyte (CTL) responses drive the selection of escape mutations that reduce viral replication capacity (VRC). Escape mutations, including those with reduced VRC, can be transmitted and accumulate in a population. Here we compared two antiretroviral therapy (ART)-naive HIV clade B-infected cohorts, in Mexico and Barbados, in which the most protective HLA alleles (HLA-B*27/57/58:01/81:01) are differentially expressed, at 8% and 34%, respectively. Viral loads were significantly higher in Mexico than in Barbados (median, 40,774 versus 14,200; P llt; 0.0001), and absolute CD4+ T-cell counts were somewhat lower (median, 380/mm3 versus 403/mm3; P = 0.007). We tested the hypothesis that the disparate frequencies of these protective HLA alleles would be associated with a higher VRC at the population level in Mexico. Analysis of VRC in subjects in each cohort, matched for CD4+ T-cell count, revealed that the VRC was indeed higher in the Mexican cohort (mean, 1.13 versus 1.03; P = 0.0025). Although CD4 counts were matched, viral loads remained significantly higher in the Mexican subjects (P = 0.04). This VRC difference was reflected by a significantly higher frequency in the Barbados cohort of HLA-B*27/57/58:01/81:01-associated Gag escape mutations previously shown to incur a fitness cost on the virus (P = 0.004), a difference between the two cohorts that remained statistically significant even in subjects not expressing these protective alleles (P = 0.01). These data suggest that viral set points and disease progression rates at the population level may be significantly influenced by the prevalence of protective HLA alleles such as HLA-B*27/57/58:01/81:01 and that CD4 count-based guidelines to initiate antiretroviral therapy may need to be modified accordingly, to optimize the effectiveness of treatment-for-prevention strategies and reduce HIV transmission rates to the absolute minimum.

    IMPORTANCE Immune control of HIV at an individual level is strongly influenced by the HLA class I genotype. HLA class I molecules mediating effective immune control, such as HLA-B*27 and HLA-B*57, are associated with the selection of escape mutants that reduce viral replicative capacity. The escape mutants selected in infected patients can be transmitted and affect the viral load and CD4 count in the recipient. These findings prompt the hypothesis that the frequency of protective alleles in a population may affect viral set points and rates of disease progression in that population. These studies in Mexico and Barbados, where the prevalence rates of protective HLA alleles are 8% and 34%, respectively, support this hypothesis. These data suggest that antiretroviral therapy (ART) treatment-for-prevention strategies will be less successful in populations such as those in Mexico, where viral loads are higher for a given CD4 count. Consideration may therefore usefully be given to ART initiation at higher absolute CD4 counts in such populations to optimize the impact of ART for prevention.

  • Virus and Host Factors Affecting the Clinical Outcome of Bluetongue Virus Infection [Pathogenesis and Immunity]

  • Bluetongue is a major infectious disease of ruminants caused by bluetongue virus (BTV), an arbovirus transmitted by Culicoides. Here, we assessed virus and host factors influencing the clinical outcome of BTV infection using a single experimental framework. We investigated how mammalian host species, breed, age, BTV serotypes, and strains within a serotype affect the clinical course of bluetongue. Results obtained indicate that in small ruminants, there is a marked difference in the susceptibility to clinical disease induced by BTV at the host species level but less so at the breed level. No major differences in virulence were found between divergent serotypes (BTV-8 and BTV-2). However, we observed striking differences in virulence between closely related strains of the same serotype collected toward the beginning and the end of the European BTV-8 outbreak. As observed previously, differences in disease severity were also observed when animals were infected with either blood from a BTV-infected animal or from the same virus isolated in cell culture. Interestingly, with the exception of two silent mutations, full viral genome sequencing showed identical consensus sequences of the virus before and after cell culture isolation. However, deep sequencing analysis revealed a marked decrease in the genetic diversity of the viral population after passaging in mammalian cells. In contrast, passaging in Culicoides cells increased the overall number of low-frequency variants compared to virus never passaged in cell culture. Thus, Culicoides might be a source of new viral variants, and viral population diversity can be another factor influencing BTV virulence.

    IMPORTANCE Bluetongue is one of the major infectious diseases of ruminants. It is caused by an arbovirus known as bluetongue virus (BTV). The clinical outcome of BTV infection is extremely variable. We show that there are clear links between the severity of bluetongue and the mammalian host species infected, while at the breed level differences were less evident. No differences were observed in the virulence of two different BTV serotypes (BTV-8 and BTV-2). In contrast, we show that the European BTV-8 strain isolated at the beginning of the bluetongue outbreak in 2006 was more virulent than a strain isolated toward the end of the outbreak. In addition, we show that there is a link between the variability of the BTV population as a whole and virulence, and our data also suggest that Culicoides cells might function as an "incubator" of viral variants.

  • Downregulation of Interleukin-18-Mediated Cell Signaling and Interferon Gamma Expression by the Hepatitis B Virus e Antigen [Virus-Cell Interactions]

  • The mechanisms by which hepatitis B virus (HBV) establishes and maintains chronic hepatitis B infection (CHB) are poorly defined. Innate immune responses play an important role in reducing HBV replication and pathogenesis. HBV has developed numerous mechanisms to escape these responses, including the production of the secreted hepatitis B e antigen (HBeAg), which has been shown to regulate antiviral toll-like receptor (TLR) and interleukin-1 (IL-1) signaling. IL-18 is a related cytokine that inhibits HBV replication in hepatoma cell lines and in the liver through the induction of gamma interferon (IFN-) by NK cells and T cells. We hypothesized that HBV or HBV proteins inhibit IFN- expression by NK cells as an accessory immunomodulatory function. We show that HBeAg protein inhibits the NF-B pathway and thereby downregulates NK cell IFN- expression. Additionally, IFN- expression was significantly inhibited by exposure to serum from individuals with HBeAg-positive but not HBeAg-negative chronic HBV infection. Further, we show that the HBeAg protein suppresses IL-18-mediated NF-B signaling in NK and hepatoma cells via modulation of the NF-B pathway. Together, these findings show that the HBeAg inhibits IL-18 signaling and IFN- expression, which may play an important role in the establishment and/or maintenance of persistent HBV infection.

    IMPORTANCE It is becoming increasingly apparent that NK cells play a role in the establishment and/or maintenance of chronic hepatitis B infection. The secreted HBeAg is an important regulator of innate and adaptive immune responses. We now show that the HBeAg downregulates NK cell-mediated IFN- production and IL-18 signaling, which may contribute to the establishment of infection and/or viral persistence. Our findings build on previous studies showing that the HBeAg also suppresses the TLR and IL-1 signaling pathways, suggesting that this viral protein is a key regulator of antiviral innate immune responses.

  • Poly(I:C) Treatment Leads to Interferon-Dependent Clearance of Hepatitis B Virus in a Hydrodynamic Injection Mouse Model [Vaccines and Antiviral Agents]

  • We have previously shown that poly(I:C) activates murine hepatic cells to produce interferon (IFN) and suppresses hepatitis B virus (HBV) replication in vitro. Therefore, we addressed whether poly(I:C) is able to induce the clearance of HBV in vivo. The chronic HBV replication mouse model was established by the hydrodynamic injection (HI) of pAAV-HBV1.2 into the tail veins of wild-type and IFN-aalpha;/bbeta;R-, IFN--, and CXCR3-deficient C57BL/6 mice. Fourteen days post-HI of pAAV-HBV1.2, mice were administered poly(I:C) by intraperitoneal injection, intramuscular injection, or HI. Only treatment of poly(I:C) by HI led to HBV clearance in wild-type C57BL/6 mice. Serum HBsAg disappeared within 40 days postinfection (dpi) in mice that received poly(I:C) by HI, and this was accompanied by the appearance of anti-HBs antibodies. HBV-specific T-cell and antibody responses were significantly enhanced by HI of poly(I:C). HBV replication intermediates and HBcAg-positive hepatocytes were eliminated in the liver. HI of poly(I:C) induced the production of IFNs in mice and enhanced the levels of cytokines, IFN-stimulated genes, and T-cell markers in the liver. Importantly, poly(I:C)-induced HBV clearance was impaired in IFN-aalpha;/bbeta;R-, IFN--, and CXCR3-deficient mice, indicating that the induction of type I IFN and the stimulation and recruitment of T cells into the liver are essential for HBV clearance in this model. Taken together, the application of poly(I:C) by HI into the liver enhances innate and adaptive immune responses and leads to HBV clearance in an HBV mouse model, implicating the potential of intrahepatic Toll-like receptor 3 (TLR3) activation for the treatment of chronic hepatitis B patients.

    IMPORTANCE It has become well accepted that immunomodulation is a potentially useful approach to treat chronic viral infection. Recently, combinations of antiviral treatment and therapeutic vaccinations were evaluated for therapies of chronic hepatitis B virus (HBV) infection. Activation of the innate immune branch may also be important for viral control and contributes to HBV clearance. Our present study demonstrated that hepatic TLR3 activation led to clearance of hepatitis B virus in an HBV mouse model. For the first time, we showed that HBV clearance in this model is dependent not only on type I interferon (IFN) but also on type II IFN, indicating a coordinated action of innate and adaptive immune responses. T-cell recruitment appeared to be critical for the success of TLR3-mediated antiviral action. These findings implicate the potential of intrahepatic TLR3 activation for the treatment of chronic HBV infection.

  • Interactions between the Influenza A Virus RNA Polymerase Components and Retinoic Acid-Inducible Gene I [Virus-Cell Interactions]

  • The influenza A virus genome possesses eight negative-strand RNA segments in the form of viral ribonucleoprotein particles (vRNPs) in association with the three viral RNA polymerase subunits (PB2, PB1, and PA) and the nucleoprotein (NP). Through interactions with multiple host factors, the RNP subunits play vital roles in replication, host adaptation, interspecies transmission, and pathogenicity. In order to gain insight into the potential roles of RNP subunits in the modulation of the host's innate immune response, the interactions of each RNP subunit with retinoic acid-inducible gene I protein (RIG-I) from mammalian and avian species were investigated. Studies using coimmunoprecipitation (co-IP), bimolecular fluorescence complementation (BiFc), and colocalization using confocal microscopy provided direct evidence for the RNA-independent binding of PB2, PB1, and PA with RIG-I from various hosts (human, swine, mouse, and duck). In contrast, the binding of NP with RIG-I was found to be RNA dependent. Expression of the viral NS1 protein, which interacts with RIG-I, did not interfere with the association of RNA polymerase subunits with RIG-I. The association of each individual virus polymerase component with RIG-I failed to significantly affect the interferon (IFN) induction elicited by RIG-I and 5' triphosphate (5'ppp) RNA in reporter assays, quantitative reverse transcription-PCR (RT-PCR), and IRF3 phosphorylation tests. Taken together, these findings indicate that viral RNA polymerase components PB2, PB1, and PA directly target RIG-I, but the exact biological significance of these interactions in the replication and pathogenicity of influenza A virus needs to be further clarified.

    IMPORTANCE RIG-I is an important RNA sensor to elicit the innate immune response in mammals and some bird species (such as duck) upon influenza A virus infection. Although the 5'-triphosphate double-stranded RNA (dsRNA) panhandle structure at the end of viral genome RNA is responsible for the binding and subsequent activation of RIG-I, this structure is supposedly wrapped by RNA polymerase complex (PB2, PB1, and PA), which may interfere with the induction of RIG-I signaling pathway. In the present study, PB2, PB1, and PA were found to individually interact with RIG-Is from multiple mammalian and avian species in an RNA-independent manner, without significantly affecting the generation of IFN. The data suggest that although RIG-I binding by RNA polymerase complex is conserved in different species, it does not appear to play crucial role in the modulation of IFN in vitro.

  • Modulation of CD163 Expression by Metalloprotease ADAM17 Regulates Porcine Reproductive and Respiratory Syndrome Virus Entry [Cellular Response to Infection]

  • As a consequence of their effects on ectodomain shedding, members of the A disintegrin and metalloprotease (ADAM) family have been implicated in the control of various cellular processes. Although ADAM family members are also involved in cancer, inflammation, and other pathologies, it is unclear whether they affect porcine reproductive and respiratory syndrome virus (PRRSV) infection. Here, we demonstrate for the first time that inhibition of ADAM17 enhances PRRSV entry in Marc-145 and porcine alveolar macrophages (PAMs). We also demonstrate that the inhibition of ADAM17 upregulates membrane CD163 expression, a putative PRRSV receptor that is exogenously expressed in BHK-21 and endogenously expressed in Marc-145 and PAMs. Furthermore, overexpression of ADAM17 induced downregulation of CD163 expression and a reduction in PRRSV infection, whereas ablation of ADAM17 expression using specific small interfering RNA resulted in upregulation of CD163 expression with a corresponding increase in PRRSV infection. These ADAM17-mediated effects were confirmed with PRRSV nonpermissive BHK-21 cells transfected with CD163 cDNA. Overall, these findings indicate that ADAM17 downregulates CD163 expression and hinders PRRSV entry. Hence, downregulation of ADAM17 particular substrates may be an additional component of the anti-infection defenses.

    IMPORTANCE ADAM17 is one of the important membrane-associated metalloproteases that mediate various cellular events, as well as inflammation, cancer, and other pathologies. Here, we investigate for the first time the role of the metalloprotease ADAM17 in PRRSV infection. By using inhibitor and genetic modification methods, we demonstrate that ADAM17 negatively regulate PRRSV entry by regulating its substrate(s). More specifically, ADAM 17 mediates the downregulation of the PRRSV cellular receptor CD163. The reduction in CD163 expression represents another component of the anti-infection response initiated by ADAM17.

  • Fine Mapping of Murine Antibody Responses to Immunization with a Novel Soluble Form of Hepatitis C Virus Envelope Glycoprotein Complex [Vaccines and Antiviral Agents]

  • The hepatitis C virus (HCV) envelope glycoprotein E1E2 complex is a candidate vaccine antigen. Previous immunization studies of E1E2 have yielded various results on its ability to induce virus-neutralizing antibodies in animal models and humans. The murine model has become a vital tool for HCV research owing to the development of humanized mice susceptible to HCV infection. In this study, we investigated the antibody responses of mice immunized with E1E2 and a novel soluble form of E1E2 (sE1E2) by a DNA prime and protein boost strategy. The results showed that sE1E2 elicited higher antibody titers and a greater breadth of reactivity than the wild-type cell-associated E1E2. However, immune sera elicited by either immunogen were only weakly neutralizing. In order to understand the contrasting results of binding and serum neutralizing activities, epitopes targeted by the polyclonal antibody responses were mapped and monoclonal antibodies (MAbs) were generated. The results showed that the majority of serum antibodies were directed to the E1 region 211 to 250 and the E2 regions 421 to 469, 512 to 539, 568 to 609, and 638 to 651, instead of the well-known immunodominant E2 hypervariable region 1 (HVR1). Unexpectedly, in MAb analysis, ~12% of MAbs isolated were specific to the conserved E2 antigenic site 412 to 423, and 85% of them cross-neutralized multiple HCV isolates. The epitopes recognized by these MAbs are similar but distinct from the previously reported HCV1 and AP33 broadly neutralizing epitopes. In conclusion, E1E2 can prime B cells specific to conserved neutralizing epitopes, but the levels of serum neutralizing antibodies elicited are insufficient for effective virus neutralization. The sE1E2 constructs described in this study can be a useful template for rational antigen engineering.

    IMPORTANCE Hepatitis C virus infects 2 to 3% of the world's population and is a leading cause of liver failures and the need for liver transplantation. The virus envelope glycoprotein complex E1E2 produced by detergent extraction of cells overexpressing the protein was evaluated in a phase I clinical trial but failed to induce neutralizing antibodies in most subjects. In this study, we designed a novel form of E1E2 which is secreted from cells and is soluble and compared it to wild-type E1E2 by DNA immunization of mice. The results showed that this new E1E2 is more immunogenic than wild-type E1E2. Detailed mapping of the antibody responses revealed that antibodies to the conserved E2 antigenic site 412 to 423 were elicited but the serum concentrations were too low to neutralize the virus effectively. This soluble E1E2 provides a new reagent for studying HCV and for rational vaccine design.

  • Role of Electrostatics in the Assembly Pathway of a Single-Stranded RNA Virus [Structure and Assembly]

  • We have recently discovered (R. D. Cadena-Nava et al., J. Virol. 86:3318nndash;3326, 2012, doi:10.1128/JVI.06566-11) that the in vitro packaging of RNA by the capsid protein (CP) of cowpea chlorotic mottle virus is optimal when there is a significant excess of CP, specifically that complete packaging of all of the RNA in solution requires sufficient CP to provide charge matching of the N-terminal positively charged arginine-rich motifs (ARMS) of the CPs with the negatively charged phosphate backbone of the RNA. We show here that packaging results from the initial formation of a charge-matched protocapsid consisting of RNA decorated by a disordered arrangement of CPs. This protocapsid reorganizes into the final, icosahedrally symmetric nucleocapsid by displacing the excess CPs from the RNA to the exterior surface of the emerging capsid through electrostatic attraction between the ARMs of the excess CP and the negative charge density of the capsid exterior. As a test of this scenario, we prepare CP mutants with extra and missing (relative to the wild type) cationic residues and show that a correspondingly smaller and larger excess, respectively, of CP is needed for complete packaging of RNA.

    IMPORTANCE Cowpea chlorotic mottle virus (CCMV) has long been studied as a model system for the assembly of single-stranded RNA viruses. While much is known about the electrostatic interactions within the CCMV virion, relatively little is known about these interactions during assembly, i.e., within intermediate states preceding the final nucleocapsid structure. Theoretical models and coarse-grained molecular dynamics simulations suggest that viruses like CCMV assemble by the bulk adsorption of CPs onto the RNA driven by electrostatic attraction, followed by structural reorganization into the final capsid. Such a mechanism facilitates assembly by condensing the RNA for packaging while simultaneously concentrating the local density of CP for capsid nucleation. We provide experimental evidence of such a mechanism by demonstrating that efficient assembly is initiated by the formation of a disordered protocapsid complex whose stoichiometry is governed by electrostatics (charge matching of the anionic RNA and the cationic N termini of the CP).

  • Changes in Protein Domains outside the Catalytic Site of the Bacteriophage Q{beta} Replicase Reduce the Mutagenic Effect of 5-Azacytidine [Genetic Diversity and Evolution]

  • The high genetic heterogeneity and great adaptability of RNA viruses are ultimately caused by the low replication fidelity of their polymerases. However, single amino acid substitutions that modify replication fidelity can evolve in response to mutagenic treatments with nucleoside analogues. Here, we investigated how two independent mutants of the bacteriophage Qbbeta; replicase (Thr210Ala and Tyr410His) reduce sensitivity to the nucleoside analogue 5-azacytidine (AZC). Despite being located outside the catalytic site, both mutants reduced the mutation frequency in the presence of the drug. However, they did not modify the type of AZC-induced substitutions, which was mediated mainly by ambiguous base pairing of the analogue with purines. Furthermore, the Thr210Ala and Tyr410His substitutions had little or no effect on replication fidelity in untreated viruses. Also, both substitutions were costly in the absence of AZC or when the action of the drug was suppressed by adding an excess of natural pyrimidines (uridine or cytosine). Overall, the phenotypic properties of these two mutants were highly convergent, despite the mutations being located in different domains of the Qbbeta; replicase. This suggests that treatment with a given nucleoside analogue tends to select for a unique functional response in the viral replicase.

    IMPORTANCE In the last years, artificial increase of the replication error rate has been proposed as an antiviral therapy. In this study, we investigated the mechanisms by which two substitutions in the Qbbeta; replicase confer partial resistance to the mutagenic nucleoside analogue AZC. As opposed to previous work with animal viruses, where different mutations selected sequentially conferred nucleoside analogue resistance through different mechanisms, our results suggest that there are few or no alternative AZC resistance phenotypes in Qbbeta;. Also, despite resistance mutations being highly costly in the absence of the drug, there was no sequential fixation of secondary mutations. Bacteriophage Qbbeta; is the virus with the highest reported mutation rate, which should make it particularly sensitive to nucleoside analogue treatments, probably favoring resistance mutations even if they incur high costs. The results are also relevant for understanding the possible pathways by which fidelity of the replication machinery can be modified.

  • Virus-Induced Tubule: a Vehicle for Rapid Spread of Virions through Basal Lamina from Midgut Epithelium in the Insect Vector [Virus-Cell Interactions]

  • The plant reoviruses, plant rhabdoviruses, tospoviruses, and tenuiviruses are transmitted by insect vectors in a persistent propagative manner. These viruses induce the formation of viral inclusions to facilitate viral propagation in insect vectors. The intestines of insect vectors are formed by epithelial cells that lie on the noncellular basal lamina surrounded by visceral muscle tissue. Here, we demonstrate that a recently identified plant reovirus, southern rice black-streaked dwarf virus (SRBSDV), exploits virus-containing tubules composed of virus-encoded nonstructural protein P7-1 to directly cross the basal lamina from the initially infected epithelium toward visceral muscle tissues in the intestine of its vector, the white-backed planthopper (Sogatella furcifera). Furthermore, such tubules spread along visceral muscle tissues through a direct interaction of P7-1 and actin. The destruction of tubule assembly by RNA interference with synthesized double-stranded RNA targeting the P7-1 gene inhibited viral spread in the insect vector in vitro and in vivo. All these results show for the first time that a virus employs virus-induced tubule as a vehicle for viral spread from the initially infected midgut epithelium through the basal lamina, facilitating the rapid dissemination of virus from the intestine of the insect vector.

    IMPORTANCE Numerous plant viruses are transmitted in a persistent manner by sap-sucking insects, including thrips, aphids, planthoppers, and leafhoppers. These viruses, ingested by the insects, establish their primary infection in the intestinal epithelium of the insect vector. Subsequently, the invading virus manages to transverse the basal lamina, a noncellular layer lining the intestine, a barrier that may theoretically hinder viral spread. The mechanism by which plant viruses cross the basal lamina is unknown. Here, we report that a plant virus has evolved to exploit virus-induced tubules to pass through the basal lamina from the initially infected midgut epithelium of the insect vector, thus revealing the previously undescribed pathway adapted by the virus for rapid dissemination of virions from the intestine of the insect vector.

  • Development of Giant Bacteriophage {phi}KZ Is Independent of the Host Transcription Apparatus [Genome Replication and Regulation of Viral Gene Expression]

  • Pseudomonas aeruginosa bacteriophage KZ is the type representative of the giant phage genus, which is characterized by unusually large virions and genomes. By unraveling the transcriptional map of the ~280-kb KZ genome to single-nucleotide resolution, we combine 369 KZ genes into 134 operons. Early transcription is initiated from highly conserved AT-rich promoters distributed across the KZ genome and located on the same strand of the genome. Early transcription does not require phage or host protein synthesis. Transcription of middle and late genes is dependent on protein synthesis and mediated by poorly conserved middle and late promoters. Unique to KZ is its ability to complete its infection in the absence of bacterial RNA polymerase (RNAP) enzyme activity. We propose that transcription of the KZ genome is performed by the consecutive action of two KZ-encoded, noncanonical multisubunit RNAPs, one of which is packed within the virion, another being the product of early genes. This unique, rifampin-resistant transcriptional machinery is conserved within the diverse giant phage genus.

    IMPORTANCE The data presented in this paper offer, for the first time, insight into the complex transcriptional scheme of giant bacteriophages. We show that Pseudomonas aeruginosa giant phage KZ is able to infect and lyse its host cell and produce phage progeny in the absence of functional bacterial transcriptional machinery. This unique property can be attributed to two phage-encoded putative RNAP enzymes, which contain very distant homologues of bacterial bbeta; and bbeta;'-like RNAP subunits.

  • A Novel Life Cycle Modeling System for Ebola Virus Shows a Genome Length-Dependent Role of VP24 in Virus Infectivity [Structure and Assembly]

  • Work with infectious Ebola viruses is restricted to biosafety level 4 (BSL4) laboratories, presenting a significant barrier for studying these viruses. Life cycle modeling systems, including minigenome systems and transcription- and replication-competent virus-like particle (trVLP) systems, allow modeling of the virus life cycle under BSL2 conditions; however, all current systems model only certain aspects of the virus life cycle, rely on plasmid-based viral protein expression, and have been used to model only single infectious cycles. We have developed a novel life cycle modeling system allowing continuous passaging of infectious trVLPs containing a tetracistronic minigenome that encodes a reporter and the viral proteins VP40, VP24, and GP1,2. This system is ideally suited for studying morphogenesis, budding, and entry, in addition to genome replication and transcription. Importantly, the specific infectivity of trVLPs in this system was ~500-fold higher than that in previous systems. Using this system for functional studies of VP24, we showed that, contrary to previous reports, VP24 only very modestly inhibits genome replication and transcription when expressed in a regulated fashion, which we confirmed using infectious Ebola viruses. Interestingly, we also discovered a genome length-dependent effect of VP24 on particle infectivity, which was previously undetected due to the short length of monocistronic minigenomes and which is due at least partially to a previously unknown function of VP24 in RNA packaging. Based on our findings, we propose a model for the function of VP24 that reconciles all currently available data regarding the role of VP24 in nucleocapsid assembly as well as genome replication and transcription.

    IMPORTANCE Ebola viruses cause severe hemorrhagic fevers in humans, with no countermeasures currently being available, and must be studied in maximum-containment laboratories. Only a few of these laboratories exist worldwide, limiting our ability to study Ebola viruses and develop countermeasures. Here we report the development of a novel reverse genetics-based system that allows the study of Ebola viruses without maximum-containment laboratories. We used this system to investigate the Ebola virus protein VP24, showing that, contrary to previous reports, it only modestly inhibits virus genome replication and transcription but is important for packaging of genomes into virus particles, which constitutes a previously unknown function of VP24 and a potential antiviral target. We further propose a comprehensive model for the function of VP24 in nucleocapsid assembly. Importantly, on the basis of this approach, it should easily be possible to develop similar experimental systems for other viruses that are currently restricted to maximum-containment laboratories.

  • Influenza A Virus Attenuation by Codon Deoptimization of the NS Gene for Vaccine Development [Vaccines and Antiviral Agents]

  • Influenza viral infection represents a serious public health problem that causes contagious respiratory disease, which is most effectively prevented through vaccination to reduce transmission and future infection. The nonstructural (NS) gene of influenza A virus encodes an mRNA transcript that is alternatively spliced to express two viral proteins, the nonstructural protein 1 (NS1) and the nuclear export protein (NEP). The importance of the NS gene of influenza A virus for viral replication and virulence has been well described and represents an attractive target to generate live attenuated influenza viruses with vaccine potential. Considering that most amino acids can be synthesized from several synonymous codons, this study employed the use of misrepresented mammalian codons (codon deoptimization) for the de novo synthesis of a viral NS RNA segment based on influenza A/Puerto Rico/8/1934 (H1N1) (PR8) virus. We generated three different recombinant influenza PR8 viruses containing codon-deoptimized synonymous mutations in coding regions comprising the entire NS gene or the mRNA corresponding to the individual viral protein NS1 or NEP, without modifying the respective splicing and packaging signals of the viral segment. The fitness of these synthetic viruses was attenuated in vivo, while they retained immunogenicity, conferring both homologous and heterologous protection against influenza A virus challenges. These results indicate that influenza viruses can be effectively attenuated by synonymous codon deoptimization of the NS gene and open the possibility of their use as a safe vaccine to prevent infections with these important human pathogens.

    IMPORTANCE Vaccination serves as the best therapeutic option to protect humans against influenza viral infections. However, the efficacy of current influenza vaccines is suboptimal, and novel approaches are necessary for the prevention of disease cause by this important human respiratory pathogen. The nonstructural (NS) gene of influenza virus encodes both the multifunctional nonstructural protein 1 (NS1), essential for innate immune evasion, and the nuclear export protein (NEP), required for the nuclear export of viral ribonucleoproteins and for timing of the virus life cycle. Here, we have generated a recombinant influenza A/Puerto Rico/8/1934 (H1N1) (PR8) virus containing a codon-deoptimized NS segment that is attenuated in vivo yet retains immunogenicity and protection efficacy against homologous and heterologous influenza virus challenges. These results open the exciting possibility of using this NS codon deoptimization methodology alone or in combination with other approaches for the future development of vaccine candidates to prevent influenza viral infections.

  • Murine Leukemia Virus Gag Localizes to the Uropod of Migrating Primary Lymphocytes [Virus-Cell Interactions]

  • B and CD4+ T lymphocytes are natural targets of murine leukemia virus (MLV). Migrating lymphocytes adopt a polarized morphology with a trailing edge designated the uropod. Here, we demonstrate that MLV Gag localizes to the uropod in polarized B cells and CD4+ T cells. The uropod localization of MLV Gag was dependent on plasma membrane (PM) association and multimerization of Gag but independent of the viral glycoprotein Env. Basic residues in MA that are required for MLV Gag recruitment to virological synapses between HEK293 and XC cells were dispensable for uropod localization in migrating B cells. Ultrastructural studies indicated that both wild-type and basic-residue mutant Gag localized to the outer surface of the PM at the uropod. Late-domain mutant virus particles were seen at the uropod in form of budding-arrested intermediates. Finally, uropods mediated contact between MLV-infected B cells and uninfected T cells to form virological synapses. Our results suggest that MLV, not unlike HIV, accumulates at the uropod of primary lymphocytes to facilitate viral spreading through the formation of uropod-mediated cell-cell contacts.

    IMPORTANCE Viruses have evolved mechanisms to coordinate their assembly and budding with cell polarity to facilitate their spreading. In this study, we demonstrated that the viral determinants for MLV Gag to localize to the uropod in polarized B cells are distinct from the requirements to localize to virological synapses in transformed cell lines. Basic residues in MA that are required for the Gag localization to virological synapses between HEK293 and XC cells are dispensable for Gag localization to the uropod in primary B cells. Rather, plasma membrane association and capsid-driven multimerization of Gag are sufficient to drive MLV Gag to the uropod. MLV-laden uropods also mediate contacts between MLV-infected B cells and uninfected T cells to form virological synapses. Our results indicate that MLV accumulates at the uropod of primary lymphocytes to facilitate viral spreading through the formation of uropod-mediated cell-cell contacts.

  • H7N9 and Other Pathogenic Avian Influenza Viruses Elicit a Three-Pronged Transcriptomic Signature That Is Reminiscent of 1918 Influenza Virus and Is Associated with Lethal Outcome in Mice [Pathogenesis and Immunity]

  • Modulating the host response is a promising approach to treating influenza, caused by a virus whose pathogenesis is determined in part by the reaction it elicits within the host. Though the pathogenicity of emerging H7N9 influenza virus in several animal models has been reported, these studies have not included a detailed characterization of the host response following infection. Therefore, we characterized the transcriptomic response of BALB/c mice infected with H7N9 (A/Anhui/01/2013) virus and compared it to the responses induced by H5N1 (A/Vietnam/1203/2004), H7N7 (A/Netherlands/219/2003), and pandemic 2009 H1N1 (A/Mexico/4482/2009) influenza viruses. We found that responses to the H7 subtype viruses were intermediate to those elicited by H5N1 and pdm09H1N1 early in infection but that they evolved to resemble the H5N1 response as infection progressed. H5N1, H7N7, and H7N9 viruses were pathogenic in mice, and this pathogenicity correlated with increased transcription of cytokine response genes and decreased transcription of lipid metabolism and coagulation signaling genes. This three-pronged transcriptomic signature was observed in mice infected with pathogenic H1N1 strains such as the 1918 virus, indicating that it may be predictive of pathogenicity across multiple influenza virus strains. Finally, we used host transcriptomic profiling to computationally predict drugs that reverse the host response to H7N9 infection, and we identified six FDA-approved drugs that could potentially be repurposed to treat H7N9 and other pathogenic influenza viruses.

    IMPORTANCE Emerging avian influenza viruses are of global concern because the human population is immunologically naive to them. Current influenza drugs target viral molecules, but the high mutation rate of influenza viruses eventually leads to the development of antiviral resistance. As the host evolves far more slowly than the virus, and influenza pathogenesis is determined in part by the host response, targeting the host response is a promising approach to treating influenza. Here we characterize the host transcriptomic response to emerging H7N9 influenza virus and compare it with the responses to H7N7, H5N1, and pdm09H1N1. All three avian viruses were pathogenic in mice and elicited a transcriptomic signature that also occurs in response to the legendary 1918 influenza virus. Our work identifies host responses that could be targeted to treat severe H7N9 influenza and identifies six FDA-approved drugs that could potentially be repurposed as H7N9 influenza therapeutics.

  • Prophylaxis with a Respiratory Syncytial Virus (RSV) Anti-G Protein Monoclonal Antibody Shifts the Adaptive Immune Response to RSV rA2-line19F Infection from Th2 to Th1 in BALB/c Mice [Vaccines and Antiviral Agents]

  • Respiratory syncytial virus (RSV) is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. In the present study, we investigated the effect of prophylactic treatment with the intact and F(ab')2 forms of an anti-G protein monoclonal antibody (MAb), 131-2G, on the humoral and cellular adaptive immune responses to RSV rA2-line19F (r19F) challenge in BALB/c mice. The F(ab')2 form of 131-2G does not decrease virus replication, but intact 131-2G does. The serum specimens for antibodies and spleen cells for memory T cell responses to RSV antigens were analyzed at 30, 45, 75, and 95 days postinfection (p.i.) with or without prior treatment with 131-2G. The ratios of Th2 to Th1 antibody isotypes at each time p.i indicated that both forms of MAb 131-2G shifted the subclass response from a Th2 (IgG1 and IgG2b) to a Th1 (IgG2A) bias. The ratio of IgG1 to IgG2A antibody titer was 3-fold to 10-fold higher for untreated than MAb-treated mice. There was also some increase in IgG (22% pplusmn; 13% increase) and neutralization (32% increase) in antibodies with MAb 131-2G prophylaxis at 75 days p.i. Treatment with 131-2G significantly (P lle; 0.001) decreased the percentage of interleukin-4 (IL-4)-positive CD4 and CD8 cells in RSV-stimulated spleen cells at all times p.i., while the percentage of interferon gamma (IFN-) T cells significantly (P lle; 0.001) increased gge;75 days p.i. The shift from a Th2- to a Th1-biased T cell response in treated compared to untreated mice likely was directed by the much higher levels of T-box transcription factor (T-bet) (gge;45% versus llt;10%) in CD4 and CD8 T cells and lower levels of Gata-3 (lle;2% versus gge; 6%) in CD4 T cells in peptide-stimulated, day 75 p.i. spleen cells. These data show that the RSV G protein affects both humoral and cellular adaptive immune responses, and induction of 131-2G-like antibodies might improve the safety and long-term efficacy of an RSV vaccine.

    IMPORTANCE The data in this report suggest that the RSV G protein not only contributes to disease but also dampens the host immune response to infection. Both effects of G likely contribute to difficulties in achieving an effective vaccine. The ability of MAb 131-2G to block these effects of G suggests that inducing antibodies similar to 131-2G should prevent disease and enhance the adaptive immune response with later RSV infection. The fact that 131-2G binds to the 13-amino-acid region conserved among all strains and that flanking sequences are conserved within group A or group B strains simplifies the task of developing a vaccine to induce 131-2G-like antibodies. If our findings in mice apply to humans, then including the 131-2G binding region of G in a vaccine should improve its safety and efficacy.

  • Identification of Conserved Residues in Hepatitis C Virus Envelope Glycoprotein E2 That Modulate Virus Dependence on CD81 and SRB1 Entry Factors [Virus-Cell Interactions]

  • In spite of the high variability of its sequence, hepatitis C virus (HCV) envelope glycoprotein E2 contains several conserved regions. In this study, we explored the structural and functional features of the highly conserved E2 segment from amino acid (aa) 502 to 520, which had been proposed as a fusion peptide and shown to strongly overlap a potential conserved neutralizing epitope. For this purpose, we used reverse genetics to introduce point mutations within this region, and we characterized the phenotypes of these mutants in the light of the recently published structure of E2. The functional analyses showed that their phenotypes are in agreement with the positions of the corresponding residues in the E2 crystal structure. In contrast, our data ruled out the involvement of this region in membrane fusion, and they indicate that alternative conformations would be necessary to expose the potential neutralizing epitope present in this segment. Of particular interest, we identified three specific mutations (Y507L, V514A, and V515A) located within this neutralizing epitope which only mildly reduced infectivity and showed no assembly defect. These mutations modulated HCV dependence on the viral receptor SRB1, and/or they also modulated virion sensitivity to neutralizing antibodies. Importantly, their characterization also showed that amino acids Y507, V514, and V515 contribute to E2 interaction with HCV receptor CD81. In conclusion, our data show that the highly conserved E2 segment from aa 502 to 520 plays a key role in cell entry by influencing the association of the viral particle with coreceptors and neutralizing antibodies.

    IMPORTANCE Hepatitis C virus (HCV) envelope proteins E1 and E2 exhibit sequence variability. However, some segments of the envelope proteins are highly conserved, suggesting that these sequences play a key role at some steps of the HCV life cycle. In this work, we characterized the function and structure of a highly conserved E2 region that is targeted by neutralizing antibodies and had been proposed as a fusion peptide. Our data ruled out the involvement of this region in membrane fusion but allowed for the identification of new residues modulating the interaction of the virus with entry factors and its sensitivity to neutralizing antibodies. Moreover, structural data suggest that alternative conformations could exist for E2, which would explain the presence of a partially masked neutralizing epitope in this segment in the currently available E2 structure. Overall, our findings highlight the importance of conserved regions in the sequences of HCV envelope proteins.

  • SnRK1 Phosphorylation of AL2 Delays Cabbage Leaf Curl Virus Infection in Arabidopsis [Virus-Cell Interactions]

  • Geminivirus AL2/C2 proteins play key roles in establishing infection and causing disease in their plant hosts. They are involved in viral gene expression, counter host defenses by suppressing transcriptional gene silencing, and interfere with the host signaling involved in pathogen resistance. We report here that begomovirus and curtovirus AL2/C2 proteins interact strongly with host geminivirus Rep-interacting kinases (GRIKs), which are upstream activating kinases of the protein kinase SnRK1, a global regulator of energy and nutrient levels in plants. We used an in vitro kinase system to show that GRIK-activated SnRK1 phosphorylates recombinant AL2/C2 proteins from several begomoviruses and to map the SnRK1 phosphorylation site to serine-109 in the AL2 proteins of two New World begomoviruses: Cabbage Leaf Curl Virus (CaLCuV) and Tomato mottle virus. A CaLCuV AL2 S109D phosphomimic mutation did not alter viral DNA levels in protoplast replication assays. In contrast, the phosphomimic mutant was delayed for symptom development and viral DNA accumulation during infection of Arabidopsis thaliana, demonstrating that SnRK1 contributes to host defenses against CaLCuV. Our observation that serine-109 is not conserved in all AL2/C2 proteins that are SnRK1 substrates in vitro suggested that phosphorylation of viral proteins by plant kinases contributes to the evolution of geminivirus-host interactions.

    IMPORTANCE Geminiviruses are single-stranded DNA viruses that cause serious diseases in many crops. Dicot-infecting geminiviruses carry genes that encode multifunctional AL2/C2 proteins that are essential for infection. However, it is not clear how AL2/C2 proteins are regulated. Here, we show that the host protein kinase SnRK1, a central regulator of energy balance and nutrient metabolism in plants, phosphorylates serine-109 in AL2 proteins of three subgroups of New World begomoviruses, resulting in a delay in viral DNA accumulation and symptom appearance. Our results support SnRK1's antiviral role and reveal a novel mechanism underlying this function. Phylogenetic analysis suggested that AL2 S109 evolved as begomoviruses migrated from the Old World to the New World and may have provided a selective advantage as begomoviruses adapted to a different environment and different plant hosts. This study provides new insights into the interaction of viral pathogens with their plant hosts at the level of viral protein modification by the host.

  • The Immunodominant Influenza A Virus M158-66 Cytotoxic T Lymphocyte Epitope Exhibits Degenerate Class I Major Histocompatibility Complex Restriction in Humans [Cellular Response to Infection]

  • Cytotoxic T lymphocytes recognizing conserved peptide epitopes are crucial for protection against influenza A virus (IAV) infection. The CD8 T cell response against the M158nndash;66 (GILGFVFTL) matrix protein epitope is immunodominant when restricted by HLA-A*02, a major histocompatibility complex (MHC) molecule expressed by approximately half of the human population. Here we report that the GILGFVFTL peptide is restricted by multiple HLA-C*08 alleles as well. We observed that M158nndash;66 was able to elicit cytotoxic T lymphocyte (CTL) responses in both HLA-A*02- and HLA-C*08-positive individuals and that GILGFVFTL-specific CTLs in individuals expressing both restriction elements were distinct and not cross-reactive. The crystal structure of GILGFVFTLnndash;HLA-C*08:01 was solved at 1.84 AAring;, and comparison with the known GILGFVFTLnndash;HLA-A*02:01 structure revealed that the antigen bound both complexes in near-identical conformations, accommodated by binding pockets shaped from shared as well as unique residues. This discovery of degenerate peptide presentation by both HLA-A and HLA-C allelic variants eliciting unique CTL responses to IAV infection contributes fundamental knowledge with important implications for vaccine development strategies.

    IMPORTANCE The presentation of influenza A virus peptides to elicit immunity is thought to be narrowly restricted, with a single peptide presented by a specific HLA molecule. In this study, we show that the same influenza A virus peptide can be more broadly presented by both HLA-A and HLA-C molecules. This discovery may help to explain the differences in immunity to influenza A virus between individuals and populations and may also aid in the design of vaccines.

  • The UL12 Protein of Herpes Simplex Virus 1 Is Regulated by Tyrosine Phosphorylation [Genome Replication and Regulation of Viral Gene Expression]

  • The herpes simplex virus 1 (HSV-1) UL12 protein (pUL12) is a nuclease that is critical for viral replication in vitro and neurovirulence in vivo. In this study, mass spectrometric analysis of pUL12 and phosphate-affinity SDS-polyacrylamide gel electrophoresis analysis identified tyrosine at pUL12 residue 371 (Tyr-371) as a pUL12 phosphorylation site: Tyr-371 is conserved in pUL12 homologs in herpesviruses in all Herpesviridae subfamilies. Replacement of Tyr-371 with phenylalanine (Y371F) in pUL12 (i) abolished its exonuclease activity in HSV-1-infected Vero, HEL, and A549 cells, (ii) reduced viral replication, cell-cell spread, and pUL12 expression in infected cells in a cell type-dependent manner, (iii) led to aberrant subcellular localization of pUL12 in infected cells in a cell type-dependent manner, and (iv) reduced HSV-1 neurovirulence in mice. The effects of the pUL12 Y371F mutation in cell cultures and mice were similar to those of a nuclease-dead double mutation in pUL12, although the Y371F mutation reduced viral replication severalfold more than the nuclease-dead double mutation in a cell type- and multiplicity-of-infection-dependent manner. Replacement of Tyr-371 with glutamic acid, which mimics constitutive phosphorylation, restored the wild-type phenotype in cell cultures and mice. These results suggested that phosphorylation of pUL12 Tyr-371 was essential for pUL12 to express its nuclease activity in HSV-1-infected cells and that this phosphorylation promoted viral replication and cell-cell spread in cell cultures and neurovirulence in mice mainly by upregulating pUL12 nuclease activity and, in part, by regulating the subcellular localization and expression of pUL12 in HSV-1-infected cells.

    IMPORTANCE Herpesviruses encode a considerable number of enzymes for their replication. Like cellular enzymes, the viral enzymes need to be properly regulated in infected cells. Although the functional aspects of herpesvirus enzymes have gradually been clarified, information on how most of these enzymes are regulated in infected cells is lacking. In the present study, we report that the enzymatic activity of the herpes simplex virus 1 alkaline nuclease pUL12 was regulated by phosphorylation of pUL12 Tyr-371 in infected cells and that this phosphorylation promoted viral replication and cell-cell spread in cell cultures and neurovirulence in mice, mainly by upregulating pUL12 nuclease activity. Interestingly, pUL12 and tyrosine at pUL12 residue 371 appeared to be conserved in all herpesviruses in the family Herpesviridae, raising the possibility that the herpesvirus pUL12 homologs may also be regulated by phosphorylation of the conserved tyrosine residue.

  • The Rhesus Rhadinovirus CD200 Homologue Affects Immune Responses and Viral Loads during In Vivo Infection [Pathogenesis and Immunity]

  • Rhesus macaque rhadinovirus (RRV) is a gammaherpesvirus of rhesus macaque (RM) monkeys that is closely related to human herpesvirus 8 (HHV-8)/Kaposi's Sarcoma-associated herpesvirus (KSHV), and it is capable of inducing diseases in simian immunodeficiency virus (SIV)-infected RM that are similar to those seen in humans coinfected with HIV and HHV-8. Both HHV-8 and RRV encode viral CD200 (vCD200) molecules that are homologues of cellular CD200, a membrane glycoprotein that regulates immune responses and helps maintain immune homeostasis via interactions with the CD200 receptor (CD200R). Though the functions of RRV and HHV-8 vCD200 molecules have been examined in vitro, the precise roles that these viral proteins play during in vivo infection remain unknown. Thus, to address the contributions of RRV vCD200 to immune regulation and disease in vivo, we generated a form of RRV that lacked expression of vCD200 for use in infection studies in RM. Our data indicated that RRV vCD200 expression limits immune responses against RRV at early times postinfection and also impacts viral loads, but it does not appear to have significant effects on disease development. Further, examination of the distribution pattern of CD200R in RM indicated that this receptor is expressed on a majority of cells in peripheral blood mononuclear cells, including B and T cells, suggesting potentially wider regulatory capabilities for both vCD200 and CD200 that are not strictly limited to myeloid lineage cells. In addition, we also demonstrate that RRV infection affects CD200R expression levels in vivo, although vCD200 expression does not play a role in this phenomenon.

    IMPORTANCE Cellular CD200 and its receptor, CD200R, compose a pathway that is important in regulating immune responses and is known to play a role in a variety of human diseases. A number of pathogens have been found to modulate the CD200-CD200R pathway during infection, including human herpesvirus 8 (HHV-8), the causative agent of Kaposi's sarcoma and B cell neoplasms in AIDS patients, and a closely related primate virus, rhesus macaque rhadinovirus (RRV), which infects and induces disease in rhesus macaque monkeys. HHV-8 and RRV encode homologues of CD200, termed vCD200, which are thought to play a role in preventing immune responses against these viruses. However, neither molecule has been studied in an in vivo model of infection to address their actual contributions to immunoregulation and disease. Here we report findings from our studies in which we analyzed the properties of a mutant form of RRV that lacks vCD200 expression in infected rhesus macaques.

  • A Non-SUMOylated Tax Protein Is Still Functional for NF-{kappa}B Pathway Activation [Transformation and Oncogenesis]

  • Whether NF-B promoter transactivation by the human T-cell leukemia virus type 1 (HTLV-1) Tax protein requires Tax SUMOylation is still a matter of debate. In this study, we revisited the role of Tax SUMOylation using a strategy based on the targeting of Ubc9, the unique E2 SUMO-conjugating enzyme. We show that either a catalytically inactive form of Ubc9 (Ubc9-C93S) or Ubc9 small interfering RNA (siRNA) dramatically reduces Tax conjugation to endogenous SUMO-1 or SUMO-2/3, demonstrating that as expected, Tax SUMOylation is under the control of the catalytic activity of Ubc9. We further report that a non-SUMOylated Tax protein produced in 293T cells is still able to activate either a transfected or an integrated NF-B reporter promoter and to induce expression of an NF-B-regulated endogenous gene. Importantly, blocking Ubc9 activity in T cells also results in the production of a non-SUMOylated Tax that is still fully functional for the activation of a NF-B promoter. These results provide the definitive evidence that Tax SUMOylation is not required for NF-B-driven gene induction.

    IMPORTANCE Human T-cell leukemia virus type 1 is able to transform CD4+ T lymphocytes. The viral oncoprotein Tax plays a key role in this process by promoting cell proliferation and survival, mainly through permanent activation of the NF-B pathway. Elucidating the molecular mechanisms involved in NF-B pathway activation by Tax is therefore a key issue to understand HTLV-1-mediated transformation. Tax SUMOylation was initially proposed to be critical for Tax-induced NF-B promoter activation, which was challenged by our later observation that a low-level-SUMOylated Tax mutant was still functional for activation of NF-B promoters. To clarify the role of Tax SUMOylation, we set up a new approach based on the inhibition of the SUMOylation machinery in Tax-expressing cells. We show that blocking the SUMO-conjugating enzyme Ubc9 abolishes Tax SUMOylation and that a non-SUMOylated Tax still activates NF-B promoters in either adherent cells or T cells.

  • Genomic Diversity of Epstein-Barr Virus Genomes Isolated from Primary Nasopharyngeal Carcinoma Biopsy Samples [Genetic Diversity and Evolution]

  • Undifferentiated nasopharyngeal carcinoma (NPC) has a 100% association with Epstein-Barr virus (EBV). However, only three EBV genomes isolated from NPC patients have been sequenced to date, and the role of EBV genomic variations in the pathogenesis of NPC is unclear. We sought to obtain the sequences of EBV genomes in multiple NPC biopsy specimens in the same geographic location in order to reveal their sequence diversity. Three published EBV (B95-8, C666-1, and HKNPC1) genomes were first resequenced using the sequencing workflow of target enrichment of EBV DNA by hybridization, followed by next-generation sequencing, de novo assembly, and joining of contigs by Sanger sequencing. The sequences of eight NPC biopsy specimen-derived EBV (NPC-EBV) genomes, designated HKNPC2 to HKNPC9, were then determined. They harbored 1,736 variations in total, including 1,601 substitutions, 64 insertions, and 71 deletions, compared to the reference EBV. Furthermore, genes encoding latent, early lytic, and tegument proteins and glycoproteins were found to contain nonsynonymous mutations of potential biological significance. Phylogenetic analysis showed that the HKNPC6 and -7 genomes, which were isolated from tumor biopsy specimens of advanced metastatic NPC cases, were distinct from the other six NPC-EBV genomes, suggesting the presence of at least two parental lineages of EBV among the NPC-EBV genomes. In conclusion, much greater sequence diversity among EBV isolates derived from NPC biopsy specimens is demonstrated on a whole-genome level through a complete sequencing workflow. Large-scale sequencing and comparison of EBV genomes isolated from NPC and normal subjects should be performed to assess whether EBV genomic variations contribute to NPC pathogenesis.

    IMPORTANCE This study established a sequencing workflow from EBV DNA capture and sequencing to de novo assembly and contig joining. We reported eight newly sequenced EBV genomes isolated from primary NPC biopsy specimens and revealed the sequence diversity on a whole-genome level among these EBV isolates. At least two lineages of EBV strains are observed, and recombination among these lineages is inferred. Our study has demonstrated the value of, and provided a platform for, genome sequencing of EBV.

  • The Phosphatidylinositol 3-Kinase (PI3K) Isoform Dependence of Tumor Formation Is Determined by the Genetic Mode of PI3K Pathway Activation Rather than by Tissue Type [Transformation and Oncogenesis]

  • Previous work has shown that prostate cancer in a Pten-null murine model is dependent on the p110bbeta; isoform of phosphatidylinositol 3-kinase (PI3K), while breast cancer driven by either polyoma middle T antigen (MT) or HER2 is p110aalpha; dependent. Whether these differences in isoform dependence arise from tissue specificity or from the nature of the oncogenic signal activating the PI3K pathway is important, given increasing interest in using isoform-specific PI3K inhibitors in cancer therapy. To approach this question, we studied the PI3K isoform dependence of our recently constructed prostate cancer model driven by MT. Since MT activates a number of signaling pathways, we first confirmed that the MT-driven prostate cancer model was actually dependent on PI3K. A newly generated transgenic prostate line expressing an MT allele (Y315F) known to be defective for PI3K binding displayed a markedly reduced ability to drive tumor formation. We next selectively ablated expression of either p110aalpha; or p110bbeta; in mice in which wild-type MT was expressed in the prostate. We found that tumor formation driven by MT was significantly delayed by the loss of p110aalpha; expression, while ablation of p110bbeta; had no effect. Since the tumor formation driven by MT is p110aalpha; dependent in the prostate as well as in the mammary gland, our data suggest that PI3K isoform dependence is driven by the mode of PI3K pathway activation rather than by tissue type.

    IMPORTANCE Middle T antigen (MT), the oncogene of polyomavirus, can drive tumor formation in a variety of cell types and tissues. Interestingly, MT has no intrinsic enzymatic activity but instead functions by binding and activating cellular signaling proteins. One of the most important of these is the lipid kinase PI3K, which was first studied in MT immunoprecipitates. Ubiquitously expressed PI3K comes in two major isoforms: p110aalpha; and p110bbeta;. Previous work in animal models showed that p110aalpha; was the key isoform in breast tumors driven by oncogenes, including MT and HER2, while p110bbeta; was key in prostate tumors driven by Pten loss. We asked the simple question of whether a prostate tumor driven by MT depends on p110aalpha;, which would suggest that the mode of activation determines p110 isoform dependence, or p110bbeta;, which would suggest that tissue type determines isoform dependence. The clear answer is that MT depends on p110aalpha; in both the prostate and breast.

  • Ectopic DNMT3L Triggers Assembly of a Repressive Complex for Retroviral Silencing in Somatic Cells [Cellular Response to Infection]

  • Mammalian genomes are replete with retrotransposable elements, including endogenous retroviruses. DNA methyltransferase 3-like (DNMT3L) is an epigenetic regulator expressed in prospermatogonia, growing oocytes, and embryonic stem (ES) cells. Here, we demonstrate that DNMT3L enhances the interaction of repressive epigenetic modifiers, including histone deacetylase 1 (HDAC1), SET domain, bifurcated 1 (SETDB1), DNA methyltransferase 3A (DNMT3A), and tripartite motif-containing protein 28 (TRIM28; also known as TIF1bbeta; and KAP1) in ES cells and orchestrates retroviral silencing activity with TRIM28 through mechanisms including, but not limited to, de novo DNA methylation. Ectopic expression of DNMT3L in somatic cells causes methylation-independent retroviral silencing activity by recruitment of the TRIM28/HDAC1/SETDB1/DNMT3A/DNMT3L complex to newly integrated Moloney murine leukemia virus (Mo-MuLV) proviral DNA. Concurrent with this recruitment, we also observed the accumulation of histone H3 lysine 9 trimethylation (H3K9me3) and heterochromatin protein 1 gamma (HP1), as well as reduced H3K9 and H3K27 acetylation at Mo-MuLV proviral sequences. Ectopic expression of DNMT3L in late-passage mouse embryonic fibroblasts (MEFs) recruited cytoplasmically localized HDAC1 to the nucleus. The formation of this epigenetic modifying complex requires interaction of DNMT3L with DNMT3A as well as with histone H3. In fetal testes at embryonic day 17.5, endogenous DNMT3L also enhanced the binding among TRIM28, DNMT3A, SETDB1, and HDAC1. We propose that DNMT3L may be involved in initiating a cascade of repressive epigenetic modifications by assisting in the preparation of a chromatin context that further attracts DNMT3A-DNMT3L binding and installs longer-term DNA methylation marks at newly integrated retroviruses.

    IMPORTANCE Almost half of the mammalian genome is composed of endogenous retroviruses and other retrotransposable elements that threaten genomic integrity. These elements are usually subject to epigenetic silencing. We discovered that two epigenetic regulators that lack enzymatic activity, DNA methyltransferase 3-like (DNMT3L) and tripartite motif-containing protein 28 (TRIM28), collaborate with each other to impose retroviral silencing. In addition to modulating de novo DNA methylation, we found that by interacting with TRIM28, DNMT3L can attract various enzymes to form a DNMT3L-induced repressive complex to remove active marks and add repressive marks to histone proteins. Collectively, these results reveal a novel and pivotal function of DNMT3L in shaping the chromatin modifications necessary for retroviral and retrotransposon silencing.

  • Comprehensive High-Throughput RNA Sequencing Analysis Reveals Contamination of Multiple Nasopharyngeal Carcinoma Cell Lines with HeLa Cell Genomes [Virus-Cell Interactions]

  • In an attempt to explore infectious agents associated with nasopharyngeal carcinomas (NPCs), we employed our high-throughput RNA sequencing (RNA-seq) analysis pipeline, RNA CoMPASS, to investigate the presence of ectopic organisms within a number of NPC cell lines commonly used by NPC and Epstein-Barr virus (EBV) researchers. Sequencing data sets from both CNE1 and HONE1 were found to contain reads for human papillomavirus 18 (HPV-18). Subsequent real-time reverse transcription-PCR (RT-PCR) analysis on a panel of NPC cell lines identified HPV-18 in CNE1 and HONE1 as well as three additional NPC cell lines (CNE2, AdAH, and NPC-KT). Further analysis of the chromosomal integration arrangement of HPV-18 in NPCs revealed patterns identical to those observed in HeLa cells. Clustering based on human single nucleotide variation (SNV) analysis of two separate HeLa cell lines and several NPC cell lines demonstrated two distinct clusters with CNE1, as well as HONE1 clustering with the two HeLa cell lines. In addition, duplex-PCR-based genotyping showed that CNE1, CNE2, and HONE1 do not have a HeLa cell-specific L1 retrotransposon insertion, suggesting that these three HPV-18+ NPC lines are likely products of a somatic hybridization with HeLa cells, which is also consistent with our RNA-seq-based gene level SNV analysis. Taking all of these findings together, we conclude that a widespread HeLa contamination may exist in many NPC cell lines, and authentication of these cell lines is recommended. Finally, we provide a proof of concept for the utility of an RNA-seq-based approach for cell authentication.

    IMPORTANCE Nasopharyngeal carcinoma (NPC) cell lines are important model systems for analyzing the complex life cycle and pathogenesis of Epstein-Barr virus (EBV). Using an RNA-seq-based approach, we found HeLa cell contamination in several NPC cell lines that are commonly used in the EBV and related fields. Our data support the notion that contamination resulted from somatic hybridization with HeLa cells, likely occurring at the point of cell line establishment. Given the rarity of NPCs, the long history of NPC cell lines, and the lack of rigorous cell line authentication, it is likely that the actual prevalence and impact of HeLa cell contamination on the EBV field might be greater. We therefore recommend cell line authentication prior to performing experiments using NPC cell lines to avoid inaccurate conclusions. The novel RNA-seq-based cell authentication approach reported here can serve as a comprehensive method for validating cell lines.

  • Snow Goose Hepatitis B Virus (SGHBV) Envelope and Capsid Proteins Independently Contribute to the Ability of SGHBV To Package Capsids Containing Single-Stranded DNA in Virions [Structure and Assembly]

  • Hepadnaviruses selectively package capsids containing mature double-stranded DNA (dsDNA) genomes in virions. Snow goose hepatitis B virus (SGHBV) is the only known hepadnavirus that packages capsids containing single-stranded DNA (ssDNA) in virions. We found that cells replicating SGHBV produce virions containing ssDNA as efficiently as virions containing mature dsDNA. We determined that SGHBV capsid and envelope proteins independently contribute to the production of virions containing ssDNA, with the capsid protein (Cp) making a larger contribution. We identified that amino acid residues 74 and 107 of SGHBV Cp contribute to this feature of SGHBV. When we changed these residues in duck hepatitis B virus (DHBV) Cp, capsids containing immature ssDNA were packaged in virions. This result suggests that residues 74 and 107 contribute to the appearance of the "capsid packaging signal" on the surface of capsids and interact with the envelope proteins during virion formation. We also found that cells replicating SGHBV package a larger fraction of the total dsDNA they synthesize into virions than do those replicating DHBV. We determined that the SGHBV envelope proteins are responsible for this property of SGHBV. Determining if the ability of SGHBV envelope proteins to cause the formation of virions containing ssDNA is related to its ability to support high levels of virion production or if these two properties are mechanistically distinct will provide insights into virion morphogenesis.

    IMPORTANCE Cells replicating hepadnaviruses contain cytoplasmic capsids that contain mature and immature genomes. However, only capsids containing mature dsDNA genomes are packaged in virions. A mechanistic understanding of this phenomenon, which is currently lacking, is critical to understanding the process of hepadnaviral virion morphogenesis. In this study, we determined that the envelope proteins contribute to the ability of hepadnaviruses to selectively produce virions containing mature dsDNA genomes. Our finding sheds new light on the mechanisms underlying virion morphogenesis and challenges the dogma that "capsid maturation," and therefore the capsid protein (Cp), is solely responsible for the selective production of virions containing mature dsDNA genomes. Further, we identified amino acid residues of Cp that contribute to its ability to cause the selective production of virions containing mature dsDNA genomes. Future studies on the role of these residues in selective secretion will broaden our understanding of this poorly understood aspect of virus replication.

  • Homeostatic Cytokines Induce CD4 Downregulation in African Green Monkeys Independently of Antigen Exposure To Generate Simian Immunodeficiency Virus-Resistant CD8{alpha}{alpha} T Cells [Pathogenesis and Immunity]

  • African green monkeys (AGMs; genus Chlorocebus) are a natural host of simian immunodeficiency virus (SIVAGM). As they do not develop simian AIDS, there is great interest in understanding how this species has evolved to avoid immunodeficiency. Adult African green monkeys naturally have low numbers of CD4 T cells and a large population of major histocompatibility complex class II-restricted CD8aalpha;dim T cells that are generated through CD4 downregulation in CD4+ T cells. Mechanisms that drive this process of CD4 downregulation are unknown. Here, we show that juvenile AGMs accelerate CD4-to-CD8aalpha;aalpha; conversion upon SIV infection and avoid progression to AIDS. The CD4 downregulation induced by SIV infection is not limited to SIV-specific T cells, and vaccination of an adult AGM who had a negligible number of CD4 T cells demonstrated that CD4 downregulation can occur without antigenic exposure. Finally, we show that the T cell homeostatic cytokines interleukin-2 (IL-2), IL-7, and IL-15 can induce CD4 downregulation in vitro. These data identify a mechanism that allows AGMs to generate a large, diverse population of T cells that perform CD4 T cell functions but are resistant to SIV infection. A better understanding of this mechanism may allow the development of treatments to induce protective CD4 downregulation in humans.

    IMPORTANCE Many African primate species are naturally infected with SIV. African green monkeys, one natural host species, avoid simian AIDS by creating a population of T cells that lack CD4, the human immunodeficiency virus/SIV receptor; therefore, they are resistant to infection. However, these T cells maintain properties of CD4+ T cells even after receptor downregulation and preserve immune function. Here, we show that juvenile AGMs, who have not undergone extensive CD4 downregulation, accelerate this process upon SIV infection. Furthermore, we show that in vivo, CD4 downregulation does not occur exclusively in antigen-experienced T cells. Finally, we show that the cytokines IL-2, IL-7, and IL-15, which induce homeostatic T cell proliferation, lead to CD4 downregulation in vitro; therefore, they can provide signals that lead to antigen-independent CD4 downregulation. These results suggest that if a similar process of CD4 downregulation could be induced in humans, it could provide a cure for AIDS.

  • Tobacco etch virus Protein P1 Traffics to the Nucleolus and Associates with the Host 60S Ribosomal Subunits during Infection [Genome Replication and Regulation of Viral Gene Expression]

  • The genus Potyvirus comprises a large group of positive-strand RNA plant viruses whose genome encodes a large polyprotein processed by three viral proteinases. P1 protein, the most amino-terminal product of the polyprotein, is an accessory factor stimulating viral genome amplification whose role during infection is not well understood. We infected plants with Tobacco etch virus (TEV; genus Potyvirus) clones in which P1 was tagged with a fluorescent protein to track its expression and subcellular localization or with an affinity tag to identify host proteins involved in complexes in which P1 also takes part during infection. Our results showed that TEV P1 exclusively accumulates in infected cells at an early stage of infection and that the protein displays a dynamic subcellular localization, trafficking in and out of the nucleus and nucleolus during infection. Inside the nucleolus, P1 particularly targets the dense granular component. Consistently, we found functional nucleolar localization and nuclear export signals in TEV P1 sequence. Our results also indicated that TEV P1 physically interacts with the host 80S cytoplasmic ribosomes and specifically binds to the 60S ribosomal subunits during infection. In vitro translation assays of reporter proteins suggested that TEV P1 stimulates protein translation, particularly when driven from the TEV internal ribosome entry site. These in vitro assays also suggested that TEV helper-component proteinase (HC-Pro) inhibits protein translation. Based on these findings, we propose that TEV P1 stimulates translation of viral proteins in infected cells.

    IMPORTANCE In this work, we researched the role during infection of tobacco etch virus P1 protease. P1 is the most mysterious protein of potyviruses, a relevant group of RNA viruses infecting plants. Our experiments showed that the viral P1 protein exclusively accumulates in infected cells at an early stage of infection and moves in and out of the nucleus of infected cells, particularly targeting the nucleolus. Our experiments also showed that P1 protein binds host ribosomes during infection. Based on these findings and other in vitro experiments we propose that P1 protein stimulates translation of viral proteins during infection.

  • The P4-P2' Amino Acids Surrounding Human Norovirus Polyprotein Cleavage Sites Define the Core Sequence Regulating Self-Processing Order [Genome Replication and Regulation of Viral Gene Expression]

  • Noroviruses (NoV) are members of the family Caliciviridae. The human NoV open reading frame 1 (ORF1) encodes a 200-kDa polyprotein which is cleaved by the viral 20-kDa 3C-like protease (Pro, NS6) into 6 nonstructural proteins that are necessary for viral replication. The NoV ORF1 polyprotein is processed in a specific order, with "early" sites (NS1/2-3 and NS3-4) being cleaved rapidly and three "late" sites (NS4-5, NS5-6, and NS6-7) processed subsequently and less efficiently. Previously, we demonstrated that the NoV polyprotein processing order is directly correlated with the efficiency of the enzyme, which is regulated by the primary amino acid sequences surrounding ORF1 cleavage sites. Using fluorescence resonance energy transfer (FRET) peptides representing the NS2-3 and NS6-7 ORF1 cleavage sites, we now demonstrate that the amino acids spanning positions P4 to P2' (P4-P2') surrounding each site comprise the core sequence controlling NoV protease enzyme efficiency. Furthermore, the NoV polyprotein self-processing order can be altered by interchanging this core sequence between NS2-3 and any of the three late sites in in vitro transcription-translation assays. We also demonstrate that the nature of the side chain at the P3 position for the NS1/2-3 (Nterm/NTPase) site confers significant influence on enzyme catalysis (kcat and kcat/Km), a feature overlooked in previous structural studies. Molecular modeling provides possible explanations for the P3 interactions with NoV protease.

    IMPORTANCE Noroviruses (NoV) are the prevailing cause of nonbacterial acute gastroenteritis worldwide and pose a significant financial burden on health care systems. Proteolytic processing of the viral nonstructural polyprotein is required for norovirus replication. Previously, the core sequence of amino acids surrounding the scissile bonds responsible for governing the relative processing order had not been determined. Using both FRET-based peptides and full-length NoV polyprotein, we have successfully demonstrated that the core sequences spanning positions P4-P2' surrounding the NS2-3, NS4-5, NS5-6, and NS6-7 cleavage sites contain all of the structural information necessary to control processing order. We also provide insight into a previously overlooked role for the NS2-3 P3 residue in enzyme efficiency. This article builds upon our previous studies on NoV protease enzymatic activities and polyprotein processing order. Our work provides significant additional insight into understanding viral polyprotein processing and has important implications for improving the design of inhibitors targeting the NoV protease.

  • TRIM13 Is a Negative Regulator of MDA5-Mediated Type I Interferon Production [Pathogenesis and Immunity]

  • Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are essential intracellular detectors of viral RNA. They contribute to the type I interferon (IFN) response that is crucial for host defense against viral infections. Given the potent antiviral and proinflammatory activities elicited by the type I IFNs, induction of the type I IFN response is tightly regulated. Members of the tripartite motif (TRIM) family of proteins have recently emerged as key regulators of antiviral immunity. We show that TRIM13, an E3 ubiquitin ligase, is expressed in immune cells and is upregulated in bone marrow-derived macrophages upon stimulation with inducers of type I IFN. TRIM13 interacts with MDA5 and negatively regulates MDA5-mediated type I IFN production in vitro, acting upstream of IFN regulatory factor 3. We generated Trim13nndash;/nndash; mice and show that upon lethal challenge with encephalomyocarditis virus (EMCV), which is sensed by MDA5, Trim13nndash;/nndash; mice produce increased amounts of type I IFNs and survive longer than wild-type mice. Trim13nndash;/nndash; murine embryonic fibroblasts (MEFs) challenged with EMCV or poly(Immiddot;C) also show a significant increase in beta IFN (IFN-bbeta;) levels, but, in contrast, IFN-bbeta; responses to the RIG-I-detected Sendai virus were diminished, suggesting that TRIM13 may play a role in positively regulating RIG-I function. Together, these results demonstrate that TRIM13 regulates the type I IFN response through inhibition of MDA5 activity and that it functions nonredundantly to modulate MDA5 during EMCV infection.

    IMPORTANCE The type I interferon (IFN) response is crucial for host defense against viral infections, and proper regulation of this pathway contributes to maintaining immune homeostasis. Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are intracellular detectors of viral RNA that induce the type I IFN response. In this study, we show that expression of the gene tripartite motif 13 (Trim13) is upregulated in response to inducers of type I IFN and that TRIM13 interacts with both MDA5 and RIG-I in vitro. Through the use of multiple in vitro and in vivo model systems, we show that TRIM13 is a negative regulator of MDA5-mediated type I IFN production and may also impact RIG-I-mediated type I IFN production by enhancing RIG-I activity. This places TRIM13 at a key junction within the viral response pathway and identifies it as one of the few known modulators of MDA5 activity.

  • Differential Responses of Plasmacytoid Dendritic Cells to Influenza Virus and Distinct Viral Pathogens [Virus-Cell Interactions]

  • Plasmacytoid dendritic cells (pDCs) are key components of the innate immune response that are capable of synthesizing and rapidly releasing vast amounts of type I interferons (IFNs), particularly IFN-aalpha;. Here we investigated whether pDCs, often regarded as a mere source of IFN, discriminate between various functionally discrete stimuli and to what extent this reflects differences in pDC responses other than IFN-aalpha; release. To examine the ability of pDCs to differentially respond to various doses of intact and infectious HIV, hepatitis C virus, and H1N1 influenza virus, whole-genome gene expression analysis, enzyme-linked immunosorbent assays, and flow cytometry were used to investigate pDC responses at the transcriptional, protein, and cellular levels. Our data demonstrate that pDCs respond differentially to various viral stimuli with significant changes in gene expression, including those involved in pDC activation, migration, viral endocytosis, survival, or apoptosis. In some cases, the expression of these genes was induced even at levels comparable to that of IFN-aalpha;. Interestingly, we also found that depending on the viral entity and the viral titer used for stimulation, induction of IFN-aalpha; gene expression and the actual release of IFN-aalpha; are not necessarily temporally coordinated. In addition, our data suggest that high-titer influenza A (H1N1) virus infection can stimulate rapid pDC apoptosis.

    IMPORTANCE Plasmacytoid dendritic cells (pDCs) are key players in the viral immune response. With the host response to viral infection being dependent on specific virus characteristics, a thorough examination and comparison of pDC responses to various viruses at various titers is beneficial for the field of virology. Our study illustrates that pDC infection with influenza virus, HIV, or hepatitis C virus results in a unique and differential response to each virus. These results have implications for future virology research, vaccine development, and virology as a whole.

  • Envelope Glycoprotein Binding to the Integrin {alpha}4{beta}7 Is Not a General Property of Most HIV-1 Strains [Pathogenesis and Immunity]

  • The HIV-1 surface glycoprotein gp120 has been reported to bind and signal through aalpha;4bbeta;7 by means of a tripeptide motif in the V2 loop that mimics structures present in the natural ligands for aalpha;4bbeta;7, suggesting that aalpha;4bbeta;7 may facilitate HIV-1 infection of CD4+ T cells in the gut. Furthermore, immune correlates in the RV144 vaccine efficacy trial generated the hypothesis that V1V2 antibodies to an epitope near the putative aalpha;4bbeta;7 binding motif may play a role in protection against HIV-1 infection. In the interest of developing an assay to detect antibodies that block gp120 binding to aalpha;4bbeta;7, we used retinoic acid (RA)-activated human peripheral blood mononuclear cells (PBMCs) and transfected HEK293T (293T) cells expressing the integrin complex to study the aalpha;4bbeta;7 binding properties of 16 HIV-1 envelope glycoproteins. The natural ligand for aalpha;4bbeta;7, mucosal addressin cell adhesion molecule-1 (MAdCAM-1), bound efficiently to RA-activated PBMCs and transfected 293T cells, and this binding was blocked by antibodies to aalpha;4. gp120 from multiple HIV-1 subtypes bound to RA-activated PBMCs from three donors in a CD4-dependent manner, but little or no aalpha;4bbeta;7 binding was detected. Similarly, little or no binding to aalpha;4bbeta;7 on transfected 293T cells was detected with multiple gp120s and gp140s, including gp120s from transmitted/founder strains, or when gp120 was produced in CHO, 293T, and 293S/GnT1nndash;/nndash; cells. Finally, we found no evidence that infectious HIV-1 virions produced in either PBMCs or 293T cells could bind aalpha;4bbeta;7 on transfected 293T cells. Infectious HIV-1 virions and most gp120s/gp140s appear to be poor ligands for the aalpha;4bbeta;7 integrin complex under the conditions tested here.

    IMPORTANCE Certain HIV-1 gp120 envelope glycoproteins have been shown to bind the gut-homing receptor aalpha;4bbeta;7, and it has been suggested that this binding facilitates mucosal transmission and virus replication in the gut mucosa. Additional evidence has generated the hypothesis that antibodies that bind near the putative aalpha;4bbeta;7 binding motif in the V2 loop of gp120, possibly disrupting gp120-aalpha;4bbeta;7 binding, may be important for HIV-1 vaccines. Our evidence indicates that infectious HIV-1 virions and many gp120s lack detectable aalpha;4bbeta;7 binding activity, suggesting that this homing receptor may play a limited role in direct HIV-1 infection of cells.

  • Influenza A and B Virus Intertypic Reassortment through Compatible Viral Packaging Signals [Structure and Assembly]

  • Influenza A and B viruses cocirculate in humans and together cause disease and seasonal epidemics. These two types of influenza viruses are evolutionarily divergent, and exchange of genetic segments inside coinfected cells occurs frequently within types but never between influenza A and B viruses. Possible mechanisms inhibiting the intertypic reassortment of genetic segments could be due to incompatible protein functions of segment homologs, a lack of processing of heterotypic segments by influenza virus RNA-dependent RNA polymerase, an inhibitory effect of viral proteins on heterotypic virus function, or an inability to specifically incorporate heterotypic segments into budding virions. Here, we demonstrate that the full-length hemagglutinin (HA) of prototype influenza B viruses can complement the function of multiple influenza A viruses. We show that viral noncoding regions were sufficient to drive gene expression for either type A or B influenza virus with its cognate or heterotypic polymerase. The native influenza B virus HA segment could not be incorporated into influenza A virus virions. However, by adding the influenza A virus packaging signals to full-length influenza B virus glycoproteins, we rescued influenza A viruses that possessed HA, NA, or both HA and NA of influenza B virus. Furthermore, we show that, similar to single-cycle infectious influenza A virus, influenza B virus cannot incorporate heterotypic transgenes due to packaging signal incompatibilities. Altogether, these results demonstrate that the lack of influenza A and B virus reassortants can be attributed at least in part to incompatibilities in the virus-specific packaging signals required for effective segment incorporation into nascent virions.

    IMPORTANCE Reassortment of influenza A or B viruses provides an evolutionary strategy leading to unique genotypes, which can spawn influenza A viruses with pandemic potential. However, the mechanism preventing intertypic reassortment or gene exchange between influenza A and B viruses is not well understood. Nucleotides comprising the coding termini of each influenza A virus gene segment are required for specific segment incorporation during budding. Whether influenza B virus shares a similar selective packaging strategy or if packaging signals prevent intertypic reassortment remains unknown. Here, we provide evidence suggesting a similar mechanism of influenza B virus genome packaging. Furthermore, by appending influenza A virus packaging signals onto influenza B virus segments, we rescued recombinant influenza A/B viruses that could reassort in vitro with another influenza A virus. These findings suggest that the divergent evolution of packaging signals aids with the speciation of influenza A and B viruses and is in part responsible for the lack of intertypic viral reassortment.

  • Dual Modulation of Type I Interferon Response by Bluetongue Virus [Virus-Cell Interactions]

  • Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus that causes an economically important disease in ruminants. BTV infection is a strong inducer of type I interferon (IFN-I) in multiple cell types. It has been shown recently that BTV and, more specifically, the nonstructural protein NS3 of BTV are able to modulate the IFN-I synthesis pathway. However, nothing is known about the ability of BTV to counteract IFN-I signaling. Here, we investigated the effect of BTV on the IFN-I response pathway and, more particularly, the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription protein (STAT) signaling pathway. We found that BTV infection triggered the expression of IFN-stimulated genes (ISGs) in A549 cells. However, when BTV-infected cells were stimulated with external IFN-I, we showed that activation of the IFN-stimulated response element (ISRE) promoter and expression of ISGs were inhibited. We found that this inhibition involved two different mechanisms that were dependent on the time of infection. After overnight infection, BTV blocked specifically the phosphorylation and nuclear translocation of STAT1. This inhibition correlated with the redistribution of STAT1 in regions adjacent to the nucleus. At a later time point of infection, BTV was found to interfere with the activation of other key components of the JAK/STAT pathway and to induce the downregulation of JAK1 and TYK2 protein expression. Overall, our study indicates for the first time that BTV is able to interfere with the JAK/STAT pathway to modulate the IFN-I response.

    IMPORTANCE Bluetongue virus (BTV) causes a severe disease in ruminants and has an important impact on the livestock economy in areas of endemicity such as Africa. The emergence of strains, such as serotype 8 in Europe in 2006, can lead to important economic losses due to commercial restrictions and prophylactic measures. It has been known for many years that BTV is a strong inducer of type I interferon (IFN-I) in vitro and in vivo in multiple cell types. However, the ability of BTV to interact with the IFN-I system remains unclear. Here, we report that BTV is able to modulate the IFN-I response by interfering with the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription protein (STAT) signaling pathway. These findings contribute to knowledge of how BTV infection interferes with the host's innate immune response and becomes pathogenic. This will also be important for the design of efficacious vaccine candidates.

  • The Histone Deacetylase Inhibitor Vorinostat (SAHA) Increases the Susceptibility of Uninfected CD4+ T Cells to HIV by Increasing the Kinetics and Efficiency of Postentry Viral Events [Virus-Cell Interactions]

  • Latently infected cells remain a primary barrier to eradication of HIV-1. Over the past decade, a better understanding of the molecular mechanisms by which latency is established and maintained has led to the discovery of a number of compounds that selectively reactivate latent proviruses without inducing polyclonal T cell activation. Recently, the histone deacetylase (HDAC) inhibitor vorinostat has been demonstrated to induce HIV transcription from latently infected cells when administered to patients. While vorinostat will be given in the context of antiretroviral therapy (ART), infection of new cells by induced virus remains a clinical concern. Here, we demonstrate that vorinostat significantly increases the susceptibility of CD4+ T cells to infection by HIV in a dose- and time-dependent manner that is independent of receptor and coreceptor usage. Vorinostat does not enhance viral fusion with cells but rather enhances the kinetics and efficiency of postentry viral events, including reverse transcription, nuclear import, and integration, and enhances viral production in a spreading-infection assay. Selective inhibition of the cytoplasmic class IIb HDAC6 with tubacin recapitulated the effect of vorinostat. These findings reveal a previously unknown cytoplasmic effect of HDAC inhibitors promoting productive infection of CD4+ T cells that is distinct from their well-characterized effects on nuclear histone acetylation and long-terminal-repeat (LTR) transcription. Our results indicate that careful monitoring of patients and ART intensification are warranted during vorinostat treatment and indicate that HDAC inhibitors that selectively target nuclear class I HDACs could reactivate latent HIV without increasing the susceptibility of uninfected cells to HIV.

    IMPORTANCE HDAC inhibitors, particularly vorinostat, are currently being investigated clinically as part of a "shock-and-kill" strategy to purge latent reservoirs of HIV. We demonstrate here that vorinostat increases the susceptibility of uninfected CD4+ T cells to infection with HIV, raising clinical concerns that vorinostat may reseed the viral reservoirs it is meant to purge, particularly under conditions of suboptimal drug exposure. We demonstrate that vorinostat acts following viral fusion and enhances the kinetics and efficiency of reverse transcription, nuclear import, and integration. The effect of vorinostat was recapitulated using the cytoplasmic histone deacetylase 6 (HDAC6) inhibitor tubacin, revealing a novel and previously unknown cytoplasmic mechanism of HDAC inhibitors on HIV replication that is distinct from their well-characterized effects of long-terminal-repeat (LTR)-driven gene expression. Moreover, our results suggest that treatment of patients with class I-specific HDAC inhibitors could induce latent viruses without increasing the susceptibility of uninfected cells to HIV.

  • Virus-Like Particle Secretion and Genotype-Dependent Immunogenicity of Dengue Virus Serotype 2 DNA Vaccine [Vaccines and Antiviral Agents]

  • Dengue virus (DENV), composed of four distinct serotypes, is the most important and rapidly emerging arthropod-borne pathogen and imposes substantial economic and public health burdens. We constructed candidate vaccines containing the DNA of five of the genotypes of dengue virus serotype 2 (DENV-2) and evaluated the immunogenicity, the neutralizing (Nt) activity of the elicited antibodies, and the protective efficacy elicited in mice immunized with the vaccine candidates. We observed a significant correlation between the level of in vitro virus-like particle secretion, the elicited antibody response, and the protective efficacy of the vaccines containing the DNA of the different DENV genotypes in immunized mice. However, higher total IgG antibody levels did not always translate into higher Nt antibodies against homologous and heterologous viruses. We also found that, in contrast to previous reports, more than 50% of total IgG targeted ectodomain III (EDIII) of the E protein, and a substantial fraction of this population was interdomain highly neutralizing flavivirus subgroup-cross-reactive antibodies, such as monoclonal antibody 1B7-5. In addition, the lack of a critical epitope(s) in the Sylvatic genotype virus recognized by interdomain antibodies could be the major cause of the poor protection of mice vaccinated with the Asian 1 genotype vaccine (pVD2-Asian 1) from lethal challenge with virus of the Sylvatic genotype. In conclusion, although the pVD2-Asian 1 vaccine was immunogenic, elicited sufficient titers of Nt antibodies against all DENV-2 genotypes, and provided 100% protection against challenge with virus of the homologous Asian 1 genotype and virus of the heterologous Cosmopolitan genotype, it is critical to monitor the potential emergence of Sylvatic genotype viruses, since vaccine candidates under development may not protect vaccinated humans from these viruses.

    IMPORTANCE Five genotype-specific dengue virus serotype 2 (DENV-2) DNA vaccine candidates were evaluated for their immunogenicity, homologous and heterologous neutralizing (Nt) antibody titers, and cross-genotype protection in a murine model. The immunity elicited by our prototype vaccine candidate (Asian 1 genotype strain 16681) in mice was protective against viruses of other genotypes but not against virus of the Sylvatic genotype, whose emergence and potential risk after introduction into the human population have previously been demonstrated. The underlying mechanism of a lack of protection elicited by the prototype vaccine may at least be contributed by the absence of a flavivirus subgroup-cross-reactive, highly neutralizing monoclonal antibody 1B7-5-like epitope in DENV-2 of the Sylvatic genotype. The DENV DNA vaccine directs the synthesis and assembly of virus-like particles (VLPs) and induces immune responses similar to those elicited by live-attenuated vaccines, and its flexibility permits the fast deployment of vaccine to combat emerging viruses, such as Sylvatic genotype viruses. The enhanced VLP secretion obtained by replacement of ectodomain I-II (EDI-II) of the Cosmopolitan genotype vaccine construct (VD2-Cosmopolitan) with the Asian 1 EDI-II elicited significantly higher total IgG and Nt antibody titers and suggests a novel approach to enhance the immunogenicity of the DNA vaccine. A DENV vaccine capable of eliciting protective immunity against viruses of existing and emerging genotypes should be the focus of future DENV vaccine development.

  • Structure Analysis of the Major Capsid Proteins of Human Polyomaviruses 6 and 7 Reveals an Obstructed Sialic Acid Binding Site [Structure and Assembly]

  • Human polyomavirus 6 (HPyV6) and HPyV7 are commonly found on human skin. We have determined the X-ray structures of their major capsid protein, VP1, at resolutions of 1.8 and 1.7 AAring;, respectively. In polyomaviruses, VP1 commonly determines antigenicity as well as cell-surface receptor specificity, and the protein is therefore linked to attachment, tropism, and ultimately, viral pathogenicity. The structures of HPyV6 and HPyV7 VP1 reveal uniquely elongated loops that cover the bulk of the outer virion surfaces, obstructing a groove that binds sialylated glycan receptors in many other polyomaviruses. In support of this structural observation, interactions of VP1 with aalpha;2,3- and aalpha;2,6-linked sialic acids could not be detected in solution by nuclear magnetic resonance spectroscopy. Single-cell binding studies indicate that sialylated glycans are likely not required for initial attachment to cultured human cells. Our findings establish distinct antigenic properties of HPyV6 and HPyV7 capsids and indicate that these two viruses engage nonsialylated receptors.

    IMPORTANCE Eleven new human polyomaviruses, including the skin viruses HPyV6 and HPyV7, have been identified during the last decade. In contrast to better-studied polyomaviruses, the routes of infection, cell tropism, and entry pathways of many of these new viruses remain largely mysterious. Our high-resolution X-ray structures of major capsid proteins VP1 from HPyV6 and from HPyV7 reveal critical differences in surface morphology from those of all other known polyomavirus structures. A groove that engages specific sialic acid-containing glycan receptors in related polyomaviruses is obstructed, and VP1 of HPyV6 and HPyV7 does not interact with sialylated compounds in solution or on cultured human cells. A comprehensive comparison with other structurally characterized polyomavirus VP1 proteins enhances our understanding of molecular determinants that underlie receptor specificity, antigenicity, and, ultimately, pathogenicity within the polyomavirus family and highlight the need for structure-based analysis to better define phylogenetic relationships within the growing polyomavirus family and perhaps also for other viruses.

  • Chemokine (C-C Motif) Receptor 1 Is Required for Efficient Recruitment of Neutrophils during Respiratory Infection with Modified Vaccinia Virus Ankara [Cellular Response to Infection]

  • Modified vaccinia virus Ankara (MVA) serves as a versatile platform in vaccine development. This highly attenuated orthopoxvirus, which cannot replicate in mammalian cells, triggers strong innate immune responses, including cell migration. Previously, we have shown that induction of chemokine (C-C motif) ligand 2 (CCL2) by MVA is necessary for the recruitment of monocytes and T cells, but not neutrophils, to the lung. Here, we identified neutrophil-attracting chemokines produced by MVA-infected primary murine lung fibroblasts and murine bone marrow-derived macrophages. We demonstrate that MVA, but not vaccinia virus (VACV) strain WR, induces chemokine expression, which is independent of Toll-like receptor 2 (TLR2) signaling. Additionally, we show that both chemokine (C-C motif) receptor 1 (CCR1) and chemokine (C-X-C motif) receptor 2 (CXCR2) are involved in MVA-induced neutrophil chemotaxis in vitro. Finally, intranasal infection of Ccr1nndash;/nndash; mice with MVA, as well as application of the CCR1 antagonist J-113863, revealed a role for CCR1 in leukocyte recruitment, including neutrophils, into the lung.

    IMPORTANCE Rapid attraction of leukocytes to the site of inoculation is unique to MVA in comparison to other VACV strains. The findings here extend current knowledge about the regulation of MVA-induced leukocyte migration, particularly regarding neutrophils, which could potentially be exploited to improve other VACV strains currently in development as oncolytic viruses and viral vectors. Additionally, the data presented here indicate that the inflammatory response may vary depending on the cell type infected by MVA, highlighting the importance of the site of vaccine application. Moreover, the rapid recruitment of neutrophils and other leukocytes can directly contribute to the induction of adaptive immune responses elicited by MVA inoculation. Thus, a better understanding of leukocyte migration upon MVA infection is particularly relevant for further development and use of MVA-based vaccines and vectors.

  • Sequence of Events in Measles Virus Replication: Role of Phosphoprotein-Nucleocapsid Interactions [Genome Replication and Regulation of Viral Gene Expression]

  • The genome of nonsegmented negative-strand RNA viruses is tightly embedded within a nucleocapsid made of a nucleoprotein (N) homopolymer. To ensure processive RNA synthesis, the viral polymerase L in complex with its cofactor phosphoprotein (P) binds the nucleocapsid that constitutes the functional template. Measles virus P and N interact through two binding sites. While binding of the P amino terminus with the core of N (NCORE) prevents illegitimate encapsidation of cellular RNA, the interaction between their C-terminal domains, PXD and NTAIL is required for viral RNA synthesis. To investigate the binding dynamics between the two latter domains, the PXD F497 residue that makes multiple hydrophobic intramolecular interactions was mutated. Using a quantitative mammalian protein complementation assay and recombinant viruses, we found that an increase in PXD-to-NTAIL binding strength is associated with a slower transcript accumulation rate and that abolishing the interaction renders the polymerase nonfunctional. The use of a newly developed system allowing conditional expression of wild-type or mutated P genes, revealed that the loss of the PXD-NTAIL interaction results in reduced transcription by preformed transcriptases, suggesting reduced engagement on the genomic template. These intracellular data indicate that the viral polymerase entry into and progression along its genomic template relies on a protein-protein interaction that serves as a tightly controlled dynamic anchor.

    IMPORTANCE Mononegavirales have a unique machinery to replicate RNA. Processivity of their polymerase is only achieved when the genome template is entirely embedded into a helical homopolymer of nucleoproteins that constitutes the nucleocapsid. The polymerase binds to the nucleocapsid template through the phosphoprotein. How the polymerase complex enters and travels along the nucleocapsid template to ensure uninterrupted synthesis of up to ~6,700-nucleotide messenger RNAs from six to ten consecutive genes is unknown. Using a quantitative protein complementation assay and a biGene-biSilencing system allowing conditional expression of two P genes copies, the role of the P-to-N interaction in polymerase function was further characterized. We report here a dynamic protein anchoring mechanism that differs from all other known polymerases that rely only onto a sustained and direct binding to their nucleic acid template.

  • Expansion of Genotypic Diversity and Establishment of 2009 H1N1 Pandemic-Origin Internal Genes in Pigs in China [Genetic Diversity and Evolution]

  • Two-way transmission of influenza viruses between humans and swine has been frequently observed, and the occurrence of the 2009 H1N1 pandemic influenza virus (pdm/09) demonstrated that swine-origin viruses could facilitate the genesis of a pandemic strain. Although multiple introductions to and reassortment in swine of the pdm/09 virus have been repeatedly reported in both Eurasia and the Americas, its long-term impact on the development of swine influenza viruses (SIVs) has not been systematically explored. Our comprehensive evolutionary studies of the complete genomes of 387 SIVs obtained from 2009 to 2012 by influenza virus surveillance in China revealed 17 reassortant genotypes with pdm/09-origin genes. Even though the entire 2009 pandemic virus and its surface genes cannot persist, its internal genes have become established and are now the predominant lineages in pigs in the region. The main persistent pdm/09-origin reassortant forms had at least five pdm/09-origin internal genes, and their surface genes were primarily of European avian-like (EA) or human H3N2-like SIV origin. These findings represent a marked change in the evolutionary patterns and ecosystem of SIVs in China. It is possible that the pdm/09-origin internal genes are in the process of replacing EA or triple-reassortant-like internal genes. These alterations in the SIV gene pool need to be continually monitored to assess changes in the potential for SIV transmission to humans.

    IMPORTANCE Shortly after the emergence of the 2009 pandemic H1N1 (pdm/09) influenza virus, it was transmitted from humans to pigs and this continues to occur around the world. Many reassortants between pdm/09-origin viruses and enzootic swine influenza viruses (SIVs) have been detected. However, the long-term impact of pdm/09-origin viruses on the SIV gene pool, which could lead to the generation of influenza viruses with the potential to infect humans, has not been systematically examined. From extensive surveillance of SIVs over a 38-month period in southern China, it was found that although neither complete pdm/09 viruses nor their surface genes could persist in pigs, their internal genes did persist. Over the survey period, these internal genes became predominant, potentially replacing those of the enzootic SIV lineages. The altered diversity of the SIV gene pool needs to be closely monitored for changes in the potential for SIV transmission to humans.

  • Detailed Study of the Interaction between Human Herpesvirus 6B Glycoprotein Complex and Its Cellular Receptor, Human CD134 [Virus-Cell Interactions]

  • Recently, we identified a novel receptor, CD134, which interacts with the human herpesvirus 6B (HHV-6B) glycoprotein (g)H/gL/gQ1/gQ2 complex and plays a key role in the entry of HHV-6B into target cells. However, details of the interaction between the HHV-6B gH/gL/gQ1/gQ2 complex and CD134 were unknown. In this study, we identified a cysteine-rich domain (CRD), CDR2, of CD134 that is critical for binding to the HHV-6B glycoprotein complex and HHV-6B infection. Furthermore, we found that the expression of HHV-6B gQ1 and gQ2 subunits was sufficient for CD134 binding, which is different from the binding of human herpesvirus 6A (HHV-6A) to its receptor, CD46. Finally, we identified a region in gQ1 critical for HHV-6B gQ1 function. These results contribute much to our understanding of the interaction between this ligand and receptor.

    IMPORTANCE We identified the domain in HHV-6B entry receptor CD134 and the components in the HHV-6B gH/gL/gQ1/gQ2 complex required for ligand-receptor binding during HHV-6B infection. Furthermore, we identified domains in gQ1 proteins of HHV-6A and -6B and a key amino acid residue in HHV-6B gQ1 required for its function. These data should be the basis for further investigation of ligand-receptor interaction in the study of HHV-6A and -6B.

  • Creation of Rift Valley Fever Viruses with Four-Segmented Genomes Reveals Flexibility in Bunyavirus Genome Packaging [Genome Replication and Regulation of Viral Gene Expression]

  • Bunyavirus genomes comprise a small (S), a medium (M), and a large (L) RNA segment of negative polarity. Although the untranslated regions have been shown to comprise signals required for transcription, replication, and encapsidation, the mechanisms that drive the packaging of at least one S, M, and L segment into a single virion to generate infectious virus are largely unknown. One of the most important members of the Bunyaviridae family that causes devastating disease in ruminants and occasionally humans is the Rift Valley fever virus (RVFV). We studied the flexibility of RVFV genome packaging by splitting the glycoprotein precursor gene, encoding the (NSm)GnGc polyprotein, into two individual genes encoding either (NSm)Gn or Gc. Using reverse genetics, six viruses with a segmented glycoprotein precursor gene were rescued, varying from a virus comprising two S-type segments in the absence of an M-type segment to a virus consisting of four segments (RVFV-4s), of which three are M-type. Despite that all virus variants were able to grow in mammalian cell lines, they were unable to spread efficiently in cells of mosquito origin. Moreover, in vivo studies demonstrated that RVFV-4s is unable to cause disseminated infection and disease in mice, even in the presence of the main virulence factor NSs, but induced a protective immune response against a lethal challenge with wild-type virus. In summary, splitting bunyavirus glycoprotein precursor genes provides new opportunities to study bunyavirus genome packaging and offers new methods to develop next-generation live-attenuated bunyavirus vaccines.

    IMPORTANCE Rift Valley fever virus (RVFV) causes devastating disease in ruminants and occasionally humans. Virions capable of productive infection comprise at least one copy of the small (S), medium (M), and large (L) RNA genome segments. The M segment encodes a glycoprotein precursor (GPC) protein that is cotranslationally cleaved into Gn and Gc, which are required for virus entry and fusion. We studied the flexibility of RVFV genome packaging and developed experimental live-attenuated vaccines by applying a unique strategy based on the splitting of the GnGc open reading frame. Several RVFV variants, varying from viruses comprising two S-type segments to viruses consisting of four segments (RVFV-4s), of which three are M-type, could be rescued and were shown to induce a rapid protective immune response. Altogether, the segmentation of bunyavirus GPCs provides a new method for studying bunyavirus genome packaging and facilitates the development of novel live-attenuated bunyavirus vaccines.

  • Diverse Specificities, Phenotypes, and Antiviral Activities of Cytomegalovirus-Specific CD8+ T Cells [Pathogenesis and Immunity]

  • CD8+ T cells specific for pp65, IE1, and IE2 are present at high frequencies in human cytomegalovirus (HCMV)-seropositive individuals, and these have been shown to have phenotypes associated with terminal differentiation, as well as both cytokine and proliferative dysfunctions, especially in the elderly. However, more recently, T cell responses to many other HCMV proteins have been described, but little is known about their phenotypes and functions. Consequently, in this study, we chose to determine the diversity of HCMV-specific CD8+ T cell responses to the products of 11 HCMV open reading frames (ORFs) in a cohort of donors aged 20 to 80 years old as well as the ability of the T cells to secrete gamma interferon (IFN-). Finally, we also tested their functional antiviral capacity using a novel viral dissemination assay. We identified substantial CD8+ T cell responses by IFN- enzyme-linked immunospot (ELISPOT) assays to all 11 of these HCMV proteins, and across the cohort, individuals displayed a range of responses, from tightly focused to highly diverse, which were stable over time. CD8+ T cell responses to the HCMV ORFs were highly differentiated and predominantly CD45RA+, CD57+, and CD28nndash;, across the cohort. These highly differentiated cells had the ability to inhibit viral spread even following direct ex vivo isolation. Taken together, our data argue that HCMV-specific CD8+ T cells have effective antiviral activity irrespective of the viral protein recognized across the whole cohort and despite viral immune evasion.

    IMPORTANCE Human cytomegalovirus (HCMV) is normally carried without clinical symptoms and is widely prevalent in the population; however, it often causes severe clinical disease in individuals with compromised immune responses. HCMV is never cleared after primary infection but persists in the host for life. In HCMV carriers, the immune response to HCMV includes large numbers of virus-specific immune cells, and the virus has evolved many mechanisms to evade the immune response. While this immune response seems to protect healthy people from subsequent disease, the virus is never eliminated. It has been suggested that this continuous surveillance by the immune system may have deleterious effects in later life. The study presented in this paper examined immune responses from a cohort of donors and shows that these immune cells are effective at controlling the virus and can overcome the virus' lytic cycle immune evasion mechanisms.

  • Type III Interferon Attenuates a Vesicular Stomatitis Virus-Based Vaccine Vector [Vaccines and Antiviral Agents]

  • Vesicular stomatitis virus (VSV) has been extensively studied as a vaccine vector and oncolytic agent. Nevertheless, safety concerns have limited its widespread use in humans. The type III lambda interferon (IFN-) family of cytokines shares common signaling pathways with the IFN-aalpha;/bbeta; family and thus evokes similar antiviral activities. However, IFN- signals through a distinct receptor complex that is expressed in a cell type-specific manner, which restricts its activity to epithelial barriers, particularly those corresponding to the respiratory and gastrointestinal tracts. In this study, we determined how IFN- expression from recombinant VSV would influence vector replication, spread, and immunogenicity. We demonstrate that IFN- expression severely attenuates VSV in cell culture. In vivo, IFN- limits VSV replication in the mouse lung after intranasal administration and reduces virus spread to other organs. Despite this attenuation, however, the vector retains its capacity to induce protective CD8 T cell and antibody responses after a single immunization. These findings demonstrate a novel method of viral vector attenuation that could be used in both vaccine and oncolytic virus applications.

    IMPORTANCE Viruses such as VSV that are used as vaccine vectors can induce protective T cell and antibody responses after a single dose. Additionally, IFN- is a potent antiviral agent that has certain advantages for clinical use compared to IFN-aalpha;/bbeta;, such as fewer patient side effects. Here, we demonstrate that IFN- attenuates VSV replication and spread following intranasal virus delivery but does not reduce the ability of VSV to induce potent protective immune responses. These findings demonstrate that the type III IFN family may have widespread applicability for improving the safety and efficacy of viral vaccine and oncolytic vectors.

  • Autographa californica Multiple Nucleopolyhedrovirus DNA Polymerase C Terminus Is Required for Nuclear Localization and Viral DNA Replication [Genome Replication and Regulation of Viral Gene Expression]

  • The DNA polymerase (DNApol) of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is essential for viral DNA replication. The DNApol exonuclease and polymerase domains are highly conserved and are considered functional in DNA replication. However, the role of the DNApol C terminus has not yet been characterized. To identify whether only the exonuclease and polymerase domains are sufficient for viral DNA replication, several DNApol C-terminal truncations were cloned into a dnapol-null AcMNPV bacmid with a green fluorescent protein (GFP) reporter. Surprisingly, most of the truncation constructs, despite containing both exonuclease and polymerase domains, could not rescue viral DNA replication and viral production in bacmid-transfected Sf21 cells. Moreover, GFP fusions of these same truncations failed to localize to the nucleus. Truncation of the C-terminal amino acids 950 to 984 showed nuclear localization but allowed for only limited and delayed viral spread. The C terminus contains a typical bipartite nuclear localization signal (NLS) motif at residues 804 to 827 and a monopartite NLS motif at residues 939 to 948. Each NLS, as a GFP fusion peptide, localized to the nucleus, but both NLSs were required for nuclear localization of DNApol. Alanine substitutions in a highly conserved baculovirus DNApol sequence at AcMNPV DNApol amino acids 972 to 981 demonstrated its importance for virus production and DNA replication. Collectively, the data indicated that the C terminus of AcMNPV DNApol contains two NLSs and a conserved motif, all of which are required for nuclear localization of DNApol, viral DNA synthesis, and virus production.

    IMPORTANCE The baculovirus DNA polymerase (DNApol) is a highly specific polymerase that allows viral DNA synthesis and hence virus replication in infected insect cells. We demonstrated that the exonuclease and polymerase domains of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) alone are insufficient for viral DNA synthesis and virus replication. Rather, we identified three features, including two nuclear localization signals and a highly conserved 10-amino-acid sequence in the AcMNPV DNApol C terminus, all three of which are important for both nuclear localization of DNApol and for DNApol activity, as measured by viral DNA synthesis and virus replication.

  • Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 4 Antagonizes Beta Interferon Expression by Targeting the NF-{kappa}B Essential Modulator [Virus-Cell Interactions]

  • Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly infectious pathogen that causes severe diseases in pigs and great economic losses to the swine industry worldwide. Type I interferons (IFNs) play a crucial role in antiviral immunity. In the present study, we demonstrated that infection with the highly pathogenic PRRSV strain JXwn06 antagonized type I IFN expression induced by poly(Immiddot;C) in both porcine alveolar macrophages (PAMs) and blood monocyte-derived macrophages (BMo). Subsequently, we showed that the inhibition of poly(Immiddot;C)-induced IFN-bbeta; production by PRRSV was dependent on the blocking of NF-B signaling pathways. By screening PRRSV nonstructural and structural proteins, we demonstrated that nonstructural protein 4 (nsp4), a viral 3C-like serine protease, significantly suppressed IFN-bbeta; expression. Moreover, we verified that nsp4 inhibited NF-B activation induced by signaling molecules, including RIG-I, VISA, TRIF, and IKKbbeta;. nsp4 was shown to target the NF-B essential modulator (NEMO) at the E349-S350 site to mediate its cleavage. Importantly, nsp4 mutants with defective protease activity abolished its ability to cleave NEMO and inhibit IFN-bbeta; production. These findings might have implications for our understanding of PRRSV pathogenesis and its mechanisms for evading the host immune response.

    IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is a major agent of respiratory diseases in pigs. Like many other viruses, PRRSV has evolved a variety of strategies to evade host antiviral innate immunity for survival and propagation. In this study, we show that PRRSV nsp4 is a novel antagonist of the NF-B signaling pathway, which is responsible for regulating the expression of type I interferons and other crucial cytokines. We then investigated the underlying mechanism used by nsp4 to suppress NF-B-mediated IFN-bbeta; production. We found that nsp4 interfered with the NF-B signaling pathway through the cleavage of NEMO (a key regulator of NF-B signaling) at the E349-S350 site, leading to the downregulation of IFN-bbeta; production induced by poly(Immiddot;C). The data presented here may help us to better understand PRRSV pathogenesis.

  • Rapid Expansion of CD8+ T Cells in Wild-Type and Type I Interferon Receptor-Deficient Mice Correlates with Protection after Low-Dose Emergency Immunization with Modified Vaccinia Virus Ankara [Vaccines and Antiviral Agents]

  • Immunization with modified vaccinia virus Ankara (MVA) can rapidly protect mice against lethal ectromelia virus (ECTV) infection, serving as an experimental model for severe systemic infections. Importantly, this early protective capacity of MVA vaccination completely depends on virus-specific cytotoxic CD8+ T cell responses. We used MVA vaccination in the mousepox challenge model using ECTV infection to investigate the previously unknown factors required to elicit rapid protective T cell immunity in normal C57BL/6 mice and in mice lacking the interferon alpha/beta receptor (IFNARnndash;/nndash;). We found a minimal dose of 105 PFU of MVA vaccine fully sufficient to allow robust protection against lethal mousepox, as assessed by the absence of disease symptoms and failure to detect ECTV in organs from vaccinated animals. Moreover, MVA immunization at low dosage also protected IFNARnndash;/nndash; mice, indicating efficient activation of cellular immunity even in the absence of type I interferon signaling. When monitoring for virus-specific CD8+ T cell responses in mice vaccinated with the minimal protective dose of MVA, we found significantly enhanced levels of antigen-specific T cells in animals that were MVA vaccinated and ECTV challenged compared to mice that were only vaccinated. The initial priming of naive CD8+ T cells by MVA immunization appears to be highly efficient and, even at low doses, mediates a rapid in vivo burst of pathogen-specific T cells upon challenge. Our findings define striking requirements for protective emergency immunization against severe systemic infections with orthopoxviruses.

    IMPORTANCE We demonstrate that single-shot low-dose immunizations with vaccinia virus MVA can rapidly induce T cell-mediated protective immunity against lethal orthopoxvirus infections. Our data provide new evidence for an efficient protective capacity of vaccination with replication-deficient MVA. These data are of important practical relevance for public health, as the effectiveness of a safety-tested, next-generation smallpox vaccine based on MVA is still debated. Furthermore, producing sufficient amounts of vaccine is expected to be a major challenge should an outbreak occur. Moreover, prevention of other infections may require rapidly protective immunization; hence, MVA could be an extremely useful vaccine for delivering heterologous T cell antigens, particularly for infectious diseases that fit a scenario of emergency vaccination.

  • Spatial Localization of the Ebola Virus Glycoprotein Mucin-Like Domain Determined by Cryo-Electron Tomography [Structure and Assembly]

  • The Ebola virus glycoprotein mucin-like domain (MLD) is implicated in Ebola virus cell entry and immune evasion. Using cryo-electron tomography of Ebola virus-like particles, we determined a three-dimensional structure for the full-length glycoprotein in a near-native state and compared it to that of a glycoprotein lacking the MLD. Our results, which show that the MLD is located at the apex and the sides of each glycoprotein monomer, provide a structural template for analysis of MLD function.

  • Acute Clearance of Human Metapneumovirus Occurs Independently of Natural Killer Cells [Pathogenesis and Immunity]

  • Human metapneumovirus (HMPV) is a major cause of respiratory disease. The role of NK cells in protection against HMPV is unclear. We show that while HMPV-infected C57BL/6 mice had higher numbers of functional lung NK cells than mock-treated mice, comparing NK cell-depleted and control mice did not reveal differences in lung viral titers, histopathology, cytokine levels, or T cell numbers or function. These data indicate that NK cells are not required for host control of HMPV.

  • The Linker Region of NS3 Plays a Critical Role in the Replication and Infectivity of Hepatitis C Virus [Genome Replication and Regulation of Viral Gene Expression]

  • Hepatitis C virus (HCV) NS3-4A is required for viral replication and assembly. We establish that virus assembly is sensitive to mutations in the linker region between the helicase and protease domains of NS3-4A. However, we find that the protease cleavage, RNA binding, and unwinding rates of NS3 are minimally affected in vitro. Thus, we conclude that the NS3 linker is critical for mediating protein-protein interactions and dynamic control rather than for modulating the enzymatic functions of NS3-4A.

  • Broadly Neutralizing Antibody VRC01 Prevents HIV-1 Transmission from Plasmacytoid Dendritic Cells to CD4 T Lymphocytes [Pathogenesis and Immunity]

  • Plasmacytoid dendritic cells (pDC) poorly replicate human immunodeficiency virus type 1 (HIV-1) but efficiently transfer HIV-1 to adjacent CD4 T lymphocytes. We found that coculture with T lymphocytes downregulates SAMHD1 expression, enhances HIV-1 replication, and increases pDC maturation and alpha interferon (IFN-aalpha;) secretion. HIV-1 transfer to T lymphocytes is inhibited by broadly neutralizing antibody VRC01 with efficiency similar to that of cell-free infection of T lymphocytes. Interestingly, prevention of HIV-1 transmission by VRC01 retains IFN-aalpha; secretion. These results emphasize the multiple functions of VRC01 in protection against HIV-1 acquisition.

  • Comparison of Effects of Inhibitors of Viral and Cellular Protein Kinases on Human Cytomegalovirus Disruption of Nuclear Lamina and Nuclear Egress [Virus-Cell Interactions]

  • Human cytomegalovirus (HCMV) kinase UL97 is required for efficient nuclear lamina disruption during nuclear egress. However, cellular protein kinase C (PKC) has been implicated in this process in other systems. Comparing the effects of UL97 and cellular kinase inhibitors on HCMV nuclear egress confirms a role for UL97 in lamina disruption and nuclear egress. A pan-PKC inhibitor did not affect lamina disruption but did reduce the number of cytoplasmic capsids more than the number of nuclear capsids.

  • Recent H3N2 Influenza Virus Clinical Isolates Rapidly Acquire Hemagglutinin or Neuraminidase Mutations When Propagated for Antigenic Analyses [Genetic Diversity and Evolution]

  • Prior to serological testing, influenza viruses are typically propagated in eggs or cell culture. Recent human H3N2 strains bind to cells with low avidity. Here, we isolated nine primary H3N2 viral isolates from respiratory secretions of children. Upon propagation in vitro, five of these isolates acquired hemagglutinin or neuraminidase mutations that increased virus binding to cell surfaces. These mutations can potentially confound serological assays commonly used to identify antigenically novel influenza viruses.

  • Human Cytomegalovirus Upregulates Expression of the Lectin Galectin 9 via Induction of Beta Interferon [Pathogenesis and Immunity]

  • Regulation of the lectin galectin 9 (Gal-9) was investigated for the first time during human cytomegalovirus (HCMV) infection. Gal-9 transcription was significantly upregulated in transplant recipients with reactivated HCMV in vivo. In vitro, Gal-9 was potently upregulated by HCMV independently of viral gene expression, with interferon beta (IFN-bbeta;) identified as the mediator of this effect. This study defines an immunoregulatory protein potently increased by HCMV infection and a novel mechanism to control Gal-9 through IFN-bbeta; induction.

  • A Substitution in the Transmembrane Region of the Glycoprotein Leads to an Unstable Attenuation of Machupo Virus [Pathogenesis and Immunity]

  • Machupo virus (MACV) is the etiologic agent of Bolivian hemorrhagic fever (BHF). Utilizing a reverse-genetics system recently developed, we report the rescue of a rationally modified recombinant MACV containing a single mutation in the transmembrane region of the glycoprotein. Following challenge of susceptible mice, we identified a significant reduction in virulence in the novel virus. We also identified an instability leading to reversion of the single mutation to a wild-type genotype.

  • Correction for Coric et al., Nuclear Magnetic Resonance Structure Revealed that the Human Polyomavirus JC Virus Agnoprotein Contains an {alpha}-Helix Encompassing the Leu/Ile/Phe-Rich Domain [Author Corrections]

  • Correction for Volume 88, no. 12, 2014, Table of Contents [Author Corrections]

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    JVI Accepts: Articles Published Ahead of Print

  • PROBING THE SITES OF INTERACTIONS OF ROTAVIRAL PROTEINS INVOLVED IN REPLICATION [PublishAheadOfPrint]

  • Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viroplasms that form during virus infection. These processes are orchestrated by yet to be understood complex networks of interactions involving non-structural proteins (NSPs) 2,5,6 and structural proteins (VPs) 1,2,3,6. The multifunctional enzyme NSP2, an octamer with RNA binding activity, is critical for viroplasm formation with its binding partner NSP5, and for genome replication/packaging through its interactions with replicating RNA, the viral polymerase VP1 and the inner core protein VP2. Using isothermal calorimetry, bio-layer interferometry and peptide-array screening, we examined the interactions between NSP2, VP1, VP2, NSP5 and NSP6. These studies provide the first evidence that NSP2 can directly bind to VP1, VP2, and NSP6 in addition to the previously known binding to NSP5. The interacting sites identified from reciprocal peptide arrays were found to be in close proximity of the RNA template entry and dsRNA exit tunnels of VP1 and near the catalytic cleft and RNA-binding grooves of NSP2; these sites are consistent with the proposed role of NSP2 in facilitating dsRNA synthesis by VP1. Peptide screening of VP2 identified NSP2-binding sites in the regions close to the inter-subunit junctions suggesting that NSP2 binding could be a regulatory mechanism for preventing the premature self-assembly of VP2. The binding sites on NSP2 for NSP6 were found to overlap with that of VP1, and the NSP5 binding sites overlap with those of VP2 and VP1 suggesting that interaction of these proteins with NSP2 is likely spatially and/or temporally regulated.

    IMPORTANCE Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viroplasms that form during virus infection and are orchestrated by complex networks of interactions involving non-structural proteins (NSPs) and structural proteins (VPs). A multifunctional RNA-binding NSP2 octamer with nucleotidyl phosphatase activity is central to viroplasm formation and RNA replication. Here we provide the first evidence that NSP2 can directly bind to VP1, VP2, and NSP6, in addition to the previously known binding to NSP5. The interacting sites identified from peptide arrays are consistent with the proposed role of NSP2 in facilitating dsRNA synthesis by VP1 and also point to NSP2's possible role in preventing the premature self-assembly of VP2 cores. Our findings lead us to propose that the NSP2 octamer with multiple enzymatic activities is a principal regulator of viroplasm formation, recruitment of viral proteins into the viroplasms and possibly genome replication.

  • Structural Definition of an Antibody-Dependent Cellular Cytotoxicity (ADCC) Response Implicated in Reduced Risk for HIV-1 Infection [PublishAheadOfPrint]

  • The RV144 vaccine trial implicated epitopes in the C1 region of gp120 (A32-like epitopes) as targets of potentially protective antibody-dependent cellular cytotoxicity (ADCC) responses. A32-like epitopes are highly immunogenic as infected or vaccinated individuals frequently elicit antibodies specific for these determinants. Antibody-binding titers as measured by ELISA against these epitopes, however, do not consistently correlate with protection. Here, we report crystal structures of CD4-stabilized gp120 cores complexed with the Fab fragments of two non-neutralizing, A32-like monoclonal antibodies (mAbs), named N5-i5 and 2.2c, that compete for antigen binding and exhibit similar binding affinities, yet mediate a 75-fold difference in ADCC potency. We find that these mAbs recognize overlapping epitopes formed by mobile layers 1 and 2 of the gp120 inner domain including the C1-C2 regions, but bind gp120 at different angles via juxtaposed VH and VL contact surfaces. A comparison of structural and immunological data further showed that antibody orientation on bound antigen and the capacity to form multivalent antigen-antibody complexes on target cells were key determinants for ADCC potency, with the latter process having the greater impact. These studies provide atomic level definition of A32-like epitopes implicated as targets of protective antibodies in RV144. Moreover, these studies establish that epitope structure and mode of antibody binding can dramatically affect the potency of Fc-mediated effector function against HIV-1. These results provide key insights for understanding, refining, and improving the outcome of HIV-vaccine trials, in which relevant immune responses are facilitated by A32-like elicited responses.

    Importance: HIV-1 Env is a primary target for antibodies elicited during infection. Although a small number of infected individuals elicit broadly neutralizing antibodies, the bulk of humoral response consists of antibodies that do not neutralize or do so with limited breadth but may effect protection through Fc receptor-dependent processes, such as antibody-dependent cellular cytotoxicity (ADCC). Understanding these non-neutralizing responses is an important aspect in elucidating the complete spectrum of immune response against HIV-1 infection. With this report we provide the first atomic level definition of non-neutralizing CD4-induced epitopes in the N-terminal region of the HIV-1 gp120 (A32-like epitopes). Further, our studies point toward the dominant role of precise epitope targeting and mode of antibody attachment in ADCC responses even when largely overlapping epitopes are involved. Such information provides key insights into the mechanisms of Fc-mediated antibody function to HIV-1 and helps understand the outcome of vaccine trials based on humoral immunity.

  • Epstein Barr Virus (EBV) Late Gene Transcription Depends on the Assembly of a Viral Specific Pre-Initiation Complex [PublishAheadOfPrint]

  • During their productive cycle, herpesviruses exhibit a strictly regulated temporal cascade of gene expression that has three general stages: immediate early (IE), early (E), and late (L). Promoter complexity differs strikingly between IE/E genes and L genes. IE and E promoters contain cis-regulating sequences upstream of a TATA box whereas L promoters comprise a unique cis element. In the case of the -herpesviruses, this element is usually a TATT motif found in the position where the consensus TATA box of eukaryotic promoters typically localizes. Epstein-Barr virus encodes a protein, called BcRF1, which has structural homology with the TATA-binding protein and interacts specifically with the TATT box. However, although necessary for the expression of the L genes, BcRF1 is not sufficient, suggesting that other viral proteins are also required. Here, we present the identification and characterization of a viral protein complex necessary and sufficient for the expression of the late viral genes. This viral complex is composed of five different proteins in addition to BcRF1 and interacts with cellular RNA polymerase II. During the viral productive cycle, this complex, that we call vPIC (for viral Pre-Initiation Complex), works in concert with the viral DNA replication machinery to activate expression of the late viral genes. The EBV vPIC components have homologs in bbeta;- and -herpesviruses but not in aalpha;-herpesviruses. Our results not only reveal that bbeta;- and -herpesviruses encode their own transcription pre-initiation complex responsible for the expression of the late viral genes but also indicate their close evolutionary history.

    IMPORTANCE Control of late gene transcription in DNA viruses is a major unsolved question in virology. In eukaryotes, the first step in transcriptional activation is the formation of a permissive chromatin which allows assembly of the pre-initiation complex (PIC) at the core promoter. Fixation of the TATA-binding protein is a key rate-limiting step in this process. This study provides evidence that EBV encodes a complex composed of six proteins necessary for the expression of the late viral genes. This complex is formed around a viral TBP-like protein and interacts with cellular RNA polymerase II, suggesting that it is directly involved in the assembly of a viral-specific PIC (vPIC).

  • Distinct Mechanisms Regulate Exposure of Neutralizing Epitopes in the V2 and V3 Loops of HIV-1 Envelope [PublishAheadOfPrint]

  • Broadly neutralizing antibodies targeting the HIV-1 envelope (Env) are key components for protection against HIV-1. However, many cross-reactive epitopes are often occluded. This study investigates the mechanisms contributing to the masking of V2i (V2-integrin) epitopes compared to V3 epitopes. V2i are conformation-dependent epitopes encompassing the integrin aalpha;4bbeta;7-binding motif on the V1V2 loop of HIV-1 Env gp120. The V2i monoclonal antibodies (mAbs) display extensive cross-reactivity with gp120 monomers from many subtypes, but neutralize only few viruses, indicating V2i's cryptic nature. First, we asked whether CD4-induced Env conformational changes affect V2i epitopes similar to V3. CD4 treatment of BaL and JRFL pseudoviruses increased their neutralization sensitivity to V3 mAbs, but not to the V2i mAbs. Second, contribution of N-glycans on masking V2i versus V3 epitopes was evaluated by testing neutralization of pseudoviruses produced in the presence of a glycosidase-inhibitor, kifunensine. Viruses grown in kifunensine were more sensitive to neutralization by V3 but not V2i mAbs. Finally, we evaluated the time-dependent dynamics of the V2i and V3 epitopes. Extending the time of virus-mAb interaction to 18 hours, before adding target cells, increased virus neutralization by some V2i mAbs and all V3 mAbs tested. Consistent with this, V2i mAb binding to Env on the surface of transfected cells also increased in time-dependent manner. Hence, V2i and V3 epitopes are highly dynamic but distinct factors modulate antibody accessibility of these epitopes. The study reveals the importance of the structural dynamics of V2i and V3 epitopes in determining HIV-1 neutralization by antibodies targeting these sites.

    Importance Conserved neutralizing epitopes are present in the V1V2 and V3 regions of HIV-1 Env, but these epitopes are often occluded from Abs. This study reveals that distinct mechanisms contribute to the masking of V3 epitopes and V2i epitopes in the V1V2 domain. Importantly, V3 mAbs and some V2i mAbs display greater neutralization against relatively resistant HIV-1 isolates when the mAbs interact with the virus for a prolonged period of time. Given their highly immunogenic nature, V3 and V2i epitopes are valuable targets that would augment the efficacy of HIV vaccines.

  • MHC class I downregulation induced by equine herpesvirus type 1 (EHV-1) pUL56 is through dynamin-dependent endocytosis [PublishAheadOfPrint]

  • Equine herpesvirus type 1 (EHV-1) downregulates cell surface expression of major histocompatibility complex class I (MHC-I) in infected cells. We have previously shown that pUL56 encoded by the EHV-1 ORF1 gene regulates the process (G. Ma, S. Feineis, N. Osterrieder, and G. R. Van de Walle, J. Virol. 86:3554-3563, 2012, doi:10.1128/JVI.06994-11). Here, we report that cell surface MHC-I in EHV-1-infected cells is internalized and degraded in the lysosomal compartment in a pUL56-dependent fashion. pUL56-induced MHC-I endocytosis required dynamin and tyrosine kinase, but was independent of clathrin or caveolin-1, the main constituents of clathrin- and raft/caveolae-mediated endocytosis pathway, respectively. Downregulation of cell surface MHC-I was significantly inhibited by the ubiquitin-activating enzyme E1 inhibitor PYR41, indicating that ubiquitination is essential for the process. Finally, we show that downregulation is not specific for MHC-I and that cell surface expression of other molecules including CD46 and CD63 is also removed from the cell surface in a pUL56-dependent fashion.

    IMPORTANCE We show that alphaherpesvirus induces MHC-I downregulation through endocytosis, which is mediated by pUL56. The dynamin-dependent endocytic pathway is responsible for MHC-I internalization in infected cells. Furthermore, we discovered that this endocytic process can be disrupted by inhibiting ubiquitin-activating E1 enzyme, which is indispensible for ubiquitination. Finally, pUL56 action extends to a number of cell surface molecules that are significant for host immunity. Therefore, the protein may exert a more general immunomodulatory effect.

  • Emergence of individual HIV-specific CD8 T cell responses during primary HIV-1 infection can determine long-term disease outcome [PublishAheadOfPrint]

  • Background. Events during primary HIV-1 infection have been shown to be critical for the subsequent rate of disease progression. Early control of viral replication, resolution of clinical symptoms and development of a viral setpoint have been associated with the emergence of HIV-specific CD8 T cell responses. Here we assessed which particular HIV-specific CD8 T cell responses contribute to long-term control of HIV-1.

    Methods. A total of 620 individuals with primary HIV-1 infection were screened by IFN Elispot for HLA class I-restricted, epitope-specific CD8 T cell responses using optimally defined epitopes approximately 2 months post initial presentation. The cohort was predominantly male(97%)Caucasian(83%) (Fiebig: II/III:[n=152], IV:[n=61], V:[n=277], VI:[n=87], nd:[n=23]). Longitudinal viral loads, CD4 count and time to ART were collected for all patients.

    Results. We observed strong associations between viral load at baseline (initial viremia) and the established early viral set points (pllt;0.0001). Both were significantly associated with HLA class I genotypes (p=0.0009). While neither the breadth nor magnitude of HIV-specific CD8 T cell responses showed an influence on the early viral set point, a broader HIV-specific CD8 T cell response targeting epitopes within HIV-1 Gag during primary HIV-1 infection was associated with slower disease progression. Moreover, the induction of certain HIV-specific CD8 T cell responses - but not others mmdash;significantly influenced the time to ART initiation.

    Conclusions. Individual epitope-specific CD8 T cell responses are significantly contributing to HIV-1 disease control, demonstrating that the specificity of the initial HIV-specific CD8 T cell response rather than the restricting HLA class I molecule alone is a critical determinant of their antiviral function.

    IMPORTANCE Understanding which factors are involved in the control of HIV infection is critical for the design of therapeutic strategies for patients living with HIV/AIDS. Here we assessed in a cohort of over 600 individuals with acute and early HIV infection in unprecedented detail the individual contribution of epitope-specific CD8 T cell responses directed against HIV to control of viremia and their impact on the overall course of disease progression.

  • Activation of Intrinsic Immune Responses and Microglial Phagocytosis in an Ex Vivo Spinal Cord Slice Culture Model of West Nile Virus Infection [PublishAheadOfPrint]

  • West Nile virus (WNV) is a neurotropic flavivirus that causes significant neuroinvasive disease involving the brain and/or spinal cord. Experimental mouse models of WNV infection have established the importance of innate and adaptive immune responses in controlling the extent and severity of central nervous system (CNS) disease. However, differentiating between immune responses that are intrinsic to the CNS and those dependent on infiltrating inflammatory cells or resulting from immune events caused by extraneural systemic infection has proven difficult. We used a murine ex vivo spinal cord slice culture (SCSC) model to determine the innate immune processes specific to the CNS during WNV infections. By 7 days post ex vivo infection of SCSCs, the majority of neurons and a substantial percentage of astrocytes were infected with WNV resulting in apoptotic cell death and astrogliosis. Microglia, the resident immune cells of the CNS, were activated by WNV infection as exemplified by their amoeboid morphology, development of filopodia and lamellipodia, and phagocytosis of WNV-infected cells and debris. Microglial cell activation was concomitant with increased expression of pro-inflammatory cytokines and chemokines, including CXCL10, CXCL1, CCL5, CCL3, CCL2, TNFaalpha;, TRAIL, and IL-6. The application of minocycline, an inhibitor of neuroinflammation, altered the WNV-induced pro-inflammatory cytokine/chemokine expression profile with inhibited production of CCL5, CCL2, and IL-6 but not of CXCL10, TRAIL, and TNFaalpha;. Our findings establish that CNS resident cells have the capacity to initiate a robust innate immune response against WNV infection in the absence of infiltrating inflammatory cells and systemic immune responses.

    IMPORTANCE There are no specific treatments of proven efficacy available for WNV neuroinvasive disease. A better understanding of the pathogenesis of WNV CNS infection is crucial for the rational development of novel therapies. Development of a spinal cord slice culture (SCSC) model facilitates study of WNV pathogenesis and allows investigation of the intrinsic immune responses of the CNS. Our studies demonstrate that robust CNS innate immune responses including microglial activation and pro-inflammatory cytokine/chemokine production develop independent of contributions from the peripheral immune system and CNS-infiltrating inflammatory cells.

  • Direct Interrogation of Viral Peptides Presented by the Class I HLA of HIV Infected T Cells [PublishAheadOfPrint]

  • Identification of CD8+ cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing overlapping peptide libraries for their reactivity with T cells in vitro. Here, we pursued Deep Ligand Sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTL by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4+ SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size exclusion filtration. The ligands were fractionated first by high pH HPLC and then subjected to separation by nano LCMS at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, 37 of which were previously undetected. These data demonstrate that (1) HIV-1 Gag and Nef are extensively sampled, (2) ligand length variants are prevalent, particularly within Gag and Nef "hot spots" where ligand sequences overlap, (3) non-canonical ligands are T cell reactive, and (4) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next generation immunotherapies must factor these nascent HIV-1 ligand length variants, and the finding that CTL reactive epitopes may be absent during infection of CD4+ T cells, into strategies designed to enhance T cell immunity.

    Importance HIV-1 epitopes catalogued by the Los Alamos National Laboratory (LANL) have yielded limited success in vaccine trials. Because the HLA of infected cells have not previously been assessed for HIV-1 ligands, the objective here was to directly characterize the viral ligands that mark infected cells. Recovery of HLA-presented peptides from HIV-1 infected CD4+ T cells and interrogation of the peptide cargo by mass spectrometric DLS shows that typical and atypical viral ligands are efficiently presented by HLA and targeted by human CTL. Nef and Gag ligands dominate the infected cell's antigenic profile, largely due to extensive ligand sampling from select "hot spots" within these viral proteins. Also, HIV-1 ligands are often longer than expected, and these length variants are quite antigenic. These findings emphasize that an HLA-based view of HIV-1 ligand presentation to CTL provides previously unrealized information that may enhance the development of immune therapies and vaccines.

  • The Raccoon Polyomavirus Genome and Tumor Antigen Transcription are Stable and Abundant in Neuroglial Tumors [PublishAheadOfPrint]

  • Raccoon polyomavirus (RacPyV) is associated with 100% of neuroglial tumors in free-ranging raccoons. Other tumor-associated polyomaviruses (PyVs), including SV40, murine PyV, and Merkel cell PyV, are found integrated in the host genome in neoplastic cells, where they constitutively express splice variants of the tumor antigen (TAg) gene. We have previously reported that RacPyV exists only as an episome (non-integrated) in neuroglial tumors. Here we have investigated TAg transcription in primary tumor tissue by transcriptome analysis, and we identified the alternatively spliced TAg transcripts for RacPyV. We also determined that TAg was highly transcribed relative to host cellular genes. We further co-localized TAg DNA and mRNA by in situ hybridization, and found that the majority of tumor cells showed positive staining. Lastly, we examined stability of the viral genome and TAg transcription by quantitative reverse-transcriptase PCR in cultured tumor cells in vitro and in a mouse xenograft model. When tumor cells were cultured in vitro, TAg transcription increased nearly two log-fold over that of parental tumor tissue by passage 17. Both episomal viral genome and TAg transcription were faithfully maintained in culture and in tumors arising from xenotransplant of cultured cells in mice. This study represents a minimal criterion for RacPyV's association with neuroglial tumors, and a novel mechanism of stability for a polyomavirus in cancer.

    Importance: The natural cycle of polyomaviruses in mammals is to persist in the host without causing disease, but can cause cancer in humans or in other animals. Because this is an unpredictable and rare event, the oncogenic potential of polyomavirus is primarily evaluated in laboratory animal models. Recently, raccoon polyomavirus (RacPyV) was identified in neuroglial tumors of free-ranging raccoons. Viral copy number was consistently high in these tumors, but was low or undetectable in non-tumor tissue or in unaffected raccoons. Unlike other oncogenic polyomaviruses, RacPyV was episomal, not integrated, in these tumors. To determine the stability of the viral genome and sustained transcription of the oncogenic tumor antigen proteins, we cultured primary raccoon tumor cells and passaged them in mice, confirming the non-integrated state of the virus and the maintenance of viral protein transcription throughout. RacPyV provides a naturally occurring and tractable model for a novel mechanism of polyomavirus-mediated oncogenesis.

  • Lack of interleukin-10-mediated anti-inflammatory signals and upregulated interferon gamma production are linked to increased intestinal epithelial cell apoptosis in pathogenic simian immunodeficiency virus infection [PublishAheadOfPrint]

  • Interleukin (IL)-10 is an immunomodulatory cytokine that is important for maintenance of epithelial cell (EC) survival and anti-inflammatory responses (AIR). The majority of HIV infections occur through the mucosal route despite mucosal epithelium acting as a barrier to HIV. Therefore understanding the role of IL-10 in maintenance of intestinal homeostasis during HIV infection is of interest for better characterization of the pathogenesis of HIV-mediated enteropathy. Here we demonstrated changes in mucosal IL-10 signaling during SIV infection in rhesus macaques. Disruption of the epithelial barrier was manifested by EC apoptosis and loss of tight junction protein ZO-1. Multiple cell types, including a limited number of ECs, produced IL-10. SIV infection resulted in increased levels of IL-10, however this was associated with increased production of mucosal IFN and TNFaalpha;, suggesting that IL-10 was not able to regulate AIR. This observation was supported by downregulation of STAT3, which is necessary to inhibit production of IFN and TNFaalpha;, and upregulation of SOCS1 and SOCS3, which are important regulatory molecules in the IL-10-mediated AIR. We also observed internalization of the IL-10 receptor (IL-10R) in mucosal lymphocytes, which could limit cellular availability of IL-10 for signaling and contribute to the loss of a functional AIR. Collectively, these findings demonstrate that internalization of IL-10R with the resultant impact on IL-10 signaling and dysregulation of the IL-10-mediated AIR might play a crucial role in EC damage and subsequent SIV/HIV pathogenesis.

    IMPORTANCE Interleukin-10 (IL-10), an important immunomodulatory cytokine plays a key role to control inflammatory function and homeostasis of the gastro-intestinal mucosal immune system. Despite recent advancements in the study of IL-10 and its role in HIV infection, the role of mucosal IL-10 in SIV/HIV infection in inducing enteropathy is not well understood. Here we demonstrated changes in mucosal IL-10 signaling during SIV infection in rhesus macaques. Disruption of the intestinal epithelial barrier was evident along with the increased levels of mucosal IL-10 production. Increased production of mucosal IFN and TNFaalpha; during SIV infection suggested that the increased level of mucosal IL-10 was not able to regulate anti-inflammatory responses. Our findings demonstrate that internalization of IL-10R with the resultant impact on IL-10 signaling and dysregulation of the IL-10-mediated anti-inflammatory responses might play a crucial role in epithelial cell damage and subsequent SIV/HIV pathogenesis.

  • A novel marine DNA virus (Singapore grouper iridovirus, SGIV) entry into host cells occurs via clathrin-mediated endocytosis and macropinocytosis in a pH-dependent manner [PublishAheadOfPrint]

  • Iridoviruses are nucleocytoplasmic DNA viruses which cause great economic losses in aquaculture industry, but also show significant threat to global biodiversity. However, a lack of host cells results in poor progress in clarifying iridovirus behaviors. We investigated the crucial events during virus entry using a combination of single-virus particle tracking and biochemical assays, based on the established virus-cell infection model for Singapore grouper iridovirus (SGIV). SGIV infection in host cells was strongly inhibited when cells were pretreated with drugs blocking clathrin-mediated endocytosis, including sucrose and chlorpromazine. Inhibition of key regulators of macropinocytosis, including Na+/H+ exchanger, Rac1 GTPase, p21-activated kinase 1 (PAK1), protein kinase C (PKC) and myosin II significantly reduced SGIV uptake. Cy5-labeled SGIV particles were observed to co-localize with clathrin and macropinosomes. In contrast, disruption of cellular cholesterol by methyl-bbeta;-cyclodextrin and nystatin had no effect on virus infection, suggesting that SGIV entered grouper cells via the clathrin-mediated endocytic pathway and macropinocytosis, but not via caveolae-dependent endocytosis. Furthermore, inhibitors of endosome acidification such as chloroquine and bafilomycin A1 blocked virus infection, indicating that SGIV entered cells in a pH-dependent manner. In addition, SGIV particles were observed to transport along both microtubules and actin filaments, and intracellular SGIV motility was remarkably impaired by depolymerization of microtubules or actin filaments. The results of this study for the first time demonstrate that not only clathrin-dependent pathway, but also macropinocytosis is involved in fish DNA enveloped virus entry, thus providing a convenient tactic for exploring the life cycle of DNA viruses.

    Importance Statement Virus entry into host cells is critically important for initiating infections and usually recognized as an ideal target for the design of antiviral strategies. Iridoviruses are large DNA viruses which cause serious threat to ecological diversity and aquaculture industry worldwide. However, the current understanding of iridovirus entry is limited and controversial. Singapore grouper iridovirus (SGIV) is a novel marine fish DNA virus which belongs to genus Ranavirus, family Iridovirdae. Here, using single-virus particle tracking technology in combination with biochemical assays, we investigated the crucial events during SGIV entry, and demonstrated that SGIV entered grouper cells via clathrin-mediated endocytic pathway in a pH-dependent manner, but not caveolae-dependent endocytosis. Furthermore, we proposed for the first time that macropinocytosis was involved in iridovirus entry. Together, this work not only contributes greatly to understating iridovirus pathogenesis, but also provides an ideal model for exploring the behaviors of DNA viruses in the living cells.

  • Possible footprints of APOBEC3F and/or other APOBEC3 deaminases -- but not APOBEC3G -- on HIV-1 from patients during acute/early and chronic infection [PublishAheadOfPrint]

  • Members of the apolipo-protein-B mRNA-editing-enzyme-catalytic polypeptide-like-3 (APOBEC3) innate cellular cytidine deaminase family, particularly APOBEC3F and APOBEC3G, can cause extensive and lethal G-to-A mutations in HIV-1 plus-strand DNA (termed hypermutation). It is unclear if APOBEC3-induced mutations in vivo are always lethal or can occur at sub-lethal levels that increase HIV-1 diversification and viral adaptation to the host. The viral accessory protein Vif counteracts APOBEC3-activity by binding to APOBEC3 and promoting proteasome degradation; however, the efficiency of this interaction varies as a range of hypermutation frequencies are observed in HIV-1 patient DNA. Therefore, we examined llsquo;footprintsrrsquo; of APOBEC3G- and APOBEC3F-activity in longitudinal HIV-1 RNA pol sequences from approximately 3,000 chronically infected patients by determining whether G-to-A mutations occurred in motifs that were favored or disfavored by these deaminases. G-to-A mutations were more frequent in APOBEC3G-disfavored than in APOBEC3G-favored contexts. By contrast, mutations in APOBEC3F-disfavored contexts were relatively rare, whereas mutations in contexts favoring APOBEC3F (and possibly other deaminases) occurred 16% more often than average G-to-A mutations. These results were supported by analyses of ggt;500 HIV-1 env sequences from acute/early infection.

    Importance (63) Collectively, our results suggest that APOBEC3G-induced mutagenesis is lethal to HIV-1, whereas mutagenesis caused by APOBEC3F and/or other deaminases may result in sub-lethal mutations that might facilitate viral diversification. Therefore, Vif-specific CTL-responses and drugs that manipulate the interplay between Vif and APOBEC3 may have beneficial or detrimental clinical effects depending on how they affect the binding of Vif to various members of the APOBEC3-family.

  • Stepwise priming by acidic pH and high K+ is required for efficient uncoating of influenza A virus cores after penetration [PublishAheadOfPrint]

  • Influenza A virus uses the low pH in late endocytic vacuoles as a cue for penetration by membrane fusion. Here, we analyzed the pre-fusion reactions that prepare the core for uncoating after it has been delivered to the cytosol. We found that this priming process occurs in two steps that are mediated by the envelope-embedded M2 ion channel. The first weakens the interactions between the matrix protein, M1, and the viral ribonucleoprotein bundle. It involves a conformational change in a linker sequence and the C-terminal domain of M1 after exposure to pH below 6.5. The second step is triggered by pH llt; 6.0 and by the influx of K+ ions. It causes additional changes in M1 as well as a loss of stability in the vRNP bundle. Our results indicated that the switch from Na+ to K+ in maturing endosomes together with the decreasing pH are both needed to prime IAV cores for efficient uncoating and infection of the host cell.

    Importance: The entry of IAV involves several steps including endocytosis and fusion at late endosomes. Entry also includes disassembly of the viral core which is composed of the viral ribonucleoproteins and the RNA genome. We have found that the uncoating process of IAV is initiated long before the core is delivered into the cytosol. M2, an ion channel in the viral membrane is activated when the virus passes through early endosomes. Here, we show that protons entering the virus through M2 cause a conformational change in the matrix protein, M1. This weakens interactions between M1 and the viral ribonucleoproteins. A second change was found to occur when the virus enters late endosomes. The pre-acidified core is now exposed to a high concentration of K+, which affects the interactions between the ribonucleoproteins. Thus, when cores are finally delivered to the cytosol they are already partially destabilized and therefore uncoating-competent and infectious.

  • Fluorescent Tagging and Cellular Distribution of the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Open Reading Frame 45 (ORF45) Tegument Protein [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is a cancer-related human virus, classified as a member of the gammaherpesvirinae subfamily. We report here the construction of a dual fluorescent-tagged KSHV genome (BAC16-mCherry-ORF45), which constitutively expresses GFP and contains the tegument multifunctional ORF45 protein as a fusion protein with monomeric Cherry fluorescent protein (mCherry). We confirmed that this virus is properly expressed and correctly replicates, and that mCherry-ORF45 protein is incorporated into the virions. Using this labeled virus, we describe the dynamics of mCherry-ORF45 expression and localization in newly infected cells as well as in latently infected cells undergoing lytic induction, and show that mCherry can be used to monitor cells undergoing the lytic viral cycle. This virus is likely to enable future studies monitoring the dynamics of viral trafficking and tegumentation during viral ingress and egress.

    Importance: The present study describes the construction and characterization of a new recombinant KSHV genome BAC16 clone which expresses mCherry-tagged ORF45. This virus enables the tracking of cells undergoing lytic infection and can be used to address issues related to the trafficking and maturation pathways of KSHV virions.

  • HIV control is mediated in part by CD8+ T-cell targeting of specific epitopes [PublishAheadOfPrint]

  • We investigate the hypothesis that the correlation between the class I HLA types of an individual and whether that individual spontaneously controls HIV-1 is mediated by the targeting of specific epitopes by CD8+ T cells. Measuring interferon- ELISPOT responses to a panel of 257 optimally defined epitopes in 341 untreated HIV- infected persons, including persons who spontaneously control viremia, we find that the correlation between HLA types and control is mediated by the targeting of specific epitopes. Moreover, we perform a graphical model-based analysis suggesting that the targeting of specific epitopes is a cause of such controlmmdash;that is, some epitopes are protective rather than merely associated with controlmmdash;and identify eight epitopes that are significantly protective. In addition, we use an in silico analysis to identify protein regions where mutations are likely to affect the stability of a protein, and find that the protective epitopes identified by the ELISPOT analysis correspond almost perfectly to such regions. This in silico analysis thus suggests a possible mechanism for control, and could be used to identify protective epitopes that are not often targeted in natural infection but that may be potentially useful in a vaccine. Our analyses thus argue for the inclusion (and exclusion) of specific epitopes in an HIV vaccine.

    Importance Some individuals naturally control HIV replication in the absence of anti-retroviral therapy, and this ability to control is strongly correlated with the HLA class I alleles that they express. Here, in a large-scale experimental study, we provide evidence that this correlation is largely mediated by the targeting of specific CD8+ T-cell epitopes, and identify eight epitopes that are likely to cause control. In addition, we provide an in silico analysis indicating that control occurs because mutations within these epitopes change the stability of the protein structures. This in silico analysis also identifies additional epitopes that are not typically targeted in natural infection, but may lead to control when included in a vaccine, provided other epitopes that would otherwise distract the immune system from targeting them are excluded from the vaccine.

  • Investigating a Mystery Disease: Tales from a Viral Detective [PublishAheadOfPrint]

  • Viral outbreak investigation is challenging logistically as well as scientifically. In the context of addressing a fictional emerging viral disease I describe the process of discovery, from the initial report of a problem through discussions of intellectual property and sample management, study design, management, experimental execution and reporting of results.

  • APOBEC3F Determinants of HIV-1 Vif Sensitivity [PublishAheadOfPrint]

  • HIV-1 Vif counteracts restrictive APOBEC3 proteins by targeting them for proteasomal degradation. To determine the regions mediating sensitivity to Vif, we compared human APOBEC3F, which is HIV-1 Vif sensitive, with rhesus APOBEC3F, which is HIV-1 Vif resistant. Rhesus/human APOBEC3F chimeras and amino acid substitution mutants were tested for sensitivity to HIV-1 Vif. This approach identified the alpha-3 and alpha-4 helices of human APOBEC3F as important determinants of the interaction with HIV-1 Vif.

  • Enhanced potency of a broadly neutralizing HIV-1 antibody in vitro improves protection against lentiviral infection in vivo [PublishAheadOfPrint]

  • Over the past five years, a new generation of highly potent and broadly neutralizing HIV-1 antibodies has been identified. These antibodies can protect against lentiviral infection in non-human primates, suggesting that passive antibody transfer would prevent HIV-1 transmission in humans. To increase the protective efficacy of such monoclonal antibodies, we employed next-generation sequencing, computational bioinformatics, and structure-guided design to enhance the neutralization potency and breadth of VRC01, an antibody that targets the CD4 binding site of the HIV-1 envelope. One variant, VRC07-523, was 5- to 8-fold more potent than VRC01, neutralized 96% of viruses tested, and displayed minimal autoreactivity. To compare its protective efficacy to VRC01 in vivo, we performed a series of simian-HIV (SHIV) challenge experiments in non-human primates and calculated the doses of VRC07-523 and VRC01 that provide 50% protection (EC50). VRC07-523 prevented infection in NHPs at a 5-fold lower concentration than VRC01. These results suggest that increased neutralization potency in vitro correlates with improved protection against infection in vivo, documenting the improved functional efficacy of VRC07-523 and its potential clinical relevance for protecting against HIV-1 infection in humans.

    IMPORTANCE In the absence of an effective HIV-1 vaccine, alternative strategies are needed to block HIV-1 transmission. Direct administration of HIV-1-neutralizing antibodies may be able to prevent HIV-1 infections in humans. This approach could be especially useful in individuals at high risk for contracting HIV-1 and could be used together with antiretroviral drugs to prevent infection. To optimize the chance of success, such antibodies can be modified to improve their potency, breadth, and in vivo half-life. Here, knowledge of the structure of a potent neutralizing antibody VRC01, that targets the CD4-binding site of the HIV-1 envelope protein, was used to engineer a next-generation antibody with 5-8 fold increased potency in vitro. When administered to non-human primates, this antibody conferred protection at a five-fold lower concentration than the original antibody. Our studies demonstrate an important correlation between in vitro assays used to evaluate therapeutic potential of antibodies and their in vivo effectiveness.

  • EBV blocks the autophagic flux and appropriates the autophagic machinery to enhance viral replication [PublishAheadOfPrint]

  • Autophagy is a catabolic pathway that helps cells to survive in stressful conditions. Cells also use autophagy to clear microbiological infections but, on the other hand, microbes have learned how to manipulate the autophagic pathway for their own benefit. The experimental evidence obtained in this study suggests that the autophagic flux is blocked at the final steps during the reactivation of EBV from latency. This is indicated by the level of LC3II that does not increase in the presence of bafilomycin and by the lack of colocalization of autophagosomes with lysosomes, which correlates with a reduced Rab7 expression.. Since the inhibition of the early phases of autophagy impaired EBV replication and viral particles were observed in autophagic vescicles in the cytoplasm of producing cells, we suggest that EBV exploits the autophagic machinery for its transportation, in order to enhance viral production. The autophagic block was not mediated by ZEBRA, an immediate early EBV lytic gene, whose transfection in Ramos, Akata and 293 cells promoted a complete autophagic flux, The block occurred only when the complete set of EBV lytic genes was expressed. We suggest that the inhibition of the early autophagic steps or finding strategies to overcome the autophagic block, allowing viral degradation into the lysosomes, could be potentially exploited to manipulate EBV replication.

    IMPORTANCE This study shows for the first time that autophagy is blocked at the final degradative steps during EBV replication, in several cell types. Through this block, EBV hijacks the autophagic vescicles for its intracellular transportation and enhances viral production. A better understanding of virus/host interactions could help to design new therapeutic approaches against EBV-associated malignancies.

  • A Synthetic Biology Approach For a Vaccine Platform Against Known and Newly Emerging Serotypes of Bluetongue Virus [PublishAheadOfPrint]

  • Bluetongue is one of the major infectious diseases of ruminants and is caused by Bluetongue virus (BTV), an arbovirus existing in nature in at least 26 distinct serotypes. Here, we describe the development of a vaccine platform for BTV. The advent of synthetic biology approaches and the development of reverse genetics systems, has allowed the rapid and reliable design and production of pathogen genomes which can be subsequently manipulated for vaccine production. We describe BTV vaccines based on "synthetic" viruses in which the outer core proteins of different BTV serotypes are incorporated into a common tissue-culture adapted backbone. As a means of validation for this approach, we selected two BTV-8 synthetic reassortants and demonstrated their ability to protect sheep against virulent BTV-8 challenge. In addition, to further highlight the possibilities of genome manipulation for vaccine production, we also designed and rescued a synthetic BTV chimera containing a VP2 protein including regions derived from both BTV-1 and BTV-8. Interestingly, while the parental viruses were neutralized only by homologous antisera, the chimeric proteins could be neutralized by both BTV-1 and BTV-8 antisera. These data suggest that neutralizing epitopes are present in different areas of the BTV VP2 and likely "bivalent" strains eliciting neutralizing antibodies for multiple strains can be obtained.

    Importance Overall, this vaccine platform can significantly reduce the time taken from the identification of new BTV strains to the development and production of new vaccines, as the viral genomes of these viruses can be entirely synthesised in vitro. In addition, these vaccines can be brought quickly in the market as they alter the approach, but not the final product, of existing commercial products.

  • Molecular Basis for Ebola Virus VP35 Suppression of Human Dendritic Cell Maturation [PublishAheadOfPrint]

  • Zaire ebolavirus (EBOV) VP35 is a double-stranded RNA (dsRNA) binding protein that inhibits RIG-I signaling and interferon (IFN)-aalpha;/bbeta; responses by both dsRNA-binding dependent and independent mechanisms. VP35 also suppresses DC maturation. Here, we define the pathways and mechanisms through which VP35 impairs DC maturation. Wild-type VP35 (VP35-WT) and two well-characterized VP35 mutants (F239A and R322A) which independently ablate dsRNA-binding and RIG-I inhibition were delivered to primary human monocyte-derived DCs using a lentivirus-based expression system. VP35-WT suppressed not only IFN-aalpha;/bbeta; but also proinflammatory responses following stimulation of MDDCs with activators of RIG-I-like receptor (RLR) signaling, including RIG-I activators such as Sendai virus (SeV) or 5'-triphosphate RNA or MDA5 activators such as encephalomyocarditis virus (EMCV) or poly(I:C). F239A and R322A exhibited greatly reduced suppression of IFN-aalpha;/bbeta; and proinflammatory cytokine production following treatment of DCs with RLR agonists. VP35-WT also blocked the upregulation of DC maturation markers and the stimulation of allogeneic T cell responses upon SeV infection, whereas the mutants did not. In contrast to the RLR activators, VP35-WT and the mutant VP35s impaired IFN-bbeta; production induced by Toll-Like Receptor (TLR) 3 or TLR4 agonists but failed to inhibit proinflammatory cytokine production induced by TLR2, TLR3, or TLR4 agonists. Further, VP35 did not prevent LPS-induced upregulation of surface markers of MDDC maturation and did not prevent LPS-triggered allogeneic T cell stimulation. Therefore, VP35 is a general antagonist of DC responses to RLR activation. However, TLR agonists can circumvent many of the inhibitory effects of VP35. This suggests strategies to counteract VP35 immune evasion functions.

    Importance. The VP35 protein, which is an inhibitor of RIG-I signaling and interferon (IFN)-aalpha;/bbeta; responses, has been implicated as an Ebola virus-encoded factor that contributes to suppression of dendritic cell (DC) function. We used wild-type and previously characterized VP35 mutants to clarify VP35-DC interactions. Our data demonstrate that VP35 is a general inhibitor of RIG-I-like receptor (RLR) signaling that blocks not only RIG-I but also MDA5-mediated induction of IFN-aalpha;/bbeta; responses. Further, in DCs VP35 also impairs RLR-mediated induction of proinflammatory cytokine production, upregulation of costimulatory markers and activation of T cells. These inhibitory activities require VP35 dsRNA binding activity, an activity previously correlated to VP35 RIG-I inhibitory function. In contrast, while VP35 can inhibit IFN-aalpha;/bbeta; production induced by TLR3 or TLR4 agonists, this occurs in a dsRNA-independent fashion, and VP35 does not inhibit TLR-mediated expression of proinflammatory cytokines. These data suggest strategies to overcome VP35 inhibition of DC function.

  • The ER stress sensor IRE1{alpha} protects cells from apoptosis induced by coronavirus infectious bronchitis virus [PublishAheadOfPrint]

  • The unfolded protein response (UPR) is a signal transduction cascade triggered by perturbation of the homeostasis of the endoplasmic reticulum (ER). UPR resolves ER stress by activating a cascade of cellular response including the induction of molecular chaperones, translational attenuation, ER-associated degradation and other mechanisms. Under prolonged and irremediable ER stress, however, UPR can also trigger apoptosis. Here we report that in cells infected with the avian coronavirus infectious bronchitis virus (IBV), ER stress was induced and the IRE1aalpha;-XBP1 pathway of UPR was activated. Knockdown and over-expression experiments demonstrated that IRE1aalpha; protects the infected cells from IBV-induced apoptosis, which required both its kinase and RNase activity. Our data also suggest that splicing of XBP1 mRNA by IRE1aalpha; appears to convert XBP1 from a pro-apoptotic XBP1u protein to a pro-survival XBP1s protein. Moreover, IRE1aalpha; antagonized IBV-induced apoptosis by modulating the phosphorylation status of the pro-apoptotic c-Jun N-terminal kinase (JNK) and the pro-survival RAC-alpha serine/threonine-protein kinase (Akt). Taken together, the ER stress sensor IRE1aalpha; is activated in IBV-infected cells and serves as a survival factor during coronavirus infection.

    Importance Animal coronaviruses are important veterinary viruses, which could cross the species barrier, becoming severe human pathogens. Molecular characterization of the interactions between coronaviruses and host cells is pivotal to the understanding of pathogenicity and species specificity of coronavirus infection. It has been well established that the endoplasmic reticulum (ER) is closely associated with coronavirus replication. Here we report that inositol-requiring protein-1 alpha (IRE1aalpha;), a key sensor of ER stress, is activated in cells infected with avian coronavirus infectious bronchitis virus (IBV). Moreover, IRE1aalpha; is shown to protect the infected cells from apoptosis by modulating the unfolded protein response (UPR) and two kinases related to cell survival. This study demonstrates that UPR activation constitutes a major aspect of coronavirus-host interactions. Manipulations of the coronavirus-induced UPR may provide novel therapeutic targets to the control of coronavirus infection and pathogenesis.

  • Gammaherpesvirus Latency Differentially Impacts The Generation Of Primary Versus Secondary Memory CD8+T Cells During Subsequent Infection [PublishAheadOfPrint]

  • Unlike laboratory animals, humans are infected with multiple pathogens, including the highly prevalent herpesviruses. The purpose of these studies was to determine the effect of gammaherpesvirus latency on T cell number and differentiation during subsequent heterologous viral infections. Mice were first infected with murine gammaherpesvirus 68 (MHV68), a model of Epstein-Barr virus (EBV) infection, and then after latency was established, they were challenged with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV). The initial replication of LCMV was lower in latently infected mice and the maturation of dendritic cells was abated. Although the number of LCMV-specific effector CD8+ T cells was not altered, they were skewed to a memory phenotype. In contrast, LCMV-specific effector CD4+ T cells were increased in latently infected mice compared to those infected solely with LCMV. When the memory phase was reached, latently infected mice had a LCMV-specific memory T cell pool that was increased relative to that found in singly infected mice. Importantly, LCMV-specific memory CD8+ T cells had decreased CD27 and increased KLRG1 expression. Upon secondary challenge, LCMV-specific secondary effector CD8+ T cells expanded and cleared the infection. But, the LCMV-specific secondary memory CD8+ T cell pool was decreased in latently infected animals, abrogating the boosting effect normally observed following rechallenge. Taken together these results demonstrate ongoing gammaherpesvirus latency affects the number and phenotype of primary versus secondary memory CD8+ T cells during acute infection.

    Importance CD8+ T cells are critical for the clearance of intracellular pathogens including viruses, certain bacteria and tumors. However, current models for memory CD8+ T cell differentiation are derived from pathogen-free laboratory mice challenged with a single pathogen or vaccine vector. Unlike laboratory animals, all humans are infected with multiple acute and chronic pathogens, including the highly prevalent herpesviruses Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex viruses (HSV), and varicella zoster virus (VZV). The purpose of these studies was to determine the effect of gammaherpesvirus latency on T cell number and differentiation during subsequent heterologous viral infections. We observed that ongoing gammaherpesvirus latency affects the number and phenotype of primary versus secondary memory CD8+ T cells during acute infection. These results suggest that unlike pathogen free laboratory mice, infection or immunization of latently infected humans may result in the generation of T cells with limited potential for long-term protection.

  • Cellular Requirements for BIV Vif-Mediated Inactivation of Bovine APOBEC3 Proteins [PublishAheadOfPrint]

  • Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) viral infectivity factor (Vif) form a CRL5 E3 ubiquitin ligase complex to suppress virus restriction by host APOBEC3 (A3) proteins. The primate lentiviral Vif complex is composed of the unique cofactor CBF-bbeta; and canonical ligase components Cullin5 (CUL5), ElonginB/C (ELOB/C), and RBX2. However, the mechanism by which the Vif protein of the related lentivirus bovine immunodeficiency virus (BIV) overcomes its host A3 proteins is less clear. In this study, we show that BIV Vif interacts with Cullin2 (CUL2), ELOB/C, and RBX1, but not with CBF-bbeta; or CUL5, to form a CRL2 E3 ubiquitin ligase and degrade the restrictive bovine A3 proteins (A3Z2Z3 and A3Z3). RNAi-mediated knockdown of ELOB or CUL2 inhibits BIV Vif-mediated degradation of these A3 proteins, whereas knockdown of CUL5 or CBF-bbeta; does not. BIV Vif with mutations in the BC box (Vif SLQ-AAA) or putative VHL box (Vif YI-AA), which cannot interact with ELOB/C or CUL2, respectively, lose the ability to counteract bovine A3 proteins. Moreover, CUL2 and UBE2M dominant-negative mutants competitively inhibit the BIV Vif-mediated degradation mechanism. Thus, although the general strategy for inhibiting A3 proteins is conserved between HIV-1/SIV and BIV, the precise mechanisms can differ substantially, with only the HIV-1/SIV Vif proteins requiring CBF-bbeta; as a cofactor, HIV-1/SIV Vif using CUL5/RBX2, and BIV Vif using CUL2/RBX1.

    IMPORTANCE Primate lentivirus HIV-1 and SIV Vif proteins form a ubiquitin ligase complex to target host anti-viral APOBEC3 proteins for degradation. However, the mechanism by which the non-primate lentivirus BIV Vif inhibits bovine APOBEC3 proteins is unclear. Here we determine the mechanism for BIV Vif-mediated degradation of bovine APOBEC3 proteins, which differs from the mechanism of HIV-1/SIV Vif by being CBF-bbeta; independent and requiring different ubiquitin ligase scaffolding proteins (CUL2/RBX1 instead of CUL5/RBX2). BIV Vif is the only known retroviral protein that can interact with CUL2. This information broadens our understanding of the distinct mechanisms by which the Vif proteins of different lentiviruses facilitate viral infection. This novel mechanism for assembly of the BIV Vif-APOBEC3 ubiquitin ligase complex advances our understanding of viral hijacking of host E3 ubiquitin ligases and illustrates the evolutionary flexibility of lentiviruses.

  • A Comprehensive Analysis of HSV-1 Entry Mediated by Zebrafish 3-O-Sulfotransferase Isoforms: Implications for the Development of a Zebrafish Model of HSV-1 Infection [PublishAheadOfPrint]

  • Binding of herpes simplex virus type-1 (HSV-1) envelope glycoprotein D (gD) to the receptor 3-O-sulfated heparan sulfate (3-OS HS) mediates viral entry. 3-O-sulfation of HS is catalyzed by 3-O-sulfotransferase (3-OST) enzyme. Multiple isoforms of 3-OST are differentially expressed in tissues in Zebrafish (ZF) embryos. Here, we performed a comprehensive analysis of the role of ZF 3-OST isoforms-1, -5, -6 and -7 in HSV-1 entry. We found that a group of 3-OST gene family (3-OST-2, -3, -4, and -6 isoforms) with conserved catalytic and substrate-binding residues of the enzyme mediates HSV-1 entry and spread, while the other group (3-OST-1, -5, and -7) lack these properties. These results demonstrate that HSV-1 entry can be recapitulated by certain ZF 3-OST enzymes, a significant step towards the establishment of a ZF model of HSV-1 infection and tissue-specific tropism.

  • Immunogenicity studies of proteins forming the T4 phage head surface [PublishAheadOfPrint]

  • Advances in phage therapy and novel applications of phages in biotechnology encourage interest in phage impact on human and animal immunity. Here we present comparative studies of immunogenic properties of T4 phage head surface proteins gp23*, gp24*, Hoc and Soc, both as elements of the phage capsid and as isolated agents. Studies comprise evaluation of specific antibodies in the human population, analysis of the proteins' impact on the primary and secondary response in mice, as well as the effect of specific antibodies on phage antibacterial activity in vitro and in vivo in mice.

    In humans, natural antibodies specific to T4-like phages were abundant (81% of investigated sera). Among those, significantly elevated levels of IgG antibodies only against major head protein (gp23*) were found, which probably reflected cross-reactions of T4 with antibodies induced by other T4-like phages. Both IgM and IgG antibodies were induced mostly by gp23* and Hoc, while weak (gp24*) and very weak (Soc) reactivity of other head proteins was noticed. Thus, T4 head proteins that markedly contribute to immunological memory to the phage are: highly antigenic outer capsid protein (Hoc) and major capsid protein (gp23*). Specific anti-gp23* and anti-Hoc antibodies substantially decreased T4 phage activity in vitro and to some extent in vivo. Cooperating with antibodies, the immune complement system also contributed to annihilating phages.

    Importance Current descriptions of phage immunogenicity and its biological consequences are still vague and incomplete thus the central problem of this work is timely and may have strong practical implications. Here, is presented the very first observation of bacteriophage proteins contribution to immunological memory of the phage. Understanding of interactions between phages and mammalian immunology may help in biotechnological adaptations of phages for therapeutic requirements as well as for better appreciation of phage ecology and their role in the biosphere.

  • Lysine 63-linked TANK-binding kinase 1 ubiquitination by mindbomb E3 ubiquitin protein ligase 2 is mediated by mitochondrial antiviral signaling protein [PublishAheadOfPrint]

  • Interferon beta (IFNbbeta;) is involved in a wide range of cellular functions, and its secretion must be tightly controlled to inhibit viral spreading while minimizing cellular damage. Intracellular viral replication triggers cellular signaling cascades leading to the activation of the transcription factors NFB and interferon regulatory factors 3 and 7 (IRF3/7), which synergistically bind to the IFNbbeta; gene promoter to induce its expression. The mitochondrial antiviral signaling protein (MAVS) is a governing adaptor protein that mediates signaling communications between virus sensing proteins and transcription factors. The activity of MAVS in the regulation of IFNbbeta; secretion is affected by many cellular factors. However, the mechanism of MAVS-mediated IRF3/7 activation is not completely understood. Here, we identified a highly conserved DLAIS motif at amino acid positions 438-442 of MAVS that is indispensable for IRF3/7 activation. Specifically, the L439S and A440R mutations suppress IRF3/7 activation. Pull-down experiments using wild-type and mutant MAVS showed that mindbomb E3 ubiquitin protein ligase 2 (MIB2) binds to the DLAIS motif. Furthermore, the DLAIS motif was found to be critical for MIB2 binding, the ligation of K63-linked ubiquitin to TANK-binding kinase 1, and phosphorylation-mediated IRF3/7 activation. Our results suggest that MIB2 plays a putative role in MAVS-mediated interferon signaling.

    Highlights Mitochondrial antiviral signaling protein (MAVS) mediates signaling from virus sensing proteins to transcription factors for the induction of interferon beta. However, the mechanism underlying MAVS-mediated interferon regulatory factors 3 and 7 (IRF3/7) activation is not completely understood. We found a highly conserved DLAIS motif in MAVS that is indispensable for IRF3/7 activation through TANK-binding kinase 1 (TBK1) and identified it as the binding site for mindbomb E3 ubiquitin protein ligase 2 (MIB2). The mutations that targeted the DLAIS motif abolished MIB2 binding, attenuated the K63-linked ubiquitination of TBK1, and decreased the phosphorylation-mediated activation of IRF3/7.

  • Chlorovirus PBCV-1 Encodes an Active Copper-Zinc Superoxide Dismutase [PublishAheadOfPrint]

  • Superoxide dismutases (SODs) are metalloproteins that protect organisms from toxic reactive oxygen species by catalyzing the conversion of superoxide anion to hydrogen peroxide and molecular oxygen. Sequence analysis of the Paramecium bursaria chlorella virus-1 (PBCV-1) genome identified a protein coding sequence that resembled a Cu-Zn SOD with all of the conserved amino acid residues for binding copper and zinc; the protein was named chlorella virus SOD (cvSOD). Recombinant cvSOD inhibited nitroblue tetrazolium reduction of superoxide anion generated in a xanthine-xanthine oxidase system in solution and also a riboflavin photochemical reduction system in a polyacrylamide gel assay. The inhibition was blocked by the Cu-Zn SOD inhibitor cyanide but not by azide, which inhibits Fe and Mn SODs. A kcat/Km value for cvSOD was determined by stop-flow spectrophotometry as 1.28 x 108 M-1s-1, suggesting that cvSOD catalyzed O2- dismutation was not a diffusion controlled encounter. The cvsod gene was expressed as a late gene and cvSOD activity was detected in purified virions. Superoxide accumulated rapidly during virus infection and circumstantial evidence indicates that cvSOD aids its decomposition to benefit virus replication. Cu-Zn SOD homologs have been described in 3 other families of large DNA viruses, poxviruses, baculoviruses and mimiviruses, which group as a clade. Interestingly, cvSOD does not group in the same clade as the other virus SODs but instead groups in an expanded clade that includes Cu-Zn SODs from many cellular organisms.

    IMPORTANCE Virus infection often leads to an increase in toxic reactive oxygen species in the host, which can be detrimental to virus replication. Viruses have developed various ways to overcome this barrier. As reported in this manuscript, the chloroviruses often encode and package a functional Cu-Zn superoxide dismutase in the virion that presumably lowers the concentration of reactive oxygen induced early during virus infection.

  • Human papillomavirus type 1 (HPV1) E1^E4 protein is a potent inhibitor of the serine-arginine (SR) protein kinase SRPK1 and inhibits phosphorylation of host SR proteins and of the viral transcription and replication regulator E2 [PublishAheadOfPrint]

  • The serine-arginine-specific protein kinase SRPK1 is a common binding partner of the E1^E4 protein.of diverse human papillomavirus types. Here, we show for the first time, that the interaction between HPV1 E1^E4 and SRPK1 leads to potent inhibition of SRPK1 phosphorylation of host SR proteins that have critical roles in mRNA metabolism, including pre-mRNA processing, mRNA export and translation. Furthermore, we show that SRPK1 phosphorylates serine residues of SR/RS dipeptides in the hinge region of the HPV1 E2 protein in in vitro kinase assays and HPV1 E1^E4 inhibits this phosphorylation. Following mutation of the putative phosphoacceptor serine residues, the localization of the E2 protein was altered in primary human keratinocytes; with a significant increase in the cell population showing intense E2 staining of the nucleolus. A similar effect was observed following co-expression of E2 and E1^E4 that is competent for inhibition of SRPK1 activity, suggesting that the nuclear localization of E2 is sensitive to E1^E4-mediated SRPK1 inhibition. Collectively, these data suggest that E1^E4 mediated inhibition of SRPK1 could affect the functions of host SR proteins and those of the virus transcription/replication regulator E2. We speculate that the novel E4 function identified here is involved in the regulation of E2 and SR protein function in posttranscriptional processing of viral transcripts.

    IMPORTANCE The HPV life cycle is tightly linked to the epithelial terminal differentiation programme, with the virion-producing phase restricted to differentiating cells. While the most abundant HPV protein expressed in this phase is the E4 protein, we do not fully understand the role of this protein. Few E4 interaction partners have been identified, but we had previously shown that E4 proteins from diverse papillomaviruses interact with the serine-arginine-specific protein kinase SRPK1, a kinase important in the replication cycles of a diverse range of DNA and RNA viruses. Here, we show that HPV1 E4 is a potent inhibitor of this host cell kinase. We show that E4 inhibits SRPK1 phosphorylation, not only of cellular SR proteins involved in regulating alternative splicing of RNA, but also the viral transcription/replication regulator E2. Our findings reveal a potential E4 function in regulation of viral late gene expression through inhibition of a host cell kinase.

  • Recombinant AAV utilizes cell-specific infectious entry mechanisms [PublishAheadOfPrint]

  • Understanding the entry and trafficking mechanism(s) of rAAV into host cells can lead to evolution in capsid and vector design and delivery methods, resulting in enhanced transduction and therapeutic gene expression. Variability of findings regarding the early entry pathway of rAAV supports the possibility that rAAV, like other viruses, can utilize more than one infectious entry pathway. We tested whether inhibition of macropinocytosis impacted rAAV transduction of HeLa cells as compared to hepatocellular carcinoma cell lines. We found that macropinocytosis inhibitor cytochalasin-D blocked rAAV transduction of HeLa cells (ggt;2-fold), but enhanced (10-fold) transduction in HepG2 and Huh7 lines. Similar results were obtained with another macropinocytosis inhibitor 5-(N-Ethyl-N-isopropyl) amiloride (EIPA). The augmented transduction was neither due to viral binding nor promoter activity, affected multiple rAAV serotypes (rAAV2, rAAV2-R585E, and rAAV8), and influenced both single stranded and self-complementary virions to comparable extents. Follow-up studies using CDC42 inhibitor ML141 and p21-activated kinase 1 (PAK1) siRNA knockdown also resulted in enhanced HepG2 transduction. Microscopy revealed that macropinocytosis inhibition correlated with expedited nuclear entry of the rAAV virions into HepG2 cells. Enhancement of hepatocellular rAAV transduction extended to the mouse liver in vivo (4-fold enhancement), but inversely blocked heart tissue transduction (13-fold). This evidence of host cell-specific rAAV entry pathways confers a potent means for controlling and enhancing vector delivery, and could help unify the divergent accounts of rAAV cellular entry mechanisms.

    IMPORTANCE There is a recognized need for improved rAAV vector targeting strategies that result in delivery of fewer total particles, averting untoward toxicity and/or an immune response against the vector. A critical step in rAAV transduction is entry and early trafficking through the host cellular machinery mmdash; the mechanisms of which are under continued study. However, should the early entry and trafficking mechanisms of rAAV differ across virus serotype or be dependent on host cell environment, this could expand our ability to target particular cells and tissue for selective transduction. Thus the observation that inhibiting macropinocytosis leads to cell-specific enhancement or inhibition of rAAV transduction that extends to the organismic level exposes a new means of modulating vector targeting.

  • Naturally arising strains of polyomaviruses with severely attenuated microRNA expression [PublishAheadOfPrint]

  • Several different polyomaviruses (PyVs) encode microRNAs (miRNAs) that regulate viral as well as host gene expression. However, the functions of polyomaviral miRNAs, particularly during in vivo infection, remain poorly understood. Here we identify rare naturally arising PyVs that are severely attenuated or null for miRNA expression. We identify hypomorphic or null strains for miRNA expression from rhesus macaque Simian Virus 40 (SV40) and human JC virus. These strains were isolated from immunocompromised hosts and derive from insertions or deletions in the viral DNA that preserve the amino acid reading frame of opposing strand large T antigen gene. Characterization of the SV40 miRNA hypomorph, K661, shows that it is inhibited at the early miRNA biogenesis step of Drosha-mediated processing. Despite having a non-rearranged enhancer, which a previous study has shown renders some PyVs more susceptible to the autoregulatory activities of the miRNA, restoring miRNA expression to K661 has little effect on virus growth in either immortalized or primary monkey kidney cells. Thus, in addition to any effect of accompanying genomic elements, these results suggest that cellular context also determines susceptibility to PyV miRNA-mediated effects. Combined, these results demonstrate that polyomaviruses lacking miRNAs can arise infrequently and that the functional importance of polyomaviral miRNAs is context-dependent, consistent with an activity connected to the immune status of the host.

    IMPORTANCE Diverse virus families encode miRNAs, yet much remains unknown about viral miRNA function and contribution to the infectious cycle. Polyomaviruses (PyVs) are small DNA viruses, long important as etiological agents of rare diseases and valuable models of DNA virus infection. Here, in immunosuppressed hosts, we uncover rare naturally-arising variants of different PyVs that have lost the ability to express miRNAs. This represents some of the only known natural viruses to have lost miRNA expression. By probing the biogenesis pathways of these variants, we uncover that miRNA expression is lost via small insertions or deletions that render the transcripts resistant to early steps of miRNA biogenesis, while preserving the reading frame of the opposing T antigen transcripts. Overall, our study informs how miRNA genes evolve/devolve in viruses, and suggests that miRNA function is exquisitely dependent not only on viral genomic context but also the cellular and host environment.

  • Dissection of the antibody response against herpes simplex virus glycoproteins in naturally infected humans [PublishAheadOfPrint]

  • Relatively little is known about the extent of the polyclonal antibody (PAb) repertoire elicited by HSV glycoproteins during natural infection and how these antibodies affect virus neutralization. Here, we examined IgGs from ten HSV-seropositive individuals originally classified as high or low virus shedders. All PAbs neutralized virus to varying extents. We determined which HSV entry glycoproteins these PAbs were directed against: glycoproteins gB, gD and gC were recognized by all sera but fewer reacted against gH/gL. We previously characterized multiple mouse monoclonal antibodies (MAbs) and mapped those with high neutralizing activity to the crystal structures of gD, gB and gH/gL. We used a biosensor competition assay to determine whether there were corresponding human antibodies to those epitopes. All ten samples had neutralizing IgGs to gD epitopes but there were variations in which epitopes were seen in individual samples. Surprisingly, only three samples contained neutralizing IgGs to gB epitopes. To further dissect the nature of these IgGs, we developed a method to select out gD- and gB-specific IgGs from four representative sera via affinity chromatography, allowing us to determine the contribution of antibodies against each glycoprotein to the overall neutralization capacity of the serum. In two cases, gD and gB accounted for all of the neutralizing activity against HSV-2, with a modest amount of HSV-1 neutralization directed against gC. In the other two samples, the dominant response was to gD.

    Importance Antibodies targeting functional epitopes on HSV entry glycoproteins mediate HSV neutralization. Virus neutralizing epitopes have been defined and characterized using murine monoclonal antibodies. However, it is largely unknown whether these same epitopes are targeted by the humoral response to HSV infection in humans. We have shown that during natural infection, virus-neutralizing antibodies are principally directed against gD and gB and to a lesser extent, gC. While several key HSV neutralizing epitopes within gD and gB are commonly targeted by human serum IgG, others fail to induce consistent responses. These data are particularly relevant to the design of future HSV vaccines.

  • Analysis of the highly diverse gene borders in Ebola virus reveals a distinct mechanism of transcriptional regulation [PublishAheadOfPrint]

  • Ebola virus (EBOV) belongs to the group of nonsegmented negative sense RNA viruses. The seven EBOV genes are separated by variable gene borders, including short (4-5 nucleotides) intergenic regions (IRs), a single long (144 nucleotides) IR and gene overlaps, where the neighboring gene end and start signals share five conserved nucleotides. The unique structure of the gene overlaps and the presence of a single long IR are conserved among all filoviruses. Here, we sought to determine the impact of the EBOV gene borders during viral transcription. We show that readthrough mRNA synthesis occurs in EBOV-infected cells irrespective of the structure of the gene border, indicating that the gene overlaps do not promote recognition of the gene end signal. However, two consecutive gene end signals at the VP24 gene might improve termination at the VP24/L gene border, ensuring efficient L gene expression. We further demonstrate that the long IR is not essential for, but regulates transcription reinitiation in a length-dependent, but sequence-independent manner. Mutational analysis of bicistronic minigenomes and recombinant EBOVs showed no direct correlation between IR length and reinitiation rates, but demonstrated that specific IR lengths, not found naturally in filoviruses, profoundly inhibit downstream gene expression. Intriguingly, although truncation of the 144 nucleotide long IR to 5 nucleotides did not substantially affect EBOV transcription, it led to a significant reduction of viral growth.

    Importance Our current understanding of EBOV transcription regulation is limited due to the requirement of high containment conditions to study this highly pathogenic virus. EBOV is thought to share many mechanistic features with well-analyzed prototype nonsegmented negative sense RNA viruses. A single polymerase entry site at the 3' end of the genome determines that transcription of the genes is mainly controlled by gene order and cis-acting signals found at the gene borders. Here, we examined the regulatory role of the structurally unique EBOV gene borders during viral transcription. Our data suggest that transcriptional regulation in EBOV is highly complex and differs from prototype viruses, and further the understanding of this most fundamental process in the filovirus replication cycle. Moreover, our results with recombinant EBOVs suggest a novel role of the long IR found in all filovirus genomes during the viral replication cycle.

  • Influenza A Virus Polymerase Is a Site for Adaptive Changes During Experimental Evolution in Bat Cells [PublishAheadOfPrint]

  • The recent identification of highly divergent influenza A viruses in bats revealed a new, geographically dispersed viral reservoir. To investigate the molecular mechanisms of host-restricted viral tropism and the potential for transmission of viruses between humans and bats, we exposed a panel of cell lines from bats of diverse species to a prototypical human-origin influenza A virus. All of the tested bat cell lines were susceptible to influenza A virus infection. Experimental evolution of human and avian-like viruses in bat cells resulted in efficient replication and created highly cytopathic variants. Deep sequencing of adapted human influenza A virus revealed a mutation in the PA polymerase subunit not previously described, M285K. Recombinant virus encoding PA M285K completely phenocopied the adapted virus. Adaptation of an avian-like virus resulted in the canonical PB2 E627K mutation that is required for efficient replication in other mammals. None of the adaptive mutations occurred in the viral hemagglutinin, a gene that frequently acquires changes to recognize host-specific variations in sialic acid receptors. We showed that human influenza A virus uses canonical sialic acid receptors to infect bat cells, even though bat influenza A viruses do not appear to use these receptors for virus entry. Our results demonstrate that bats are unique hosts that select for both a novel and a well-known adaptive mutation in the viral polymerase to support replication.

    IMPORTANCE Bats constitute well-known reservoirs for viruses that may be transferred into human populations, sometimes with fatal consequences. Influenza A viruses have recently been identified in bats, dramatically expanding the known host range of this virus. Here we investigate the replication of human influenza A virus in bat cell lines and the barriers the virus faces in this new host. Human influenza A and B viruses infected cells from geographically and evolutionarily diverse New and Old World bats. Viruses mutated during infections in bat cells resulting in increased replication and cytopathic effects. These mutations were mapped to the viral polymerase and shown to be solely responsible for adaptation to bat cells. Our data suggest that replication of human influenza A viruses in a non-native host drives evolution of new variants and may be an important source of genetic diversity.

  • Novel Denisovan and Neanderthal Retroviruses [PublishAheadOfPrint]

  • Following the recent availability of high-coverage genomes for Denisovan and Neanderthal man, we conducted a screen for endogenised retroviruses, identifying six novel, previously unreported HERV-K(HML2) elements. These elements are absent from the human genome (hg38), and appear to be unique to archaic hominids. These findings provide further evidence supporting the recent activity of the HERV-K(HML2) group, which has been implicated in human disease. They will also provide insights into the evolution of archaic hominids.

  • Trafficking of Bluetongue virus visualized by recovery of tetracysteine-tagged virion particles [PublishAheadOfPrint]

  • Bluetongue virus (BTV), a member of the Orbivirus genus in the Reoviridae family, is a double-capsid insect-borne virus enclosing a genome of 10 double-stranded RNA segments. As with other members of the family, BTV virions are non-enveloped particles containing two architecturally complex capsids. The two proteins of the outer capsid, VP2 and VP5, are involved in BTV entry and the delivery of the transcriptionally active core in the cell cytoplasm. Although the importance of endocytic pathway in BTV entry has been reported, a detailed analysis of entry and the role of each protein on virus trafficking have not been possible due to unavailability of a tagged virus. Here for the first time we report on the successful manipulation of a segmented genome of a non-enveloped capsid virus by the introduction of tags that were subsequently fluorescently visualized in infected cells. The genetically engineered fluorescent BTV particles were observed to enter live cells immediately after virus adsorption. Further, we showed separation of VP2 from VP5 during virus entry and confirmed that while VP2 is shed from virions in early endosomes, virus particles still consisting of VP5 were trafficked sequentially from early to late endosomes. Since BTV infects both mammalian and insect cells, the generation of tagged viruses will allow visualization of further downstream trafficking of BTV in different host cells. In addition, the tagging technology also has potential for transferable application on other non-enveloped complex viruses.

    Importance Live virus trafficking in host cells has been highly informative in understanding interactions between virus and host cells. Although insertion of fluorescent markers in viral genome have made it possible to study trafficking of enveloped viruses, the physical constraints of architecturally complex capsid viruses have led to practical limitations. In this study, we have successfully genetically engineered the segmented RNA genome of Bluetongue virus (BTV), a complex non-enveloped virus belonging to the Reoviridae family. The resulting fluorescent virus particles could be visualized in virus entry studies for both live and fixed cells. This is the first time a structurally complex capsid virus has been successfully genetically manipulated to generate virus particles that could be visualized in infected cells.

  • Intrabronchial infection of rhesus macaques with simian varicella virus results in a robust immune response in the lungs. [PublishAheadOfPrint]

  • Varicella zoster virus (VZV) is the etiological agent of varicella (chickenpox) and herpes zoster (shingles). Primary VZV infection is believed to occur via the inhalation of virus either in respiratory droplets, from shedding varicella lesions or by direct contact with infectious vesicular fluid. However, the ensuing immune response in the lungs remains incompletely understood. We have shown that intrabronchial inoculation of rhesus macaques with simian varicella virus (SVV), a homolog of VZV, recapitulates the hallmarks of acute and latent VZV infection in humans. In this study, we performed an in-depth analysis of the host immune response to acute SVV infection in the lungs and peripheral blood. We report that acute SVV infection results in a robust innate immune response in the lungs, characterized by the production of inflammatory cytokines, chemokines, and growth factors as well as an increased frequency of plasmacytoid DCs that corresponded with IFNaalpha; production and a rapid decrease in viral loads in the lungs. This is followed by T and B cell proliferation, antibody production, T cell differentiation and cytokine production, which correlate with the complete cessation of viral replication. Although terminally differentiated CD8 T cells became the predominant T cell population in bronchoalveolar lavage cells, a higher percentage of CD4 T cells were SVV-specific, which suggests a critical role for these cells in the resolution of primary SVV infection in the lungs. Given the homology between SVV and VZV, our data provide insight into the immune response to VZV within the lung.

    Importance Although primary VZV infection occurs primarily via the respiratory route, our understanding of the host response in the lungs and its contribution to the cessation of viral replication and establishment of latency remains poorly understood. The difficulty in accessing lung tissue and washes from individuals infected with VZV has hampered efforts to address this knowledge gap. SVV infection of rhesus macaques is an important model of VZV infection of humans; therefore we utilized this animal model to gain a comprehensive view of the kinetics of the immune response to SVV in the lung and its relationship to the resolution of acute infection in respiratory tissues. These data not only advance our understanding of host immunity to VZV, a critical step in developing new vaccines, but also provide additional insight into immunity to respiratory pathogens.

  • IMPACT OF CLADE, GEOGRAPHY AND AGE OF THE EPIDEMIC ON HIV-1 NEUTRALIZATION BY ANTIBODIES [PublishAheadOfPrint]

  • Neutralizing antibodies (nAbs) are a high priority for vaccines that aim to prevent the acquisition of HIV-1 infection. Vaccine effectiveness will depend on the extent to which induced antibodies neutralize the global diversity of circulating HIV-1 variants. Using large panels of genetically and geographically diverse HIV-1 Env-pseudotyped viruses and chronic infection plasma samples, we unambiguously show that cross-clade nAb responses are commonly induced in response to infection by any virus clade. Nonetheless, neutralization was significantly greater when the plasma clade matched the clade of the virus being tested. This within-clade advantage was diminished in older, more diverse epidemics in southern Africa, the US and Europe compared to more recent epidemics in Asia. It was most pronounced for circulating recombinant form (CRF) 07_BC, which is common in China and is the least divergent lineage studied; this was followed by the slightly more diverse Asian CRF01_AE. We found no evidence that transmitted/founder viruses are generally more susceptible to neutralization and are therefore easier targets for vaccination than chronic viruses. Features of the gp120 V1V2 loop, in particular length, net charge and number of N-linked glycans, were associated with Env susceptibility and plasma neutralization potency in a manner consistent with neutralization-escape being a force that drives viral diversification and plasma neutralization breadth. The overall susceptibility of Envs and potencies of plasmas were highly predictive of the neutralization outcome of any single virus/plasma combination. These findings highlight important considerations for the design and testing of candidate HIV-1 vaccines that aim to elicit effective nAbs.

    IMPORTANCE An effective HIV-1 vaccine will need to overcome the extraordinary variability of the virus, which is most pronounced in the envelope glycoproteins (Env) that are the sole targets for neutralizing antibodies (nAbs). Distinct genetic lineages, or clades, of HIV-1 occur in different locales that may require special consideration when designing and testing vaccines candidates. We show that nAb responses to HIV-1 infection are generally active across clades but are most potent within clades. Because effective vaccine-induced nAbs are likely to share these properties, optimal coverage of a particular clade or combination of clades may require clade-matched immunogens. Optimal within-clade coverage might be easier to achieve in regions such as China and Thailand, where the epidemic is more recent and the virus less diverse than in southern Africa, the US and Europe. Finally, features of the first and second hypervariable regions of gp120 (V1V2) may be critical for optimal vaccine design.

  • Residue 82 of the Chikungunya Virus E2 Attachment Protein Modulates Viral Dissemination and Arthritis in Mice [PublishAheadOfPrint]

  • Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has reemerged to cause profound epidemics of fever, rash, and arthralgia throughout sub-Saharan Africa, Southeast Asia, and the Caribbean. Like other arthritogenic alphaviruses, mechanisms of CHIKV pathogenesis are not well defined. Using the attenuated CHIKV strain 181/25 and virulent strain AF15561, we identified a residue in the E2 viral attachment protein that is a critical determinant of viral replication in cultured cells and pathogenesis in vivo. Viruses containing an arginine at E2 residue 82 displayed enhanced infectivity in mammalian cells but reduced infectivity in mosquito cells and diminished virulence in a mouse model of CHIKV disease. Mice inoculated with virus containing an arginine at this position exhibited reduced swelling at the site of inoculation with a concomitant decrease in the severity of necrosis in joint-associated tissues. Viruses containing a glycine at E2 residue 82 produced higher titers in the spleen and serum at early times postinfection. Using wildtype and glycosaminoglycan (GAG)-deficient Chinese hamster ovary (CHO) cell lines and soluble GAGs, we found that an arginine at residue 82 conferred greater dependence on GAGs for infection of mammalian cells. These data suggest that CHIKV E2 interactions with GAGs diminish dissemination to lymphoid tissue, establishment of viremia, and activation of inflammatory responses early in infection. Collectively, these results suggest a function for GAG utilization in regulating CHIKV tropism and host responses that contribute to arthritis.

    IMPORTANCE CHIKV is a reemerging alphavirus of global significance with high potential to spread into new, immunologically naiiuml;ve populations. The severity of CHIKV disease, particularly its propensity for chronic musculoskeletal manifestations, emphasizes the need for identification of genetic determinants that dictate CHIKV virulence in the host. To better understand mechanisms of CHIKV pathogenesis, we probed the function of an amino acid polymorphism in the E2 viral attachment protein using a mouse model of CHIKV musculoskeletal disease. In addition to influencing glycosaminoglycan utilization, we identified roles for this polymorphism in differential infection of mammalian and mosquito cells and targeting of CHIKV to specific tissues within infected mice. These studies demonstrate a correlation between CHIKV tissue tropism and virus-induced pathology modulated by a single polymorphism in E2, which in turn illuminates potential targets for vaccine and antiviral drug development.

  • Sapovirus Translation Requires an Interaction Between VPg and the Cap Binding Protein eIF4E [PublishAheadOfPrint]

  • Sapoviruses of the Caliciviridae family of small RNA viruses, are emerging pathogens that cause gastroenteritis in humans and animals. Molecular studies on human sapovirus have been hampered due to the lack of a cell culture system. In contrast, porcine sapovirus (PSaV) can be grown in cell culture making it a suitable model for understanding the infectious cycle of sapoviruses and related-enteric caliciviruses. Caliciviruses are known to use a novel mechanism of protein synthesis that relies on the interaction of cellular translation initiation factors with the virus encoded VPg protein that is covalently linked to the 5' end of the viral genome. Using PSaV as a representative member of the Sapovirus genus, we characterized the role of the viral VPg protein in sapovirus translation. As observed for other caliciviruses, the PSaV genome was found to be covalently linked to VPg and this linkage was required for the translation and the infectivity of viral RNA. The PSaV VPg protein was associated with the eIF4F complex in infected cells and bound directly to the eIF4E protein. As have been previously demonstrated for feline calicivirus, a member of the Vesivirus genus, PSaV translation required eIF4E and the interaction between eIF4E and eIF4G. Overall our study provides new insights into the novel mechanism of sapovirus translation suggesting that sapovirus VPg can hijack the cellular translation initiation mechanism by recruiting the eIF4F complex through a direct eIF4E interaction.

    IMPORTANCE Sapoviruses, of the Caliciviridae family, are one of the causative agents of viral gastroenteritis in humans. However, human sapovirus remains non cultivable in the cell culture hampering the ability to characterize the virus infectious cycle. Here, we show that the VPg protein from porcine sapovirus, the only cultivatable sapovirus, is essential for viral translation and functions via a direct interaction with the cellular translation initiation factor eIF4E. This work provides new insights into the novel protein primed mechanism of calicivirus VPg-dependent translation initiation.

  • Establishment and characterization of a lethal mouse model for the Angola strain of Marburg virus [PublishAheadOfPrint]

  • Infections with Marburg (MARV) and Ebola virus (EBOV) cause severe hemorrhagic fever in humans and non-human primates (NHPs) with fatality rates up to 90%. A number of experimental vaccine and treatment platforms have previously been shown to be protective against EBOV infection. However, the rate of development for prophylactics and therapeutics against MARV has been slower in comparison, possibly because a small animal model is not widely available. Here we report the development of a mouse model for studying the pathogenesis of MARV Angola (MARV/Ang), the most virulent strain of MARV. Infection with the wild-type virus does not cause disease in mice, but the adapted virus (MARV/Ang-MA) recovered from liver homogenates after 24 serial passages in severe combined immunodeficient (SCID) mice caused severe disease when administered intranasally (IN) or intraperitoneally (IP). The LD50 was determined to be 0.015 TCID50 MARV/Ang-MA in SCID mice, and IP infection at a dose of 1000 x LD50 resulted in death between 6-8 days post-infection (dpi) in SCID mice. Similar results were obtained with immunocompetent BALB/c and C57BL/6 mice challenged IP with 2000 x LD50 of MARV/Ang-MA. Virological and pathological analyses in MARV/Ang-MA infected BALB/c mice revealed that the associated pathology was reminiscent of observations made in NHPs with MARV/Ang. MARV/Ang-MA infected mice showed most of the clinical hallmarks observed with Marburg hemorrhagic fever, including lymphopenia, thrombocytopenia, marked liver damage and uncontrolled viremia. Virus titers reached 108 TCID50/ml in the blood, and between 106-10 TCID50/g tissue in the intestines, kidney, lungs, brain, spleen, and liver. This model provides an important tool to screen candidate vaccines and therapeutics against MARV infections.

    Importance The Angola strain of Marburg virus (MARV/Ang) was responsible for the largest outbreak ever documented for Marburg viruses. At a 90% fatality rate, it is similar to Ebola virus, which makes it one of the most lethal viruses known to humans. There are currently no approved interventions for Marburg virus, in part because a small animal model that is vulnerable to MARV/Ang infection is not available to screen and test potential vaccines and therapeutics in a quick and economical manner. To address this need, we have adapted MARV/Ang so that it causes illness in mice resulting in death. The signs of disease in these mice are reminiscent of wild-type MARV/Ang infections in humans and nonhuman primates. We believe this will be of help in accelerating the development of life-saving measures against Marburg virus infections.

  • Epstein-Barr Virus Glycoproteins gB and gHgL Can Mediate Fusion and Entry in Trans; Heat Can Act as a Partial Surrogate for gHgL and Trigger a Conformational Change in gB [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) fusion with an epithelial cell requires virus glycoproteins gHgL and gB and is triggered by an interaction between gHgL and integrins aalpha;vbbeta;5, aalpha;vbbeta;6 or aalpha;vbbeta;8. Fusion with a B cell requires gHgL, gp42 and gB and is triggered by an interaction between gp42 and HLA class II. We report here that like alpha and betaherpesviruses, EBV, a gammaherpesvirus, can mediate cell fusion if gB and gHgL are expressed in trans. Entry of a gH-null virus into an epithelial cell is possible if the epithelial cell expresses gHgL and entry of the same virus, which phenotypically lacks gHgL and gp42, into a B cell expressing gHgL is possible in the presence of a soluble integrin. Heat is capable of inducing fusion of cells expressing only gB and the proteolytic digestion pattern of gB in virions changes in the same way following exposure of virus to heat or to soluble integrins. It is suggested that the Gibbs free energy released as a result of the high affinity interaction of gHgL with an integrin may contribute to the activation energy required to cause refolding of gB from a prefusion to a postfusion conformation.

    IMPORTANCE The core fusion machinery of herpesviruses consists of glycoproteins gB and gHgL. We demonstrate that as in alpha and betaherpesvirus, gB and gHgL of the gammaherpesvirus EBV can mediate fusion and entry when expressed in trans in opposing membranes implicating interactions between the ectodomains of the proteins in activation of fusion. We further show that heat and exposure to a soluble integrin, both of which activate fusion, result in the same changes in the proteolytic digestion pattern of gB possibly representing the refolding of gB from its prefusion to its post fusion conformation.

  • The HSV-2 Virion-associated Ribonuclease, vhs, Interferes with Stress Granule Formation [PublishAheadOfPrint]

  • In a previous study, it was observed that cells infected with herpes simplex virus type 2 (HSV-2) failed to accumulate stress granules (SGs) in response to oxidative stress induced by arsenite treatment. As a follow up to this observation, we demonstrate here that disruption of arsenite-induced SG formation by HSV-2 is mediated by a virion component. Through studies on SG formation in cells infected with HSV-2 strains carrying defective forms of UL41, the gene that encodes vhs, we identify vhs as a virion component required for this disruption. Cells infected with HSV-2 strains producing defective forms of vhs form SGs spontaneously late in infection. In addition to core SG components, these spontaneous SGs contain the viral immediate early protein ICP27 as well as the viral serine/threonine kinase Us3. As part of these studies, we re-examined the frameshift mutation known to reside within the UL41 gene of HSV-2 strain HG52. We demonstrate that this mutation is unstable and can rapidly revert to restore wild type UL41 following low multiplicity passaging. Identification of the involvement of virion-associated vhs in the disruption of SG formation will enable mechanistic studies on how HSV-2 is able to counteract antiviral stress responses early in infection. In addition, the ability of Us3 to localize to stress granules may indicate novel roles for this viral kinase in regulation of translation.

    IMPORTANCE Eukaryotic cells respond to stress by rapidly shutting down protein synthesis and storing mRNAs in cytoplasmic stress granules (SGs). Stoppages in protein synthesis are problematic for all viruses as they rely on host cell machinery to synthesize viral proteins. Thus, many viruses target SGs for disruption or modification. Infection by herpes simplex virus type 2 (HSV-2) was previously observed to disrupt SG formation induced by oxidative stress. In this follow up study, we identify virion host shutoff protein (vhs) as a viral protein involved in this disruption. Identification of a specific viral protein involved in disrupting SG formation is a key step towards understanding how HSV-2 interacts with these antiviral structures. Additionally, this understanding may provide insights into the biology of SGs that may find application in studies on human motor neuron degenerative diseases, like amyotrophic lateral sclerosis (ALS), which may arise as a result of disregulation of SG formation.

  • Organization of Capsid-associated Tegument Components in Kaposi's Sarcoma-associated Herpesvirus [PublishAheadOfPrint]

  • Capsid-associated tegument proteins have been identified in alpha- and beta-herpesviruses to play an essential role in viral DNA-packaging. Whether and how such tegument proteins exist in gamma-herpesviruses have been a mystery. Here we report a 6AAring; resolution cryoEM structure of Kaposi's sarcoma-associated herpesvirus (KSHV) virion, a member of the oncogenic gamma-herpesvirus subfamily. The KSHV virion structure reveals for the first time how capsid-associated tegument proteins are organized in a gamma-herpesvirus mmdash; with five tegument densities cap each penton vertex mmdash; a pattern highly similar to that in alpha-herpesvirus but completely different from that in beta-herpesvirus. Each KSHV tegument density can be divided into three prominent regions: a penton-binding globular region, a helix-bundle stalk region and a bbeta;-sheet-rich triplex-binding region. Fitting of crystal structure of the truncated HSV-1 UL25 (the KSHV ORF19 homolog) and secondary structure analysis of the full-length ORF19 establish that ORF19 constitutes the globular region with an N-terminal, 60 amino acid long helix extending into the stalk region. Matching secondary structural features resolved in the cryoEM density with secondary structures predicted by sequence analysis identifies the triplex-binding region to be ORF32, a homolog of alpha-herpesvirus UL17. Despite high-level of tegument structural similarities between KSHV and alpha-herpesvirus, an ORF19 monomer in KSHV, in contrast to a UL25 dimer in alpha-herpesviruses, binds each penton subunit, an observation that correlates with conformational difference in their pentons. This newly discovered organization of triplex-ORF32-ORF19 also resolves a long-standing mystery surrounding the virion location and conformation of the alpha-herpesvirus UL25.

    Importance Several capsid-associated tegument proteins have been identified in the alpha- and beta-herpesvirus subfamilies of the herpesviridae. These tegument proteins play essential roles in viral propagation and are potential drug targets for curbing herpesvirus infections. However, no such tegument proteins have been identified for gamma-herpesviruses, the third herpesvirus subfamily that contains members causing several human cancers. Here, by high-resolution cryo electron microscopy (cryoEM), we show the three-dimensional structure of the capsid-associated tegument proteins in the prototypical member of gamma-herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV). The cryoEM structure reveals that the organization of KSHV capsid-associated tegument proteins is highly similar to that in alpha-herpesvirus, but completely different from that in beta-herpesvirus. Structural analyses further localize ORF19 and ORF32 proteins (the alpha-herpesvirus UL25 and UL17 homologs in KSHV respectively) in the KSHV capsid-associated tegument cryoEM structure. These findings also resolve a long-standing mystery regarding the location and conformation of alpha-herpesvirus UL25 inside the virion.

  • Autographa californica multiple nucleopolyhedrovirus orf132 encodes a nucleocapsid-associated protein required for budded virus and multiply enveloped occlusion-derived virus production [PublishAheadOfPrint]

  • Autographa californica multiple nucleopolyhedrovirus orf132, named ac132, has homologs in all genome-sequenced group I nucleopolyhedroviruses. Its role in viral replication cycle is unknown. In this study, ac132 was shown to express a protein of around 28 kD, which was determined to be associated with the nucleocapsids of both occlusion-derived virus and budded virus. Confocal microscopy showed that AC132 protein appeared in central region of the nucleus as early as 12 hours post infection with the virus. It formed a ring zone at the periphery of the nucleus by 24 hours post infection. To investigate its role in virus replication, ac132 was deleted from the viral genome by using a bacmid system. In the Sf9 cell culture transfected by the ac132-knochout bacmid, infection was restricted to single cells, and the titer of infectious budded virus reduced to undetectable level. However, viral DNA replication and the expression of late genes vp39 and odv-e25 and a reporter gene under control of the very late gene p10 promoter were unaffected. Electron microscopy showed that nucleocapsids, virions, and occlusion bodies were synthesized in the cells transfected by an ac132-knockout bacmid, but the formation of the virogenic stroma and occlusion bodies was delayed, the numbers of enveloped nucleocapsid was reduced and the occlusion bodies contained mainly singly enveloped nucleocapsids. AC132 was found to interact with envelope protein ODV-E18 and the viral DNA-binding protein P6.9. The data from this study suggest that ac132 possibly plays an important role in the assembly and envelopment of nucleocapsids.

    Importance To our knowledge, this is the first report on a functional analysis of ac132. The data presented here demonstrate that ac132 is required for production of the budded virus and multiply enveloped occlusion-derived virus of Autographa californica multiple nucleopolyhedrovirus. The paper revealed unique phenotypic changes induced by ac132 deletion on the virus and multiple new findings on ac132.

  • Catalytic Function and Substrate Specificity of the PLpro Domain of nsp3 from the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) [PublishAheadOfPrint]

  • The papain-like protease (PLpro) domain from the deadly Middle East Respiratory Syndrome coronavirus (MERS-CoV) was over-expressed and purified. MERS-CoV PLpro constructs with or without the putative ubiquitin-like (UBL) domain at the N-terminus were found to possess protease, deubiquitinating, deISGylating, and interferon antagonism activities in transfected HEK293T cells. The quaternary structure and substrate preferences of MERS-CoV PLpro were determined and compared to those of SARS-CoV PLpro, revealing prominent differences between these closely related enzymes. Steady-state kinetic analyses of purified MERS-CoV and SARS-CoV PLpros uncover significant differences in their rates of hydrolysis of 5-aminomethyl coumarin (AMC) from C-terminally labeled peptide, ubiquitin and ISG15 substrates, as well as in their rates of isopeptide bond cleavage of K48- and K63-linked polyubiquitin chains. MERS-CoV PLpro was found to have an 8-fold and 3,500-fold higher catalytic efficiency for hydrolysis of the ISG15-AMC over the Ub-AMC and Z-RLRGG-AMC substrates respectively. A similar trend is observed for SARS-CoV PLpro although it is much more efficient than MERS-CoV PLpro towards ISG15-AMC and peptide-AMC substrates. MERS-CoV PLpro was found to process K48- and K63-linked polyubiquitin chains with similar rates and debranching patterns producing monoubiquitin species. However, SARS-CoV PLpro much prefers K48-linked polyubiquitin chains to K63-linked chains, and it rapidly produces di-ubiquitin molecules from K48-linked chains. Finally, potent inhibitors of SARS-CoV PLpro were found to have no effect on MERS-CoV PLpro. A homology model of MERS-CoV PLpro structure was generated and compared to the X-ray structure of SARS-CoV PLpro to provide plausible explanations for differences in substrate and inhibitor recognition.

    IMPORTANCE Unlocking the secrets of how coronavirus (CoV) papain-like proteases (PLpros) perform their multifunctional roles during viral replication entails a complete mechanistic understanding of their substrate recognition and enzymatic activities. We show that the PLpro domains from the MERS and SARS coronaviruses can recognize and process the same substrates but with different catalytic efficiencies. The differences in substrate recognition between these closely related PLpros suggest that neither enzyme can be used as a generalized model to explain the kinetic behavior of all CoV PLpros. As a consequence, decoding the mechanisms of PLpro-mediated antagonism of the host innate immune response and the development of anit-CoV PLpro enzyme inhibitors will be a challenging undertaking. The results from this study provide valuable information for understanding how MERS-CoV PLpro-mediated antagonism of the host innate immune response is orchestrated and insight into the design of inhibitors against MERS-CoV PLpro./Background

  • Role of the hypervariable region 1 (HVR1) for the interplay of hepatitis C virus with entry factors and lipoproteins [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) particles associate with lipoproteins and infect cells using at least four cell entry factors. These include the scavenger receptor class B type I (SR-BI), CD81, claudin 1 (CLDN1) and occludin (OCLN). Little is known about specific functions of individual host factors during HCV cell entry and viral domains that mediate interaction with these factors. The HVR1 within the viral envelope protein 2 (E2) is involved in usage of SR-BI and conceals the viral CD81 binding site. Moreover, deletion of this domain alters the density of virions. We compared lipoprotein interaction, surface attachment, receptor usage and cell entry between wild type HCV and a viral mutant lacking this domain. Deletion of HVR1 did not affect CD81, CLDN1 and OCLN usage. However, unlike wild type HCV, HVR1-deleted viruses were not neutralized by antibodies and small molecules targeting SR-BI. Nevertheless, modulation of SR-BI cell surface expression altered infection efficiency of both viruses to similar levels. Analysis of affinity purified virions revealed comparable levels of ApoE incorporation into viruses with or without HVR1. However, ApoE incorporated into these viruses was differentially recognized by ApoE-specific antibodies. Thus, SR-BI has at least two functions during cell entry. One of them can be neutralized by SR-BI-targeting molecules and it is critical only for wild type HCV. The other one is important for both viruses, but apparently is not inactivated by those SR-BI-binding antibodies and small molecules evaluated here. In addition, HVR1 modulates the conformation and/or epitope exposure of virus particle assocated ApoE.

    Importance HCV cell entry is SR-BI-dependent irrespective of the presence or absence of HVR1. Moreover, this domain modulates the properties of ApoE on the surface of virus particles. These findings have implications for the development of SR-BI targeting antivirals. Furthermore, they highlight separable functions of SR-BI during HCV cell entry and reveal a novel role of HVR1 for the properties of virus associated lipoproteins.

  • Multiple groups of endogenous epsilon-like endogenous retroviruses conserved across primates. [PublishAheadOfPrint]

  • Several types of cancer in fish are caused by retroviruses, including those responsible for major outbreaks of disease, such as walleye dermal sarcoma virus and salmon swim bladder sarcoma virus. These viruses form a phylogenetic group often described as the "epsilonretrovirus" genus. Epsilon-like retroviruses have become endogenous retroviruses (ERVs) on several occasions, integrating into germline cells to become part of the host genome, and sections of fish and amphibian genomes are derived from epsilon-like retroviruses. However, epsilon-like ERVs have been identified in very few mammals.

    We have developed a pipeline to screen full genomes for ERVs and using this pipeline, we have located over 800 endogenous epsilon-like ERV fragments in primate genomes. Genomes from 32 species of mammals and birds were screened and epsilon-like ERV fragments were found in all primate and tree shrew genomes but no others. These viruses appear to have entered the genome of a common ancestor of old and new world monkeys between 42 million and 65 million years ago

    Based on these results, there is an ancient evolutionary relationship between epsilon-like retroviruses and primates. Clearly, these viruses had the potential to infect the ancestors of primates and were at some point a common pathogen in these hosts. Therefore, this result raises questions about the potential of epsilonretroviruses to infect humans and other primates and about the evolutionary history of these retroviruses.

    Importance Epsilonretroviruses are a group of retroviruses which cause several important diseases in fish. Retroviruses have the ability to become a permanent part of the DNA of their host by entering the germline as endogenous retroviruses (ERVs), where they lose their infectivity over time but can be recognised as retroviruses for millions of years. Very few mammals are known to have epsilon-like ERVs, however, we have identified over 800 fragments of endogenous epsilon-like ERVs in the genomes of all major groups of primates, including humans. These viruses seem to have circulated and infected primate ancestors 42 to 65 million years ago. We are now interested in how these viruses have evolved and whether they have the potential to infect modern humans or other primates.

  • Immunodeficient Mouse Models with Different Disease Profiles by in vivo Infection with the Same Clinical Isolate of Enterovirus 71 [PublishAheadOfPrint]

  • Like poliovirus, severe infection with enterovirus 71 (EV71) can cause neuropathology. Unlike poliovirus, EV71 is often associated with hand, foot, and mouth disease (HFMD). Here, we established three mouse models for experimental infection with the same clinical isolate of EV71. The NOD/SCID mouse model is unique in developing skin rash, an HFMD-like symptom. While the NOD/SCID model developed limb paralysis and death at near 100% efficiency, the interferon-gamma receptor knockout (ifngr KO) and the stat-1 knockout mice exhibited paralysis and death rates near 78% and 30%, respectively. Productive infection with EV71 depends on viral dose, host age, and inoculation routes. Infectious EV71, and VP1-specific RNA and protein in muscle, brain, and spinal cord, were compared side-by-side between NOD/SCID and stat-1 models before, during, and after disease onset. Spleen fibrosis and muscle degeneration are common in NOD/SCID and stat-1 models. Main differences between these two models include their disease manifestations and cytokine/chemokine profiles. Pathology of the NOD/SCID model includes 1) inflammation and expression of viral VP1 antigen in muscle; 2) increased neutrophils and decreased eosinophils and lymphocytes; 3) hair loss and skin rash. Characteristic pathology of the stat-1 model includes 1) a strong tropism of EV71 for central nervous system; 2) detection of VP1 protein in the Purkinje layer of cerebellar cortex, pons, brainstem, and spinal cord; 3) amplification of microglial cells; 4) dystrophy of intestinal villi. Our comparative studies on these new models by oral or intraperitoneal (i.p.) infection underscored the contribution of host immunity, including interferon-gamma receptor, to EV71 pathogenesis.

    IMPORTANCE In the past decade, enterovirus 71 (EV71) has emerged as a major threat to the public health in the Asia-Pacific regions. Disease manifestations include subclinical infection, common cold-like syndromes, hand-foot-and-mouth disease (HFMD), uncomplicated brainstem encephalitis, severe dysregulated autonomic nerve system, fatal pulmonary edema, and cardiopulmonary collapse. To date, no effective vaccine or treatment is available. A user-friendly and widely accessible animal model for researching EV71 infection and pathogenesis is urgently needed by the global community, both academia and industry.

  • Kinetic and phenotypic analysis of CD8+ T cell responses after priming with alphavirus replicons and homologous or heterologous booster immunizations [PublishAheadOfPrint]

  • Alphavirus replicons are potent inducers of CD8+ T cell responses and thus constitute an attractive vaccine vector platform for developing novel vaccines. However, the kinetics and memory phenotype of CD8+ T cell responses induced by alphavirus replicons are not well characterized. Furthermore, little is known how priming with alphavirus replicons affects booster immune responses induced by other vaccine modalities. We demonstrate that a single immunization with an alphavirus replicon, administered as viral particles or naked DNA, induced an antigen-specific CD8+ T cell response that had a sharp peak, followed by a rapid contraction. Administering a homologous boost before contraction had occurred did not further increase the response. In contrast, boosting after contraction when CD8+ T cells had obtained a memory phenotype (based on CD127/CD62L expression), resulted in maintenance of CD8+ T cells with a high recall capacity (based on CD27/CD43 expression). Increasing the dose of replicon particles promoted T effector memory (Tem) and inhibited T central memory (Tcm) development. Moreover, infection with a replicating alphavirus induced a similar distribution of CD8+ T cells as the replicon vector. Lastly, the distribution of T cell subpopulations induced by a DNA-launched alphavirus replicon could be altered by heterologous boosts. For instance, boosting with a poxvirus vector (MVA) favored expansion of the Tem compartment. In summary, we have characterized the antigen-specific CD8+ T cell response induced by alphavirus replicon vectors and demonstrated how it can be altered by homologous and heterologous boost immunizations.

    Importance Alphavirus replicons are promising vaccine candidates against a number of diseases and are by themselves developed as vaccines against for example chikungunya virus infection. Replicons are also considered to be used for priming followed by booster immunization using different vaccine modalities. In order to rationally design prime-boost immunization schedules with these vectors, characterization of the magnitude and phenotype of CD8+ T cell responses induced by alphavirus replicons is needed. Here, we demonstrate how factors such as timing and dose affect the phenotype of the memory T cell populations induced by immunization with alphavirus replicons. These findings are important for designing future clinical trials with alphaviruses, as they can be used to tailor vaccination regimens in order to induce a CD8+ T cell response that is optimal for control and/or clearance of a specific pathogen.

  • The temperature sensitive and attenuation phenotypes conferred by mutations in the influenza PB2, PB1, and NP genes are influenced by the species of origin of the PB2 gene in reassortant viruses derived from influenza A/California/07/2009 and A/WSN/33. [PublishAheadOfPrint]

  • Live attenuated influenza vaccines in the US are derived from a human virus that is temperature sensitive (ts), characterized by restricted (gge; 100-fold) replication at 39ddeg;C. The ts genetic signature (ts sig) has been mapped to 5 loci in 3 genes: PB1 (391E, 581G, and 661T), PB2 (265S) and NP (34G). However, when transferred into avian and swine influenza viruses, only partial ts and attenuation phenotypes occur. To investigate the reason for this, we introduced the ts sig into the human-origin virus A/WSN/33 (WSN), the avian-origin virus A/Vietnam/1203/04 (VN04), and the swine-origin triple reassortant 2009 pandemic H1N1 virus A/California/07/2009 (CA07), which contains gene segments from human-, avian- and swine-viruses. The VN04 ts sig and CA07 ts sig viruses replicated efficiently in MDCK cells at 39ddeg;C, but the replication of WSN ts sig was restricted gge; 100-fold compared to 33ddeg;C. Reassortant CA07 ts sig viruses were generated with individual polymerase gene segments from WSN, and vice versa. Only ts sig viruses with a PB2 gene segment derived from WSN were restricted in replication gge; 100-fold at 39ddeg;C. In ferrets, the CA07 ts sig virus replicated in the upper and lower respiratory tracts, but the replication of a reassortant CA07 ts sig virus with a WSN PB2 gene was severely restricted in the lungs. Taken together, these data suggest that the origin of the PB2 gene segment influences the ts phenotype in vitro and attenuation in vivo. This could have implications for the design of novel live vaccines against animal-origin influenza viruses.

    Importance Live attenuated influenza vaccines (LAIV) on temperature sensitive (ts) backbones derived from animal-origin influenza viruses are being sought for use in the poultry and swine industries and to protect people against animal-origin influenza. However, inserting the ts genetic signature from a licensed LAIV backbone fails to fully attenuate these viruses. Our data indicate this is associated with the presence of a PB2 gene segment derived from an avian influenza virus. We show that a reassortant 2009 pandemic H1N1 virus with the ts signature from a licensed LAIV donor virus is ts in vitro and attenuated in vivo when the PB2 gene is derived from a human-origin virus but not from an avian virus. Our study provides information that could benefit the rational design of alternative LAIV backbones against animal-origin influenza viruses.

  • HIV Vaccine Trial Exploits a Dual and Central Role for Innate Immunity [PublishAheadOfPrint]

  • Limited understanding of what protects from HIV hinders development of an efficacious vaccine. Lehner and colleagues now report that vaginal immunization with an HIVgp140 vaccine linked to the 70kD heat shock protein down-regulated the HIV co-receptor, CCR5, and increased expression of the HIV resistance factor, APOBEC3G, in women. These effects correlated with HIV suppression ex vivo. Thus, vaccine-induced innate responses not only facilitate adaptive immunity nndash; they may prove to be critical for preventing HIV transmission.

  • Inhibition of breast cancer cell proliferation through disturbance of the Calcineurin/NFAT pathway by Human Herpesvirus-6B U54 tegument protein [PublishAheadOfPrint]

  • Nuclear factor of activated T cell (NFAT) proteins are key regulators involved in multiple physiological mechanisms such as immune response or cell growth. Selective calcineurin/NFAT inhibitors already demonstrated their capacity to decrease NFAT-dependent cancer cell progression, particularly in breast cancer. In this study, we report a role for the human herpesvirus 6B (HHV-6B) U54 tegument protein in inhibiting MCF-7 breast cancer cell line proliferation by inhibiting NFAT activation.

  • The Composition of West Nile virus Lipid Envelope Unveils a Role of Sphingolipid Metabolism on Flavivirus Biogenesis [PublishAheadOfPrint]

  • West Nile virus (WNV) is an emerging zoonotic mosquito-borne flavivirus responsible for outbreaks of febrile illness and meningoencephalitis. The replication of WNV takes place on virus-modified membranes from the endoplasmic reticulum of the host cell and virions acquire their envelope by budding into this organelle. Consistent with this view, the cellular biology of this pathogen is intimately ligated to modifications of the intracellular membranes, and the requirement of specific lipids such as cholesterol and fatty acids has been documented. In this study, we evaluated the impact of WNV infection on two important components of cellular membranes, glycerophospholipids and sphingolipids, by mass spectrometry of infected cells. A significant increase in the content of several glycerophospholipids (phosphatidylcholine, plasmalogens and lysophospholipids) and sphingolipids (ceramide, dihidroceramide and sphingomyelin) was noticed in WNV-infected cells, suggesting functional roles of these lipids during WNV infection. Furthermore, the analysis of the lipid envelope of WNV virions and recombinant virus-like particles revealed a unique composition of their envelopes that were enriched in sphingolipids (sphingomyelin) and showed reduced levels of phosphatidylcholine, in a manner similar to that of sphingolipid enriched lipid microdomains. Inhibition of neutral sphingomyelinase (which catalyzes the hydrolysis of sphingomyelin into ceramide), either by pharmacological approaches or siRNA mediated silencing, reduced the release of flavivirus virions as well as virus-like particles, suggesting a role of sphingomyelin to ceramide conversion in flavivirus budding and confirming the importance of sphingolipids in the biogenesis of WNV.

    IMPORTANCE West Nile virus (WNV) is a neurotropic flavivirus spread by mosquitoes that can infect multiple vertebrate hosts including humans. There is no specific vaccine or therapy licensed for human use against this pathogen. Since the multiplication of this virus is associated to rearrangements of host cell membranes, we analyzed the effect of WNV infection on different cellular lipids that constitute important membrane components. Multiple lipid species were increased in infected cells, pointing to major alterations of cellular lipid metabolism induced by WNV infection. Interestingly, certain sphingolipids, which were increased in infected cells, were also enriched in the lipid envelope of the virus, thus suggesting a potential role during virus assembly. We further verified the role of sphingolipids in the production of WNV by means of functional analyses. This study provides new insight in the formation of flavivirus infectious particles and the involvement of sphingolipids in the WNV life cycle.

  • Regulation of autophagic activation by Rta of Epstein-Barr virus via ERK-kinase pathway [PublishAheadOfPrint]

  • Autophagy is an intracellular degradation pathway that provides a host defense mechanism against intracellular pathogens. However, many viruses exploit this mechanism to promote their replication. This study shows that lytic induction of Epstein-Barr virus (EBV) increases the membrane-bound form of LC3 (LC3-II) and LC3-containing punctate structures in EBV-positive cells. Transfecting 293T cells with a plasmid that expresses Rta also induces autophagy, revealing that Rta is responsible for autophagic activation. The activation involves Atg5, a key component of autophagy, but not the mTOR pathway. The expression of Rta also activates the transcription of the genes that participate in the formation of autophagosomes, including LC3A, LC3B and ATG9B genes, as well as those that are involved in the regulation of autophagy, including the genes that encode TNF, IRGM and TRAIL. Additionally, treatment with U0126 inhibits the Rta-induced autophagy and the expression of autophagy genes, indicating that the autophagic activation is caused by the activation of ERK signaling by Rta. Finally, the inhibition of autophagic activity by an autophagy inhibitor, 3-methyladenine, or Atg5 siRNA, reduces the expression of EBV lytic proteins and the production of viral particles, revealing that autophagy is critical to EBV lytic progression. This investigation reveals how an EBV-encoded transcription factor promotes autophagy to affect viral lytic development.

    Importance Autophagy is a cellular process that degrades and recycles nutrients under stress conditions to promote cell survival. Although autophagy commonly serves as a defense mechanism against viral infection, many viruses exploit this mechanism to promote their replication. This study finds that a transcription factor that is encoded by Epstein-Barr virus (EBV), Rta, activates autophagy, the inhibition of autophagy reduces the ability of the virus to express viral lytic proteins and to generate progenies. Unlike other virus-encoded proteins that modulate autophagy by interacting with proteins that are involved in the autophagic pathway, Rta activates the transcription of the autophagy-related genes via the ERK pathway. The results of this study reveal how the virus manipulates autophagy to promote its lytic development.

  • Inhibition of Interleukin-2 Gene Expression by Human Herpesvirus 6B U54 Tegument Protein [PublishAheadOfPrint]

  • Human Herpesvirus 6B (HHV-6B) is a ubiquitous pathogen causing life-long infections in approximately 95% of humans worldwide. To persist within its host, HHV-6B developed several immune evasion mechanisms such as latency during which minimal proteins are expressed and also by disturbing innate and adaptive immune responses. The primary cellular targets of HHV-6B are CD4+ T cells. Previous studies by Flamand et al. reported on the capacity of HHV-6A, as well as UV-irradiated HHV-6A at inhibiting interleukin-2 (IL-2) synthesis in CD4+ lymphocytes suggesting that viral structural components could be responsible for this effect. In the present study, we have identified the HHV-6B U54 tegument protein (U54) to be capable of inhibiting IL-2 expression. U54 binds the calcineurin (CaN) phosphatase enzyme causing improper dephosphorylation and nuclear translocation of nuclear factor of activated T cells (NFAT) proteins, resulting in sub-optimal IL-2 gene transcription. The U54 GISIT motif (aa 293-297), analogous to the NFAT PxIxIT motif, contributed to the inhibition of NFAT activation.

    IMPORTANCE Human herpesvirus-6A (HHV-6A) and HHV-6B are associated with an increasing number of pathologies. These viruses have developed strategies to avoid the immune response allowing them to persist into host. Several studies have illustrated mechanisms by which HHV-6A and HHV-6B are able to disrupt host defenses. Previous work informed us that HHV-6A is able to suppress interleukin-2 (IL-2) synthesis, a key immune growth factor essential for adequate T lymphocyte proliferation and expansion. We provide evidence that HHV-6B also inhibits IL-2 gene expression and identified the mechanisms by which it does so. Our work led us to the identification of U54, a virion-associated tegument protein, as being responsible for suppression of IL-2. Consequently, we have identified HHV-6B U54 protein as playing a role in immune evasion. These results further contribute to our understanding of HHV-6 interactions with its human host and the efforts deployed to ensure its long-term persistence.

  • Core-binding factor subunit beta is not required for non-primate lentiviral Vif mediated APOBEC3 degradation [PublishAheadOfPrint]

  • Viral infectivity factor (Vif) is required for lentivirus fitness and pathogenicity, except in equine infectious anemia virus (EIAV). Vif enhances viral infectivity by a Cullin5mmdash;Elongin B/C E3 complex to inactivate the host restriction factor APOBEC3. Core-binding factor subunit beta (CBF-bbeta;) is a cell factor that was recently shown to be important for the primate lentiviral Vif function. Non-primate lentiviral Vif also degrades APOBEC3 through the proteasome pathway. However, it is unclear whether CBF-bbeta; is required for the non-primate lentiviral Vif function. In this study, we demonstrated that the Vif of non-primate lentiviruses, including feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), caprine arthritis encephalitis virus (CAEV), and maedi-visna virus (MVV), do not interact with CBF-bbeta;. In addition, CBF-bbeta; did not promote the stability of FIV, BIV, CAEV, and MVV Vifs. Furthermore, CBF-bbeta; silencing or overexpression did not affect non-primate lentiviral Vif-mediated APOBEC3 degradation. Our results suggest that non-primate lentiviral Vif induces APOBEC3 degradation through a different mechanism compared with primate lentiviral Vif.

    Importance The APOBEC3 protein family members are host restriction factors that block retrovirus replication. Vif, an accessory protein of lentivirus, degrades APOBEC3 to rescue viral infectivity by forming Cullin5-Elongin B/C based E3 complex. CBF-bbeta; was proved to be a novel regulator of the primate lentiviral Vif function. Here, we found that CBF-bbeta; knockdown or overexpression did not affect FIV Vif's function, which induced polyubiquitination and degradation of APOBEC3 by recruiting the E3 complex in a manner similar to that of HIV-1 Vif. We also showed that other non-primate lentiviral Vifs did not require CBF-bbeta; to degrade APOBEC3. CBF-bbeta; did not interact with non-primate lentiviral Vifs or promote their stability. These results suggest that a different mechanism exists for the Vifnndash;APOBEC interaction and that non-primates are not suitable animal models for exploring pharmacological interventions that disrupt the Vifnndash;CBF-bbeta; interaction.

  • The VP4 peptide of Hepatitis A Virus (HAV) Ruptures Membranes Through Formation of Discrete Pores [PublishAheadOfPrint]

  • Membrane active peptides, components of capsid structural proteins, assist viruses in overcoming the host membrane barrier in the initial stages of infection. Several such peptides have been identified and their roles in membrane fusion or disruption have been characterized through biophysical studies. In several members of the picornaviridae family, the role of the VP4 structural peptide in cellular membrane penetration is well established. However, there is not much information on the membrane penetrating capsid components of Hepatitis A Virus (HAV), an unusual member of this family. The VP4 peptide of HAV differs from its analogues in other picornaviruses by being significantly shorter in length, and also by lacking a signal for myristoylation, thought to be a critical requisite for VP4-mediated membrane penetration. Here we report, for the first time, that the atypical VP4 in HAV contains significant membrane-penetrating activity. Using a combination of biophysical assays and molecular dynamics simulation studies, we show that VP4 integrates into membrane vesicles through its N-terminal region, to finally form discrete pores of 5-9 nm diameter, which induces leakage in the vesicles without altering their overall size or shape. We further demonstrate that the membrane activity of VP4 is specific towards vesicles mimicking the lipid content of late endosomes, at acidic pH. Taken together, our data indicates that VP4 might be essential for the penetration of host endosomal membranes and release of genome during HAV entry.

    Importance Hepatitis A Virus (HAV) causes acute hepatitis in humans through the faecal-oral route, and is particularly prevalent in underdeveloped regions with poor hygienic conditions. Although a vaccine for HAV exists, its high cost makes it unsuitable for universal application in developing countries. Studies on host-virus interaction for HAV have been hampered due to lack of starting material, since the virus is extremely slow growing in culture. Among the unknown aspects of the HAV life cycle is its manner of host membrane penetration, which is one of the most important initial steps in viral infection. Here, we present data to suggest that a small peptide VP4, a component of the HAV structural polyprotein, might be essential in helping the viral genome cross cell membranes during entry. It is hoped that this work might help in elucidating the manner of initial host cell interaction by HAV.

  • Computational prediction of vaccine strains for human influenza A (H3N2) viruses [PublishAheadOfPrint]

  • Human influenza A viruses are rapidly evolving pathogens that cause substantial morbidity and mortality in seasonal epidemics around the globe. To ensure continued protection, the strains used for the production of the seasonal influenza vaccine have to be regularly updated, which involves data collection and analysis by numerous experts worldwide. Computer-guided analysis is becoming increasingly important in this problem due to the vast amounts of generated data. We here describe a computational method for selecting a suitable strain for production of the human influenza A virus vaccine. It interprets available antigenic and genomic sequence data based on measures of antigenic novelty and rate of propagation of the viral strains throughout the population. For viral isolates sampled between 2002 and 2007 we used this method to predict the antigenic evolution of the H3N2 viruses in retrospective testing scenarios. When seasons are scored as true or false predictions, our method returned six true positives, three false negatives, eight true negatives and one false positive prediction or 77% accuracy overall. In comparison to the recommendations by the WHO, we identified the correct antigenic variant once at the same time and twice one season ahead. Even though it cannot be ruled out that practical reasons such as lack of a sufficiently well-growing candidate strain may in some cases have prevented recommendation of the best matching strain by the WHO, our computational decision procedure allows to quantitatively interpret the growing amounts of data and may help to match the vaccine better to predominating strains in seasonal influenza epidemics.

    Importance Human influenza A viruses continuously change antigenically to circumvent the immune protection evoked by vaccination or previously circulating viral strains. To maintain vaccine protection and thereby reduce the mortality and morbidity caused by infections, regular updates of the vaccine strains are required. We have developed a data-driven framework for vaccine strain prediction which facilitates the computational analysis of genetic and antigenic data and does not rely on explicit evolutionary models. Our computational decision procedure generated good matches of the vaccine strain to the circulating predominant strain for most seasons and could be used to support the expert-guided prediction made by the WHO and may allow to further increase vaccine efficiency.

  • Increased replicative fitness can lead to decreased drug sensitivity of hepatitis C virus [PublishAheadOfPrint]

  • Passage of hepatitis C virus (HCV) in human hepatoma cells resulted in populations that displayed partial resistance to IFN-aalpha;, telaprevir, daclatasvir, cyclosporine A and ribavirin, despite no prior exposure to these drugs. Mutant spectrum analyses and kinetics of virus production in the absence and presence of drugs indicate that resistance is not due to the presence of drug-resistance mutations in the mutant spectrum of the initial or passaged populations, but to increased replicative fitness acquired during passage. Fitness increase did not alter host factors that lead to shutoff of general host cell protein synthesis and preferential translation of HCV RNA. The results imply viral replicative fitness as a mechanism of multidrug resistance in HCV.

    Importance Viral drug resistance is usually attributed to the presence of amino acid substitutions in the protein targeted by the drug. In the present study with HCV we show that high viral replicative fitness can confer a general drug resistance phenotype to the virus. The results exclude that genomes with drug resistance mutations are responsible for the observed phenotype. The fact that replicative fitness can be a determinant of multidrug resistance may explain why in prolonged chronic HCV infections that favor replicative fitness increase, the virus is less sensitive to drug treatments.

  • Emergence of broadly neutralizing antibodies and viral co-evolution in two subjects during the early stages of infection with the human immunodeficiency virus type 1 [PublishAheadOfPrint]

  • Delineating the key early events that lead to the development of broadly neutralizing anti-HIV-1 antibodies during natural infection may help guide the development of immunogens and vaccine regimens to prevent HIV-1 infection. In this study, we followed two HIV-1 positive subjects, VC20013 and VC10014, over the course of infection from before they developed broadly neutralizing antibody (bNAb) activity until several years after breadth was detected in the plasma. Both subjects developed bNAb activity after approximately one year post infection, which ultimately mapped to the membrane proximal external region (MPER) in VC20013 and an epitope that overlaps the CD4 receptor binding site in VC10014. In subject VC20013, we were able to identify anti-MPER activity in the earliest plasma sample that exhibited no bNAb activity, indicating that this epitope specificity was acquired very early on, but that it was initially not able to mediate neutralization. Escape mutations within the bNAb epitopes did not arise in the circulating envelopes until bNAb activity was detectable in the plasma, indicating that this early response was not sufficient to drive viral escape. As bNAb activity began to emerge in both subjects, we observed a simultaneous increase in autologous anti-Envelope antibody binding affinity, indicating that antibody maturation was occurring as breadth was developing. Our findings illustrate one potential mechanism by which bNAbs develop during natural infection in which an epitope target is acquired very early on during the course of infection, but requires time and maturation to develop into broadly neutralizing activity.

    Importance One major goal of HIV-1 vaccine research is the development of a vaccine that can elicit broadly neutralizing antibodies (bNAbs). Although no such vaccine exists, bNAbs develop in approximately twenty percent of HIV-1-infected subjects, providing prototype of the bNAbs that must be re-elicited by vaccine. Thus, there is significant interest in understanding the mechanisms by which bNAbs develop during the course of infection. We studied the timing, the epitope specificity, and the evolution of the bNAb responses in two HIV-1 positive patients who developed bNAb activity within the first several years after infection. In one subject, antibodies to a broadly neutralizing epitope developed very early but were non-neutralizing. After several months neutralizing activity developed and the virus mutated to escape their activity. Our study highlights one mechanism for the development of bNAbs where early epitope acquisition followed by sufficient time for antibody maturation drives the epitope-specific antibody response toward broadly neutralizing activity.

  • Probing the functions of the paramyxovirus glycoproteins F and HN with a panel of synthetic antibodies (sABs) [PublishAheadOfPrint]

  • Paramyxoviruses are enveloped negative-strand RNA viruses that are significant human and animal pathogens. Most paramyxoviruses infect host cells via the concerted action of a tetrameric attachment protein (variously called HN, H, or G) that either binds sialic acid or protein receptors on target cells and a trimeric fusion protein (F) that merges the viral envelope with the plasma membrane at neutral pH. F initially folds to a metastable prefusion conformation that becomes activated via a cleavage event during cellular trafficking. Upon receptor binding the attachment protein, which consists of a globular head anchored to the membrane via a helical tetrameric stalk, triggers a major conformation change in F which results in fusion of virus and host cell membranes. We recently proposed a model for F activation in which the attachment protein head domains move following receptor binding to expose HN-stalk residues critical for triggering F. To test the model in the context of wild type viral glycoproteins, we used a restricted diversity combinatorial Fab library and phage display to rapidly generate synthetic antibodies (sABs) against multiple domains of the paramyxovirus parainfluenza 5 (PIV5) pre- and postfusion F and HN. As predicted by the model, sABs that bind to the critical F-triggering region of the HN stalk do not disrupt receptor binding or NA activity, but are potent inhibitors of fusion. An inhibitory prefusion F specific sAB recognized a quaternary antigenic site and may inhibit fusion by preventing F refolding or blocking the F-HN interaction.

    IMPORTANCE The paramyxovirus family of negative strand RNA virus cause significant disease in humans and animals. The viruses bind to cells via their receptor binging protein and then enter cells by fusion of their envelope with the host cell plasma membrane, a process mediated by a metastable viral fusion (F) protein. To understand the steps in viral membrane fusion a library of synthetic antibodies to F protein and the receptor binding protein was generated in bacteriophage. These antibodies bound to different regions of the F protein and the receptor binding protein and the location of antibody binding affected different processes in viral entry into cells.

  • Stearoyl-CoA Desaturase 1 Is Associated with Hepatitis C Virus Replication Complex and Regulates Viral Replication [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) life cycle is tightly regulated by lipid metabolism of host cells. In order to identify host factors involved in HCV propagation, we have recently screened the small interfering RNA (siRNA) library targeting host genes that control lipid metabolism and lipid droplet formation using cell culture grown HCV (HCVcc)-infected cells. We selected and characterized the gene encoding stearoyl-CoA desaturase 1 (SCD1). siRNA-mediated knockdown or pharmacological inhibition of SCD1 abrogated HCV replication in both subgenomic replicon and Jc1-infected cells, while exogenous supplementation of either oleate or palmitoleate, products of SCD1 activity, resurrected HCV replication in SCD1 knockdown cells. SCD1 was coimmunoprecipitated with HCV nonstructural proteins, and colocalized with both dsRNA and HCV nonstructural proteins, indicating that SCD1 is associated with HCV replication complex. Moreover, SCD1 was fractionated and enriched with HCV nonstructural proteins at detergent-resistant membrane. Electron microscopy data showed that SCD1 is required for NS4B-mediated intracellular membrane rearrangement. These data further support that SCD1 is associated with HCV replication complex and its products may contribute to the proper formation and maintenance of membranous web structures in HCV replication complex. Collectively, these data suggest that manipulation of SCD1 activity may represent a novel host-targeted antiviral strategy for the treatment of HCV infection.

    IMPORTANCE Stearoyl-CoA desaturase 1, a liver-specific enzyme, regulates HCV replication through its enzyme activity. HCV nonstructural proteins are associated with SCD1 at detergent-resistant membranes and SCD1 is enriched on the lipid raft by HCV infection. Therein SCD1 supports NS4B-mediated membrane rearrangement to provide a suitable microenvironment for HCV replication. We demonstrated that either genetic or chemical knockdown of SCD1 abrogated HCV replication in both replicon cells and HCV-infected cells. These findings provide novel mechanistic insights into the roles of SCD1 in HCV replication.

  • Apolipoprotein E likely contributes to a maturation step of infectious hepatitis C virus particles and interacts with viral envelope glycoproteins [PublishAheadOfPrint]

  • Assembly of infectious hepatitis C virus (HCV) particles is tightly linked to components of the very-low-density lipoprotein (VLDL) pathway. We and others have shown that apolipoprotein E (ApoE) plays a major role in production of infectious HCV particles. However, the mechanism by which ApoE contributes to virion assembly/release and how it gets associated with the HCV particle is poorly understood. We found that knock-down of ApoE reduces titers of infectious intra- and extracellular HCV, but not of the related Dengue virus. ApoE depletion also reduced amounts of extracellular HCV core protein without affecting intracellular core amounts. Moreover, we found that ApoE depletion neither affected formation of nucleocapsids nor their envelopment suggesting that ApoE acts at a late step of assembly such as particle maturation and infectivity. Importantly, we demonstrate that ApoE interacts with the HCV envelope glycoproteins, most notably E2. This interaction was independent from other viral proteins and required the transmembrane domain of E2 that was also required for recruitment of HCV envelope glycoproteins to detergent-resistant membrane fractions. These results suggest that ApoE plays an important role in HCV particle maturation, presumably by direct interaction with viral envelope glycoproteins.

    Importance The hepatitis C virus (HCV) replication cycle is tightly linked to host cell lipid pathways and components. This is best illustrated by the dependency of HCV assembly on lipid droplets and the very-low-density lipoprotein (VLDL) component apolipoprotein E (ApoE). Although the role of ApoE for production of infectious HCV particles is well established, it is still poorly understood how ApoE contributes to virion formation and how it gets associated with HCV particles. Here, we provide experimental evidence that ApoE likely is required for an intracellular maturation step of HCV particles. Moreover, we demonstrate that ApoE associates with the viral envelope glycoproteins. This interaction appears to be dispensable for envelopment of virus particles, but likely contributes to a quality control of secreted infectious virions. These results shed new light onto the exploitation of host cell lipid pathways by HCV and the link of viral particle assembly to the VLDL component ApoE.

  • Visualization of retroviral envelope spikes in complex with the V3 loop antibody 447-52D on intact viruses by cryo-electron tomography [PublishAheadOfPrint]

  • The gp120 portion of the envelope spike on human immunodeficiency virus -1 (HIV-1) plays a critical role in viral entry into host cells and is a key target for the humoral immune response, yet many structural details remain elusive. We have used cryoelectron tomography to visualize the binding of the broadly neutralizing monoclonal antibody (MAb) 447-52D to intact envelope spikes on virions of HIV-1 MN strain. Antibody 447-52D has previously been shown to bind to the tip of the V3 loop. Our results show antibody arms radiating from the sides of the gp120 protomers at a range of angles and place the antibody-bound V3 loop in an orientation that differs from that predicted by most current models but consistent with the idea that antibody binding dislodges the V3 loop from its location in the Env spike and making it flexible and disordered. These data reveal information on the position of the V3 loop, its relative flexibility, and suggest that 447-52D neutralizes HIV-1 MN by capturing the V3 loop, blocking its interaction with the co-receptor and altering the structure of the envelope spike.

    IMPORTANCE Antibody neutralization is one of the primary ways that the body fights infection with HIV. Because HIV is a highly mutable virus, the body must constantly produce new antibodies to counter new strains of HIV that the body itself is producing. Consequently, antibodies capable of neutralizing multiple HIV strains are comparatively few. An improved understanding of the mechanism of antibody neutralization might advance the development of immunogens. Most neutralizing antibodies target the Env glycoprotein spikes found on the virus surface. The broadly neutralizing antibody 447-52D targets the highly conserved bbeta;-turn of variable loop 3 (V3) of gp120. The importance of V3 lies in its contribution to the coreceptor binding site on the target cell. Here we show that 447-52D binding to V3 converts the Env conformation from closed to open and makes the V3 loop highly flexible, implying disruption of coreceptor binding and attachment to the target cell.

  • The N-terminus of the influenza B virus nucleoprotein is essential for virus viability, nuclear localization and optimal transcription and replication of the viral genome. [PublishAheadOfPrint]

  • The nucleoprotein (NP) of influenza viruses is a multifunctional protein with essential roles throughout viral replication. Despite influenza A and B viruses belonging to separate genera of the Orthomyxoviridae family, their NP proteins share a relatively high level of sequence conservation. However NP of influenza B viruses (BNP) contains an evolutionarily conserved N-terminal 50 amino acid extension that is absent from NP of influenza A viruses. There is conflicting evidence as to the functions of the BNP N-terminal extension, however this has never been assessed in the context of viral infection. We have used reverse genetics to assess the significance of this region on the functions of BNP and virus viability. Truncation of more than three amino acids prevented virus recovery suggesting that the N-terminal extension is essential for virus viability. Mutational analysis indicated that multiple regions of the protein are involved in nuclear localization of BNP with the entire N-terminal extension required for this to function efficiently. Viruses containing mutations in the first ten residues of BNP demonstrated little differences in nuclear localization, however the viruses exhibited significant reductions in viral mRNA transcription and genome replication resulting in significantly attenuated phenotypes. Mutations introduced to ablate a previously reported nuclear localization signal also resulted in a significant decrease in mRNA production during early stages of viral replication. Overall our results demonstrate that the N-terminal extension of BNP is essential to virus viability not only for directing nuclear localization of BNP, but also for regulating viral mRNA transcription and genome replication.

    Importance The multifunctional nucleoprotein (NP) of influenza viruses has roles throughout the viral replication cycle and is therefore essential for virus viability. Despite high levels of homology between the NP proteins of influenza A and B viruses the NP of influenza B virus (BNP) contains an evolutionarily conserved 50 amino acid N-terminal extension that is absent from the NP of influenza A viruses. In this study we show that this N-terminal extension is essential for virus viability and we confirm and expand upon recent findings that this region of BNP is required for nuclear localization of the protein. Furthermore we demonstrate for the first time that the N-terminus of BNP is involved in regulating viral mRNA transcription and replication of the viral genome. As the NP of influenza A virus lacks this N-terminal extension it suggests that these viruses have evolved separate mechanisms to regulate these processes.

  • Counteracting effects of cellular Notch and Epstein-Barr virus EBNA2: implications for stromal effects on virus-host interactions [PublishAheadOfPrint]

  • A number of diverse environmental cues have been linked to B lymphocyte differentiation and activation. One such cue, Notch-2, may be particularly relevant to the biology of Epstein Barr virus (EBV) infection which colonizes the B cell compartment. Activated Notch and EBV nuclear antigen, EBNA2, both function as transcriptional activators by virtue of their interactions with the transcription factor RBP-J. Although EBNA2 and activated Notch appear to have partially overlapping functions, we now report that activated Notch counteracts a crucial EBNA2 function in both newly-infected primary B cells and in lymphoblastoid cell lines (LCLs). EBNA2 is directly responsible for the initiation of transcription of the majority of EBV proteins associated with type III latency, leading to the outgrowth of LCLs. One of the key proteins driving this outgrowth is latent membrane protein 1 (LMP1), which is regulated by an EBNA2-responsive element within its ED-L1 promoter. Activation of Notch-2 via Delta-like ligand-1 inhibits EBNA2 mediated initiation of LMP1 transcription. Furthermore, ligated Notch-2 also efficiently turns off LMP1 from the ED-L1 promoter in LCLs already expressing LMP1. Modulation of EBV gene expression by Notch was not confined to EBNA2-dependent events. Activated Notch-2 also inhibited EBV entry into lytic cycle in a B-cell non-Hodgkin lymphoma line by up-regulating the cellular transcription factor Zeb2, which represses transcription of BZLF1.

    These results support the concept that in vivo, cumulative signals from the microenvironment down-regulate EBV gene expression in B cells to the latency 0 gene expression profile observed in B cells entering the peripheral blood.

    Importance Experimental infection of resting B cells by Epstein Barr virus leads to the growth transformation programme of virus gene expression and outgrowth of lymphoblastoid cell lines. Previous studies at the single cell level revealed complex cellular and viral signaling networks regulating transcription of the viral genome. This study demonstrates that viral gene expression can also be radically altered by molecules expressed on stromal cells in the microenvironment of lymphoid tissue; specifically, Delta-like ligand-1 on stromal cells ligating Notch-2 on infected B cells. Activation of Notch interferes with the transactivation function of EBNA2, downregulates expression of LMP1 and LMP2a, and inhibits activation of lytic virus replication in B-cell non-Hodgkin lymphoma line by preventing expression of BZLF1. The significance of these observations is that they indicate new mechanisms whereby the microenvironment in normal lymphoid tissue may facilitate the repression of viral gene expression enabling establishment of true latency in memory B cells.

  • DESC1 and MSPL activate influenza A viruses and emerging coronaviruses for host cell entry [PublishAheadOfPrint]

  • The type II transmembrane serine protease (TTSP) TMPRSS2 cleaves and activates the surface proteins of influenza and coronaviruses. Expression of TMPRSS2 is essential for spread and pathogenesis of H1N1 influenza viruses in mice. In contrast, H3N2 viruses are less dependent on TMPRSS2 for viral amplification, suggesting that these viruses might employ other TTSPs for their activation. Here, we analysed TTSPs, reported to be expressed in the respiratory system, for their ability to activate influenza and coronaviruses. We found that MSPL and, to a lesser degree DESC1, are expressed in human lung tissue and cleave and activate the spike proteins of the MERS- and SARS-coronavirus for cell-cell and virus-cell fusion. In addition, we show that these proteases support the spread of all influenza virus subtypes previously pandemic in humans. In sum, we identified two host cell proteases which could promote the amplification of influenza and emerging coronaviruses in humans and might constitute targets for antiviral intervention.

    IMPORTANCE Activation of influenza viruses by host cell proteases is essential for viral infectivity and the responsible enzymes are potential targets for antiviral intervention. The present study demonstrates that two cellular serine proteases, DESC1 and MSPL, activate influenza viruses and emerging coronaviruses in cell culture and, due to their expression in human lung tissue, might promote viral spread in the infected host. Antiviral strategies aiming to prevent viral activation might thus need to encompass inhibitors targeting MSPL and DESC1.

  • High frequencies of polyfunctional CD8+ NK cells in chronic HIV-1 infection are associated with slower disease progression [PublishAheadOfPrint]

  • Summary: Natural killer (NK) cells are effector and regulatory innate immune cells and play a critical role in the first line of defense against various viral infections. Although previous reports indicated vital contributions of NK cells to HIV-1 immune control, non-genetic NK cell parameters directly associated with slower disease progression have not been defined yet. In a longitudinal, retrospective study of 117 untreated HIV-infected subjects we show that higher frequencies as well as absolute numbers of CD8+CD3- lymphocytes are linked to delayed HIV-1 disease progression. We show that the majority of these cells are well-described blood NK cells. In a subsequent cross-sectional study we demonstrate a significant loss of CD8+ NK cells in untreated HIV-infected individuals, which correlated with HIV viral loads and inversely correlated with CD4+ T cell counts. CD8+ NK cells had modestly higher frequencies of CD57-expressing cells compared to CD8- cells but no differences in the expression of a number of activating and inhibiting NK cell receptors. However, CD8+ NK cells exhibited a more functional profile as detected by cytokine production and degranulation.

    Importance: We demonstrate that the frequency of highly functional CD8+ NK cells is inversely associated with HIV-related disease markers and linked with delayed disease progression. These results thus indicate that CD8+ NK cells represent a novel NK cell-derived, innate immune correlate with an improved clinical outcome in HIV infection.

  • Combined alphavirus replicon particle vaccine induces durable and cross-protective immune responses against equine encephalitis viruses [PublishAheadOfPrint]

  • Alphavirus replicons were evaluated as potential vaccine candidates for Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV) or eastern equine encephalitis virus (EEEV) when given individually or in combination (V/W/E) to mice or cynomolgus macaques. Individual replicon vaccines or the combination V/W/E replicon vaccine elicited strong neutralizing antibodies in mice to their respective alphavirus. Protection from either subcutaneous or aerosol challenge with VEEV, WEEV or EEEV was demonstrated out to 12 months after vaccination in mice. Individual replicon vaccines or the combination V/W/E replicon vaccine elicited strong neutralizing antibodies in macaques and demonstrated good protection against aerosol challenge with an epizootic VEEV- IAB virus, Trinidad Donkey. Similarly, the EEEV replicon and V/W/E combination vaccine elicited neutralizing antibodies against EEEV and protected against aerosol exposure to a North American-variety EEEV. Both the WEEV replicon and combination V/W/E vaccination; however, elicited poor neutralizing antibodies to WEEV in macaques and the protection conferred was not as strong. These results demonstrate that a combination V/W/E vaccine is possible for protection against aerosol challenge and that cross-interference between the vaccines is minimal.

    Importance Three related viruses belonging to the genus Alphavirus cause severe encephalitis in humans: Venezuelan Equine Encephalitis (VEE), Western Equine Encephalitis (WEE) and Eastern Equine Encephalitis (EEE). Normally transmitted by mosquitoes, these viruses can cause disease when inhaled so there is concern that these viruses could be used as a biological weapon. Prior reports have suggested that vaccines for these three viruses might interfere with one another. We have developed a combined vaccine for Venezuelan Equine Encephalitis, Western Equine Encephalitis and Eastern Equine Encephalitis expressing the surface proteins of all three viruses. In this report we demonstrate in both mice and macaques that this combined vaccine is safe, generates a strong immune response and protects against aerosol challenge with the viruses that cause Venezuelan Equine Encephalitis, Western Equine Encephalitis and Eastern Equine Encephalitis.

  • Polyoma Small T Antigen Interacts with YES-Associated Protein (YAP) to Regulate Cell Survival and Differentiation [PublishAheadOfPrint]

  • Murine polyomavirus small T antigen (PyST) regulates cell cycle, cell survival, apoptosis, differentiation and cooperates with middle T antigen (MT) to transform primary cells in vitro and in vivo. Like all polyoma virus T antigens, PyST functions largely via its interactions with host cell proteins. Here we show that PyST binds both Yes-associated proteins YAP1 and YAP2, integral parts of the Hippo signaling pathway, which is a subject of increasing interest in human cancer. The transcription factor TEAD, which is a known target of YAP, is also found in PyST complexes. PyST enhanced YAP association with protein phosphatase 2A (PP2A) leading to decreased YAP phosphorylation. PyST increased YAP levels by decreasing its degradation. This effect was mediated by a reduction in YAP association with bbeta;TRCP, which is known to regulate YAP turnover in a phosphorylation dependent manner. Genetic analysis has identified PyST mutants defective in YAP binding. These mutants demonstrated that YAP binding is important for PyST to block myoblast differentiation and to synergize with the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) to promote cell death in 3T3L1 preadipocytes placed under differentiation conditions. In addition to YAP binding, both of these phenotypes require PyST binding to PP2A.

    IMPORTANCE The Hippo/YAP pathway is a highly conserved cascade important for tissue development and homeostasis. Defects in this pathway are increasingly being associated with cancer. Polyoma small T antigen is a viral oncogene that cooperates with middle T antigen in transformation. On its own, small T controls cell survival and differentiation. By binding YAP, small T brings it together with protein phosphatase 2A. This work shows how this association of small T with YAP is important for its effects on cell phenotype. It also suggests that PyST can be used to characterize cellular processes that are regulated by YAP.

  • Epitope Mapping of the Hemagglutinin Molecule of A/(H1N1)pdm09 Virus by Using Monoclonal Antibody Escape Mutants [PublishAheadOfPrint]

  • We determined the antigenic structure of pandemic influenza A(H1N1)pdm09 virus hemagglutinin (HA) using 599 escape mutants that were selected using 16 anti-HA monoclonal antibodies (MAbs) against A/Narita/1/2009. The sequencing of mutant HA genes revealed 43 amino acid substitutions at 24 positions in three antigenic sites, Sa, Sb and Ca2, which were previously mapped onto A/Puerto Rico/8/34 (A/PR/8/34) HA (A.J. Caton, G.G. Brownlee, J.W. Yewdell, and W. Gerhard, Cell 31:417-427, 1982), and an undesignated site, i.e., amino acid residues 141, 142, 143, 171, 172, 174, 177 and 180 in the Sa site; residues 170, 173, 202, 206, 210, 211 and 212 in the Sb site; residues 151, 154, 156, 157, 158, 159, 200 and 238 in the Ca2 site; and residue 147 in the undesignated site (numbering begins at the first methionine). Sixteen MAbs were classified into four groups based on their cross-reactivity with the panel of escape mutants in the hemagglutination inhibition test. Among them, six MAbs targeting the Sa and Sb sites recognized both residues at positions 172 and 173. MAb n2 lost reactivity when mutations were introduced at positions 147, 159 (site Ca2), 170 (site Sb) and 172 (site Sa). We designated the site consisting of these residues as site Pa. From 2009 to 2013, no antigenic drift was detected for the A(H1N1)pdm09 viruses. However, if a novel variant carrying a mutation at a position involved in the epitopes of several MAbs, such as 172, appeared, such a virus would have the advantage of becoming a drift strain.

    IMPORTANCE The first influenza pandemic of the 21st century occurred in 2009 with the emergence of a novel virus originating from swine influenza, A(H1N1)pdm09. Although HA of A(H1N1)pdm09 has a common origin (1918 H1N1) with seasonal H1N1, the antigenic divergence of HA between the seasonal H1N1 and A(H1N1)pdm09 viruses gave rise to the influenza pandemic in 2009. To take precautions against the antigenic drift of the A(H1N1)pdm09 virus in the near future, it is important to identify its precise antigenic structure. To obtain various mutants that are not neutralized by MAbs, it is important to neutralize several plaque-cloned parent viruses rather than only a single parent virus. We characterized 599 escape mutants that were obtained by neutralizing four parent viruses of A(H1N1)pdm09 in the presence of 16 MAbs. Consequently, we were able to determine the details of the antigenic structure of HA, including a novel epitope.

  • Equine viperin restricts equine infectious anemia virus replication by inhibiting the production and/or release of viral Gag, Env and receptor by distorting the endoplasmic reticulum [PublishAheadOfPrint]

  • Viperin is an endoplasmic reticulum (ER)-associated multifunctional protein that regulates virus replication and possesses broad antiviral activity. In many cases, viperin interferes with the trafficking and budding of viral structural proteins by distorting the membrane transportation system. The lentivirus equine infectious anemia virus (EIAV) has been studied extensively. In this study, we examined the restrictive effect of equine viperin (eViperin) on EIAV replication and investigated the possible molecular basis of this restriction to obtain insights into the effect of this cellular factor on retroviruses. We demonstrated that EIAV infection of primary equine monocyte-derived macrophages (eMDMs) up-regulated the expression of eViperin. The overexpression of eViperin significantly inhibited the replication of EIAV in eMDMs, and knockdown of eViperin transcription enhanced the replication of EIAV in eMDMs by approximate 45.8%. Further experiments indicated that eViperin restricts EIAV at multiple steps of viral replication. The overexpression of eViperin inhibited EIAV Gag release. Both the aalpha;-helix domain and radical SAM domain were required for this activity. However, the essential motifs in SAM were different from those reported for the inhibition of HIV-1 Gag by human viperin. Furthermore, eViperin disrupted the synthesis of both EIAV Env and receptor, which consequently inhibited viral production and entry, respectively, and this disruption was dependent on the eViperin aalpha;-helix domain. Using immunofluorescence assays and electron microscopy, we demonstrated that the aalpha;-helix domain is responsible for the distortion of the endoplasmic reticulum (ER). Finally, EIAV did not exhibit counteracting eViperin at the protein level.

    IMPORTANCE In previously studies, viperin was indicated restricting virus replications primarily by the inhibition of virus budding. Here, we show that viperin may have multiple antiviral mechanisms including the reduction of EIAV Gag budding and Env expression, and these activities are dependent on different viperin domains. Especially, we demonstrate that overexpression of viperin inhibits EIAV entry by decreasing the virus receptor. Therefore, viperin restriction of viruses is largely determined by the dependence of virus on cellular membrane transportation system.

  • Type I interferon protects mice from fatal neurotropic infection with Langat virus by systemic and local anti-viral response [PublishAheadOfPrint]

  • Vector-borne flaviviruses such as tick-borne encephalitis virus (TBEV), West Nile virus and dengue virus cause millions of infections in humans. TBEV causes a broad range of pathological symptoms ranging from meningitis to severe encephalitis or even hemorrhagic fever with high mortality. Despite the availability of an effective vaccine, incidence of TBEV infections is increasing. Not much is known about the role of the innate immune system in the control of TBEV infections. Here, we show that the type I interferon (IFN) system is essential for protection against TBEV and Langat virus (LGTV) in mice. In the absence of a functional IFN system, mice rapidly develop neurological symptoms and succumb to LGTV and TBEV infections. Type I IFN system deficiency results in severe neuro-inflammation in LGTV-infected mice characterized by breakdown of the blood-brain barrier and infiltration of macrophages into the central nervous system (CNS). Using mice with tissue-specific IFN receptor deletions, we show that a coordinated activation of the type I IFN system in peripheral tissues as well as in the CNS is indispensable for viral control and protection against virus induced inflammation and fatal encephalitis.

    Importance The type I interferon (IFN) system is important to control viral infections, however, the interactions between tick-borne encephalitis virus (TBEV) and the type I IFN system is poorly characterized. TBEV causes severe infections in humans that are characterized by fever and debilitating encephalitis, which can progress to chronic illness or death. No treatment options are available. An improved understanding of antiviral innate immune responses is pivotal for the development of effective therapeutics. We show that type I IFN, an effector molecule of the innate immune system is responsible for the extended survival of TBEV and Langat virus (LGTV), an attenuated member of the TBE serogroup. IFN production and signaling appeared to be essential in two different phases during infection: first in the periphery, by reducing systemic LGTV replication and spreading into the central nervous system (CNS). Secondly, the local IFN response in the CNS prevents virus-induced inflammation and the development of encephalitis.

  • The matrix gene segment destabilizes the acid and thermal stability of the hemagglutinin of pandemic live attenuated influenza virus vaccines [PublishAheadOfPrint]

  • The threat of future influenza pandemics and their potential for rapid spread, morbidity, and mortality has led to the development of pandemic vaccines. We generated 7 reassortant pandemic live attenuated influenza vaccines (pLAIV) with the hemagglutinin (HA) and neuraminidase (NA) genes derived from animal influenza viruses on the backbone of the 6 internal protein gene segments of the temperature sensitive, cold-adapted (ca) A/Ann Arbor/60 (H2N2) virus (AA/60 ca) of the licensed seasonal LAIV. The pLAIVs were moderately to highly restricted in replication in seronegative adults; we sought to determine the biological basis for this restriction. Avian influenza viruses generally replicate at higher temperatures than human influenza viruses and although they shared the same backbone, the pLAIVs had a lower shut off temperature than seasonal LAIVs, suggesting the HA and NA influence the degree of temperature sensitivity. The pH of HA activation of highly pathogenic avian influenza viruses was greater than human and low pathogenicity avian influenza viruses, as reported by others. However, pLAIVs had a consistently higher pH of HA activation and reduced HA thermostability compared with the corresponding wild-type parental viruses. From studies with single gene reassortant viruses bearing one gene segment from the AA/60 ca virus in recombinant H5N1 or pH1N1 viruses, we found that the lower HA thermal stability and increased pH of HA activation were associated with the AA/60 M gene. Together, the impaired HA acid and thermal stability and temperature sensitivity likely contributed to the restricted replication of the pLAIVs we observed in seronegative adults.

    Significance: There is increasing evidence that HA stability of influenza viruses depends on the virus strain and host species and that HA stability can influence replication, virulence, and transmission of influenza A viruses in different species. We investigated the HA stability of pandemic live attenuated influenza vaccines (pLAIVs) and observed that the pLAIVs consistently had a less stable HA than the corresponding wild-type influenza viruses. The reduced HA stability and temperature sensitivity of the pLAIVs may account for their restricted replication in clinical trials.

  • Methyltransferase-defective avian metapneumovirus vaccines provide complete protection against challenge with homologous Colorado strain and heterologous Minnesota strain [PublishAheadOfPrint]

  • Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis virus, is the causative agent of turkey rhinotracheitis, and is associated with swollen head syndrome in chickens. Since its discovery in the 1970s, aMPV has been recognized as an economically important pathogen in the poultry industry worldwide. The conserved region VI (CR-VI) of the large (L) polymerase proteins of paramyxoviruses catalyzes methyltransferase (MTase) activities that typically methylate viral mRNAs at guanine N-7 (G-N-7) and ribose 2'-O positions. In this study, we generated a panel of recombinant aMPV (raMPV) Colorado strains carrying mutations in the S-adenosyl methionine (SAM) binding site in the CR-VI of L protein. These recombinant viruses were specifically defective in ribose 2'-O, but not G-N-7 methylation, and were genetically stable and highly attenuated in cell culture and viral replication in the upper and lower respiratory tracts of specific-pathogen-free (SPF) young turkeys. Importantly, turkeys vaccinated with these MTase-defective raMPVs triggered a high level of neutralizing antibody and were completely protected from challenge with homologous aMPV Colorado strain and heterologous aMPV Minnesota strain. Collectively, our results indicate that (i) aMPV lacking 2'-O methylation is highly attenuated in vitro and in vivo, and (ii) inhibition of mRNA cap MTase can serve as a novel target to rationally design live attenuated vaccines for aMPV, and perhaps other paramyxoviruses.

    Importance Paramyxoviruses include many economically and agriculturally important viruses such as avian metapneumovirus (aMPV), and Newcastle disease virus (NDV); human pathogens such as human respiratory syncytial virus, human metapneumovirus, human parainfluenza virus type 3, and measles virus; and highly lethal emerging pathogens such as Nipah virus, and Hendra virus. For many of them, there is no effective vaccine or antiviral drug. These viruses share common strategy for viral gene expression and replication. During transcription, paramyxoviruses produce capped, methylated, and polyadenylated mRNAs. Using aMPV as a model, we found that viral ribose 2'-O methyltransferase (MTase) is a novel approach to rationally attenuate the virus for vaccine purpose. Recombinant aMPV (raMPV) lacked 2'-O MTase were not only highly attenuated in turkeys, but also provided complete protection against the challenge of homologous and heterologous aMPV strains. This novel approach can be applicable to other avian and human paramyxoviruses for rationally designing live attenuated vaccines.

  • Modulation of hepatitis C virus genome replication by glycosphingolipids and FAPP2 protein [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) assembles its replication complex on cytosolic membrane vesicles often clustered in a membranous web (MW). During infection, HCV NS5A protein activates PI4KIIIaalpha; enzyme, causing massive production and redistribution of phosphatidylinositol 4-phosphate (PI4P) lipid to the replication complex. However, the role of PI4P in HCV lifecycle is not well understood. We postulated that PI4P recruits host effectors to modulate HCV genome replication or virus particles production. To test this hypothesis, we generated cell lines for doxycycline-inducible expression of shRNAs targeting PI4P effector, four-phosphate adaptor protein 2 or FAPP2. FAPP2 depletion attenuated HCV infectivity and impeded HCV RNA synthesis. Indeed, FAPP2 has two functional lipid-binding domains specific for PI4P and glycosphingolipids. While expression of the PI4P-binding mutant protein was expected to inhibit HCV replication, a marked drop in replication efficiency was unexpectedly observed with the glycosphingolipid-binding mutant protein. These data suggest that both domains are crucial for the role of FAPP2 in HCV genome replication. We also found that HCV significantly increases the level of some glycosphingolipids, whereas adding these lipids to FAPP2 depleted cells partially rescued replication, further arguing for the importance of glycosphingolipids in HCV RNA synthesis. Interestingly, FAPP2 is redistributed to the replication complex (RC) characterized by HCV NS5A, NS4B or dsRNA foci. Additionally, FAPP2 depletion disrupts the RC and alters the co-localization of HCV replicase proteins. Altogether, our study implies that HCV co-opts FAPP2 for virus genome replication via PI4P-binding and glycosphingolipids transport to the HCV RC.

    IMPORTANCE Like most viruses with a positive sense RNA genome, HCV replicates its RNA on remodeled host membranes composed of lipids hijacked from various internal membrane compartments. During infection, HCV induces massive production and retargeting of the PI4P lipid to its replication complex. However, the role of PI4P in HCV replication is not well understood. In this study, we have shown that FAPP2, a PI4P effector and glycosphingolipid-binding protein, is recruited to the HCV replication complex, required for HCV genome replication and replication complex formation. More importantly, this study demonstrates for the first time, the crucial role of glycosphingolipids in HCV lifecycle and suggests a link between PI4P and glycosphingolipids in HCV genome replication.

  • Cross-clade Ultrasensitive PCR-based Assays to Measure HIV Persistence in Large Cohort Studies [PublishAheadOfPrint]

  • A small pool of infected cells persists in HIV-infected individuals receiving antiretroviral therapy (ART). Here, we developed ultrasensitive assays to precisely measure the frequency of cells harbouring total HIV DNA, integrated HIV DNA and 2-LTR circles. These assays are performed on cell lysates, which circumvents the labour intensive step of DNA extraction and rely on the co-quantification of each HIV molecular form together with CD3 gene sequences to precisely measure cell input. Using primary isolates from HIV subtypes A, B, C, D, and CRF01_AE, we demonstrate that these assays can efficiently quantify low target copy numbers from diverse HIV subtypes. We further used these assays to measure total HIV DNA, integrated HIV DNA and 2-LTR circles in CD4+ T cells from HIV-infected subjects infected with subtype B. All samples obtained from ART-naiiuml;ve subjects were positive for the three HIV molecular forms (n=15). In ART-suppressed individuals, total HIV DNA, integrated HIV DNA and 2-LTR circles were detected in 100%, 94% and 77% of the samples. Higher levels of total HIV DNA and 2-LTR circles were detected in untreated subjects when compared to individuals on ART (p=0.0003 and p=0.0004, respectively), while the frequency of CD4+ T cells harbouring integrated HIV DNA did not differ between the two groups. These results demonstrate that these novel assays have the ability to quantify very low levels of HIV DNA from multiple HIV subtypes without the need of nucleic acid extraction, making them well-suited for monitoring viral persistence in large populations of HIV-infected individuals.

    Importance Since the discovery of viral reservoirs in HIV-infected subjects receiving suppressive ART, measuring the degree of viral persistence has been one of the greatest challenges in the field of HIV research. Here, we report the development and validation of ultrasensitive assays to measure HIV persistence in HIV-infected individuals from multiple geographical regions. These assays are relatively inexpensive, do not require DNA extraction and can be completed in a single day. Therefore, they are perfectly adapted to monitor HIV persistence in large cohorts of HIV infected individuals, and given their sensitivity, can be used to monitor the efficacy of therapeutic strategies aimed at interfering with HIV persistence after prolonged ART.

  • Cellular Visualization of Macrophage Pyroptosis and IL1{beta} Release in a Viral Hemorrhagic Infection in Zebrafish Larvae [PublishAheadOfPrint]

  • Hemorrhagic viral diseases are distributed worldwide with important pathogens, such as Dengue virus or Hantaviruses. The lack of adequate in vivo infection models has limited the research on viral pathogenesis and the current understanding of the underlying infection mechanisms. Although hemorrhages have been associated with the infection of endothelial cells, other cellular types could be the main targets for hemorrhagic viruses. Our objective was to take advantage of the use of zebrafish larvae in the study of viral hemorrhagic diseases, focusing on the interaction between viruses and host cells. Cellular processes, such as transendothelial migration of leukocytes, viral-induced pyroptosis of macrophages and Il1bbeta; release, could be observed in individual cells, providing a deeper knowledge of the immune mechanisms implicated in the disease. Furthermore, the application of these techniques to other pathogens will improve the current knowledge of host-pathogen interactions and increase the potential for the discovery of new therapeutic targets.

    IMPORTANCE Pathogenic mechanisms of hemorrhagic viruses are diverse and most of the research regarding interactions between viruses and host cells has been performed in cell lines that might not be major targets during natural infections. Thus, viral pathogenesis research has been limited because of the lack of adequate in vivo infection models. The understanding of the relative pathogenic roles of the viral agent and the host response to the infection is crucial. This will be facilitated by the establishment of in vivo infection models using organisms as zebrafish, which allows the study of the diseases in the context of complete individual. The use of this animal model with other pathogens could improve the current knowledge on host-pathogen interactions and increase the potential for the discovery of new therapeutic targets against diverse viral diseases.

  • High Eomesodermin expression among CD57+ CD8+ T cells identifies a CD8+ T cell subset associated with viral control during chronic human immunodeficiency virus infection [PublishAheadOfPrint]

  • During HIV infection, increase in CD57 expression among CD8+ T cells has been associated to immune senescence and defective immune responses. Interestingly, CD57 expressing CD8+ T cells exhibit dual profile being simultaneously highly cytotoxic (terminally differentiated effectors) and poorly proliferative (replicative senescent). Recent publications point towards a positive role of CD57 expressing CD8+ T cell subsets, presumably due to their high cytolytic activity. We further investigated the phenotype of CD57 expressing CD8+ T cells in healthy donors and during HIV infection combining CD57 expression to Eomesodermin (EOMES), a T-box transcription factor which determine, coordinately with T-bet, effector and memory CD8+ T cell differentiation. We defined in healthy donors two functionally distinct CD57 expressing CD8+ T cell subsets exhibiting different levels of EOMES expression: EOMEShiCD57+ and EOMESintCD57+ CD8+ T cells. EOMEShiCD57+ cells exhibited low cytotoxic activity but preserved proliferative capacity and IL-7 receptor expression, whereas EOMESintCD57+ exhibited obvious cytotoxic functions and more terminal differentiated phenotype. We next performed a similar analysis in different contexts of HIV infection: primary infected patients, long-term viremic patients, aviremic patients treated with antiretroviral therapy and HIV controllers, demonstrating higher percentage of CD57 expressing cells in all HIV infected patients regardless of virological status. When heterogeneity in EOMES expression among CD57 cells was taken into account, we detected significantly higher proportions of EOMEShiCD57+ cells among HIV-specific and non-specific CD8+ T cells from HIV-controllers compared to aviremic antiretroviral-treated patients and viremic patients. Importantly, such peculiar non-terminally differentiated EOMEShiCD57+ phenotypic profile was associated with viral control.

    IMPORTANCE SECTION This study demonstrates that functional heterogeneity exist among CD57 expressing CD8 T cells which include both terminally differentiated, highly cytotoxic EomesintCD57+ CD8+ T cells and less differentiated EomeshiCD57+ CD8 T cells which do not exhibit immediate cytotoxic functions but present high proliferative capacity. Interestingly, HIV controllers present a high proportion of EomeshiCD57 among CD57 expressing HIV-specific CD8 T cells compared to both long-term viremic and aviremic ART-treated patients, suggesting a beneficial role for this cell subset in viral control.

  • Hepatitis E Virus Inhibits Type I Interferon Induction by ORF1 Product [PublishAheadOfPrint]

  • Hepatitis E virus (HEV) causes both endemic and epidemic human hepatitis by fecal-oral transmission in many parts of the world. Zoonotic transmission of HEV from animals to human has been reported. Due to the lack of an efficient cell culture system, the molecular mechanisms for HEV infection remain largely unknown. In this study, we found that HEV replication in hepatoma cells inhibited poly(I:C)-induced interferon (IFN)-bbeta; expression and that HEV ORF1 product was responsible for this inhibition. Two domains, X and the papain-like cysteine protease domain (PCP), from HEV ORF1 were identified as the putative IFN antagonists. When overexpressed in HEK293T cells, the X domain (or Macro domain) inhibited poly(I:C)-induced phosphorylation of interferon regulatory factor 3 (IRF-3), which is the key transcription factor for IFN induction. The PCP domain was shown to have deubiquitinase activity for both RIG-I and TBK-1, whose ubiquitination is a key step in their activation in poly(I:C)-induced IFN induction. Furthermore, replication of a HEV replicon containing GFP (E2-GFP) in hepatoma cells led to impaired phosphorylation of IRF-3 and reduced ubiquitination of RIG-I and TBK-1, which confirmed our observations of X and PCP inhibitory effects in HEK293T cells. Taken together, our study identified the IFN antagonists within the HEV ORF1 polyprotein and expanded our understanding of the function of several of the HEV ORF1 products as well as the mechanisms of HEV pathogenesis.

    IMPORTANCE Type I interferons (IFNs) are important components in innate immunity and play a crucial role against viral infection. They also serve as key regulators to evoke adaptive immune response. Virus infection can induce the synthesis of interferons, however, viruses have evolved many strategies to antagonize the induction of interferons. There is little knowledge about how hepatitis E virus inhibits induction of host IFNs though the viral genome was sequenced more than two decades ago. This is the first report for identification of the potential IFN antagonists encoded by HEV. By screening all the domains in ORF1 polyprotein, we identified two IFN antagonists and performed further study to figure out which step in IFN induction pathway and how they antagonize host IFN induction. Our work provides valuable information about HEV virus-cell interaction and pathogenesis.

  • An Eriophyid Mite-transmitted Plant Virus Contains Eight Genomic RNA Segments with Unusual Heterogeneity in the Nucleocapsid Protein [PublishAheadOfPrint]

  • Eriophyid mite-transmitted, multipartite, negative-sense RNA plant viruses with membrane-bound spherical virions are classified in the genus Emaravirus. We report here that the eriophyid mite-transmitted Wheat mosaic virus (WMoV), an Emaravirus, contains eight genomic RNA segments, the most in a known negative-sense RNA plant virus. Remarkably, two RNA 3 consensus sequences, encoding the nucleocapsid protein, were found with 12.5% sequence divergence, while no heterogeneity was observed in the consensus sequences of additional genomic RNA segments. The RNA-dependent RNA polymerase, glycoprotein precursor, nucleocapsid and P4 proteins of WMoV exhibited limited sequence homology with the ortholog proteins of other emaraviruses, while proteins encoded by additional genomic RNA segments displayed no significant homology with proteins reported in GenBank, suggesting that the genus Emaravirus evolved further with a divergent octapartite genome. Phylogenetic analyses revealed that WMoV formed an evolutionary-link between members of the Emaravirus and Bunyaviridae. Furthermore, genomic-length virus- and virus complementary (vc)-sense strands of all WMoV genomic RNAs accumulated asymmetrically in infected wheat, with 10- to 20-fold more virus-sense genomic RNAs compared to vc-sense RNAs. These data further confirm the octapartite negative-sense polarity nature of the WMoV genome. In WMoV-infected wheat, subgenomic-length mRNAs of vc-sense were detected for genomic RNAs 3, 4, 7, and 8 but not for other RNA species, suggesting that the ORFs present in the complementary sense of genomic RNAs are expressed through subgenomic- or near genomic-length vc-sense mRNAs.

    IMPORTANCE Wheat mosaic virus (WMoV), an Emaravirus, is the causal agent of High Plains disease of wheat and maize. In this study, we demonstrated that the genome of WMoV comprises eight negative-sense RNA segments with an unusual sequence polymorphism in an RNA encoding the nucleocapsid protein but not in the additional genomic RNA segments. WMoV proteins displayed weak or no homology with reported emaraviruses, suggesting that the genus Emaravirus further evolved with divergent octapartite genome. The current study also examined the profile of WMoV RNA accumulation in wheat and provided evidence for the synthesis of subgenomic-length mRNAs of virus complementary-sense. This is the first report to demonstrate that emaraviruses produce subgenomic-length mRNAs that are most likely utilized for genome expression. Importantly, this study facilitates the examination of genes functions and virus diversity and the development of effective diagnostic methods and management strategies for an economically important but poorly understood virus.

  • Inactivation of the human papillomavirus E6 or E7 gene in cervical carcinoma cells using a bacterial CRISPR/Cas RNA-guided endonuclease [PublishAheadOfPrint]

  • High-risk human papillomaviruses (HPVs), including HPV-16 and HPV-18, are the causative agents of cervical carcinomas as well as being linked to several other tumors of the anogenital and oropharyngeal regions. The majority of HPV-induced tumors contain integrated copies of the normally episomal HPV genome that invariably retain intact forms of the two HPV oncogenes E6 and E7. E6 induces degradation of the cellular tumor suppressor p53, while E7 destabilizes the retinoblastoma (Rb) protein. Previous work has shown that loss of E6 function in cervical cancer cells induces p53 expression as well as downstream effectors that induce apoptosis and cell cycle arrest. Similarly, loss of E7 allows increased Rb expression, leading to cell cycle arrest and senescence.

    Here, we demonstrate that expression of a bacterial Cas9 RNA-guided endonuclease, together with single guide RNAs (sgRNAs) specific for E6 or E7, is able to induce cleavage of the HPV genome, resulting in the introduction of inactivating deletion and insertion mutations into the E6 or E7 gene. This results in induction of p53 or Rb, leading to cell cycle arrest and eventual cell death. Both HPV-16 and HPV-18-transformed cells were found to be responsive to targeted HPV genome-specific DNA cleavage. These data provide proof of principle for the idea that vector-delivered Cas9/sgRNA combinations could represent effective treatment modalities for HPV-induced cancers.

    IMPORTANCE Human papillomaviruses (HPVs) are the causative agent of almost all cervical carcinomas and many other tumors, including many head and neck cancers. In these cancer cells, the HPV DNA genome is integrated into the cellular genome where it expresses high levels of two viral oncogenes, called E6 and E7, that are required for cancer cell growth and viability. Here, we demonstrate that the recently described bacterial CRISPR/Cas RNA-guided endonuclease can be reprogrammed to target and destroy the E6 or E7 gene in cervical carcinoma cells transformed by HPV, resulting in cell cycle arrest leading to cancer cell death. We propose that viral vectors designed to deliver E6- and/or E7-specific CRISPR/Cas to tumor cells could represent a novel and highly effective tool to treat and eliminate HPV-induced cancers.

  • EBV-encoded LMP2A Promotes EMT in Nasopharyngeal Carcinoma via MTA1 and mTOR Signaling Induction [PublishAheadOfPrint]

  • Epstein-Barr Virus-encoded latent membrane protein 2A (LMP2A) promotes the epithelial-mesenchymal transition (EMT) of nasopharyngeal carcinoma (NPC), thereby increasing tumor invasion. Recently, the dysregulation of metastatic tumor antigen 1 (MTA1) was found to enhance tumor metastasis in a variety of cancers. A molecular connection between these two proteins has been proposed but not firmly established. In this study, we reported the overexpression of MTA1 in 29/60 (48.3%) NPC patients and the overexpression of MTA1 significantly correlated with tumor metastasis. The overexpression of MTA1 promoted EMT via the Wnt1 pathway and bbeta;-catenin activation. We demonstrated that LMP2A reinforces the expression of MTA1 via the mechanistic target of rapamycin (mTOR) pathway to promote EMT in NPC. Furthermore, by knocking-down of 4EBP1 in combination with the new mTOR inhibitor INK-128 treatment, we discovered that LMP2A expression activates the 4EBP1-eIF4E axis and increases the expression of MTA1 at the translational level, partial independent of c-myc. These findings provided novel insights into the correlation between the LMP2A and MTA1 proteins and reveal a novel function of the 4EBP1-eIF4E axis in EMT of nasopharyngeal carcinoma.

    Importance statement: Prevention of the recurrence and metastasis of NPC is critical to achieving a successful NPC treatment. As we all know, epithelial-mesenchymal transition (EMT) acts a vital role in metastasis of malignancies. LMP2A, oncoprotein of EBV, a well-known NPC activator, induces EMT and has been proved to exert a promoting-effect in tumor metastasis. Our study demonstrated that LMP2A could induce EMT by activating MTA1 at translational level via activating the mTOR signaling and the 4EBP1-eIF4E axis. Taken together, our findings bridged the gap between the NPC-specific cell surface molecule and the final phenotype of the NPC cells. Additionally, our findings indicate that LMP2A and mTOR may serve as potential targets for NPC therapy in the future.

  • Induction of CD8 T Cell Heterologous Protection by a Single Dose of Single-cycle Infectious Influenza Virus [PublishAheadOfPrint]

  • The effector functions of specific CD8 T cells are crucial in mediating influenza heterologous protection. However, new approaches for influenza vaccines that can trigger effective CD8 T cell responses have not been extensively explored. Here, we report the generation of single cycle infectious influenza virus that lacks a functional HA gene on an X31 genetic background and demonstrate its potential for triggering protective CD8 T cell immunity against heterologous influenza challenge. In vitro, X31-sciIV can infect MDCK cells, but infectious virions are not produced unless HA is trans-complimented. In vivo, intranasal immunization with X31-sciIV does not cause any clinical symptoms in mice but generate influenza specific CD8 T cells in lymphoid (MLN and spleen) and non-lymphoid tissues including lung and BAL as measured by H2-Db NP366 and PA224 tetramer staining. In addition, a significant proportion of X31-sciIV induce, antigen specific respiratory CD8 T cells expressed VLA-1, a marker that is associated with heterologous influenza protection. Further, these influenza specific CD8 T cells produce antiviral cytokines when stimulated with NP366 and PA224 peptides, indicating CD8 T cells triggered by X31-sciIV are functional. When challenged with a lethal dose of heterologous PR8 virus, X31-sciIV primed mice were fully protected from death. However, when CD8 T cells were depleted after priming or before priming, mice could not effectively control virus replication or survive the lethal challenge, indicating X31-sciIV induced memory CD8 T cells mediate the heterologous protection. Thus, our results demonstrate the potential for sciIV as a CD8 T cell inducing vaccine.

    IMPORTANCE One of the challenges for influenza prevention is its existence of multiple subtypes and variants and the fact that new strains could be emerged yearly. Numerous studies have indicated the effector functions of specific CD8 T cells are crucial in mediating influenza heterologous protection. However, influenza vaccines that can trigger effective CD8 T cell responses for heterologous protection have not been developed. Here, we report the generation of an X31 (H3N2) virus derived single cycle infectious influenza virus, X31-sciIV. One dose immunization with X31-sciIV is capable of inducing functional influenza specific CD8 T cells that can be recruited into respiratory tissues and provide protection for lethal heterologous challenge. Without these cells, protection against lethal challenge was essentially lost. Our data indicate influenza vaccine that primarily relies on CD8 T cells for protection could be developed.

  • Co-infection with Streptococcus pneumoniae modulates the B cell response to influenza virus [PublishAheadOfPrint]

  • Pathogen-specific antibodies (Abs) protect against respiratory infection with influenza A virus (IAV) and Streptococcus pneumoniae (Sp) and are the basis of effective vaccines. Sequential or overlapping co-infections with both pathogens are common, yet the impact of co-infection on the generation and maintenance of Ab responses is largely unknown. We report here that the B cell response to IAV is altered in IAV-Sp co-infected mice and that this response differs depending on the order of pathogen exposure. In mice exposed to Sp prior to IAV, the initial virus-specific germinal center (GC) B cell response is significantly enhanced in the lung-draining mediastinal lymph node and spleen, and there is an increase in CD4+ T follicular helper (TFH) cell numbers. In contrast, secondary Sp infection exaggerates early anti-viral antibody secreting cell formation, and at later times GCs, TFH cells and anti-viral serum IgG are elevated. Mice exposed to Sp prior to IAV do not maintain the initially robust GC response in secondary lymphoid organs, and exhibit reduced anti-viral serum IgG with diminished virus-neutralization activity a month after infection. Our data suggest that the history of pathogen exposures can critically affect the generation of protective anti-viral Abs and may partially explain the differential susceptibility and disease outcomes to IAV infection in humans.

    IMPORTANCE Respiratory tract co-infections, specifically those involving influenza A viruses and Streptococcus pneumoniae, remain a top global health burden. We sought to determine how Sp co-infection modulates the B cell immune response to influenza virus since antibodies are key mediators of protection.

  • Effect of formaldehyde inactivation on poliovirus [PublishAheadOfPrint]

  • Inactivated polio vaccines, which have been used in many countries for more than 50 years, are produced by treatment of live poliovirus (PV) with formaldehyde. However, the molecular mechanisms underlying virus inactivation are not well understood. Infection by PV is initiated by virus binding to specific cell receptors which results in viral particles undergoing sequential conformational changes that generate altered structural forms (135S and 80S particles) and leads to virus cell entry. We have analysed the ability of inactivated PV to bind to the human poliovirus receptor (hPVR) using various techniques such as ultracentrifugation, fluorescent-activated cell sorting (FACS) flow cytometry and real-time RT-PCR. The results showed that although retaining the ability to bind to hPVR, inactivated PV bound less efficiently in comparison to live PV. We also found that inactivated PV showed resistance to structural conversion in vitro as judged by measuring changes in antigenicity, ability to bind to hPVR and viral RNA release at high temperature. Furthermore, viral RNA from inactivated PV was shown to be modified, as cDNA yields obtained by RT-PCR amplification were severely reduced and no infectious virus was recovered after RNA transfection into susceptible cells.

    IMPORTANCE This study represents a novel insight into the molecular mechanisms responsible for poliovirus inactivation. We have shown that inactivation with formaldehyde has an effect on early steps of viral replication as it reduces the ability of PV to bind to hPVR, decreases the sensitivity of PV to convert to 135S particles and abolishes the infectivity of its viral RNA. These changes are likely responsible for the loss of infectivity shown by PV following inactivation. Techniques used in this study represent new approaches for the characterisation of inactivated PV products and could be useful to help developing improved methods for the production and quality control testing of inactivated polio vaccines. Measuring the antigenicity, capsid stabilty and RNA integrity of inactivated PV samples could help establishing the optimal balance between loss of infectivity and preservation of virus antigenicity during inactivation.

  • A Chimeric Virus-Mouse Model System for Evaluating the Function and Inhibition of Papain-like Proteases of Emerging Coronaviruses [PublishAheadOfPrint]

  • To combat emerging coronaviruses, developing safe and efficient platforms to evaluate viral protease activities and the efficacy of protease inhibitors is a high priority. Here we exploit a biosafety level 2 (BSL-2) chimeric Sindbis virus system to evaluate protease activities and the efficacy of inhibitors directed against the papain-like protease (PLpro) of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), a biosafety level 3 (BSL-3) pathogen. We engineered Sindbis virus to co-express PLpro and a substrate, murine interferon stimulated gene 15 (ISG15), and found that PLpro mediates removal of ISG15 (deISGylation) from cellular proteins. Mutation of the catalytic cysteine residue of PLpro or addition of a PLpro inhibitor blocked deISGylation in virus-infected cells. Thus, deISGylation is a marker of PLpro activity. Infection of Interferon-alpha/beta receptor knockout (IFNARnndash;/nndash;) mice with these chimeric viruses revealed that PLpro deISGylation activity removed the ISG15-mediated protection during viral infection. Importantly, administration of a PLpro inhibitor protected these mice from lethal infection demonstrating the efficacy of a coronavirus protease inhibitor in a mouse model. However, this PLpro inhibitor was not sufficient to protect mice from lethal infection with SARS-CoV MA15, suggesting that further optimization of the delivery and stability of PLpro inhibitors is needed. We extended the chimeric virus platform to evaluate papain-like protease/deISGylating activity of Middle East Respiratory Syndrome Coronavirus (MERS-CoV), to provide a small animal model to evaluate PLpro protease inhibitors to this recently emerged pathogen. This platform has the potential to be universally adaptable to other viral and cellular enzymes that have deISGylating activity.

    Importance Evaluating viral protease inhibitors in a small animal model is a critical step in the pathway toward antiviral drug development. We modified a biosafety level 2 chimeric virus system to facilitate evaluation of inhibitors directed against highly pathogenic coronaviruses. We used this system to demonstrate the in vivo efficacy of an inhibitor of the papain-like protease of Severe Acute Respiratory Syndrome Coronavirus. Furthermore, we demonstrate that the chimeric virus system can be adapted to study the proteases of emerging human pathogens such as Middle East Respiratory Syndrome Coronavirus. This system provides an important tool to rapidly assess the efficacy of protease inhibitors targeting existing and emerging human pathogens as well as other enzymes capable of removing ISG15 from cellular proteins.

  • Trans-protease activity and structural insights into the active form of the alphavirus capsid protease [PublishAheadOfPrint]

  • The alphavirus capsid protein (CP) is a serine protease that possesses cis-proteolytic activity essential for its release from the nascent structural polyprotein. The released CP further participates in viral genome encapsidation, nucleocapsid core formation followed by its attachment to glycoproteins and virus budding. Thus, protease activity of the alphavirus capsid is a potential anti-alphaviral target to arrest capsid release, maturation and structural polyprotein processing. However, the discovery of capsid protease inhibitors has been hampered due to the lack of a suitable screening assay and crystal structure in its active form. Here we report the development of a trans-proteolytic activity assay for Aura virus capsid protease (AVCP) based on fluorescence resonance energy transfer (FRET) for screening protease inhibitors. Kinetic parameters using fluorogenic peptide substrates were estimated, and a KM value was found to be 2.63 pplusmn; 0.62 mmu;M, and a Kcat/KM value was 4.97 x 104 Mnndash;1 minnndash;1. Also, the crystal structure of the trans-active form of AVCP has been determined to 1.81 AAring; resolution. Structural comparisons of the active form with the crystal structures of available substrate-bound mutant and inactive blocked forms of the capsid protease identifies conformational changes in the active site, the oxyanion hole and the substrate specificity pocket residues, which could be critical for rational drug design.

    IMPORTANCE The alphavirus capsid protease is an attractive antiviral therapeutic target. In this study, we have described the formerly unappreciated trans-proteolytic activity of the enzyme and for the first time have developed a FRET based protease assay for screening capsid protease inhibitors. Our structural studies unveil the structural features of the trans-active protease which has been previously proposed to exist in the natively unfolded form (1). The different enzymatic forms have been structurally compared to reveal conformational variations in the active and substrate binding site. The flexible active site residue Ser218, the disordered C-terminal residues after His261, and the presence of a water molecule in the oxyanion hole of AVCP2 reveal the effect of the C-terminal Trp267 deletion on enzyme structure. New structural data reported in this study along with the fluorogenic assay will be useful in substrate specificity characterization, high-throughput protease inhibitor screening and structure-based development of antiviral drugs.

  • Influenza A virus Acquires Enhanced Pathogenicity and Transmissibility After Serial Passages in Swine [PublishAheadOfPrint]

  • Genetic and phylogenetic analyses suggest that the pandemic H1N1/2009 virus was derived from well-established swine influenza lineages; however, there is no convincing evidence that the pandemic virus was generated from a direct precursor in pigs. Furthermore, the evolutionary dynamics of influenza virus in pigs have not been well documented. Here, we subjected a recombinant virus (rH1N1) with the same constellation makeup as the pandemic H1N1/2009 virus to nine serial passages in pigs. Severity of infection sequentially increased with each passage. Deep sequencing of viral quasi-species from the ninth passage found five consensus amino acid mutations: PB1 A469T, PA 1129T, NA N329D, NS1 N205K and NEP T48N. Mutations in the HA protein, however, differed greatly between the upper and lower respiratory tracts. Three representative viral clones with the five consensus mutations were selected for functional evaluation. Relative to the parental virus, the three viral clones showed enhanced replication and polymerase activity in vitro, and enhanced replication, pathogenicity and transmissibility in pigs, guinea pigs and ferrets in vivo. Specifically, two mutants of rH1N1 (PB1 A469T, and combined NS1 N205K and NEP T48N) were identified as determinants of transmissibility in guinea pigs. Crucially, one mutant viral clone with the five consensus mutations, which also carried D187E, K211E and S289N mutations in its hemagglutinin (HA), was additionally able to infect ferrets by airborne transmission as effectively the pandemic virus. Our findings demonstrate that influenza virus can acquire viral characteristics that are similar to those of the pandemic virus after limited serial passages in pigs.

    Importance We demonstrated here an engineered reassortant swine influenza virus, with the same gene constellation pattern as the pandemic H1N1/2009 virus, subjected to only nine serial passages in pigs acquired greatly enhanced virulence and transmissibility. In particular, one representative pathogenic passaged virus clone, which carried three mutations in the HA gene and five consensus mutations in PB1, PA, NA, NS1, and NEP genes, was additionally able to confer respiratory droplet transmission as effectively as the pandemic H1N1/2009 virus. Our findings suggest that pigs can readily induce adaptive mutational changes to a precursor pandemic-like virus to transform it into a highly virulent and infectious form akin to that of the pandemic H1N1/2009 virus which underlines the potential direct role of pigs in promoting influenza A virus pathogenicity and transmissibility.

  • Characterization of the Polyproline Region of the Hepatitis E Virus in Immunocompromised Patients [PublishAheadOfPrint]

  • Little is known about virus adaptation in immunocompromised patients with chronic genotype 3 hepatitis E virus (HEV3) infections. Virus-host recombinant strains have been isolated recently from chronically infected patients. The nature and incidence of such recombinant events occuring during infections of solid-organ transplant (SOT) recipients are essentially unknown.

    The polyproline region (PPR) of strains isolated from SOT patients were sequenced during the acute infection phase (n=59) and during follow-up of patients whose infections became chronic (n= 27). These 27 HEV strains included 3 (11%) that showed recombinant events 12, 34, 48 or 88 months after infection. In one strain parts of the PPR and the RNA-dependent RNA polymerase were concomitantly inserted. In the second a fragment of human tyrosine aminotransferase gene (TAT) was inserted first, followed by a fragment of PPR. A fragment of the human inter-aalpha;-trypsin inhibitor gene (ITI) was inserted in the third. All the inserted sequences were rich in aliphatic and basic amino acids. In vitro growth experiments suggest that the ITI insert promoted more vigorous virus growth. In silico studies showed that the inserted sequences could provide potential acetylation, ubiquitination and phosphorylation sites.

    We find that recombinant events occur in the HEV PPR in approximately 11% of the strains isolated from chronically infected transplant patients followed-up in Toulouse University Hospital. These inserted fragments come from the HEV genome or a human gene and could enhance virus replication.

    IMPORTANCE Hepatitis E virus (HEV) can cause chronic infections in immunocompromised patients, including solid organ transplant (SOT) recipients. Two strains that had undergone recombination with human ribosomal genes were described recently. The strains with inserted sequences replicated better in vitro. Little is known about the frequency of such recombinant events or how such an insertion enhances replication. We therefore investigated 59 SOT patients infected with HEV and found 3 strains with 4 recombinant events in 27 of these patients whose infection became chronic. The 4 inserted sequences were of different origins (human gene or HEV genome) but all were enriched in aliphatic and basic amino acid and provided potential regulation sites.

    Our data indicate that recombinant events occur in approximately 11% of strains isolated from chronically infected patients. The structures of the inserted sequences provide new clues as to how the inserted sequences could foster virus replication.

  • The attachment protein G of an African bat henipavirus is differentially restricted in chiropteran and non-chiropteran cells [PublishAheadOfPrint]

  • Henipaviruses are associated with pteropodid reservoir hosts. The glycoproteins G and F of an African henipavirus (strain M74) have been reported to induce syncytium formation in kidney cells derived from a Hypsignathus monstrosus bat (HypNi/1.1), but not in the non-chiropteran cells BHK-21 and Vero76. Here we show that syncytia are also induced in two other pteropodid cell lines from Hypsignathus monstrosus and Eidolon helvum bats upon co-expression of the M74 glycoproteins. The G protein was transported to the surface of transfected chiropteran cells, whereas surface expression in the non-chiropteran cells was detectable only in a fraction of cells. By contrast, the G protein of Nipah virus is transported efficiently to the surface of both chiropteran and non-chiropteran cells. Even in chiropteran cells, M74-G was predominantly expressed in the ER as indicated by colocalization with marker proteins. This result is consistent with the finding that all N-glycans of the M74-G proteins are of the mannose-rich type as indicated by sensitivity to endo H treatment. These data indicate that the surface transport of M74-G is impaired in available cell culture systems with larger amounts of viral glycoprotein present on chiropteran cells. The restricted surface expression of M74-G explains the reduced fusion activity of the glycoproteins of the African henipavirus. Our results suggest strategies for the isolation of infectious viruses which is necessary to assess the risk of zoonotic virus transmission

    Importance Henipaviruses are highly pathogenic zoonotic viruses associated with pteropodid bat hosts. Whether the recently described African bat henipaviruses have a similarly high zoonotic potential as their Asian and Australian relatives is unknown. We show that surface expression of the attachment protein G of an African henipavirus, M74, is restricted in comparison to the G protein of the highly pathogenic Nipah virus. Transport to the cell surface is more restricted in non-chiropteran cells than it is in chiropteran cells explaining the differential fusion activity of the M74 surface proteins in these cells. Our results imply that surface expression of viral glycoproteins may serve as a major marker to assess the zoonotic risk of emerging henipaviruses.

  • Coronaviruses Resistant to a 3C-like Protease Inhibitor Are Attenuated for Replication and Pathogenesis Revealing a Low Genetic Barrier but High Fitness Cost of Resistance [PublishAheadOfPrint]

  • Viral protease inhibitors are remarkably effective at blocking the replication of viruses such as human immunodeficiency virus and hepatitis C virus, but inevitably lead to the selection of inhibitor resistant mutants, which may contribute to ongoing disease. Protease inhibitors blocking the replication of coronavirus (CoV), including the causative agents of Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS), provide a promising foundation for the development of anti-coronaviral therapeutics. However, the selection and consequences of inhibitor resistant CoVs are unknown. Here, we exploit the model coronavirus, mouse hepatitis virus (MHV) to investigate the genotype and phenotype of MHV quasispecies selected for resistance to a broad-spectrum CoV 3C-like protease (3CLpro) inhibitor. Clonal sequencing identified single or double mutations within the 3CLpro coding sequence of inhibitor resistant virus. Using reverse genetics to generate isogenic viruses with mutant 3CLpros, we found that viruses encoding double mutant 3CLpros are fully resistant to the inhibitor and exhibit a significant delay in proteolytic processing of the viral replicase polyprotein. The inhibitor resistant viruses also exhibited postponed and reduced production of infectious virus particles. Biochemical analysis verified double mutant 3CLpro enzyme as impaired for protease activity and exhibiting reduced sensitivity to the inhibitor, and revealed a delayed kinetics of inhibitor hydrolysis and activity restoration. Furthermore, the inhibitor resistant virus was shown to be highly attenuated in mice. Our study provides the first insight into the pathogenicity and mechanism of 3CLpro inhibitor resistant CoV mutants, revealing a low genetic barrier but high fitness cost of resistance.

    Importance RNA viruses are infamous for their ability to evolve in response to selective pressure, such as the presence of antiviral drugs. For coronaviruses such as the causative agent of Middle East Respiratory Syndrome (MERS), protease inhibitors have been developed and shown to block virus replication, but the consequences of selection of inhibitor resistant mutants has not been studied. Here, we report the low genetic barrier and relatively high deleterious consequences of CoV resistance to a 3CLpro protease inhibitor in a coronavirus model system, mouse hepatitis virus (MHV). We found that although mutations that confer resistance arise quickly, the resistant viruses replicate slowly and do not cause lethal disease in mice. Overall, our study provides the first analysis of the low barrier but high cost of resistance to a CoV 3CLpro inhibitor, which will facilitate the further development of protease inhibitors as anti-coronavirus therapeutics.

  • Mutagenesis of the catalytic and cleavage site residues of the hypovirus papain-like proteases p29 and p48 reveals alternative processing and contributions to optimal viral RNA accumulation. [PublishAheadOfPrint]

  • The positive-stranded RNA genome of the prototypic virulence-attenuating hypovirus CHV-1/EP713 contains two open reading frames (ORF), each encoding an autocatalytic papain-like leader protease. Protease p29, derived from the N-terminal portion of ORF A, functions as a suppressor of RNA silencing while protease p48, derived from the N-terminal portion of ORF B, is required for viral RNA replication. The catalytic and cleavage site residues required for autoproteolytic processing have been functionally mapped in vitro for both proteases, but not confirmed in the infected fungal host. Here we report the mutagenesis of the CHV-1/EP713 infectious cDNA clone to define the requirements for p29 and p48 cleavage and the role of autoprotelysis in the context of hypovirus replication. Mutation of the catalytic cysteine and histidine residues for either p29 or p48 was tolerated, but reduced viral RNA accumulation to approximately 20-50% of the wild-type level. Mutation of the p29 catalytic residues caused an accumulation of unprocessed ORF A product p69. Surprisingly, the release of p48 from the ORF B-encoded polyprotein was not prevented by mutation of the p48 catalytic and cleavage site residues and was independent of p29. The results show that, while dispensable for hypovirus replication, the autocatalytic processing of the leader proteases p29 and p48 contributes to optimal virus RNA accumulation. The role of the predicted catalytic residues in autoproteolytic processing of p29 was confirmed in the infected host, while p48 was found to also undergo alternative processing independent of the encoded papain-like protease activities.

    IMPORTANCE Hypoviruses are positive strand RNA mycoviruses that attenuate virulence of their pathogenic fungal hosts. The prototypic hypovirus CHV-1/EP713, that infects the chestnut bight fungus Cryphonetria parasitica, encodes two papain-like autocatalytic leader proteases, p29 and p48, that also play important functions in suppressing the RNA silencing antiviral defense response and in viral RNA replication, respectively. The mutational analysis of the CHV-1/EP713 infectious cDNA clone, reported here, define the requirements for p29 and p48 cleavage and the functional importance of autoproteolysis in the context of hypovirus replication and exposed an alternative p48 processing pathway independent of the encoded papain-like protease activities. These findings provide additional insights into hypovirus gene expression, replication and evolution and inform ongoing efforts to engineer hypoviruses for interrogating and modulating fungal virulence.

  • The 3' UTR of Pea Enation Mosaic Virus Contains Two T-Shaped, Ribosome-Binding Cap-Independent Translation Enhancers [PublishAheadOfPrint]

  • Many plant viruses without 5rrsquo; caps or 3rrsquo; polyA tails contain 3rrsquo; proximal, cap-independent translation enhancers (3rrsquo; CITEs) that bind to ribosomal subunits or translation factors thought to assist in ribosome recruitment. Most 3rrsquo; CITEs participate in a long-distance kissing-loop interaction with a 5rrsquo; proximal hairpin to deliver ribosomal subunits to the 5rrsquo; end for translation initiation. Pea enation mosaic virus (PEMV) contains two adjacent 3rrsquo; CITEs in the center of its 703-nt 3rrsquo; UTR, the ribosome-binding, kissing-loop T-shaped structure (kl-TSS) and eIF4E-binding Panicum mosaic virus-like translation enhance (PTE). We now report that PEMV contains a third, independent 3rrsquo; CITE located near the 3rrsquo; terminus. This 3rrsquo; CITE is composed of three hairpins and two pseudoknots, similar to the TSS 3rrsquo; CITE of Carmovirus Turnip crinkle virus (TCV). As with the TCV TSS, the PEMV 3rrsquo; TSS is predicted to fold into a T-shaped structure that binds to 80S ribosomes and 60S ribosomal subunits. A small hairpin (kl-H) upstream of the 3rrsquo; TSS contains an apical loop capable of forming a kissing-loop interaction with a 5rrsquo; proximal hairpin and is critical for accumulation of full-length PEMV in protoplasts. Although the kl-H and 3rrsquo; TSS are dispensable for translation of a reporter construct containing the complete PEMV 3rrsquo; UTR in vitro, deleting the normally required kl-TSS and PTE 3rrsquo; CITEs and placing the kl-H and 3rrsquo; TSS proximal to the reporter termination codon restores translation to near WT levels. This suggests that PEMV requires three 3rrsquo; CITEs for proper translation and that additional translation enhancers may have been missed if reporter constructs were used in 3rrsquo; CITE identification.

    Importance The rapid life-cycle of viruses requires efficient translation of viral-encoded proteins. Many plant RNA viruses contain 3rrsquo; cap-independent translation enhancers (3rrsquo; CITEs) to effectively compete with ongoing host translation. Since only single 3rrsquo; CITEs have been identified for the vast majority of individual virus, it is widely accepted that this is sufficient for a virus's translational needs. Pea enation mosaic virus possesses a ribosome-binding 3rrsquo; CITE that can connect to the 5rrsquo; end through an RNA:RNA interaction and an adjacent eIF4E-binding 3rrsquo; CITE. We report the identification of a third 3rrsquo; CITE that binds weakly to ribosomes and requires an upstream hairpin to form a bridge between the 3rrsquo; and 5rrsquo; ends. Although both ribosome-binding 3rrsquo; CITEs are critical for virus accumulation in vivo, only the CITE closest to the termination codon of a reporter ORF is active, suggesting that artificial constructs used for 3rrsquo; CITE identification may underestimate the number of CITEs that participate in translation.

  • Specific ARGONAUTES bind selectively small RNAs derived from potato spindle tuber viroid and attenuate viroid accumulation in vivo [PublishAheadOfPrint]

  • The identification of viroid-derived small RNAs (vd-sRNAs) of 21-24 nucleotides (nt) in plants infected by viroids (infectious non-protein-coding RNAs of just 250-400 nt), supports their targeting by dicer-like enzymes, the first host RNA silencing barrier. However, whether viroids, like RNA viruses, are also targeted by the RNA-induced silencing complex (RISC) remains controversial. At the RISC core is one Argonaute (AGO) protein that, guided by endogenous or viral sRNAs, targets complementary RNAs. To examine whether AGO proteins also load vd-sRNAs, leaves of Nicotiana benthamiana infected by potato spindle tuber viroid (PSTVd) were agroinfiltrated with plasmids expressing epitope-tagged versions of AGO1, AGO2, AGO3, AGO4, AGO5, AGO6, AGO7, AGO9 and AGO10 from Arabidopsis thaliana. Immunoprecipitation analyses of the agroinfiltrated halos revealed that all AGOs, except AGO6, AGO7 and AGO10, associated with vd-sRNAs: AGO1, AGO2 and AGO3 preferentially with those of 21 and 22 nt, while AGO4, AGO5 and AGO9 additionally bound those of 24 nt. Deep sequencing analyses showed that sorting of vd-sRNAs into AGO1, AGO2, AGO4 and AGO5 depended essentially on their 5'-terminal nucleotide, with the profiles of the corresponding AGO-loaded vd-sRNAs adopting specific hot spot distributions along the viroid genome. Furthermore, agroexpression of AGO1, AGO2, AGO4 and AGO5 on PSTVd-infected tissue attenuated the level of the genomic RNAs, suggesting that they, or their precursors, are RISC-targeted. In contrast to RNA viruses, PSTVd infection of N. benthamiana did not affect miR168-mediated regulation of the endogenous AGO1, which loaded vd-sRNAs with specificity similar to its A. thaliana counterpart.

    IMPORTANCE To contain invaders, particularly RNA viruses, plants have evolved an RNA silencing mechanism relying on the generation by Dicer-like (DCL) enzymes of virus-derived small RNAs of 21-24 nucleotides (nt) that load and guide Argonaute (AGO) proteins to target and repress viral RNA. Viroids, despite their minimal genomes (non-protein-coding RNAs of only 250-400 nt), infect and incite disease in plants. The accumulation in these plants of 21-24 nt viroid-derived small RNAs (vd-sRNAs) supports that DCLs also target viroids, but does not clarify whether vd-sRNAs activate one or more AGOs. Here, we show that in leaves of Nicotiana benthamiana infected by potato spindle tuber viroid, the endogenous AGO1 and distinct AGOs from Arabidopsis thaliana that were overexpressed, associated with vd-sRNAs displaying the same properties (5'-terminal nucleotide and size) previously established for endogenous and viral small RNAs. Overexpression of AGO1, AGO2, AGO4 and AGO5 attenuated viroid accumulation, supporting their role in antiviroid defense.

  • The Genomes, Proteomes and Structure of Three Novel Phages that Infect the Bacillus Cereus Group and Carry Putative Virulence Factors [PublishAheadOfPrint]

  • This report includes the study of three novel bacteriophages, JL, Shanette and Basilisk, which infect the pathogen B. cereus and carry genes that may contribute to its pathogenesis. We analyzed host range and superinfection ability, mapped their genomes, and characterized phage structure by mass spectrometry and transmission electron microscopy (TEM). The JL and Shanette genomes were 96% similar and contained 217 ORFs and 220 ORFs respectively, while Basilisk has an unrelated genome containing 138 ORFs. Mass spectrometry revealed 23 phage particle proteins for JL and 15 for Basilisk, while only 11 and four, respectively, were predicted to be present by sequence analysis. Structural protein homology to well-characterized phages predicted JL and Shanette as Myoviridiae, which was confirmed by TEM. The third phage, Basilisk, was similar to only uncharacterized phages and is an unrelated siphovirus. Cryogenic electron microscopy of this novel phage revealed a T=9 icosahedral capsid structure with the major capsid protein (MCP) likely having the same fold as bacteriophage HK97 MCP despite the lack of sequence similarity. Several putative virulence factors were encoded by these phage genomes including genes TerC and TerD for tellurium resistance. Host range analysis of all three phages supports genetic transfer of such factors within the B. cereus group, including B. cereus, B. anthracis and B. thuringiensis. The results of our study provide a basis for understanding these and other related phages as well as their contributions to the evolution and pathogenicity of B. cereus bacteria.

    IMPORTANCE The B. cereus group of bacteria contains several human and plant pathogens, including B. cereus, B. anthracis, and B. thuringiensis. Phage are intimately linked to the evolution of their bacterial hosts and often provide virulence factors, making the study of B. cereus phages important to understanding the evolution of pathogenic strains. Herein, we provide the detailed study of three novel B. cereus phages, two highly related myoviruses (JL and Shanette) and an unrelated siphovirus (Basilisk). The detailed characterization of host range and superinfection, together with genomic, proteomic, and structural analysis, reveal several putative virulence factors as well as the ability of these phages to infect different pathogenic species.

  • The Epstein-Barr virus (EBV) glycoprotein B (gB) cytoplasmic C-terminal tail domain (CTD) regulates the energy requirement for EBV-induced membrane fusion [PublishAheadOfPrint]

  • The entry of enveloped viruses into host cells is preceded by membrane fusion, which in Epstein-Barr virus (EBV) is thought to be mediated by the refolding of glycoptrotein B (gB) from a pre-fusion to a post-fusion state. In our current studies, we characterized a gB C-terminal tail domain (CTD) truncation mutant at amino acid 843 (gB843). This truncation mutant is hyperfusogenic as monitored by syncytium formation and in a quantitative fusion assay and is dependent on gH/gL for fusion activity. gB843 can rescue fusion function of other glycoprotein mutants that have null or decreased fusion activity in epithelial and B cells. In addition, gB843 requires less gp42 and gH/gL for fusion and can function in fusion at lower temperature indicative of a lower energy requirement gB for fusion activation. Since a key step in fusion is the conversion of gB from pre-fusion to active post-fusion state by gH/gL, gB843 may access this activated gB state more readily. Our studies indicate that the gB CTD may participate in fusion function by maintaining gB in an inactive pre-fusion form prior to activation by receptor binding.

    IMPORTANCE Diseases resulting from Epstein-Barr virus (EBV) infection in humans range from the fairly benign infectious mononucleosis to life threatening cancer. As an enveloped virus, EBV must fuse with a host cell membrane for entry and infection using glycoproteins gH/gL, gB and gp42. Among these glycoproteins, gB is thought to be the protein that executes fusion. To further characterize the function of the EBV gB cytoplasmic C-terminal tail domain (CTD) in fusion, we used a previously constructed CTD truncation mutants and studied fusion activity in the context of other EBV glycoprotein mutants. From these studies, we find that the gB CTD regulates fusion by altering the energy requirements for the triggering of fusion mediated by gH/gL or gp42. Overall, our studies may lead to a better understanding of EBV fusion and entry which may result in new and novel therapies that target the EBV entry step.

  • Inhibition of enterovirus 71 by adenosine analog NITD008 [PublishAheadOfPrint]

  • Enterovirus 71 (EV71) is a major viral pathogen in China and Southeast Asia. There is no clinically approved vaccine or antiviral therapy for EV71 infection. NITD008, an adenosine analog, is an inhibitor of flavivirus that blocks viral RNA synthesis. Here we report that NITD008 has a potent antiviral activity against EV71. In cell culture, the compound inhibits EV71 with an EC50 of 0.67 mmu;M and CC50 of 119.97 mmu;M. When dosed at 5 mg/kg in an EV71 mouse model, the compound reduced viral load in various organs and completely prevented clinical symptoms and death. To study the antiviral mechanism and drug resistance, we selected escape mutant viruses by culturing EV71 with increasing concentration of NITD008. Resistance mutations were reproducibly mapped to viral 3A and 3D polymerase regions. Resistance analysis using recombinant viruses demonstrated that either 3A or 3D mutation alone could lead to resistance to NITD008. Combination of both 3A and 3D mutations conferred higher resistance, suggesting a collaborative interplay between 3A and 3D proteins during viral replication. The resistance results underline the importance of combination therapy required for EV71 treatment.

    Significance Human enterovirus 71 (EV71) has emerged as a major cause of viral encephalitis in children worldwide, especially in the Asia-Pacific regions. Vaccines and antivirals are urgently needed to prevent and treat EV71 infections. In this study, we report the in vitro and in vivo efficacy of NITD008 (an adenosine analog) to inhibit EV71. The efficacy results validated that nucleoside analogs have potential as antiviral drugs for EV71 infections. Mechanistically, we showed that mutations in viral 3A and 3D polymerase alone or in combination could confer resistance to NITD008. The resistance results suggest an intrinsic interaction between viral proteins 3A and 3D during replication as well as the importance for combination therapy for treatment of EV71 infections.

  • Human Rhinovirus-16 causes Golgi fragmentation without blocking protein secretion. [PublishAheadOfPrint]

  • Replication of picornaviruses has been described to cause fragmentation of the Golgi that blocks the secretory pathway. The inhibition of MHC class I upregulation and cytokine, chemokine and interferon secretion may have important implications for host defence. Previous studies have shown that disruption of the secretory pathway can be replicated by expression of individual non-structural proteins; however the situation with different serotypes of human Rhinovirus (HRV) is unclear. Expression of 3A protein from HRV14 or HRV2 did not cause Golgi disruption or a block in secretion whereas other studies showed that infection of cells with HRV1A did cause Golgi disruption which was replicated by expression of 3A. HRV16 is the serotype most widely used in clinical HRV challenge studies; consequently to address the issue of Golgi disruption for HRV16 we have systematically and quantitatively examined the effect of HRV16 on both Golgi fragmentation and protein secretion in HeLa cells. First we expressed each individual non-structural protein and examined their cellular localization and their disruption of endoplasmic reticulum and Golgi architecture. We quantified their effects on the secretory pathway by measuring secretion of the reporter protein Gaussia luciferase. Finally, we examined the same outcomes following infection of cells with live virus. We demonstrate that expression of HRV16 3A and 3AB and to a lesser extent 2B, caused dispersal of the Golgi structure and these three non-structural proteins also inhibited protein secretion. Infection of cells with HRV16 also caused significant Golgi dispersal, however this did not result in inhibition of protein secretion.

    Importance The ability of replicating picornaviruses to influence the function of the secretory pathway has important implications for host defence. However, there appears to be differences between different members of the family and inconsistent results when comparing infection with live virus to expression of individual non-structural proteins. We demonstrate that individual non-structural HRV16 proteins when expressed in HeLa cells can both fragment the Golgi and block secretion whereas viral infection fragments the Golgi without blocking secretion. This has major implications for how we interpret mechanistic evidence derived from expression of single viral proteins.

  • The M33 GPCR encoded by MCMV is dispensable for hematogenous dissemination but is required for growth within the salivary gland [PublishAheadOfPrint]

  • Human Cytomegalovirus (HCMV) is a pathogen found worldwide and is a serious threat to immunocompromised individuals and developing fetuses. Due to the species specificity of CMVs, murine cytomegalovirus (MCMV) has been used as a model for in vivo studies of HCMV pathogenesis. MCMV like other beta- and gamma- herpesviruses encodes G protein-coupled receptors (GPCRs) that modulate host signaling pathways presumably to facilitate viral replication and dissemination. Among these viral receptors, the MCMV encoded M33 GPCR is an activator of CREB, NF-B and PLC-bbeta; signaling pathways and has been implicated in aspects of pathogenesis in vivo including persistence in the salivary glands of BALB/c mice. In this study we used immunocompetent non-obese diabetic (NOD) and immunocompromised NOD-scid-gamma (NSG) mice to further investigate the salivary gland defect exhibited by M33 deficiency. Interestingly, we demonstrate that virus deleted of M33 (M33) can replicate in salivary gland of immunocompromised animals, albeit with a 400x defect. Moreover, we determined that M33 does not have a role in cell-associated hematogenous dissemination but is required for viral amplification once the virus reaches the gland. We conclude that the reduced replicative capacity of the M33 virus is due to a specific defect occurring within the localized environment of the salivary gland. Importantly, since salivary glands represent an essential site for persistence and horizontal transmission, an understanding of the mechanisms of viral replication within this site could lead to the generation of novel therapeutics useful for the prevention of HCMV spread.

    Importance Human cytomegalovirus infects the majority of the American people and can reside silently in infected individuals for the duration of their lifetime. Under a number of circumstances, the virus can "reactivate" leading to a variety of diseases in both adults and developing babies and therefore identifying the function of viral proteins is essential to understand how the virus spreads and causes disease. We aim to utilize animal models to study the function of an important class of viral proteins termed G-protein coupled receptors with the ultimate goal of developing inhibitors to these proteins that could one day be used to prevent viral spread.

  • Inhibition of the Human Respiratory Syncytial Virus Small Hydrophobic Protein and Structural variations in a bicelle environment. [PublishAheadOfPrint]

  • The small hydrophobic (SH) protein is a 64-amino acid polypeptide encoded by the human respiratory syncytial virus (hRSV). SH protein has a single aalpha;-helical transmembrane (TM) domain that forms pentameric ion channels. Herein, we report the first inhibitor of the SH protein channel, pyronin B, and we have mapped its binding site to a conserved surface of the RSV SH pentamer, at the C-terminal end of the trasnmembrane domain. The validity of the SH protein structural model used has been confirmed by using a bicellar membrane-mimicking environment. However, in bicelles the aalpha;-helical stretch of the TM domain extends up to His-51, and by comparison with previous models both His-22 and His-51 adopt an interhelical/lumenal orientation relative to the channel pore. Neither His residue was found to be essential for channel activity, although His-51 protonation reduced channel activity at low pH, with His-22 adopting a more structural role. These latter results are in contrast with previous patch clamp data showing channel activation at low pH, which could not be reproduced in the present work. Overall, these results establish a solid ground for future drug development targeting this important viroporin.

    IMPORTANCE The human respiratory syncytial virus (hRSV) is responsible for 64 million reported cases of infection and 160,000 deaths each year. Lack of adequate antivirals fuels the search for new targets for treatment. The small hydrophobic (SH) protein is a 64-amino acid polypeptide encoded by hRSV and other paramyxoviruses, and its absence leads to viral attenuation in vivo and early apoptosis in infected cells. SH protein forms pentameric ion channels that may constitute novel drug targets, but no inhibitor for this channel activity has been reported so far. A small molecule inhibitor, pyronin B, can reduce SH channel activity and its likely binding site on the SH protein channel has been identified. Black lipid membrane (BLM) experiments confirm that protonation of both Histidine residues reduces stability and channel activity. These results contrast with previous patch clamp data that showed low pH activation, which we have not been able to reproduce.

  • Gene expression profiling to predict and assess the consequences of therapy-induced virus eradication in chronic HCV [PublishAheadOfPrint]

  • Systems biology has proven to be a powerful tool to identify reliable predictors for treatment response in chronic HCV. In the present study, we studied chronic HCV patients who responded to IFN-based therapy as evidenced by absence of HCV RNA at the end of treatment, and focused on two issues that have not received much attention. Firstly, we evaluated whether specific genes or gene expression patterns in blood were able to distinguish responder patients with a viral relapse from responder patients who remained virus-negative after cessation of treatment. We found that chronic HCV patients who were sustained responders and relapsers to IFN-based therapy showed comparable baseline clinical parameters and immune composition in blood. However, at baseline, the gene expression profiles of a set of 18 genes predicted treatment outcome with an accuracy of 94%. Secondly, we examined whether patients with successful therapy-induced clearance of HCV still exhibited gene expression patterns characteristic for HCV, or whether normalization of their transcriptome was observed. We observed that the relatively high expression of IFN-stimulated genes (ISG) in chronic HCV patients prior to therapy was reduced after successful IFN-based antiviral therapy (at 24 weeks follow-up). These ISG included CXCL10, OAS1, IFI6, DDX60, TRIM5 and STAT1. In addition, 1428 differentially expressed non-ISG genes were identified in paired pre- and post-treatment samples from sustained responders, which included genes involved in TGF-bbeta; signaling, apoptosis, autophagy, and nucleic acid and protein metabolism. Interestingly, 1424 genes were identified with altered expression in responder patients after viral eradication in comparison to normal expression levels in healthy individuals. Additionally, aberrant expression of a subset of these genes, including IL-32, IL-16, CCND3 and RASSF1, was also observed at baseline.

    Our findings indicate that successful antiviral therapy of chronic HCV patients does not lead to normalization of their blood transcriptional signature. The altered transcriptional activity may reflect HCV-induced liver damage in previously infected individuals.

    Importance Tools to predict the efficacy of antiviral therapy of chronic HCV patients are important to select the optimal therapeutic strategy. Using a systems biology approach, we identify a set of 18 genes expressed in blood that predicts the recurrence of HCV RNA after cessation of therapy consisting of peginterferon and ribavirin. This set of genes may be applicable as a useful biomarker in clinical decision-making since the number of genes included in the predictor is small and the correct prediction rate is high (94%). In addition, we observe that the blood transcriptional profile in chronic HCV patients who were successfully treated is not normalized to the status observed in healthy individuals. Even 6 months after therapy-induced elimination of HCV RNA, gene expression profiles in blood are still altered in these chronic HCV patients, strongly suggesting long-term modulation of immune parameters in previously infected patients.

  • Isolation of dengue virus-specific memory B cells with live virus antigen from human subjects following natural infection reveals the presence of diverse novel functional groups of antibody clones [PublishAheadOfPrint]

  • Natural dengue virus (DENV) infection in humans induces antibodies (Abs) that neutralize the serotype of infection in a potent and type-specific manner, however most Abs generated in response to infection are serotype cross-reactive and poorly neutralizing. Such cross-reactive Abs may enhance disease during subsequent infection with virus of a different DENV serotype. Previous screening assays for DENV-specific human B cells and antibodies using viral and recombinant antigens mainly led to the isolation of dominant non-neutralizing B cell clones. To improve upon our ability to recover and study rare but durable and potently neutralizing DENV-specific Abs, we isolated human DENV-specific B cells using a primary screen of binding to live virus, followed by a secondary screen with a high-throughput flow cytometry-based neutralization assay to identify DENV-specific B cell lines prior to generation of hybridomas. Using this strategy, we identified several new classes of serotype-specific and serotype cross-neutralizing anti-DENV mAbs, including ultra-potent inhibitory antibodies with neutralizing activity concentrations less than 10 ng/mL. We isolated serotype-specific neutralizing Abs that target diverse regions of the E protein including epitopes present only on the intact fully-assembled viral particle. We also isolated a number of serotype cross-neutralizing mAbs, most of which recognized a region in the E protein domain I/II containing the fusion loop. These data provide insights into targets of the protective Ab-mediated immune response to natural DENV infection, which will prove valuable in the design and testing of new experimental DENV vaccines.

    Importance. Dengue virus infection is one of the most common mosquito-borne diseases and occurs in most countries of the world. Infection of humans with dengue virus induces a small number of antibodies that inhibit the infecting strain, but also induces a large number of antibodies that can bind but do not inhibit dengue virus strains of other serotypes. We used a focused screening strategy to discover a large number of rare potently inhibiting antibodies, and we mapped the regions on the virus that were recognized by such antibodies. The studies revealed that humans have the potential to generate very potent antibodies directed to diverse regions of the dengue virus surface protein. The studies provide important new information about protection from dengue infection that will be useful in the design and testing of new experimental dengue vaccines for humans.

  • Dynamic and Nucleolin-Dependent Localization of Human Cytomegalovirus UL84 to the Periphery of Viral Replication Compartments and Nucleoli [PublishAheadOfPrint]

  • Protein-protein and protein-nucleic acid interactions within sub-cellular compartments are required for viral genome replication. To understand the localization of the human cytomegalovirus viral replication factor UL84 relative to other proteins involved in viral DNA synthesis and to replicating viral DNA in infected cells, we created a recombinant virus expressing a FLAG tagged version of UL84 (UL84FLAG) and used this virus in immunofluorescence assays. UL84FLAG localization differed at early and late times of infection, transitioning from diffuse distribution throughout the nucleus to exclusion from the interior of replication compartments with some concentration at the periphery of replication compartments with newly labeled DNA and the viral DNA polymerase subunit UL44. Early in infection, UL84FLAG co-localized with the viral single stranded DNA binding protein UL57, but co-localization became less prominent as infection progressed. A portion of UL84FLAG also co-localized with the host nucleolar protein nucleolin, at the periphery of both replication compartments and nucleoli. siRNA mediated knockdown of nucleolin resulted in a dramatic elimination of UL84FLAG from replication compartments and other parts of the nucleus and its accumulation in the cytoplasm. Reciprocal co-immunoprecipitation of viral proteins from infected cell lysates revealed association of UL84, UL44 and nucleolin. These results indicate that UL84 localization during infection is dynamic, which is likely relevant to its functions, and suggest that its nuclear and subnuclear localization is highly dependent on direct or indirect interactions with nucleolin.

    IMPORTANCE The protein-protein interactions among viral and cellular proteins required for replication of the human cytomegalovirus (HCMV) DNA genome are poorly understood. We sought to understand how an enigmatic human cytomegalovirus (HCMV) protein critical for virus replication, UL84, localizes relative to other viral and cellular proteins required for HCMV genome replication and replicating viral DNA. We found that UL84 localizes with viral proteins, viral DNA and the cellular nucleolar protein nucleolin in the sub-nuclear replication compartments in which viral DNA replication occurs. Unexpectedly, we also found localization of UL84 with nucleolin in nucleoli, and showed that the presence of nucleolin is involved in localization of UL84 to the nucleus. These results add to previous work showing the importance of nucleolin in replication compartment architecture and viral DNA synthesis, and are relevant to understanding UL84 function.

  • Murine gammaherpesvirus 68 reactivation from B cells requires IRF4, but not XBP-1 [PublishAheadOfPrint]

  • Gammaherpesviruses display tropism for B cells and, like all known herpesviruses, exhibit distinct lytic and latent life cycles. One well established observation among members of the gammaherpesvirus family is the link between viral reactivation from latently infected B cells and plasma cell differentiation. Importantly, a number of studies have identified a potential role for a CREB/ATF family member, X-box binding protein 1 (XBP-1), in trans-activating the immediate-early BZLF-1 or BRLF1/gene 50 promoters of EBV and KSHV, respectively. XBP-1 is required for the unfolded protein response and has been identified as a critical transcription factor in plasma cells. Here we demonstrate that XBP-1 is capable of trans-activating the MHV68 RTA promoter in vitro, consistent with previous observations for EBV and KSHV. However, we show that in vivo there does not appear to be a requirement for XBP-1 expression in B cells for virus reactivation. The MHV68 M2 gene product under some experimental conditions plays an important role in virus reactivation from B cells. M2 has been shown to drive B cell differentiation to plasma cells, as well as IL-10 production, both of which are dependent on M2 induction of Interferon Regulatory Factor 4 (IRF4) expression. IRF4 is required for plasma cell differentiation and, consistent with a role for plasma cells in MHV68 reactivation from B cells, we show that IRF4 expression in B cells is required for efficient reactivation of MHV68 from splenocytes. Thus, the latter analyses are consistent with previous studies linking plasma cell differentiation to MHV68 reactivation from B cells. The apparent independence of MHV68 reactivation from XBP-1 expression in plasma cells may reflect redundancy among CREB/ATF family members, or the involvement of other plasma cell-specific transcription factors. Regardless, these findings underscore the importance of in vivo studies in assessing the relevance of observations made in tissue culture models.

    IMPORTANCE All known herpesvirus establish a chronic infection of their respective host, persisting for the life of the individual. A critical feature of these viruses is their ability to reactivate from a quiescent form of infection (latency) and generate progeny virus. In the case of gammaherpesviruses, which are associated with the development of lymphoproliferative disorders including lymphomas, reactivation from latently infected B lymphocytes occurs upon terminal differentiation of these cells to plasma cells mmdash; the cell type that produces antibodies. A number of studies have linked a plasma cell transcription factor, XBP-1, to the induction of gammaherpesvirus reactivation and we show here that indeed in tissue culture models this cellular transcription factor can trigger expression of the murine gammaherpesvirus gene involved in driving virus reactivation. However, surprisingly, when we examined the role of XBP-1 in the setting of infection of mice mmdash; using mice that lack a functional XBP-1 gene in B cells nndash; we failed to observe a role for XBP-1 in virus reactivation. However, we show that another cellular factor essential for plasma cell differentiation, IRF4, is critical for virus reactivation. Thus, these studies point out the importance of studies in animal models to validate findings from studies carried out in cell lines passaged in vitro.

  • Kinetic Models for Receptor-catalyzed Conversion of Coxsackievirus B3 to A-particles [PublishAheadOfPrint]

  • The immunoglobulin superfamily protein receptors for poliovirus, human rhinovirus, and coxsackievirus B (CVB) serve to bind the viruses to target cells and to facilitate the release of virus genome by catalyzing the transition from the mature infectious virus to the A-particle uncoating-intermediate. Receptor binding sites characterized by two equilibrium dissociation constants have been identified. The site with higher affinity is best observed at warmer temperatures and appears to correlate with the reversible conformational state in which the capsid is permeable to small molecules, and peptides that are buried in the crystal structures are exposed. Measurements of CVB conversion to inactive particles over time in the presence of varied concentrations of soluble coxsackievirus and adenovirus receptor showed that the observed first-order rate constant varies with receptor concentration. The dose-response data, previously modeled as the sum of first-order reactions, have been used to evaluate models for the receptor-catalyzed conversion of CVB that include the high- and low-affinity binding sites associated with capsid breathing. Allosteric models wherein receptor binding shifts the equilibrium toward the open capsid conformation in which the high-affinity binding site is available best fit the data.

    Importance This paper derives and compares models that relate the structural, mechanistic, and kinetic details of receptor-virus interactions known from previous work with human enteroviruses to recent results from receptor-catalyzed conversion of coxsackievirus B3 to non-infectious A-particles. Of those considered, the acceptable models include the capsid breathing cycle and two conformation-dependent receptor binding sites. The results indicate that the receptor enhancement of virus conversion to A-particles involves allostery through conformation selection.

  • Human Herpesvirus 8 Viral Interleukin-6 Signaling Through gp130 Promotes Virus Replication in Primary Effusion Lymphoma and Endothelial Cells [PublishAheadOfPrint]

  • The contributions of human herpesvirus 8 (HHV-8) viral interleukin-6 (vIL-6) to virus biology remain unclear. Here we examined the role of vIL-6/gp130 signaling in HHV-8 productive replication in primary effusion lymphoma and endothelial cells. Depletion and depletion-complementation experiments revealed that endoplasmic reticulum-localized vIL-6 activity via gp130 and gp130-activated STAT signaling, but not ERK activation, were critical for vIL-6 pro-replication activity. Our data significantly extend current understanding of vIL-6 function and associated mechanisms in HHV-8 biology.

  • The 19S proteasome activator promotes human cytomegalovirus immediate early gene expression through proteolytic and non-proteolytic mechanisms [PublishAheadOfPrint]

  • Proteasomes are large, multi-subunit complexes that support normal cellular activities by executing the bulk of protein turnover. During infection, many viruses have been shown to promote viral replication by using proteasomes to degrade cellular factors that restrict viral replication. For example, the human cytomegalovirus (HCMV) pp71 protein induces the proteasomal degradation of Daxx, a cellular transcriptional repressor that can silence viral immediate early (IE) gene expression. We previously showed this degradation requires both the proteasome catalytic 20S core particle (CP) and the 19S regulatory particle (RP). The 19S RP associates with the 20S CP to facilitate protein degradation, but also plays a 20S CP-independent role promoting transcription. Here, we present a non-proteolytic role of the 19S RP in HCMV IE gene expression. We demonstrate that 19S RP subunits are recruited to the Major Immediate Early Promoter (MIEP) that directs IE transcription. Depletion of 19S RP subunits generated a defect in RNA Polymerase II elongation through the MIE locus during HCMV infection. Our results reveal that HCMV commandeers proteasome components for both proteolytic and non-proteolytic roles to promote HCMV lytic infection.

    Importance Proteasome inhibitors decrease or eliminate 20S CP activity and are garnering increasing interest as chemotherapeutics. However, an increasing body of evidence implicates 19S RP subunits in important proteolytic-independent roles during transcription. Thus, pharmacological inhibition of the 20S CP as a means to modulate proteasome function towards therapeutic effect is an incomplete capitalization on the potential of this approach. Here we provide an additional example of non-proteolytic 19S RP function in promoting HCMV transcription. This data provides a novel system with which to study the roles of different proteasome components during transcription, a rationale for previously described shifts in 19S RP subunit localization during HCMV infection, and a potential therapeutic intervention point at a pre-immediate early stage for the inhibition of HCMV infection.

  • Gene expression of five different iteradensoviruses: BmDV, CeDV, PpDV, SfDV and DpIDV [PublishAheadOfPrint]

  • Iteradensoviruses are 5 kb parvoviruses with typical J-shaped inverted-terminal repeats of about 250 nucleotides and terminal hairpins of about 165 nucleotides. The single-stranded DNA genome contains several open reading frames but their expression strategy is still unknown. Here the transcription maps and expression of the viruses in this genus were explored. As for brevidensoviruses, the two NS genes were expressed by overlapping promoters with alternate transcription starts at both sides of the NS1 start codon.

  • Classic nuclear localization signals and a novel nuclear localization motif are required for nuclear transport of porcine parvovirus capsid proteins [PublishAheadOfPrint]

  • Nuclear targeting of capsid proteins (VPs) is important for genome delivery and precedes assembly in the replication cycle of porcine parvovirus (PPV). Clusters of basic amino acids, corresponding to potential nuclear localization signals (NLS), were found only in the unique region of VP1 (VP1up). Of the five identified basic regions (BR), three were important for nuclear localization of VP1up: BR1 was a classic Pat7 NLS and the combination of BR4 and BR5 was a classic bipartite NLS. These NLS were essential for viral replication. VP2, the major capsid protein, lacked these NLS and contained no region with more than two basic amino acids in proximity. However, three regions of basic clusters were identified in the folded protein, assembled into a trimeric structure. Mutagenesis experiments showed that only one of these three regions was involved in VP2 transport to the nucleus. This structural NLS, termed the nuclear localization motif (NLM), was located inside the assembled capsid, and thus can be used to transport trimers to the nucleus in late steps of infection but not for virions in initial infection steps. The two NLS of VP1up are located in the N-terminal part of the protein, externalized from the capsid during endosomal transit, exposing them for the nuclear targeting during early steps of infection. Globally, the determinants of nuclear transport of structural protein of PPV were different from those of closely-related parvoviruses.

    IMPORTANCE Most DNA viruses use the nucleus for their replication cycle. Thus, structural proteins need to be targeted to this cellular compartment at two distinct steps of the infection: in early steps to deliver viral genomes to the nucleus and in the late steps to assemble new viruses. Nuclear targeting of proteins depends on the recognition of a stretch of basic amino acids by cellular transport proteins. This study reports the identification of two classic nuclear localization signals in the minor capsid protein (VP1) of porcine parvovirus. The major protein (VP2) nuclear localization was shown to depend on a complex structural motif. This motif can be used as a strategy by the virus to avoid transport of incorrectly folded proteins and selectively import assembled trimers into the nucleus. Structural nuclear localization motifs can also be important for nuclear proteins without classic basic amino acids stretch, including multimeric cellular proteins.

  • Immunodominant CD4+ T-cell Responses to Influenza A Virus in Healthy Individuals Focus on Matrix 1 and Nucleoprotein [PublishAheadOfPrint]

  • Antigen-specific CD4+ T cells are essential for effective virus-specific host responses, with recent human challenge studies (in volunteers) establishing their importance for influenza A viruses (IAV)-specific immunity. However, while many IAV CD4+ T cell epitopes have been identified, few are known to stimulate immunodominant CD4+ T cell responses. Moreover, much remains unclear concerning the major antigen(s) responded to by the human CD4+ T cells and the extent and magnitude of these responses. We initiated a systematic screen of immunodominant CD4+ T cell responses to IAV in healthy individuals. Using in vitro expanded multi-specificity IAV-specific T cell lines and individual IAV protein antigens produced by recombinant vaccinia viruses, we found that the internal Matrix protein 1 (M1) and Nucleoprotein (NP) were the immunodominant targets of CD4+ T cell responses. Ten epitopes derived from M1 and NP were definitively characterized. Furthermore, epitope sequence conservation analysis established that immunodominance correlated with an increased frequency of mutations, reflecting that these prominent epitopes are under greater selective pressure. Such evidence that particular CD4+ T cells are important for protection/recovery is of value for the development of novel IAV vaccines, and for our understanding of differential susceptibility profiles to these major pathogens.

    Importance statement: Influenza virus causes half a million deaths annually. CD4+ T cell responses have been shown to be important for influenza protection and recovery. CD4+ T cell responses are also critical for efficient CD8+ T cell response and antibody response. As immunodominant T cells generally play more important role, characterizing these immunodominant responses is critical for influenza vaccine development. We show here that the internal Matrix protein 1 (M1) and Nucleoprotein (NP), rather than the surface proteins reported previously, are the immunodominant targets of CD4+ T cell responses. Interestingly, these immunodominant epitope regions accumulated many mutations overtime, which likely indicates increased immune pressure. These findings have significant implications for the design of T cell based influenza vaccines.

  • Engineering Recombinant Orsay Virus Directly in the Metazoan Host C. elegans [PublishAheadOfPrint]

  • The recent identification of Orsay virus, the first virus that is capable of naturally infecting Caenorhabditis elegans, provides a unique opportunity to explore host-virus interaction studies in this invaluable model organism. A key feature of this system is the robust genetic tractability of the host, C. elegans, which would ideally be complemented by the ability to genetically manipulate Orsay virus in parallel. To this end, we developed a plasmid-based reverse genetics system for Orsay virus by creating transgenic C. elegans strains harboring Orsay virus cDNAs. Both wild type and mutant Orsay viruses, including a FLAG epitope tagged recombinant Orsay virus, were generated by use of the reverse genetics system. This is the first plasmid-based virus reverse genetics system in the metazoan C. elegans. The Orsay virus reverse genetics we established will serve as a fundamental tool in host-virus interaction studies in the model organism C. elegans.

    Importance To date, Orsay virus is the first and the only identified virus capable of naturally infecting Caenorhabditis elegans. C. elegans is a simple multi-cellular model organism that mimics many fundamental features of human biology and has been used to define many biological properties conserved through evolution. Thus, the Orsay virus-C. elegans infection system provides a unique opportunity to study host-virus interactions. In order to take maximal advantage of this system, the ability to genetically engineer mutant forms of Orsay virus would be highly desirable. Most efforts to engineer viruses have been done in cultured cells. Here we describe the creation of mutant viruses directly in the multi-cellular organism C. elegans without the use of cell culture. We engineered a virus expressing a genetically tagged protein that could be detected in C. elegans. This provides proof of concept for modifying Orsay virus, which will greatly facilitate studies in this experimental system.

  • A Single Amino Acid Substitution in the Novel H7N9 Influenza A Virus NS1 Protein Increases CPSF30 Binding and Virulence [PublishAheadOfPrint]

  • Although an effective interferon antagonist in human and avian cells, the novel H7N9 influenza virus NS1 protein is defective at inhibiting CPSF30. An I106M substitution in H7N9 NS1 can restore CPSF30 binding together with the ability to block host gene expression. Furthermore, a recombinant virus expressing H7N9 NS1-I106M replicates to higher titers in vivo, and is subtly more virulent, than parental. Natural polymorphisms in H7N9 NS1 that enhance CPSF30 binding may be cause for concern.

  • Re-emergence of chikungunya virus [PublishAheadOfPrint]

  • Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes acute fever and acute and chronic musculoskeletal pain in humans. Since 2004, CHIKV has caused millions of disease cases in the Indian Ocean region and has emerged in new areas, including Europe, the Middle East, and the Pacific region. The mosquito vectors for this virus are globally distributed in tropical and temperate zones, providing the opportunity for CHIKV to continue to expand into new geographic regions. In October of 2013 locally acquired cases of CHIKV infection were identified on the Caribbean island of St. Martin, signaling the arrival of the virus in the Western Hemisphere. In just nine months, CHIKV has spread to 22 countries in the Caribbean and Central and South America, resulting in hundreds of thousands of cases. CHIKV disease can be highly debilitating and large epidemics have severe economic consequences. Thus, there is an urgent need for continued research into the epidemiology, pathogenesis, prevention, and treatment of these infections.

  • Herpes Simplex Virus 1 Counteracts Viperin via Its Virion Host Shutoff Protein UL41 [PublishAheadOfPrint]

  • The IFN-inducible viperin restricts a broad range of viruses. However, whether viperin plays a role during HSV-1 infection is poorly understood. In the present study, it was shown for the first time that wild type (WT) HSV-1 infection couldn't induce viperin production and ectopicly expressed viperin inhibited the replication of UL41-null HSV-1, but not WT viruses. The underlying molecular mechanism is that UL41 counteracts viperin's antiviral activity by reducing its mRNA accumulation.

  • AUF1 p45 promotes West Nile virus replication by an RNA chaperone activity that supports cyclization of the viral genome [PublishAheadOfPrint]

  • A central aspect of current virology is to define the function of cellular proteins (llsquo;host factorsrrsquo;) that support the viral multiplication process. This study aimed at characterizing cellular proteins that assist the RNA replication process of the prevalent human pathogen West Nile virus (WNV). Using in vitro and cell-based approaches, we defined the p45 isoform of the RNA-binding protein AUF1 as a host factor that enables efficient WNV replication. It was demonstrated that AUF1 p45 has an RNA chaperone activity, which aids the structural rearrangement and cyclization of the WNV RNA that is required by the viral replicase to initiate RNA replication. The obtained data suggest the RNA chaperone activity of AUF1 p45 as an important determinant of the WNV life-cycle.

    Importance In this study, we identified a cellular protein, AUF1 (other name hnRNPD) acting as a helper (llsquo;host factorrrsquo;) of the multiplication process of the important human pathogen West Nile virus. Several different variants of AUF1 exist in the cell, and one variant, AUF1 p45, was shown to support viral replication most significantly. Interestingly, we obtained a set of experimental data indicating that a main function of AUF1 p45 is to modify and thus llsquo;preparerrsquo; the West Nile virus genome in such a way that the viral enzyme that generates progeny genomes is empowered to do this considerably more efficiently than in the absence of the host factor. The capability of AUF1 p45 to re-arrange the West Nile virus genome was thus identified to be an important aspect of a West Nile virus infection.

  • A monomeric uncleaved RSV F antigen retains pre-fusion-specific neutralizing epitopes [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) is the leading cause of severe respiratory disease in infants and a major cause of respiratory illness in the elderly. It remains an unmet vaccine need despite decades of research. Insufficient potency, homogeneity and stability of previous RSV fusion protein (F) subunit vaccine candidates have hampered vaccine development. RSV F and related parainfluenza virus (PIV) F proteins are cleaved by furin during intracellular maturation, producing disulfide-linked F1 and F2 fragments. During cell entry, the cleaved Fs rearrange from pre-fusion trimers to post-fusion trimers. Using RSV F constructs with mutated furin cleavage sites, we isolated an uncleaved RSV F ectodomain that is predominantly monomeric and requires specific cleavage between F1 and F2 for self-association and rearrangement into stable post-fusion trimers. The uncleaved RSV F monomer is folded, homogenous and displays at least two key RSV neutralizing epitopes shared between the pre-fusion and post-fusion conformations. Unlike the cleaved trimer, the uncleaved monomer binds the pre-fusion-specific monoclonal antibody D25 and human neutralizing immunoglobulins that do not bind to post-fusion F. These observations suggest that the uncleaved RSV F monomer has a prefusion-like conformation and is a potential pre-fusion subunit vaccine candidate.

    Importance RSV is the leading cause of severe respiratory disease in infants and a major cause of respiratory illness in the elderly. Development of an RSV vaccine was stymied when a clinical trial using a formalin inactivated RSV virus made disease, following RSV infection, more severe. Recent studies have defined the structures that the RSV F envelope glycoprotein adopts before and after virus entry (pre-fusion and post-fusion conformation, respectively). Key neutralization epitopes of pre-fusion and post-fusion RSV F have been identified, and a number of current vaccine development efforts are focused on generating easily produced subunit antigens that retain these epitopes. Here we show that a simple modification in the F ectodomain results in a homogeneous protein that retains critical pre-fusion neutralizing epitopes. These results improve our understanding of RSV F protein folding and structure and can guide further vaccine design efforts.

  • Parvovirus-derived endogenous viral elements in two South American rodent genomes [PublishAheadOfPrint]

  • We describe endogenous viral elements (EVEs) derived from parvoviruses (family Parvoviridae) in the long-tailed chinchilla (Chinchilla lanigera) and degu (Octodon degus) genomes. The novel EVEs include Dependovirus-related elements, and representatives of a clearly distinct parvovirus lineage that also has endogenous representatives in marsupial genomes. In the degu, one dependovirus-derived EVEs was found to encode an intact reading frame, and was differentially expressed in vivo, with increased expression in the liver.

  • Combined effects of the structural heterogeneity and dynamics of flaviviruses on antibody recognition [PublishAheadOfPrint]

  • Flaviviruses are thought to sample an ensemble of structures at equilibrium. One consequence of a structurally dynamic virion is the observed time-dependent increases in neutralization sensitivity that can occur after prolonged incubation with antibody. Differences in how virus strains "breathe" may affect epitope exposure and contribute to the underlying mechanisms of strain-dependent neutralization sensitivity. Beyond the contribution of structural dynamics, flaviviruses exist as a structurally heterogeneous population due to an inefficient virion maturation process. Herein we investigate the interplay between virion maturation and structural dynamics that contribute to antibody-mediated neutralization. Using West Nile (WNV) and dengue (DENV) viruses produced under conditions that modify the extent of virion maturation, we investigated time-dependent changes in neutralization sensitivity associated with structural dynamics. Our results identify distinct patterns of neutralization against viruses that vary markedly with respect to the extent of virion maturation. Reducing the efficiency of virion maturation resulted in greater time-dependent changes in neutralization potency and a marked reduction in the stability of the particle at 37ddeg;C as compared to more mature virus. That neutralization sensitivity of WNV and DENV did not increase after prolonged incubation in the absence of antibody, regardless of virion maturation, suggests that the dynamic processes that govern epitope accessibility on infectious viruses are reversible. Against the backdrop of heterogeneous flavivirus structures, differences in the pathways by which viruses "breathe" represent an additional layer of complexity towards understanding maturation state-dependent patterns of antibody recognition.

    Importance: Flaviviruses exist as a group of related structures at equilibrium that arise from the dynamic motion of E proteins that comprise the antigenic surface of the mature virion. This process has been characterized for numerous viruses and is referred to as viral "breathing". Additionally, flaviviruses are structurally heterogeneous due to an inefficient maturation process responsible for cleaving prM on the virion surface. Both of these mechanisms vary the exposure of antigenic sites available for antibody binding and impact the ability of antibodies to neutralize infection. We demonstrate that virions with inefficient prM cleavage "breathe" differently than their more mature counterparts, resulting in distinct patterns of neutralization sensitivity. Additionally, the maturation state was found to impact virus stability in solution. Our findings provide insight into the complex flavivirus structures that contribute to infection with the potential to impact antibody recognition.

  • Toll-like receptor 9-mediated protection of enterovirus 71 infection in mice is due to the release of danger-associated molecular patterns [PublishAheadOfPrint]

  • Enterovirus 71 (EV71), a positive-stranded RNA virus, is the major cause of hand, foot and mouth disease (HFMD) with severe neurological symptoms. Anti-viral type I interferon responses initiated from innate receptor signaling are inhibited by EV71-encoded proteases. It is less understood whether EV71-induced apoptosis provides a signal to activate type I interferon (IFNaalpha;/bbeta;) responses as a host defensive mechanism. In this report, we found that EV71 alone cannot activate toll-like receptor 9 (TLR9) signaling, but supernatant from EV71-infected cells is capable of activating TLR9. We hypothesized that TLR9-activating signaling from pDCs may contribute to host defense mechanisms. To test our hypothesis, Flt3 ligand-cultured DCs (Flt3L-DCs) from both wild-type (WT) and TLR9 knockout (TLR9KO) mice were infected with EV71. More viral particles were produced in TLR9KO mice than WT mice. In contrast, interferon-alpha (IFN-aalpha;), monocyte chemotactic protein 1(MCP-1), tumor necrosis factor-alpha (TNF-aalpha;), IFN-, interleukin 6 (IL-6) and IL-10 levels were increased in Flt3L-DCs from WT mice infected with EV71 compared with TLR9KO mice. Seven-day-old TLR9KO mice infected with a non-mouse adapted EV71 strain develop neurological lesion-related symptoms, including hindlimb paralysis, slowness, ataxia and lethargy, but WT mice did not present with these symptoms. Lung, brain, small intestine, forelimb and hindlimb tissue collected from TLR9KO mice exhibit significantly higher viral loads than equivalent tissues collected from WT mice. Histopathologic damage was observed in brain, small intestine, forelimb and hindlimb tissues collected from TLR9KO mice infected with EV71. Our findings demonstrate that TLR9 is an important host defense molecule during EV71 infection.

    Importance The host innate immune system is equipped with pattern recognition receptors (PRRs), which are useful for defending the host against invading pathogens. During EV71 infection, the innate immune system is activated by pathogen-associated molecular patterns (PAMPs), which include viral RNA or DNA, and these PAMPs are recognized by PRRs. Toll-like receptor 3 (TLR3) and TLR7/8 recognize viral nucleic acids, and TLR9 senses unmethylated CpG DNA or pathogen-derived DNA. These PRRs stimulate the production of type I IFNs to counteract viral infection, and they are the major source of anti-viral IFN-aalpha; production in pDCs, which can produce 200- to 1000-fold more IFN-aalpha; than any other immune cell type. In addition to PAMPs, danger-associated molecular patterns (DAMPs) are known to be potent activators of innate immune signaling, including TLR9. We found that EV71 induces cellular apoptosis, resulting in tissue damage; the endogenous DNA from dead cells may activate the innate immune system through TLR9. Therefore, our study provides new insights into EV71-induced apoptosis, which stimulates TLR9 in EV71-associated infections.

  • Duplication of the A17L Locus of Vaccinia Virus Provides an Alternate Route to Rifampin Resistance [PublishAheadOfPrint]

  • Specific gene duplications can enable double-stranded DNA viruses to adapt rapidly to environmental pressures despite the low mutation rate of their high fidelity DNA polymerases. We report on the rapid, positive selection of a novel vaccinia virus genomic duplication mutant in the presence of the assembly inhibitor rifampin. Until now, all known rifampin-resistant vaccinia virus isolates contain missense mutations in the D13L gene, which encodes a capsid-like scaffold protein required for stabilizing membrane curvature during the early stage of virion assembly. Here we describe a second pathway to rifampin-resistance involving A17, a membrane protein that binds and anchors D13 to the immature virion. After one-round of selection, a rifampin-resistant virus that contained a genomic duplication in the A17L-A21L region was recovered. The mutant had both C-terminal truncated and full-length A17L open reading frames. Expression of the truncated A17 protein was retained when the virus was passaged in the presence of rifampin but was lost in the absence of drug suggesting that the duplication decreased general fitness. Both forms of A17 were bound to the virion membrane and associated with D13. Moreover, insertion of an additional truncated or inducible full-length A17L open reading frame into the genome of wild-type virus was sufficient to confer rifampin-resistance. In summary, this report contains the first evidence of an alternate mechanism for resistance of poxviruses to rifampin, indicates a direct relationship between A17 levels and the resistance phenotype and provides further evidence for the ability of double-stranded DNA viruses to acquire drug-resistance through gene duplication.

    IMPORTANCE TO THE FIELD The present study provides the first evidence for a new mechanism of resistance by a poxvirus to the antiviral drug rifampin. In addition, it affirms the importance of the interaction between the D13 scaffold protein and the A17 membrane protein for assembly of virus particles. Resistance to rifampin was linked to a partial duplication of the gene encoding the A17 protein, similar to the resistance to hydroxyurea enabled by duplication of the gene encoding the small subunit of ribonucletotide reductase and of the K3L gene to allow adaptation to the antiviral action of protein kinase R. Gene duplication may provide a way for poxviruses and other DNA viruses with high fidelity DNA polymerases to adjust rapidly to changes in the environment.

  • Conformational properties of prion strains can be transmitted to recombinant prion protein fibrils in real-time quaking-induced conversion [PublishAheadOfPrint]

  • The phenomenon of prion strains with distinct biological characteristics has been hypothesized to be involved in the structural diversity of abnormal prion protein (PrPSc). However, the molecular basis of the transmission of strain properties remains poorly understood. Real-time quaking-induced conversion (RT-QUIC) is a cell-free system that uses E. coli-derived recombinant PrP (rPrP) for the sensitive detection of PrPSc. To investigate whether properties of various prion strains can be transmitted to amyloid fibrils consisting of rPrP (rPrP-fibrils) using RT-QUIC, we examined the secondary structure, conformational stability and infectivity of rPrP-fibrils seeded with PrPSc derived from either the Chandler or 22L strain. In the first round of the reaction there were differences in the secondary structures, especially in bands attributed to bbeta;-sheets, as determined by infrared spectroscopy, and conformational stability between Chandler-seeded (1st-rPrP-fibCh) and 22L-seeded rPrP-fibrils (1st-rPrP-fib22L). Of note, specific identifying characteristics of the two rPrP-fibril-types seen in the bbeta;-sheets resembled those of the original PrPSc. Furthermore, the conformational stability of 1st-rPrP-fibCh was significantly higher than that of 1st-rPrP-fib22L, as with Chandler- and 22L-PrPSc. The survival periods in mice inoculated with 1st-rPrP-fibCh or 1st-rPrP-fib22L were significantly shorter than those of the mice inoculated with mock 1st-QUIC mixtures. In contrast, these biochemical characteristics were no longer evident in subsequent rounds, suggesting that nonspecific uninfected rPrP-fibrils became predominant probably because of their rapid growth rate. Together, these findings show that can be transmitted to rPrP-fibrils and unknown cofactors or environmental conditions in the RT-QUIC may be required for further conservation.

    Importance The phenomenon of prion strains with distinct biological characteristics is assumed to result from the conformational variations in the abnormal prion protein (PrPSc). However, important questions remain about the mechanistic relationship between the conformational differences and the strain diversity, including how to transmit strain-specific conformations. In this study, we investigated whether properties of diverse prion strains can be transmitted to amyloid fibrils consisting of E. coli-derived recombinant PrP (rPrP) generated in the real-time quaking-induced conversion (RT-QUIC), a recently-developed in vitro PrPSc formation method. We demonstrate that at least some of the strain-specific conformational properties can be transmitted to rPrP-fibrils in the first round of RT-QUIC by examining the secondary structure, conformational stability and infectivity of rPrP-fibrils seeded with PrPSc derived from either the Chandler or 22L prion strain. We believe that these findings will advance our understanding of the conformational basis underlying prion strain diversity.

  • Comparison of Genotypes I and III in Japanese encephalitis virus reveal distinct differences in their genetic and host diversity [PublishAheadOfPrint]

  • Japanese encephalitis (JE) is an arthropod-borne disease associated with the majority of viral encephalitis cases in the Asia-Pacific region. The causative agent, Japanese encephalitis virus (JEV), has been phylogenetically divided into five genotypes. Recent surveillance data indicates that genotype I (GI) is gradually replacing genotype III (GIII) as the dominant genotype.

    To investigate the mechanism behind the genotype shift and the potential consequences in terms of vaccine efficacy, human cases and virus dissemination, we collected (i) all full length and partial JEV molecular sequences and (ii) associated genotype and host information comprising a dataset of 873 sequences. We then examined differences between the two genotypes at the genetic and epidemiological level by investigating amino acid mutations, positive selection and host range.

    We found that although GI is dominant, it has fewer sites predicted to be under positive selection, a narrower host range and significantly fewer human isolates. For the E protein, the sites under positive selection define a haplotype set for each genotype that shows striking differences in their composition and diversity, with GIII showing significantly more variety than GI. Our results suggest that GI has displaced GIII by achieving a replication cycle that is more efficient but is also more restricted in its host range.

    Importance Japanese encephalitis is an arthropod-borne disease associated with the majority of viral encephalitis cases in the Asia-Pacific region. The causative agent, Japanese encephalitis virus (JEV), has been divided into five genotypes based on sequence similarity. Recent data indicates that genotype I (GI) is gradually replacing genotype III (GIII) as the dominant genotype. Understanding the reasons behind this shift and the potential consequences in terms of vaccine efficacy, human cases and virus dissemination are important for controlling the spread of the virus and reducing human fatalities. We collected all available full length and partial JEV molecular sequences and associated genotype and host information. We then examined differences between the two genotypes at the genetic and epidemiological level by investigating amino acid mutations, positive selection and host range. Our results suggest that GI has displaced GIII by achieving a replication cycle that is more efficient but more restricted in host range

  • Inhibition of pSTAT1 Nuclear Translocation and Antiviral Protein Expression in Human Brain Vascular Adventitial Fibroblasts Infected with Varicella Zoster Virus [PublishAheadOfPrint]

  • In VZV-infected primary human brain vascular adventitial fibroblasts (BRAFs), IFN-bbeta;, STAT1 and STAT2 transcripts, as well as STAT1 and STAT2 protein were decreased. IFN-aalpha; transcripts were increased but not secreted IFN-aalpha; protein. Compared to IFN-aalpha;-treated controls, in VZV-infected BRAFs, phosphorylated STAT1 did not translocate to the nucleus, resulting in impaired downstream expression of interferon-inducible antiviral Mx1. Overall, VZV interference with the type I interferon pathway may promote virus persistence in cerebral arteries.

  • Careers in Virology: Teaching at a Primarily Undergraduate Institution [PublishAheadOfPrint]

  • A faculty position at a primarily undergraduate institution requires working with undergraduates both in the classroom and research lab. Graduate students and postdoctoral fellows who are interested in such a career should understand that faculty at these institutions need to teach broadly and devise research questions that can be addressed safely and with limited resources compared to a Research I university. Aspects of, and ways to prepare for, this career will be reviewed herein.

  • Virome analysis of Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis ticks reveals novel highly divergent vertebrate and invertebrate viruses [PublishAheadOfPrint]

  • A wide range of bacterial pathogens have been described in ticks, yet the diversity of viruses in ticks is largely unexplored. In the United States, Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis are among the principal tick species associated with pathogen transmission. We used high-throughput sequencing to characterize the viromes of these tick species and identified the presence of Powassan virus and eight novel viruses. These included the most divergent nairovirus described to date, two new clades of tick-borne phleboviruses, a mononegavirus, and viruses with similarity to plant and insect viruses. Our analysis revealed that ticks are reservoirs to a wide range of viruses and suggests that discovery and characterization of tick-borne viruses will have implications on viral taxonomy and may provide insight into tick-transmitted diseases.

    Importance Ticks are implicated as vectors of a wide array of human and animal pathogens. To better understand the extent of tick-borne diseases, it is crucial to uncover the full range of microbial agents associated with ticks. Our current knowledge of the diversity of tick-associated viruses is limited, in part due to the lack of investigation of tick viromes. In this study we examined the virome of three tick species from the United States. We found that ticks are hosts to highly divergent viruses across several taxa, including ones previously associated with human disease. Our data underscore the diversity of tick-associated viruses and provides the foundation for further studies into viral etiology of tick-borne diseases.

  • Short Self-interacting N-terminal Region of Rubella Virus Capsid Protein Is Essential for Cooperative Actions of Capsid and Nonstructural p150 Proteins [PublishAheadOfPrint]

  • Nucleocapsid formation is a primary function of the rubella virus capsid protein, which also promotes viral RNA synthesis via an unknown mechanism. The present study demonstrates that in infected cells, the capsid protein is associated with the nonstructural p150 protein via the short self-interacting N-terminal region of the capsid protein. Mutational analyses indicated that hydrophobic amino acids in this N-terminal region are essential for its N-terminal self-interaction, which is critical for the capsidmmdash;p150 association. An analysis based on a subgenomic replicon system demonstrated that the self-interacting N-terminal region of the capsid protein plays a key role in promoting viral gene expression. Analyses using a virus-like particle (VLP) system also showed that the self-interacting N-terminal region of the capsid protein is not essential for VLP production, but is critical for VLP infectivity. These results demonstrate that the close cooperative actions of the capsid protein and p150 require the short self-interacting N-terminal region of the capsid protein during the life cycle of the rubella virus.

    IMPORTANCE The capsid protein of rubella virus promotes viral RNA replication via an unknown mechanism. This protein interacts with the nonstructural protein p150, but the importance of this interaction is also unclear. In this study, we demonstrate that the short N-terminal region of the capsid protein forms a homo-oligomer that is critical for the capsidmmdash;p150 interaction. These interactions are required for the viral-gene-expression-promoting activity of the capsid protein, allowing efficient viral growth. These findings provide information about the mechanisms underlying the regulation of rubella virus RNA replication via the cooperative actions of the capsid protein and p150.

  • Characterization of the mode-of-action of a potent Dengue virus capsid inhibitor [PublishAheadOfPrint]

  • Dengue viruses (DV) represent a significant global health burden, with up to 400 million infections every year and around 500,000 infected individuals developing life-threatening disease. In spite of attempts in developing vaccine candidates and anti-viral drugs, there is currently a lack of approved therapeutics for the treatment of DV infection. We have previously reported the identification of ST-148, a small molecule inhibitor exhibiting a broad and potent antiviral activity against DV in vitro and in vivo. In the present study, we investigated the mode-of-action of this promising compound by using a combination of biochemical, virological and imaging-based techniques. We confirmed that ST-148 targets the capsid protein and obtained evidence of a bi-modal antiviral activity affecting both assembly/release and entry of infectious DV particles. Importantly, by using a robust bioluminescence resonance energy transfer-based assay we observed a ST-148-dependent increase of capsid self-interaction. These results were corroborated by molecular modeling studies that also revealed a plausible model for compound binding to capsid protein and inhibition by a distinct resistance mutation. These results suggest that ST-148-enhanced capsid protein self-interaction perturbs assembly and disassembly of DV nucleocapsids probably by inducing structural rigidity. Thus, as previously reported for other enveloped viruses, stabilization of capsid protein structure is an attractive therapeutic concept also applicable for flaviviruses.

    Importance Dengue viruses (DV) are arthropod-borne viruses representing a significant global health burden. They infect up to 400 million people and are endemic in subtropical and tropical areas of the world. Currently, there are neither vaccines nor approved therapeutics for the prophylaxis or the treatment of DV infections, respectively. This study reports the characterization of the mode-of-action of ST-148, a small-molecule capsid inhibitor with potent antiviral activity against all DV serotypes. Our results demonstrate that ST-148 stabilizes capsid protein self-interaction, thereby likely perturbing assembly and disassembly of viral nucleocapsids by inducing structural rigidity. This, in turn, might interfere with the release of viral RNA from incoming nucleocapsids (llsquo;uncoatingrrsquo;) as well as assembly of progeny virus particles. As previously reported for other enveloped viruses, we propose capsid as a novel tractable target for flavivirus inhibitors.

  • Antiviral Regulation in Porcine Monocytic Cells at Different Activation States [PublishAheadOfPrint]

  • Monocytic cells, including macrophages and dendritic cells, exist in different activation states that are critical to the regulation of antimicrobial immunity. Many pandemic viruses are monocytotropic, including porcine reproductive and respiratory syndrome virus (PRRSV), which directly infects subsets of monocytic cells and interferes in antiviral responses. To study antiviral responses in PRRSV-infected monocytic cells, we characterized inflammatory cytokine responses and genome-wide profiled signature genes to investigate response pathways in uninfected and PRRSV-infected monocytic cells at different activation states. Our findings showed suppressed interferon (IFN) production in macrophages at non-antiviral states and an arrest of lipid metabolic pathways in macrophages at antiviral states. Importantly, porcine monocytic cells at different activation states were susceptible to PRRSV and responded differently to viral infection. Based on gene ontology analysis, two approaches were used to potentiate antiviral activity: 1) pharmaceutical modulation of cellular lipid metabolism and 2) in situ PRRSV replication-competent expression of IFN-aalpha;; both approaches significantly suppressed exogenous viral infection in monocytic cells. In particular, the engineered IFN-expressing PRRSV strain eliminated exogenous virus infection and sustained cell viability at 4 days post infection in macrophages. These findings suggest an intricate interaction of viral infection with activation status of porcine monocytic cells. An understanding and integration of antiviral infection with activation status of monocytic cells may provide a means of potentiating antiviral immunity.

    IMPORTANCE: Activation statuses of monocytic cells including monocytes, macrophages (Ms) and dendritic cells (DCs) are critically important for antiviral immunity. Unfortunately, the activation status of porcine monocytic cells or how cell activation status functionally interacts with antiviral immunity remains largely unknown. This is a significant omission because many economically important porcine viruses are monocytotropic including our focus PRRSV, which alone causes near $800M economic loss annually in the US swine industries. PRRSV is ideal for deciphering how monocytic cell activation statuses interact with antiviral immunity, because it directly infects subsets of monocytic cells and subverts overall immune responses. In this study, we systematically investigate the activation status of porcine monocytic cells to determine intricate interaction of viral infection with activation statuses, and functionally regulate antiviral immunity within the framework of the activation paradigm. Our findings may provide a means of potentiating antiviral immunity and leading to novel vaccines for PRRS prevention.

  • Exosomes from Human Immunodeficiency virus type-1 (HIV-1)-infected cells license quiescent CD4+ T lymphocytes to replicate HIV-1 through a Nef- and ADAM17-dependent mechanism [PublishAheadOfPrint]

  • Resting CD4+ T lymphocytes resist HIV infection. Here, we provide evidence that exosomes from HIV-1 infected cells render resting human primary CD4+ T lymphocytes permissive to HIV-1 replication. These results were obtained with trans-well co-cultures of HIV-1 infected cells with quiescent CD4+ T lymphocytes in the presence of inhibitors of exosome release, and confirmed using exosomes purified from supernatants of HIV-1 infected primary CD4+ T lymphocytes. We found that the expression of HIV-1 Nef in exosome-producing cells is both necessary and sufficient for cell activation as well as HIV-1 replication in target CD4+ T lymphocytes. We also identified a Nef domain important for the effects we observed, i.e., the 62EEEE65 acidic cluster domain. In addition, we observed that ADAM17, i.e., a disintegrin and metalloprotease converting pro-TNFaalpha; in its mature form, associates with exosomes from HIV-1 infected cells, and plays a key role in the HIV-1 replication in quiescent CD4+ T lymphocytes. Treatment with an inhibitor of ADAM17 abolished both activation and HIV-1 replication in resting CD4+ T lymphocytes. TNFaalpha; is the downstream effector of ADAM17 since the treatment of resting lymphocytes with anti-TNFaalpha; antibodies blocked the HIV-1 replication. The here presented data are consistent with a model where Nef induces intercellular communication through exosomes to activate bystander quiescent CD4+ T lymphocytes thus stimulating viral spread.

    Importance Overall, our findings support the idea that HIV evolved to usurp the exosome-based intercellular communication network to favor its spread in infected host.

  • Stability determinants of Murine Cytomegalovirus long non-coding RNA7.2 [PublishAheadOfPrint]

  • Cytomegalovirus is a ubiquitous herpesvirus that persistently replicates in glandular epithelial tissue. Murine Cytomegalovirus expresses a 7.2 kb long non-coding RNA, (RNA7.2) that is a determinant of viral persistence in the salivary gland. RNA7.2 is an extremely long-lived intron, yet the basis of its stability is unknown. We present data that localize key sequence determinants of RNA stability to the 3rrsquo; end of RNA7.2, and suggest that stability is a result of sustained lariat conformation.

  • HIV-1 infection leads to increased transcription of HERV-K (HML-2) proviruses in vivo but not to increased virion production. [PublishAheadOfPrint]

  • Recent studies suggest that human endogenous retrovirus group K (HERV-K) provirus expression plays a role in the pathogenesis of HIV-1 infection. In particular, RNA from the HML-2 subgroup of HERV-K proviruses has been reported to be highly expressed at the cellular level and detectable in the plasma of HIV-1 infected patients, suggestive of virion production and, perhaps, replication. In this study, we developed an HML-2 specific quantitative PCR assay, which detects 51 of the 89 known HML-2 proviruses in the human genome. Plasma and peripheral blood mononuclear cells (PBMCs) from HIV-negative controls and HIV-1 infected patients were collected for analysis of HML-2 RNA expression. Contrary to previous reports, we did not detect high levels of HML-2 RNA in the plasma of HIV-1 infected patients, but did observe a significant increase of HML-2 RNA in total PBMCs as compared to HIV-negative controls. The level of HML-2 expression in PBMCs does not appear to be related to patient use of antiretrovirals or to HIV-1 plasma RNA, cellular RNA or cellular DNA levels. To investigate the source of HML-2 RNA expression, patient PBMCs were sorted into CD3+ CD4+, CD3+CD8+, CD3-CD14+ and CD3-CD20+ cell subsets and then analyzed for HML-2 RNA levels. No single cell subset was enriched for HML-2 RNA expression in HIV-1 infected patients, but there appears to be substantial variability in the level of HML-2 expression dependent on the cell type.

    Importance Here, we report that HERV-K (HML-2) proviruses are expressed at significantly higher levels in PBMCs from patients with HIV-1 infection compared to uninfected individuals. However, contrary to previous reports, this expression did not lead to detectable virions in the plasma of these patients. In addition, we found that HML-2 proviruses were expressed in multiple blood cell types from HIV-1 infected individuals, and the magnitude of HML-2 expression was not related to HIV-1 disease markers in this patient cohort. These findings may have implications for HML-2 based therapies targeting HIV-1 infection.

  • Virus-specific memory CD8 T cells provide substantial protection from lethal SARS-CoV infection [PublishAheadOfPrint]

  • The Severe Acute Respiratory Syndrome-coronavirus (SARS-CoV) caused an acute human respiratory illness with high morbidity and mortality in 2002-2003. Several studies have demonstrated the role of neutralizing antibodies induced by the spike (S) glycoprotein in protecting susceptible hosts from lethal infection. However, the anti-SARS-CoV antibody response is short-lived in SARS-recovered patients making it critical to develop additional vaccine strategies. SARS-CoV-specific memory CD8 T cells persisted for up to six years after SARS-CoV infection, a time at which memory B cells and anti-virus antibodies were undetectable in SARS-recovered individuals. Here, we assessed the ability of virus-specific memory CD8 T cells to mediate protection against infection in the absence of SARS-CoV-specific memory CD4 T or B cells. We demonstrate that memory CD8 T cells specific for a single immunodominant epitope (S436 or S525) substantially protected 8-10 month old mice from lethal SARS-CoV infection. Intravenous immunization with peptide-loaded dendritic cells (DCs) followed by intranasal boosting with recombinant vaccinia virus (rVV) encoding S436 or S525 resulted in accumulation of virus-specific memory CD8 T cells in alveolar lavage fluid (BAL), lungs and spleen. Upon challenge with a lethal dose of SARS-CoV, virus-specific memory CD8 T cells efficiently produced multiple effector cytokines (IFN-, TNF-aalpha; and IL-2) and cytolytic molecules (granzyme B) and reduced lung viral loads. Overall, our results show that SARS-CoV-specific memory CD8 T cells protect susceptible hosts from lethal SARS-CoV infection, but also suggest that SARS-CoV specific CD4 T cell and antibody responses are necessary for complete protection.

    IMPORTANCE: Virus-specific CD8 T cells are required for pathogen clearance following primary SARS-CoV infection. However, the role of SARS-CoV-specific memory CD8 T cells in mediating protection after SARS-CoV challenge has not been previously investigated. Here, using a prime-boost immunization approach, we show that virus-specific CD8 T cells protect susceptible 8-10 month old mice from lethal SARS-CoV challenge. Thus, future vaccines against emerging coronaviruses should emphasize the generation of a memory CD8 T cell response for optimal protection.

  • Evasion of siRNA-directed antiviral silencing in Musa acuminata persistently infected with six distinct banana streak pararetroviruses [PublishAheadOfPrint]

  • Vegetatively-propagated crop plants often suffer from infections with persistent RNA and DNA viruses. Such viruses appear to evade the plant defenses that normally restrict viral replication and spread. The major antiviral defense mechanism is based on RNA silencing generating viral short interfering RNAs (siRNAs) that can potentially repress viral genes post-transcriptionally through RNA cleavage and transcriptionally through DNA cytosine methylation. Here we examined the RNA silencing machinery of banana plants persistently infected with six pararetroviruses after many years of vegetative propagation. Using deep sequencing, we reconstructed consensus master genomes of the viruses and characterized virus-derived and endogenous small RNAs. Consistent with the presence of endogenous siRNAs that can potentially establish and maintain DNA methylation, the banana genomic DNA was extensively methylated in both healthy and virus-infected plants. A novel class of abundant 20-nucleotide (nt) endogenous small RNAs with 5rrsquo; -terminal guanosine was identified. In all virus-infected plants, 21-24 nt viral siRNAs accumulated at relatively high levels (up to 22% of total small RNA population) and covered the entire circular viral DNA genomes in both orientations. The hotspots of 21-nt and 22-nt siRNAs occurred within ORFs I, II and 5rrsquo; -portion of ORF III, while 24-nt siRNAs were more evenly distributed along the viral genome. Despite the presence of abundant viral siRNAs of different size-classes, the viral DNA was largely free of cytosine methylation. Thus, the virus is able to evade siRNA-directed DNA methylation and thereby avoid transcriptional silencing. This evasion of silencing likely contributes to the persistency of pararetroviruses in banana plants.

    Importance We report that DNA pararetroviruses in Musa acuminata banana plants are able to evade DNA cytosine methylation and transcriptional gene silencing, despite being targeted by the host silencing machinery generating abundant 21-24 nucleotide short interfering RNAs. At the same time the banana genomic DNA is extensively methylated in both healthy and virus-infected plants. Our findings shed light on the siRNA-generating gene silencing machinery of banana and provide possible explanation why episomal pararetroviruses can persist in plants, while true retroviruses having an obligatory genome-integration step in their replication cycle do not exist in plants.

  • Downregulation of miR-526a by Enterovirus Inhibits RIG-I-Dependent Innate Immune Response [PublishAheadOfPrint]

  • Retinoic acid-inducible gene I (RIG-I) is an intracellular RNA virus sensor that induces type I interferon-mediated host protective innate immunity against viral infection. Although cylindromatosis (CYLD) has been shown to negatively regulate innate antiviral response by removing K-63-linked polyubiquitin from RIG-I, the regulation of its expression and the underlying regulatory mechanisms are still incompletely understood. Here we show that RIG-I activity is regulated by miR-526a-mediated inhibition of CYLD expression. We found that viral infection specifically upregulates miR-526a expression in macrophages via IRF-dependent mechanisms. In turn, miR-526a positively regulates virus-triggered type I Interferon (IFN-I) production, thus suppressing viral replication, the underlying mechanism of which is the enhancement of RIG-I K63-linked ubiquitination by miR-526a via suppressing the expression of CYLD. Remarkably, viral-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein, while ectopic miR-526a expression inhibits the replication of EV71 virus. The collective results of this study suggest a novel mechanism of the regulation of RIG-I activity during RNA virus infection by miR-526a and propose a novel mechanism for the evasion of innate immune response controlled by EV71.

    Importance RNA virus infection upregulates the expression of miR-526a in macrophages through IRF-dependent pathways. In turn, miR-526a positively regulates virus-triggered type I IFN production and inhibits viral replication, the underlying mechanism of which is the enhancement of RIG-I K-63 ubiquitination by miR-526a via suppressing the expression of CYLD. Remarkably, viral-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein, cells with overexpressed miR-526a were highly resistant to EV71 infection. The collective results of this study suggest a novel mechanism of the regulation of RIG-I activity during RNA virus infection by miR-526a and propose a novel mechanism for the evasion of innate immune response controlled by EV71.

  • New insights into the non-conserved non-coding region of the subtype-determinant HA and NA segments of influenza A viruses [PublishAheadOfPrint]

  • The non-coding regions (NCRs) of the eight RNA segments of influenza A virus consist of the highly conserved promoter region and the non-conserved segment-specific NCRs at both the 3' and 5' ends. The roles of the segment-specific NCRs of the eight segments have been extensively studied. However, the diversities in the same region of the two subtype-determinant HA and NA segments received little attention. In this report, we bioinformatically analyzed all available NCRs of HA and NA vRNAs of influenza A viruses and found that nucleotides in the segment-specific NCRs of HA and NA vRNAs are subtype-specific and varied significantly in sequence and length at both the 3' and 5' ends among different subtypes. We then systematically studied the biological significance of the HA subtype-specific NCRs (HA ssNCRs) of those common HA subtypes (H1-H7 and H9) in the context of WSN (H1N1) reverse genetics system. We found that the HA ssNCRs play a critical role in HA vRNA virion incorporation. Upon the HA vRNA incorporation, the 3' end HA ssNCR plays a more critical role than the 5' end HA ssNCR and no stringent compatibility between the two ends is required. Furthermore, our data imply that, in addition to particular nucleotide(s), the length of the HA ssNCR is involved in regulating HA vRNA incorporation efficiency. These results provide new insights into the HA segment virion incorporation that is critical for the emergence of epidemic and pandemic influenza A virus strains.

    IMPORTANCE The non-conserved non-coding regions (NCRs) of the eight segments of influenza A virus have been extensively studied whereas the diversities in the non-conserved NCRs of the two subtype-determinant segments HA and NA received little attention. In this report, we bioinformatically analyzed all available NCRs of HA and NA vRNAs and discovered that HA and NA vRNAs contain key subtype signatures in the NCR. Our functional studies of the HA ssNCRs of those common HA subtypes in the context of WSN virus (H1N1) demonstrated that the HA ssNCR modulates virus replication efficiency by influencing HA segment virion incorporation. Moreover, we revealed important features of the HA ssNCR in determining HA vRNA incorporation efficiency. These data not only show new genetic characteristics to influenza A viruses, but also provide further evidence in understanding the selective genome packaging of influenza virus required for the emergence of epidemic and pandemic influenza virus strains.

  • Deep sequencing of norovirus genomes defines evolutionary patterns in an urban tropical setting [PublishAheadOfPrint]

  • Norovirus is a highly transmissible infectious agent that causes epidemic gastroenteritis in susceptible children and adults. Norovirus infections can be severe and can be initiated from an exceptionally small number of viral particles. Detailed genome sequence data are useful for tracking norovirus transmission and evolution. To address this need we have developed a whole-genome deep sequencing method that generates entire genome sequences from small amounts of clinical specimens. This novel approach employs an algorithm for reverse transcription and PCR amplification primer design using all publically-available norovirus sequence data. Deep sequencing and de novo assembly were used to generate norovirus genomes from a large set of diarrheal patients attending 3 hospitals in Ho Chi Minh City, Vietnam, over a 2.5-year period. Positive selection analysis and direct examination of protein changes in the virus over time identified codons in the regions encoding proteins VP1, p48 (NS1-2) and p22 (NS4) under positive selection and expands the known targets of norovirus evolutionary pressure.

    Importance The high transmissibility and rapid evolution rate of norovirus, combined with short-lived host immune responses, are thought to be responsible for the virus causing a majority of pediatric viral diarrhea cases. The evolutionary patterns of this RNA virus have only been described in detail for a portion of the virus genome and never from a detailed urban tropical setting. We have developed robust deep sequencing methods for generating complete genome sequences directly from small amounts of patient fecal material. We use this method to provide a detailed sequence description of the noroviruses circulating in three Ho Chi Minh City hospitals over a 2.5-year period. The study identified patterns of virus change in known sites of host immune response and identified three additional regions of the virus genome under selection that were not previously recognized. In addition, the methods described here provide a robust full-genome sequencing platform for community-based virus surveillance.

  • Contribution of silent mutations to thermal adaptation of RNA bacteriophage Q{beta} [PublishAheadOfPrint]

  • Changes in protein function and other biological properties, such as RNA structure, are crucial for adaptation of organisms to novel or inhibitory environments. To investigate how mutations that do not alter amino acid sequence may be positively selected, we performed a thermal adaptation experiment using the single-stranded RNA bacteriophage Qbbeta; where the culture temperature was increased from 37.2ddeg;C to 41.2ddeg;C and finally to an inhibitory temperature of 43.6ddeg;C in a stepwise manner in three independent lines. Whole-genome analysis revealed 31 mutations, including 14 mutations that did not result in amino acid sequence alterations, in this thermal adaptation. Eight of the 31 mutations were observed in all three lines. Reconstruction and fitness analyses of Qbbeta; strains containing only mutations observed in all three lines indicated that five mutations that did not result in amino acid sequence changes but increased the amplification ratio appeared in the course of adaptation to growth at 41.2ddeg;C. Moreover, these mutations provided a suitable genetic background for subsequent mutations, altering the fitness contribution from deleterious to beneficial. These results clearly showed that mutations that do not alter the amino acid sequence play important roles in adaptation of this single-stranded RNA virus to elevated temperature.

    Importance Recent studies using whole-genome analysis technology suggested the importance of mutations that do not alter the amino acid sequence for adaptation of organisms to novel environmental conditions. It is necessary to investigate how these mutations may be positively selected and to determine to what degree such mutations that do not alter amino acid sequences contribute to adaptive evolution. Here, we report the roles of these silent mutations in thermal adaptation of RNA bacteriophage Qbbeta; based on experimental evolution where Qbbeta; showed adaptation to growth at an inhibitory temperature. Intriguingly, four synonymous mutations and one mutation in the untranslated region that spread widely in the Qbbeta; population during the adaptation process at moderately high temperature provided a suitable genetic background to alter the fitness contribution of subsequent mutations from deleterious to beneficial at a higher temperature.

  • Immunodominance Changes as a Function of the Infecting Dengue Virus Serotype and Primary versus Secondary Infection [PublishAheadOfPrint]

  • Dengue virus (DENV) is the causative agent of dengue fever (DF). Disease can be caused by any of four DENV serotypes (DENV1-4), sharing 67-75% sequence homology with one another. The effect of subsequent infections with different serotypes on the T cell repertoire is not fully understood. We utilized mice transgenic for human leukocyte antigens (HLA) lacking the IFN -aalpha;/bbeta; receptor to study responses to heterologous DENV infection. First, we defined the primary T cell response to DENV3 in the context of a wide range of HLA molecules. The primary DENV3 immune response recognized epitopes derived from all 10 DENV proteins, with a significant fraction of the response specific for structural proteins. This is in contrast to primary DENV2 infection where structural proteins are a minor component of the response, suggesting differential antigen immunodominance as a function of the infecting serotype. We next investigated the effect of secondary heterologous DENV infection on the T cell repertoire. In the case of both DENV2/3 and DENV3/2 heterologous infections, recognition of conserved/cross-reactive epitopes was either constant or expanded, as compared to homologous infection. Furthermore, in heterologous infection, previous infection with a different serotype impaired the development of responses directed to serotype specific but not conserved epitopes. Thus, a detrimental effect of previous heterotypic responses might not be due to dysfunctional and weakly crossreactive epitopes dominating the response. Rather, responses to the original serotype might limit the magnitude of responses directed against epitopes that are either crossreactive or specific for the most recently infecting serotype.

    Importance DENV transmission occurs in more than 100 countries and is an increasing public health problem in tropical and sub-tropical regions. At present, no effective antiviral therapy or licensed vaccine exists, and treatment is largely supportive in nature. Disease can be caused by any of the four DENV serotypes (DENV1-4), which share a high degree of sequence homology with one another. In this study we have addressed the question how the T cell repertoire changes as a function of infections with different serotypes and of subsequent heterologous secondary infections. This is of particular interest in the field of dengue viruses (DENV) where secondary infections with a different DENV serotypes increase the risk of severe disease. Our results on the evolution of the immune response after primary and secondary infection provides new insights into HLA-restricted T cell responses against DENV relevant for vaccine design against DENV

  • Genome-wide DNA methylation as an epigenetic consequence of Epstein-Barr virus infection of immortalized keratinocytes [PublishAheadOfPrint]

  • The oral cavity is a persistent reservoir for Epstein-Barr virus (EBV) with lifelong infection of resident epithelial and B cells. Infection of these cell types results in distinct EBV gene expression patterns regulated by epigenetic modifications involving DNA methylation and chromatin structure. Regulation of EBV gene expression relies on viral manipulation of the host epigenetic machinery that may result in long-lasting host epigenetic reprogramming. To identify epigenetic events following EBV infection, a transient infection model was established to map epigenetic changes in telomerase-immortalized oral keratinocytes. EBV-infected oral keratinocytes exhibited a predominantly latent viral gene expression program with some lytic or abortive replication. Calcium and methylcellulose-induced differentiation was delayed in EBV-positive and in clones that lost EBV compared to uninfected controls, indicating a functional consequence of EBV epigenetic modifications. Analysis of global cellular DNA methylation identified over 13,000 differentially methylated CpG residues in cells exposed to EBV compared to uninfected controls, with CpG island hypermethylation observed at several cellular genes. Although the vast majority of the DNA methylation changes were silent, 65 cellular genes that acquired CpG methylation showed altered transcript levels. Genes with increased transcript levels frequently acquired DNA methylation within the gene body while those with decreased transcript levels acquired DNA methylation near the transcription start site. Treatment with the DNA methyltransferase inhibitor, decitabine, restored expression of some hypermethylated genes in EBV-infected and EBV-negative transiently-infected clones. Overall, these observations suggested that EBV infection of keratinocytes leaves a lasting epigenetic imprint that can enhance the tumorigenic phenotype of infected cells.

    Importance Here we show that EBV infection of oral keratinocytes led to CpG island hypermethylation as an epigenetic scar of prior EBV infection that was retained after loss of the virus. Such EBV-induced epigenetic modification recapitulated the hypermethylated CpG island methylator phenotype (CIMP) observed in EBV-associated carcinomas. These epigenetic alterations not only impacted gene expression but resulted in delayed calcium and methylcellulose-induced keratinocyte differentiation. Importantly, these epigenetic changes occurred in cells that were not as genetically unstable as carcinoma cells indicating that EBV infection induced an epigenetic mutator phenotype. The impact of this work is that we have provided a mechanistic framework for how a tumor virus using the epigenetic machinery can act in a "hit-and-run fashion" with retention of epigenetic alterations after loss of the virus. Unlike genetic alterations, these virally-induced epigenetic changes can be reversed pharmacologically providing therapeutic interventions to EBV-associated malignancies.

  • Rational design of human metapneumovirus live attenuated vaccine candidates by inhibiting viral messenger RNA cap methyltransferase [PublishAheadOfPrint]

  • The paramyxoviruses, human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), and human parainfluenza virus 3 (hPIV3) are responsible for the majority of pediatric respiratory diseases and inflict significant economic loss, health costs, and emotional burdens. Despite major efforts, there are no vaccines available for these viruses. The conserved region VI (CR-VI) of the large (L) polymerase proteins of paramyxoviruses catalyzes methyltransferase (MTase) activities that typically methylate viral mRNAs at positions guanine N-7 (G-N-7) and ribose 2rrsquo; -O. In this study, we generated a panel of recombinant hMPVs carrying mutations in the S-adenosyl methionine (SAM) binding site in the CR-VI of L protein. These recombinant viruses were specifically defective in ribose 2rrsquo; -O, but not G-N-7 methylation, and were genetically stable and highly attenuated in cell culture and viral replication in the upper and lower respiratory tracts of cotton rats. Importantly, cotton rats vaccinated with these MTase-defective rhMPVs triggered a high level of neutralizing antibody and were completely protected from challenge with wild-type rhMPV. Collectively, our results indicate that (i) amino acid residues in the SAM binding site in hMPV L protein are essential for 2rrsquo; -O methylation, and (ii) inhibition of mRNA cap. MTase can serve as a novel target to rationally design live attenuated vaccines for hMPV, and perhaps other paramyxoviruses such as RSV and PIV3.

    Importance Human paramyxoviruses including RSV, hMPV, and PIV3 cause the majority of acute upper and lower respiratory tract infection in humans, particularly in infants, children, the elderly, and immunocompromised individuals. Currently, there is no licensed vaccine available. A formalin-inactivated vaccine is not suitable for these viruses because it causes enhanced lung damage upon reinfection with the same virus. A live attenuated vaccine is the most promising vaccine strategy for human paramyxoviruses. However, it remains a challenge to identify an attenuated virus strain that has an optimal balance between attenuation and immunogenicity. Using reverse genetics, we generated a panel of recombinant hMPVs that were specifically defective in ribose 2rrsquo; -O, but not G-N-7 methyltransferase (MTase). These MTase-defective hMPVs were genetically stable and sufficiently attenuated but retained high immunogenicity. This work highlights a critical role of 2rrsquo; -O MTase in paramyxovirus replication and pathogenesis, and a new avenue for the development of safe and efficacy live attenuated vaccines for hMPV and other human paramyxoviruses.

  • Kaposi's sarcoma-associated herpesvirus ORF18 and ORF30 are essential for late gene expression during lytic replication [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several human malignances. As saliva is likely the major vehicle for KSHV transmission, we studied in vitro KSHV infection of oral epithelial cells. Through infecting two types of oral epithelial cells, normal human oral keratinocytes (NHOKs) and papilloma-immortalized human oral keratinocyte (HOK16B) cells, we found that KSHV can undergo robust lytic replication in oral epithelial cells. By employing de novo lytic infection of HOK16B cells, we studied the function of two previously uncharacterized genes, ORF18 and ORF30, during the KSHV lytic cycle. For this purpose, an ORF18-deficient virus and an ORF30-deficient virus were generated using a mutagenesis strategy based on bacterial artificial chromosome (BAC) technology. We found that neither ORF18 nor ORF30 is required for immediately early, early gene expression, or viral DNA replication, but each is essential for late gene expression during both de novo lytic replication and reactivation. This critical role of ORF18 and ORF30 in late gene expression was also observed during KSHV reactivation. In addition, global analysis of viral transcripts by RNA-sequencing indicated that ORF18 and ORF30 control the same set of viral genes. Therefore, we suggest that these two viral ORFs are involved in the same mechanism or pathway that co-regulates the viral late genes as a group.

    Importance While KSHV can infect multiple cell types in vitro, only a few can support a full lytic replication cycle with progeny virions produced. Consequently, KSHV lytic replication is mostly studied through reactivation, which requires chemicals to induce the lytic cycle or over-expression of the viral transcriptional activator, RTA. In this study, we presented a robust de novo lytic infection system based on oral epithelial cells. Using this system, we demonstrated the role of two viral ORFs, ORF18 and ORF30, in regulating the viral gene expression during KSHV lytic replication. As the major route of KSHV transmission is thought to be via saliva, this new KSHV lytic replication system will have important utility in the field.

  • Plasmacytoid dendritic cells mediate synergistic effects of HIV and LPS on CD27+IgD- memory B cell apoptosis [PublishAheadOfPrint]

  • Background: The effects of heightened microbial translocation on B cells during HIV infection are unknown.

    Methods: We examined the in vitro effects of HIV and LPS on apoptosis of CD27+IgDnndash; memory B (mB) cell from healthy controls. In vivo analysis was conducted on a cohort of 82 HIV+ donors and 60 healthy controls.

    Results: In vitro exposure of PBMCs to LPS and HIV led to mB cell death via the Fas/Fas ligand (FasL) pathway. Plasmacytoid dendritic cells (pDCs) produced FasL in response to HIV via binding to CD4 and chemokine co-receptors. HIV and LPS increased Fas expression on mB cells in PBMCs which was dependent on the presence of pDCs and monocytes. Furthermore, mB cells purified from PBMCs and pretreated with both HIV and LPS were more sensitive to apoptosis when co-cultured with HIV-treated pDCs. Blocking the interferon receptor (IFNR) prevented HIV-stimulated FasL production in pDCs, HIV+LPS-induced Fas expression, and apoptosis of mB cells. In vivo or ex vivo, HIV+ donors have higher levels of plasma LPS, Fas expression on mB cells, and mB cell apoptosis compared to controls. Correspondingly, in HIV+ donors but not in controls, a positive correlation was found between plasma levels of FasL and HIV RNA, and between Fas expression on mB cells and plasma levels of LPS.

    Conclusion: This work reveals a novel mechanism of mB cell apoptosis mediated by LPS and HIV through the Fas/FasL pathway, with key involvement of pDCs and type I IFN, suggesting a role for microbial translocation in HIV pathogenesis.

    Importance: This study demonstrates that LPS and type I IFN, play an important role in memory B cell apoptosis in HIV infection. It reveals a previously unrecognized role of microbial translocation in HIV pathogenesis.

  • Inducible gene-expression in tumors colonized by modified oncolytic vaccinia virus strains [PublishAheadOfPrint]

  • Exogenous gene induction of therapeutic, diagnostic and safety mechanism could be a considerable improvement in oncolytic virotherapy. Here, we introduced a doxycycline-inducible promoter system (comprised of a tetracycline-repressor, several promoter constructs and a tet-operator sequence) into oncolytic recombinant vaccinia viruses (rVACV), which was further characterized in detail. Experiments in cell culture as well as in tumor bearing mice were analyzed to determine the role of the inducible system components. To accomplish this, we took advantage of the optical reporter construct, which resulted in the production of click-beetle luciferase as well as a red fluorescent protein. The results indicated that each of the system components could be used to optimize the induction rates and had influence on the background expression levels. Depending on a given gene to be induced in rVACV colonized tumors of patients, we discuss the doxycycline-inducible promoter system adjustment and further optimization.

    IMPORTANCE Oncolytic virotherapy of cancer can greatly benefit from the expression of heterologous genes. It is reasonable that some of those heterologous gene products could have detrimental effects on either the cancer patient or on the oncolytic virus itself if expressed at the wrong time or if the expression levels are too high. Therefore, exogenous control of gene expression levels by administration of a non-toxic inducer will have positive effects on the safety as well as the therapeutic outcome of oncolytic virotherapy. In addition, it paves the way for introduction of new therapeutic genes into the genome of oncolytic viruses that could not have been tested otherwise.

  • Molecular Characterization of a Lizard Adenovirus Reveals The First Atadenovirus with two Fiber Genes, and the First Adenovirus with Either One Short or Three Long Fibers per Penton [PublishAheadOfPrint]

  • Although adenoviruses have been found in a wide variety of reptiles including numerous squamate species, turtles and crocodiles, the number of reptilian adenovirus isolates is still scarce. The only fully sequenced reptilian adenovirus, snake adenovirus 1 (SnAdV-1), belongs to the Atadenovirus genus. Recently, two new atadenoviruses were isolated from a captive Gila monster (Heloderma suspectum) and Mexican beaded lizards (H. horridum). Here we report the full genomic and proteomic characterization of the latter, designated as lizard adenovirus 2 (LAdV-2). The dsDNA genome of LAdV-2 is 32,965 bp long with an average G+C content of 44.16%. The overall arrangement and gene content of the LAdV-2 genome was largely concordant with those in other atadenoviruses, except for four novel ORFs at the right end of the genome. Phylogeny reconstructions and plesiomorphic traits, shared with SnAdV-1, further supported the assignment of LAdV-2 to the Atadenovirus genus. Surprisingly, two fiber genes were found for the first time in an atadenovirus. After optimizing the production of LAdV-2 in cell culture, we determined the protein composition of the virions. The two fiber genes produce two fiber proteins of different size that are incorporated into the viral particles. Interestingly, the two different fiber proteins assemble as either one short or three long fiber projections per vertex. Stoichiometry estimations indicate that the long fiber triplet is present at only one or two vertices per virion. Neither triple fibers, nor a mixed number of fibers per vertex, had previously been reported for adenoviruses, or any other virus.

    IMPORTANCE Here we show that a lizard adenovirus, LAdV-2, has a penton architecture never observed before. LAdV-2 expresses two fiber proteins, one short and one long. In the virion, most vertices have one short fiber, but a few of them have three long fibers attached to the same penton base. This observation raises new intriguing questions on virus structure. How can the triple fiber attach to a pentameric vertex? What determines the number and location of each vertex type in the icosahedral particle? Since fibers are responsible for primary attachment to the host, this novel architecture also suggests a novel mode of cell entry for LAdV-2. Adenoviruses have a recognized potential in nanobiomedicine, but only a few of the more than 200 types found so far in nature have been characterized in detail. Exploring the taxonomic wealth of adenoviruses should improve our chances to successfully use them as therapeutic tools.

  • Phosphorylation of hepatitis C virus RNA polymerase Ser29 and Ser42 by PRK2 regulates viral RNA replication [PublishAheadOfPrint]

  • The hepatitis C virus (HCV) nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase (RdRp), is the key enzyme for HCV RNA replication. We previously showed that HCV RdRp is phosphorylated by protein kinase C-related kinase 2 (PRK2). In the present study, we used biochemical and reverse genetic approaches to demonstrate that HCV NS5B phosphorylation is crucial for viral RNA replication in cell culture. Two-dimensional phosphoamino acid analysis revealed that PRK2 phosphorylates NS5B exclusively at its serine residues in vitro and in vivo. Using in vitro kinase assays and mass spectrometry, we identified two phosphorylation sites, Ser29 and Ser42, on the 1 finger loop region that interacts with the thumb subdomain of NS5B. Colony-forming assays using drug-selectable HCV subgenomic RNA replicons revealed that preventing phosphorylation by Ala substitution at either Ser29 or Ser42 impairs HCV RNA replication. Furthermore, reverse genetic studies using HCV infectious clones encoding phosphorylation-defective NS5B confirmed the crucial role of these PRK2 phosphorylation sites in viral RNA replication. Molecular modeling studies predicted that the phosphorylation of NS5B stabilizes the interactions between its 1 loop and thumb subdomain, which are required for the formation of the closed conformation of NS5B known to be important for de novo RNA synthesis. Collectively, our results provide evidence that HCV NS5B phosphorylation has a positive regulatory role in HCV RNA replication.

    IMPORTANCE While the role of RdRps in viral RNA replication is clear, little is known about their functional regulation by phosphorylation. In this study, we addressed several important questions about the function and structure of phosphorylated HCV NS5B protein. Reverse genetic studies with HCV replicons encoding phosphorylation-defective NS5B mutants and analysis of their RdRp activity revealed previously unidentified NS5B protein features related to HCV replication and NS5B phosphorylation. These attributes most likely reflect potential structural changes induced by phosphorylation at the 1 finger loop region of NS5B with two identified phosphate acceptor sites, Ser29 and Ser42, which may transiently affect the closed conformation of NS5B. Elucidating the effects of dynamic changes in NS5B phosphorylation status during viral replication and its impact on RNA synthesis will improve our understanding of the molecular mechanisms of NS5B phosphorylation-mediated regulation of HCV replication.

  • The Epstein-Barr Virus EBNA1 Protein Regulates Viral latency Through Effects on Let-7 microRNA and Dicer [PublishAheadOfPrint]

  • The EBNA1 protein of Epstein-Barr virus (EBV) plays multiple roles in EBV latent infection including altering cellular pathways relevant for cancer. Here we have used microRNA cloning coupled with high throughput sequencing to identify the effects of EBNA1 on cellular miRNAs in two nasopharyngeal carcinoma cell lines. EBNA1 affected a small percentage of cellular miRNAs in both cell lines, in particular up-regulating multiple let-7 family miRNAs, including let-7a. EBNA1 effects on let-7a were verified by demonstrating that EBNA1 silencing in multiple EBV-positive carcinomas down-regulated let-7a. Accordingly, the let-7a target, Dicer, was found to be partially down-regulated by EBNA1 expression (at mRNA and protein levels) and up-regulated by EBNA1 silencing in EBV-positive cells. Reporter assays based on the Dicer 3rrsquo; UTR with and without let-7a target sites indicated that the effects of EBNA1 on Dicer were mediated by let-7a. EBNA1 was also found to induce the expression of let-7a primary RNAs in a manner dependent on the EBNA1 transcriptional activation region, suggesting that EBNA1 induces let-7a by transactivating the expression of its primary transcripts. Consistent with previous reports that Dicer promotes EBV reactivation, we found that a let-7a mimic inhibited EBV reactivation to the lytic cycle while a let-7 sponge increased reactivation. The results provide a mechanism by which EBNA1 could promote EBV latency by inducing let-7 miRNAs.

    IMPORTANCE The EBNA1 protein of Epstein-Barr virus (EBV) contributes in multiple ways to the latent mode of EBV infection that leads to life-long infection. In this study we identify a mechanism by which EBNA1 helps to maintain EBV infection in a latent state. This involves induction of a family of microRNAs (let-7 miRNA) that in turn decrease the level of the cellular protein Dicer. We demonstrate that let-7 miRNAs inhibit the reactivation of latent EBV, providing an explanation for our previous observation that EBNA1 promotes latency. In addition, since decreased levels of Dicer have been associated with metastatic potential, EBNA1 may increase metastases by down-regulating Dicer.

  • Dynamic regulation of host restriction factor expression over the course of HIV-1 infection in vivo [PublishAheadOfPrint]

  • In this study, we investigated the expression levels of host restriction factors in six untreated HIV-1-positive patients over the course of infection. We found that the host restriction factor gene expression profile consistently increased over time and significantly associated with CD4+ T cell activation and viral load. Our data are among the first to demonstrate the dynamic nature of host restriction factors in vivo over time.

  • Structural insights into the human metapneumovirus Glycoprotein ectodomain [PublishAheadOfPrint]

  • Human metapneumovirus is a major cause of respiratory tract infections worldwide. Previous reports have shown that the viral glycoprotein (G) modulates innate and adaptive immune responses, leading to incomplete immunity and promoting reinfection. Using bioinformatics analyses, static light scattering and small-angle x-ray scattering, we show that the extracellular region of G behaves as a heavily glycosylated, instrinsically disordered polymer. We discuss potential implications of these findings for the modulation of immune responses by G.

  • Novel indole-2-carboxamide compounds are potent broad spectrum antivirals active against western equine encephalitis virus in vivo [PublishAheadOfPrint]

  • Neurotropic alphaviruses, including western, eastern, and Venezuela equine encephalitis viruses, cause serious and potentially fatal central nervous system infections in humans, for which no currently approved therapies exist. We previously identified a series of thieno[3,2-b]pyrrole derivatives as novel inhibitors of neurotropic alphavirus replication using a cell-based phenotypic assay, and subsequently developed second and third generation indole-2-carboxamide derivatives with improved potency, solubility, and metabolic stability. In this report, we describe the antiviral activity of the most promising third generation lead compound, CCG205432, and closely related analogs, CCG206381 and CCG209023. These compounds have half maximal inhibitory concentrations ~1 mmu;M and selectivity indices ggt;100 in cell-based assays using western equine encephalitis virus replicons. Furthermore, CCG205432 retains similar potency against fully infectious virus in cultured human neuronal cells. These compounds show broad inhibitory activity against a range of RNA viruses in culture, including members of the Togaviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae families. Although their exact molecular target remains unknown, mechanism of action studies reveal that these novel indole-based compounds target a host factor that modulates cap-dependent translation. Finally, we demonstrate that both CCG205432 and CCG209023 dampen clinical disease severity and enhance survival of mice given a lethal western equine encephalitis virus challenge. These studies demonstrate that indole-2-carboxamide compounds are viable candidates for continued preclinical development as inhibitors of neurotropic alphaviruses, and potentially other RNA viruses.

    IMPORTANCE There are currently no approved drugs to treat infections with alphaviruses. We previously identified a novel series of compounds with activity against these potentially devastating pathogens. We have now produced third generation compounds with enhanced potency, and this manuscript provides detailed information on the antiviral activity of these advanced generation compounds, including activity in an animal model. The results of this study represent a notable achievement in the continued development of this novel class of antiviral inhibitors.

  • Herpes simplex virus gE/gI extracellular domains promote axonal transport and spread from neurons to epithelial cells. [PublishAheadOfPrint]

  • Following reactivation from latency, there are two distinct steps in the spread of herpes simplex virus (HSV) from infected neurons to epithelial cells: i) anterograde axonal transport of virus particles from neuron cell bodies to axon tips and ii) exocytosis and spread of extracellular virions across cell junctions into adjacent epithelial cells. HSV heterodimeric glycoprotein gE/gI is important for anterograde axonal transport and gE/gI cytoplasmic domains play important roles in sorting of virus particles into axons. However, the roles of the large (~400 residue) gE/gI extracellular (ET) domains in both axonal transport and neuron-to-epithelial cell spread have not been characterized. Two gE mutants: gE-277 and gE-348 contain small insertions in the gE ET domain, fold normally, form gE/gI heterodimers and are incorporated into virions. Both gE-277 and gE-348 did not function in anterograde axonal transport, there was markedly reduced numbers of viral capsids and glycoproteins, compared with wild type HSV. The defects in axonal transport were manifest in neuronal cell bodies involving missorting of HSV capsids before entry into proximal axons. Although there were diminished numbers of mutant gE-348 capsids and glycoproteins in distal axons, there was efficient spread to adjacent epithelial cells, similar to wild type HSV. By contrast, virus particles produced by HSV gE-277 spread poorly to epithelial cells, despite similar numbers of virus particles as gE-348. These results genetically separate the two steps in HSV spread from neurons to epithelial cells and demonstrate that the gE/gI ET domains functions in both processes.

    IMPORTANCE An essential phase of the life cycle of herpes simplex virus (HSV) and other aalpha;-herpesviruses is the capacity to reactivate from latency then spread from infected neurons to epithelial tissues. This spread involves at least two steps: i) anterograde transport to axon tips, followed by ii) exocytosis and extracellular spread from axons to epithelial cells. HSV gE/gI is a glycoprotein that facilitates this virus spread, although by poorly understood mechanisms. Here, we show that the extracellular (ET) domains of gE/gI promote the sorting of viral structural proteins into proximal axons to begin axonal transport. However, the gE/gI ET domains also participate in the extracellular spread from axon tips across cell junctions to epithelial cells. Understanding the molecular mechanisms involved in gE/gI-mediated sorting of virus particles into axons and extracellular spread to adjacent cells is fundamentally important in terms of identifying novel targets to reduce aalpha;-herpesvirus disease.

  • In Vivo Reactivation of Latent Herpes Simplex Virus 1 in Mice Can Occur in the Brain before in the Trigeminal Ganglion [PublishAheadOfPrint]

  • Herpes simplex virus 1 (HSV-1) establishes latency in neurons of brains and sensory ganglia of humans and experimentally infected mice. The latent virus can reactivate to cause recurrent infection. Both primary and recurrent infections can induce diseases, such as encephalitis. In humans, the majority of encephalitis cases occur as a recurrent infection. However, in the past, numerous mouse studies documented that viral reactivation occurs efficiently in the ganglion, but extremely rarely in the brain, when assessed ex vivo by cultivating minced tissue explants. Here we compare the brains and the trigeminal ganglia of mice latently infected with HSV-1 (strain 294.1 or McKrae) for levels of viral genomes and in vivo reactivation. The amounts of 294.1 and McKrae genomes in the brain stem were significantly higher than those in the trigeminal ganglion. Most importantly, 294.1 and McKrae reactivation was detected in the brain stem before in the trigeminal ganglion of mice treated with hyperthermia to reactivate latent virus in vivo. In addition, the brain stem yielded reactivated virus with a high frequency when compared with the trigeminal ganglion, especially in mice latently infected with 294.1 after hyperthermia treatment. These results provide evidence that the recurrent brain infection can be induced by the reactivation of latent virus in the brain in situ.

    IMPORTANCE HSV-1 establishes latency in neurons of brains and sensory ganglia of humans and experimentally infected mice. The latent virus can reactivate to cause recurrent infection. In the past, studies of viral reactivation focused on the ganglion, because efficient viral reactivation was detected in the ganglion, but not in the brain, when assessed ex vivo by cultivating mouse tissue explants. In this study, we report that the brain contains more viral genomes than the trigeminal ganglion of latently infected mice. Notably, the brain yields reactivated virus early and efficiently when compared with the trigeminal ganglion after mice are stimulated to reactivate latent virus. Our findings raise the potential importance of HSV-1 latent infection and reactivation in the brain.

  • A viral protein mediates superinfection exclusion at the whole organism level while is not required for exclusion at the cellular level [PublishAheadOfPrint]

  • Superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by the same or closely related virus, has been described for different viruses, including important pathogens of humans, animals, and plants. Citrus tristeza virus (CTV), a positive-sense RNA virus, represents a valuable model system for studying SIE due to the existence of several phylogenetically distinct strains. Furthermore, CTV allows for the possibility of examining SIE at the whole organism level. Previously we demonstrated that SIE by CTV is a virus-controlled function that requires the viral protein p33. In this study, we show that p33 mediates SIE at the whole organism level while it is not required for exclusion at the cellular level. Primary infection of a host with a fluorescent protein-tagged CTV variant lacking p33 did not interfere with the establishment of a secondary infection by the same virus labeled with a different fluorescent protein. However, cellular coinfection by both viruses was rare. The obtained observations along with estimates of the cellular multiplicity of infection (MOI) and MOI model selection suggested that low levels of cellular coinfection appear to be best explained by exclusion at the cellular level. Based on these results, we propose that SIE by CTV is operated at two levels mmdash; the cellular and the whole organism levels mmdash; by two distinct mechanisms that could function independently. This novel aspect of viral SIE highlights the intriguing complexity of this phenomenon further understanding of which may open up new avenues to manage virus diseases.

    Importance Many viruses exhibit superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by related viruses. SIE plays an important role in pathogenesis and evolution of virus populations. The observations described here suggest that SIE could be controlled independently at different levels of the host: the whole organism or individual cells. The p33 protein encoded by Citrus tristeza virus (CTV), an RNA virus, was shown to mediate SIE at the whole organism level, while it appeared not to be required for exclusion at the cellular level. SIE by CTV is, therefore, highly complex and appears to use different mechanisms than those proposed for other viruses. A better understanding of this phenomenon may lead to the development of new strategies for controlling viral diseases in human populations and agro-ecosystems.

  • Seed-sequence matched controls reveal limitations of siRNA knockdown in functional and structural studies of HCV NS5A-MOBKL1B interaction [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) is a widespread human pathogen causing liver cirrhosis and cancer. Similar to other viruses, HCV depends on host and viral factors to complete its life cycle. We used proteomic and yeast two-hybrid approaches to elucidate host factors involved in HCV nonstructural protein NS5A function and found that MOBKL1B interacts with NS5A. Initial experiments with siRNA knockdown suggesting a role in HCV replication led us to examine the interaction using biochemical and structural approaches. As revealed by a co-crystal structure of a core MOBKL1Bmmdash;NS5A peptide complex at 1.95 AAring;, NS5A binds to a hydrophobic patch on the MOBKL1B surface. Biosensor binding assays identified a highly conserved, 18 amino acid binding site in domain II of NS5A, which encompasses residues implicated in cyclophilin A (CypA)-dependent HCV RNA replication. However, a CypA-independent HCV variant had reduced replication in MOBKL1B knockdown cells, even though its NS5A does not interact with MOBKL1B. These discordant results prompted more extensive studies of MOBKL1B gene knockdowns, which included additional siRNAs and specifically matched seed-sequence siRNA controls. We found that reduced virus replication after treating cells with MOBKL1B siRNA was actually due to off-target inhibition, and indicated that the initial finding of virus replication dependence on the MOBKL1B-NS5A interaction was incorrect. Ultimately, using several approaches we found no relationship of the MOBKL1B-NS5A interaction to virus replication. These findings collectively serve as a reminder to investigators and scientific reviewers of the pervasive impact of siRNA off-target effects on interpretation of biological data.

    Importance Our study illustrates an underappreciated shortcoming of siRNA gene knockdown technology. We initially identified a cellular protein, MOBKL1B, as a binding partner with the NS5A protein of hepatitis C virus (HCV). MOBKL1B siRNA, but not irrelevant RNA treatment was associated with both reduced virus replication and the absence of MOBKL1B. Believing that HCV replication depended on the MOBKL1B-NS5A interaction, we carried out structural and biochemical analyses. Unexpectedly, an HCV variant lacking the MOBKL1B-NS5A interaction could not replicate after cells were treated with MOBKL1B siRNA. By repeating the MOBKL1B siRNA knockdowns, and including seed sequence-matched siRNA instead of irrelevant siRNA as a control, we found that the MOBKL1B siRNAs utilized had off-target inhibitory effects on virus replication. Collectively, our results suggest that stricter controls must be utilized in all RNAi-mediated gene knockdown experiments to ensure sound conclusions and a reliable scientific knowledge database.

  • Amino acid substitutions in PB1 of avian influenza viruses influence pathogenicity and transmissibility in chickens [PublishAheadOfPrint]

  • Amino acid substitutions in PB1 were introduced to characterize the interaction between polymerase activity and pathogenicity. Previously, we demonstrated that pathogenicity of the highly pathogenic avian influenza virus (HPAIV) H5N1 in chickens is regulated by the PB1 gene, using recombinant viruses containing the hemagglutinin (HA) and neuraminidase genes from an H5N1 strain, and other internal genes from two low-pathogenicity avian influenza viruses isolated from chicken (LP) and wild bird (WB) hosts (Y. Uchida et al., J. Virol. 86: 2686nndash;2695, 2012, doi: 10.1128/JVI.06374-11). In this study, we introduced a C38Y substitution into PB1 of WB and demonstrated that this substitution increased polymerase activity in vitro in DF-1 cells, as well as pathogenicity of the recombinant viruses in chickens. The V14A substitution in PB1 of LP reduced polymerase activity, but did not affect pathogenicity in chickens. Interestingly, the V14A substitution reduced viral shedding and transmissibility. These studies demonstrate that increasing polymerase activity directly correlates with enhanced pathogenicity, while decreased polymerase activity does not always correlate with pathogenicity and requires further analysis.

    IMPORTANCE We identified 2 novel amino acid substitutions in the PB1 gene of avian influenza virus that affect viral characteristics of highly pathogenic avian influenza virus (HPAIV) H5N1, such as viral replication and polymerase activity in vitro and pathogenicity and transmissibly in chickens. Amino acid substitution at residue 38 in PB1 directly affected pathogenicity in chickens, and was associated with changes in polymerase activity in vitro. Substitution at residue 14 reduced polymerase activity in vitro, while its effects on pathogenicity and transmissibility depended on the constellation of the internal genes.

  • Interferon regulatory factor 5 (IRF5)-dependent immune responses in the draining lymph node protect against West Nile virus infection [PublishAheadOfPrint]

  • Upon activation of Toll-like and RIG-I-like receptor signaling pathways, the transcription factor IRF5 translocates to the nucleus and induces antiviral immune programs. The recent discovery of a homozygous mutation in the immunoregulatory gene guanine exchange factor dedicator of cytokinesis 2 (Dock2mu/mu) in several Irf5-/- mouse colonies has complicated interpretation of immune functions previously ascribed to IRF5. To define the antiviral functions of IRF5 in vivo, we infected backcrossed Irf5-/- x Dock2wt/wt mice (hereafter called Irf5-/- mice) and independently-generated CMV-Cre Irf5fl/fl mice with West Nile virus (WNV), a pathogenic neurotropic flavivirus. Compared to congenic wild-type animals, Irf5-/- and CMV-Cre Irf5fl/fl mice were more vulnerable to WNV infection and this phenotype was associated with increased infection in peripheral organs, which resulted in higher viral titers in the central nervous system. The loss of IRF5, however, was associated with only small differences in the type I IFN response systemically and in the draining lymph node during WNV infection. Instead, lower levels of several other proinflammatory cytokines and chemokines, as well as fewer and less activated immune cells, were detected in the draining lymph node two days after WNV infection. WNV-specific antibody responses in Irf5-/- mice also were blunted in the context of live or inactivated virus infection and this was associated with fewer antigen-specific memory B cells and long-lived plasma cells. Our results with Irf5-/- mice establish a key role for IRF5 in shaping the early innate immune response in the draining lymph node, which impacts the spread of virus infection, optimal B cell immunity, and disease pathogenesis.

    IMPORTANCE Although the roles of IRF3 and IRF7 in orchestrating innate and adaptive immunity after viral infection are established, the function of the related transcription factor IRF5 remains less certain. Prior studies in Irf5-/- mice reported conflicting results as to the contribution of IRF5 in regulating type I IFN and adaptive immune responses. The lack of clarity may stem from a recently discovered homozygous loss-of-function mutation of the immunoregulatory gene Dock2 in several colonies of Irf5-/- mice. Here, using a mouse model with a deficiency in IRF5 and wild-type Dock2 alleles, we investigated how IRF5 modulates West Nile virus (WNV) pathogenesis and host immune responses. Our in vivo studies indicate that IRF5 has a key role in shaping the early pro-inflammatory cytokine response in the draining lymph node, which impacts immunity and control of WNV infection.

  • High risk HPV E6 protein promotes reprogramming of Fanconi Anemia patient cells through repression of p53 but does not allow for sustained growth of iPSC [PublishAheadOfPrint]

  • DNA repair plays a crucial role in embryonic and somatic stem cell biology and cell reprogramming. The Fanconi Anemia pathway, which promotes error-free repair of DNA double strand breaks, is required for somatic cell reprogramming to iPSC. Thus, cells from Fanconi Anemia patients, which lack this critical pathway, fail to reprogram to iPSC under standard conditions unless the defective FA gene is complemented. In this study, we utilized the oncogenes of high risk human papillomavirus type 16 to overcome the resistance to reprogramming of FA patient cells. We found that E6, but not E7, recovers FA iPS colony formation, and furthermore, that p53 inhibition is necessary and sufficient for this activity. The iPS colonies resulting from each of these approaches stained positive for alkaline-phosphatase, NANOG, and Tra-1-60, indicating that they were fully reprogrammed into pluripotent cells. However, FA iPSC were incapable of outgrowth into stable iPSC lines regardless of p53 suppression, whereas their FA-complemented counterparts grew efficiently. Thus, we conclude that the FA pathway is required for the growth of iPSC beyond reprogramming, and that p53-independent mechanisms are involved.

    Importance: A novel approach is described whereby HPV oncogenes are used as tools to uncover DNA repair-related molecular mechanisms affecting somatic cell reprogramming. The findings indicate that p53-dependent mechanisms block FA cells from reprogramming but also uncover a previously unrecognized defect in FA iPSC proliferation independent of p53.

  • Rooting the phylogenetic tree of MERS-Coronavirus by characterization of a conspecific virus from an African Bat [PublishAheadOfPrint]

  • The emerging Middle East respiratory syndrome-coronavirus (MERS-CoV) causes lethal respiratory infections mainly on the Arabian Peninsula. The evolutionary origins of MERS-CoV are unknown. We determined the full genome sequence of a CoV directly from fecal material obtained from a South African Neoromicia capensis bat (NeoCoV). NeoCoV shared essential details of genome architecture with MERS-CoV. 85% of the NeoCoV genome was identical to MERS-CoV on nucleotide level. Based on taxonomic criteria, NeoCoV and MERS-CoV belonged to one viral species. Presence of a genetically divergent S1 subunit within the NeoCoV Spike gene indicated that intra-Spike recombination events may have been involved in the emergence of MERS-CoV. NeoCoV constitutes a sister taxon to MERS-CoV, placing the MERS-CoV root between a recently-described virus from African camels and all other viruses. This suggests a higher viral diversity in camels than in humans. Together with serologic evidence for widespread MERS-CoV infection in camelids sampled up to 20 years back in Africa and the Arabian Peninsula, the genetic data indicates that camels act as sources of virus for humans rather than vice versa. The majority of camels on the Arabian Peninsula is imported from the greater Horn of Africa, where several Neoromicia species occur. The acquisition of MERS-CoV by camels from bats might have taken place in Sub-Saharan Africa. Camelids may represent mixing vessels for MERS-CoV and other mammalian CoVs.

    Importance It is unclear how, when and where the highly pathogenic MERS-CoV emerged. We characterized the full genome of an African bat virus closely related to MERS-CoV and show that human, camel and bat viruses belong to the same viral species. The bat virus roots the phylogenetic tree of MERS-CoV, providing evidence for an evolution of MERS-CoV in camels that preceded that in humans. The revised tree suggests that humans are infected by camels rather than vice versa. Although MERS-CoV cases occur mainly on the Arabian Peninsula, the data from this study together with serologic and molecular investigations of African camels indicate that the initial host switch from bats may have taken place in Africa. The emergence of MERS-CoV likely involved exchange of genetic elements between different viral ancestors. These exchanges may have taken place either in bat ancestors or camels acting as mixing vessels for viruses from different hosts.

  • Potent Neutralization of Vaccinia Virus by Divergent Murine Antibodies Targeting a Common Site of Vulnerability in L1 Protein [PublishAheadOfPrint]

  • Vaccinia virus (VACV) L1 is an important target for viral neutralization and has been included in multicomponent DNA or protein vaccines against orthopoxviruses. To further understand the protective mechanism of the anti-L1 antibodies, we generated five murine anti-L1 monoclonal antibodies (mAbs), which clustered into 3 distinct epitope groups. While two groups of anti-L1 failed to neutralize, one group of 3 mAbs potently neutralized VACV in an isotype- and complement-independent manner. This is in contrast to neutralizing antibodies against major VACV envelope proteins such as H3, D8 or A27, which failed to completely neutralize VACV unless the antibodies are of complement-fixing isotypes and complement is present. Compared to non-neutralizing anti-L1 mAbs, the neutralization antibodies bound to the recombinant L1 protein with a significantly higher affinity and could also bind to virions. By using a variety of techniques including the isolation of neutralization escape mutants, hydrogen/deuterium exchange mass spectrometry, and X-ray crystallography, the epitope of the neutralizing antibodies was mapped to a conformational epitope with Asp35 as the key residue. This epitope is similar to the epitope of 7D11, a previously described potent VACV neutralizing antibody. The epitope was recognized mainly by CDR1 and CDR2 of the heavy chain, which are highly conserved among antibodies recognizing the epitope. These antibodies, however, had divergent light chain and heavy chain CDR3 sequences. Our study demonstrates that the conformational L1 epitope with Asp35 is a common site of vulnerability for potent neutralization by a divergent group of antibodies.

    Importance Vaccinia virus, the live vaccine for smallpox, is one of the most successful vaccines in human history but presents a level of risk that has become unacceptable for the current population. Studying the immune protection mechanism of smallpox vaccine is important for understanding the basic principle of successful vaccines and the development of next generation, safer vaccines for highly pathogenic orthopoxviruses. We studied antibody targets in smallpox vaccine by developing potent neutralizing antibodies against vaccinia virus and comprehensively characterizing their epitopes. We found a site in vaccinia virus L1 protein as the target of a group of highly potent murine neutralizing antibodies. The analysis of antibody:antigen complex structure and the sequences of the antibody genes shed light on how these potent neutralizing antibodies are elicited from immunized mice.

  • Virulent bioluminescent and fluorescent dual reporter Marek's Disease virus (MDV) unveils an alternative MDV's spreading pathway in addition to cell-to-cell contact. [PublishAheadOfPrint]

  • Marek disease virus (MDV) is a growing threat for the poultry industry. Unfortunately, despite successful vaccination against the disease, MDV remains in circulation within vaccinated flocks, leading to the selection of increasingly virulent pathotypes. Detailed knowledge of the virus biology and the host/virus interaction is required to improve the vaccine efficiency. In the present study we have engineered an original, dual reporter MDV virus to track and quantify virus replication in vitro and in vivo.

  • Annexin V incorporated into influenza virus particles inhibits interferon-{gamma} signaling and promotes viral replication [PublishAheadOfPrint]

  • During the budding process, influenza A viruses (IAVs) incorporate multiple host cell membrane proteins. However, for most of them, their significance in viral morphogenesis and infectivity remains unknown. Here we demonstrate that the expression of annexin V (A5) is up-regulated at the cell surface upon IAV infection and that a substantial proportion of the protein is present in lipid rafts, the site of virus budding. Western blotting and immunogold analysis of highly purified IAV particles showed the presence of A5 in the virion. Significantly, interferon- (IFN-) induced stat-phosphorylation and IFN--induced protein 10 kDa (IP-10) production in macrophage-derived THP-1 cells was inhibited by purified IAV particles. Disruption of the IFN- signaling pathway was A5 dependent as down-regulation of its expression or its blockage reversed the inhibition and resulted in decreased viral replication, in vitro. The functional significance of these results was also observed in vivo. Thus, IAVs can subvert the IFN- antiviral immune response by incorporating A5 in their envelope during the budding process.

    IMPORTANCE Many enveloped viruses including influenza A viruses bud from the plasma membrane of their host cell and incorporate cellular surface proteins into viral particles. However, for the vast majority of these proteins, only the observation of their incorporation has been reported. In this manuscript, we demonstrated that the host protein annexin 5 is specifically incorporated into influenza virus particles during the budding process. Importantly, we showed that packaged annexin 5 counteracted the antiviral activity of interferon-gamma in vitro and in vivo. Thus, these results showed that Annexin 5 incorporated in the viral envelope of influenza viruses allow viral escape from immune surveillance. Understanding the role of host incorporated protein into virions may reveal how enveloped RNA viruses hijack the host cell machinery for their own purposes.

  • AAV Type 2 Wild-Type and Vector-Mediated Genomic Integration Profiles in Human Diploid Fibroblasts Analyzed by 3rd Generation PacBio DNA Sequencing [PublishAheadOfPrint]

  • Genome-wide analysis of adeno-associated virus (AAV) type 2 integration in HeLa cells has shown that wild-type AAV integrates at numerous genomic sites, including AAVS1 on chromosome 19q13.42. Multiple GAGY/C repeats, resembling consensus AAV Rep-binding sites are preferred, whereas rep-deficient AAV vectors (rAAV) regularly show a random integration profile. This study is the first study to analyze wild-type AAV integration in diploid human fibroblasts. Applying high-throughput 3rd generation PacBio-based DNA sequencing, integration profiles of wild-type AAV and rAAV are compared side by side. Bioinformatic analysis reveals that both, wild-type AAV and rAAV prefer open chromatin regions. Although genomic features of AAV integration largely reproduce previous findings, the pattern of integration hotspots differs from that described in HeLa cells before. DNase-Seq data for human fibroblasts and for HeLa cells reveal variant chromatin accessibility at preferred AAV integration hotspots that correlates with variant hotspot preferences. DNase-Seq patterns of these sites in human tissues including liver, muscle, heart, brain, skin and embryonic stem cells further underline variant chromatin accessibility. In summary, AAV integration is dependent on cell-type-specific, variant chromatin accessibility leading to random integration profiles for rAAV, whereas wild-type AAV integration sites cluster near GAGY/C repeats.

    Importance Adeno-associated virus type 2 (AAV) is assumed to establish latency by chromosomal integration of its DNA. This is the first genome-wide analysis of wild-type AAV2 integration in diploid human cells and the first to compare wild-type to recombinant AAV vector integration side by side under identical experimental conditions. Major determinants of wild-type AAV integration represent open chromatin regions with accessible consensus AAV Rep-binding sites. The variant chromatin accessibility of different human tissues or cell types will have impact on vector targeting to be considered during gene therapy.

  • Up to Four Distinct Polypeptides are produced from the {gamma}34.5 Open Reading Frame of Herpes Simplex Virus 2 [PublishAheadOfPrint]

  • The HSV-1 ICP34.5 protein strongly influences neurovirulence and regulates several cellular antiviral responses. Despite the clinical importance of HSV-2, relatively little is known about its ICP34.5 ortholog. We have found that HSV-2 produces up to four distinct forms of ICP34.5 in infected cells: full-length protein, one shorter form sharing the N-terminus, and two shorter forms sharing the C-terminus. These forms appeared with similar kinetics and accumulated in cells over much of the replication cycle. We confirmed that the N-terminal form is translated from the primary unspliced transcript to a stop codon within the intron unique to HSV-2 34.5. We found that the N-terminal form was produced in a variety of cell types, and by 9 of 10 clinical isolates. ICP27 influenced but was not required for expression of the N-terminal form. Western blot and reverse transcription PCR indicated the C-terminal forms did not contain the N-terminus, and were not products of alternative splicing or internal transcript initiation. Expression plasmids encoding methionine at amino acids 56 and 70 generated products which co-migrated in SDS-PAGE with the C1 and C2 forms, respectively, and mutation of these sites abolished C1 and C2. Using a recombinant HSV-2 encoding HA-tagged ICP34.5, we demonstrated that the C-terminal forms were also produced during infection of many human and mouse cell types, but were not detectable in mouse primary neurons. The protein diversity generated from the HSV-2 34.5 open reading frame implies additional layers of cellular regulation through potential independent activities associated with the various forms of ICP34.5.

    Importance The HSV-1 protein ICP34.5, encoded by the 34.5 gene, interferes with several host defense mechanisms by binding cellular proteins that would otherwise stimulate the cell's autophagic, translational arrest, and type I interferon responses to virus infection. ICP34.5 also plays a crucial role in determining the severity of nervous system infections with HSV-1 and HSV-2. The HSV-2 34.5 gene contains an intron not present in HSV-1 34.5. A shorter amino-terminal form of HSV-2 ICP34.5 can be translated from the unspliced 34.5 mRNA. Here we show that two additional forms consisting of the carboxy-terminal portion of ICP34.5 are generated in infected cells. Production of these N- and C-terminal forms is highly conserved among HSV-2 strains including many clinical isolates, and they are broadly expressed in several cell types but not mouse primary neurons. Multiple ICP34.5 polypeptides add additional complexity to potential functional interactions influencing HSV-2 neurovirulence.

  • P2X-selective purinergic antagonists are strong inhibitors of HIV-1 fusion during both cell-to-cell and cell-free infection [PublishAheadOfPrint]

  • Human Immunodeficiency Virus (HIV-1) is a chronic and incurable infection. Antiretroviral drugs effectively suppress replication; however, persistent activation of inflammatory pathways remains a key cause of morbidity. Recent studies proposed that purinergic signaling is required for HIV-1 viral infection. Purinergic receptors are distributed through a wide variety of tissue types that detect extracellular ATP as a danger signal released from dying cells. We have explored how these pathways are involved in transmission of HIV-1 from cell-to-cell through virological synapses. Infection of CD4+ T lymphocytes with HIV-1 in the presence of an inhibitor of P2X receptors effectively inhibited HIV-1 infection through both cell-free and cell-to-cell contact in a dose-dependent manner. Inhibition of direct cell-to-cell infection does not affect the formation of virological synapses or the subsequent cell-to-cell transfer of HIV-1. During both cell-free and cell-to-cell CD4+ T lymphocyte infection, purinergic antagonists blocked infection at the level of viral membrane fusion. During cell-to-cell transmission, within target lymphocytes we observed CXCR4 colocalization with the newly internalized virus particles, and found that the purinergic antagonists did not impair the recruitment of coreceptor CXCR4 to the site of Gag internalization in the target cell. In a screen of a library of purinergic antagonists, we found that the most potent inhibitors of HIV-1 fusion were those that target P2X receptors, while P2Y receptor antagonists or adenosine receptor antagonists were ineffective. Our results suggest that P2X receptors may provide a therapeutic target with potent antiviral activity against infection of CD4+ T lymphocytes by both cell-free and cell-to-cell infection.

    Importance This study identifies purinergic antagonists as potent inhibitors of HIV-1 cell-free and cell-to-cell mediated infection and provides stepwise determination of these compounds as inhibitors of HIV-1 viral membrane fusion. These data provide a rationale for the development of novel antiretroviral therapies that have a dual role in both direct anti-viral activity as well as HIV-associated inflammation. Purinergic antagonists are shown here to have equivalent efficacy in inhibiting HIV infection via cell-free and cell-to-cell infection and could provide an attractive therapeutic anti-HIV target that might avoid resistance by targeting a host signaling pathway that potently regulates HIV-infection. The high throughput screen of HIV-1 fusion inhibitors further defines P2X-selective compounds among purinergic compounds as being the most potent HIV entry inhibitors. Clinical studies on these drugs for other inflammatory indications suggest they are safe and thus, if developed for as an anti-HIV, they could reduce both HIV replication and HIV-related inflammation.

  • Inhibition of Antiviral Innate Immunity by Birnavirus VP3 Protein via Blockage of Viral dsRNA Binding to the Host Cytoplasmic RNA Detector MDA5 [PublishAheadOfPrint]

  • Chicken MDA5 (chMDA5), the sole known pattern recognition receptor for cytoplasmic viral RNA in chickens, initiates type I interferon (IFN) production. Infectious bursal disease virus (IBDV) evades host innate immunity but the mechanism is unclear. We report here that IBDV inhibited antiviral innate immunity via the chMDA5-dependent signaling pathway. IBDV infection did not induce efficient type I IFN production but antagonized the antiviral activity of IFN-bbeta; in DF-1 cells pretreated with IFN-aalpha;/bbeta;. Dual-luciferase assays and inducible expression systems demonstrated that IBDV protein VP3 significantly inhibited IFN-bbeta; expression stimulated by naked IBDV genomic dsRNA. VP3 protein competed strongly with chMDA5 to bind IBDV genomic dsRNA in vitro and in vivo, and VP3 from other birnaviruses also bound dsRNA. Site-directed mutagenesis confirmed that deletion of the VP3 dsRNA binding domain restored IFN-bbeta; expression. Our data demonstrate that VP3 inhibits antiviral innate immunity by blocking binding of viral genomic dsRNA to MDA5.

    IMPORTANCE MDA5, a known pattern recognition receptor and cytoplasmic viral RNA sensor, plays a critical role in host antiviral innate immunity. Many pathogens escape or inhibit the host antiviral immune response, but the mechanisms involved are unclear for most pathogens. We report here that birnaviruses inhibit host antiviral innate immunity via the MDA5-dependent signaling pathway. The antiviral innate immune system involving IFN-bbeta; did not function effectively during birnavirus infection, and the viral protein VP3 significantly inhibited IFN-bbeta; expression stimulated by naked viral genomic dsRNA. We also showed that VP3 blocked MDA5 binding to viral genomic dsRNA in vitro and in vivo. Our data reveal that birnavirus-encoded viral protein VP3 is an inhibitor of antiviral innate immune response and inhibits the antiviral innate immune response via the MDA5-dependent signaling pathway.

  • Prolidase (PEPD) is required for early trafficking events during influenza A virus entry [PublishAheadOfPrint]

  • Influenza A virus (IAV) entry is a multi-step process that requires the interaction of the virus with numerous host factors. In this study, we demonstrate that prolidase (PEPD) is a cellular factor required by IAV for successful entry into target cells. PEPD was selected as a candidate during an entry screen performed on non-validated primary hits from previously published genome-wide siRNA screens. siRNA-mediated depletion of PEPD resulted in decreased growth of IAV during mono- and multi-cycle growth. This growth defect was independent of cell type or virus strain. Furthermore, IAV restriction was apparent as early as 3h post-infection and experiments in the absence of protein biosynthesis revealed that nuclear import of viral ribonucleoprotein complexes (vRNPs) was already blocked in the absence of PEPD. These results led us to investigate which step during entry was affected. Receptor expression, IAV attachment or internalization were not dependent on the presence of PEPD. However, when looking at the distribution of incoming IAV particles in PEPD knockdown cells, we found a localization pattern that differed compared to control cells: IAV mostly localized to the cell periphery and consequently, viral particles displayed reduced co-localization with early and late endosome markers and fusion between viral and endosomal membranes was strongly reduced. Finally, experiments using a competitive inhibitor of PEPD catalytic activity suggest that the enzymatic function of the dipeptidase is required for its proviral effect on IAV entry. In sum, this study establishes PEPD as a novel entry factor required for early endosomal trafficking of IAV.

    Importance Influenza A virus (IAV) continues to be a constant threat to public health. As IAV relies on its host cell for replication the identification of host factors required by the virus is of importance: First, such studies often reveal novel functions of cellular factors and can extend our knowledge of cellular processes. Second, we can further our understanding of processes that are required for entry of IAV into target cells. Third, the identification of host factors that contribute to IAV entry will enlarge the number of potential targets for the development of novel antiviral drugs that are of urgent need. Our study identifies prolidase (PEPD) as a novel entry factor of IAV required for correct routing within the endosomal compartment following virus internalization. Thereby, we link PEPD which has been shown to play a role during collagen recycling and growth factor signaling, to early events of viral infection.

  • Histone deacetylase 6 inhibits influenza A virus release by downregulating the trafficking of viral components to the plasma membrane via its substrate acetylated microtubules [PublishAheadOfPrint]

  • Mammalian cells produce many proteins such as IFITM3, ISG15, MxA, and viperin that inhibit influenza A virus (IAV) infection. Here we show that a new class of host protein, histone deacetylase 6 (HDAC6) inhibits IAV infection. We found that the HDAC6-overexpressing cells release about 3-fold less IAV progeny, whereas the HDAC6-depleted cells release about 6-fold more IAV progeny. The deacetylase activity of HDAC6 played a role in its anti-IAV function as tubacin, a specific small-molecule inhibitor of HDAC6, increased the release of IAV progeny in a dose-dependent manner. Further, as visualized by the electron microscopy, tubacin-treated cells showed an increase in IAV budding at the plasma membrane, the site of IAV assembly. Tubacin is a domain-specific inhibitor and binds to one of the two HDAC6 catalytic domains possessing the tubulin deacetylase activity. This indicated the potential involvement of acetylated microtubules in the trafficking of viral components to the plasma membrane. Indeed, as quantified by flow cytometry, there was about 2.0 to 2.5-fold increase and about 2-fold decrease in the amount of viral envelope protein hemagglutinin present on the plasma membrane of tubacin-treated, HDAC6-depleted, and HDAC6-overexpressing cells, respectively. In addition, the viral ribonucleoprotein complex was co-localized with acetylated microtubule filaments and viral nucleoprotein co-immunoprecipitated with acetylated tubulin. Together, our findings indicate that HDAC6 is an anti-IAV host factor and exerts its anti-IAV function by negatively regulating the trafficking of viral components to the host cell plasma membrane via its substrate acetylated microtubules.

    Importance Host cells produce many proteins that have the natural ability to restrict the influenza virus infection. Herein, we discovered that another host protein, histone deacetylase 6 (HDAC6) inhibits the influenza virus infection. We demonstrate that HDAC6 exerts its anti-influenza function by negatively regulating the trafficking of viral components to the site of influenza virus assembly via its substrate acetylated microtubules. HDAC6 is a multi-substrate enzyme and regulates multiple cellular pathways including the ones leading to various cancers, neurodegenerative diseases, and inflammatory disorders. Therefore, several drugs targeting HDAC6 are under clinical development for the treatment of wide range of diseases. Influenza virus continues to be a major global public health problem due to regular emergence of drug-resistant and novel influenza strains in humans. As an alternative antiviral strategy, HDAC6 modulators could be employed to stimulate the anti-influenza potential of endogenous HDAC6 to inhibit influenza virus infection.

  • Expression of porcine fusion protein IRF7/3(5D) efficiently controls foot-and-mouth disease virus replication [PublishAheadOfPrint]

  • Several studies have demonstrated that administration of type I, II, or III interferons (IFNs) delivered using a replication-defective human adenovirus 5 (Ad5) vector can effectively control foot-and-mouth disease (FMD) in cattle and swine during experimental infections. However, relatively high doses are required to achieve protection. In this study, we identified the functional properties of a porcine fusion protein, poIRF7/3(5D), as a biotherapeutic and enhancer of IFN activity against FMD virus (FMDV). We showed that poIRF7/3(5D) is a potent inducer of type I IFNs including IFNaalpha;, bbeta;, and but not type III IFN (IL28B), without inducing cytotoxicity. Expression of poIRF7/3(5D) significantly and steadily reduced FMDV viral titers by up to 6 log10 in swine and bovine cell lines. Treatment with an IFN receptor inhibitor (B18R) combined with an anti-IFNaalpha; antibody neutralized the antiviral activity in the supernatants of Ad5-poIRF7/3(5D) transduced cells. However, several transcripts with known antiviral function, and including type I IFNs, were still highly up-regulated (ranging from 8 to over 500 fold increase) by poIRF7/3(5D) in the presence of B18R. Furthermore, mice treated with Ad5-poIRF7/3(5D) showed antiviral activity in sera that was associated with high induction of IFNaalpha; and resulted in complete protection against FMDV challenge at 6, 24 or 48 hours post-treatment. This study, highlights for the first time, the antiviral potential of Ad5-poIRF7/3(5D) in vitro and in vivo against FMDV.

    Importance: FMD remains one of the most devastating diseases that affect livestock worldwide. Effective vaccine formulations are available but are serotype specific and require approximately 7 days for protective immunity. We have shown that vector-delivered IFN is an option to protect animals against many FMDV serotypes as soon as 24 h and for about 4 days post administration. Here we demonstrate that delivery of a constitutively active transcription factor that induces the production of endogenous IFNs and potentially other antiviral genes is a viable strategy to protect against FMD.

  • New Small Molecule Inhibitors Effectively Blocking Picornavirus Replication [PublishAheadOfPrint]

  • Few drugs targeting picornaviruses are available, making the discovery of antivirals a high priority. Here, we identified and characterized three compounds from a library of kinase inhibitors that block replication of poliovirus, Coxsackie virus B3 and encephalomyocarditis virus. Using an in vitro translation-replication system, we showed that these drugs inhibit different stages of the poliovirus lifecycle. A4 (1) inhibited both formation and functioning of the replication complexes while E5 (1) and E7 (2) were most effective during the formation but not the functioning step. Neither of the compounds significantly inhibited VPg uridylylation. Poliovirus resistant to E7 (2) had a mutation G5318A in the 3A protein. This mutation was previously found to confer resistance to enviroxime-like compounds which target a PI4KIIIbbeta;-dependent step in the viral replication. Analysis of host proteins recruitment showed that E7 (2) reduced amount of GBF1on the replication complexes, however the level of PI4KIIIbbeta; remained intact. E7 (2) as well as another enviroxime-like compound GW5074 interfered with the viral polyprotein processing affecting both 3C and 2A-dependent cleavages, and the resistant mutation G5318A partially rescued this defect. Moreover E7 (2) induced abnormal recruitment to membranes of the viral proteins, thus enviroxime-like compounds likely severely compromise interaction of the viral polyprotein with membranes. A4 (1) demonstrated partial protection from paralysis in a murine model of poliomyelitis. Multiple attempts to isolate resistant mutants in the presence of A4 (1) or E5 (1) were unsuccessful, showing that effective broad-spectrum antivirals could be developed on the basis of these compounds.

    Importance Diverse picornaviruses can trigger multiple human maladies, yet currently only hepatitis A virus and poliovirus can be controlled with vaccination. The development of anti-picornaviral therapeutics is also facing significant difficulties because these viruses readily generate resistance to compounds targeting either viral or cellular factors. Here we describe three novel compounds that effectively block replication of distant picornaviruses with minimal toxicity to the cells. The compounds prevent viral RNA replication after the synthesis of the uridylylated VPg primer. Importantly, two of the inhibitors are strongly refractory to the emergence of resistant mutants making them promising candidates for further broad-spectrum therapeutic development. Evaluation of one of the compounds in an in vivo model of poliomyelitis demonstrated partial protection from the onset of paralysis.

  • Age-dependent myeloid dendritic cell responses mediate resistance to La Crosse Virus induced neurological disease. [PublishAheadOfPrint]

  • La Crosse Virus (LACV) is the major cause of pediatric viral encephalitis in the USA; however, the mechanisms responsible for age-related susceptibility in the pediatric population are not well understood. Our current studies in a mouse model of LACV infection indicated that differences in myeloid dendritic cell (mDC) responses between weanling and adult mice accounted for susceptibility to LACV-induced neurological disease. We found that type I interferon (IFN) responses were significantly stronger in adult versus weanling mice. Production of these IFNs required both endosomal toll-like receptors (TLR) and cytoplasmic RIG-I like receptors (RLR). Surprisingly, IFN expression was not dependent on plasmacytoid DCs (pDCs) but rather mDCs, which were found in greater number and induced stronger IFN responses in adults than weanlings. Inhibition of these IFN responses in adults resulted in susceptibility to LACV-induced neurological disease, whereas post-infection treatment with type I IFN provided protection in young mice. These studies provide a definitive mechanism for age-related susceptibility to LACV encephalitis, where mDCs in young mice are insufficiently activated to control peripheral virus replication, thereby allowing virus to persist and eventually cause CNS disease.

    IMPORTANCE La Crosse Virus (LACV) is the primary cause of pediatric viral encephalitis in the United States. Although the virus infects both adults and children, over 80% of the reported neurological disease cases are in children. To understand why LACV primarily causes neurological disease in young animals, we used a mouse model where weanling mice, but not adult mice, develop neurological disease following virus infection. We found that an early immune response cell type, myeloid dendritic cells, was critical for protection in adult animals, and that these cells were reduced in young animals. Activation of these cells during virus infection or post-treatment with type I interferon in young animals provided protection from LACV. Thus, this study demonstrates a reason for susceptibility to LACV infection in young animals and shows that early therapeutic treatment in young animals can prevent neurological disease.

  • The effect of vaginal immunization in women with HIVgp140 and HSP70 on HIV-1 replication, innate and T cell adaptive immunity [PublishAheadOfPrint]

  • The international effort to prevent HIV-1 infection by vaccination has failed to develop an effective vaccine. The aim of this vaccine trial in women was to administer by the vaginal mucosal route a vaccine consisting of HIV-1gp140 linked to the chaperone 70kD heat shock protein (HSP70). The primary objective was to determine safety of the vaccine. The secondary objective was to examine HIV-1 infectivity ex-vivo and innate and adaptive immunity to HIV-1. Protocol defined female volunteers were recruited. HIV-1 CN54gp140 linked to HSP70 was administered by the vaginal route. Significant adverse reactions were not detected. HIV-1 was significantly inhibited ex vivo in the post- compared with the pre-immunization CD4+ T cells. The innate anti-viral restrictive factor APOBEC3G was significantly upregulated, as were CC-chemokines, which induce down-regulation of CCR5 in CD4+ T cells. Indeed, significant inverse correlation was found between the proportion of CCR5+ T cells and the concentration of CCL-3 or CCL-5. Importantly, APOBEC3G showed a significant inverse correlation, whereas CCR5 exhibited a trend to correlating with inhibition of HIV-1 infection (r=0.51). Furthermore, specific CD4+ and CD8+ T cell proliferative responses were significantly increased and CD4+ T cells showed a trend to inverse correlation with the viral load (r=-0.60). However, HIVgp140 specific IgG or IgA antibodies were not detected. The results provide proof of concept that an innate mechanism consisting of CC-chemokines, and APOBEC 3G, and adaptive immunity by CD4 and CD8 T cells might be involved in controlling HIV-1 infectivity following vaginal mucosal immunization in women.

    Importance Vaginal immunization of women with a vaccine consisting of HIVgp140 linked to the 70kD heat shock protein (HSP70) elicited ex vivo significant inhibition of HIV-1 replication in the post- compared with pre-immunization PBMC. There were no significant adverse events. The vaccine induced significant upregulation of CC chemokines and downmodulation of CCR5 expression in CD4+ T cells, as well as an inverse correlation between them. Furthermore, CCR5 was directly correlated with the viral load, consistent with the protective mechanism of a decrease in CCR5 molecules on CD4+ T cells decreasing HIV-1 envelope binding. The anti-viral restriction factor APOBEC3G was inversely correlated with the viral load, suggesting that it may inhibit intracellular HIV-1 replication. Both CD4+ and CD8+ T cells showed HIVgp140 and HSP70 specific proliferation. A strong inverse correlation was found between the CC-chemokine modulated CCR5 expressing CD4+ T cells and CD4+ or CD8+ T cells stimulated proliferation with HIVgp140, demonstrating a significant interaction between innate and adaptive immunity.

  • Impact of sequence variation in a dominant HLA-A*02-restricted epitope in HCV on priming and cross-reactivity of CD8+ T cells [PublishAheadOfPrint]

  • CD8+ T cells are an essential component of successful adaptive immune responses against hepatitis C virus (HCV). A major obstacle to vaccine design against HCV is the inherent viral sequence diversity. Here, we test the hypothesis, if different sequence variants of an immunodominant CD8+ T cell epitope all binding with high affinity to HLA class I target different T cell receptor repertoires and thereby influence the quality of the CD8+ T cell response. The impact of sequence differences in the HLA-A*02-restricted HCV NS31406-1415 epitope on in vitro priming of naiiuml;ve CD8+ T cells from seronegative donors and cross-reactivity of primed T cells with other epitope variants were characterized. Despite that the six epitope variants tested were all high-affinity binders to HLA-A*02:01, substantial differences in priming and cross-reactivity of CD8+ T cells were observed. The variant associated with the most reproducible priming and induction of T cells with broad cross-reactivity was a genotype 1b variant (KLSALGLNAV) that is more common in HCV isolates collected in Asia but is rare in sequences from Europe and North America. The superior immunogenicity and cross-reactivity of this relatively rare epitope variant was confirmed in HCV-specific memory CD8+ T cells from people who inject drugs, frequently exposed to HCV. Collectively, the data suggest that sequence differences at the epitope level between HCV isolates substantially impact CD8+ T cell priming and the degree of cross-reactivity with other epitope variants.

    Importance: The results have important implications for vaccine design against highly variable pathogens and suggest that evidence-based selection of the vaccine antigen sequence may improve immunogenicity and T cell cross-reactivity. Cross-reactive CD8+ T cells are likely beneficial for immune control of transmitted viruses carrying epitope variants and for prevention of immune escape during acute infection. To this end, rare epitope variants and potentially even altered epitope sequences associated with priming of broadly cross-reactive T cell receptors should be considered for vaccine design and need further testing.

  • A Mutation in the DNA Polymerase Accessory Factor of Herpes Simplex Virus 1 Restores Viral DNA Replication in the Presence of Raltegravir [PublishAheadOfPrint]

  • Previous reports showed that Raltegravir, a recently approved antiviral compound that targets HIV integrase, can inhibit the nuclease function of human cytomegalovirus (HCMV) in vitro. In this study, subtoxic levels of Raltegravir were shown to inhibit the replication of four different herpesviruses: HSV-1, HSV-2, HCMV and MCMV (mouse cytomegalovirus) by 30-700 fold depending on the dose and virus tested. Southern blot and qPCR revealed that Raltegravir inhibits DNA replication of HSV-1 rather than cleavage of viral DNA. A Raltegravir-resistant HSV-1 mutant was generated by repeated passage in the presence of 200 mmu;M Raltegravir. The genomic sequence of the resistant virus, designated clone 7, contains mutations in 16 open reading frames. Of these, mutations in UL15 (F198S, encoding the large terminase subunit), UL32 (A374V, required for DNA cleavage and packaging), UL42 (V296I, encoding the DNA polymerase accessory factor), and UL54 (A224S, encoding ICP27, an important transcriptional regulator) were introduced independently into the wild type HSV-1(F) genome and the recombinant viruses were tested for Raltegravir resistance. Viruses bearing both the UL15 and UL32 mutations inserted within the genome of the UL42 mutant were also tested. While the UL15, UL32 and UL54 mutant viruses were fully susceptible to Raltegravir, any virus bearing the UL42 mutation was as resistant to Raltegravir as clone 7. Overall, these results suggest that Raltegravir may be a valuable therapeutic agent against herpesviruses, and the antiviral activity targets the DNA polymerase accessory factor rather than the nuclease activity of the terminase.

    Importance This paper is important because it shows that Raltegravir, and anti-retrovirus drug targeting integrase, is effective against various herpesviruses. Drug resistance mapped to the herpesvirus DNA polymerase accessory factor which was an unexpected finding.

  • HIV-1 GP120 ACTIVATES the STAT3/IL-6 AXIS in PRIMARY HUMAN MONOCYTE-DERIVED DENDRITIC CELLS [PublishAheadOfPrint]

  • Dendritic cells (DCs) are fundamental for the initiation of immune responses and are important players in AIDS immunopathogenesis. The modulation of DC functional activities represents a strategic mechanism for HIV-1 to evade immune surveillance. Impairment of DC function may result from bystander effects of HIV-1 envelope proteins independently of direct HIV-1 infection. In this study, we report that exposure of immature monocyte-derived DCs (MDDCs) to HIV-1 R5 gp120 resulted in the CCR5-dependent production of IL-6 via MAPK/NF-kB pathways. IL-6 in turn activated STAT3 by an autocrine loop. Concomitantly, gp120 promoted an early activation of STAT3 that further contributed to IL-6 induction. This activation paralleled a concomitant up-regulation of the STAT3 inhibitor PIAS3. Notably, STAT3/IL-6 pathway activation was not affected by the CCR5 specific ligand CCL4.

    These results identify STAT3 as a key signaling intermediate activated by gp120 in MDDCs and highlight the existence of a viral-induced dysregulation of the IL-6/STAT3 axis. HIV-1 gp120 signaling through STAT3 may provide an explanation for the impairment of DC function observed upon HIV exposure.

    Importance This study provides new evidence for the molecular mechanisms and signaling pathways triggered by HIV-1 gp120 in human DCs, in the absence of productive infection, emphasizing a role of aberrant signaling in early virus-host interaction, contributing to viral pathogenesis. We identified STAT3 as a key component in the gp120-mediated signaling cascade involving MAPK and NF-kB components, and ultimately leading to IL-6 secretion.

    STAT3 is now recognized as a key regulator of DC functions. Thus, the identification of this transcription factor as a signaling molecules mediating some of the gp120 biological effects, unveils a new mechanism by which HIV-1 may deregulate DC functions and contribute to AIDS pathogenesis.

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