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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]

  • Viruses and RNA Interference: Issues and Controversies [Gem]

  • The question of whether any mammalian cells are able to mount an effective RNA interference-mediated antiviral innate immune response has remained highly controversial. In this Gem, I review recent data addressing this important issue and propose a testable hypothesis that can explain many of the apparently contradictory results published in this area of research.

  • HIV Control Is Mediated in Part by CD8+ T-Cell Targeting of Specific Epitopes [Pathogenesis and Immunity]

  • We investigated 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. By measuring gamma interferon enzyme-linked immunosorbent spot (ELISPOT) assay responses to a panel of 257 optimally defined epitopes in 341 untreated HIV-infected persons, including persons who spontaneously control viremia, we found that the correlation between HLA types and control is mediated by the targeting of specific epitopes. Moreover, we performed a graphical model-based analysis that suggested 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 identified 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 we found 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 antiretroviral 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 mediated largely by the targeting of specific CD8+ T-cell epitopes, and we 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 identified additional epitopes that are not typically targeted in natural infection but may lead to control when included in a vaccine, provided that other epitopes that would otherwise distract the immune system from targeting them are excluded from the vaccine.

  • Envelope Variants Circulating as Initial Neutralization Breadth Developed in Two HIV-Infected Subjects Stimulate Multiclade Neutralizing Antibodies in Rabbits [Vaccines and Antiviral Agents]

  • Identifying characteristics of the human immunodeficiency virus type 1 (HIV-1) envelope that are effective in generating broad, protective antibodies remains a hurdle to HIV vaccine design. Emerging evidence of the development of broad and potent neutralizing antibodies in HIV-infected subjects suggests that founder and subsequent progeny viruses may express unique antigenic motifs that contribute to this developmental pathway. We hypothesize that over the course of natural infection, B cells are programmed to develop broad antibodies by exposure to select populations of emerging envelope quasispecies variants. To test this hypothesis, we identified two unrelated subjects whose antibodies demonstrated increasing neutralization breadth against a panel of HIV-1 isolates over time. Full-length functional env genes were cloned longitudinally from these subjects from months after infection through 2.6 to 5.8 years of infection. Motifs associated with the development of breadth in published, cross-sectional studies were found in both subjects. We compared the immunogenicity of envelope vaccines derived from time points obtained during and after broadening of neutralization activity within these subjects. Rabbits were coimmunized four times with selected multiple gp160 DNAs and gp140-trimeric envelope proteins. The affinity of the polyclonal response increased as a function of boosting. The most rapid and persistent neutralization of multiclade tier 1 viruses was elicited by envelopes that were circulating in plasma at time points prior to the development of 50% neutralization breadth in both human subjects. The breadth elicited in rabbits was not improved by exposure to later envelope variants. These data have implications for vaccine development in describing a target time point to identify optimal envelope immunogens.

    IMPORTANCE Vaccine protection against viral infections correlates with the presence of neutralizing antibodies; thus, vaccine components capable of generating potent neutralization are likely to be critical constituents in an effective HIV vaccine. However, vaccines tested thus far have elicited only weak antibody responses and very modest, waning protection. We hypothesized that B cells develop broad antibodies by exposure to the evolving viral envelope population and tested this concept using multiple envelopes from two subjects who developed neutralization breadth within a few years of infection. We compared different combinations of envelopes from each subject to identify the most effective immunogens and regimens. In each subject, use of HIV envelopes circulating during the early development and maturation of breadth generated more-potent antibodies that were modestly cross neutralizing. These data suggest a new approach to identifying envelope immunogens that may be more effective in generating protective antibodies in humans.

  • Emergence of Broadly Neutralizing Antibodies and Viral Coevolution in Two Subjects during the Early Stages of Infection with Human Immunodeficiency Virus Type 1 [Pathogenesis and Immunity]

  • 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 monitored 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 neutralizing breadth was detected in plasma. Both subjects developed bNAb activity after approximately 1 year postinfection, 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 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 antienvelope 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 require 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 20% of HIV-1-infected subjects, providing a prototype of the bNAbs that must be reelicited by vaccine. Thus, there is significant interest in understanding the mechanisms by which bNAbs develop during the course of infection. We studied the timing, epitope specificity, and 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 nonneutralizing. 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.

  • Human H7N9 and H5N1 Influenza Viruses Differ in Induction of Cytokines and Tissue Tropism [Pathogenesis and Immunity]

  • Since emerging in 2013, the avian-origin H7N9 influenza viruses have resulted in over 400 human infections, leading to 115 deaths to date. Although the epidemiology differs from human highly pathogenic avian H5N1 influenza virus infections, there is a similar rapid progression to acute respiratory distress syndrome. The aim of these studies was to compare the pathological and immunological characteristics of a panel of human H7N9 and H5N1 viruses in vitro and in vivo. Although there were similarities between particular H5N1 and H7N9 viruses, including association between lethal disease and spread to the alveolar spaces and kidney, there were also strain-specific differences. Both H5N1 and H7N9 viruses are capable of causing lethal infections, with mortality correlating most strongly with wider distribution of viral antigen in the lungs, rather than with traditional measures of virus titer and host responses. Strain-specific differences included hypercytokinemia in H5N1 infections that was not seen with the H7N9 infections regardless of lethality. Conversely, H7N9 viruses showed a greater tropism for respiratory epithelium covering nasal passages and nasopharynx-associated lymphoid tissue than H5N1 viruses, which may explain the enhanced transmission in ferret models. Overall, these studies highlight some distinctive properties of H5N1 and H7N9 viruses in different in vitro and in vivo models.

    IMPORTANCE The novel avian-origin H7N9 pandemic represents a serious threat to public health. The ability of H7N9 to cause serious lung pathology, leading in some cases to the development of acute respiratory distress syndrome, is of particular concern. Initial reports of H7N9 infection compared them to infections caused by highly pathogenic avian (HPAI) H5N1 viruses. Thus, it is of critical importance to understand the pathology and immunological response to infection with H7N9 compared to HPAI H5N1 viruses. We compared these responses in both in vitro and in vivo models, and found that H5N1 and H7N9 infections exhibit distinct pathological, immunological, and tissue tropism differences that could explain differences in clinical disease and viral transmission.

  • Direct Interrogation of Viral Peptides Presented by the Class I HLA of HIV-Infected T Cells [Cellular Response to Infection]

  • Identification of CD8+ cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of 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 CTLs 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 first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)nndash;mass spectrometry (MS) 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, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) 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 show that typical and atypical viral ligands are efficiently presented by HLA and targeted by human CTLs. 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 CTLs provides previously unrealized information that may enhance the development of immune therapies and vaccines.

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

  • 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 that are dependent on infiltrating inflammatory cells 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 after 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, the development of filopodia and lamellipodia, and phagocytosis of WNV-infected cells and debris. Microglial cell activation was concomitant with increased expression of proinflammatory cytokines and chemokines, including CXCL10, CXCL1, CCL5, CCL3, CCL2, tumor necrosis factor alpha (TNF-aalpha;), TNF-related apoptosis-inducing ligand (TRAIL), and interleukin-6 (IL-6). The application of minocycline, an inhibitor of neuroinflammation, altered the WNV-induced proinflammatory cytokine/chemokine expression profile, with inhibited production of CCL5, CCL2, and IL-6. 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 the 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 proinflammatory cytokine/chemokine production, develop independently of contributions from the peripheral immune system and CNS-infiltrating inflammatory cells.

  • 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 [Pathogenesis and Immunity]

  • Interleukin-10 (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 human immunodeficiency virus (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. We demonstrated here changes in mucosal IL-10 signaling during simian immunodeficiency virus (SIV) infection in rhesus macaques. Disruption of the epithelial barrier was manifested by EC apoptosis and loss of the 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 gamma interferon (IFN-) and tumor necrosis factor alpha (TNF-aalpha;), suggesting that IL-10 was not able to regulate AIR. This observation was supported by the downregulation of STAT3, which is necessary to inhibit production of IFN- and TNF-aalpha;, and the 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 gastrointestinal 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. 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 TNF-aalpha; 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.

  • Stepwise Priming by Acidic pH and a High K+ Concentration Is Required for Efficient Uncoating of Influenza A Virus Cores after Penetration [Virus-Cell Interactions]

  • Influenza A virus (IAV) uses the low pH in late endocytic vacuoles as a cue for penetration by membrane fusion. Here, we analyzed the prefusion 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 a pH below 6.5. The second step is triggered by a pH of 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 viral ribonucleoprotein bundle. Our results indicate that both the switch from Na+ to K+ in maturing endosomes and the decreasing pH are 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 preacidified core is then 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.

  • Entry of a Novel Marine DNA Virus, Singapore Grouper Iridovirus, into Host Cells Occurs via Clathrin-Mediated Endocytosis and Macropinocytosis in a pH-Dependent Manner [Virus-Cell Interactions]

  • Iridoviruses are nucleocytoplasmic DNA viruses which cause great economic losses in the aquaculture industry but also show significant threat to global biodiversity. However, a lack of host cells has resulted in poor progress in clarifying iridovirus behavior. We investigated the crucial events during virus entry using a combination of single-virus 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 colocalize 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 caveola-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 be transported 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 the clathrin-dependent pathway but also macropinocytosis are involved in fish DNA enveloped virus entry, thus providing a convenient tactic for exploring the life cycle of DNA viruses.

    IMPORTANCE Virus entry into host cells is critically important for initiating infections and is usually recognized as an ideal target for the design of antiviral strategies. Iridoviruses are large DNA viruses which cause serious threats to ecological diversity and the 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 Iridoviridae. Here, using single-virus tracking technology in combination with biochemical assays, we investigated the crucial events during SGIV entry and demonstrated that SGIV entered grouper cells via the clathrin-mediated endocytic pathway in a pH-dependent manner but not via caveola-dependent endocytosis. Furthermore, we propose for the first time that macropinocytosis is involved in iridovirus entry. Together, this work not only contributes greatly to understating iridovirus pathogenesis but also provides an ideal model for exploring the behavior of DNA viruses in living cells.

  • The Highly Conserved Proline at Position 438 in Pseudorabies Virus gH Is Important for Regulation of Membrane Fusion [Virus-Cell Interactions]

  • Membrane fusion in herpesviruses requires viral glycoproteins (g) gB and gH/gL. While gB is considered the actual fusion protein but is nonfusogenic per se, the function of gH/gL remains enigmatic. Crystal structures for different gH homologs are strikingly similar despite only moderate amino acid sequence conservation. A highly conserved sequence motif comprises the residues serine-proline-cysteine corresponding to positions 437 to 439 in pseudorabies virus (PrV) gH. The PrV-gH structure shows that proline438 induces bending at the end of an alpha-helix, thereby placing cysteine404 and cysteine439 in juxtaposition to allow formation of a strictly conserved disulfide bond. However, PrV vaccine strain Bartha unexpectedly carries a serine at this conserved position. To test the influence of this substitution, we constructed different gH chimeras carrying proline or serine at position 438 in gH derived from either PrV strain Kaplan or strain Bartha. Mutants expressing gH with serine438 showed reduced fusion activity in transient-fusion assays and during infection, with delayed penetration kinetics and a small-plaque phenotype which indicates that proline438 is important for efficient fusion. A more drastic effect was observed when disulfide bond formation was completely blocked by mutation of cysteine404 to serine. Although PrV expressing gHC404S was viable, plaque size and penetration kinetics were drastically reduced. Alteration of serine438 to proline in gH of strain Bartha enhanced cell-to-cell spread and penetration kinetics, but restoration of full activity required additional alteration of aspartic acid to valine at position 59.

    IMPORTANCE The role of the gH/gL complex in herpesvirus membrane fusion is still unclear. Structural studies predicted a critical role for proline438 in PrV gH to allow the formation of a conserved disulfide bond and correct protein folding. Functional analyses within this study corroborated these structural predictions: mutation of this residue resulted in a drastic impairment of membrane fusion kinetics not only in vitro in transient transfection-fusion assays but also during virus infection. Elimination of formation of the disulfide bond yielded the same phenotype in transient assays but had a more drastic effect on virus replication. Thus, our studies add important information to structure-function analyses of herpesvirus gH.

  • Identification of a High-Efficiency Baculovirus DNA Replication Origin That Functions in Insect and Mammalian Cells [Genome Replication and Regulation of Viral Gene Expression]

  • The p143 gene from Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) has been found to increase the expression of luciferase, which is driven by the polyhedrin gene promoter, in a plasmid with virus coinfection. Further study indicated that this is due to the presence of a replication origin (ori) in the coding region of this gene. Transient DNA replication assays showed that a specific fragment of the p143 coding sequence, p143-3, underwent virus-dependent DNA replication in Spodoptera frugiperda IPLB-Sf-21 (Sf-21) cells. Deletion analysis of the p143-3 fragment showed that subfragment p143-3.2a contained the essential sequence of this putative ori. Sequence analysis of this region revealed a unique distribution of imperfect palindromes with high AT contents. No sequence homology or similarity between p143-3.2a and any other known ori was detected, suggesting that it is a novel baculovirus ori. Further study showed that the p143-3.2a ori can replicate more efficiently in infected Sf-21 cells than baculovirus homologous regions (hrs), the major baculovirus ori, or non-hr oris during virus replication. Previously, hr on its own was unable to replicate in mammalian cells, and for mammalian viral oris, viral proteins are generally required for their proper replication in host cells. However, the p143-3.2a ori was, surprisingly, found to function as an efficient ori in mammalian cells without the need for any viral proteins. We conclude that p143 contains a unique sequence that can function as an ori to enhance gene expression in not only insect cells but also mammalian cells.

    IMPORTANCE Baculovirus DNA replication relies on both hr and non-hr oris; however, so far very little is known about the latter oris. Here we have identified a new non-hr ori, the p143 ori, which resides in the coding region of p143. By developing a novel DNA replication-enhanced reporter system, we have identified and located the core region required for the p143 ori. This ori contains a large number of imperfect inverted repeats and is the most active ori in the viral genome during virus infection in insect cells. We also found that it is a unique ori that can replicate in mammalian cells without the assistance of baculovirus gene products. The identification of this ori should contribute to a better understanding of baculovirus DNA replication. Also, this ori is very useful in assisting with gene expression in mammalian cells.

  • Chemical Induction of Unfolded Protein Response Enhances Cancer Cell Killing through Lytic Virus Infection [Virus-Cell Interactions]

  • Cancer cells are susceptible to oncolytic viruses, albeit variably. Human adenoviruses (HAdVs) are widely used oncolytic agents that have been engineered to produce progeny within the tumor and elicit bystander effects. We searched for host factors enhancing bystander effects and conducted a targeted RNA interference screen against guanine nucleotide exchange factors (GEFs) of small GTPases. We show that the unfolded protein response (UPR), which is readily inducible in aggressive tumor cells, enhances melanoma or epithelial cancer cell killing upon HAdV infection. UPR was triggered by knockdown of Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF-1) or the GBF-1 inhibitor golgicide A (GCA) and stimulated HAdV infection. GBF-1 is a GEF for ADP ribosylation factors (Arfs) regulating endoplasmic reticulum (ER)-to-Golgi apparatus and intra-Golgi apparatus membrane transport. Cells treated with GCA enhanced HAdV-induced cytopathic effects in epithelial and melanoma cancer cells but not normal cells, if the drug was applied several hours prior to HAdV inoculation. This was shown by real-time label-free impedance measurements using the xCELLigence system. GCA-treated cells contained fewer incoming HAdVs than control cells, but GCA treatment boosted HAdV titers and spreading in cancer cells. GCA enhanced viral gene expression or transgene expression from the cytomegalovirus promoter of B- or C-species HAdVs but did not enhance viral early region 1A (E1A) expression in uninfected cell lines or cells transfected with plasmid reporter DNA. The UPR-enhanced cell killing required the nuclease activity of the UPR sensor inositol-requiring enzyme 1 (IRE-1) and X box binding protein 1 (XBP-1), which alleviate ER stress. The collective results show that chemical UPR induction and viruses boost tumor cell killing by enhancing oncolytic viral efficacy.

    IMPORTANCE Cancer is difficult to combat. A wide range of oncolytic viruses show promise for killing cancer cells, yet the efficacy of oncolytic killing is low. We searched for host factors enhancing adenovirus cancer cell killing and found that the knockdown of Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF-1) or chemical inhibition of GBF-1 enhanced adenovirus infection by triggering the IRE-1/XBP-1 branch of the unfolded protein response (UPR). IRE-1/XBP-1 promote cell survival and enhanced the levels of the adenoviral immediate early gene product E1A, virus spreading, and killing of cancer cells. Aggressive tumor cells depend on a readily inducible UPR and, hence, present prime targets for a combined strategy involving adenoviruses and small chemicals inducing UPR.

  • Matrix Proteins of Nipah and Hendra Viruses Interact with Beta Subunits of AP-3 Complexes [Virus-Cell Interactions]

  • Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections.

    IMPORTANCE Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people, and smaller outbreaks have since occurred in Bangladesh and India. In this study, we have defined, for the first time, host factors that interact with henipavirus M proteins and contribute to viral particle assembly. We have also defined a new host protein-viral protein binding interface that can potentially be targeted for the inhibition of paramyxovirus infections.

  • Molecular Characterization of a Novel Temperate Sinorhizobium Bacteriophage, FELM21, Encoding DNA Methyltransferase with CcrM-Like Specificity [Genetic Diversity and Evolution]

  • LM21 is a temperate phage isolated from Sinorhizobium sp. strain LM21 (Alphaproteobacteria). Genomic analysis and electron microscopy suggested that LM21 is a member of the family Siphoviridae. The phage has an isometric head and a long noncontractile tail. The genome of LM21 has 50,827 bp of linear double-stranded DNA encoding 72 putative proteins, including proteins responsible for the assembly of the phage particles, DNA packaging, transcription, replication, and lysis. Virion proteins were characterized using mass spectrometry, leading to the identification of the major capsid and tail components, tape measure, and a putative portal protein. We have confirmed the activity of two gene products, a lytic enzyme (a putative chitinase) and a DNA methyltransferase, sharing sequence specificity with the cell cycle-regulating methyltransferase (CcrM) of the bacterial host. Interestingly, the genome of Sinorhizobium phage LM21 shows very limited similarity to other known phage genome sequences and is thus considered unique.

    IMPORTANCE Prophages are known to play an important role in the genomic diversification of bacteria via horizontal gene transfer. The influence of prophages on pathogenic bacteria is very well documented. However, our knowledge of the overall impact of prophages on the survival of their lysogenic, nonpathogenic bacterial hosts is still limited. In particular, information on prophages of the agronomically important Sinorhizobium species is scarce. In this study, we describe the isolation and molecular characterization of a novel temperate bacteriophage, LM21, of Sinorhizobium sp. LM21. Since we have not found any similar sequences, we propose that this bacteriophage is a novel species. We conducted a functional analysis of selected proteins. We have demonstrated that the phage DNA methyltransferase has the same sequence specificity as the cell cycle-regulating methyltransferase CcrM of its host. We point out that this phenomenon of mimicking the host regulatory mechanisms by viruses is quite common in bacteriophages.

  • Increased Replicative Fitness of a Dengue Virus 2 Clade in Native Mosquitoes: Potential Contribution to a Clade Replacement Event in Nicaragua [Genetic Diversity and Evolution]

  • The four dengue virus (DENV) serotypes (DENV serotype 1 [DENV-1] to DENV-4) are transmitted by Aedes aegypti and A. albopictus mosquitoes, causing up to 390 million DENV infections worldwide each year. We previously reported a clade replacement of the DENV-2 Asian-American genotype NI-1 clade by the NI-2B clade in Managua, Nicaragua. Here, we describe our studies of the replicative ability of NI-1 and NI-2B viruses in an A. aegypti cell line (Aag2) and A. aegypti mosquitoes reared from eggs collected in Managua. In coinfection experiments, several different pairs of NI-1 and NI-2B clinical isolates were used to infect Aag2 cells or blood-fed A. aegypti mosquitoes. Results consistently showed a significant replicative advantage of NI-2B over NI-1 viruses early after infection in vitro, and in mosquitoes, NI-2B viruses attained a higher replicative index than NI-1 isolates 3 to 7 days postinfection (dpi). At 7 dpi, NI-2B viruses displayed a significantly higher replicative index in legs and salivary glands; however, this advantage was lost by 14 and 21 dpi. We also found that the percentage of mosquitoes in which NI-2B viruses were dominant was significantly higher than that in which NI-1 viruses were dominant on day 7 but not at later time points. Taken together, these data demonstrate that clade NI-2B holds a replicative advantage over clade NI-1 early in infection that wanes at later time points. This early fitness advantage of NI-2B viruses over NI-1 viruses in the native vector, A. aegypti, suggests a shorter extrinsic incubation period for NI-2B viruses, which could have contributed to the clade replacement event in Managua.

    IMPORTANCE Dengue virus (DENV), one of the most medically important arthropod-borne viruses, is transmitted to humans by Aedes aegypti and A. albopictus mosquitoes in tropical and subtropical regions worldwide. Dengue epidemics continue to increase in frequency, geographic range, and severity and are a major public health concern. This is due to globalization, unplanned urbanization, and climate change, as well as host genetics and immune responses and viral genetic changes. DENV consists of four serotypes, in turn composed of genotypes and genetically distinct clades. What drives the frequent replacement of a previously circulating DENV clade by another is unclear. Here, we investigate the replicative fitness of two clades of DENV serotype 2 in Aedes aegypti cells and mosquitoes collected from the region where the viruses circulated and conclude that increased replicative fitness could have contributed to a DENV clade replacement event in Nicaragua. These findings provide insight into vector-driven evolution of DENV epidemics.

  • Induction of Viral, 7-Methyl-Guanosine Cap-Independent Translation and Oncolysis by Mitogen-Activated Protein Kinase-Interacting Kinase-Mediated Effects on the Serine/Arginine-Rich Protein Kinase [Genome Replication and Regulation of Viral Gene Expression]

  • Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g., upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for plus-strand RNA viruses, where immediate translation of incoming viral RNAs shapes host-virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38aalpha; MAPK-interacting kinases MNK1/2 and their role in controlling 7-methyl-guanosine (m7G) "cap"-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNK's known functions as eukaryotic initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser/Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity, and viral proliferation in infected cells.

    IMPORTANCE We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A phase I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the antineoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral cap-independent translation in infected host cells.

  • Mitogen-Activated Protein Kinase-Interacting Kinase Regulates mTOR/AKT Signaling and Controls the Serine/Arginine-Rich Protein Kinase-Responsive Type 1 Internal Ribosome Entry Site-Mediated Translation and Viral Oncolysis [Genome Replication and Regulation of Viral Gene Expression]

  • Translation machinery is a major recipient of the principal mitogenic signaling networks involving Raf-ERK1/2 and phosphoinositol 3-kinase (PI3K)-mechanistic target of rapamycin (mTOR). Picornavirus internal ribosomal entry site (IRES)-mediated translation and cytopathogenic effects are susceptible to the status of such signaling cascades in host cells. We determined that tumor-specific cytotoxicity of the poliovirus/rhinovirus chimera PVSRIPO is facilitated by Raf-ERK1/2 signals to the mitogen-activated protein kinase (MAPK)-interacting kinase (MNK) and its effects on the partitioning/activity of the Ser/Arg (SR)-rich protein kinase (SRPK) (M. C. Brown, J. D. Bryant, E. Y. Dobrikova, M. Shveygert, S. S. Bradrick, V. Chandramohan, D. D. Bigner, and M, Gromeier, J. Virol. 22:13135nndash;13148, 2014, doi: Here, we show that MNK regulates SRPK via mTOR and AKT. Our investigations revealed a MNK-controlled mechanism acting on mTORC2-AKT. The resulting suppression of AKT signaling attenuates SRPK activity to enhance picornavirus type 1 IRES translation and favor PVSRIPO tumor cell toxicity and killing.

    IMPORTANCE Oncolytic immunotherapy with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES, is demonstrating early promise in clinical trials with intratumoral infusion in recurrent glioblastoma (GBM). Our investigations demonstrate that the core mechanistic principle of PVSRIPO, tumor-selective translation and cytotoxicity, relies on constitutive ERK1/2-MNK signals that counteract the deleterious effects of runaway AKT-SRPK activity in malignancy.

  • Activation of NF-{kappa}B by the Kaposi's Sarcoma-Associated Herpesvirus K15 Protein Involves Recruitment of the NF-{kappa}B-Inducing Kinase, I{kappa}B Kinases, and Phosphorylation of p65 [Virus-Cell Interactions]

  • Kaposi's sarcoma herpesvirus (KSHV) (or human herpesvirus 8) is the cause of Kaposi's sarcoma, primary effusion lymphoma (PEL), and the plasma cell variant of multicentric Castleman's disease (MCD). The transmembrane K15 protein, encoded by KSHV, has been shown to activate NF-B and the mitogen-activated protein kinases (MAPKs) c-jun-N-terminal kinase (JNK) and extracellular signal-regulated kinase (Erk) as well as phospholipase C gamma (PLC) and to contribute to KSHV-induced angiogenesis. Here we investigate how the K15 protein activates the NF-B pathway. We show that activation of NF-B involves the recruitment of NF-B-inducing kinase (NIK) and IKK aalpha;/bbeta; to result in the phosphorylation of p65/RelA on Ser536. A K15 mutant devoid in NIK/IKK recruitment fails to activate NF-B but remains proficient in the stimulation of both NFAT- and AP1-dependent promoters, showing that the structural integrity of the mutant K15 protein has not been altered dramatically. Direct recruitment of NIK represents a novel way for a viral protein to activate and manipulate the NF-B pathway.

    IMPORTANCE KSHV K15 is a viral protein involved in the activation of proinflammatory and angiogenic pathways. Previous studies reported that K15 can activate the NF-B pathway. Here we show the molecular mechanism underlying the activation of this signaling pathway by K15, which involves direct recruitment of the NF-B-inducing kinase NIK to K15 as well as NIK-mediated NF-B p65 phosphorylation on Ser536. K15 is the first viral protein shown to activate NF-B through direct recruitment of NIK. These results indicate a new mechanism whereby a viral protein can manipulate the NF-B pathway.

  • A Leucine Residue in the C Terminus of Human Parainfluenza Virus Type 3 Matrix Protein Is Essential for Efficient Virus-Like Particle and Virion Release [Genome Replication and Regulation of Viral Gene Expression]

  • Paramyxovirus particles, like other enveloped virus particles, are formed by budding from membranes of infected cells, and matrix (M) proteins are critical for this process. To identify the M protein important for this process, we have characterized the budding of the human parainfluenza virus type 3 (HPIV3) M protein. Our results showed that expression of the HPIV3 M protein alone is sufficient to initiate the release of virus-like particles (VLPs). Electron microscopy analysis confirmed that VLPs are morphologically similar to HPIV3 virions. We identified a leucine (L302) residue within the C terminus of the HPIV3 M protein that is critical for M protein-mediated VLP production by regulating the ubiquitination of the M protein. When L302 was mutated into A302, ubiquitination of M protein was defective, the release of VLPs was abolished, and the membrane binding and budding abilities of M protein were greatly weakened, but the ML302A mutant retained oligomerization activity and had a dominant negative effect on M protein-mediated VLP production. Furthermore, treatment with a proteasome inhibitor also inhibited M protein-mediated VLP production and viral budding. Finally, recombinant HPIV3 containing the ML302A mutant could not be rescued. These results suggest that L302 acts as a critical regulating signal for the ubiquitination of the HPIV3 M protein and virion release.

    IMPORTANCE Human parainfluenza virus type 3 (HPIV3) is an enveloped virus with a nonsegmented negative-strand RNA genome. It can cause severe respiratory tract diseases, such as bronchiolitis, pneumonia, and croup in infants and young children. However, no valid antiviral therapy or vaccine is currently available. Thus, further elucidation of its assembly and budding will be helpful in the development of novel therapeutic approaches. Here, we show that a leucine residue (L302) located at the C terminus of the HPIV3 M protein is essential for efficient production of virus-like particles (VLPs). Furthermore, we found L302 regulated M protein-mediated VLP production via regulation of M protein ubiquitination. Recombinant HPIV3 containing the ML302A mutant is growth defective. These findings provide new insight into the critical role of M protein-mediated VLP production and virion release of a residue that does not belong to L domain and may advance our understanding of HPIV3 viral assembly and budding.

  • A Histidine Residue of the Influenza Virus Hemagglutinin Controls the pH Dependence of the Conformational Change Mediating Membrane Fusion [Pathogenesis and Immunity]

  • The conformational change of the influenza virus hemagglutinin (HA) protein mediating the fusion between the virus envelope and the endosomal membrane was hypothesized to be induced by protonation of specific histidine residues since their pKas match the pHs of late endosomes (pKa of ~6.0). However, such critical key histidine residues remain to be identified. We investigated the highly conserved His184 at the HA1-HA1 interface and His110 at the HA1-HA2 interface of highly pathogenic H5N1 HA as potential pH sensors. By replacing both histidines with different amino acids and analyzing the effect of these mutations on conformational change and fusion, we found that His184, but not His110, plays an essential role in the pH dependence of the conformational change of HA. Computational modeling of the protonated His184 revealed that His184 is central in a conserved interaction network possibly regulating the pH dependence of conformational change via its pKa. As the propensity of histidine to get protonated largely depends on its local environment, mutation of residues in the vicinity of histidine may affect its pKa. The HA of highly pathogenic H5N1 viruses carries a Glu-to-Arg mutation at position 216 close to His184. By mutation of residue 216 in the highly pathogenic as well as the low pathogenic H5 HA, we observed a significant influence on the pH dependence of conformational change and fusion. These results are in support of a pKa-modulating effect of neighboring residues.

    IMPORTANCE The main pathogenic determinant of influenza viruses, the hemagglutinin (HA) protein, triggers a key step of the infection process: the fusion of the virus envelope with the endosomal membrane releasing the viral genome. Whereas essential aspects of the fusion-inducing mechanism of HA at low pH are well understood, the molecular trigger of the pH-dependent conformational change inducing fusion has been unclear. We provide evidence that His184 regulates the pH dependence of the HA conformational change via its pKa. Mutations of neighboring residues which may affect the pKa of His184 could play an important role in virus adaptation to a specific host. We suggest that mutation of neighboring residue 216, which is present in all highly pathogenic phenotypes of H5N1 influenza virus strains, contributed to the adaptation of these viruses to the human host via its effect on the pKa of His184.

  • Characterization of Simian Immunodeficiency Virus (SIV) That Induces SIV Encephalitis in Rhesus Macaques with High Frequency: Role of TRIM5 and Major Histocompatibility Complex Genotypes and Early Entry to the Brain [Pathogenesis and Immunity]

  • Although nonhuman primate models of neuro-AIDS have made tremendous contributions to our understanding of disease progression in the central nervous system (CNS) of human immunodeficiency virus type 1 (HIV-1)-infected individuals, each model holds advantages and limitations. In this study, in vivo passage of SIVsmE543 was conducted to obtain a viral isolate that can induce neuropathology in rhesus macaques. After a series of four in vivo passages in rhesus macaques, we have successfully isolated SIVsm804E. SIVsm804E shows efficient replication in peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs) in vitro and induces neuro-AIDS in high frequencies in vivo. Analysis of the acute phase of infection revealed that SIVsm804E establishes infection in the CNS during the early phase of the infection, which was not observed in the animals infected with the parental SIVsmE543-3. Comprehensive analysis of disease progression in the animals used in the study suggested that host major histocompatibility complex class I (MHC-I) and TRIM5aalpha; genotypes influence the disease progression in the CNS. Taken together, our findings show that we have successfully isolated a new strain of simian immunodeficiency virus (SIV) that is capable of establishing infection in the CNS at early stage of infection and causes neuropathology in infected rhesus macaques at a high frequency (83%) using a single inoculum, when animals with restrictive MHC-I or TRIM5aalpha; genotypes are excluded. SIVsm804E has the potential to augment some of the limitations of existing nonhuman primate neuro-AIDS models.

    IMPORTANCE Human immunodeficiency virus (HIV) is associated with a high frequency of neurologic complications due to infection of the central nervous system (CNS). Although the use of antiviral treatment has reduced the incidence of severe complications, milder disease of the CNS continues to be a significant problem. Animal models to study development of neurologic disease are needed. This article describes the development of a novel virus isolate that induces neurologic disease in a high proportion of rhesus macaques infected without the need for prior immunomodulation as is required for some other models.

  • African Great Apes Are Naturally Infected with Roseoloviruses Closely Related to Human Herpesvirus 7 [Genetic Diversity and Evolution]

  • Primates are naturally infected with herpesviruses. During the last 15 years, the search for homologues of human herpesviruses in nonhuman primates allowed the identification of numerous viruses belonging to the different herpesvirus subfamilies and genera. No simian homologue of human herpesvirus 7 (HHV7) has been reported to date. To investigate the putative existence of HHV7-like viruses in African great apes, we applied the consensus-degenerate hybrid oligonucleotide primers (CODEHOP) program-mediated PCR strategy to blood DNA samples from the four common chimpanzee subspecies (Pan troglodytes verus, P. t. ellioti, P. t. troglodytes, and P. t. schweinfurthii), pygmy chimpanzees (Pan paniscus), as well as lowland gorillas (Gorilla gorilla gorilla). This study led to the discovery of a novel roseolovirus close to HHV7 in each of these nonhuman primate species and subspecies. Generation of the partial glycoprotein B (1,111-bp) and full-length DNA polymerase (3,036/3,042-bp) gene sequences allowed the deciphering of their evolutionary relationships. Phylogenetic analyses revealed that HHV7 and its African great ape homologues formed well-supported monophyletic lineages whose topological resemblance to the host phylogeny is suggestive of virus-host codivergence. Notably, the evolutionary branching points that separate HHV7 from African great ape herpesvirus 7 are remarkably congruent with the dates of divergence of their hosts. Our study shows that African great apes are hosts of human herpesvirus homologues, including HHV7 homologues, and that the latter, like other DNA viruses that establish persistent infections, have cospeciated with their hosts.

    IMPORTANCE Human herpesviruses are known to possess simian homologues. However, surprisingly, none has been identified to date for human herpesvirus 7 (HHV7). This study is the first to describe simian homologues of HHV7. The extensive search performed on almost all African great ape species and subspecies, i.e., common chimpanzees of the four subspecies, bonobos, and lowland gorillas, has allowed characterization of a specific virus in each. Genetic characterization of the partial glycoprotein B and full-length DNA polymerase gene sequences, followed by their phylogenetic analysis and estimation of divergence times, has shed light on the evolutionary relationships of these viruses. In this respect, we conclusively demonstrate the cospeciation between these new viruses and their hosts and report cases of cross-species transmission between two common chimpanzee subspecies in both directions.

  • Human Coronavirus NL63 Utilizes Heparan Sulfate Proteoglycans for Attachment to Target Cells [Virus-Cell Interactions]

  • Human coronavirus NL63 (HCoV-NL63) is an alphacoronavirus that was first identified in 2004 in the nasopharyngeal aspirate from a 7-month-old patient with a respiratory tract infection. Previous studies showed that HCoV-NL63 and the genetically distant severe acute respiratory syndrome (SARS)-CoV employ the same receptor for host cell entry, angiotensin-converting enzyme 2 (ACE2), but it is largely unclear whether ACE2 interactions are sufficient to allow HCoV-NL63 binding to cells. The present study showed that directed expression of angiotensin-converting enzyme 2 (ACE2) on cells previously resistant to HCoV-NL63 renders them susceptible, showing that ACE2 protein acts as a functional receptor and that its expression is required for infection. However, comparative analysis showed that directed expression or selective scission of the ACE2 protein had no measurable effect on virus adhesion. In contrast, binding of HCoV-NL63 to heparan sulfates was required for viral attachment and infection of target cells, showing that these molecules serve as attachment receptors for HCoV-NL63.

    IMPORTANCE ACE2 protein was proposed as a receptor for HCoV-NL63 already in 2005, but an in-depth analysis of early events during virus infection had not been performed thus far. Here, we show that the ACE2 protein is required for viral entry but that it is not the primary binding site on the cell surface. Conducted research showed that heparan sulfate proteoglycans function as adhesion molecules, increasing the virus density on cell surface and possibly facilitating the interaction between HCoV-NL63 and its receptor. Obtained results show that the initial events during HCoV-NL63 infection are more complex than anticipated and that a newly described interaction may be essential for understanding the infection process and, possibly, also assist in drug design.

  • Two Novel Simian Arteriviruses in Captive and Wild Baboons (Papio spp.) [Genetic Diversity and Evolution]

  • Since the 1960s, simian hemorrhagic fever virus (SHFV; Nidovirales, Arteriviridae) has caused highly fatal outbreaks of viral hemorrhagic fever in captive Asian macaque colonies. However, the source(s) of these outbreaks and the natural reservoir(s) of this virus remain obscure. Here we report the identification of two novel, highly divergent simian arteriviruses related to SHFV, Mikumi yellow baboon virus 1 (MYBV-1) and Southwest baboon virus 1 (SWBV-1), in wild and captive baboons, respectively, and demonstrate the recent transmission of SWBV-1 among captive baboons. These findings extend our knowledge of the genetic and geographic diversity of the simian arteriviruses, identify baboons as a natural host of these viruses, and provide further evidence that baboons may have played a role in previous outbreaks of simian hemorrhagic fever in macaques, as has long been suspected. This knowledge should aid in the prevention of disease outbreaks in captive macaques and supports the growing body of evidence that suggests that simian arterivirus infections are common in Old World monkeys of many different species throughout Africa.

    IMPORTANCE Historically, the emergence of primate viruses both in humans and in other primate species has caused devastating outbreaks of disease. One strategy for preventing the emergence of novel primate pathogens is to identify microbes with the potential for cross-species transmission in their natural state within reservoir species from which they might emerge. Here, we detail the discovery and characterization of two related simian members of the Arteriviridae family that have a history of disease emergence and host switching. Our results expand the phylogenetic and geographic range of the simian arteriviruses and define baboons as a natural host for these viruses. Our findings also identify a potential threat to captive macaque colonies by showing that simian arteriviruses are actively circulating in captive baboons.

  • On the Laws of Virus Spread through Cell Populations [Virus-Cell Interactions]

  • The dynamics of viral infections have been investigated extensively, often with a combination of experimental and mathematical approaches. Mathematical descriptions of virus spread through cell populations are well established in the literature and have yielded important insights, yet the formulation of certain fundamental aspects of virus dynamics models remains uncertain and untested. Here, we investigate the process of infection and, in particular, the effect of varying the target cell population size on the number of productively infected cells generated. Using an in vitro single-round HIV-1 infection system, we find that the established modeling framework cannot accurately fit the data. If the model is fit to data with the lowest number of cells and is used to predict data generated with larger cell populations, the model significantly overestimates the number of productively infected cells generated. Interestingly, this deviation becomes stronger under experimental conditions that promote mixing of cells and viruses. The reason for the deviation is that the standard model makes certain oversimplifying assumptions about the fate of viruses that fail to find a cell in their immediate proximity. We derive from stochastic processes a different model that assumes simultaneous access of the virus to multiple target cells. In this scenario, if no cell is available to the virus at its location, it has a chance to interact with other cells, a process that can be promoted by mixing of the populations. This model can accurately fit the experimental data and suggests a new interpretation of mass action in virus dynamics models.

    IMPORTANCE Understanding the principles of virus growth through cell populations is of fundamental importance to virology. It helps us make informed decisions about intervention strategies aimed at preventing virus growth, such as drug treatment or vaccination approaches, e.g., in HIV infection, yet considerable uncertainty remains in this respect. An important variable in this context is the number of susceptible cells available for virus replication. How does the number of susceptible cells influence the growth potential of the virus? Besides the importance of such information for clinical responses, a thorough understanding of this is also important for the prediction of virus levels in patients and the estimation of crucial patient parameters through the use of mathematical models. This paper investigates the relationship between target cell availability and the virus growth potential with a combination of experimental and mathematical approaches and provides significant new insights.

  • Interaction of Adenovirus Type 5 E4orf4 with the Nuclear Pore Subunit Nup205 Is Required for Proper Viral Gene Expression [Virus-Cell Interactions]

  • Adenovirus type 5 E4orf4 is a multifunctional protein that regulates viral gene expression. The activities of E4orf4 are mainly mediated through binding to protein phosphatase 2A (PP2A). E4orf4 recruits target phosphoproteins into complexes with PP2A, resulting in dephosphorylation of host factors, such as SR splicing factors. In the current study, we utilized immunoprecipitation followed by mass spectrometry to identify novel E4orf4-interacting proteins. In this manner we identified Nup205, a component of the nuclear pore complex (NPC) as an E4orf4 interacting partner. The arginine-rich motif (ARM) of E4orf4 was required for interaction with Nup205 and for nuclear localization of E4orf4. ARMs are commonly found on viral nuclear proteins, and we observed that Nup205 interacts with three different nuclear viral proteins containing ARMs. E4orf4 formed a trimolecular complex containing both Nup205 and PP2A. Furthermore, Nup205 complexed with E4orf4 was hypophosphorylated, suggesting that the protein is specifically targeted for dephosphorylation. An adenovirus mutant that does not express E4orf4 (Orf4nndash;) displayed elevated early and reduced late gene expression relative to that of the wild type. We observed that knockdown of Nup205 resulted in the same phenotype as that of the Orf4nndash; virus, suggesting that the proteins function as a complex to regulate viral gene expression. Furthermore, knockdown of Nup205 resulted in a more than a 4-fold reduction in the replication of wild-type adenovirus. Our data show for first time that Ad5 E4orf4 interacts with and modifies the NPC and that Nup205-E4orf4 binding is required for normal regulation of viral gene expression and viral replication.

    IMPORTANCE Nuclear pore complexes (NPCs) are highly regulated conduits in the nuclear membrane that control transport of macromolecules between the nucleus and cytoplasm. Viruses that replicate in the nucleus must negotiate the NPC during nuclear entry, and viral DNA, mRNA, and proteins must then be exported from the nucleus. Several types of viruses restructure the NPC to facilitate replication, and the current study shows that adenovirus type 5 (Ad5) utilizes a novel mechanism to modify NPC function. We demonstrate that a subunit of the NPC, Nup205, is a phosphoprotein that is actively dephosphorylated by the Ad5-encoded protein E4orf4. Moreover, Nup205 is required by Ad5 to regulate viral gene expression and efficient viral replication. Nup205 is a nonstructural subunit that is responsible for the gating functions of the NPC, and this study suggests for the first time that the NPC is regulated by phosphorylation both during normal physiology and viral infection.

  • Induction of Broadly Reactive Anti-Hemagglutinin Stalk Antibodies by an H5N1 Vaccine in Humans [Vaccines and Antiviral Agents]

  • Influenza virus infections are a major public health concern and cause significant morbidity and mortality worldwide. Current vaccines are effective but strain specific due to their focus on the immunodominant globular head domain of the hemagglutinin (HA). It has been hypothesized that sequential exposure of humans to hemagglutinins with divergent globular head domains but conserved stalk domains could refocus the immune response to broadly neutralizing epitopes in the stalk. Humans have preexisting immunity against H1 (group 1 hemagglutinin), and vaccination with H5 HA (also group 1)mmdash;which has a divergent globular head domain but a similar stalk domainmmdash;represents one such sequential-exposure scenario. To test this hypothesis, we used novel reagents based on chimeric hemagglutinins to screen sera from an H5N1 clinical trial for induction of stalk-specific antibodies by quantitative enzyme-linked immunosorbent assay (ELISA) and neutralization assays. Importantly, we also investigated the biological activity of these antibodies in a passive transfer in a mouse challenge model. We found that the H5N1 vaccine induced high titers of stalk-reactive antibodies which were biologically active and protective in the passive-transfer experiment. The induced response showed exceptional breadth toward divergent group 1 hemagglutinins but did not extend to group 2 hemagglutinins. These data provide evidence for the hypothesis that sequential exposure to hemagglutinins with divergent globular head domains but conserved stalk domains can refocus the immune response toward the conserved stalk domain. Furthermore, the results support the concept of a chimeric hemagglutinin universal influenza virus vaccine strategy that is based on the same principle.

    IMPORTANCE Influenza virus vaccines have to be reformulated and readministered on an annual basis. The development of a universal influenza virus vaccine could abolish the need for this cumbersome and costly process and would also enhance our pandemic preparedness. This study addressed the following questions, which are essential for the development of a hemagglutinin stalk-based universal influenza virus vaccine. (i) Can stalk-reactive antibodies be boosted by vaccination with divergent HAs that share conserved epitopes? (ii) How long-lived are these vaccine-induced stalk-reactive antibody responses? (iii) What is the breadth of this reactivity? (iv) Are these antibodies functional and protective? Our results further strengthen the concept of induction of stalk-reactive antibodies by sequential exposure to hemagglutinin immunogens with conserved stalk and divergent head domains. A universal influenza virus vaccine based on the same principles seems possible and might have a significant impact on global human health.

  • Accumulation of Human-Adapting Mutations during Circulation of A(H1N1)pdm09 Influenza Virus in Humans in the United Kingdom [Genetic Diversity and Evolution]

  • The influenza pandemic that emerged in 2009 provided an unprecedented opportunity to study adaptation of a virus recently acquired from an animal source during human transmission. In the United Kingdom, the novel virus spread in three temporally distinct waves between 2009 and 2011. Phylogenetic analysis of complete viral genomes showed that mutations accumulated over time. Second- and third-wave viruses replicated more rapidly in human airway epithelial (HAE) cells than did the first-wave virus. In infected mice, weight loss varied between viral isolates from the same wave but showed no distinct pattern with wave and did not correlate with viral load in the mouse lungs or severity of disease in the human donor. However, second- and third-wave viruses induced less alpha interferon in the infected mouse lungs. NS1 protein, an interferon antagonist, had accumulated several mutations in second- and third-wave viruses. Recombinant viruses with the third-wave NS gene induced less interferon in human cells, but this alone did not account for increased virus fitness in HAE cells. Mutations in HA and NA genes in third-wave viruses caused increased binding to aalpha;-2,6-sialic acid and enhanced infectivity in human mucus. A recombinant virus with these two segments replicated more efficiently in HAE cells. A mutation in PA (N321K) enhanced polymerase activity of third-wave viruses and also provided a replicative advantage in HAE cells. Therefore, multiple mutations allowed incremental changes in viral fitness, which together may have contributed to the apparent increase in severity of A(H1N1)pdm09 influenza virus during successive waves.

    IMPORTANCE Although most people infected with the 2009 pandemic influenza virus had mild or unapparent symptoms, some suffered severe and devastating disease. The reasons for this variability were unknown, but the numbers of severe cases increased during successive waves of human infection in the United Kingdom. To determine the causes of this variation, we studied genetic changes in virus isolates from individual hospitalized patients. There were no consistent differences between these viruses and those circulating in the community, but we found multiple evolutionary changes that in combination over time increased the virus's ability to infect human cells. These adaptations may explain the remarkable ability of A(H1N1)pdm09 virus to continue to circulate despite widespread immunity and the apparent increase in severity of influenza over successive waves of infection.

  • Interactome Analysis of the Influenza A Virus Transcription/Replication Machinery Identifies Protein Phosphatase 6 as a Cellular Factor Required for Efficient Virus Replication [Virus-Cell Interactions]

  • The negative-sense RNA genome of influenza A virus is transcribed and replicated by the viral RNA-dependent RNA polymerase (RdRP). The viral RdRP is an important host range determinant, indicating that its function is affected by interactions with cellular factors. However, the identities and the roles of most of these factors remain unknown. Here, we employed affinity purification followed by mass spectrometry to identify cellular proteins that interact with the influenza A virus RdRP in infected human cells. We purified RdRPs using a recombinant influenza virus in which the PB2 subunit of the RdRP is fused to a Strep-tag. When this tagged subunit was purified from infected cells, copurifying proteins included the other RdRP subunits (PB1 and PA) and the viral nucleoprotein and neuraminidase, as well as 171 cellular proteins. Label-free quantitative mass spectrometry revealed that the most abundant of these host proteins were chaperones, cytoskeletal proteins, importins, proteins involved in ubiquitination, kinases and phosphatases, and mitochondrial and ribosomal proteins. Among the phosphatases, we identified three subunits of the cellular serine/threonine protein phosphatase 6 (PP6), including the catalytic subunit PPP6C and regulatory subunits PPP6R1 and PPP6R3. PP6 was found to interact directly with the PB1 and PB2 subunits of the viral RdRP, and small interfering RNA (siRNA)-mediated knockdown of the catalytic subunit of PP6 in infected cells resulted in the reduction of viral RNA accumulation and the attenuation of virus growth. These results suggest that PP6 interacts with and positively regulates the activity of the influenza virus RdRP.

    IMPORTANCE Influenza A viruses are serious clinical and veterinary pathogens, causing substantial health and economic impacts. In addition to annual seasonal epidemics, occasional global pandemics occur when viral strains adapt to humans from other species. To replicate efficiently and cause disease, influenza viruses must interact with a large number of host factors. The reliance of the viral RNA-dependent RNA polymerase (RdRP) on host factors makes it a major host range determinant. This study describes and quantifies host proteins that interact, directly or indirectly, with a subunit of the RdRP. It increases our understanding of the role of host proteins in viral replication and identifies a large number of potential barriers to pandemic emergence. Identifying host factors allows their importance for viral replication to be tested. Here, we demonstrate a role for the cellular phosphatase PP6 in promoting viral replication, contributing to our emerging knowledge of regulatory phosphorylation in influenza virus biology.

  • Broad Protection against Avian Influenza Virus by Using a Modified Vaccinia Ankara Virus Expressing a Mosaic Hemagglutinin Gene [Vaccines and Antiviral Agents]

  • A critical failure in our preparedness for an influenza pandemic is the lack of a universal vaccine. Influenza virus strains diverge by 1 to 2% per year, and commercially available vaccines often do not elicit protection from one year to the next, necessitating frequent formulation changes. This represents a major challenge to the development of a cross-protective vaccine that can protect against circulating viral antigenic diversity. We have constructed a recombinant modified vaccinia virus Ankara (MVA) that expresses an H5N1 mosaic hemagglutinin (H5M) (MVA-H5M). This mosaic was generated in silico using 2,145 field-sourced H5N1 isolates. A single dose of MVA-H5M provided 100% protection in mice against clade 0, 1, and 2 avian influenza viruses and also protected against seasonal H1N1 virus (A/Puerto Rico/8/34). It also provided short-term (10 days) and long-term (6 months) protection postvaccination. Both neutralizing antibodies and antigen-specific CD4+ and CD8+ T cells were still detected at 5 months postvaccination, suggesting that MVA-H5M provides long-lasting immunity.

    IMPORTANCE Influenza viruses infect a billion people and cause up to 500,000 deaths every year. A major problem in combating influenza is the lack of broadly effective vaccines. One solution from the field of human immunodeficiency virus vaccinology involves a novel in silico mosaic approach that has been shown to provide broad and robust protection against highly variable viruses. Unlike a consensus algorithm which picks the most frequent residue at each position, the mosaic method chooses the most frequent T-cell epitopes and combines them to form a synthetic antigen. These studies demonstrated that a mosaic influenza virus H5 hemagglutinin expressed by a viral vector can elicit full protection against diverse H5N1 challenges as well as induce broader immunity than a wild-type hemagglutinin.

  • Early Preservation of CXCR5+ PD-1+ Helper T Cells and B Cell Activation Predict the Breadth of Neutralizing Antibody Responses in Chronic HIV-1 Infection [Cellular Response to Infection]

  • Much is known about the characteristics of broadly neutralizing antibodies (bNAbs) generated during HIV-1 infection, but little is known about immunological mechanisms responsible for their development in only a minority of those infected by HIV-1. By monitoring longitudinally a cohort of HIV-1-infected subjects, we observed that the preservation of CXCR5+ CD4+ T helper cell frequencies and activation status of B cells during the first year of infection correlates with the maximum breadth of plasma neutralizing antibody responses during chronic infection independently of viral load. Although, during the first year of infection, no differences were observed in the abilities of peripheral CXCR5+ CD4+ T helper cells to induce antibody secretion by autologous naive B cells, higher frequencies of class-switched antibodies were detected in cocultures of CXCR5+ CD4+ T and B cells from the subjects who later developed broadly neutralizing antibody responses than those who did not. Furthermore, B cells from the former subjects had higher expression of AICDA than B cells from the latter subjects, and transcript levels correlated with the frequency of CXCR5+ CD4+ T cells. Thus, the early preservation of CXCR5+ CD4+ T cells and B cell function are central to the development of bNAbs. Our study provides a possible explanation for their infrequent generation during HIV-1 infection.

    IMPORTANCE Broadly neutralizing antibodies are developed by HIV-1-infected subjects, but so far (and despite intensive efforts over the past 3 decades) they have not been elicited by immunization. Understanding how bNAbs are generated during natural HIV-1 infection and why only some HIV-1-infected subjects generate such antibodies will assist our efforts to elicit bNAbs by immunization. CXCR5+ PD-1+ CD4+ T cells are critical for the development of high-affinity antigen-specific antibody responses. In our study, we found that the HIV-1-infected subjects who develop bNAbs have a higher frequency of peripheral CXCR5+ PD-1+ CD4+ T cells in early infection and also that this frequency mirrored what was observed in uninfected subjects and correlated with the level of B cell activation across subjects. Our study highlights the critical role helper T cell function has in the elicitation of broadly neutralizing antibody responses in the context of HIV infection.

  • Expression Library Immunization Can Confer Protection against Lethal Challenge with African Swine Fever Virus [Vaccines and Antiviral Agents]

  • African swine fever is one of the most devastating pig diseases, against which there is no vaccine available. Recent work from our laboratory has demonstrated the protective potential of DNA vaccines encoding three African swine fever viral antigens (p54, p30, and the hemagglutinin extracellular domain) fused to ubiquitin. Partial protection was afforded in the absence of detectable antibodies prior to virus challenge, and survival correlated with the presence of a large number of hemagglutinin-specific CD8+ T cells in blood. Aiming to demonstrate the presence of additional CD8+ T-cell determinants with protective potential, an expression library containing more than 4,000 individual plasmid clones was constructed, each one randomly containing a Sau3AI restriction fragment of the viral genome (p54, p30, and hemagglutinin open reading frames [ORFs] excluded) fused to ubiquitin. Immunization of farm pigs with the expression library yielded 60% protection against lethal challenge with the virulent E75 strain. These results were further confirmed by using specific-pathogen-free pigs after challenging them with 104 hemadsorbing units (HAU) of the cell culture-adapted strain E75CV1. On this occasion, 50% of the vaccinated pigs survived the lethal challenge, and 2 out of the 8 immunized pigs showed no viremia or viral excretion at any time postinfection. In all cases, protection was afforded in the absence of detectable specific antibodies prior to challenge and correlated with the detection of specific T-cell responses at the time of sacrifice. In summary, our results clearly demonstrate the presence of additional protective determinants within the African swine fever virus (ASFV) genome and open up the possibility for their future identification.

    IMPORTANCE African swine fever is a highly contagious disease of domestic and wild pigs that is endemic in many sub-Saharan countries, where it causes important economic losses and is currently in continuous expansion across Europe. Unfortunately, there is no treatment nor an available vaccine. Early attempts using attenuated vaccines demonstrated their potential to protect pigs against experimental infection. However, their use in the field remains controversial due to safety issues. Although inactive and subunit vaccines did not confer solid protection against experimental ASFV infection, our DNA vaccination results have generated new expectations, confirming the key role of T-cell responses in protection and the existence of multiple ASFV antigens with protective potential, more of which are currently being identified. Thus, the future might bring complex and safe formulations containing more than a single viral determinant to obtain broadly protective vaccines. We believe that obtaining the optimal vaccine formulation it is just a matter of time, investment, and willingness.

  • Prime-Boost Immunization Strategies against Chikungunya Virus [Vaccines and Antiviral Agents]

  • Chikungunya virus (CHIKV) is a reemerging mosquito-borne alphavirus that causes debilitating arthralgia in humans. Here we describe the development and testing of novel DNA replicon and protein CHIKV vaccine candidates and evaluate their abilities to induce antigen-specific immune responses against CHIKV. We also describe homologous and heterologous prime-boost immunization strategies using novel and previously developed CHIKV vaccine candidates. Immunogenicity and efficacy were studied in a mouse model of CHIKV infection and showed that the DNA replicon and protein antigen were potent vaccine candidates, particularly when used for priming and boosting, respectively. Several prime-boost immunization strategies eliciting unmatched humoral and cellular immune responses were identified. Further characterization by antibody epitope mapping revealed differences in the qualitative immune responses induced by the different vaccine candidates and immunization strategies. Most vaccine modalities resulted in complete protection against wild-type CHIKV infection; however, we did identify circumstances under which certain immunization regimens may lead to enhancement of inflammation upon challenge. These results should help guide the design of CHIKV vaccine studies and will form the basis for further preclinical and clinical evaluation of these vaccine candidates.

    IMPORTANCE As of today, there is no licensed vaccine to prevent CHIKV infection. In considering potential new vaccine candidates, a vaccine that could raise long-term protective immunity after a single immunization would be preferable. While humoral immunity seems to be central for protection against CHIKV infection, we do not yet fully understand the correlates of protection. Therefore, in the absence of a functional vaccine, there is a need to evaluate a number of different candidates, assessing their merits when they are used either in a single immunization or in a homologous or heterologous prime-boost modality. Here we show that while single immunization with various vaccine candidates results in potent responses, combined approaches significantly enhance responses, suggesting that such approaches need to be considered in the further development of an efficacious CHIKV vaccine.

  • Occurrence and Reassortment of Avian Influenza A (H7N9) Viruses Derived from Coinfected Birds in China [Genetic Diversity and Evolution]

  • Over the course of two waves of infection, H7N9 avian influenza A virus has caused 436 human infections and claimed 170 lives in China as of July 2014. To investigate the prevalence and genetic diversity of H7N9, we surveyed avian influenza viruses in poultry in Jiangsu province within the outbreak epicenter. We found frequent occurrence of H7N9/H9N2 coinfection in chickens. Molecular clock phylogenetic analysis confirms coinfection by H7N9/H9N2 viruses and also reveals that the identity of the H7N9 outbreak lineage is confounded by ongoing reassortment between outbreak viruses and diverse H9N2 viruses in domestic birds. Experimental inoculation of a coinfected sample in cell culture yielded two reassortant H7N9 strains with polymerase segments from the original H9N2 strain. Ongoing reassortment between the H7N9 outbreak lineage and diverse H9N2 viruses may generate new strains with the potential to infect humans, highlighting the need for continued viral surveillance in poultry and humans.

    IMPORTANCE We found frequent occurrence of H7N9/H9N2 coinfection in chickens. The H7N9 outbreak lineage is confounded by ongoing reassortment between H7N9 and H9N2 viruses. The importance of H9N2 viruses as the source of novel avian influenza virus infections in humans requires continuous attention.

  • Hallmarks of Hepatitis C Virus in Equine Hepacivirus [Genetic Diversity and Evolution]

  • Equine hepacivirus (EHcV) has been identified as a closely related homologue of hepatitis C virus (HCV) in the United States, the United Kingdom, and Germany, but not in Asian countries. In this study, we genetically and serologically screened 31 serum samples obtained from Japanese-born domestic horses for EHcV infection and subsequently identified 11 PCR-positive and 7 seropositive serum samples. We determined the full sequence of the EHcV genome, including the 3' untranslated region (UTR), which had previously not been completely revealed. The polyprotein of a Japanese EHcV strain showed approximately 95% homology to those of the reported strains. HCV-like cis-acting RNA elements, including the stem-loop structures of the 3' UTR and kissing-loop interaction were deduced from regions around both UTRs of the EHcV genome. A comparison of the EHcV and HCV core proteins revealed that Ile190 and Phe191 of the EHcV core protein could be important for cleavage of the core protein by signal peptide peptidase (SPP) and were replaced with Ala and Leu, respectively, which inhibited intramembrane cleavage of the EHcV core protein. The loss-of-function mutant of SPP abrogated intramembrane cleavage of the EHcV core protein and bound EHcV core protein, suggesting that the EHcV core protein may be cleaved by SPP to become a mature form. The wild-type EHcV core protein, but not the SPP-resistant mutant, was localized on lipid droplets and partially on the lipid raft-like membrane in a manner similar to that of the HCV core protein. These results suggest that EHcV may conserve the genetic and biological properties of HCV.

    IMPORTANCE EHcV, which shows the highest amino acid or nucleotide homology to HCV among hepaciviruses, was previously reported to infect horses from Western, but not Asian, countries. We herein report EHcV infection in Japanese-born horses. In this study, HCV-like RNA secondary structures around both UTRs were predicted by determining the whole-genome sequence of EHcV. Our results also suggest that the EHcV core protein is cleaved by SPP to become a mature form and then is localized on lipid droplets and partially on lipid raft-like membranes in a manner similar to that of the HCV core protein. Hence, EHcV was identified as a closely related homologue of HCV based on its genetic structure as well as its biological properties. A clearer understanding of the epidemiology, genetic structure, and infection mechanism of EHcV will assist in elucidating the evolution of hepaciviruses as well as the development of surrogate models for the study of HCV.

  • Cholesterol-Dependent Membrane Fusion Induced by the gp41 Membrane-Proximal External Region-Transmembrane Domain Connection Suggests a Mechanism for Broad HIV-1 Neutralization [Pathogenesis and Immunity]

  • The HIV-1 glycoprotein 41 promotes fusion of the viral membrane with that of the target cell. Structural, biochemical, and biophysical studies suggest that its membrane-proximal external region (MPER) may interact with the HIV-1 membrane and induce its disruption and/or deformation during the process. However, the high cholesterol content of the envelope (ca. 40 to 50 mol%) imparts high rigidity, thereby acting against lipid bilayer restructuring. Here, based on the outcome of vesicle stability assays, all-atom molecular dynamics simulations, and atomic force microscopy observations, we propose that the conserved sequence connecting the MPER with the N-terminal residues of the transmembrane domain (TMD) is involved in HIV-1 fusion. This junction would function by inducing phospholipid protrusion and acyl-chain splay in the cholesterol-enriched rigid envelope. Supporting the functional relevance of such a mechanism, membrane fusion was inhibited by the broadly neutralizing 4E10 antibody but not by a nonneutralizing variant with the CDR-H3 loop deleted. We conclude that the MPER-TMD junction embodies an envelope-disrupting C-terminal fusion peptide that can be targeted by broadly neutralizing antibodies.

    IMPORTANCE Fusion of the cholesterol-enriched viral envelope with the cell membrane marks the beginning of the infectious HIV-1 replicative cycle. Consequently, the Env glycoprotein-mediated fusion function constitutes an important clinical target for inhibitors and preventive vaccines. Antibodies 4E10 and 10E8 bind to one Env vulnerability site located at the gp41 membrane-proximal external region (MPER)nndash;transmembrane domain (TMD) junction and block infection. These antibodies display broad viral neutralization, which underscores the conservation and functionality of the MPER-TMD region. In this work, we combined biochemical assays with molecular dynamics simulations and microscopy observations to characterize the unprecedented fusogenic activity of the MPER-TMD junction. The fact that such activity is dependent on cholesterol and inhibited by the broadly neutralizing 4E10 antibody emphasizes its physiological relevance. Discovery of this functional element adds to our understanding of the mechanisms underlying HIV-1 infection and its blocking by antibodies.

  • Herpes Simplex Virus Internalization into Epithelial Cells Requires Na+/H+ Exchangers and p21-Activated Kinases but neither Clathrin- nor Caveolin-Mediated Endocytosis [Virus-Cell Interactions]

  • Herpes simplex virus 1 (HSV-1) is an alphaherpesvirus that has been reported to infect some epithelial cell types by fusion at the plasma membrane but others by endocytosis. To determine the molecular mechanisms of productive HSV-1 cell entry, we perturbed key endocytosis host factors using specific inhibitors, RNA interference (RNAi), or overexpression of dominant negative proteins and investigated their effects on HSV-1 infection in the permissive epithelial cell lines Vero, HeLa, HEp-2, and PtK2. HSV-1 internalization required neither endosomal acidification nor clathrin- or caveolin-mediated endocytosis. In contrast, HSV-1 gene expression and internalization were significantly reduced after treatment with 5-(N-ethyl-N-isopropyl)amiloride (EIPA). EIPA blocks the activity of Na+/H+ exchangers, which are plasma membrane proteins implicated in all forms of macropinocytosis. HSV-1 internalization furthermore required the function of p21-activated kinases that contribute to macropinosome formation. However, in contrast to some forms of macropinocytosis, HSV-1 did not enlist the activities of protein kinase C (PKC), tyrosine kinases, C-terminal binding protein 1, or dynamin to activate its internalization. These data suggest that HSV-1 depends on Na+/H+ exchangers and p21-activated kinases either for macropinocytosis or for local actin rearrangements required for fusion at the plasma membrane or subsequent passage through the actin cortex underneath the plasma membrane.

    IMPORTANCE After initial replication in epithelial cells, herpes simplex viruses (HSVs) establish latent infections in neurons innervating these regions. Upon primary infection and reactivation from latency, HSVs cause many human skin and neurological diseases, particularly in immunocompromised hosts, despite the availability of effective antiviral drugs. Many viruses use macropinocytosis for virus internalization, and many host factors mediating this entry route have been identified, although the specific perturbation profiles vary for different host and viral cargo. In addition to an established entry pathway via acidic endosomes, we show here that HSV-1 internalization depended on sodium-proton exchangers at the plasma membrane and p21-activated kinases. These results suggest that HSV-1 requires a reorganization of the cortical actin cytoskeleton, either for productive cell entry via pH-independent fusion from macropinosomes or for fusion at the plasma membrane, and subsequent cytosolic passage to microtubules that mediate capsid transport to the nucleus for genome uncoating and replication.

  • Characterization of pH-Sensitive Molecular Switches That Trigger the Structural Transition of Vesicular Stomatitis Virus Glycoprotein from the Postfusion State toward the Prefusion State [Structure and Assembly]

  • Vesicular stomatitis virus (VSV; the prototype rhabdovirus) fusion is triggered at low pH and mediated by glycoprotein G, which undergoes a low-pH-induced structural transition. A unique feature of rhabdovirus G is that its conformational change is reversible. This allows G to recover its native prefusion state at the viral surface after its transport through the acidic Golgi compartments. The crystal structures of G pre- and postfusion states have been elucidated, leading to the identification of several acidic amino acid residues, clustered in the postfusion trimer, as potential pH-sensitive switches controlling the transition back toward the prefusion state. We mutated these residues and produced a panel of single and double mutants whose fusion properties, conformational change characteristics, and ability to pseudotype a virus lacking the glycoprotein gene were assayed. Some of these mutations were also introduced in the genome of recombinant viruses which were further characterized. We show that D268, located in the segment consisting of residues 264 to 273, which refolds into postfusion helix F during G structural transition, is the major pH sensor while D274, D395, and D393 have additional contributions. Furthermore, a single passage of recombinant virus bearing the mutation D268L (which was demonstrated to stabilize the G postfusion state) resulted in a pseudorevertant with a compensatory second mutation, L271P. This revealed that the propensity of the segment of residues 264 to 273 to refold into helix F has to be finely tuned since either an increase (mutation D268L alone) or a decrease (mutation L271P alone) of this propensity is detrimental to the virus.

    IMPORTANCE Vesicular stomatitis virus enters cells via endocytosis. Endosome acidification induces a structural transition of its unique glycoprotein (G), which mediates fusion between viral and endosomal membranes. G conformational change is reversible upon increases in pH. This allows G to recover its native prefusion state at the viral surface after its transport through the acidic Golgi compartments. We mutated five acidic residues, proposed to be pH-sensitive switches controlling the structural transition back toward the prefusion state. Our results indicate that residue D268 is the major pH sensor, while other acidic residues have additional contributions, and reveal that the propensity of the segment consisting of residues 264 to 273 to adopt a helical conformation is finely regulated. This segment might be a good target for antiviral compounds.

  • A Bivalent Vaccine Based on a Replication-Incompetent Influenza Virus Protects against Streptococcus pneumoniae and Influenza Virus Infection [Vaccines and Antiviral Agents]

  • Streptococcus pneumoniae is a major causative pathogen in community-acquired pneumonia; together with influenza virus, it represents an important public health burden. Although vaccination is the most effective prophylaxis against these infectious agents, no single vaccine simultaneously provides protective immunity against both S. pneumoniae and influenza virus. Previously, we demonstrated that several replication-incompetent influenza viruses efficiently elicit IgG in serum and IgA in the upper and lower respiratory tracts. Here, we generated a replication-incompetent hemagglutinin knockout (HA-KO) influenza virus possessing the sequence for the antigenic region of pneumococcal surface protein A (PspA). Although this virus (HA-KO/PspA virus) could replicate only in an HA-expressing cell line, it infected wild-type cells and expressed both viral proteins and PspA. PspA- and influenza virus-specific antibodies were detected in nasal wash and bronchoalveolar lavage fluids and in sera from mice intranasally inoculated with HA-KO/PspA virus, and mice inoculated with HA-KO/PspA virus were completely protected from lethal challenge with either S. pneumoniae or influenza virus. Further, bacterial colonization of the nasopharynx was prevented in mice immunized with HA-KO/PspA virus. These results indicate that HA-KO/PspA virus is a promising bivalent vaccine candidate that simultaneously confers protective immunity against both S. pneumoniae and influenza virus. We believe that this strategy offers a platform for the development of bivalent vaccines, based on replication-incompetent influenza virus, against pathogens that cause respiratory infectious diseases.

    IMPORTANCE Streptococcus pneumoniae and influenza viruses cause contagious diseases, but no single vaccine can simultaneously provide protective immunity against both pathogens. Here, we used reverse genetics to generate a replication-incompetent influenza virus carrying the sequence for the antigenic region of pneumococcal surface protein A and demonstrated that mice immunized with this virus were completely protected from lethal doses of infection with either influenza virus or Streptococcus pneumoniae. We believe that this strategy, which is based on a replication-incompetent influenza virus possessing the antigenic region of other respiratory pathogens, offers a platform for the development of bivalent vaccines.

  • Modified Vaccinia Virus Ankara Encoding Influenza Virus Hemagglutinin Induces Heterosubtypic Immunity in Macaques [Vaccines and Antiviral Agents]

  • Current influenza virus vaccines primarily aim to induce neutralizing antibodies (NAbs). Modified vaccinia virus Ankara (MVA) is a safe and well-characterized vector for inducing both antibody and cellular immunity. We evaluated the immunogenicity and protective efficacy of MVA encoding influenza virus hemagglutinin (HA) and/or nucleoprotein (NP) in cynomolgus macaques. Animals were given 2 doses of MVA-based vaccines 4 weeks apart and were challenged with a 2009 pandemic H1N1 isolate (H1N1pdm) 8 weeks after the last vaccination. MVA-based vaccines encoding HA induced potent serum antibody responses against homologous H1 or H5 HAs but did not stimulate strong T cell responses prior to challenge. However, animals that received MVA encoding influenza virus HA and/or NP had high frequencies of virus-specific CD4+ and CD8+ T cell responses within the first 7 days of H1N1pdm infection, while animals vaccinated with MVA encoding irrelevant antigens did not. We detected little or no H1N1pdm replication in animals that received vaccines encoding H1 (homologous) HA, while a vaccine encoding NP from an H5N1 isolate afforded no protection. Surprisingly, H1N1pdm viral shedding was reduced in animals vaccinated with MVA encoding HA and NP from an H5N1 isolate. This reduced shedding was associated with cross-reactive antibodies capable of mediating antibody-dependent cellular cytotoxicity (ADCC) effector functions. Our results suggest that ADCC plays a role in cross-protective immunity against influenza. Vaccines optimized to stimulate cross-reactive antibodies with ADCC function may provide an important measure of protection against emerging influenza viruses when NAbs are ineffective.

    IMPORTANCE Current influenza vaccines are designed to elicit neutralizing antibodies (NAbs). Vaccine-induced NAbs typically are effective but highly specific for particular virus strains. Consequently, current vaccines are poorly suited for preventing the spread of newly emerging pandemic viruses. Therefore, we evaluated a vaccine strategy designed to induce both antibody and T cell responses, which may provide more broadly cross-protective immunity against influenza. Here, we show in a translational primate model that vaccination with a modified vaccinia virus Ankara encoding hemagglutinin from a heterosubtypic H5N1 virus was associated with reduced shedding of a pandemic H1N1 virus challenge, while vaccination with MVA encoding nucleoprotein, an internal viral protein, was not. Unexpectedly, this reduced shedding was associated with nonneutralizing antibodies that bound H1 hemagglutinin and activated natural killer cells. Therefore, antibody-dependent cellular cytotoxicity (ADCC) may play a role in cross-protective immunity to influenza virus. Vaccines that stimulate ADCC antibodies may enhance protection against pandemic influenza virus.

  • In Vivo Cellular Tropism of Gorilla Simian Foamy Virus in Blood of Infected Humans [Virus-Cell Interactions]

  • Simian foamy viruses (SFV) are retroviruses that are widespread among nonhuman primates. SFV can be transmitted to humans, giving rise to a persistent infection. Only a few data are available concerning the distribution of SFV in human blood cells. Here we purified blood mononuclear cell subsets from 11 individuals infected with a Gorilla gorilla SFV strain and quantified SFV DNA levels by quantitative PCR. SFV DNA was detected in the majority of the CD8+, CD4+, and CD19+ lymphocyte samples and rarely in CD14+ monocyte and CD56+ NK lymphocyte samples. The median (interquartile range [IQR]) SFV DNA counts were 16.0 (11.0 to 49.8), 11.3 (5.9 to 28.3), and 17.2 (2.0 to 25.2) copies/105 cells in CD8+ T lymphocytes, CD4+ T lymphocytes, and CD19+ B lymphocytes, respectively. In the CD4 compartment, SFV DNA was detected in both memory and naive CD4+ T lymphocytes. SFV DNA levels in CD4+ T cells were positively correlated with the duration of the infection. Our study shows with a quantitative method that CD8+, CD4+, and B lymphocytes are major cellular targets of SFV in the blood of infected humans.

    IMPORTANCE Investigation of SFV infections in humans is important due to the origin of human immunodeficiency viruses (HIV) and human T cell lymphotropic viruses (HTLV) from cross-species transmission of their simian counterparts to humans. Surprisingly little is known about many aspects of the biology of SFV in infected humans, including quantitative data concerning the cellular targets of SFV in vivo. Here we show that the distribution of SFV DNA among the different leukocyte populations is not homogeneous and that viral load in CD4+ T lymphocytes is correlated with the duration of infection. These new data will help in understanding the biology of retroviral infections in humans and can be useful in the growing field of SFV-based gene therapy.

  • Mutations to PB2 and NP Proteins of an Avian Influenza Virus Combine To Confer Efficient Growth in Primary Human Respiratory Cells [Virus-Cell Interactions]

  • Influenza pandemics occur when influenza A viruses (IAV) adapted to other host species enter humans and spread through the population. Pandemics are relatively rare due to host restriction of IAV: strains adapted to nonhuman species do not readily infect, replicate in, or transmit among humans. IAV can overcome host restriction through reassortment or adaptive evolution, and these are mechanisms by which pandemic strains arise in nature. To identify mutations that facilitate growth of avian IAV in humans, we have adapted influenza A/duck/Alberta/35/1976 (H1N1) (dk/AB/76) virus to a high-growth phenotype in differentiated human tracheo-bronchial epithelial (HTBE) cells. Following 10 serial passages of three independent lineages, the bulk populations showed similar growth in HTBE cells to that of a human seasonal virus. The coding changes present in six clonal isolates were determined. The majority of changes were located in the polymerase complex and nucleoprotein (NP), and all isolates carried mutations in the PB2 627 domain and regions of NP thought to interact with PB2. Using reverse genetics, the impact on growth and polymerase activity of individual and paired mutations in PB2 and NP was evaluated. The results indicate that coupling of the mammalian-adaptive mutation PB2 E627K or Q591K to selected mutations in NP further augments the growth of the corresponding viruses. In addition, minimal combinations of three (PB2 Q236H, E627K, and NP N309K) or two (PB2 Q591K and NP S50G) mutations were sufficient to recapitulate the efficient growth in HTBE cells of dk/AB/76 viruses isolated after 10 passages in this substrate.

    IMPORTANCE Influenza A viruses adapted to birds do not typically grow well in humans. However, as has been seen recently with H5N1 and H7N9 subtype viruses, productive and virulent infection of humans with avian influenza viruses can occur. The ability of avian influenza viruses to adapt to new host species is a consequence of their high mutation rate that supports their zoonotic potential. Understanding of the adaptation of avian viruses to mammals strengthens public health efforts aimed at controlling influenza. In particular, it is critical to know how readily and through mutation to which functional components avian influenza viruses gain the ability to grow efficiently in humans. Our data show that as few as three mutations, in the PB2 and NP proteins, support robust growth of a low-pathogenic, H1N1 duck isolate in primary human respiratory cells.

  • A Unique Nodavirus with Novel Features: Mosinovirus Expresses Two Subgenomic RNAs, a Capsid Gene of Unknown Origin, and a Suppressor of the Antiviral RNA Interference Pathway [Genetic Diversity and Evolution]

  • Insects are a reservoir for many known and novel viruses. We discovered an unknown virus, tentatively named mosinovirus (MoNV), in mosquitoes from a tropical rainforest region in Coocirc;te d'Ivoire. The MoNV genome consists of two segments of positive-sense RNA of 2,972 nucleotides (nt) (RNA 1) and 1,801 nt (RNA 2). Its putative RNA-dependent RNA polymerase shares 43% amino acid identity with its closest relative, that of the Pariacoto virus (family Nodaviridae). Unexpectedly, for the putative capsid protein, maximal pairwise identity of 16% to Lake Sinai virus 2, an unclassified virus with a nonsegmented RNA genome, was found. Moreover, MoNV virions are nonenveloped and about 50 nm in diameter, larger than any of the known nodaviruses. Mature MoNV virions contain capsid proteins of ~56 kDa, which do not seem to be cleaved from a longer precursor. Northern blot analyses revealed that MoNV expresses two subgenomic RNAs of 580 nt (RNA 3) and 292 nt (RNA 4). RNA 4 encodes a viral suppressor of RNA interference (RNAi) that shares its mechanism with the B2 RNAi suppressor protein of other nodaviruses despite lacking recognizable similarity to these proteins. MoNV B2 binds long double-stranded RNA (dsRNA) and, accordingly, inhibits Dicer-2-mediated processing of dsRNA into small interfering RNAs (siRNAs). Phylogenetic analyses indicate that MoNV is a novel member of the family Nodaviridae that acquired its capsid gene via reassortment from an unknown, distantly related virus beyond the family level.

    IMPORTANCE The identification of novel viruses provides important information about virus evolution and diversity. Here, we describe an unknown unique nodavirus in mosquitoes, named mosinovirus (MoNV). MoNV was classified as a nodavirus based on its genome organization and on phylogenetic analyses of the RNA-dependent RNA polymerase. Notably, its capsid gene was acquired from an unknown virus with a distant relationship to nodaviruses. Another remarkable feature of MoNV is that, unlike other nodaviruses, it expresses two subgenomic RNAs (sgRNAs). One of the sgRNAs expresses a protein that counteracts antiviral defense of its mosquito host, whereas the function of the other sgRNA remains unknown. Our results show that complete genome segments can be exchanged beyond the species level and suggest that insects harbor a large repertoire of exceptional viruses.

  • Specific Cells in the Primary Salivary Glands of the Whitefly Bemisia tabaci Control Retention and Transmission of Begomoviruses [Virus-Cell Interactions]

  • The majority of plant viruses are vectored by arthropods via persistent-circulative or noncirculative transmission. Previous studies have shown that specific binding sites for noncirculative viruses reside within the stylet or foregut of insect vectors, whereas the transmission mechanisms of circulative viruses remain ambiguous. Here we report the critical roles of whitefly primary salivary glands (PSGs) in the circulative transmission of two begomoviruses. The Middle East Asia Minor 1 (MEAM1) species of the whitefly Bemisia tabaci complex efficiently transmits both Tomato yellow leaf curl China virus (TYLCCNV) and Tomato yellow leaf curl virus (TYLCV), whereas the Mediterranean (MED) species transmits TYLCV but not TYLCCNV. PCR and fluorescence in situ hybridization experiments showed that TYLCCNV efficiently penetrates the PSGs of MEAM1 but not MED whiteflies. When a fragment of the coat protein of TYLCCNV was exchanged with that of TYLCV, mutated TYLCCNV accumulated in the PSGs of MED whiteflies, while mutant TYLCV was nearly undetectable. Confocal microscopy revealed that virion transport in PSGs follows specific paths to reach secretory cells in the central region, and the accumulation of virions in the secretory region of PSGs was correlated with successful virus transmission. Our findings demonstrate that whitefly PSGs, in particular the cells around the secretory region, control the specificity of begomovirus transmission.

    IMPORTANCE Over 75% of plant viruses are transmitted by insects. However, the mechanisms of virus transmission by insect vectors remain largely unknown. Begomoviruses and whiteflies are a complex of viruses and vectors which threaten many crops worldwide. We investigated the transmission of two begomoviruses by two whitefly species. We show that specific cells of the whitefly primary salivary glands control viral transmission specificity and that virion transport in the glands follows specific paths to reach secretory cells in the central region and then to reach the salivary duct. Our results indicate that the secretory cells in the central region of primary salivary glands determine the recognition and transmission of begomoviruses. These findings set a foundation for future research not only on circulative plant virus transmission but also on other human and animal viruses transmitted by arthropod vectors.

  • Adenovirus E1A Targets the DREF Nuclear Factor To Regulate Virus Gene Expression, DNA Replication, and Growth [Virus-Cell Interactions]

  • The adenovirus E1A gene is the first gene expressed upon viral infection. E1A remodels the cellular environment to maximize permissivity for viral replication. E1A is also the major transactivator of viral early gene expression and a coregulator of a large number of cellular genes. E1A carries out its functions predominantly by binding to cellular regulatory proteins and altering their activities. The unstructured nature of E1A enables it to bind to a large variety of cellular proteins and form new molecular complexes with novel functions. The C terminus of E1A is the least-characterized region of the protein, with few known binding partners. Here we report the identification of cellular factor DREF (ZBED1) as a novel and direct binding partner of E1A. Our studies identify a dual role for DREF in the viral life cycle. DREF contributes to activation of gene expression from all viral promoters early in infection. Unexpectedly, it also functions as a growth restriction factor for adenovirus as knockdown of DREF enhances virus growth and increases viral genome copy number late in the infection. We also identify DREF as a component of viral replication centers. E1A affects the subcellular distribution of DREF within PML bodies and enhances DREF SUMOylation. Our findings identify DREF as a novel E1A C terminus binding partner and provide evidence supporting a role for DREF in viral replication.

    IMPORTANCE This work identifies the putative transcription factor DREF as a new target of the E1A oncoproteins of human adenovirus. DREF was found to primarily localize with PML nuclear bodies in uninfected cells and to relocalize into virus replication centers during infection. DREF was also found to be SUMOylated, and this was enhanced in the presence of E1A. Knockdown of DREF reduced the levels of viral transcripts detected at 20 h, but not at 40 h, postinfection, increased overall virus yield, and enhanced viral DNA replication. DREF was also found to localize to viral promoters during infection together with E1A. These results suggest that DREF contributes to activation of viral gene expression. However, like several other PML-associated proteins, DREF also appears to function as a growth restriction factor for adenovirus infection.

  • HBZ Stimulates Brain-Derived Neurotrophic Factor/TrkB Autocrine/Paracrine Signaling To Promote Survival of Human T-Cell Leukemia Virus Type 1-Infected T Cells [Transformation and Oncogenesis]

  • Brain-derived neurotrophic factor (BDNF) is a neurotrophin that promotes neuronal proliferation, survival, and plasticity. These effects occur through autocrine and paracrine signaling events initiated by interactions between secreted BDNF and its high-affinity receptor, TrkB. A BDNF/TrkB autocrine/paracrine signaling loop has additionally been implicated in augmenting the survival of cells representing several human cancers and is associated with poor patient prognosis. Adult T-cell leukemia (ATL) is a fatal malignancy caused by infection with the complex retrovirus human T-cell leukemia virus type 1 (HTLV-1). In this study, we found that the HTLV-1-encoded protein HBZ activates expression of BDNF, and consistent with this effect, BDNF expression is elevated in HTLV-1-infected T-cell lines compared to uninfected T cells. Expression of TrkB is also higher in HTLV-1-infected T-cell lines than in uninfected T cells. Furthermore, levels of both BDNF and TrkB mRNAs are elevated in peripheral blood mononuclear cells (PBMCs) from ATL patients, and ATL patient sera contain higher concentrations of BDNF than sera from noninfected individuals. Finally, chemical inhibition of TrkB signaling increases apoptosis in HTLV-1-infected T cells and reduces phosphorylation of glycogen synthase kinase 3bbeta; (GSK-3bbeta;), a downstream target in the signaling pathway. These results suggest that HBZ contributes to an active BDNF/TrkB autocrine/paracrine signaling loop in HTLV-1-infected T cells that enhances the survival of these cells.

    IMPORTANCE Infection with human T-cell leukemia virus type 1 (HTLV-1) can cause a rare form of leukemia designated adult T-cell leukemia (ATL). Because ATL patients are unresponsive to chemotherapy, this malignancy is fatal. As a retrovirus, HTLV-1 integrates its genome into a host cell chromosome in order to utilize host factors for replication and expression of viral proteins. However, in infected cells from ATL patients, the viral genome is frequently modified to block expression of all but a single viral protein. This protein, known as HBZ, is therefore believed to modulate cellular pathways necessary for the leukemic state and the chemotherapeutic resistance of the cell. Here we provide evidence to support this hypothesis. We found that HBZ promotes a BDNF/TrkB autocrine/paracrine signaling pathway that is known to enhance the survival and chemotherapeutic resistance of other types of cancer cells. It is possible that inhibition of this pathway may improve treatments for ATL.

  • Circulating Clinical Strains of Human Parainfluenza Virus Reveal Viral Entry Requirements for In Vivo Infection [Virus-Cell Interactions]

  • Human parainfluenza viruses (HPIVs) cause widespread respiratory infections, with no vaccines or effective treatments. We show that the molecular determinants for HPIV3 growth in vitro are fundamentally different from those required in vivo and that these differences impact inhibitor susceptibility. HPIV infects its target cells by coordinated action of the hemagglutinin-neuraminidase receptor-binding protein (HN) and the fusion envelope glycoprotein (F), which together comprise the molecular fusion machinery; upon receptor engagement by HN, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. Peptides derived from key regions of F can potently inhibit HPIV infection at the entry stage, by interfering with the structural transition of F. We show that clinically circulating viruses have fusion machinery that is more stable and less readily activated than viruses adapted to growth in culture. Fusion machinery that is advantageous for growth in human airway epithelia and in vivo confers susceptibility to peptide fusion inhibitors in the host lung tissue or animal, but the same fusion inhibitors have no effect on viruses whose fusion glycoproteins are suited for growth in vitro. We propose that for potential clinical efficacy, antivirals should be evaluated using clinical isolates in natural host tissue rather than lab strains of virus in cultured cells. The unique susceptibility of clinical strains in human tissues reflects viral inhibition in vivo.

    IMPORTANCE Acute respiratory infection is the leading cause of mortality in young children under 5 years of age, causing nearly 20% of childhood deaths worldwide each year. The paramyxoviruses, including human parainfluenza viruses (HPIVs), cause a large share of these illnesses. There are no vaccines or drugs for the HPIVs. Inhibiting entry of viruses into the human cell is a promising drug strategy that blocks the first step in infection. To develop antivirals that inhibit entry, it is critical to understand the first steps of infection. We found that clinical viruses isolated from patients have very different entry properties from those of the viruses generally studied in laboratories. The viral entry mechanism is less active and more sensitive to fusion inhibitory molecules. We propose that to interfere with viral infection, we test clinically circulating viruses in natural tissues, to develop antivirals against respiratory disease caused by HPIVs.

  • Binding Interactions between the Encephalomyocarditis Virus Leader and Protein 2A [Genome Replication and Regulation of Viral Gene Expression]

  • The leader (L) and 2A proteins of cardioviruses are the primary antihost agents produced during infection. For encephalomyocarditis virus (EMCV), the prototype of the genus Cardiovirus, these proteins interact independently with key cellular partners to bring about inhibition of active nucleocytoplasmic trafficking and cap-dependent translation, respectively. L and 2A also bind each other and require this cooperation to achieve their effects during infection. Recombinant L and 2A interact with 1:1 stoichiometry at a KD (equilibrium dissociation constant) of 1.5 mmu;M. The mapped contact domains include the amino-proximal third of 2A (first 50 amino acids) and the central hinge region of L. This contact partially overlaps the L segment that makes subsequent contact with Ran GTPase in the nucleus, and Ran can displace 2A from L. The equivalent proteins from Theiler's murine encephalomyelitis virus (TMEV; BeAn) and Saffold virus interact similarly in any subtype combination, with various affinities. The data suggest a mechanism whereby L takes advantage of the nuclear localization signal in the COOH region of 2A to enhance its trafficking to the nucleus. Once there, it exchanges partners in favor of Ran. This required cooperation during infection explains many observed codependent phenotypes of L and 2A mutations.

    IMPORTANCE Cardiovirus pathogenesis phenotypes vary dramatically, from asymptomatic, to mild gastrointestinal (GI) distress, to persistent demyelination and even encephalitic death. Leader and 2A are the primary viral determinants of pathogenesis, so understanding how these proteins cooperate to induce such a wide variety of outcomes for the host is of great important and interest to the field of virology, especially to those who use TMEV as a murine model for multiple sclerosis.

  • Common Features of Mucosal and Peripheral Antibody Responses Elicited by Candidate HIV-1 Vaccines in Rhesus Monkeys [Vaccines and Antiviral Agents]

  • Human immunodeficiency virus type 1 (HIV-1) vaccines that elicit protective antibody responses at mucosal sites would be highly desirable. Here, we report that intramuscular immunization of candidate HIV-1 vaccine vectors and purified Env proteins elicited potent and durable humoral immune responses in colorectal mucosa in rhesus monkeys. The kinetics, isotypes, functionality, and epitope specificity of these mucosal antibody responses were similar to those of peripheral responses in serum. These data suggest a close immunological relationship between mucosal and systemic antibody responses following vaccination in primates.

  • Correction for Trott et al., Immune Activation and Regulation in Simian Immunodeficiency Virus-Plasmodium fragile-Coinfected Rhesus Macaques [Author Corrections]

  • Correction for Ruwona et al., Fine Mapping of Murine Antibody Responses to Immunization with a Novel Soluble Form of Hepatitis C Virus Envelope Glycoprotein Complex [Author Corrections]

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    JVI Accepts: Articles Published Ahead of Print

  • Entry mechanisms of Herpes Simplex Virus Type 1 into murine epidermis: Involvement of nectin-1 and HVEM as cellular receptors [PublishAheadOfPrint]

  • Skin keratinocytes represent a primary entry site for herpes simplex virus type 1 (HSV-1) in vivo. The cellular proteins nectin-1 and HVEM act as efficient receptors for both serotypes of HSV and are sufficient for disease development mediated by HSV-2 in mice. How HSV-1 enters skin, and whether both nectin-1 and HVEM are involved, is not known. We addressed the impact of nectin-1 during entry of HSV-1 into murine epidermis and investigated the putative contribution of HVEM. Using ex vivo infection of murine epidermis we showed that HSV-1 entered the basal keratinocytes of the epidermis very efficiently. In nectin-1 deficient epidermis entry was strongly reduced. Almost no entry was observed, however, in nectin-1 deficient keratinocytes grown in culture. This observation correlated with the presence of HVEM on the keratinocyte surface in epidermis and with the lack of HVEM expression in nectin-1 deficient primary keratinocytes. Our results suggest that nectin-1 is the primary receptor in epidermis while HVEM has a more limited role. In primary murine keratinocytes where nectin-1 acts as single receptor, electron microscopy suggested that HSV-1 can enter both by direct fusion with the plasma membrane and via endocytic vesicles. Thus, we conclude that nectin-1 directs internalization into keratinocytes via alternative pathways. In summary, HSV-1 entry into epidermis was shown to strongly depend on the presence of nectin-1, however, the restricted presence of HVEM can potentially replace nectin-1 as receptor illustrating the flexibility employed by HSV-1 to efficiently invade tissue in vivo.

    Importance Herpes simplex virus (HSV) can cause a range of diseases in humans from uncomplicated mucocutaneous lesions to life-threatening infections. The skin is one target tissue of HSV and the question of how the virus overcomes the protective skin barrier and penetrates into the tissue to reach its receptors is still open. Previous studies analyzing entry into cells grown in vitro revealed nectin-1 and HVEM as HSV receptors. To explore the contribution of nectin-1 and HVEM to entry into a natural target tissue, we established an ex vivo infection model. Using nectin-1 or HVEM deficient mice, we demonstrated the distinct involvement of nectin-1 and HVEM for HSV type 1 (HSV-1) entry into epidermis and characterized the internalization pathways. Such advances in understanding the involvement of receptors in tissue are essential preconditions for unravelling HSV invasion skin, which, in turn, allows the development of antiviral reagents.

  • Selective acquisition of host-derived ICAM-1 by HIV-1 is a matrix-dependent process [PublishAheadOfPrint]

  • HIV-1 acquires an impressive number of foreign components during its formation. Despite all the previous efforts spent studying the nature and functionality of virus-anchored host molecules, the exact mechanism(s) through which such constituents are acquired by HIV-1 is still unknown. However, in the case of ICAM-1, one of the most extensively studied transmembrane protein found associated with mature virions, the Pr55Gag precursor polyprotein appears as a potential interaction partner. We investigated and characterized at the molecular level the process of ICAM-1 incorporation using initially a Pr55Gag-based virus-like particle (VLP) model. Substitution of various domains of Pr55Gag, such as the nucleocapsid, SP2, or p6, had no effect on the acquisition of ICAM-1. We found that the structural Matrix protein (MA) is mandatory for ICAM-1 incorporation within VLPs, and we confirmed this novel observation with the replication-competent HIV-1 molecular clone NL4.3. Additional studies suggest that the C-terminal two thirds of MA are important, and especially thirteen amino acids positioned inside the fifth aalpha;-helix. Moreover, based on 3D-modeling of protein-protein interactions (i.e. protein-protein docking) and further validation by a virus capture assay, we found that a series of acidic residues in the MA domain interact with basic amino acids located in the ICAM-1 cytoplasmic tail. Our findings provide new insight on the molecular mechanism governing the acquisition of ICAM-1, a host molecule known to enhance HIV-1 infectivity in a significant manner. Altogether these observations offer a new avenue for the development of anti-viral therapeutics that are directed at a target of host origin.

    Importance Intercellular adhesion molecule I (ICAM-1) is a cell surface host component known to be efficiently inserted within emerging HIV-1 particles. It has been demonstrated that host-derived ICAM-1 molecules act as a strong attachment factor and increase substantially HIV-1 infectivity. Despite all the previous efforts spent studying virus-associated host molecules, the precise mechanism(s) through which such constituents are inserted within emerging HIV-1 particles still remain(s) obscure. Previous data suggest that the Pr55Gag precursor polyprotein appears as a potential interaction partner with ICAM-1. In the present study, we demonstrate that the HIV-1 matrix domain plays a key role in the ICAM-1 incorporation process. Some observations were confirmed in whole virus preparations amplified in primary human cells, thereby providing physiological significance to our data.

  • Identification of TRIM27 as a Novel Degradation Target of Herpes Simplex Virus 1 ICP0 [PublishAheadOfPrint]

  • The herpes simplex virus 1 immediate-early protein ICP0 performs many functions during infection, including transactivation of viral gene expression, suppression of innate immune responses, and modification as well as eviction of histones from viral chromatin. Although these functions of ICP0 have been characterized, the detailed mechanisms underlying ICP0's complex role during infection warrant further investigation. We thus undertook an unbiased proteomic approach to identifying viral and cellular proteins that interact with ICP0 in the infected cell. Cellular candidates resulting from our analysis included the ubiquitin-specific protease USP7, the transcriptional repressor TRIM27, DNA repair proteins NBS1 and MRE11, regulators of apoptosis including BIRC6, and the proteasome. We also identified two HSV-1 early proteins involved in nucleotide metabolism, UL39 and UL50, as novel candidate interactors of ICP0. Because TRIM27 was the most statistically significant cellular candidate, we investigated the relationship between TRIM27 and ICP0. We observed rapid, ICP0-dependent loss of TRIM27 during HSV-1 infection. TRIM27 protein levels were restored by disrupting the RING domain of ICP0 or by inhibiting the proteasome, arguing that TRIM27 is a novel degradation target of ICP0. A mutant ICP0 lacking E3 ligase activity interacted with endogenous TRIM27 during infection as demonstrated by reciprocal co-immunoprecipitation and supported by immunofluorescence data. Surprisingly, ICP0-null mutant virus yields decreased upon TRIM27 depletion, arguing that TRIM27 has a positive effect on infection despite being targeted for degradation. These results illustrate a complex interaction between TRIM27 and viral infection with potential positive or negative effects of TRIM27 on HSV under different infection conditions.

    Importance During productive infection a virus must simultaneously redirect multiple cellular pathways to replicate itself while evading detection by the host's defenses. To orchestrate such complex regulation, viruses including herpes simplex virus 1 (HSV-1) rely on multifunctional proteins such as the E3 ubiquitin ligase ICP0. This protein regulates various cellular pathways concurrently by targeting a diverse set of cellular factors for degradation. While some of these targets have been previously identified and characterized, we undertook a proteomic screen to identify additional targets of this activity to further characterize ICP0's role during infection. We describe a set of candidate interacting proteins of ICP0 identified through this approach as well as our characterization of the most statistically significant result, the cellular transcriptional repressor TRIM27. We present TRIM27 as a novel degradation target of ICP0 and describe the relationship of these two proteins during infection.

  • Kaposi's Sarcoma-Associated Herpesvirus-Encoded RTA Impairs Innate Immunity via Ubiquitin-mediated Degradation of MyD88 [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is a human -herpesvirus with latent and lytic reactivation cycles. The mechanism by which KSHV evades the innate immune system to establish latency has not yet been precisely elucidated. Toll-like receptors (TLRs) are the first line of defense against viral infections. Myeloid differentiation factor 88 (MyD88) is a key adaptor that interacts with all TLRs except TLR3 to produce inflammatory factors and type I interferons (IFNs), which are central components of innate immunity against microbial infection. Here, we found that KSHV replication and transcription activator (RTA), which is an immediate-early master switch protein of viral cycles, downregulates MyD88 expression at the protein level by degrading MyD88 through the ubiquitin (Ub)-proteasome pathway. We identified the interaction between RTA and MyD88 in vitro and in vivo and demonstrated that RTA functions as an E3 ligase to ubiquitinate MyD88. MyD88 was also repressed at the early stage of de novo infection as well as in lytic reactivation. We also found that RTA inhibited lipopolysaccharide (LPS)-triggered activation of the TLR4 pathway by reducing IFNs production and NF-B activity. Finally, we showed that MyD88 promoted the production of IFNs and inhibited KSHV LANA-1 gene transcription. Taken together, our results suggest that KSHV RTA facilitates the virus to evade innate immunity through degradation of MyD88, which might be critical for viral latency control.

    Importance: MyD88 is an adaptor for all TLRs other than TLR3 and mediates inflammatory factors and IFNs production. Our study demonstrated that the KSHV RTA protein functions as an E3 ligase to degrade MyD88 through the ubiquitin-proteasome pathway and block the transmission of TLRs signals. Moreover, we found that KSHV inhibited MyD88 expression during the early stage of de novo infection as well as in lytic reactivation. These results provide a potential mechanism for the virus to evade innate immunity.

  • Nucleocapsid Protein from Fig Mosaic Virus Forms Cytoplasmic Agglomerates That Are Hauled by Endoplasmic Reticulum Streaming [PublishAheadOfPrint]

  • Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while non-enveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated as NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant-negative inhibition of the class XI myosins, XI-1, XI-2 and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localized in the cytoplasm but in close proximity to the ER membrane to form enveloped particles, and that this caused passive movements of cytoplasmic NBs by ER streaming.

    IMPORTANCE Intracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors, but have failed to provide evidence for the mechanism or biological significance of their motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly throughout the cell, and performed live imaging and ultrastructural analysis to identify the mechanism of motility. We provide evidence that cytoplasmic protein agglomerates were passively dragged by actomyosin-mediated streaming of the endoplasmic reticulum (ER) in plant cells. In virus-infected cells, NP agglomerates were surrounded by the ER membranes, indicating that NP agglomerates form the basis of enveloped virus particles in close proximity to the ER. Our work provides a sophisticated model of macromolecular trafficking in plant cells and improves our understanding of the formation of enveloped particles of negative-strand RNA viruses.

  • Grouper heat-shock cognate protein 70 (GHSC70) is involved in the cellular entry of nervous necrosis virus (NNV) [PublishAheadOfPrint]

  • Nervous necrosis virus (NNV) is a devastating pathogen of cultured marine fish, and has affected more than 40 fish species. NNV belongs to the betanodavirus of Nodaviridae and is a non-enveloped icosahedral particle with 2 single-stranded positive-sense RNAs. To date, the knowledge regarding NNV entry into the host cell remains limited, and no NNV-specific receptor protein has been published. Using grouper fin cell line GF-1 and purified NNV capsid protein in a virus overlay protein binding assay (VOPBA), grouper heat-shock cognate protein 70 (GHSC70) and grouper voltage-dependent anion selective channel protein 2 (GVDAC2) were presumably to be NNV receptor protein candidates. We cloned, sequenced, and expressed the genes of GHSC70 and GVDAC2 in Escherichia coli for anti-serum preparation. The expression knockdown of GHSC70 and GVDAC2 genes with specific short interfering RNA (siRNA) significantly downregulated viral RNA expression in NNV-infected GF-1 cells. After an immuno-precipitation assay, we confirmed that GHSC70 interacted with NNV capsid protein, while VDAC2 did not. Immunofluorescence staining and flow cytometry analysis revealed the GHSC70 protein on the cell surface. After a blocking assay, we detected the NNV RNA2 level after 1 h of adsorption to GF-1 cells, which was significantly lower in the cells pretreated with the GHSC70 antiserum than in non-treated cells. Therefore, we suggest that GHSC70 participates in the NNV entry of GF-1 cells, likely functions as NNV receptor or co-receptor protein.

    IMPORTANCE Fish nodavirus has caused mass mortality of more than 40 fish species worldwide, and resulted in huge economical lost in the past 20 years. Among the four genotypes of fish nodaviruses, red-spotted grouper nervous necrosis virus (RGNNV) genotype exhibits the widest host range. In our previous study, we have developed monoclonal antibodies with high neutralizing efficiency against grouper NNV in GF-1 cells, indicating NNV-specific receptor(s) may exist on GF-1 cell membrane. However, no NNV receptor protein has been published. In this study, we found GHSC70 to be NNV receptor (or co-receptor) candidate through VOBPA, and then provided several lines of evidence to demonstrate that GHSC70 protein was enrolled in the NNV entry step of GF-1 cells. To the best of our knowledge, this is the first report identifying grouper HSC70 and its role in NNV entry into GF-1 cells.

  • Swine IFITM proteins potently inhibit influenza A virus replication [PublishAheadOfPrint]

  • Human IFITMs were identified as restriction factors of influenza A virus (IAV). Given the important role of pigs in the zoonotic cycle of IAV we cloned swine IFITMs (swIFITM) and found two IFITM1-like proteins, one homologue of IFITM2 and one of IFITM3. We show that swIFITM2 and swIFITM3 localize to endosomes and display potent antiviral activity. Knockdown of swIFITMs strongly reduced virus inhibition by interferon establishing the swIFITMs as potent restriction factors in porcine cells.

  • Phosphorylation of Herpes Simplex Virus 1 dUTPase Regulates Viral Virulence and Genome Integrity by Compensating for Low Cellular dUTPase Activity in the Central Nervous System [PublishAheadOfPrint]

  • A mutation in herpes simplex virus 1 dUTPase (vdUTPase), which precluded its phosphorylation at Ser-187, decreased viral neurovirulence and increased mutation frequency in progeny virus genomes in the brains of mice where endogenous cellular dUTPase activity was relatively low, and overexpression of cellular dUTPase restored viral neurovirulence and mutation frequency altered by the mutation. Thus, phosphorylation of vdUTPase appeared to regulate viral virulence and genome integrity by compensating for low cellular dUTPase activity in vivo.

    Importance Many DNA viruses encode a homolog of host cell dUTPases, which are known to function in accurate replication of cellular DNA genomes. The viral dUTPase activity has long been assumed to play a role in viral replication by preventing mutations in progeny virus genomes if cellular dUTPase activity was not sufficient. Here we showed that a mutation in herpes simplex virus 1 dUTPase, which precluded its phosphorylation at Ser-187 and reduced its activity, decreased viral neurovirulence and increased mutation frequency in progeny virus genomes in the brains of mice where endogenous cellular dUTPase activity was relatively low. In contrast, overexpression of cellular dUTPase restored viral neurovirulence and mutation frequency altered by the mutation in the brains of mice. This is the first report, to our knowledge, directly showing that viral dUTPase activity regulates viral genome integrity and pathogenicity by compensating for insufficient cellular dUTPase activity in vivo.

  • Profiling and characterization of potentially multidrug-resistant influenza neuraminidase 1 (N1) strains against neuraminidase inhibitors [PublishAheadOfPrint]

  • Neuraminidase inhibitors (NAIs) have been widely used to control influenza infection, but their increased use could promote global emergence of resistant variants. Although various mutations associated with NAI resistance have been identified, the amino acid substitutions that confer multidrug resistance with undiminished viral fitness remain poorly understood. We therefore screened known mutation(s) that could confer multidrug resistance to currently approved NAIs oseltamivir, zanamivir, and peramivir by assessing recombinant viruses with mutant NA genes (catalytic residues, R152K and R292K; framework residues, E119A/D/G, D198N, H274Y, and N294S) in the backbones of the 2009 pandemic H1N1 (pH1N1) and highly pathogenic avian influenza (HPAI) H5N1 viruses. Of the 14 single- and double-mutant viruses recovered in the backbone of pH1N1, 4 variants (E119D, E119A/D/G-H274Y) exhibited reduced inhibition to all the NAIs and 2 variants (E119D and E119D-H274Y) retained the overall properties of gene stability, replicative efficiency, pathogenicity, and transmissibility in vitro and in vivo. Of the 9 recombinant H5N1 viruses, 4 variants (E119D, E119A/D/G-H274Y) also showed reduced inhibition to all the NAIs, though their overall viral fitness was impaired in vitro and/or in vivo. Thus, single mutations or certain combination of the established mutations could confer potential multidrug resistance to pH1N1 or HPAI H5N1 viruses. Our findings emphasize the urgency of developing alternative drugs against influenza virus infection.

    Importance There has been a widespread emergence of influenza virus strains with reduced susceptibility to neuraminidase inhibitors (NAIs). We screened multidrug-resistant viruses by studying the viral fitness of neuraminidase mutants in vitro and in vivo. We found that recombinant E119D and E119A/D/G/-H274Y mutant viruses demonstrated reduced inhibition to all the NAIs tested in both the backbone of the 2009 H1N1 pandemic (pH1N1) and highly pathogenic avian influenza H5N1 viruses. Furthermore, E119D and E119D-H274Y mutants in the pH1N1 background maintained overall fitness properties in vitro and in vivo. Our study highlights the importance of vigilance and continued surveillance of potential NAI multidrug-resistant influenza variants as well as the development of alternative therapeutics.

  • Murine cytomegalovirus is not restricted to the house mouse Mus musculus domesticus: prevalence and genetic diversity in the European house mouse hybrid zone. [PublishAheadOfPrint]

  • The murine cytomegalovirus (MCMV) is a betaherpesvirus of the house mouse, Mus musculus domesticus (Mmd). It is a common infection in wild mice and a highly studied pathogen of the laboratory mouse. Betaherpesviruses are specific to their hosts and it is unknown if other Mus taxa carry the MCMV or if it is restricted to Mmd. We sampled mice over a 145km transect of Bavaria-Bohemia crossing an hybrid zone between Mmd and M. m. musculus (Mmm), to investigate the occurrence of MCMV in two Mus subspecies and test the limit of specificity of the virus for its host. We hypothesized that if the two subspecies carry MCMV and if the virus is highly specific to its host, divergent MCMV lineages would have co-diverged with their hosts and would have a geographical distribution constrained by the host genetic background. 520 mice were tested by ELISA and/or nested PCR targeting the M94 gene. Seropositive and PCR positive individuals were found in both Mus subspecies. Seroprevalence was high, 79.4%, but viral DNA was only detected in 41.7% of mice. Sequencing revealed 20 haplotypes clustering in 3 clades that match the host genetic structure in the hybrid zone showing one and two MCMV lineages in Mmd and Mmm, respectively. The estimated MCMV time to most recent common ancestor (1.1 Mya) matches that of their hosts. In conclusion, MCMV has co-evolved with these hosts suggesting its diversity in nature may be underappreciated as other members of the subgenus Mus likely carry different MCMVs.

    Importance The murine cytomegalovirus (MCMV) is a betaherpesvirus of the house mouse, Mus musculus domesticus, an important lab model for human cytomegalovirus (HCMV) infection. The majority of lab studies are based on only two strains of MCMVs isolated from M. m. domesticus, Smith and K181, itself derived from repeated passage of Smith in mouse submaxillary glands. The presence of MCMV in other members of the Mus subgenus had not even been investigated. By screening mouse samples collected in the European house mouse hybrid zone between M. m. domesticus and M. m. musculus, we show that MCMV is not restricted to the M. m. domesticus subspecies and that MCMV likely co-diverged with their Mus hosts. Thus the diversity of MCMV in nature may be seriously underappreciated since other members of the subgenus Mus likely carry their own MCMV lineages.

  • Slaying the Trojan horse: Natural killer cells exhibit robust anti-HIV-1 antibody-dependent activation and cytolysis against allogeneic T-cells [PublishAheadOfPrint]

  • Many attempts to design prophylactic HIV-1 vaccines have focused on the induction of neutralizing antibodies (Abs) that block infection by free virions. Despite the focus on viral particles, virus-infected cells, which can be found within mucosal secretions, are more infectious than free virus both in vitro and in vivo. Furthermore, assessment of human transmission couples suggests infected seminal lymphocytes might be responsible for a proportion of HIV-1 transmissions. Although vaccines that induce neutralizing Abs are sought, only some broadly neutralizing Abs efficiently block cell-to-cell transmission of HIV-1. As HIV-1 vaccines need to elicit immune responses capable of controlling both free and cell-associated virus, we evaluated the potential of NK cells to respond in an Ab-dependent manner to allogeneic T-cells bearing HIV-1 antigens. This manuscript presents data measuring Ab-dependent anti-HIV-1 NK cell responses to primary and transformed allogeneic T-cell targets. We found that NK cells are robustly activated in an anti-HIV-1 Ab-dependent manner against allogeneic targets, and that tested target cells are subject to Ab-dependent cytolysis. Furthermore, the educated KIR3DL1+ NK cell subset from HLA-Bw4+ individuals exhibits an activation advantage over the KIR3DL1nndash; subset that contains both NK cells educated through other receptor/ligand combinations and uneducated NK cells. These results are intriguing and important for understanding the regulation of Ab-dependent NK cell responses, and are potentially valuable for designing Ab-dependent therapies and/or vaccines.

    Importance: NK cell-mediated anti-HIV-1 antibody-dependent functions have been associated with protection from infection and disease progression, however their role in protecting from infection with allogeneic cells infected with HIV-1 is unknown. We found that HIV-1-specific ADCC antibodies bound to allogeneic cells infected with HIV-1 or coated with HIV-1 gp120 were capable of activating NK cells and/or trigging cytolysis of the allogeneic target cells. This suggests ADCC may be able to assist in preventing infection with cell-associated HIV-1. In order to fully utilize NK cell-mediated Ab-dependent effector functions, it might also be important that educated NK cells, which hold the highest activation potential, can become activated against targets bearing HIV-1 antigens and expressing the ligands for self inhibitory receptors. Here we show that with Ab-dependent stimulation NK cells expressing inhibitory receptors can mediate robust activation against targets expressing the ligands for those receptors.

  • Contact-induced mitochondria polarization supports HIV-1 virological synapse formation [PublishAheadOfPrint]

  • Rapid HIV-1 spread between CD4 T lymphocytes occurs at retrovirus-induced immune cell contacts called virological synapses (VS). VS are associated with striking T cell polarization and localized virus budding at the site of contact that facilitates cell-cell spread. In addition to this, spatial clustering of organelles including mitochondria to the contact zone has been previously shown. However, whether cell-cell contact specifically induces dynamic T cell remodeling during VS formation and what regulates this process remains unclear. Here we report that contact between an HIV-1 infected T cell and an uninfected target T cell specifically triggers polarization of mitochondria concomitant with recruitment of the major HIV-1 structural protein Gag to the site of cell-cell contact. Using fixed and live cell imaging we show that mitochondria and Gag polarization in HIV-1 infected T cells occurs within minutes of contact with target T cells, requires the formation of stable cell-cell contacts and is an active, calcium-dependent process. We also find that perturbation of mitochondria polarization impairs cell-cell spread of HIV-1 at the VS. Taken together these data suggest that HIV-1 infected T cells are able to sense and respond to contact with susceptible target cells and undergo dynamic cytoplasmic remodeling to create a synaptic environment that supports efficient HIV-1 VS formation between CD4 T lymphocytes.

    IMPORTANCE HIV-1 remains one of the major global health challenges of modern times. The capacity of HIV-1 to cause disease depends on the virus's ability to spread between immune cells, most notably CD4 T lymphocytes. Cell-cell transmission is the most efficient way of HIV-1 spread and occurs at the Virological Synapse (VS). The VS forms at the site of contact between an infected cell and an uninfected cell and is characterized by polarized assembly and budding of virions and clustering of cellular organelles including mitochondria. Here we show that cell-cell contact induces rapid recruitment of mitochondria to the contact site and that this supports efficient VS formation and consequently cell-cell spread. Additionally, we observed that cell-cell contact induces a mitochondria-dependent increase in intracellular calcium, indicative of cellular signalling. Taken together, our data suggest that VS formation is a regulated process and thus a potential target to block HIV-1 cell-cell spread.

  • Differentiation of Neurons Restricts Arbovirus Replication and Increases Expression of the Alpha Isoform of IRF-7 [PublishAheadOfPrint]

  • Susceptibility to alphavirus infection is age-dependent and host maturation is associated with decreased virus replication and less severe encephalitis. To identify factors associated with maturation-dependent restriction of virus replication, we studied AP-7 rat olfactory bulb neuronal cells that can be differentiated in vitro. Differentiation was associated with a 150- to 1000-fold decrease in replication of Sindbis and Venezuelan equine encephalitis alphaviruses, as well as La Crosse bunyavirus. Differentiation delayed synthesis of SINV RNA and protein, but did not alter the susceptibility of neurons to infection or virion maturation. Additionally, differentiation slowed virus-induced translation arrest and death of infected cells. Differentiation of uninfected AP-7 neurons was associated with changes in expression of antiviral genes. Expression of key transcription factors was increased, including interferon regulatory factor-3 and -7 (IRF-3 and IRF-7) and STAT-1, suggesting that neuronal maturation may enhance the capacity for antiviral signaling upon infection. IRF-7 produced by undifferentiated AP-7 neurons was exclusively the short dominant-negative isoform while that produced by differentiated neurons was the full-length aalpha; isoform. A similar switch in IRF-7 isoforms also occurred in the brains of maturing C57BL/6J mice. Silencing of IRF expression did not improve virus multiplication in differentiated neurons. Therefore, neuronal differentiation is associated with up regulation of transcription factors that activate antiviral signaling, but this alone does not account for maturation-dependent restriction of virus replication.

    IMPORTANCE Viral encephalomyelitis is an important cause of age-dependent morbidity and mortality. Because mature neurons are not readily regenerated, recovery from encephalitis suggests that mature neurons utilize unique antiviral mechanisms to block infection and/or clear virus. To identify maturational changes in neurons that may improve outcome, we compared immature and mature cultured neurons for susceptibility to three encephalitic arboviruses and found that replication of Old and New World alphaviruses and a bunyavirus were reduced in mature compared to immature neurons. Neuronal maturation was associated with increased baseline expression of interferon regulatory factors -3 and -7 mRNAs, and production of distinct isoforms of interferon regulatory factor-7 protein. Overall, our studies identify maturational changes in neurons that likely contribute to assembly of immunoregulatory factors prior to infection, a more rapid antiviral response, increased resistance to virus infection, and improved survival.

  • Identification of a neutralizing epitope within antigenic domain 5 of glycoprotein B of human cytomegalovirus [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) is an important, ubiquitous pathogen that causes severe clinical disease in immunocompromised individuals such as organ transplant recipients and infants infected in-utero. The envelope glycoprotein B (gB) of HCMV is a major antigen for the induction of virus neutralizing antibodies. We have begun to define target structures within gB that are recognized by virus neutralizing antibodies. Antigenic domain 5 (AD-5) of gB has been identified as an important target for neutralizing antibodies in studies using human monoclonal antibodies (MAbs). Anti-AD-5 MAbs share a common target site on gB despite originating from different, healthy HCMV-infected donors. Mutational analysis of AD-5 identified tyrosine 280 in combination with other surface-exposed residues (the YNND epitope) as critical for antibody binding. The YNND epitope is strictly conserved among different HCMV strains. Recombinant viruses carrying YNND-mutations in AD-5 were resistant to virus neutralizing MAbs. Competition ELISAs with human HCMV-convalescent sera from unselected donors confirmed the conserved antibody responses for the YNND epitope in HCMV infected individuals and, because a significant fraction of the gB-AD-5 response was directed against the YNND epitope, further argued that this epitope was a major target of anti-AD-5 antibody responses. In addition, affinity-purified polyclonal anti-AD-5 antibodies prepared from individual sera showed similar reactivity to AD-5 and comparable neutralization activity towards gB-mutant viruses as AD-5 specific MAbs. Taken together, our data indicated that the YNND epitope represents an important target for anti-gB antibody responses as well for anti-AD-5 virus neutralizing antibodies.

    Importance HCMV is a major global health concern and a vaccine to prevent HCMV disease is a widely recognized medical need. Glycoprotein B of HCMV is an important target for neutralizing antibodies and hence an interesting molecule for intervention strategies, e.g. vaccination. Mapping the target structures of neutralizing antibodies induced by naturally-occurring HCMV infection can aid in defining the properties required for a protective capacity of vaccine antigens. The data presented here extend our knowledge of neutralizing epitopes within gB to include AD-5. Collectively, our data will contribute to optimal vaccine design and development of antibody-based therapies.

  • Assessment of the internal genes of influenza A (H7N9) virus contributing to the high pathogenicity in mice [PublishAheadOfPrint]

  • The recently identified H7N9 influenza A virus has caused severe economic losses and worldwide public concern. Genetic analysis indicates that its six internal genes all originated from H9N2 viruses. However, the H7N9 virus is more highly pathogenic in humans than H9N2, which suggests that the internal genes of H7N9 have mutated. To analyze which H7N9 virus internal genes contribute to its high pathogenicity, a series of reassortants were generated by reverse genetics, each containing a single internal gene of the typical A/Anhui/1/2013(H7N9) virus in the genetic background of the A/chicken/Shandong/lx1023/2007(H9N2) virus. Their replication ability, polymerase activity, and pathogenicity were then evaluated in vitro and in vivo. These recombinants displayed high genetic compatibility, and the H7N9-derived PB2, M, and NP genes were identified as the virulence genes for the reassortants in mice. Further investigation confirmed PB2-K627 is critical for the high pathogenicity of the H7N9 virus and the reassortant containing the H7N9-derived PB2 segment (H9N2-AH/PB2). Notably, the H7N9-derived PB2 gene displayed a greater compatibility with the H9N2 genome than that of H7N9, endowing the H9N2-AH/PB2 reassortant with greater viability and virulence than the parental H7N9 virus. In addition, the H7N9 virus, with the exception of the H9N2 reassortants, could effectively replicate in human A549 cells. Our results indicate that PB2, M, and NP are the key virulence genes, together with the surface HA and NA proteins, contributing to the high infectivity of the H7N9 virus in humans.

    Importance To date, the novel H7N9 influenza A virus has caused 437 human infections, with approximately 30% mortality. Previous work has primarily focused on the two viral surface proteins, HA and NA, but the contribution of the six internal genes to the high pathogenicity of H7N9 has not been systematically studied. Here, the H9N2 virus was used as a genetic backbone to evaluate the virulence genes of H7N9 virus in vitro and in vivo. Our data indicate the PB2, M, and NP genes play important roles in viral infection in mice and, together with HA and NA, contribute to the high infectivity of the H7N9 virus in humans.

  • Topical HSV-2 Vaccination with HPV Vectors Expressing gB/gD Ectodomains Induces Genital Tissue Resident Memory CD8+ T cells and Reduces Genital Disease and Viral Shedding After HSV-2 Challenge [PublishAheadOfPrint]

  • No Herpes simplex virus type-2 (HSV-2) vaccine has been licensed for use in humans. HSV-2 glycoproteins B (gB) and D (gD) are targets of neutralizing antibodies and T cells, but clinical trials involving intramuscular injection of HSV-2 gB and gD proteins in adjuvants have not been effective. Here we evaluated intravaginal (ivag) genetic immunization of C57BL/6 mice with a replication-defective human papillomavirus pseudovirus (HPV PsV) expressing HSV-2 gB (HPV-gB) or gD (HPV-gD) constructs to target different subcellular compartments. HPV PsV expressing a secreted ectodomain of gB (gBsec) and gD (gDsec), but not PsV expressing cytoplasmic or membrane-bound forms, induced circulating and intravaginal tissue resident memory CD8+ T cells able to secrete IFN- and TNF-aalpha; and moderate levels of serum HSV neutralizing antibodies. Combined immunization with HPV-gBsec/gDsec vaccines conferred improved survival after HSV-2 vaginal challenge compared to HPV-gBsec or HPV-gDsec alone. HPV-gBsec/gDsec ivag vaccination was associated with reduced severity of genital lesions and lower viral shedding in the genital tract after HSV-2 challenge. In contrast, intramuscular vaccination with soluble HSV-2 gD in alum and monophosphoryl lipid A (gD2t/Alum/MPL) elicited high neutralizing antibody titers and improved survival, but did not reduce genital lesions and viral shedding. Vaccination combining HPV-gBsec/gDsec ivag and gD2t/Alum/MPL i.m. improved survival and reduced genital lesions and viral shedding. Finally, circulating HSV-2-specific CD8+ T cells, not serum antibodies, correlated with reduced viral shedding. Together our data underscore the potential of HPV PsV as a platform for a topical mucosal vaccine to control local manifestations of primary HSV-2 infection.

    Importance: Genital herpes is a highly prevalent chronic disease caused by HSV infection. To date, there is no licensed vaccine against HSV infection. This study describes intravaginal vaccination with a non-replicating HPV-based vector expressing HSV glycoprotein antigens. The data presented in this study underscore the potential of HPV based vectors as a platform to induce genital tissue resident memory T cell responses and to control local manifestations of primary HSV infection.

  • Super-infection with woodchuck hepatitis virus (WHV) strain WHVNY of the livers chronically infected with the strain WHV7. [PublishAheadOfPrint]

  • The determinants of the maintenance of chronic hepadnaviral infection are yet to be fully understood. A long-standing unresolved argument in hepatitis B virus (HBV) research field suggests that during chronic hepadnaviral infection, cell-to-cell spread of hepadnavirus is at least very inefficient (if it occurs at all); virus super-infection is an unlikely event; and chronic hepadnavirus infection can be maintained exclusively via division of infected hepatocytes in the absence of virus spread. Super-infection exclusion was previously shown for duck HBV, but it was not demonstrated for HBV or HBV-related woodchuck hepatitis virus (WHV). Three woodchucks, which were chronically infected with the strain WHV7 and already developed WHV-induced HCCs, were super-infected with another WHV strain, WHVNY. Six weeks after the super-infection, the woodchucks were sacrificed and tissues of the livers and HCCs were examined. The WHVNY super-infection was demonstrated by using WHV strain-specific PCR assays, and (i) finding WHVNY relaxed circular DNA in the serum samples collected from all super-infected animals during weeks one through six after the super-infection; (ii) detecting replication-derived WHVNY RNA in the tissue samples of the livers and HCCs collected from three super-infected woodchucks; and (iii) finding WHVNY DNA replication intermediates in tissues harvested after the super-infection. The results are consistent with the occurrence of continuous, but inefficient hepadnavirus cell-to-cell spread and super-infection during chronic infection, and suggest that replication space occupied by the super-infecting hepadnavirus in chronically infected livers is limited. The findings are discussed in the context of the mechanism of chronic hepadnavirus infection.

    IMPORTANCE. The study aimed to better understand the determinants of the maintenance of chronic hepadnavirus infection. The generated data suggest that in the livers chronically infected with woodchuck hepatitis virus, (i) hepadnavirus super-infection and cell-to-cell spread likely continue to occur; and (ii) the virus spread is apparently inefficient, which is consistent with the interpretation that a limited number of cells in the livers facilitates the spread of hepadnavirus. The limitations of the cell-to-cell virus spread are most likely mediated at the level of the cells, and do not reflect the properties of the virus. Our results further advance the understanding of the mechanism of chronic hepadnavirus infection. The significance of the continuous, but limited hepadnavirus spread and super-infection for the maintenance of the chronic state of infection should be further evaluated in follow-up studies in order to determine whether blocking the virus spread would facilitate the suppression of chronic hepadnavirus infection.

  • Malawi polyomavirus is a prevalent human virus that interacts with known tumor suppressors [PublishAheadOfPrint]

  • Malawi polyomavirus (MWPyV) is a recently identified human polyomavirus. Serology for MWPyV VP1 indicates that infection frequently occurs in childhood and reaches a prevalence of 75% in adults. MWPyV ST binds PP2A and LT binds pRb, p107, p130 and p53. However, MWPyV LT was less stable than SV40 LT and unable to promote the growth of normal cells. This study confirms that MWPyV is a widespread human virus expressing T antigens with low transforming potential.

  • Merkel cell polyomavirus small T antigen mediates microtubule destabilisation to promote cell motility and migration [PublishAheadOfPrint]

  • Merkel cell carcinoma (MCC) is an aggressive skin cancer of neuroendocrine origin with a high propensity of recurrence and metastasis. Merkel cell polyomavirus (MCPyV) causes the majority of MCC cases due to the expression of the MCPyV Small and Large Tumour (ST and LT) antigens. Although a number of molecular mechanisms have been attributed to MCPyV tumour antigen-mediated cellular transformation or replication, to date, no studies have investigated any potential link between MCPyV T antigen expression and the highly metastatic nature of MCC. Here we use a quantitative proteomic approach to show that MCPyV ST promotes differential expression of cellular proteins implicated in microtubule-associated cytoskeletal organisation and dynamics. Intriguingly, we demonstrate that MCPyV ST expression promotes microtubule destabilisation leading to a motile and migratory phenotype. We further highlight the essential role of the microtubule-associated protein stathmin in MCPyV ST-mediated microtubule destabilisation and cell motility and implicate the cellular phosphatase catalytic subunit PP4C in the regulation of this process. These findings suggest a possible molecular mechanism for the highly metastatic phenotype associated with MCC.

    IMPORTANCE Merkel Cell Polyomavirus (MCPyV) causes the majority of cases of Merkel cell carcinoma (MCC), an aggressive skin cancer with a high metastatic potential. However, the molecular mechanisms leading to virally-induced cancer development are yet to be fully elucidated. In particular, no studies have investigated any potential link between the virus and the highly metastatic nature of MCC. We demonstrate that the MCPyV Small Tumour (ST) antigen promotes the destabilisation of the host cell microtubule network which leads to a more motile and migratory cell phenotype. We further show that MCPyV ST induces this process by regulating the phosphorylation status of the cellular microtubule-associated protein stathmin, by its known association with the cellular phosphatase catalytic subunit PP4C. These findings highlight stathmin as a possible biomarker of MCC and as a target for novel anti-tumoral therapies.

  • An interleukin-1 beta encoding retrovirus exhibits enhanced replication in vivo. [PublishAheadOfPrint]

  • Interleukin-1 beta (IL-1bbeta;) is an inflammatory cytokine that is secreted in response to inflammasome activation by innate microbe-sensing pathways. Although some retroviruses can trigger IL-1bbeta; secretion through the DNA-sensing molecule IFI16, the effect of IL-1bbeta; on the course of infection is unknown. To test whether IL-1bbeta; secretion affects retroviral replication in vivo, I constructed a novel murine leukemia virus strain (FMLV-IL1bbeta;) that encodes the mature form of IL-1bbeta;. This virus replicated with kinetics similar to wild-type virus in tissue culture, but caused a dramatically more aggressive infection of both C57BL/6 and BALB/c mice. By 7dpi, mice infected with FMLV-IL1bbeta; exhibited splenomegaly and viral loads three hundred-fold higher than mice infected with wild-type FMLV. Furthermore, the enlarged spleens of FMLV-IL1bbeta;-infected mice correlated with a large expansion of Gr-1+CD11b+ myeloid-derived suppressor cells, as well as elevated levels of immune activation. Although FMLV-IL1bbeta; infection was controlled by C57BL/6 mice by 14dpi, FMLV-IL1bbeta; was able to establish a significant persistent infection and immune activation in BALB/c mice. These results demonstrate that IL-1bbeta; secretion is a powerful positive regulator of retroviral infection, and that FMLV-IL1bbeta; represents a new model of pro-inflammatory retroviral infection.

    Importance Interleukin-1 beta (IL-1bbeta;) is an inflammatory cytokine released in response to activation of innate pathogen-sensing pathways during microbial infection. To examine the potential impact of IL-1bbeta; on retroviral replication in vivo, I constructed a novel mouse retrovirus strain (FMLV-IL1bbeta;) that encodes IL-1bbeta; and promotes abundant IL-1bbeta; secretion from infected cells. This virus replicates with normal kinetics in cultured cells, but displays a dramatically enhanced ability to replicate in mice, and caused persistent infection and immune activation in the BALB/c strain of mice. These results establish IL-1bbeta; as a positive regulator of retroviral replication and pathogenesis, and suggest that targeting this pathway may have therapeutic benefits for pro-inflammatory retroviruses. This virus can also be used to further study the impact of pro-inflammatory pathways on retroviral infection.


  • The emerging zoonotic pathogens Hendra virus (HeV) and Nipah virus (NiV) are in the genus Henipaviridae family Paramyxoviridae. HeV and NiV infections can be highly fatal to humans and livestock. The goal of this study was to develop candidate vaccines against henipaviruses utilizing two well-established rhabdoviral vaccine vector platforms: recombinant rabies virus (RABV) and recombinant vesicular stomatitis virus (VSV), expressing either the codon-optimized or the wild-type HeV glycoprotein (G). The RABV vector expressing the codon-optimized HeV G showed a 2 to 3-fold increase in incorporation compared to the RABV vector expressing wild-type (wt) HeV G. There was no significant difference in HeV G incorporation in the VSV vectors expressing either wt or codon-optimized HeV G. Mice inoculated intranasally with any of these live recombinant viruses showed no signs of disease, including weight loss, indicating that HeV G expression and incorporation did not increase the neurotropism of the vaccine vector. To test immunogenicity of the vaccine candidates, we immunized mice intramuscularly with either one dose of the live vaccines or 3 doses of 10mmu;g chemically inactivated viral particles. Increased codon-optimized HeV G incorporation into RABV virions resulted in higher antibody titers against HeV G compared to inactivated RABV virions expressing wt HeV G. The live VSV vectors induced more HeV G-specific antibodies as well as higher levels of HeV neutralizing antibodies than the RABV vectors. In the case of killed particles, HeV neutralizing serum titers were very similar between the two platforms. These results indicated that killed RABV with codon-optimized HeV G should be the vector of choice as a dual vaccine in areas where rabies is endemic.

    Importance Scientists have been tracking two new viruses carried by the Pteropid fruit bats: Hendra virus (HeV) and Nipah virus (NiV). Both viruses can be fatal to humans and also pose a serious risk to domestic animals. A recent escalation in the frequency of outbreaks has increased the need for a vaccine that prevents HeV and NiV infections. In this study we performed an extensive comparison of live and killed particles of two recombinant rhabdoviral vectors, rabies virus and vesicular stomatitis virus (VSV) expressing wild-type or codon-optimized HeV glycoprotein, with the goal to develop a candidate vaccine against HeV. Based on our data from the presented mouse immunogenicity studies, we conclude that a killed RABV vaccine would be highly effective against HeV infections and would make an excellent vaccine candidate in areas where both RABV and henipaviruses pose a threat to human health.

  • G2/M cell cycle arrest correlates with primate lentiviral Vpr interaction with the SLX4 complex [PublishAheadOfPrint]

  • The accessory gene vpr, common to all primate lentiviruses, induces a potent G2/M arrest in cycling cells. A recent study showed that HIV-1 Vpr mediates this through activation of the SLX4/MUS81/EME1 exonuclease complex that forms part of the Fanconi Anemia DNA repair pathway. To confirm these observations, we have examined the G2/M arrest phenotypes of a panel of SIV Vpr proteins. We show that SIV Vpr proteins vary in their ability to promote cell-cycle arrest in human cells. Whilst this is dependent on the DCAF1/DDB1/CUL4 Ubiquitin ligase complex, interaction with human DCAF1 does not predict G2/M arrest activity of SIV Vpr in human cells. In all cases, SIV Vpr-mediated cell cycle arrest in human cells correlated with interaction with huSLX4 and could be abolished by siRNA depletion of any member of the SLX4 complex. By contrast, all but one of the HIV/SIV Vpr proteins tested, including those that lacked activity in human cells, were competent for G2/M arrest in Grivet cells. Correspondingly, here cell-cycle arrest correlated with interaction with the Grivet orthologues of the SLX4 complex, suggesting a level of host adaptation in these interactions. Phylogenetic analyses strongly suggest that G2/M arrest/SLX4 interactions are ancestral activities of primate lentiviral Vpr proteins, and that the ability to dysregulate the Fanconi Anemia DNA repair pathway is an essential function of Vpr in vivo.

    Importance The Vpr protein of HIV-1 and its related viruses is essential for the virus in vivo. The ability of Vpr to block the cell cycle at mitotic entry is well known, but the importance of this function for viral replication is unclear. Recent data has shown that HIV-1 Vpr targets the Fanconi Anemia DNA repair pathway by interacting with and activating an endonuclease complex, SLX4/MUS81/EME1, that processes interstrand DNA crosslinks. Here we show that the ability of a panel of SIV Vpr proteins to mediate cell-cycle arrest correlates with species-specific interactions with the SLX4 complex in human and primate cells. These studies suggest that the SLX4 complex is a conserved target of primate lentiviral Vpr proteins, and that the ability to dysregulate members of the Fanconi Anemia DNA repair pathway is essential for HIV/SIV replication in vivo.

  • Venezuelan equine encephalitis virus variants lacking transcription inhibitory functions demonstrate highly attenuated phenotype [PublishAheadOfPrint]

  • Alphaviruses represent a significant public health threat worldwide. They are transmitted by mosquitoes and cause a variety of human diseases ranging from severe meningoencephalitis to polyarthritis. To date, no efficient and safe vaccines have been developed against any alphavirus infection. However, in recent years, significant progress has been made in understanding the mechanism of alphavirus replication and virus-host interactions. These data have provided the possibility for development of new rationally designed alphavirus vaccine candidates, which combine efficient immunogenicity, high safety and inability to revert to pathogenic phenotype. New attenuated variants of Venezuelan equine encephalitis virus (VEEV) designed in this study, combine a variety of characteristics which independently contribute to a reduction of virulence. These constructs encode a noncytopathic VEEV capsid protein, which is incapable of interfering with the innate immune response. The capsid-specific mutations strongly affect neurovirulence of the virus. In other constructs, they were combined with changes in control of capsid translation and an extensively mutated packaging signal. These modifications additionally affected the residual neurovirulence of the virus, but it remained immunogenic, and a single immunization protected mice against subsequent infection with epizootic VEEV. Similar approaches of attenuation can be applied to other encephalitogenic New World alphaviruses.

    SIGNIFICANCE Venezuelan equine encephalitis virus (VEEV) is an important human and animal pathogen, which causes periodic outbreaks of highly debilitating disease. Despite a continuous public health threat, no safe and efficient vaccine candidates have been developed to date. In this study, we applied accumulated knowledge about the mechanism of VEEV replication, RNA packaging and interaction with the host to design new VEEV vaccine candidates, which demonstrate exceptionally high levels of safety due to a combination of extensive modifications in the viral genome. The introduced mutations did not affect RNA replication or structural protein synthesis, but had deleterious effects on VEEV neuroinvasion and virulence. In spite of dramatically reduced virulence, the designed mutants remained highly immunogenic and protected mice against subsequent infection with epizootic VEEV. Similar methodologies can be applied for attenuation of other encephalitogenic New World alphaviruses.

  • Tetramer-enrichment reveals presence of phenotypically diverse hepatitis C virus specific CD8+ T cells in chronic infection [PublishAheadOfPrint]

  • Virus-specific CD8+ T cells are rarely detectable ex vivo by conventional methods during chronic hepatitis C virus (HCV) infection. In this study, however, we were able to detect and characterize HCV-specific CD8+ T cells in all chronically genotype 1a infected, HLA-A*02:01+ patients analyzed by performing major histocompatibility complex (MHC) class I tetramer enrichment. Two thirds of these enriched HCV-specific CD8+ T-cell populations displayed an effector-memory phenotype whereas, surprisingly, one third displayed a naiiuml;ve-like phenotype despite ongoing viral replication. CD8+ T cells with an effector-memory phenotype could not expand in vitro, suggesting exhaustion of these cells. Interestingly, some of the naiiuml;ve-like CD8+ T cells proliferated vigorously upon in vitro priming, whereas others did not. These differences were linked to the corresponding viral sequences in the respective patients. Indeed, naiiuml;ve-like CD8+ T cells from patients with consensus sequence in the corresponding T-cell epitope did not expand in vitro. In contrast, in patients displaying sequence variations, we were able to induce HCV-specific CD8+ T-cell proliferation, which may indicate infection with a variant virus. Collectively, these data reveal the presence of phenotypically and functionally diverse HCV-specific CD8+ T cells at very low frequencies that are detectable in all chronically infected patients despite viral persistence.

    Importance In this study, we analyzed CD8+ T-cell responses specific for HLA-A*02:01 restricted epitopes in chronically HCV infected patients, using MHC class I tetramer enrichment. Importantly, we could detect HCV-specific CD8+ T-cell populations in all patients. To further characterize these HCV-specific CD8+ T-cell populations that are not detectable using conventional techniques, we performed phenotypic, functional and viral sequence analyses. These data revealed different mechanisms for CD8+ T-cell failure in HCV infection, including T-cell exhaustion, viral escape and functional impairment of naiiuml;ve-like HCV-specific CD8+ T cells.

  • Quantitative proteomics identifies host factors modulated during acute hepatitis E infection in swine model [PublishAheadOfPrint]

  • Hepatitis E virus (HEV) causes acute enterically-transmitted hepatitis. In industrialized countries, it is a zoonotic disease, swine being the major reservoir of human HEV contaminations. The occurrence and severity of the disease are variable, ranging from asymptomatic to self-limiting acute hepatitis, chronic infection or fulminant hepatitis. In the absence of a robust cell culture system or small animal models, the HEV life cycle and pathological process remain unclear. To characterize HEV pathogenesis and virulence mechanisms, a quantitative proteomic analysis was carried out to identify cellular factors and pathways modulated during acute infection of swine. Three groups of pigs were inoculated with 3 different strains of swine HEV to evaluate the possible role of viral determinants in pathogenesis. Liver samples were analyzed by a differential proteomic approach, 2D-DIGE and 61 modulated proteins were identified by mass spectroscopy. The results obtained show that the 3 HEV strains replicate similarly in swine and that they modulate several cellular pathways suggesting that HEV impairs several cellular processes, which can account for various disease expressions. Several proteins such as Heterogeneous nuclear ribonucleoprotein K, Apolipoprotein E and Prohibitin, known to be involved in other viral life cycles, were up-regulated in HEV-infected livers. Some differences were observed between the 3 strains, suggesting that HEV's genetic variability may induce variation in pathogenesis. This comparative analysis of liver proteome modulated during HEV infection with 3 different strains of genotype 3 provides an important basis for further investigations on factors involved in HEV replication and the mechanism of HEV pathogenesis.

    Importance Hepatitis E virus (HEV) is responsible for acute hepatitismmdash;ranging from asymptomatic to self-limiting acute hepatitismmdash;chronic infection, or fulminant hepatitis. In industrialized countries, HEV is considered an emerging zoonotic disease, swine being the principal reservoir for human contaminations. The viral and cellular factors involved in the replication and/or pathogenesis of HEV are still not fully known. Here we report that several cellular pathways involved in cholesterol, lipid metabolism or cell survival were modulated during HEV infection in swine model. Moreover, we observed a difference between the different swine strains, suggesting that HEV's genetic variability could play a role in pathogenesis. We also identified some proteins known to be involved in other viral cycles. Our study provides insight into the mechanisms modulated during HEV infection and constitutes a useful reference for future work on HEV pathogenesis and virulence.

  • A 2.5 kb deletion contaning a cluster of nine microRNAs in the LAT locus of the pseudorabies virus affects the host response of porcine trigeminal ganglia during established latency. [PublishAheadOfPrint]

  • The alphaherpesvirus Pseudorabies virus (PrV) establishes latency primarily in neurons of trigeminal ganglia when only transcription of the latency-associated transcript (LAT) locus is detected. Eleven microRNAs (miRNAs) cluster within LAT, suggesting a role in establishment and/or maintenance of latency.

    We generated a mutant PrV (M) deleted of nine miRNA genes which displayed almost identical properties with the parental PrV (WT) during propagation in vitro. Fifteen pigs were experimentally infected with either WT, M or mock infected.

    Similar levels of virus excretion and host antibody response were observed in all infected animals. At 62 days post infection trigeminal ganglia were excised and profiled by deep sequencing and RT-qPCR.

    Latency was established in all infected animals without evidence of viral reactivation demonstrating that miRNAs are not mandatory for this process. Lower levels of Large Latency Transcript (LLT) were found in ganglia infected by M compared to WT PrV. All PrV miRNAs were expressed, with highest expression found for prv-miR-LLT1, prv-miR-LLT2 (in WT-ganglia) and prv-miR-LLT10 (in both WT and M-ganglia). No evidence of differentially expressed porcine miRNAs was found. Fifty-four porcine genes were differentially expressed between WT, M and control ganglia. Both viruses triggered a strong host immune response, but in M- ganglia gene upregulation was prevalent. Pathway analyses indicated that several biofunctions, including those related to cell-mediated immune response and migration of dendritic cells, were impaired in M- ganglia. These findings are consistent with a function of the LAT locus in the modulation of host response for maintaining a latent state.

    Importance This study provides a thorough reference on the establishment of latency by PrV in its natural host, the pig. Our results corroborate the evidence obtained from the study of several LAT mutants of other alphaherpesviruses encoding miRNAs from their LAT regions. Neither PrV miRNA expression nor high LLT expression levels are essential to achieve latency in trigeminal ganglia. Once latency is established by PrV the only remarkable differences are found in the pattern of host response. This indicates that, LAT functions as an immune evasion locus.

  • Basic motifs target PSGL-1, CD43, and CD44 to plasma membrane sites where HIV-1 assembles [PublishAheadOfPrint]

  • HIV-1 incorporates various host membrane proteins during particle assembly at the plasma membrane; however the mechanisms mediating this incorporation process remain poorly understood. We previously showed that the HIV-1 structural protein Gag localizes to the uropod, a rear-end structure of polarized T cells, and that assembling Gag copatches with a subset, but not all, of uropod-directed proteins, i.e., PSGL-1, CD43, and CD44, in non-polarized T cells. The latter observation suggests the presence of a mechanism promoting virion incorporation of these cellular proteins. To address this possibility and identify molecular determinants, in the present study we examined coclustering between Gag and the transmembrane proteins in T and HeLa cells using quantitative two-color super-resolution localization microscopy. Consistent with the T-cell copatching study, we found that basic residues within the matrix domain of Gag are required for Gag-PSGL-1 coclustering. Notably, the presence of a polybasic sequence in the PSGL-1 cytoplasmic domain significantly enhanced this coclustering. We also found that polybasic motifs present in the cytoplasmic tails of CD43 and CD44 also promote their coclustering with Gag. ICAM-1 and ICAM-3, uropod-directed proteins that do not copatch with Gag in T cells, and CD46, a non-uropod-directed protein, showed no or little coclustering with Gag. However, replacing their cytoplasmic tails with that of PSGL-1 significantly enhanced their coclustering with Gag. Altogether, these results identify a novel mechanism for host membrane protein association with assembling HIV-1 Gag in which polybasic sequences present in the cytoplasmic tails of the membrane proteins and in Gag are the major determinants.

    Importance Nascent HIV-1 particles incorporate many host plasma membrane proteins during assembly. However, it is largely unknown what mechanisms promote association of these proteins with virus assembly sites within the plasma membrane. Notably, our previous study showed that HIV-1 structural protein Gag colocalizes with a group of uropod-directed transmembrane proteins, PSGL-1, CD43, and CD44, at the plasma membrane of T cells. The results obtained in the current study using super-resolution localization microscopy suggest the presence of a novel molecular mechanism promoting association of PSGL-1, CD43, and CD44 with assembling HIV-1, which relies on polybasic sequences in HIV-1 Gag and in cytoplasmic domains of the transmembrane proteins. This information advances our understanding of virion incorporation of host plasma membrane proteins, some of which modulate virus spread positively or negatively, and suggests a possible new strategy to enrich HIV-1-based lentiviral vectors with a desired transmembrane protein.

  • Interaction of NS2 with AIMP2 Facilitates the Switch from Ubiquitination to SUMOylation of M1 in Influenza A Virus-Infected Cells [PublishAheadOfPrint]

  • Influenza A viruses (IAVs) rely on host factors to support their life cycle as viral proteins could "hijack" or interact with cellular proteins to execute their functions. Identification and understanding of these factors would increase the knowledge of molecular mechanisms manipulated by the viruses. In this study, we searched for novel binding partners of influenza NS2 protein, the nuclear export protein responsible for overcoming host-range restriction, by a yeast two-hybrid screening, GST pull-down and co-immunoprecipitation assays, and identified AIMP2, a potent tumor suppressor that usually functions to regulate protein stability, as one of the major NS2-binding candidates. We found that the presence of NS2 protected AIMP2 from ubiquitin-mediated degradation in NS2-transfected cells and AIMP2 functioned as a positive regulator for IAV replication. Interestingly, AIMP2 had no significant effect on NS2 but enhanced stability of the matrix protein M1. We further provided evidence that AIMP2 recruitment switched the modification of M1 from ubiquitination to SUMOylation occurring on the same attachment site K242 on M1, and thereby promoted M1-mediated vRNPs nuclear export to increase viral replication. Collectively, our results reveal a new mechanism of AIMP2 in mediating influenza virus replication.

    IMPORTANCE Although ubiquitination of M1 during IAV infection has been observed, the precise modification site and the molecular consequences of this modification remain obscure. Here, we demonstrate for the first time that ubiquitin and SUMO compete for the same lysine K242 on M1 and interaction of NS2 with AIMP2 facilitates the switch of M1 modification from ubiquitination to SUMOylation thus increasing viral replication.

  • The human CD8+ T cell responses induced by a live attenuated tetravalent dengue vaccine are directed against highly conserved epitopes [PublishAheadOfPrint]

  • The incidence of infection with any of the four dengue virus serotypes (DENV 1-4) has increased dramatically in the last few decades, and the lack of a treatment or vaccine has contributed to significant morbidity and mortality worldwide. A recent comprehensive analysis of the human T cell response against wild-type DENV suggested an HLA-linked protective role for CD8+ T cells. We have collected one-unit blood donations from study participants receiving the monovalent or tetravalent live attenuated DENV vaccine (DLAV), developed by the U.S. National Institutes of Health. PBMCs from these donors were screened in IFN ELISPOT assays with pools of predicted, HLA matched, class I binding peptides covering the entire DENV proteome. Here, we characterize for the first time CD8+ T cell responses after live attenuated dengue vaccination and show that CD8+ T cell responses in vaccinees were readily detectable and comparable to natural dengue infection. Interestingly, while broad responses to structural and non-structural (NS) proteins were observed after monovalent vaccination, T cell responses following tetravalent vaccination were, dramatically, focused towards the highly conserved NS proteins. Epitopes were highly conserved in a vast variety of field isolates and able to elicit multifunctional T cell responses. Detailed knowledge of the T cell response will contribute to the identification of robust correlates of protection in natural immunity and following vaccination against DENV.

    Importance The development of effective vaccination strategies against DENV infection and clinically significant disease is a task of high global public health value and significance, while also being a challenge of significant complexity. A recent efficacy trial of the most advanced dengue vaccine candidate, demonstrated only partial protection against all four DENV serotypes, despite three subsequent immunizations and detection of measurable neutralizing antibodies to each serotype in most subjects. These results challenge the hypothesis that sero-conversion is the only reliable correlate of protection. Here, we show that CD8+ T cell responses in vaccinees were readily detectable and comparable to natural dengue infection. Detailed knowledge of the T cell response may further contribute to the identification of robust correlates of protection in natural immunity and vaccination against DENV.

  • A novel mode of ISG15 mediated protection against influenza A virus and Sendai virus in mice [PublishAheadOfPrint]

  • ISG15 is a diubiquitin-like modifier and one of the most rapidly induced genes upon type I interferon stimulation. Hundreds of host proteins and a number of viral proteins have been shown to be ISGylated, and understanding how these modifications affect the interferon response and virus replication has been of considerable interest. ISG15nndash;/nndash; mice exhibit increased susceptibility to viral infection, and in the case of influenza B virus and vaccinia virus, ISG15 conjugation has been shown to restrict virus replication in vivo. A number of studies have also found that ISG15 is capable of antagonizing replication of some viruses in tissue culture. However, recent findings have demonstrated that ISG15 can protect mice from Chikungunya virus infection without affecting virus burden. In order to better understand the function of ISG15 in vivo, we characterized the pathogenesis of influenza A virus and Sendai virus in ISG15nndash;/nndash; mice. We found that ISG15 protects mice from virus induced lethality by a conjugation dependent mechanism in both of these models. However, surprisingly, we found that ISG15 had minimal effect on virus replication, and did not have an obvious role in the modulation of the acute immune response to infection. Instead, we observed an increase in the number of diseased small airways in mice lacking ISG15. This ability of ISG15 to protect mice in a conjugation-dependent, but non-antiviral, manner from respiratory virus infection represents a previously undescribed role for ISG15 and demonstrates the importance of further characterization of ISG15 in vivo.

    Importance It has previously been demonstrated that ISG15nndash;/nndash; mice are more susceptible to a number of viral infections. As one of the most strongly induced genes after type I interferon stimulation, analysis of ISG15 function has largely focused on its role as an antiviral molecule during acute infection. While a number of studies have shown that ISG15 does have a small effect on virus replication in tissue culture, few studies have confirmed this mechanism of protection in vivo. In these studies we have found that while ISG15nndash;/nndash; mice are more susceptible to influenza A virus and Sendai virus infections, ISGylation does not appear to mediate this protection through the direct inhibition of virus replication or the modulation of the acute immune response. Thus in addition to showing a novel mode of ISG15 mediated protection from virus infection, this study demonstrates the importance of studying the role of ISG15 in vivo.

  • Perturbation in the conserved methyltransferase-polymerase interface of flavivirus NS5 differentially affects polymerase initiation and elongation [PublishAheadOfPrint]

  • The flavivirus NS5 is a natural fusion of a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP). Analogous to DNA-dependent RNA polymerases, the NS5 polymerase initiates RNA synthesis through a de novo mechanism and then makes a transition to a processive elongation phase. However, whether and how the MTase affects polymerase activities through intra-molecular interactions remains elusive. By solving the crystal structure of Japanese encephalitis virus (JEV) NS5, we recently identified an MTase-RdRP interface containing a set of six hydrophobic residues highly conserved among flaviviruses. To dissect the functional relevance of this interface, we made a series of JEV NS5 constructs with mutations of these hydrophobic residues and/or with the N-terminal 261-303 residues deleted. Comparing to the wild type (WT) NS5, full-length NS5 variants exhibited consistent up- or down-regulation of the initiation activities in two types of polymerase assays. Five representative full-length NS5 constructs were then tested in an elongation assay from which the apparent single-nucleotide incorporation rate constant was estimated. Interestingly, two constructs exhibited different elongation kinetics from the WT NS5, with an effect rather opposite to what was observed at initiation. Moreover, constructs with MTase and/or the linker region (residues 266-275) removed still retained polymerase activities, albeit at overall lower levels. However, further removal of the N-terminal extension (residues 276-303) abolished regular template-directed synthesis. Together, our data showed that the MTase-RdRP interface was relevant in both polymerase initiation and elongation, likely with different regulation mechanisms in these two major phases of RNA synthesis.

    IMPORTANCE The flavivirus NS5 is very unique in placing a methyltransferase (MTase) to the immediate N-terminus of its RNA-dependent RNA polymerase (RdRP). We recently solved the crystal structure of the full-length NS5 that reveals a conserved interface between MTase and RdRP. Building on this discovery, here we carried out in vitro polymerase assays to address the functional relevance of the interface interactions. By explicitly probing polymerase initiation and elongation activities, we found that perturbation in the MTase-RdRP interface had different impacts on different phases of synthesis, suggesting that the roles and contribution of the interface interactions may change upon phase transitioning. By comparing the N-terminal truncated enzymes with the full-length NS5, we collected data to indicate the indispensability to regular polymerase activities of a region that was functionally un-clarified previously. Taken together, we have provided biochemical evidences and mechanistic insights for the crosstalk between the two enzyme modules of flavivirus NS5.

  • Proinflammatory Effects of Interferon Gamma in Mouse Adenovirus Type 1 Myocarditis [PublishAheadOfPrint]

  • Adenoviruses are frequent causes of pediatric myocarditis. Little is known about the pathogenesis of adenovirus myocarditis, and the species-specificity of human adenoviruses has limited the development of animal models, which is a significant barrier to strategies for prevention or treatment. We have developed a mouse model of myocarditis following mouse adenovirus type 1 (MAV-1) infection to study the pathogenic mechanisms of this important cause of pediatric myocarditis. Following intranasal infection of neonatal C57BL/6 mice, we detected viral replication and induction of interferon-gamma (IFN-) in the hearts of infected mice. MAV-1 caused myocyte necrosis and induced substantial cellular inflammation that was predominantly composed of CD3+ T lymphocytes. Depletion of IFN- during acute infection reduced cardiac inflammation in MAV-1-infected mice without affecting viral replication. We observed decreased contractility during acute infection of neonatal mice, and persistent viral infection in the heart was associated with cardiac remodeling and hypertrophy in adulthood. IFN- is a proinflammatory mediator during adenovirus-induced myocarditis, and persistent adenovirus infection may contribute to ongoing cardiac dysfunction.

    Importance Studying the pathogenesis of myocarditis caused by different viruses is essential in order to characterize both virus-specific and generalized factors that contribute to disease. Very little is known about the pathogenesis of adenovirus myocarditis, which is a significant impediment to the development of treatment or prevention strategies. We used MAV-1 to establish a mouse model of human adenovirus myocarditis, providing the means to study host and pathogen factors contributing to adenovirus-induced cardiac disease during acute and persistent infection. The MAV-1 model will enable fundamental studies of viral myocarditis, including IFN- modulation, as a therapeutic strategy.

  • Insight into the Mechanism of Inhibition of Recombinant Adeno-Associated Virus by the Mre11/Rad50/Nbs1 Complex [PublishAheadOfPrint]

  • Adeno-Associated virus (AAV) is a dependent virus of the family parvoviridae. Gene expression and replication of AAV and derived recombinant vectors (rAAV) are severely limited (ggt;10-fold) by the cellular DNA damage sensing complex made up of Mre11, Rad50, and Nbs1 (MRN). AAV does not encode the means to circumvent this block to productive infection, but relies on co-infecting helper-virus to do so. Using adenovirus helper proteins E1B55k andE4orf6, which enhance transduction of AAV via degradation of MRN, we investigate the mechanism through which this DNA damage complex inhibits gene expression from rAAV. We test substrate specificity of inhibition and the contribution of different functions of the MRN complex. Our results demonstrate that both single- and double-stranded rAAV vectors are inhibited by MRN, which is in contrast to the predominant model that inhibition is the result of a block to second-strand synthesis. Exploring the contribution of known functions of MRN, we found inhibition of rAAV does not require downstream DNA damage response factors, including signaling kinases ATM and ATR. The nuclease domain of Mre11 appears to play only a minor role in inhibition, while the DNA-binding domain makes a greater contribution. Additionally, mutation of the inverted terminal repeat of the rAAV genome, which has been proposed to be the signal for interaction with MRN, is tolerated by the mechanism of inhibition. These results articulate a model of inhibition of gene expression in which physical interaction is more important than enzymatic activity and several key downstream damage repair factors are dispensable.

    IMPORTANCE Many viruses modulate the host DNA damage response (DDR) in order to create a cellular environment permissive for infection. The MRN complex is a primary sensor of damage in the cell, but also responds to invading viral genomes, often posing a block to infection. AAV is greatly inhibited by MRN and dependent on co-infecting helper-virus, such as Adenovirus, to remove this factor. Currently, the mechanism through which MRN inhibits AAV and other viruses is poorly understood. Our results reform the predominant model that inhibition of rAAV by MRN is due to limiting second-strand DNA synthesis. Instead, a novel mechanism of inhibition of gene expression independent of a block in rAAV DNA synthesis or downstream damage factors is indicated. These findings have clear implications toward understanding this restriction to transduction of AAV and rAAV vectors, which have high therapeutic relevance, and likely translate to other viruses that must navigate the DDR.

  • Activation of p90 Ribosomal S6 Kinases (RSKs) by ORF45 of Kaposi Sarcoma-Associated Herpesvirus is Critical for Optimal Production of Infectious Viruses [PublishAheadOfPrint]

  • We have previously shown that ORF45, an immediate-early and tegument protein of Kaposi's sarcoma associated herpesvirus (KSHV), causes sustained activation of p90 ribosomal S6 kinases (RSKs) and ERK. We have now identified the critical region of ORF45 that is involved in RSK interaction and activation. Alanine scanning mutagenesis of this region revealed that a single F66A point mutation abolished binding of ORF45 to RSK or ERK, and consequently its ability to activate the kinases. We introduced the F66A mutation into BAC16 (a bacterial artificial chromosome clone containing the entire infectious KSHV genome), producing BAC16-45F66A. In parallel, we also repaired the mutation and obtained a revertant BAC16-45A66F. Reconstitution of these mutants in iSLK cells demonstrated that the ORF45-F66A mutant failed to cause sustained ERK and RSK activation during lytic reactivation, resulting in dramatic differences in the phosphoproteomic profile between the wild-type virus-infected cells and the mutant virus-infected cells. ORF45 mutation or deletion was also accompanied by noticeably decreased viral gene expression during lytic reactivation. The ORF45-F66A mutant consequently produced significantly fewer infectious progeny virions than the wild type or the revertant. These results suggest a critical role for ORF45-mediated RSK activation in KSHV lytic replication.

    IMPORTANCE STATEMENT Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three human malignancies. KSHV pathogenesis is intimately linked to its ability to modulate the host cell microenvironment, and facilitate efficient production of progeny viral particles. We have previously described the mechanism by which the KSHV lytic protein ORF45 activates the cellular kinases ERK and RSK. We now have mapped the critical region of ORF45 responsible for binding and activation of ERK/RSK to a single residue, F66. We mutated this amino acid of ORF45 (F66A) and introduced the mutation into a newly developed bacterial artificial chromosome containing the KSHV genome (BAC16). This system has provided us with a useful tool to characterize the functions of ORF45-activated RSK upon KSHV lytic reactivation. We show that viral gene expression and virion production are significantly reduced by F66A mutation, indicating a critical role for ORF45-activated RSK during KSHV lytic replication.

  • Variation of pH sensitivity, acid stability, and fusogenicity of three influenza H3 subtypes [PublishAheadOfPrint]

  • Influenza A virus strains adapt to achieve successful entry into host species. Entry is mediated by the viral membrane protein, hemagglutinin (HA), which triggers membrane fusion and genome release under acidic conditions in the endosome. In addition to changes in the receptor binding domain, the acid stability of HA has been linked to successful transmission of virus between avian and human hosts. However, to fully understand the connection between changes in HA and host tropism, additional factors relevant to HA structure-function and membrane fusion are also likely to be important. Using single particle tracking (SPT) techniques, individual membrane fusion events can be observed under specific conditions, which provides detailed information regarding HA pH sensitivity, acid stability, and the rate and extent of membrane fusion. This provides a comparative way to characterize and distinguish influenza fusion properties among virus strains. We used SPT to quantify the fusion properties of three H3 influenza strains: A/Aichi/68/H3N2 (X:31), A/Udorn/72/H3N2 (Udorn) and A/Brisbane/07/H3N2 (Brisbane). The rate of fusion for the most clinically relevant strain, Brisbane, is generally insensitive to decreasing pH, while fusion of the egg-adapted strains, Udorn and X:31, are strongly dependent on pH (and faster) as pH decreases. All strains exhibit similar acid stability (the length of time they remain fusogenic in an acidic environment) at higher pH's, but the egg-adapted strains become less acid stable at lower pH's. Thus, it appears that the lab-adapted H3 strains tested may have evolved to compensate for the faster HA deactivation at low pH with a commensurate increase in the rate of fusion and number of proteins facilitating fusion, relative to the Brisbane strain.

    Importance The ability of influenza virus to release its genome under different acidic conditions has recently been linked to transmission of influenza virus between different species. However, it is yet to be determined how acid-induced membrane fusion varies with virus strain and influences tropism. The results presented here are an intra-H3 subtype study of acid stability and fusion kinetics. Using a single particle tracking (SPT) technique, we show here that the highest pH that initiates fusion is not necessarily the pH where the kinetics of fusion is fastest and most abundant for a given strain. Strains exhibit different fusion behaviors, as evidenced by their unique kinetic trends; pH sensitivities, as evidenced by the differences when the first fusion events commence; and HA stabilities, as evidenced by the length of time virions can persist in acidic environment and still be fusion competent.

  • The putative HSV-1 chaperone protein UL32 modulates disulfide bond formation during infection. [PublishAheadOfPrint]

  • During DNA encapsidation, HSV-1 procapsids are converted to DNA-containing capsids by a process involving activation of the viral protease, expulsion of the scaffold proteins and the uptake of viral DNA. Encapsidation requires six minor capsid proteins (UL6, UL15, UL17, UL25, UL28 and UL33) and one viral protein, UL32, not found associated with capsids. Although functions have been assigned to each of the minor capsid proteins, the role of UL32 in encapsidation has remained a mystery. Using an HSV-1 variant containing a functional HA-tagged UL32, we demonstrated that UL32 was synthesized with true late kinetics and that it exhibited a previously unrecognized localization pattern. At 6 nndash; 9 h post infection (hpi), UL32 accumulated in viral replication compartments in the nucleus of the host cell while at 23 hpi it was additionally found in the cytoplasm. A newly generated UL32 null mutant was used to confirm that although B-capsids containing wild-type levels of capsid proteins were synthesized, these procapsids were unable to initiate the encapsidation process. Furthermore, we showed that UL32 is redox sensitive and identified two highly conserved oxidoreductase-like C-X-X-C motifs that are essential for protein function. In addition, the disulfide bond profiles of the viral proteins UL6, UL25, VP19C and the viral protease, VP24, were altered in the absence of UL32, suggesting that UL32 may act to modulate disulfide bond formation during procapsid assembly and maturation.

    Importance Although functions have been assigned to six of the seven required packaging proteins of HSV, the role of UL32 in encapsidation has remained a mystery. UL32 is a cysteine-rich viral protein that contains C-X-X-C motifs reminiscent of proteins that participate in the regulation of disulfide bond formation. We have previously demonstrated that disulfide bonds are required for the formation and stability of the viral capsids and are also important for the formation and stability of the UL6 portal ring. In this report, we demonstrate that the disulfide bond profiles of the viral proteins UL6, UL25, VP19C and the viral protease, VP24, are altered in cells infected with a newly isolated UL32 null mutant virus, suggesting that UL32 acts as a chaperone capable of modulating disulfide bond formation. Furthermore, these results suggest that proper regulation of disulfide bonds is essential for initiating encapsidation.

  • Complex TCR repertoire dynamics underlie the CD8+ T-cell response to HIV-1 [PublishAheadOfPrint]

  • Although CD8+ T-cells are important for the control of HIV-1 in vivo, the precise correlates of immune efficacy remain unclear. In this study, we conducted a comprehensive analysis of viral sequence variation and T-cell receptor (TCR) repertoire composition across multiple epitope specificities in a group of antiretroviral treatment-naiiuml;ve individuals chronically infected with HIV-1. A negative correlation was detected between changes in antigen-specific TCR repertoire diversity and CD8+ T-cell response magnitude, reflecting clonotypic expansions and contractions related to alterations in cognate viral epitope sequences. These patterns were independent of the individual, evidenced by discordant clonotype-specific evolution against different epitopes in single subjects. Moreover, long-term asymptomatic HIV-1 infection was characterized by evolution of the TCR repertoire in parallel with viral replication. Collectively, these data suggest a continuous bidirectional process of adaptation between HIV-1 and virus-specific CD8+ T-cell clonotypes orchestrated at the TCR/antigen interface.

    Importance We describe a relation between viral epitope mutation, antigen-specific T-cell expansion and the repertoire of responding clonotypes in chronic HIV-1 infection. This work provide insights into the process of co-adaptation between the human immune system and a rapidly evolving lentivirus.

  • The Autographa californica multiple nucleopolyhedrovirus ORF11 is essential for BV production and ODV envelopment [PublishAheadOfPrint]

  • ORF11 (ac11) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene with unknown function. To determine the role of ac11 in the baculovirus life cycle, an ac11-knockout mutant of AcMNPV, Ac11KO, was constructed. Northern blot and 5'-RACE analysis revealed that ac11 is an early gene in the life cycle. Microscopy, titration assays, and Western blot analysis revealed that budded viruses (BVs) were not produced in the Ac11KO-transfected Sf9 cells. However, qPCR analysis demonstrated that the deletion of ac11 did not affect viral DNA replication. Furthermore, electron microscopy revealed that there was no nucleocapsid in the cytoplasm or plasma membrane of the Ac11KO-transfected cells, which demonstrates that the defect in BV production from the Ac11KO-transfected cells is due to the inefficient egress of nucleocapsids from the nucleus to the cytoplasm. In addition, the electron microscopy observations showed that the nucleocapsids in the nucleus were not enveloped to form ODVs, and that their subsequent embedding into OBs was also blocked in the Ac11KO-transfected cells, demonstrating that ac11 is required for ODV envelopment. These results therefore demonstrate that ac11 is an early gene that is essential for BV production and ODV envelopment.

    IMPORTANCE Baculoviruses have been extensively used not only as specific, environmentally benign insecticides but also as helper-independent protein expression vector. Although the function of baculovirus genes in viral replication has been studied using gene knock-out technology, the function of more than one-third of viral genes which include some highly conserved genes are still unknown. In this study, ac11 was proved to play a crucial role in BV production and ODV envelopment. These results will lead to a better understanding of baculovirus infection cycles.

  • NS5A and human RPA increase the processivity of HCV NS5B polymerase activity in vitro [PublishAheadOfPrint]

  • The precise role(s) and topological organization of different factors in the hepatitis C virus (HCV) RNA replication complex are not well understood. In order to elucidate the role of viral and host proteins in HCV replication, we have developed a novel in vitro replication system that utilizes a rolling circle RNA template. Under close-to-physiological salt conditions, HCV NS5B21, the RNA dependent RNA polymerase, has poor affinity for the RNA template. Human replication protein A (RPA) and HCV NS5A, recruit NS5B21 to the template. Subsequently, NS3 is recruited to the replication complex by NS5B21 resulting in RNA synthesis stimulation by helicase. Both RPA and NS5A(S25-C447), but not NS5A(S25-K215), enabled the NS5B21bbull;NS3 helicase complex to be stably associated with the template and synthesize RNA product in a highly processive manner in vitro. This new in vitro HCV replication system is a useful tool that may facilitate the study of other replication factors and aid in the discovery of novel inhibitors of HCV replication.

    Importance The molecular mechanism of hepatitis C virus (HCV) replication is not fully understood but viral and host proteins collaborate in this process. Using a rolling circle RNA template, we have reconstituted an in vitro HCV replication system that allows us to interrogate the role of viral and host proteins in HCV replication and delineate the molecular interactions. We showed that HCV NS5A(S25-C447) and cellular replication protein A (RPA) functionally cooperate as a processivity factor to stimulate HCV replication by HCV NS5B21 polymerase and NS3 helicase. This system paves the way to test other proteins and may be used as an assay for discovery of HCV inhibitors

  • Structure-Function Relationships Underlying the Replication Fidelity of Viral RNA-dependent RNA Polymerases [PublishAheadOfPrint]

  • Viral RNA-dependent RNA polymerases are considered to be low fidelity enzymes, providing for high mutation rates that allow for the rapid adaptation of RNA viruses to different host cell environments. Fidelity is tuned to provide the proper balance of virus replication rates, pathogenesis, and tissue tropism needed for virus growth. Using our structures of picornaviral polymerase-RNA elongation complexes, we have previously engineered over a dozen coxsackievirus B3 polymerase mutations that significantly altered virus replication rates and in vivo fidelity, and also provided a set of secondary adaptation mutations after tissue culture passage. Here we report a biochemical analysis of these mutations based on rapid stopped-flow kinetics to determine elongation rates and nucleotide discrimination factors. The data show a spatial separation of fidelity and replication rate effects within the polymerase structure. Mutations in the palm domain have the greatest effects on in vitro nucleotide discrimination and these are strongly correlated with elongation rates and in vivo mutation frequencies, with faster polymerases having lower fidelity. Mutations located at the top of the fingers domain, on the other hand, primarily affect elongation rates and have relatively minor effects on fidelity. Similar modulation effects are seen in poliovirus polymerase, an inherently lower fidelity enzyme where analogous mutations now increase nucleotide discrimination. The findings further our understanding of viral RNA-dependent RNA polymerase structure-function relationships and suggest that (+) strand RNA viruses retain a unique palm domain based active site closure mechanism to fine tune replication fidelity.

    IMPORTANCE Positive strand RNA viruses represent a major class of human and animal pathogens with significant health and economic impacts. These viruses replicate using a virally encoded RNA-dependent RNA polymerase enzyme that has low fidelity, generating many mutations that allow for the rapid adaptation of these viruses to different tissue types and host cells. In this work we use a structure-based approach to engineer mutations in viral polymerases and study their effects on in vitro nucleotide discrimination as well as virus growth and genome replication fidelity. The results show that mutation rates can be drastically increased or decreased as a result of single mutations at several key residues in the polymerase palm domain, and this can significantly attenuate virus growth in vivo. The findings provide a pathway for developing live attenuated virus vaccines based on engineering the polymerase to reduce virus fitness.

  • Expression of IFN{gamma} by a recombinant rabies virus strongly attenuates the pathogenicity of the virus via induction of type I interferon [PublishAheadOfPrint]

  • Previous animal model experiments have shown a correlation between interferon gamma (IFN) expression and both survival from infection with attenuated rabies virus and reduction of neurological sequelae. Therefore, we hypothesized that rapid production of murine IFN by the rabies virus itself would induce a more robust antiviral response than would occur naturally in mice. To test this hypothesis, we used reverse engineering to clone the mouse IFN gene into a pathogenic rabies virus backbone, SPBN, to produce the recombinant rabies virus designated SPBN. Morbidity and mortality were monitored in mice infected intranasally with SPBN or SPBN(-) control virus to determine the degree of attenuation caused by the expression of IFN. Incorporation of IFN into the rabies genome highly attenuated the virus. SPBN has an LD50 more than 100 fold greater than SPBN(-). In vitro and in vivo mouse experiments show that SPBN infection enhances the production of type I interferons. Furthermore, knockout mice lacking the ability to signal through the type I interferon receptor (IFNARnndash;/nndash;) cannot control the SPBN infection and rapidly die. These data suggest that IFN production has antiviral effects in rabies, largely due to the induction of type I interferons.

    IMPORTANCE Survival from rabies is dependent upon the early control of virus replication and spread. Once the virus reaches the CNS this becomes highly problematic. Studies of CNS immunity to RABV have shown that control of replication begins at the onset of T cell entry and IFN production in the CNS prior to the appearance of virus-neutralizing antibodies. Moreover antibody deficient mice are able to control but not clear attenuated RABV from the CNS. We find here that IFN triggers the early production of type I interferons with the expected antiviral effects. We also show that engineering a lethal rabies virus to express IFN directly to the infected tissue reduces rabies virus replication and spread limiting its pathogenicity in normal and immunocompromised mice. Therefore, vector delivery of IFN to the brain may have the potential to treat individuals who would otherwise succumb to infection with rabies virus.

  • Compensatory Substitutions in the HIV-1 Capsid Reduce the Fitness Cost Associated with Resistance to a Capsid-Targeting Small-Molecule Inhibitor [PublishAheadOfPrint]

  • The HIV-1 capsid plays multiple roles in infection and is an emerging therapeutic target. The small molecule HIV-1 inhibitor PF-3450074 (PF74) blocks HIV-1 at an early postentry stage by binding the viral capsid and interfering with its function. Selection for resistance resulted in accumulation of five amino acid changes in the viral CA protein, which collectively reduced binding of the compound to HIV-1 particles. In the present study, we dissected the individual and combinatorial contributions of each of the five substitutions Q67H, K70R, H87P, T107N, and L111I to PF74 resistance, PF74 binding, and HIV-1 infectivity. Q67H, K70R, and T107N each conferred low-level resistance to PF74, and collectively conferred strong resistance. The substitutions K70R and L111I impaired HIV-1 infectivity, which was partially restored by the other substitutions at positions 67 and 107. PF74 binding to HIV-1 particles was reduced by the Q67H, K70R, and T107N substitutions, consistent with the location of these positions in the inhibitor-binding pocket. Replication of the 5Mut virus was markedly impaired in cultured macrophages, reminiscent of the previously reported N74D CA mutant. 5Mut substitutions also reduced the binding of the host protein CPSF6 to assembled CA complexes in vitro and permitted infection of cells expressing the inhibitory protein CPSF6-358. Our results demonstrate that strong resistance to PF74 requires accumulation of multiple substitutions in CA to inhibit PF74 binding and compensate for fitness impairments associated with some of the sequence changes.

    IMPORTANCE The HIV-1 capsid is an emerging drug target, and several small molecule compounds have been reported to inhibit HIV-1 infection by targeting the capsid. Here we show that resistance to the capsid-targeting inhibitor PF74 requires multiple amino acid substitutions in the binding pocket of the CA protein. Three changes in CA were necessary to inhibit binding of PF74 while maintaining viral infectivity. Replication of the PF74-resistant HIV-1 mutant was impaired in macrophages, likely owing to altered interactions with host cell factors. Our results suggest that HIV-1 resistance to capsid-targeting inhibitors will be limited by functional constraints on the viral capsid protein. Therefore, this work enhances the attractiveness of the HIV-1 capsid as a therapeutic target.

  • Blocking ESCRT-mediated envelopment inhibits microtubule-dependent trafficking of alphaherpesviruses in vitro. [PublishAheadOfPrint]

  • Herpes simplex virus and, as reported here, Pseudorabies virus utilize the ESCRT-apparatus to drive cytoplasmic envelopment of their capsids. Here we demonstrate that blocking ESCRT-mediated envelopment using the dominant negative inhibitor Vps4A-EQ reduced the ability of HSV and PRV particles to subsequently traffic along microtubules in vitro. HSV and PRV capsid-associated particles with bound GFP-Vps4A-EQ were readily detected by fluorescence microscopy in cytoplasmic extracts of infected cells. These Vps4A-EQ-associated capsid-containing particles bound to microtubules in vitro but were unable to traffic along them. Using a PRV strain expressing a fluorescent capsid and a fluorescently tagged form of the envelope protein gD, we found that similar numbers of gD-positive and gD-negative capsid-associated particles accumulated in cytoplasmic extracts under our conditions. Both classes of PRV particle bound to microtubules in vitro with comparable efficiency, and similar results were obtained for HSV using anti-gD immunostaining. The gD-positive and gD-negative PRV capsids were both capable of trafficking along microtubules in vitro, however motile gD-positive particles were less numerous and their trafficking was more sensitive to the inhibitory effects of Vps4A-EQ. We discuss our data in the context of microtubule-mediated trafficking of naked and enveloped alphaherpesvirus capsids.

    Importance The alphaherpesviruses include several important human pathogens. These viruses utilize microtubule-mediated transport to travel through the cell cytoplasm, however the molecular mechanisms of trafficking are not well understood. In this study we have used a cell-free system to examine the requirements for microtubule-trafficking, and have attempted to distinguish between the movement of "naked" and membrane-associated cytoplasmic alphaherpesvirus capsids.

  • Complex expression of the UL136 gene of human cytomegalovirus results in multiple protein isoforms with unique roles in replication [PublishAheadOfPrint]

  • Human cytomegalovirus is a complex DNA virus with a 230-kilobase genome encoding 170 to 750 proteins. The upper limit of this coding capacity suggests the evolution of complex mechanisms to substantially increase the coding potential from the 230-kb genome. Our work examines the complexity of one gene, UL136, encoded within the ULb' region of the genome that is lost during serial passage of HCMV in cultured fibroblasts. UL136 is expressed as five protein isoforms. We mapped these isoforms and demonstrate that they originate from both a complex transcriptional profile and possibly the usage of multiple translation initiation sites. Intriguingly, the pUL136 isoforms exhibit distinct subcellular distributions with varying association with the Golgi apparatus. The subcellular localization of membrane bound isoforms of UL136 differ when they are expressed exogenous compared to in the context of viral infection, suggesting that the trafficking of these isoforms is mediated by infection specific factors. While UL136, as most ULb' genes, was dispensable for replication in fibroblasts, the soluble 23-/19-kDa isoforms suppressed virus replication. In CD34+ hematopoietic progenitor cells (HPCs) infected in vitro, disruption of the 23-/19-kDa isoforms resulted in increased replication and a loss of latency phenotype, similar to the UL138 latency determinant encoded within the same genetic locus. Our work suggests complex interplay between the UL136 isoforms, which balances viral replication in multiple cell types and likely contributes to the cell-type dependent phenotypes of the UL133/8 locus and the outcome of HCMV infection.

    SIGNIFICANCE HCMV is a significant cause of morbidity in immunocompromised individuals, including transplant patients. Life-long persistence of the virus results in a high worldwide seroprevalence and may contribute to age related pathologies, such as atherosclerosis. Mechanisms of viral persistence are poorly understood; however, understanding the molecular basis of persistence is imperative for the development of new treatments. In this current work, we characterize a complex HCMV gene, UL136, which is expressed as five protein isoforms. These isoforms arise predominately from complex transcriptional mechanisms, which contribute to an increased coding capacity of the virus. Further, the UL136 isoforms oppose the activity of one another to balance HCMV replication in multiple cell types. We identify soluble isoforms of UL136 that function to suppress virus replication in fibroblasts and in CD34+ HPCs for latency.

  • Unabated Adenovirus replication following activation of the cGAS/STING dependent antiviral response in human cells [PublishAheadOfPrint]

  • The cGAS/STING DNA sensing complex has recently been established as a predominant pathogen recognition receptor (PRR) for DNA directed type I interferon innate immune activation. Using replication defective Adenovirus vectors and replication competent wildtype adenovirus, we have modeled the influence of the cGAS/STING cascade in permissive human cell lines (A549, HeLa, ARPE19, THP1). Wildtype adenovirus induces efficient early activation of the cGAS/STING cascade in a cell specific manner. In all responsive cell lines, cGAS/STING shRNA knockdown results in a loss of TBK1 and IRF3 activation, a lack of interferon bbeta; transcript induction, loss of interferon dependent STAT1 activation, and diminished induction of interferon-stimulated genes (ISGs). Adenoviruses that infect through the CAR (Ad2, Ad5), CD46 (Ad35), and Desmoglein-2 (Ad7) viral receptors all induce the cGAS/STING/TBK1/IRF3 cascade. The magnitude of the IRF3/IFN/ISG antiviral response, was strongly influenced by serotype with Ad35ggt;Ad7ggt;Ad2. For each serotype no enhancement of viral DNA replication or virus production occurred in cGAS or STING shRNA targeted cell line pools. We found no replication advantage in permissive cell lines that do not trigger the cGAS/STING cascade following infection. The cGAS/STING/TBK1/IRF3 cascade was not a direct target of viral anti-host strategies and we found no evidence that Ad stimulation of the cGAS/STING DNA response had an impact on viral replication efficiency.

    Significance statement: This study shows for the first time that the cGAS DNA sensor directs a dominant IRF3/IFN/ISG antiviral response to Adenovirus in human cell lines. Activation of cGAS occurs with viruses that infect through different high affinity receptors (CAR, CD46, and Desmoglein-2) and the magnitude of the cGAS/STING DNA response cascade is influenced by serotype specific functions. Furthermore, activation of the cGAS cascade occurred in a cell specific manner. Activation of the cGAS/STING response did not impact viral replication, and viral immune evasion strategies did not target the cGAS/STING/TBK1/IRF3 cascade. These studies provide novel insight into the early innate recognition response to adenovirus.

  • Putative Domain-Domain Interactions in the Vesicular Stomatitis Virus L Polymerase Protein Appendage Region [PublishAheadOfPrint]

  • The multidomain polymerase protein (L) of nonsegmented negative strand (NNS) RNA viruses catalyzes transcription and replication of the virus genome. The N-terminal half of the protein forms a ring-like polymerase structure while the C-terminal half encoding viral mRNA transcript modifications consists of a flexible appendage with three distinct globular domains. To gain insight into putative transient interactions between L domains during viral RNA synthesis, we exchanged each of the four distinct regions encompassing the appendage region of vesicular stomatitis virus (VSV) Indiana serotype L protein with their counterparts from VSV New Jersey, and analyzed effects on virus polymerase activity in a minigenome system. The methyltransferase domain exchange yielded a fully active polymerase protein, which functioned as well as wild type L in the context of a recombinant virus. Exchange of the downstream C-terminal non-conserved region abolished activity, but co-exchanging it with the methyltransferase domain generated a polymerase favoring replicase over transcriptase activity, providing strong evidence of interaction between these two regions. Exchange of the capping enzyme domain, or the adjacent non-conserved region thought to function as an "unstructured" linker, also abrogated polymerase activity, even when co-exchanged with other appendage domains. Further probing of the putative linker segment using in-frame EGFP insertions likewise abrogated activity. We discuss the implications of these findings with regard to L protein appendage domain structure and putative domain-domain interactions required for polymerase function.

    IMPORTANCE NNS viruses include many well-known human pathogens (e.g., rabies, measles, and Ebola viruses), as well as emerging viral threats (e.g., Nipah and Hendra viruses). These viruses all encode a large L polymerase protein similarly organized into multiple domains that work in concert to enable virus genome transcription and replication. But how the unique L protein carries out the multiplicity of individual steps in these two distinct processes is poorly understood. Using two different approaches, i.e., exchanging individual domains in the C-terminal appendage region of the protein between two closely related VSV serotypes, and inserting unrelated protein domains, we shed light on requirements for domain-domain interactions and domain contiguity in polymerase function. These findings further our understanding of the conformational dynamics of NNS L polymerase proteins, which play an essential role in pathogenic properties of these viruses and represent attractive targets for the development of antiviral measures.

  • Recombinant MVA generating excess early double-stranded RNA transiently activates PKR and triggers enhanced innate immune responses [PublishAheadOfPrint]

  • Double-stranded (ds) RNA is an important molecular pattern associated with viral infection and is detected by various extra- and intracellular recognition molecules. Poxviruses have evolved to avoid producing dsRNA early in infection but generate significant amounts of dsRNA late in infection due to convergent transcription of late genes. Protein kinase R (PKR) is activated by double-stranded (ds) RNA and triggers major cellular defenses against viral infection including protein synthesis shutdown, apoptosis and type I interferon (IFN-I) production. The poxviral E3 protein binds and sequesters viral dsRNA and is a major antagonist of the PKR pathway. We found that the highly replication-restricted modified vaccinia virus Ankara (MVA) engineered to produce excess amounts of dsRNA early in infection showed enhanced induction of IFN-bbeta; in murine and human cells in the presence of an intact E3L gene. IFN-bbeta; induction required a minimum overlap length of 300 bp between early complementary transcripts and was strongly PKR-dependent. Excess early dsRNA produced by MVAs activated PKR early but transiently in murine cells and induced enhanced systemic levels of IFN-aalpha;, IFN-, and other cytokines and chemokines in mice in a largely PKR-dependent manner. Replication-competent chorioallantois vaccinia virus Ankara (CVA) generating excess early dsRNA also enhanced IFN-I production and was apathogenic in mice even at very high doses, but showed no in vitro host range defect. Thus, genetically adjuvanting MVA and CVA to generate excess early dsRNA is an effective method to enhance innate immune stimulation by orthopoxvirus vectors and to attenuate replicating vaccinia virus in vivo.

    Importance Efficient cellular sensing of pathogen-specific components including double-stranded RNA (dsRNA) is an important prerequisite of an effective antiviral immune response. The prototype poxvirus vaccinia virus (VACV) and its derivative modified vaccinia virus Ankara (MVA) produce dsRNA as a byproduct of viral transcription. We found that inhibition of cellular dsRNA recognition established by the virus-encoded proteins E3 and K3 can be overcome by directing viral overexpression of dsRNA early in infection without compromising replication of MVA in permissive cells. Early dsRNA induced transient activation of the cellular dsRNA sensor protein kinase R (PKR), resulting in enhanced production of interferons and cytokines in cells and mice. Enhancing the capacity of MVA to activate the innate immune system is an important approach to further improve the immunogenicity of this promising vaccine vector.

  • The VP40 protein of Marburg virus exhibits impaired budding and increased sensitivity to human tetherin following mouse-adaptation [PublishAheadOfPrint]

  • The Marburg virus VP40 protein is a viral matrix protein that spontaneously buds from cells. It also functions as an interferon (IFN) signaling antagonist by targeting janus kinase 1 (JAK1). A previous study demonstrated that the VP40 protein of the Ravn strain of Marburg virus (Ravn virus or RAVV) failed to block IFN signaling in mouse cells, whereas the mouse-adapted RAVV (maRAVV) VP40 acquired the ability to inhibit IFN responses in mouse cells. The increased IFN-antagonist function of maRAVV VP40 mapped to residues 57 and 165, which were mutated during the mouse adaptation process. In the present study, we demonstrate that maRAVV VP40 lost the capacity to efficiently bud from human cell lines, despite the fact that both parental and maRAVV VP40s bud efficiently from mouse cell lines. The impaired budding in human cells corresponds with the appearance of protrusions on the surface of maRAVV VP40-expressing Huh7 cells and with an increased sensitivity of maRAVV VP40 to restriction by human tetherin but not mouse tetherin. However, transfer of the human tetherin cytoplasmic tail to mouse tetherin restored restriction of maRAVV VP40. Residues 57 and 165 were demonstrated to contribute to the failure of maRAVV VP40 to bud from human cells, and residue 57 was demonstrated to alter VP40 oligomerization, as assessed by co-precipitation assay, and to determine sensitivity to human tetherin. This suggests that RAVV VP40 acquired, during adaptation to mice, changes in its oligomerization potential that enhanced IFN-antagonist function. However, this new capacity impaired RAVV VP40 budding from human cells.

    IMPORTANCE Filoviruses, which include Marburg viruses and Ebola viruses, are zoonotic pathogens which cause severe disease in humans and non-human primates but do not cause similar disease in wild-type laboratory strains of mice unless first adapted to these animals. Although mouse adaptation has been used as a method to develop small animal models of pathogenesis, the molecular determinants associated with filovirus mouse-adaptation are poorly understood. Our study demonstrates how genetic changes which accrued during mouse-adaptation of the Ravn strain of Marburg virus have impacted the budding function of the viral VP40 matrix protein. Strikingly, we find impairment of mouse-adapted VP40 budding function in human but not mouse cell lines, and we correlate the impairment with an increased sensitivity of VP40 to restriction by human but not mouse tetherin and with changes in VP40 oligomerization. These data suggest that there are functional costs associated with filovirus adaptation to new hosts and implicate tetherin as a filovirus host restriction factor.

  • TNF-{alpha} and IL-6 facilitate corneal lymphangiogenesis in response to HSV-1 infection [PublishAheadOfPrint]

  • HSV-1 is a common human pathogen of clinical significance due to its association with vision impairment and encephalitis. In a mouse model of ocular neovascularization, we have previously identified HSV-1 elicits the genesis of lymphatic vessels into the cornea proper through epithelial cell expression of VEGFA dependent upon expression of VEGFR2 during acute infection. We hypothesized other factors may be involved in lymphangiogenesis with pro-inflammatory cytokines as the leading candidates. In the absence of infection or inflammation, intrastromal administration of TNF-aalpha; coupled with VEGFA elicited lymphatic vessel genesis significantly above either factor alone as well as vehicle control. Consistent with this observation, anti-TNF-aalpha; Ab blocked HSV-1-mediated corneal lymphangiogenesis within the first five days post infection. However, TNF-aalpha; deficient (TNF-aalpha;nndash;/nndash;) mice displayed a similar level of corneal vessel growth as wild type (WT) controls. To investigate the likely redundant nature of cytokines, PCR array analysis of HSV-1-infected TNF-aalpha;nndash;/nndash; mice revealed several factors elevated above that found in HSV-1-infected WT mice including IL-1bbeta;, platelet-derived growth factor, angiopoietin 2, insulin-like growth factor 2, and IL-6. Subconjunctival administration of neutralizing Ab to IL-6 blocked lymphangiogenesis in TNF-aalpha;nndash;/nndash; mice. Whereas the cornea levels of IL-6 were significantly reduced, there was no appreciable change in the level of IL-1bbeta; or other pro-angiogenic factors analyzed. Collectively, the results suggest in addition to VEGFA, TNF-aalpha; and IL-6 promotes and likely synergize with VEGFA in corneal lymphangiogenesis during acute HSV-1 infection.

    Importance We have identified at least two pro-inflammatory cytokines expressed locally that are involved in the genesis of lymphatic vessels in the normally avascular cornea in response to HSV-1 infection. This finding provides the basis to target IL-6 and TNF-aalpha; as additional pro-angiogenic factors in the cornea during the development of herpetic stromal keratitis as a means to alleviate further neovascularization and tissue pathology associated with the host immune response to the pathogen.

  • The Human Cytomegalovirus UL26 protein antagonizes NF{kappa}B activation [PublishAheadOfPrint]

  • Viral infection frequently triggers activation of host innate immune pathways that attempt to limit viral spread. The NFB pathway is a critical component that governs this response. We have found that the Human Cytomegalovirus (HCMV) UL26 protein antagonizes NFB activation. Upon infection, an HCMV strain lacking the UL26 gene (UL26) induced the nuclear translocation of the NFB RelB subunit, and activated expression and secretion of IL-6, an NFB target gene. The UL26 mutant was also more sensitive to challenge with TNFaalpha;, a canonical NFB inducer. Further, expression of UL26 in the absence of other viral proteins blocked NFB activation induced by either TNFaalpha; treatment or infection with Sendai virus (SeV). Our results indicate that UL26 expression is sufficient to block TNFaalpha;-induced NFB nuclear translocation and IB degradation. Lastly, UL26 blocks TNFaalpha;-induced IKappaB-kinase (IKK) phosphorylation, a key step in NFB activation. Combined, our results indicate that UL26 is part of a viral program to antagonize innate immunity through modulation of NFB signaling.

    Importance The NFB signaling pathway regulates innate immunity, an integral host process that limits viral pathogenesis. Viruses have evolved mechanisms to modulate NFB signaling to ensure their replication. Human Cytomegalovirus (HCMV) is a major cause of birth defects and disease in immunosuppressed populations. HCMV is known to actively target the NFB pathway, which is important for HCMV infection. Our results indicate that the HCMV UL26 gene is a key modulator of NFB pathway activity. We find the UL26 gene is both necessary and sufficient to block NFB activation upon challenge with anti-viral cytokines. Further, UL26 attenuates the phosphorylation and activation of a key NFB activating kinase complex, IKK. Our study provides new insight into how HCMV targets the NFB pathway. Given its importance to viral infection, the mechanisms through which viruses target the NFB pathway highlight areas of vulnerability that could be therapeutically targeted to attenuate viral replication.

  • RNA Populations in Immunocompromised Patients as Reservoirs for Novel Norovirus Variants [PublishAheadOfPrint]

  • Noroviruses are the leading cause of acute gastroenteritis outbreaks worldwide. The majority of norovirus outbreaks are caused by genogroup II.4 (GII.4) noroviruses. Novel GII.4 noroviruses emerge every 2-4 years and replace older variants as the dominant norovirus. The process of the emergence of novel variants is believed to be caused by a combination of recombination, genetic drift, and selection driven by population immunity, but how or where these novel variants emerge is not known. We detected two previously unknown novel GII.4 variants, termed GII.4 UNK1 and GII.4 UNK2, and a diverse norovirus population in fecal specimens from immunocompromised individuals with diarrhea after they had undergone bone-marrow transplantation. We hypothesized that immunocompromised individuals can serve as reservoirs for novel norovirus variants. To test our hypothesis, metagenomic analysis of viral RNA populations was combined with a full genome bioinformatic analysis of publicly available GII.4 noroviruses sequences from 1974 nndash; 2014 to identify converging sites. Localization analysis indicated that variable sites were more likely to be within two amino acids (Pllt; 0.05) of positively selected sites. Further analysis indicated polymorphic site distribution was random and its proximity to positively selected sites was dependent on the size of the norovirus genome and the number of positively selected sites. The results indicate that random mutations can have a positive impact on driving norovirus evolution and that immunocompromised individuals have the ability to serve as a potential reservoirs for novel GII.4 strains.

    Importance Norovirus is the most common cause of viral gastroenteritis in the US. Every two to three years novel norovirus variants emerge and rapidly disseminate throughout the world. The continual emergence of novel noroviruses is believed to be caused by a combination of genetic drift, population immunity, and recombination, but exactly how this emergence occurs remains unknown. In this study we identified two novel GII.4 variants in immunocompromised bone marrow transplant patients. Using metagenomic and bioinformatics analysis, we show that most genetic polymorphisms in the novel variants occur near, 0-2 amino acids, of positively selected sites, but the distribution of mutations was random; clustering of polymorphisms with positively selected sites was a result of genome size, number of mutations and positively selected sites. This study shows that immunocompromised patients can harbor infectious novel norovirus variants and although mutations in viruses are random they can have a positive effect in viral evolution.

  • Determinants of efficient degradation of APOBEC3 restriction factors by HIV-1 Vif [PublishAheadOfPrint]

  • The APOBEC3 deoxycytidine deaminases can restrict the replication of HIV-1 in cell culture to differing degrees. The effects of APOBEC3 enzymes are largely suppressed by HIV-1 Vif that interacts with host proteins to form a Cullin5-Ring E3 ubiquitin ligase that induces 48K-linked polyubiquitination (poly-Ub) and proteasomal degradation of APOBEC3 enzymes. Vif variants have differing abilities to induce degradation of APOBEC3 enzymes and the underlying biochemical mechanisms for these differences is not fully understood. We hypothesized that by characterizing the interaction of multiple APOBEC3 enzymes and Vif variants we could identify common features that resulted in Vif-mediated degradation and further define the determinants required for efficient Vif-mediated degradation of APOBEC3 enzymes. We used Vifs from HIV-1 NL4-3 (IIIB) and HXB2 to characterize their induced degradation of and interaction with APOBEC3G, APOBEC3G D128K, APOBEC3H, and APOBEC3B in 293T cells. We quantified the APOBEC3G-Vif and APOBEC3H-Vif interaction strengths in vitro using rotational anisotropy. Our biochemical and cellular analyses of the interactions support a model in which the degradation efficiency of VifIIIB and VifHXB2 correlated with both the binding strength of the APOBEC3-Vif interaction and the APOBEC3-Vif interface, which differs for APOBEC3G and APOBEC3H. Notably, Vif bound to APOBEC3H and APOBEC3B in the natural absence of Vif-induced degradation and the interaction resulted in 63K-linked poly-Ub of APOBEC3H and APOBEC3B, demonstrating additional functionality of the APOBEC3-Vif interaction apart from induction of proteasomal degradation.

    Importance APOBEC3 enzymes can potently restrict the replication of HIV-1 in the absence of HIV-1 Vif. Vif suppresses APOBEC3 action by inducing their degradation through a direct interaction with APOBEC3 enzymes and other host proteins. Vif variants from different HIV-1 strains have different effects on APOBEC3 enzymes. We used differing Vif degradation capacities of two Vif variants and various APOBEC3 enzymes with differential sensitivities to Vif to delineate determinants of the APOBEC3-Vif interaction that are required for inducing efficient degradation. Using a combined biochemical and cellular approach we identified that the strength of the APOBEC3-Vif binding interaction and the APOBEC3-Vif interface are determinants for degradation efficiency. Our results highlight the importance of using Vif variants with different degradation potential when delineating mechanisms of Vif-induced APOBEC3 degradation and identify features important for consideration in the development of HIV-1 therapies that disrupt the APOBEC3-Vif interaction.

  • Role of the short telomeric repeat region in Marek's disease virus (MDV) replication, genomic integration and lymphomagenesis [PublishAheadOfPrint]

  • Marek's disease virus (MDV) is a cell-associated alphaherpesvirus that causes generalized polyneuritis and T-cell lymphomas in chickens. MDV is able to integrate its genome into host telomeres, but the mechanism of integration is poorly understood. The MDV genome harbors two arrays of telomeric repeats (TMR) at the ends of its linear genome: multiple telomeric repeats (mTMR), with a variable number up to 100 repeats, and short telomeric repeats (sTMR), with a fixed number of 6 repeats. The mTMR have recently been shown to play an important role in MDV integration and tumor formation; however, the functions of the sTMR have remained unknown. In this study, we demonstrate that deletion of the sTMR in the MDV genome abrogates virus replication, while extensive mutation of the sTMR did not, indicating that presence but not the sTMR sequence itself is important. Furthermore, we generated a panel of truncation mutants to determine the minimal length of the sTMR and observed a direct correlation between sTMR length and MDV replication. To address the role of sTMR in MDV replication, integration and tumorigenesis, sTMR sequences were substituted by a scrambled repeated sequence (vsTMR_mut). vsTMR_mut replicated comparable to parental and revertant virus in vitro. In vivo, however, a significant reduction in disease and tumor incidence was observed in chickens infected with vsTMR_mut that also correlated with a reduced number of viral integration sites in tumor cells. Taken together, our data demonstrate that the sTMR play a central role in MDV genome replication, pathogenesis and MDV-induced tumor formation.

    Importance Marek's disease virus (MDV) is highly oncogenic alphaherpesvirus that infects chickens and causes high economic losses in poultry industry. MDV integrates its genetic material into host telomeres, a process that is crucial for efficient tumor formation. The MDV genome harbors two arrays of telomeric repeats (TMR) identical to host telomeres at the ends of its linear genome, termed mTMR and sTMR. mTMR have been recently shown to be involved in MDV integration, while the functions of sTMR remain unknown. Here we demonstrate that presence and length of sTMR sequence are crucial for MDV replication, but not the exact nucleotide sequence. Furthermore, the sTMR contribute to the high integration frequency of MDV and is important for MDV pathogenesis and tumor formation. As number of herpesviruses harbor arrays of telomeric repeats (TMR), MDV serves as a model to determine the role of the herpesvirus TMR in replication, integration and pathogenesis.

  • Linking pig-tailed macaque major histocompatibility complex class I haplotypes and cytotoxic T lymphocyte escape mutations in SIV infection [PublishAheadOfPrint]

  • The influence of MHC-I alleles on HIV diversity has been well characterised in humans at the population level. MHC-I alleles likely affect viral diversity in the SIV-infected pig-tailed macaque (M. nemestrina) model, but this is poorly characterised. We studied the evolution of SIV in pig-tailed macaques with a range of MHC-I haplotypes. SIVmac251 genomes were amplified from the plasma of 44 pig-tailed macaques infected with SIVmac251 at 4-10 months after infection and characterized by Illumina deep sequencing. MHC-I typing was performed on cellular RNA using Roche/454 pyrosequencing. MHC-I haplotypes and viral sequence polymorphisms were linked using in-house bioinformatics pipelines, both at individual mutations and groups of mutations spanning 10 amino acid segments, since CTL escape can occur at different amino acids within the same epitope in different animals. The approach successfully identified 6 known CTL escape mutations within 3 Mane-A1*084-restricted epitopes. The approach also identified over 70 new SIV polymorphisms linked to a variety of MHC-I haplotypes. Using functional CD8 T cell assays we confirmed that one of these associations, a Mane-B028 haplotype-linked mutation in Nef, corresponded to a CTL epitope. We also identified mutations associated with the Mane-B017 haplotype that were previously described as CTL epitopes restricted by Mamu-B*017:01 in rhesus macaques. This detailed study of pig-tailed macaque MHC-I genetics and SIV polymorphisms will enable a refined level of analysis for future vaccine design and treatment strategies for HIV.

    Importance Cytotoxic T lymphocytes select for virus escape mutants of HIV and SIV and this limits the effectiveness of vaccines and immunotherapies against these viruses. Patterns of immune escape variants are similar in HIV-1 infected human subjects that share the same MHC-I genes, but this has not been studied for SIV infection of macaques. By studying SIV sequence diversity in 44 MHC-typed SIV-infected pigtail macaques, we defined over 70 sites within SIV where mutations were common in macaques sharing particular MHC-I genes. Further, pigtail macaques sharing near-identical MHC-I genes with rhesus macaques responded to the same CTL epitope and forced immune escape. This allows many reagents developed to study rhesus macaque reagents to also be used to study pigtail macaques. Overall, our study defines sites of immune escape in SIV in pigtailed macaques and this enables a more refined level of analysis of future vaccine design and treatment strategies for HIV.

  • Genome analysis of the first Marseilleviridae representative from Australia indicates that most of its genes contribute to the virus fitness. [PublishAheadOfPrint]

  • The Marseilleviridae family consists of Acanthamoeba-infecting large DNA viruses with icosahedral particles ~0.2 micrometer in diameter and genome sizes in the 346-380 kb range. Since the isolation of Marseillevirus from a cooling tower in Paris (France) in 2009, the Marseilleviridae family rapidly expanded with representatives from Europe and Africa. Five members have been fully sequenced that are distributed among 3 emerging Marseilleviridae lineages. One comprises Marseillevirus and Cannes8virus, another one includes Insectomime and Tunisvirus, the third one corresponding to the more distant Lausannevirus. We now report the genomic characterization of Melbournevirus, the first Marseilleviridae representative isolated in Australia, from a fresh water pond in Melbourne. Despite the large distance separating this sampling point from France, Melbournevirus is remarkably similar to Cannes8virus and Marseillevirus with most orthologous genes exhibiting more than 98% identical nucleotide sequences. We took advantage of this optimal evolutionary distance to evaluate the selection pressure as the ratio of non-synonymous over synonymous mutations for various categories of genes. This ratio was found to be smaller than one for all of them, including those solely shared by the closest Melbournevirus and Cannes8virus isolates and absent from Lausannevirus. This suggests that most of the 403 protein coding genes composing the large Melbournevirus genome are under negative/purifying selection and must thus significantly contribute to the virus fitness. This conclusion contrasts with the more common view that many of the genes of the usually more diverse large DNA viruses might be (almost) dispensable.

    Importance A pervasive view is that viruses are fast evolving parasites and carry the smallest possible genomic information required to highjack the host cell machinery and perform their replication. This notion, probably inherited from the study of RNA viruses, is being gradually undermined by the discovery of DNA viruses with increasingly large gene content. These viruses also encode a variety of DNA repair functions presumably slowing down their evolution by preserving their genomes from random alterations. On the other hand, these viruses also encode a majority of proteins without cellular homologs, including many only shared between closest members of the same family. One may thus question the actual contribution of these anonymous and/or quasi-orphan genes to the virus fitness. Genomic comparisons of Marseilleviridae, including a new Marseillevirus isolated in Australia, demonstrate that most of their genes, irrespective of their functions and conservation across families, are evolving under negative selection.

  • Different roles of the three loops forming the adhesive interface of nectin-4 in measles virus binding and cell entry, nectin-4 homodimerization, and heterodimerization with nectin-1 [PublishAheadOfPrint]

  • Many viruses utilize cell adhesion molecules of the immunoglobulin superfamily as receptors. In particular, viruses of different classes exploit nectins. The large DNA viruses, herpes simplex and pseudorabies, use ubiquitous nectins 1 and 2. The negative strand RNA virus measles (MeV) uses tissue-specific nectin-4, and the positive strand RNA virus polio uses nectin-like 5 (necl-5), also known as poliovirus receptor. These viruses contact the BC, C'C'' and FG loops on the upper tip of their receptor's most membrane-distal domain. This location corresponds to the newly defined canonical adhesive interface of nectins, but how viruses utilize this interface has remained unclear. Here we show that the same key residues in the BC and FG loops of nectin-4 govern binding to the MeV attachment protein hemagglutinin (H) and cell entry, nectin-4 homodimerization, and heterodimerization with nectin-1. On the other hand, residues in the C'C'' loop necessary for homo- and heterotypic interactions are dispensable for MeV-induced fusion and cell entry. Remarkably, the C'C'' loop governs dissociation of the nectin-4 and H ectodomains. We provide formal proof that H can interfere with the formation of stable nectin-1/nectin-4 heterodimers. Finally, while developing an alternative model to study MeV spread, we observed that polarized primary pig airway epithelial sheets cannot be infected. We show that a single amino acid variant in the BC loop of pig nectin-4 fully accounts for restricted MeV entry. Thus, the three loops forming the adhesive interface of nectin-4 have different roles in supporting MeV H association and dissociation, and MeV-induced fusion.

    Importance Different viruses utilize nectins as receptors. Nectins are immunoglobulin superfamily glycoproteins that mediate cell-cell adhesion in vertebrate tissues. They interact through an adhesive interface located at the top of their membrane-distal domain. How viruses utilize the three loops forming this interface has remained unclear. We demonstrate that while nectin-nectin interactions require residues in all three loops, the association of nectin-4 with the measles virus hemagglutinin only requires the BC and FG loops. However, we discovered that residues in the C'C'' loop modulate the dissociation of nectin-4 from the viral hemagglutinin. Analogous mechanisms may support cell entry of other viruses that utilize nectins or other cell adhesion molecules of the immunoglobulin superfamily as receptors.

  • Differential contribution of adeno-associated virus type 2 Rep protein expression and nucleic acid elements to the inhibition of adenoviral replication in cis and in trans [PublishAheadOfPrint]

  • The helper-dependent adeno-associated virus (AAV-2) exhibits complex interactions with its helper adenovirus. Whereas AAV-2 is dependent on adenoviral functions for productive replication, it conversely inhibits adenoviral replication, both when its genome is present in trans after coinfection with both viruses or when it is present in cis as in the production of recombinant adenovirus (rAd)/AAV-2 hybrid vectors. The notion that AAV-mediated inhibition of adenoviral replication is predominantly due to the expression of the AAV-2 Rep proteins has recently been challenged by successful Rep78 expression in a rAd5 vector through recoding of the Rep ORF. We closely analyzed the relative contribution of AAV-2 nucleic acid elements and Rep protein expression to the inhibition of adenoviral replication in both of the above scenarios. When present in cis, a sequence element in the 3'-part of the rep gene comprising only the AAV-2 p40 promoter and the AAV-2 intron sequence, which we termed RIS-Ad, completely blocks adenoviral replication. p5/p19 promoter-driven Rep protein expression, on the other hand, only weakly inhibits rAd/AAV-2 vector propagation and by inactivation of the RIS-Ad it is feasible to generate first-generation rAd vectors expressing functional Rep proteins. The RIS-Ad plays no role in the inhibition of adenoviral replication in trans in a model closely mimicking AAV-2/Ad co-infection. Here expression of the Rep proteins is required, but additionally the presence of an amplifiable ITR containing template. Thus very different AAV-2 elements and mechanisms are involved in inhibition of adenoviral replication during rAd/AAV-2 vector propagation and after Ad/AAV coinfection.

    Importance This is the first study to systematically compare the contribution of AAV-2 protein expression and AAV-2 nucleic acid elements to the inhibition of adenoviral replication in rAd/AAV-2 hybrid vector generation and in AAV-2/adenovirus coinfection. It shows that the two inhibitory processes are very different with regard to the AAV-2 functions and the mechanisms involved. Whereas inhibition of rAd/AAV-2 hybrid vector propagation mostly involves a 3'-nucleic acid element in the rep gene, inhibition of an adenoviral genome in trans requires the Rep proteins and the AAV-ITRs. These findings have important implications both for the basic understanding of the AAV replication cycle and for generation of rAd/AAV-2 hybrid vectors expressing the nonstructural and structural proteins of AAV-2.

  • Evolution of co-circulating varicella zoster virus genotypes during a chickenpox outbreak in Guinea Bissau [PublishAheadOfPrint]

  • Varicella zoster virus (VZV), a double-stranded DNA alphaherpesvirus, is associated with seasonal outbreaks of varicella in non-immunised populations. Little is known about whether these outbreaks are associated with a single or multiple viral genotypes and if new mutations rapidly accumulate during transmission. Here, we take advantage of a well-characterised population cohort in Guinea-Bissau and produce a unique set of 23 full length genome sequences, collected over seven months from eight households. Comparative sequence analysis reveals that four distinct genotypes co-circulated amongst the population, three of which were present during the first week of the outbreak, although no patients were co-infected, which indicates that exposure to infectious virus from multiple sources is common during VZV outbreaks. Transmission of VZV was associated with length polymorphisms in the R1 repeat region and the origin of DNA replication. In two cases, these were associated with the formation of distinct lineages and point to the possible co-evolution of these loci, despite the lack of any known functional link in VZV or related herpesviruses. We show that these and all other sequenced clade 5 viruses possess a distinct R1 repeat motif that increases the acidity of an ORF 11p protein domain and postulate that this has either arisen or been lost following divergence of the major clades. Thus, sequencing of whole VZV genomes collected during an outbreak has provided novel insights into VZV biology, transmission patterns and (recent) natural history.

    Importance VZV is a highly infectious virus and the causative agent of chickenpox and shingles, the latter being particularly associated with the risk of painful complications. Seasonal outbreaks of chickenpox are very common amongst young children, yet little is known about the dynamics of the virus during person-to-person to transmission or whether multiple distinct viruses seed and/or co-circulate during an outbreak. In this study we have sequenced chickenpox viruses from an outbreak in Guinea Bissau that are supported by detailed epidemiological data. Our data show that multiple different virus strains seeded and were maintained throughout the six month outbreak period, and that viruses transmitted between individuals accumulated new mutations in specific genomic regions. Of particular interest is the potential co-evolution of two distinct parts of the genomes and our calculations of the rate of viral mutation, both of which increase our understanding of how VZV evolves over short periods of time in human populations.

  • HIV-1 Nef and Vpu are Functionally Redundant Broad-Spectrum Modulators of Cell Surface Receptors Including Tetraspanins [PublishAheadOfPrint]

  • HIV-1 Nef and Vpu are thought to optimize virus replication in the infected host, at least in part via their ability to interfere with vesicular host cell trafficking. Despite the use of distinct molecular mechanisms, Nef and Vpu share the specificity for some molecules such as CD4 and MHC-I, while disruption of intracellular transport of the host cell restriction factor CD317/tetherin represents a specialized activity of Vpu not exerted by HIV-1 Nef. To establish a profile of host cell receptors whose intracellular transport is affected by Nef, Vpu or both, we comprehensively analyzed the effect of these accessory viral proteins on cell surface receptors levels on A3.01 T lymphocytes. 36 out of 105 detectable receptors were significantly downregulated by HIV-1 Nef, revealing a previously unappreciated scope with which HIV-1 Nef remodels the cell surface of infected cells. Remarkably, the effects of HIV-1 Vpu on host cell receptor exposure largely matched those of HIV-1 Nef in breadth and specificity (32 of 105, all also targeted by Nef), even though the magnitude was generally less pronounced. Of particular note, cell surface exposure of all members of the tetraspanin (TSPAN) protein family analyzed was reduced by both Nef and Vpu, and the viral proteins triggered the enrichment of TSPANs in a perinuclear area of the cell. While Vpu displayed significant colocalization and physical association with TSPANs, interactions of Nef with TSPANs were less robust. TSPANs thus emerge as a major target of deregulation in host cell vesicular transport by HIV-1 Nef and Vpu. The conservation of this activity in two independent accessory proteins suggests its importance for spread of HIV-1 in the infected host.

    Importance In this manuscript we define that HIV-1 Nef and Vpu display a surprising functional overlap and affect the cell surface exposure of a previously unexpected breadth of cellular receptors. Our analyses furthermore identify the tetraspanin protein family as a previously unrecognized target of Nef and Vpu activity. These findings have implications for the interpretation of effects detected for these accessory gene products on individual host cell receptors and illustrate the co-evolution of Nef and Vpu function.

  • Role of Mason-Pfizer monkey virus CA-NC spacer peptide-like domain in assembly of immature particles [PublishAheadOfPrint]

  • The hexameric lattice of an immature retroviral particle consists of Gag polyprotein, which is the precursor of all viral structural proteins. Lentiviral and alpharetroviral Gag contains a peptide sequence called the spacer peptide (SP), which is localized between the capsid (CA) and nucleocapsid (NC) domains. SP plays a critical role in intermolecular interactions during the assembly of immature particles of several retroviruses. Published models of supramolecular structures of immature particles suggest that in lentiviruses and alpharetroviruses, SP adopts a rod-like six-helix bundle organization. In contrast, Mason-Pfizer monkey virus (M-PMV), a betaretrovirus that assembles in the cytoplasm, does not contain a distinct SP sequence, and the CA-NC connecting region is not organized into a clear rod-like structure. Nevertheless, the CA-NC junction comprises a sequence critical for assembly of immature M-PMV particles. In the present work, we characterize this region, called the SP-like domain, in detail. We provide biochemical data confirming the critical role of M-PMV SP-like domain in immature particle assembly, release, processing and infectivity. Circular dichroism spectroscopy revealed that, in contrast to the SP regions of other retroviruses, a short SP-like domain-derived peptide (SPLP) does not form a purely helical structure in aqueous or helix-promoting solution. Using the 8 AAring; cryo-electron microscopy density maps of immature M-PMV particles, we prepared computational models of the SP-like domain and indicate the structural features required for M-PMV immature particle assembly.

    IMPORTANCE Retroviruses such as HIV-1 are of great medical importance. Using Mason-Pfizer monkey virus (M-PMV) as a model retrovirus, we provide biochemical and structural data confirming the general relevance of a short segment of the structural polyprotein Gag for retrovirus assembly and infectivity. Although this segment is critical for assembly of immature particles of lentiviruses, alpharetroviruses and betaretroviruses, the organization of this domain is strikingly different. A previously published electron microscopic structure of an immature M-PMV particle allowed us to model this important region into the electron density map. The data presented here help explain the different packing of the Gag segment of various retroviruses, such as HIV, RSV and M-PMV. Such knowledge contributes to understanding the importance of this region and its structural flexibility among retroviral species. The region might play a key role in Gag-Gag interactions, leading to different morphological pathways of immature particle assembly.

  • Anterograde Glycoprotein Dependent Transport of Newly Generated Rabies Virus in Dorsal Root Ganglion Neurons [PublishAheadOfPrint]

  • Rabies virus (RABV) spread is widely accepted to occur only by retrograde axonal transport. However, examples of anterograde RABV spread in peripheral neurons such as dorsal root ganglion (DRG) neurons indicated a possible bidirectional transport by an uncharacterized mechanism. Here, we analyzed the axonal transport of fluorescence labeled RABV in DRG neurons by live-cell microscopy. Both, entry-related retrograde transport of RABV after infection at axon endings and post-replicative transport of newly formed virus were visualized in compartmentalized DRG neuron cultures. Whereas entry-related transport at 1.5 mmu;m/sec occurred only retrogradually, after two days of infection multiple particles were observed in axons moving in both the anterograde and retrograde directions. The dynamics of post-replicative retrograde transport (1.6 mmu;m/sec) were similar to entry-related retrograde transport. In contrast, anterograde particle transport at 3.4 mmu;m/sec was faster, indicating active particle transport. Interestingly, RABV missing the glycoproteins did not move anterogradually within the axon. Thus, anterograde RABV particle transport depended on the RABV glycoprotein. Moreover, co-localization of GFP-labeled ribonucleoproteins (RNP) and glycoprotein in distal axonal regions as well as co-transport of labeled RNPs with membrane anchored mCherry reporter confirmed that either complete enveloped virus particles, or vesicle associated RNPs were transported. Our data show that anterograde RABV movement in peripheral DRG neurons occurs by active motor protein dependent transport. We propose two models for post-replicative long distance transport in peripheral neurons: either transport of complete virus particles or co-transport of RNPs and G-containing vesicles through axons to release virus at distal sites of infected DRG neurons.

    Importance Rabies virus retrograde axonal transport by dynein motors supports virus spread over long distances and lethal infection of the central nervous system. Though active rabies virus has been widely accepted to be unidirectional, evidence for anterograde spread in peripheral neurons supports the hypothesis that in some neurons RABV also enters the anterograde pathway by so far unknown mechanisms.

    By live microscopy we visualized fast anterograde axonal transport of rabies virus. The velocities exceeded those of retrograde movements, suggesting that active, most likely kinesin-dependent transport machineries are involved. Dependency of anterograde transport on the expression of virus glycoprotein G and co-transport with vesicles further suggest, that complete enveloped virus particles or co-transport of virus ribonucleoprotein and G-containing vesicles occurred. These data provide first insight in the mechanism of anterograde rabies virus transport and substantially contributes to the understanding of RABV replication and spread of newly formed virus in peripheral neurons.

  • HIV-1 Envelope Glycan Moieties Modulate HIV-1 Transmission [PublishAheadOfPrint]

  • HIV-1 envelope protein (Env) is heavily glycosylated with approximately 50% of the Env molecular mass contributed by N-glycans. HIV-1 Env N-glycans shield the protein backbone and have been shown to play key roles in determining Env structure, surface exposure, and, consequently, antigenicity, infectivity, antibody neutralization, and carbohydrate and receptor binding. Studies of HIV-1 glycosylation have focused mainly on the position of glycosylation, rather than the types of glycans. Also, the role of Env glycan moieties on HIV-1 transmission has not been systematically defined. Using viruses with modified Env glycan content and heterogeneity, we examined the effects of Env glycan moieties on the major events of HIV-1 transmission. Compared to viruses with less oligomannose and more complex Env glycans, viruses with more oligomannose and less complex glycans more efficiently (a) transcytose across an epithelial cell monolayer, (b) attach to monocyte-derived macrophages (MDMs), (c) bind monocyte-derived dendritic cells (MoDCs), and (d) trans-infect primary lymphocytes via MoDCs. However, viruses with more oligomannose and less complex glycans displayed impaired infectivity in TZMbl cells, MDMs, primary lymphocytes and fresh human intestinal tissue. Thus, N-linked Env glycans display discordant effects on the major events of HIV-1 transmission, with mature oligosaccharide structures on Env playing crucial role in HIV-1 infection. Env glycosylation should be taken into consideration in the development of vaccine strategies to interdict HIV-1 transmission.

    IMPORTANCE HIV-1 Env N-glycans shield the protein backbone and play key roles in determining Env structure and surface exposure, thereby impacting Env vantigenicity, infectivity, antibody neutralization, and carbohydrate and receptor binding. Studies of HIV-1 glycosylation have focused mainly on the position of glycosylation, rather than the types of glycans. In this report, we investigated systematically the role of Env glycan moieties on HIV-1 transmission. We show that N-linked Env glycans display discordant effects on the major events of HIV-1 transmission. These data indicate that Env glycan moieties impact HIV-1 transmission and that modulation of Env glycan moieties offers a potential strategy for the development of therapeutic or prophylactic vaccines against HIV-1.

  • Vaccine-Induced CD107a+ CD4+ T Cells Are Resistant to Depletion Following AIDS Virus Infection [PublishAheadOfPrint]

  • CD4+ T-cell responses are crucial for effective antibody and CD8+ T-cell induction following virus infection. However, virus-specific CD4+ T cells can be preferential targets for human immunodeficiency virus (HIV) infection. HIV-specific CD4+ T-cell induction by vaccination may thus result in enhancement of virus replication following infection. In the present study, we show that vaccine-elicited CD4+ T cells expressing CD107a are relatively resistant to depletion in a macaque AIDS model. Comparison of virus-specific CD107a, macrophage inflammatory protein-1bbeta;, interferon-, tumor necrosis factor-aalpha;, and interleukin-2 responses in CD4+ T cells of vaccinated macaques pre- and 1-week post-challenge showed a significant reduction in the CD107anndash; but not the CD107a+ subset after virus exposure. Those vaccinees that failed to control viremia showed a more marked reduction and exhibited significantly higher viral loads at week 1 than unvaccinated animals. Our results indicate that vaccine-induced CD107anndash; CD4+ T cells are depleted following virus infection, suggesting a rationale for avoiding virus-specific CD107anndash; CD4+ T-cell induction in HIV vaccine design.

    Importance Induction of effective antibody and/or CD8+ T-cell responses is a principal vaccine strategy against human immunodeficiency virus (HIV) infection. CD4+ T-cell responses are crucial for effective antibody and CD8+ T-cell induction. However, virus-specific CD4+ T cells can be preferential targets for HIV infection. Here, we show that vaccine-induced virus-specific CD107anndash; CD4+ T cells are largely depleted following infection in a macaque AIDS model. While CD4+ T-cell responses are important in viral control, our results indicate that virus-specific CD107anndash; CD4+ T-cell induction by vaccination may not lead to efficient CD4+ T-cell responses following infection but rather be detrimental and accelerate viral replication in the acute phase. This suggests that HIV vaccine design should avoid virus-specific CD107anndash; CD4+ T-cell induction. Conversely, this study found that vaccine-induced CD107a+ CD4+ T cells are relatively resistant to depletion following virus challenge, implying that induction of these cells may be an alternative approach toward HIV control.

  • Exposure of epitope residues on the outer face of the chikungunya virus envelope trimer determines antibody neutralizing efficacy [PublishAheadOfPrint]

  • Chikungunya virus (CHIKV) is a re-emerging alphavirus that causes a debilitating arthritic disease, infects millions of people, and has no specific treatment. Like many alphaviruses, the structural targets on CHIKV that elicit a protective humoral immune response in humans are poorly defined. Here we used phage display against virus-like particles (VLPs) to isolate seven human monoclonal antibodies (MAbs) against the CHIKV envelope glycoproteins E2 and E1. One MAb, IM-CKV063, was highly neutralizing (IC50 7.4 ng/ml), demonstrated high-affinity binding (320 pM), and was capable of therapeutic and prophylactic protection in multiple animal models, up to 24 h post-exposure. Epitope mapping using a comprehensive shotgun mutagenesis library of 910 E2/E1 alanine mutations demonstrated that IM-CKV063 binds to an inter-subunit conformational epitope on domain A, a functionally important region of E2. MAbs against the highly conserved fusion loop have not previously been reported, but were also isolated in our studies. Fusion loop MAbs were broadly cross-reactive against diverse alphaviruses, but were non-neutralizing. Fusion loop MAb reactivity was affected by temperature and reactivity conditions, suggesting that the fusion loop is hidden in infectious virions. Visualizing the binding sites of 15 different MAbs on the structure of E2/E1 reveals that all epitopes are located at the membrane distal region of the E2/E1 spike. Interestingly, epitopes on the exposed top-most and outer surfaces of the E2/E1 trimer structure are neutralizing whereas epitopes facing the interior of the trimer are not, providing a rationale for vaccine design and therapeutic MAb development using the intact CHIKV E2/E1 trimer.

    IMPORTANCE CHIKV is the most important alphavirus affecting humans, resulting in a chronic arthritic condition that can persist for months or years. In recent years, millions of people have been infected globally and spread to the Americas is now beginning, with over 100,000 cases in the Caribbean within six months of arrival. Our study reports seven human MAbs against CHIKV envelope, including a highly protective MAb and rarely isolated fusion loop MAbs. Epitope mapping of these MAbs demonstrates how some E2/E1 epitopes are exposed or hidden from the human immune system and suggests a structural mechanism by which these MAbs protect (or fail to protect) against CHIKV infection. Our results suggest that the membrane distal end of CHIKV E2/E1 is the primary target for the humoral immune response to CHIKV and antibodies targeting the exposed top-most and outer surfaces of the E2/E1 trimer determine the neutralizing efficacy of this response.

  • A recombinant HCV envelope glycoprotein vaccine elicits antibodies targeting multiple epitopes on the envelope glycoproteins associated with broad cross-neutralization [PublishAheadOfPrint]

  • Although effective Hepatitis C Virus (HCV) antivirals are on the horizon, a global prophylactic vaccine for HCV remains elusive. The diversity of the virus is a major concern for vaccine development; there are 7 major genotypes of HCV found globally. Therefore, a successful vaccine will need to protect against HCV infection of all genotypes. Despite the diversity, many monoclonal antibodies (mAbs) with broadly cross-neutralizing activity have been described suggesting the presence of conserved epitopes that can be targeted to prevent infection. Similarly, a vaccine comprising recombinant envelope glycoproteins (rE1E2) derived from the genotype 1a HCV-1 strain has been shown to be capable of eliciting cross-neutralizing antibodies in guinea pigs, chimpanzees, and healthy human volunteers. In order to investigate the basis for this cross-neutralization, epitope mapping of anti-E1E2 antibodies present within antisera from goats and humans immunized with HCV-1 rE1E2 was conducted through peptide mapping and competition studies with a panel of cross-neutralizing mAbs targeting various epitopes within E1E2. The immunized goat antisera was shown to compete with the binding of all mAbs tested (AP33, HC33.4, HC84.26, 1:7, AR3B, AR4A, AR5A, IGH526, A4). Antisera showed the best competition against HC84.26/AR3B and the weakest competition against AR4A. Furthermore, antisera from five immunized human vaccinees were shown to compete with five pre-selected mAbs (AP33, AR3B, AR4A, AR5A, IGH526). These data show that immunization with HCV-1 rE1E2 elicits antibodies targeting multiple cross-neutralizing epitopes. Our results further support the use of such a vaccine antigen to induce cross-genotype neutralization.

    Importance An effective prophylactic vaccine for HCV is needed for optimal control of the disease burden. The high diversity of HCV has posed a challenge for developing vaccines that elicit neutralizing antibodies for protection against infection. Despite this, we have previously shown that a vaccine comprising recombinant envelope glycoproteins derived from a single genotype 1a strain was capable of eliciting a cross-neutralizing antibody response in human volunteers. Here, we have used competition binding assays and peptide binding assays to show that antibodies present in the vaccinated antisera bind epitopes overlapping with those of a variety of well-characterized cross-neutralizing monoclonal antibodies. This provides a mechanism for the cross-neutralizing human antisera: antibodies present in the antisera bind to conserved regions associated with cross-neutralization. Importantly, this work provides further support for a vaccine comprising recombinant envelope glycoproteins mmdash; perhaps in a formulation with a vaccine component eliciting strong anti-HCV CD4+ and CD8+ T cell responses.

  • Cooperativity Among Rev-Associated Nuclear Export Signals Regulates HIV-1 Gene Expression and is a Determinant of Virus Species Tropism [PublishAheadOfPrint]

  • Murine cells exhibit a profound block to HIV-1 virion production that was recently mapped to a species-specific structural attribute of the murine version of the CRM1 (mCRM1) nuclear export receptor, and rescued by expression of human CRM1 (hCRM1). In human cells, the HIV-1 Rev protein recruits hCRM1 to intron-containing viral mRNAs encoding the Rev response element (RRE), thereby facilitating viral late gene expression. Here we exploited murine 3T3 fibroblasts as a gain-of-function system to study hCRM1's species-specific role in regulating Rev's effector functions. We show that Rev is rapidly exported from the nucleus by mCRM1 despite only weak contributions to HIV-1's post-transcriptional stages. Indeed, Rev preferentially accumulates in the cytoplasm of murine 3T3 cells with or without hCRM1 expression, in contrast to human HeLa cells where Rev exhibits striking en masse transitions between the nuclear and cytoplasmic compartments. Efforts to bias Rev's trafficking either into or out of the nucleus revealed that Rev encoding a second CRM1 binding domain (Rev-2xNES), or Rev-dependent viral gag-pol mRNAs bearing tandem RREs (GP-2xRRE), rescue virus particle production in murine cells even in the absence of hCRM1. Combined, these results suggest a model wherein Rev-associated nuclear export signals cooperate to regulate the number or quality of CRM1's interactions with viral Rev/RRE ribonucleoprotein complexes in the nucleus. This mechanism regulates CRM1-dependent viral gene expression and is a determinant of HIV-1's capacity to produce virions in non-human cell types.

    Importance Cells derived from mice and other non-human species exhibit profound blocks to HIV-1 replication. Here we elucidate a block to HIV-1 gene expression attributable to the murine version of the CRM1 (mCRM1) nuclear export receptor. In human cells, hCRM1 regulates the nuclear export of viral intron-containing mRNAs through the activity of the viral Rev adapter protein that forms a multimeric complex on these mRNAs prior to recruiting hCRM1. We demonstrate that Rev-dependent gene expression is poor in murine cells despite the finding that, surprisingly, the bulk of Rev interacts efficiently with mCRM1 and is rapidly exported from the nucleus. Instead, we map the mCRM1 defect to the apparent inability of this factor to engage Rev multimers in the context of large viral Rev/RNA ribonucleoprotein complexes. These findings shed new light on HIV-1 gene regulation and could inform the development of novel antiviral strategies that target viral gene expression.

  • Viral Reprogramming of the Daxx-Histone H3.3 Chaperone During EBV Early Infection [PublishAheadOfPrint]

  • Host chromatin assembly can function as a barrier to viral infection. Epstein-Barr virus (EBV) establishes latent infection as chromatin-assembled episomes in which all but a few viral genes are transcriptionally silent. The factors that control chromatin assembly and guide transcription regulation during the establishment of latency are not well understood. Here, we demonstrate that the EBV tegument protein BNRF1 binds the histone H3.3 chaperone Daxx to modulate histone mobility and chromatin assembly on the EBV genome during the early stages of primary infection. We demonstrate that BNRF1 substitutes for the repressive co-chaperone ATRX to form a ternary complex of BNRF1-Daxx-H3.3-H4, using co-immunoprecipitation and size exclusion chromatography with highly purified components. Fluorescence recovery after photobleaching (FRAP) assays were used to demonstrate that BNRF1 promotes global mobilization of cellular histone H3.3. Mutation of putative nucleotide binding motifs on BNRF1 attenuates the displacement of ATRX from Daxx. We also show by immunofluorescence (IF) combined with fluorescence in situ hybridization (FISH) that BNRF1 is important for the dissociation of ATRX and Daxx from nuclear bodies during de novo infection of primary B-lymphocytes. Virion-delivered BNRF1 suppresses Daxx-ATRX-mediated H3.3 loading on viral chromatin as measured by chromatin immunoprecipitation (ChIP) assays and enhances viral gene expression during early infection. We propose that EBV tegument protein BNRF1 replaces ATRX to reprogram Daxx-mediated H3.3 loading, in turn generating chromatin suitable for latent gene expression.

    Importance Epstein-Barr Virus (EBV) is a human herpesvirus that efficiently establishes latent infection in primary B-lymphocytes. Cellular chromatin assembly plays an important role in regulating the establishment of EBV latency. We show that the EBV tegument protein BNRF1 functions to regulate chromatin assembly on the viral genome during early infection. BNRF1 alters the host cellular chromatin assembly to prevent anti-viral repressive chromatin, and establish chromatin structure permissive for viral gene expression and the establishment of latent infection.

  • The 2'-5' oligoadenylate synthetase 3 (OAS3) enzyme potently synthesizes the 2'-5' oligoadenylates required for RNase L activation [PublishAheadOfPrint]

  • The oligoadenylate synthetase (OAS) family of proteins are antiviral restriction factors that target a wide range of RNA and DNA viruses. They function as intracellular double stranded RNA (dsRNA) sensors that upon binding to dsRNA undergo a conformational change and are activated to synthesize 2'-5' linked oligoadenylates (2-5A). 2-5As of sufficient length act as second messengers to activate RNase L and thereby restrict viral replication. We expressed human OAS3 using the baculovirus system and purified it to homogeneity. We show that recombinant OAS3 is activated at a substantially lower concentration of dsRNA compared to OAS1, making it a potent in vivo sensor of dsRNA. Moreover, we find that OAS3 synthesizes considerably longer 2-5As than previously reported and that OAS3 can activate RNase L intracellularly. The combined high affinity for dsRNA and the capability to produce 2-5As of sufficient length to activate RNase L suggests that OAS3 is a potent activator of RNase L. In addition, we provide experimental evidence to support one active site of OAS3 located in the C-terminal OAS domain and generate a low resolution structure of OAS3 using SAXS.

    IMPORTANCE We are the first to purify the OAS3 enzyme to homogeneity, which allowed us to characterize the mechanism utilized by OAS3 and identify the active site. We provide compelling evidence that OAS3 can produce 2'-5' oligoadenylates of sufficient length to activate RNase L. This is contrary to what is described in the current literature but agrees with recent in vivo data showing that OAS3 harbors an antiviral activity requiring RNase L. Thus our work redefines our understanding of the biological role of OAS3. Furthermore, we used a combination of mutagenesis and small angle X-ray scattering to describe the active site and low resolution structure of OAS3.

  • A Combination of HA and PA Mutations Enhances Virulence in a Mouse-adapted H6N6 Influenza A Virus [PublishAheadOfPrint]

  • H6N6 viruses are commonly isolated from domestic ducks and avian-to-swine transmissions of H6N6 viruses have been detected in China. Whether subsequent adaptation of H6N6 viruses in mammals would increase their pathogenicity towards humans is not known. To address this, we generated a mouse-adapted swine influenza H6N6 virus (GDK6-MA) which exhibited greater virulence than the wild-type virus (GDK6). Amino acid substitutions in PB2 (E627K), PA (I38M) and HA (L111F, H156N and S263R) occurred in GDK6-MA. HA H156N resulted in enlarged plaque sizes on MDCK cells and enhanced early stage viral replication in mammalian cells. PA I38M raised polymerase activity in vitro but did not change virus replication in either mammalian cells or mice. These single substitutions had only limited effects on virulence, however, a combination of HA H156N, S263R and PA I38M in the GDK6 backbone led to a significantly more virulent variant. This suggests these substitutions can compensate for the lack of PB2-627K and modulate virulence, revealing a new determinant of pathogenicity for H6N6 viruses in mice, which might also pose a threat to human health.

    Importance Avian H6N6 influenza viruses are enzootic in domestic ducks and have been detected in swine in China. Infections of mammals by H6N6 viruses raise the possibility of viral adaptation and increasing pathogenicity in the new hosts. To examine the molecular mechanisms of adaptation, a mouse-adapted avian-origin swine influenza H6N6 virus (GDK6-MA), which had higher virulence than its parental virus, was generated. Specific mutations were found in PB2 (E627K), PA (I38M) and HA (L111F, H156N, S263R) and were assessed for their virulence in mice. The combination of HA H156N, S263R and PA I38M compensated for the lack of PB2-627K and showed increased pathogenicity in mice, revealing a novel mechanism that can affect the virulence of influenza viruses. H6N6 viruses should be monitored for more virulent forms in the field that could threaten human health.

  • Kaposi's Sarcoma Herpesvirus downregulates Transforming Growth Factor-{beta}2 to promote the enhanced stability of capillary-like tube formation [PublishAheadOfPrint]

  • Kaposi's Sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's Sarcoma (KS), the most common tumor of AIDS patients world-wide. A key characteristic of KS tumors is extremely high levels of vascular slits and extravasated red blood cells making neo-angiogenesis a key component of the tumor. The main KS tumor cell is the spindle cell, a cell of endothelial origin that maintains KSHV predominantly in the latent state. In cultured endothelial cells, latent KSHV infection induces angiogenic phenotypes including longer term stabilization of capillary-like tube formation in matrigel, a basement membrane matrix. The present studies show that KSHV infection of endothelial cells strongly downregulates TGF-bbeta;2. This downregulation allows the stabilization of capillary-like tube formation during latent infection, as the addition of exogenous TGF-bbeta;2 inhibits the KSHV-induced stability of these structures. While two KSHV microRNAs are sufficient to downregulate TGF-bbeta;2 in endothelial cells, they are not required during KSHV infection. However, activation of the gp130 cell surface receptor is both necessary and sufficient for downregulation of TGF-bbeta;2 in KSHV infected cells.

    Importance: Kaposi's Sarcoma is a highly vascularized, endothelial cell based tumor supporting large amounts of angiogenesis. There is evidence that KSHV, the etiologic agent of KS, induces aberrant angiogenesis. For example, KSHV induces stabilization of capillary-like tube formation in cultured endothelial cells. A clearer understanding of how KSHV regulates angiogenesis could provide potential therapeutic targets for KS. We found that KSHV downregulates TGF-bbeta;2, a cytokine related to TGF-bbeta;1, that is known to inhibit angiogenesis. The downregulation of this inhbitor promotes the stability of capillary-like tube formation insofar as adding back TGF-bbeta;2 to infected cells blocks KSHV induced long-term tubule stability. Therefore, KSHV downregulation of TGF-bbeta;2 may increase aberrent vascularization in KS tumors through increased capillary formation and thereby aid in KS tumor promotion

  • Expression of the Human Cytomegalovirus UL11 Glycoprotein in Viral Infection and Evaluation of its Effect on Virus-Specific CD8 T Cells [PublishAheadOfPrint]

  • The human cytomegalovirus (CMV) UL11 open reading frame (ORF) encodes a putative type I transmembrane glycoprotein, which displays remarkable amino acid sequence variability among different CMV isolates, suggesting that it represents an important virulence factor. In a previous study we have shown that UL11 can interact with the cellular receptor tyrosine phosphatase CD45, which has a central role for signal transduction in T cells, and treatment of T cells with high amounts of a soluble UL11 protein inhibited their proliferation. In order to analyze UL11 expression in CMV-infected cells, we constructed CMV recombinants that either encode tagged UL11 versions or carry a stop mutation in the UL11 ORF. Moreover, we examined whether UL11 affects the function of virus-specific cytotoxic T lymphocytes (CTL). We found that the UL11 ORF gives rise to several proteins due to both posttranslational modification and alternative translation initiation sites. Biotin labelling of surface proteins on infected cells indicated that only highly glycosylated UL11 forms are present at the plasma membrane, whereas low glycosylated UL11 forms were found in the endoplasmic reticulum. We did not find evidence of UL11 cleavage and secretion of a soluble UL11 version. Co-cultivation of CTLs recognizing different CMV epitopes with fibroblasts infected with a UL11 deletion mutant or the parental strain revealed that under the conditions applied UL11 did not influence the activation of CMV-specific CD8 T cells. For further studies we propose to investigate the interaction of UL11 with CD45 and the functional consequences in other immune cells expressing CD45.

    IMPORTANCE Human cytomegalovirus belongs to those viruses that extensively interfere with the host immune response. Yet, the precise function of many putative immunomodulatory CMV proteins remains elusive. Previously, we have shown that the CMV UL11 protein interacts with the leukocyte common antigen CD45, a cellular receptor tyrosine phosphatase with a central role for signal transduction in T cells. Here, we examined the proteins expressed by the UL11 gene in CMV infected cells and found that at least one form of UL11 is present at the cell surface, enabling it to interact with CD45 on immune cells. Surprisingly, CMV-expressed UL11 did not affect the activity of virus-specific CD8 T cells. This finding warrants to investigate the impact of UL11 on CD45 functions in other leukocyte subpopulations.

  • Nipah Virion Entry Kinetics, Composition, and Conformational Changes Determined by Enzymatic VLPs and New Flow Virometry Tools [PublishAheadOfPrint]

  • Viral-cell membrane fusion is essential for enveloped virus infections. However, mechanistic viral membrane fusion studies have predominantly focused on cell-cell fusion models, largely due to the low availability of technologies capable of characterizing actual viral-cell membrane fusion. Although cell-cell fusion assays are valuable, they do not fully recapitulate all the variables of viral-cell membrane fusion. Drastic differences between viral vs. cellular membrane lipid and protein compositions and curvatures exist. For BSL4 pathogens such as the deadly Nipah virus (NiV), viral-cell fusion mechanistic studies are notably cumbersome. To circumvent these limitations we used enzymatic Nipah virus-like-particles (NiVLPs) and developed new flow virometric tools. NiV's attachment (G) and fusion (F) envelope glycoproteins mediate viral binding to the ephrinB2/ephrinB3 cell receptors and viral-cell membrane fusion, respectively. The NiV matrix protein (M) can autonomously induce NiV assembly and budding. Using a bbeta;-Lactamase (bbeta;La) reporter/NiV-M chimeric protein, we produced NiVLPs expressing NiV-G and wild-type or mutant NiV-F on their surfaces. By pre-loading target cells with the bbeta;La fluorescent substrate CCF2-AM, we obtained viral entry kinetic curves that correlated with the NiV-F fusogenic phenotypes, validating NiVLPs as suitable viral entry kinetic tools, and suggesting overall relatively slower viral entry than cell-cell fusion kinetics. Additionally, the proportions of F and G on individual NiVLPs and the extent of receptor-induced conformational changes in NiV-G were measured via flow virometry, allowing the proper interpretation of the viral entry kinetic phenotypes. The significance of these findings in the viral entry field extends beyond NiV to other paramyxoviruses and enveloped viruses.

    IMPORTANCE Viral-cell membrane fusion is essential for enveloped virus infections. However, mechanistic viral membrane fusion studies have predominantly focused on cell-cell fusion models, largely due to the low availability of technologies capable of characterizing actual viral-cell membrane fusion. Although cell-cell fusion assays are valuable, they do not fully recapitulate all the variables of viral-cell membrane fusion. For example, drastic differences between viral vs. cellular membrane lipid and protein compositions and curvatures exist. For biosafety level 4 (BSL4) pathogens such as the deadly Nipah virus (NiV), viral-cell fusion mechanistic studies are especially cumbersome. To circumvent these limitations we used enzymatic Nipah virus-like-particles (NiVLPs) and developed new flow virometric tools. Our new tools allowed us the high-throughput measurement of viral entry kinetics, glycoprotein proportions on individual viral particles, and receptor-induced conformational changes in viral glycoproteins on viral surfaces. The significance of these findings extends beyond NiV to other paramyxoviruses and enveloped viruses.

  • Hyperglycosylated stable core immunogens designed to present the CD4 binding site preferentially recognized by broadly neutralizing antibodies [PublishAheadOfPrint]

  • The HIV-1 surface envelope glycoprotein (Env) trimer mediates entry into CD4+/CCR5+ host cells. Env possesses conserved antigenic determinants, such as the gp120 primary receptor CD4 binding site (CD4bs), a known neutralization target. Env also contains variable regions and protein surfaces occluded within the trimer that elicit non-neutralizing antibodies. Here, we engineered additional N-linked glycans onto a cysteine-stabilized gp120 core (0G) deleted of its major variable regions to preferentially expose the conformationally fixed CD4bs. Three, 6, 7 and 10 new NXT/S glycan (G) motifs were engineered into 0G to encode 3G, 6G, 7G and 10G cores. Following purification, most glycoproteins were recognized by broadly neutralizing CD4bs-directed antibodies except for 10G. Gel and glycan mass-spectrometry confirmed that additional N-glycans were post-translationally added to the redesigned cores. Binding kinetics revealed high-affinity recognition by seven broadly neutralizing CD4bs-directed antibodies and low-to-no binding by non-broadly-neutralizing CD4bs-directed antibodies. Rabbits inoculated with the hyperglycosylated cores elicited IgM and IgG responses to each given protein that were similar to those elicited by parental 0G. Site-specific glycan masking effects were detected in the elicited sera and the anti-sera competed with b12 for CD4bs-directed binding specificity. However, the core-elicited sera showed very limited neutralization activity. Trimer priming or boosting of the core immunogens elicited tier 1-level neutralization that mapped to both the CD4bs and V3 and appeared solely trimer-dependent. Fine-mapping at the CD4bs indicated that conformational stabilization of the cores and addition of N-glycans altered the molecular surface, suggesting why the elicited neutralization was not improved by this rational-design strategy.

    Importance Major obstacles to develop an effective HIV-1 vaccine are the variability of its surface proteins and its high-density shield generated by incorporation of host (human) glycans (sugars). HIV-1 does harbor highly conserved sites on the exposed envelope protein surface, gp120, one of which is the virus receptor (CD4) binding site. Several broadly neutralizing antibodies elicited from HIV patients do target this gp120 CD4 binding site (CD4bs); however, gp120 immunogens do not elicit broadly neutralizing antibodies. In this study, we targeted the CD4bs by conformational stabilization and additional glycan-masking. We used high-resolution structure to re-engineer gp120 cores to preferentially present the cysteine-stabilized CD4bs and to glycan-mask non-neutralizing determinants. Importantly, glycan masking did successfully focus antibody responses to the CD4bs; however, the elicited CD4bs-directed antibodies did not neutralize HIV or bind to unmodified gp120, presumably due to the structure-guided modifications of this conserved region.

  • Overlapping and distinct molecular determinants dictating the antiviral activities of TRIM56 against flaviviruses and coronavirus [PublishAheadOfPrint]

  • The tripartite motif-containing (TRIM) proteins have emerged as a new class of host antiviral restriction factors, with several demonstrating roles in regulating innate antiviral responses. Of ggt;70 known TRIMs, TRIM56 inhibits replication of bovine viral diarrhea virus, a ruminant pestivirus of the family Flaviviridae, but has no appreciable effect on VSV, a rhabdovirus. Yet, the antiviral spectrum of TRIM56 remains undefined. In particular, how TRIM56 impacts human-pathogenic viruses is unknown. Also unclear are the molecular determinants governing the antiviral activities of TRIM56. Herein, we show that TRIM56 poses a barrier to infections by yellow fever virus (YFV), dengue virus serotype-2 (DENV2), and human coronavirus virus (HCoV)-OC43 but not encephalomyocarditis virus (EMCV). Moreover, by engineering cell lines conditionally expressing various TRIM56 mutants, we demonstrate that TRIM56's anti-flavivirus effects required both the E3 ligase activity that lies in the N-terminal RING domain and the integrity of its C-terminal portion, while the restriction of HCoV-OC43 relied upon the TRIM56 E3 ligase activity alone. Furthermore, TRIM56 was revealed to impair YFV and DENV2 propagation by suppressing intracellular viral RNA accumulation but to compromise HCoV-OC43 infection at a later step in the viral life cycle, suggesting that distinct TRIM56 domains accommodate differing antiviral mechanisms. Altogether, TRIM56 is a versatile antiviral host factor that confers resistance to YFV, DENV2 and HCoV-OC43 through overlapping and distinct molecular determinants.

    Importance We previously reported tripartite-motif protein 56 (TRIM56) as a host restriction factor of bovine viral diarrhea virus, a ruminant pathogen. However, the impact of TRIM56 on human-pathogenic RNA viruses is unknown. Herein, we demonstrate that TRIM56 restricts two medically-important flaviviruses, yellow fever virus (YFV) and dengue virus serotype-2 (DENV2), and a human coronavirus, HCoV-OC43, but not encephalomyocarditis virus, a picornavirus. Further, we show that TRIM56-mediated inhibition of HCoV-OC43 multiplication solely depends on its E3 ligase activity whereas its restriction of YFV and DENV2 requires both the E3 ligase activity and integrity of the C-terminal portion. The differing molecular determinants appear to accommodate distinct antiviral mechanisms TRIM56 adopts to target different families of virusesmmdash; while TRIM56 curbs intracellular YFV/DENV2 RNA replication, it acts at a later step in HCoV-OC43 life cycle. These novel findings illuminate the molecular basis of the versatility and specificity of TRIM56's antiviral activities against positive-strand RNA viruses.

  • DnaJA1/Hsp40 is co-opted by influenza A virus to enhance its viral RNA polymerase activity [PublishAheadOfPrint]

  • The RNA-dependent RNA polymerase (RdRp) of influenza A virus is a heterotrimeric complex composed by PB1, PB2 and PA subunits. The interplay between host factors and the three subunits of the RdRp is critical to enable viral RNA synthesis to occur in the nucleus of infected cells. In this report, we newly identified a host factor DnaJA1, a member of the type I DnaJ/Hsp40 family, acting as a positive regulator for influenza virus replication. We found that DnaJA1 associates with PB2 and PA subunits and enhances viral RNA synthesis both in vivo and in vitro. Moreover, DnaJA1 could be translocated from cytoplasm into the nucleus upon influenza virus infection. The translocation of DnaJA1 is specifically accompanied by the PB1-PA nuclear import. Interestingly, we observed that the effect of DnaJA1 on viral RNA synthesis is mainly dependent on its C-terminal substrate-binding domain but not on its typical J-domain, while the J-domain normally mediates the Hsp70-DnaJ interaction required for regulating Hsp70 ATPase activity. Therefore, we propose that DnaJA1 is co-opted by the influenza A virus to entre nucleus and to enhance its RNA polymerase activity in an Hsp70 cochaperone-independent manner.

    Importance The interplay between host factors and influenza virus RNA polymerase plays a critical role in determining virus pathogenicity and host adaption. In this report, we newly identified a host protein DnaJA1/Hsp40 that is co-opted by influenza A virus RNA polymerase to enhance its viral RNA synthesis in the nucleus of infected cells. We found that DnaJA1 associates with both PB2 and PA subunits and translocates into nucleus along with the nuclear import of PB1-PA dimer during influenza virus replication. Interestingly, the effect of DnaJA1 is mainly dependent on its C-terminal substrate-binding domain but not on its typical J-domain that is required for its Hsp70 cochaperone function. To our knowledge, this is the first report on a member of Hsp40s that is specifically involved in regulating influenza virus RNA polymerase. Targeting the interactions between polymerase subunits and DnaJA1 may provide a novel strategy to develop antiviral drugs.

  • Latency-associated viral IL-10 encoded by human cytomegalovirus modulates cellular IL-10 and CCL8 secretion during latent infection through changes in the cellular miRNA, hsa-miR-92a. [PublishAheadOfPrint]

  • The UL111A gene of human cytomegalovirus encodes a viral homologue of the cellular immunomodulatory cytokine interleukin IL-10 (cIL-10) which, due to alternative splicing, results in expression of two isoforms designated LAcmvIL-10 (expressed during both lytic and latent infection) or cmvIL-10 (identified only during lytic infection).

    We have analysed the functions of LAcmvIL-10 during latent infection of primary myeloid progenitor cells and find that LAcmvIL-10 is responsible, at least in part, for the known increase in secretion of cellular IL-10 and CCL8 in the secretome of latently infected cells. This latency-associated increase in CCL8 expression results from a concomitant LAcmvIL-10-mediated suppression of the expression of cellular miRNA hsa-miR-92a which targets CCL8 directly.

    Taken together, we show that the previously observed downregulation of hsa-miR-92a and upregulation of CCL8 during HCMV latent infection of myeloid cells are intimately linked via the latency-associated expression of LAcmvIL-10.

    Importance HCMV latency causes significant morbidity and mortality in immune compromised individuals yet is carried silently llsquo;latentlyrrsquo; in 50-90% of the population. Understanding of how HCMV maintains infection for the lifetime of an infected individual is critical for the treatment of the immune compromised suffering with disease as a result of HCMV. In this study we analyse one of the proteins which are expressed during the llsquo;latentrrsquo; phase of HCMV, LAcmvIL-10 and find that the expression of this gene modulates the microenvironment of the infected cell leading to evasion of the immune system.

  • Type I interferon signaling enhances CD8+ T cell effector function and differentiation during MHV68 infection [PublishAheadOfPrint]

  • CD8+ T cell responses are critical to the control of replication and reactivation associated with gammaherpesvirus infection. Type I interferons have been shown to have direct and indirect roles in supporting CD8+ T cell development and function during viral infection; however, the role of type I interferons during latent viral infection has not been examined. Mice deficient in type I IFN signaling (IFNAR1nndash;/nndash; mice) have high levels of reactivation during infection with MHV68, a murine gammaherpesvirus model for Epstein-Barr virus. We hypothesized that type I IFNs function to enhance the anti-gammaherpesvirus CD8+ T cell response. To test this, IFNAR1nndash;/nndash; mice were infected with MHV68 and the CD8+ T cell response was analyzed. In the absence of type I IFN signaling there was a marked increase in short-lived effector CD8+ T cells, and MHV68-specific CD8+ T cells had upregulated expression of PD-1 and reduced TNF-aalpha;, IFN-, and IL-2 production. Suppressing MHV68 replication early in infection using the antiviral cidofovir rescued CD8+ T cell cytokine production and reduced PD-1 expression. However, suppressing high levels of reactivation in IFNAR1nndash;/nndash; mice failed to improve CD8+ T cell cytokine production during latency. T cell-specific abrogation of type I IFN signaling showed that the effects of type I IFNs on the CD8+ T cell response during MHV68 infection are independent of direct type I IFN signaling on T cells. Our findings support a model in which type I IFNs likely suppress MHV68 replication, thus limiting viral antigen and facilitating an effective gammaherpesvirus-directed CD8+ T cell response.

    Importance The murine gammaherpesvirus, MHV68, has both genetic and biologic homology to the human gammaherpesvirus Epstein-Barr virus (EBV) which infects over 90% of humans. Latent EBV infection and reactivation are associated with various life-threatening diseases and malignancies. Host suppression of gammaherpesvirus latency and reactivation requires both CD8+ T cells as well as type I interferon signaling. Type I IFNs have been shown to critically support the antiviral CD8+ T cell response in other viral models. Here, we identify an indirect role for type I IFN signaling in enhancing gammaherpesvirus-specific CD8+ T cell cytokine production. Further, this function of type I IFN signaling can be partially rescued by suppressing viral replication during early MHV68 infection. Our data suggest that type I IFN signaling on non-T cells can enhance CD8+ T cell function during gammaherpesvirus infection, potentially through suppressing MHV68 replication.

  • B cell response to herpesvirus infection of the olfactory neuroepithelium [PublishAheadOfPrint]

  • Viruses commonly infect the respiratory tract. Analyses of host defence have focussed on the lungs and the respiratory epithelium. Spontaneously inhaled Murid Herpesvirus-4 (MuHV-4) and Herpes simplex virus type 1 (HSV-1) infect instead the olfactory epithelium, where neuronal cilia are exposed to environmental antigens and provide a route across the epithelial mucus. We used MuHV-4 to define how B cells respond to virus replication in this less well characterized site. Olfactory infection elicited generally weaker acute responses than lung infection, particularly in the spleen, reflecting slower viral replication and spread. Few virus-specific antibody-forming cells (AFCs) were found in the nasal-associated lymphoid tissue (NALT), a prominent response site for respiratory epithelial infection. Instead they appeared first in the superficial cervical lymph nodes. The focus of the AFC response then moved to the spleen, matching the geography of virus dissemination. Little virus-specific IgA response was detected until late on in the bone marrow. Neuroepithelial HSV-1 infection also elicited no significant AFC response in the NALT and a weak IgA response. Thus olfactory herpesvirus infection differed immunologically from an infection of the adjacent respiratory epithelium. Poor IgA induction may help herpesviruses to transmit via long-term mucosal shedding.

    Importance Herpesviruses are widespread, persistent pathogens against which vaccines have had limited success. We need to understand better how they interact with host immunity. MuHV-4 and HSV-1 inhaled by alert mice infect the olfactory neuroepithelium, suggesting that this is a natural entry route. Its immunology is almost completely unknown. The antibody response to neuroepithelial herpesvirus infection started in the cervical lymph nodes, and unlike respiratory influenza virus infection did not involve significantly the nasal-associated lymphoid tissue. MuHV-4 and HSV-1 infections also elicited little virus-specific IgA. Therefore vaccine-induced IgA might provide a defence that herpesviruses are ill-equipped to meet.

  • Hijacking Discs Large 1 for Oncogenic Phosphatidylinositol 3-Kinase Activation in Human Epithelial Cells Is a Conserved Mechanism of Human Adenovirus E4-ORF1 Proteins [PublishAheadOfPrint]

  • The E4-ORF1 gene of human adenoviruses encodes a 14-kDa protein that promotes viral replication as well as cellular metabolic reprogramming, survival, and transformation by constitutively activating cellular phosphatidylinositol 3-kinase (PI3K). We recently reported that the E4-ORF1 protein from subgroup D human adenovirus type 9 upregulates and oncogenically activates PI3K by a novel mechanism involving separate interactions of E4-ORF1 with cellular Discs large 1 (Dlg1) and PI3K to form a ternary complex that translocates to the plasma membrane (K. Kong, M. Kumar, M. Taruishi, and R. T. Javier, PLoS Pathog. 10:e1004102, 2014, doi:10.1371/journal.ppat.1004102). The current study was carried out to investigate whether other human adenovirus E4-ORF1 proteins share this mechanism of action. Results showed that in human MCF10A epithelial cells, stable expression of E4-ORF1 proteins encoded by representative human adenovirus serotypes from subgroups A to D induce ternary complex formation, Dlg1-dependent PI3K activation, PI3K protein elevation, Dlg1 and PI3K membrane recruitment, and PI3K-dependent cellular transformation. The first three of these E4-ORF1 activities were also observed in MCF10A cells infected with each wild-type human adenovirus from subgroups A to D. Our findings indicate that most, if not all, human adenovirus E4-ORF1 proteins share a conserved molecular mechanism of PI3K activation, which confers a common capacity to promote oncogenic transformation in human epithelial cells.

    IMPORTANCE PI3K activation by the adenovirus E4-ORF1 protein mediates oncogenic cellular transformation by human adenovirus type 9, augments viral protein expression and replication by human adenovirus type 5, and dysregulates cellular glucose and lipid metabolism by human adenovirus type 36. For the first time, we report that E4-ORF1 proteins from human adenoviruses in subgroups A to D evolved a conserved molecular mechanism to mediate constitutive PI3K activation that can provoke oncogenic transformation in human epithelial cells. The results raise potential safety concerns about the use of vectors encoding the E4-ORF1 gene in human gene therapy and vaccination. Our findings further suggest that the conserved mechanism revealed here may be targeted for development of therapeutic drugs to treat and prevent adenovirus-associated human diseases.

  • Drosophila C virus systemic infection leads to intestinal obstruction [PublishAheadOfPrint]

  • Drosophila C virus (DCV) is a positive-sense RNA virus belonging to the Dicistroviridae family. This natural pathogen of the model organism Drosophila melanogaster is commonly used to investigate antiviral host-defense in flies, which involves both RNA interference and inducible responses. Although lethality is routinely used as readout for the efficiency of the antiviral immune response in these studies, virus-induced pathologies in flies are still poorly understood. Here, we characterize the pathogenesis associated with systemic DCV infection. Comparison of the transcriptome of flies infected with DCV or two other positive-sense RNA viruses, Flock House virus and Sindbis virus, reveals that DCV infection, unlike those of the other two viruses, represses the expression of a large number of genes. Several of these genes are specifically expressed in the midgut, and are also repressed by starvation. We show that systemic DCV infection triggers a nutritional stress in Drosophila, which result from intestinal obstruction with accumulation of peritrophic matrix at the entry of the midgut, and accumulation of the food ingested in the crop, a blind muscular food storage organ. The related virus Cricket paralysis virus (CrPV), which efficiently grows in Drosophila, does not trigger this pathology. We show that DCV, but not CrPV, infects the smooth muscles surrounding the crop, causing extensive cytopathology and strongly reducing the rate of contractions. We conclude that the pathogenesis associated with systemic DCV infection results from the tropism of the virus for an important organ within the foregut of Dipteran insects, the crop.

    Importance DCV is one of the few identified natural viral pathogens affecting the model organism Drosophila melanogaster. As such, it is an important virus for the deciphering of host-virus interactions in insects. We characterize here the pathogenesis associated with DCV infection in flies, and show that it results from the tropism of the virus for an essential but as yet poorly characterized organ in the digestive tract, the crop. Our results may have relevance for other members of the Dicistroviridae, some of which are pathogenic to beneficial or pest insect species.

  • Activation of RIG-I pathway during influenza vaccination enhances germinal center reactions, T follicular helper cell induction and, provides dose-sparing effect and protective immunity [PublishAheadOfPrint]

  • Pattern Recognition Receptors (PRR) sense certain molecular patterns uniquely expressed by pathogens. Retinoic-acid-inducible gene I (RIG-I) is a cytosolic PRR that senses viral nucleic acids and induces innate immune activation and secretion of type-I IFNs. Here, using influenza vaccine antigens, we investigated the consequences of activating the RIG-I pathway on antigen-specific adaptive immune responses. We found that mice immunized with influenza vaccine antigens co-administered with 5rrsquo; ppp-dsRNA, a RIG-I ligand, developed robust levels of hemagglutination inhibiting antibodies, enhanced germinal center reaction and T follicular helper cell responses. In addition, RIG-I activation enhanced antibody affinity maturation and plasma cell responses in draining lymph nodes, spleen, and bone marrow and conferred protective immunity against virus challenge. Importantly, activation of RIG-I pathway was able to reduce the antigen requirement by 10-100 folds in inducing optimal influenza-specific cellular and humoral responses including protective immunity. The effects induced by 5rrsquo; ppp-dsRNA were significantly dependent on type-I IFN and IPS-1 (adapter protein downstream of RIG-I pathway) signaling, but were independent of MyD88 or TLR3 mediated pathways. Our results show that activation of RIG-I-like receptor pathway programs the innate immunity to achieve qualitatively and quantitatively enhanced protective cellular adaptive immune responses even at low antigen doses and thus, indicate the potential utility of RIG-I ligands as molecular adjuvants for the viral vaccines.

    Study Importance Statement The recently discovered RNA helicase family of RIG-I-like receptors (RLRs) is a critical component of host defense mechanisms responsible for detecting viruses and triggering innate anti-viral cytokines that help control viral replication and dissemination. In this study we show that the RLR-pathway can be effectively exploited for enhancing adaptive immunity and protective immune memory against viral infection. Our results show that activation of RIG-I pathway along with influenza vaccination programs the innate immunity to induce qualitatively and quantitatively superior protective adaptive immunity against pandemic influenza viruses. More importantly, the RIG-I activation at the time of vaccination allows induction of robust adaptive responses even at sparing vaccine antigen doses. These results highlight the potential utility of exploiting RIG-I pathway for enhancing viral vaccine specific immunity and have broader implications for designing better vaccines in general.

  • Vaccinia virus mutations in the L4R gene encoding a virion structural protein produce abnormal mature particles lacking a nucleocapsid [PublishAheadOfPrint]

  • Electron micrographs from the 1960s revealed the presence of an S-shaped tubular structure in the center of the vaccinia virion core. Recently, we showed that packaging of virus transcription enzymes is necessary for the formation of the tubular structure, suggesting that the structure is equivalent to a nucleocapsid. Based on this study and on what is known about nucleocapsids of other viruses, we hypothesized that in addition to transcription enzymes, the tubular structure also contains the viral DNA and a structural protein as a scaffold. The vaccinia virion structural protein L4 stands out as the best candidate for the role of a nucleocapsid structural protein because it is abundant, it is localized in the center of the virion core and it binds DNA. In order to gain more insight into the structure and relevance of the nucleocapsid, we analyzed thermosensitive and inducible mutants in the gene L4R. Using a cryo-fixation method for electron microscopy (high pressure freezing followed by freeze substitution) to preserve labile structures like the nucleocapsid, we were able to demonstrate that in the absence of functional L4, mature particles with defective internal structure are produced under non-permissive conditions. These particles do not contain a nucleocapsid. In addition, the core wall of these virions is abnormal. This suggests that the nucleocapsid interacts with the core wall and that the nucleocapsid structure might be more complex than originally assumed.

    Importance The vaccinia virus nucleocapsid has been neglected since the 1960s due to a lack of electron microscopy techniques to preserve this labile structure. With the advent of cryo-fixation techniques, like High Pressure Freezing/Freeze Substitution, we are now able to consistently preserve and visualize the nucleocapsid. Because vaccinia virus early transcription is coupled to the viral core structure, detailing the structure of the nucleocapsid is indispensable for determining the mechanisms of vaccinia virus core-directed transcription. The present study represents our second attempt to understand the structure and biological significance of the nucleocapsid. We demonstrate the importance of the protein L4 for the formation of the nucleocapsid and reveal in addition that the nucleocapsid and the core wall may be associated, suggesting a higher level of complexity of the nucleocapsid than predicted. In addition, we prove the utility of high pressure freezing in preserving the vaccinia nucleocapsid.

  • Chlorovirus Skp1-binding Ankyrin-Repeat Protein Interplay and Mimicry of Cellular Ubiquitin Ligase Machinery [PublishAheadOfPrint]

  • The ubiquitin-proteasome system is targeted by many viruses that have evolved strategies to redirect host ubiquitination machinery. Members of the genus Chlorovirus are proposed to share a common ancestral lineage with a broader group of related viruses, Nucleo-Cytoplasmic Large DNA Viruses (NCLDV). Chloroviruses encode a Skp1 homolog and ankyrin-repeat (ANK) proteins. Several chlorovirus encoded ANK-repeats contain C-terminal domains characteristic of cellular F-boxes or related NCLDV chordopox PRANC domains. These observations suggested that this unique combination of Skp1 and ANK-repeat proteins might form complexes analogous to the cellular Skp1-Cul1-F-box (SCF) ubiquitin ligase complex. We identified two ANK proteins from the prototypic chlorovirus PBCV-1 that functioned as binding partners for the virus-encoded Skp1, proteins A682L and A607R. These ANK proteins had a C-terminal Skp1 interactional motif that functioned like cellular F-box domains. A C-terminal motif of ANK protein A682L binds Skp1 proteins from widely divergent species. Yeast two-hybrid analyses using serial domain deletion constructs confirmed the C-terminal localization of the Skp1 interactional motif in PBCV-1 A682L. ANK protein A607R represents an ANK family with one member present in all 41 sequenced chloroviruses. A comprehensive phylogenetic analysis of these related ANK and viral Skp1 proteins suggested partnered function tailored to the host alga or common ancestral heritage. Here, we show protein-protein interaction between corresponding family clusters of virus-encoded ANK and Skp1 proteins from three chlorovirus genera. Collectively, our results indicate that chloroviruses have evolved complimenting Skp1 and ANK proteins that mimic cellular SCF-associated proteins.

    Importance Viruses have evolved ways to direct ubiquitination events in order to create environments conducive to their replication. As reported in this manuscript, the large chloroviruses encode several components involved in the SCF ubiquitin ligase complex including a viral Skp1 homolog. Studies on how chloroviruses manipulate their host algal ubiquitination system will provide insights towards viral protein mimicry, substrate recognition, and key interactive domains controlling selective protein degradation. These findings may also help to understand the evolution of other large DNA viruses, like poxviruses, that are reported to share the same monophyly lineage as chloroviruses.

  • Variation of the Specificity of the Human Antibody Responses after Tick-Borne Encephalitis Virus Infection and Vaccination [PublishAheadOfPrint]

  • Tick-borne encephalitis (TBE) virus is an important human-pathogenic flavivirus endemic in large parts of Europe, Central and Eastern Asia. Neutralizing antibodies specific for the viral envelope protein E are believed to mediate long-lasting protection after natural infection and vaccination. To study the specificity and individual variation of human antibody responses we developed immunoassays with recombinant antigens representing viral surface protein domains and domain combinations. These allowed us to dissect and quantify antibody populations of different fine specificities in sera of TBE patients and vaccinees.

    Post-infection and post-vaccination sera both displayed strong individual variation of antibody titers as well as the relative proportions of antibodies to different domains of E, indicating that the immunodominance patterns observed were strongly influenced by individual-specific factors. The contributions of these antibody populations to virus neutralization were quantified by serum depletion analyses and revealed a significantly biased pattern. Antibodies to domain III mmdash; in contrast to what was found in mouse immunization studies with TBE and other flaviviruses mmdash; did not play any role in the human neutralizing antibody response which was dominated by antibodies to domains I and II.

    Importantly, most of the neutralizing activity could be depleted from sera by a dimeric soluble form of the E protein which is the building block of the icosahedral herringbone-like shell of flaviviruses, suggesting that antibodies to more complex quaternary epitopes involving residues from adjacent dimers play only a minor role in the total response to natural infection and vaccination in humans.

    Importance Tick-borne encephalitis (TBE) virus is a close relative of yellow fever, dengue, Japanese encephalitis and West Nile viruses and distributed in large parts of Europe, Central and Eastern Asia. Antibodies to the viral envelope protein E prevent viral attachment and entry into cells and thus mediate virus neutralization and protection from disease. However, the fine specificity and individual variation of neutralizing antibody responses is currently not known. We have therefore developed new in vitro assays for dissecting the antibody populations present in blood serum and determining their contribution to virus neutralization. In our analysis of human post-infection and post-vaccination sera, we found an extensive variation of the antibody populations present in sera, indicating substantial influences of individual-specific factors that control the specificity of the antibody response. Our study provides new insights into the immune response to an important human pathogen that is of relevance for the design of novel vaccines.

  • Evolution of Bluetongue Virus Serotype 1 in Northern Australia over 30 Years [PublishAheadOfPrint]

  • BTV 1 was first isolated in Australia from cattle blood collected in 1979 at Beatrice Hill Farm (BHF), Northern Territory (NT). From long term surveillance programs (1977 to 2011), 2487 isolations of ten BTV serotypes were made. The most frequently isolated serotype was BTV 1 (41%, 1019) followed by BTV 16 (17.5%, 436) and BTV 20 (14%, 348). In three years no BTVs were isolated and in twelve years no BTV 1 was isolated. Seventeen BTV 1 isolates were sequenced and analyzed in comparison with ten Australian prototype serotypes. BTV 1 showed an episodic pattern of evolutionary change characterized by four distinct periods. Each period consisted primarily of slow genetic drift which was punctuated from time to time by genetic shifts generated by segment reassortment and the introduction of new genome segments. Evidence was found for co-evolution of BTV genome segments. Evolutionary dynamics and selection pressures estimates showed strong temporal and clock-like molecular evolutionary dynamics of six Australian BTV genome segments. Bayesian coalescent estimates of mean substitution rates clustered in the range of 3.5 to 5.3 x 10-4 substitutions per site per year. All BTV genome segments evolved under strong purifying (negative) selection with only three sites identified as under pervasive diversifying (positive) selection. The obligate replication in alternate hosts (insect vector and vertebrate host) imposed strong evolutionary constraints. The dominant mechanism generating genetic diversity of BTV 1 at BHF was through the introduction of new viruses and reassortment of genome segments with existing viruses.

    IMPORTANCE Bluetongue virus (BTV) is the causative agent of bluetongue disease in ruminants. It is a disease of concern globally and is transmitted by biting midges (Culicoides species). Analysis of the evolutionary and selection pressures on BTV 1 at a single surveillance site in northern Australia showed strong temporal and clock-like dynamics. Obligate replication in alternate hosts of insect and vertebrate imposed strong evolutionary constraints with all BTV genome segments evolving under strong purifying (negative) selection. Generation of genetic diversity of BTV 1 in northern Australia is through genome segment reassortment and the introduction of new serotypes.

  • The CD8+ memory stem T cell (TSCM) subset is associated with improved prognosis in chronic HIV-1 infection [PublishAheadOfPrint]

  • Memory stem T cells (TSCM) constitute a long-lived, self-renewing lymphocyte population essential for the maintenance functional immunity. The hallmarks of HIV-1 pathogenesis are CD4+ T cell depletion and abnormal cellular activation. We investigated the impact of HIV-1 infection on the TSCM compartment, as well as any protective role these cells may have in disease progression, by characterizing this subset in a cohort of 113 subjects with variable degrees of viral control on and off highly active antiretroviral therapy (HAART). We observed that the frequency of CD8+ TSCM is decreased in all individuals with chronic, untreated HIV-1 infection, and that HAART has a restorative effect on this subset. In contrast, natural controllers of HIV-1 had the highest absolute number of CD4+ TSCM cells among all of the infected groups. The frequency of CD4+ TSCM predicted higher CD8+ TSCM frequencies, consistent with a role for the CD4+ subset in helping to maintain CD8+ memory T cells. In addition, TSCM appeared to be progenitors for effector T cells (TEM), as these two compartments were inversely correlated. Increased frequencies of CD8+ TSCM predicted lower viral loads, higher CD4+ counts and less CD8+ T cell activation. Finally, we found that TSCM express the mucosal homing integrin aalpha;4bbeta;7 and can be identified in the gastrointestinal-associated lymphoid tissue (GALT). The frequency of mucosal CD4+ TSCM was inversely correlated with that in the blood, potentially reflecting the ability of these self-renewing cells to migrate to a crucial site of ongoing viral replication and CD4+ T cell depletion.

    Importance: HIV-1 infection leads to profound impairment of the immune system. TSCM constitute a recently identified lymphocyte subset with stem-cell like qualities including the ability to generate other memory T cell subtypes, and are therefore likely to play an important role in controlling viral infection. We investigated the relationship between the size of the CD8+ TSCM compartment and HIV-1 disease progression in a cohort of chronically infected individuals. Our results suggest that HAART restores a normal frequency of CD8+ TSCM and that the natural preservation of this subset in the setting of untreated HIV-1 infection is associated with improved viral control and immunity. Therefore, the CD8+ TSCM population may represent a correlate of protection in chronic HIV-1 infection that is directly relevant to the design of T cell-based vaccines, adoptive immunotherapy approaches or the pharmacologic induction of TSCM.

  • Exonuclease domain of the Lassa virus nucleoprotein is critical to avoid RIG-I signaling and to inhibit the innate immune response. [PublishAheadOfPrint]

  • Lassa virus (LASV), which causes a viral hemorrhagic fever, inhibits the innate immune response. The exonuclease (ExoN) domain of its nucleoprotein (NP) is implicated in the suppression of RIG-I signaling. We show here that a LASV in which ExoN function has been abolished strongly activates innate immunity and that this effect is dependent on RIG-I signaling. These results highlight the key role of NP ExoN function in the immune evasion that occurs during LASV infection.

  • The exonuclease activity of Lassa virus nucleoprotein is involved in APC-mediated NK cell responses [PublishAheadOfPrint]

  • Lassa virus is an Old World Arenavirus which causes for Lassa hemorrhagic fever in humans mostly in West Africa. Lassa fever is an important public health problem and a safe and effective vaccine is urgently needed. The infection causes immunosuppression, probably due to the absence of activation of antigen-presenting cells (dendritic cells and macrophages), and in low type I IFN production and deficient NK cell function. However, a recombinant Lassa virus carrying D389A and G392A substitutions in the nucleoprotein that abolish the exonuclease activity and IFN activation loses its inhibitory activity and induces strong type I IFN production by dendritic cells and macrophages. We show here that during infection by this mutant Lassa virus, antigen-presenting cells trigger efficient human NK cell responses in vitro including production of IFN- and cytotoxicity. NK cell activation involves close contact with both antigen-presenting cells and soluble factors. We report that infected dendritic cells and macrophages express the NKG2D ligands MHC class I-related chains A and B and they may produce IL-12, IL-15 and IL-18, all involved in NK cell functions. NK cell degranulation is significantly increased in cocultures suggesting that NK cells seem to kill infected dendritic cells and macrophages. This work confirms the inhibitory function of Lassa virus nucleoprotein. Importantly, we demonstrate for the first time that Lassa virus nucleoprotein is involved in the inhibition of antigen presenting cell-mediated NK cell responses.

    Importance Of The Study The pathogenesis and immune responses induced by Lassa virus are poorly known. Recently, an exonuclease domain contained in the viral nucleoprotein has been shown to be able to inhibit type I IFN response by avoiding the recognition of viral RNA by cell sensors. Here, we studied the responses of NK cells to dendritic cells and macrophages infected with a recombinant Lassa virus in which the exonuclease functions have been abolished and demonstrated that NK cells are strongly activated and presented effective functions. These results show that the strategy developed by Lassa virus to evade innate immunity is also effective on NK cells, explaining the weak NK cell activation observed with the wild-type virus. By providing a better understanding of the interactions between Lassa virus and the host immune system, these results are important for the field of arenavirus biology and may be useful for a vaccine approach against Lassa fever.

  • The quantitation of replication-competent HIV-1 in populations of resting CD4+ T cells [PublishAheadOfPrint]

  • Central memory (TCM) CD4+ T cells are the principal reservoir of latent HIV-1 infection that persists despite durable, successful antiretroviral therapy (ART). In a study that measured HIV DNA in 34 patients and replication-competent HIV in four patients, pools of resting and activated transitional memory (TTM) CD4+ T cells were found to be a reservoir for HIV infection. As defective viruses account for the majority of integrated HIV DNA and do not reflect the actual frequency of latent, replication-competent proviral infection, we assessed the specific contribution of resting TTM cells to latent HIV infection. We measured the frequency of replication-competent HIV in purified resting memory cell subpopulations by a limiting dilution, quantitative viral outgrowth assay (QVOA). HIV was routinely detected within the resting central memory compartment, but was infrequently detected within the resting TTM compartment. These observations suggest that prolonged ART may limit persistent latent infection in the TTM compartment. Our results confirm the importance of latent infection within the TCM compartment, and again focus attention on these cells as the most important latent viral reservoir. While proliferation may drive expansion of detectable viral genomes in cells, the frequency of replication-competent HIV must be carefully assessed. Latent infection appears to wane within the transitional memory compartment in patients who have sustained successful viral suppression via ART, or were treated very early in infection.

    IMPORTANCE Antiretroviral therapy (ART) has led to a significant decrease in morbidity and mortality among HIV-infected patients. However, HIV integrates into the genome of CD4+ T cells generating pools of long-lived cells that are reservoirs of latent HIV. Two main subsets of CD4+ T cells, central memory and transitional memory cells were reported to be major reservoirs of HIV infection. However, this study primarily measured the HIV DNA content, which also includes defective proviruses that would not be able to replicate and initiate new rounds of infection. By analyzing the replication-competent virus in both cell subsets we show that transitional memory cells may not be a durable reservoir in patients on successful ART.

  • Structurally Similar Woodchuck and Human Hepadnavirus Core Proteins Have Distinctly Different Temperature Dependences of Assembly [PublishAheadOfPrint]

  • Woodchuck hepatitis virus (WHV), a close relative of human hepatitis B virus (HBV), has been a key model for disease progression and clinical studies. Sequences of the assembly domain of WHV and HBV core proteins (wCp149 and hCp149, respectively) have 65% identity, suggesting similar assembly behavior. We report a cryo-electron microscopy structure of the WHV capsid at sub-nanometer resolution and a characterization of wCp149 assembly. At this resolution, the T=4 capsid structures of WHV and HBV are practically identical. In contrast to their structural similarity, wCp149 demonstrates enhanced assembly kinetics and stronger dimer-dimer interactions compared to hCp149: at 23ddeg;C and 100 mM ionic strength, the pseudo-critical concentrations of assembly of wCp149 and hCp149 are 1.8 mmu;M and 43.3 mmu;M, respectively. Transmission electron microscopy reveals that wCp149 assembles into predominantly T=4 capsids with a sizeable population of larger, non-icosahedral structures. Charge detection mass spectrometry indicates that T=3 particles are extremely rare compared to the ~5% observed in hCp149 reactions. Unlike hCp149, wCp149 capsid assembly is favorable over a temperature range of 4 ddeg;C to 37 ddeg;C; van't Hoff analyses relate the differences in temperature dependence to the large positive values for heat capacity, enthalpy, and entropy of wCp149 assembly. Because the final capsids are so similar, these findings suggest that free wCp149 and hCp149 undergo different structural transitions leading to assembly. The difference in the temperature dependence of wCp149 assembly may be related to the temperature range of its hibernating host.

    IMPORTANCE: In this paper we present a cryo-EM structure of a WHV capsid showing its similarity to HBV. We then observe that assembly properties of the two homologous proteins are very different. Unlike human HBV, the capsid protein of WHV has evolved to function in a non-homeostatic environment. These studies yield insight into the interplay between core protein self-assembly and host environment, which may also be particularly relevant to plant viruses and viruses with zoonotic cycles involving insect vectors.

  • Modeling the timing of anti-latency drug administration during HIV treatment [PublishAheadOfPrint]

  • Latently infected cells are considered a major barrier to cure of HIV infection, as they are long-lived under antiretroviral treatment (ART) and cause viral replication to restart soon after stopping ART. In the last decade different types of anti-latency drugs have been explored with the aim of reactivating and purging this latent reservoir, and the hope of achieving a cure. Because of toxicity and safety considerations, anti-latency drugs can only be given for a short time to patients on long term ART, with small effect.

    We recently investigated the turnover of latently infected cells during active infection, and have found that it was strongly correlated with viral load. This implies that although latently infected cells had long lifespans in the setting of low viral load (as during ART), they turned over fast under high viral load. Possible reasons could be that increased viral load causes increased activation or death of CD4+ T cells, including those that are latently infected. Taking these results into account, we developed a mathematical model to study the most appropriate timing of anti-latency drugs in relationship to the initiation of ART. We found that the best timing of a short-term anti-latency drug would be the start of ART, when viral load, CD4+ T cell activation and latent cell turnover are high. These results have important implications for the design of HIV cure-related clinical trials.

    Importance The antiretroviral therapy of HIV-infected patients currently needs to be life-long, because the cells latently infected with HIV start new rounds of infection as soon as the treatment is stopped. In the last decade, a number of different types of anti-latency drugs have been explored with the aim of "reactivating" and "purging" this latent reservoir, and hoping to achieve a cure. These drugs have so far been tested on patients only after long term ART, and have shown a small or no effect. We use mathematical modeling to argue that the most efficacious timing of a short-term anti-latency treatment may be the start of ART, because of possible interactions of anti-latency drugs with natural activation pathways.

  • p130Cas Scaffolds the Signalosome to Direct Adaptor-Effector Crosstalk During Kaposi's Sarcoma-Associated Herpesvirus Trafficking in Human Microvascular Dermal Endothelial Cells [PublishAheadOfPrint]

  • KSHV interacts with cell surface receptors, such as heparan sulfate, integrins (aalpha;3bbeta;1, aalpha;Vbbeta;3, and aalpha;Vbbeta;5) and EphrinA2 (EphA2), and activates FAK, Src, PI3-K, c-Cbl, and RhoA GTPase signal molecules early during lipid raft (LR) dependent productive macropinocytic entry into human dermal microvascular endothelial cells. Our recent studies have identified CIB1 as a signal amplifier facilitating EphA2 phosphorylation and subsequent cytoskeletal cross-talk during KSHV macropinocytosis. Although CIB1 lacks an enzymatic activity and traditional adaptor domain or known interacting sequence, it associated with the KSHV entry signal complex and the CIB1-KSHV association was sustained over 30 minutes post-infection. To identify factors scaffolding the EphA2-CIB1 signal axis, the role of major cellular scaffold protein p130Cas (Crk associated substrate of Src) was investigated. Inhibitor and siRNA studies demonstrated that KSHV induced p130Cas in an EphA2, CIB1, and Src dependent manner. p130Cas and Crk were associated with KSHV, LRs, EphA2, and CIB1 early during infection. Live cell microscopy and biochemical studies demonstrated that p130Cas knockdown did not affect KSHV entry but significantly reduced productive nuclear trafficking of viral DNA and routed KSHV to lysosomal degradation. p130Cas aided in scaffolding adaptor Crk to downstream guanine nucleotide exchange factor phospho-C3G possibly to coordinate GTPase signaling during KSHV trafficking. Collectively, these studies demonstrate that p130Cas acts as a bridging molecule between the KSHV induced entry signal complex and the downstream trafficking signalosome in endothelial cells, and suggests that simultaneous targeting of KSHV entry receptors with p130Cas would be an attractive potential avenue for therapeutic intervention of KSHV infection.

    IMPORTANCE Eukaryotic cell adaptor molecules, without any intrinsic enzymatic activity, are well known to allow a great diversity of specific and coordinated protein-protein interactions imparting signal amplification to different networks for physiological and pathological signaling. They are involved in integrating signals from growth factors, extra cellular matrix molecules, bacterial pathogens, and apoptotic cells. The present study identifies HMVEC-d cellular scaffold protein p130Cas (Crk associated substrate) as a platform to promote KSHV trafficking. Early during KSHV de novo infection p130Cas associates with lipid rafts and scaffolds EphA2 associated critical adaptor members to downstream effector molecules, promoting successful nuclear delivery of the KSHV genome. Hence, simultaneous targeting of the receptor EphA2 and scaffolding action of p130Cas can potentially uncouple the signal crosstalk between the KSHV entry associated upstream signal complex from the immediate downstream trafficking associated signalosome, consequently, routing KSHV towards lysosomal degradation and eventually blocking KSHV infection and associated malignancies.

  • HIV-1 entry in SupT1-R5, CEM-ss and primary CD4+ T-cells occurs at the plasma membrane and does not require endocytosis [PublishAheadOfPrint]

  • Cytoplasmic entry of HIV-1 requires binding of the viral glycoproteins to the cellular receptor and co-receptor leading to fusion of viral and cellular membranes. Early studies suggested that productive HIV-1 infection occurs by direct fusion at the plasma membrane. Endocytotic uptake of HIV-1 was frequently observed, but was considered to constitute an unspecific dead-end pathway. More recent evidence suggested that endocytosis contributes to productive HIV-1 entry and may even represent the predominant or exclusive route of infection. We have analyzed HIV-1 binding, endocytosis, cytoplasmic entry and infection in T-cell lines and in primary CD4+ T-cells. Efficient cell binding and endocytosis required viral glycoproteins and CD4, but not the co-receptor. The contribution of endocytosis to cytoplasmic entry and infection was assessed by two strategies: (i) Expression of dominant-negative dynamin-2 efficiently blocked HIV-1 endocytosis, but did not affect fusion or productive infection. (ii) Making use of the fact that HIV-1 fusion is blocked at temperatures below 23ddeg;C, cells were incubated with HIV-1 at 22ddeg;C for varying times, and endocytosis was quantified by parallel analysis of transferrin and fluorescent HIV-1 uptake. Subsequently, plasma membrane entry was blocked by high concentrations of the peptidic fusion inhibitor T-20, which does not reach previously endocytosed particles. HIV-1 infection was scored after shifting to 37ddeg;C in the presence of T-20. These experiments revealed that productive HIV-1 entry occurs predominantly at the plasma membrane in SupT1-R5, CEM-ss and primary CD4+ T-cells, with little if any contribution from endocytosed virions.

    Importance HIV-1, like all enveloped viruses, reaches the cytoplasm by fusion of the viral and cellular membranes. Many viruses enter the cytoplasm by endosomal uptake and fusion from the endosome, while cell entry can also occur by direct fusion at the plasma membrane in some cases. Conflicting evidence has been reported regarding the site of HIV-1 fusion with some studies claiming fusion to occur predominantly at the plasma membrane, while others suggested predominant or even exclusive fusion from the endosome. We have revisited HIV-1 entry using a T-cell line that exhibits HIV-1 endocytosis dependent on the viral glycoproteins and the cellular CD4 receptor; results with this cell line were confirmed for another T-cell line and primary CD4+ T-cells. Our studies show that fusion and productive entry occur predominantly at the plasma membrane, and we conclude that endocytosis is dispensable for HIV-1 infectivity in these T-cell lines and in primary CD4+ T-cells.

  • Suppressors of cytokine signaling 1 and 3 are up-regulated in brain resident cells in response to virus induced inflammation of the CNS via at least two distinctive pathways [PublishAheadOfPrint]

  • Suppressors of cytokine signaling (SOCS) proteins are intracellular proteins that inhibit cytokine signaling in a variety of cell types. A number of viral infections have been associated with SOCS up-regulation; however, not much is known about the mechanisms regulating SOCS expression during viral infection.

    In this study, we have used two pathologically distinct intracerebral (i.c.) infection models to characterize temporal and spatial aspects of SOCS expression in the virus-infected CNS, and by employing various knockout mouse models, we have sought to identify regulatory mechanisms that may underlie a virus induced up-regulation of SOCS in the CNS. We found that i.c. infection with either lymphocytic choriomeningitis virus (LCMV) or yellow fever virus (YF) results in gradual up-regulation of SOCS1/3 mRNA expression peaking at day 7 post infection (p.i.). In the LCMV model, SOCS mRNA was expressed in brain resident cells including astrocytes and some neurons, and for SOCS1 in particular this up-regulation was almost entirely mediated by IFN- produced by infiltrating T cells. Following infection with YF, we also found SOCS expression to be up-regulated in brain resident cells with a peak on day 7 p.i., but in this model, the up-regulation was only partially dependent on IFN- and T cells, indicating that at least one other mediator was involved in the up-regulation of SOCS following YF infection. We conclude that virus induced inflammation of the CNS is associated with up-regulation of SOCS1/3 mRNA expression in brain resident cells, and that at least two distinctive pathways can lead to this up-regulation.

    Importance In the present report, we have studied the induction of SOCS1 and SOCS3 expression in the context of virus-induced CNS infection. We find that both a non-cytolytic and a cytolytic virus induce marked up-regulation of SOCS1 and-3 expression. Notably, the kinetics of the observed up-regulation follows that of activity within pro-inflammatory signalling pathways and, interestingly, type II IFN, which is also a key inducer of inflammatory mediators, seems to be essential in initiating this counter-inflammatory response. Another key observation is that not only cells of the immune system, but also CNS resident cells are actively involved in both the pro- and counter-inflammatory immune circuits; thus e. g. astrocytes up-regulates both CXCL10 and SOCS when exposed to type II IFN in vivo.

  • Trunkloads of viruses [PublishAheadOfPrint]

  • Elephant populations are under intense pressure internationally from habitat destruction and poaching for ivory and meat. They also face pressure from infectious agents, including elephant endotheliotropic herpesvirus 1 (EEHV-1), which kills ~20% of Asian elephants (Elephas maximus) born in zoos and causes disease in the wild. EEHV-1 is one of at least 6 distinct EEHV in a phylogenetic lineage that appears to represent a new subfamily (the Deltaherpesvirinae) in family Herpesviridae.

  • Vaginal memory T cells induced by intranasal vaccination are critical for protective T cell recruitment and prevention of genital HSV-2 disease [PublishAheadOfPrint]

  • Protective immunity against genital pathogens causing chronic infections such as herpes simplex virus type 2 (HSV-2) or human immunodeficiency virus requires the induction of cell-mediated immune responses locally in the genital tract. Intranasal immunization with a thymidine kinasemmdash;deficient (TKmmdash;) mutant of HSV-2 effectively induces HSV-2-specific IFN-secreting memory T-cell production and protective immunity against intravaginal challenge with wild-type HSV-2. However, the precise mechanism by which intranasal immunization induces protective immunity in the distant genital mucosa more effectively than does systemic immunization is unknown. Here, we showed that intranasal immunization with live HSV-2 TKmmdash; induced the production of effector T cells and their migration to, and retention in, the vaginal mucosa, whereas systemic vaccination barely established a local effector T cell pool, even when it induced the production of circulating memory T cells in the systemic compartment. The long-lasting HSV-2-specific local effector T cells induced by intranasal vaccination provided superior protection against intravaginal wild-type HSV-2 challenge by starting viral clearance at the entry site earlier than with intraperitoneal immunization. Intranasal immunization is an effective strategy for eliciting high levels of cell-mediated protection of the genital tract by providing long-lasting Ag-specific local effector T cells without introducing topical infection or inflammation.

    Importance Intranasal (IN) vaccines against sexually transmitted diseases, which are caused by viruses, such as herpes simplex virus type 2 (HSV-2), have long been in development, but no vaccine candidate is currently available. Understanding the cellular mechanisms of immune responses in a distant vaginal mucosa induced by IN immunization with HSV-2 will contribute to designing the vaccine. Our study demonstrated that IN immunization with an attenuated strain of HSV-2 generated long-lasting IFN- secreting T cells in vaginal mucosa more effectively than systemic immunization. We found that these vaginal effector memory T cells are critical for the early stage of viral clearance at natural infection sites, and prevent severe vaginal inflammation and herpes encephalitis.

  • The Conserved Disulfide Bond within Domain II (D-II) of Epstein-Barr Virus (EBV) gH has Divergent Roles in Membrane Fusion with Epithelial Cells and B Cells [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) infects target cells via fusion with cellular membranes. For entry into epithelial cells, EBV requires the herpesvirus conserved core fusion machinery composed of glycoprotein B (gB) and gH/gL. In contrast, for B cell fusion it requires gB and gH/gL with gp42 serving as a cell tropism switch. The available crystal structures for gH/gL allow the targeted analysis of structural determinants of gH to identify functional regions critical for membrane fusion. Domain II of EBV gH contains two disulfide bonds (DB), the first is unique for EBV and closely related -herpesviruses. The second is conserved across the bbeta;- and -herpesviruses and is positioned to stabilize a putative syntaxin-like bundle motif. To analyze the role of these DBs in membrane fusion, gH was mutated by amino acid substitution of the DB cysteines. Mutation of the EBV-specific DB resulted in diminished gH/gL cell surface expression that correlated with diminished B cell and epithelial cell fusion. In contrast, mutation of the conserved DB resulted in wild-type-like B cell fusion whereas epithelial cell fusion was greatly reduced. The gH mutants bound well to gp42 but had diminished binding to epithelial cells. Tyrosine 336, located adjacent to cysteine 335 of the conserved DB, was also found to be important for DB stabilization and gH/gL function. We conclude that the conserved DB has a cell type specific function, since it is important for the binding of gH to epithelial cells initiating epithelial cell fusion but not for fusion with B cells and gp42 binding.

    Importance EBV predominantly infects epithelial and B cells in humans, which can result in EBV-associated cancers such as Burkitt and Hodgkin lymphoma as well as nasopharyngeal carcinoma. EBV is also associated with a variety of lymphoproliferative disorders, typically of B cell origin, observed in immunosuppressed individuals such as post-transplant or HIV/AIDS patients. The gH/gL complex plays an essential but still poorly characterized role as an important determinant for EBV cell tropism. In our current studies, we found that mutants in the DB C278/C335 and a neighboring tyrosine 336 have cell type specific functional deficits with selective decreases in epithelial cell, but not B cell, binding and fusion. The present study brings new insights into the gH function as determinant for epithelial cell tropism during herpesvirus induced membrane fusion and highlights a specific gH motif required for epithelial cell fusion.

  • Induced maturation of human immunodeficiency virus [PublishAheadOfPrint]

  • HIV-1 assembles at the plasma membrane of virus producing cells as an immature, non-infectious particle. Processing of the Gag and Gag-Pol polyproteins by the viral protease (PR) activates the viral enzymes and results in dramatic structural rearrangements within the virion mmdash; termed maturation mmdash; that are a prerequisite for infectivity. Despite its fundamental importance for viral replication, little is currently known about the regulation of proteolysis and about the dynamics and structural intermediates of maturation. This is mainly due to the fact that HIV-1 release and maturation occur asynchronously both at the level of individual cells and at the level of particle release from a single cell. Here, we report a method to synchronize HIV-1 proteolysis in vitro based on protease inhibitor (PI) wash-out from purified immature virions, thereby temporally uncoupling virus assembly and maturation. Drug wash-out resulted in the induction of proteolysis with cleavage efficiencies correlating with koff of the respective PR-PI complex. Proteolysis of Gag was nearly complete and yielded the correct products with an optimal t1/2 of ~5 h, but viral infectivity was not recovered. Failure to gain infectivity following PI wash-out may be explained by the observed formation of aberrant viral capsids and/or by pronounced defects in processing of the RT heterodimer associated with a lack of RT activity. Based on our results, we hypothesize that both the polyprotein processing dynamics and the tight temporal coupling of immature particle assembly and PR activation are essential for correct polyprotein processing and morphological maturation, and thus for HIV-1 infectivity.

    Importance Cleavage of the HIV-1 polyproteins Gag and Gag-Pol into their functional subunits by the viral protease activates the viral enzymes and causes major structural rearrangements essential for HIV-1 infectivity. This proteolytic maturation occurs concomitant with virus release and investigation of its dynamics is hampered by the fact that virus populations in tissue culture contain particles at all stages of assembly and maturation. Here we developed an inhibitor wash-out strategy to synchronize activation of protease in wild-type virus. We demonstrated that nearly complete Gag processing and resolution of the immature virus architecture is accomplished under optimized conditions. Nevertheless, most of the resulting particles displayed irregular morphologies, Gag-Pol processing was not faithfully reconstituted and infectivity was not recovered. These data show that HIV-1 maturation is sensitive to the dynamics of processing, and also that a tight temporal link between virus assembly and PR activation is required for correct polyprotein processing.

  • Transcriptome responses of the host, Trichoplusia ni, to infection by the baculovirus, Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) [PublishAheadOfPrint]

  • Productive infection of insect Trichoplusia ni cells by the baculovirus AcMNPV leads to expression of ~156 viral genes and results in dramatic cell remodeling. How the cell transcriptome responds to viral infection was unknown due to the lack of a reference genome and transcriptome for T. ni. We used an ~60 Gb RNA-seq dataset from infected and uninfected T. ni cells, to generate and annotate a de novo transcriptome assembly of approximately 70,322 T. ni unigenes, representing the 48 hour infection cycle. Using differential gene expression analysis, we found that the majority of host transcripts were down-regulated after 6 hours post infection (h p.i.) and throughout the remainder of the infection. In contrast, 5.7% (4028) of the T. ni unigenes were up-regulated during the early period (0-6 h p.i.), followed by a decrease through the remainder of the infection cycle. Also, a small subset of genes related to metabolism and stress response showed a significant elevation of transcript levels at 18 and 24 h p.i., but a decrease thereafter. We also examined the responses of genes belonging to a number of specific pathways of interest, including stress responses, apoptosis, immunity, and protein trafficking. We identified specific pathways members that were up-regulated during the early phase of the infection. Combined with the parallel analysis of AcMNPV expression, these results provide both a broad and a detailed view of how baculovirus infection impacts the host cell transcriptome to evade cellular defensive responses, to modify cellular biosynthetic pathways, and to remodel cell structure.

    IMPORTANCE Baculoviruses are insect-specific DNA viruses that are highly pathogenic to their insect hosts. In addition to their use for biological control of certain insects, baculoviruses also serve as viral vectors for numerous biotechnological applications, such as mammalian cell transduction and protein expression for vaccine production. While there is considerable information regarding viral gene expression in infected cells, little is known regarding responses of the host cell to baculovirus infection. In these studies, we assembled a cell transcriptome from the host Trichoplusia ni and used that transcriptome to analyze changes in host cell gene expression throughout the infection cycle. The study was performed in parallel with a prior study of changes in viral gene expression. Combined, these studies provide an unprecedented new level of detail and an overview of events in the infection cycle, and will stimulate new experimental approaches to understand, modify, and utilize baculoviruses for a variety of applications.

  • Effects of Human Anti-Spike Protein Receptor Binding Domain Antibodies on SARS-Coronavirus Neutralization Escape and Fitness [PublishAheadOfPrint]

  • The receptor binding domain (RBD) of the spike (S) glycoprotein of SARS-CoV is a major target of protective immunity in vivo. Although a large number of neutralizing antibodies (nAbs) have been developed, it remains unclear if a single RBD-targeting nAb or two in combination can prevent neutralization escape and if not, attenuate viral virulence in vivo. In this study, we used a large panel of human nAbs against an epitope which overlaps the interface between the RBD and its receptor, ACE2, to assess their cross-neutralization activities against a panel of human and zoonotic SARS-CoVs and neutralization escape mutants. We also investigated the neutralization escape profiles of these nAbs, and evaluated their effects on receptor binding and virus fitness in vitro and in mice. We found that some nAbs had great potency and breadth in neutralizing multiple viral stains including neutralization escape viruses derived from other nAbs, however no single nAb nor a combination of two blocked neutralization escape. Interestingly, in mice the neutralization escape mutant viruses showed either attenuation (Urbani-background) or increased virulence (GD03-background) consistent with the different binding affinities between their RBDs and the mouse ACE2. We conclude that using either single or dual nAb combinations to target a SARS-CoV RBD epitope that shows plasticity may have limitations for preventing neutralization escape during in vivo immunotherapy. However, RBD-directed nAbs may be useful for providing broad neutralization and prevention of escape variants when combined with other nAbs that target a second conserved epitope with less plasticity and more structural constraint.

    Importance The emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) in 2002 and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in 2012 have resulted in severe human respiratory disease with high death rates. Their zoonotic origins highlight the likelihood of reemergence or further evolution into novel human coronavirus pathogens. Broadly neutralizing antibodies (nAbs) that prevent infection of related viruses represent an important immuno-strategy for combating coronaviruses infections, however for this strategy to succeed, it is essential to uncover nAb-mediated escape pathways and to pioneer strategies that prevent escape. Here, we used SARS-CoV as a research model and examined the escaping pathways of broadly nAbs that target the receptor binding domain (RBD) of the virus. We found that neither single nor two nAbs in combination blocked escape. Our results suggest that targeting conserved regions with less plasticity and more structural constraint rather than SARS-CoV RBD-like region(s) should have broader utility for antibody-based immunotherapy.

  • Structural Plasticity of the Coiled-coil Domain of Rotavirus NSP4 [PublishAheadOfPrint]

  • Rotavirus (RV) nonstructural protein NSP4 is a virulence factor that disrupts cellular Ca2+ homeostasis and plays multiple roles regulating RV replication and the pathophysiology of RV-induced diarrhea. Although its native oligomeric state is unclear, crystallographic studies of the coiled-coil domain (CCD) of NSP4 from two different strains suggest that it functions as a tetramer or a pentamer. While the CCD of simian SA11 NSP4 forms a tetramer that binds Ca2+ at its core, the CCD of a human strain ST3 forms a pentamer lacking the bound Ca2+ despite the residues (E120 and Q123) that coordinate Ca2+-binding being conserved. In these previous studies, while the tetramer crystallized at neutral pH, the pentamer crystallized at low pH suggesting that preference for a particular oligomeric state is pH-dependent and that pH could influence Ca2+-binding. Here, we sought to examine if the CCD of NSP4 from a single RV strain can exist in two oligomeric states regulated by Ca2+ or pH. Biochemical, biophysical and crystallographic studies show that while the CCD of SA11 NSP4 exhibits high affinity binding to Ca2+ at neutral pH and forms a tetramer, at low pH, it does not bind Ca2+ and forms a pentamer, and the transition from tetramer to pentamer is reversible with pH. Mutational analysis shows that Ca2+ binding is necessary for the tetramer formation as an E120A mutant forms a pentamer. We propose that the structural plasticity of NSP4 regulated by pH and Ca2+ may form a basis for its pleiotropic functions during RV replication.

    Importance The non-structural protein NSP4 of rotavirus is a multifunctional protein that plays an important role in virus replication, morphogenesis and pathogenesis. Previous crystallography studies of the coiled-coil domain (CCD) of NSP4 from two different rotavirus strains showed two distinct oligomeric states - a Ca2+-bound tetrameric state and a Ca2+-free pentameric state. Whether NSP4 CCD from the same strain can exist in different oligomeric states, and what factors might regulate its oligomeric preferences are not known. This study used a combination of biochemical, biophysical and crystallography techniques and found that the NSP4 CCD can undergo a reversible transition from a Ca2+-bound tetramer to a Ca2+-free pentamer in response to changes in pH. From these studies, we hypothesize that this remarkable structural adaptability of the CCD forms a basis for the pleiotropic functional properties of NSP4.

  • HIV-1 Uncoating is Facilitated by Dynein and Kinesin-1 [PublishAheadOfPrint]

  • Following entry into the target cell, HIV-1 must reverse transcribe its RNA genome to DNA and traffic to the nuclear envelope, where the viral genome is translocated into the nucleus for subsequent integration into the host cell chromosome. During this time, the viral core, which houses the genome, undergoes a poorly understood process of disassembly, known as uncoating. Collectively, many studies suggest that uncoating is tightly regulated to allow nuclear import of the genome while minimizing the exposure of the newly synthesized DNA to cytosolic DNA sensors. However, whether host cellular proteins facilitate this process remain poorly understood. Here, we report that intact microtubules facilitate HIV-1 uncoating in target cells. Disruption of microtubules with Nocodazole substantially delays HIV-1 uncoating, as revealed with three different assay systems. This defect in uncoating did not correlate with defective reverse transcription at early times post-infection, demonstrating that microtubule facilitated uncoating is distinct from the previously reported role for viral reverse transcription on the uncoating process. . We also find that pharmacological or siRNA mediated inhibition of cytoplasmic dynein and the kinesin-1 heavy chain KIF5B delay uncoating, providing detailed insight into how microtubules facilitate the uncoating process. These studies reveal a previously unappreciated role for microtubules and microtubule motor function in HIV-1 uncoating, establishing a functional link between viral trafficking and uncoating. Targeted disruption of capsid/motor interaction may reveal novel mechanisms to inhibit viral infection or provide opportunities to activate cytoplasmic antiviral responses directed against capsid or viral DNA.

    Importance During HIV-1 infection, fusion of viral and target cell membranes dispenses the viral ribonucleoprotein complex into the cytoplasm of target cells. During this time, the virus must reverse transcribe its RNA genome, traffic from the location of fusion to the nuclear membrane, and undergo the process of uncoating, whereby the viral capsid core disassembles to allow the subsequent nuclear import of the viral genome. Numerous cellular restriction factors target the viral capsid, suggesting that perturbation of the uncoating process represents an excellent antiviral target. However, this uncoating process, and cellular factors which facilitate uncoating, remain poorly understood. The main observation from this study is that normal uncoating requires intact microtubules and is facilitated by dynein and kinesin motors. These factors may either directly inhibit infection or delays them enough to trigger mediators of intrinsic immunity which recognize cytoplasmic capsid or DNA and subsequently induce an antiviral state in these cells.

  • Sendai virus pathogenesis in mice is prevented by Ifit2 and exacerbated by interferon [PublishAheadOfPrint]

  • The Type I/III interferon (IFN) system has major roles in regulating viral pathogenesis, usually ameliorating pathogenesis by impairing virus replication through the antiviral actions of one or more IFN-induced proteins. Ifit2 is one such protein which can be induced by IFN or virus infection and is responsible for protecting mice from neuropathogenesis caused by vesicular stomatitis virus. Here, we show that Ifit2 also protects mice from pathogenesis caused by the respirovirus, Sendai virus (SeV). Mice lacking Ifit2 (Ifit2-/-) suffered severe weight loss and succumbed to intranasal infection with SeV strain 52, at a dose that killed only few wild-type mice. Viral RNA was detectable only in lungs and SeV titers were higher in Ifit2-/- mice, compared to wild-type mice. Similar infiltration of immune cells was found in the lungs of both mouse lines, corresponding to similar levels of many induced cytokines and chemokines. In contrast, IFN-bbeta; and IFN-3 expression were considerably higher in the lungs of Ifit2-/- mice. Surprisingly, type I IFN receptor knock-out (IFNAR-/-) mice were less susceptible to SeV than Ifit2-/- mice, although their pulmonary virus titers were similarly high. To test the intriguing possibility that type I IFN-action enhances pathogenesis in the context of elevated SeV replication in lungs, we generated Ifit2/IFNAR-/- double knock-out mice. These mice were less susceptible to SeV than Ifit2-/- mice, although viral titers in their lungs were even higher. Our results indicate that high SeV replication in the lungs of infected Ifit2-/- mice cooperates with elevated IFN-bbeta; induction to cause disease.

    IMPORTANCE The IFN system is an innate defense against virus infections. It is triggered quickly in infected cells, which then secrete IFN. Via their cell surface receptors on surrounding cells, they induce transcription of numerous IFN-stimulated genes (ISG), which in turn protect these cells by inhibiting virus life cycles. Hence, IFNs are commonly considered as beneficial during virus infections. Here, we report two key findings. Firstly, lack of a single ISG in mice, Ifit2, resulted in high mortality after SeV infection of the respiratory tract, following higher virus loads and higher IFN production in Ifit2-/- lungs. Secondly, mortality of Ifit2-/- mice was reduced when mice also lacked the type I IFN receptor, while SeV loads in lungs were still high. This indicates that type I IFN exacerbates pathogenesis in the SeV model, and that limitation of both viral replication and IFN production is needed for effective prevention of disease.

  • Molecular mimicry between dengue virus and coagulation factors induces antibodies to inhibit thrombin activity and enhance fibrinolysis [PublishAheadOfPrint]

  • Dengue virus (DENV) is the most common cause of viral hemorrhagic fever, and it may lead to life-threating dengue hemorrhagic fever and shock syndrome (DHF/DSS). Because most cases of DHF/DSS occur in patients with secondary DENV infection, anti-DENV antibodies are generally considered to play a role in the pathogenesis of DHF/DSS. Previously, we have found that anti-thrombin antibodies (ATAs) with both anti-thrombotic and profibrinolytic activities are present in the sera of dengue patients. However, the mechanism by which these autoantibodies are induced is unclear. In this study, we demonstrated that antibodies induced by DENV immunization in mice and rabbits could bind to DENV antigens as well as to human thrombin and plasminogen (Plg). The binding of anti-DENV antibodies to thrombin and Plg was inhibited by pre-adsorption with DENV nonstructural protein 1. In addition, affinity-purified ATAs from DENV-immunized rabbit sera could inhibit thrombin activity and enhance Plg activation both in vitro and in vivo. Taken together, our results suggest that molecular mimicry between DENV and coagulation factors can induce the production of autoantibodies with biological effects similar to those of ATAs found in dengue patients. These coagulation factor cross-reactive anti-DENV antibodies can interfere with the balance of coagulation and fibrinolysis, which may lead to the tendency of DHF/DSS patients to bleed.

    Importance Dengue virus (DENV) infection is the most common mosquito-borne viral disease in tropical and sub-tropical areas. Over 50 million DENV infection cases develop each year, and more than 2.5 billion people are at risk of dengue-induced hemorrhagic fever and shock syndrome. Currently, there is no vaccine or drug treatment for DENV. In the present study, we demonstrated that DENV immunization could induce thrombin and plasminogen (Plg) cross-reactive antibodies, which were able to inhibit thrombin activity and enhance Plg activation. These results suggest that molecular mimicry between DENV antigens, thrombin, and Plg may elicit antibodies that disturb hemostasis. The selection of appropriate candidate antigens for use in DENV vaccines should prevent these potentially dangerous autoimmune responses.

  • Establishment and characterization of an air-liquid canine corneal organ culture model to study acute herpes keratitis [PublishAheadOfPrint]

  • Despite the clinical importance of herpes simplex virus (HSV)-induced ocular disease, the underlying pathophysiology of this disease remains poorly understood, in part due to the lack of adequate virus-natural host models in which to study the cellular and viral factors involved in acute corneal infection. We developed an air-liquid canine corneal organ culture model and evaluated its susceptibility to canine herpesvirus type 1 (CHV-1) in order to study ocular herpes in a physiologically relevant natural host model. Canine corneas were maintained in culture at an air-liquid interface for up to 25 days and no degenerative changes were observed in the corneal epithelium during cultivation using histology for morphometric analyses, TUNEL assays and transmission electron microscopy (TEM). Next, canine corneas were inoculated with CHV-1 for 48 hours and at that time point post infection, viral plaques could be visualized in the corneal epithelium and viral DNA copies were detected in both the infected corneas as well as in culture supernatants. In addition, we found that canine corneas produced proinflammatory cytokines in response to CHV-1 infection similarly to what has been described for HSV-1. This emphasizes the value of our model as a virus-natural host model to study ocular herpesvirus infections.

    Importance This study is the first to describe the establishment of an air-liquid canine corneal organ culture model as a useful model to study ocular herpesvirus infections. Advantages of this physiologically relevant model include that it (i) provides a system in which ocular herpes can be studied in a virus-natural host setting and (ii) reduces the number of experimental animals needed. In addition, this long-term explant culture model may facilitate research in other fields also, where non-infectious and infectious ocular diseases of dogs and men are being studied.

  • Drift of the HIV-1 envelope glycoprotein gp120 toward increased neutralization resistance over the course of the epidemic: a comprehensive study using the most potent and broadly neutralizing monoclonal antibodies [PublishAheadOfPrint]

  • Extending our previous analyses to the most recently described broadly neutralizing monoclonal antibodies (bNAbs) we confirm a drift of HIV-1 clade B variants over two decades toward higher resistance to bNAbs targeting almost all the identified gp120 neutralizing epitopes. In contrast, the sensitivity to bNAbs targeting the gp41 MPER remained stable, suggesting a selective pressure on gp120 preferentially. Despite this evolution, selected combinations of bNAbs remain capable to neutralize efficiently most of the circulating variants.

  • Characterization of an alphamesonivirus 3C-like protease defines a special group of nidovirus main proteases [PublishAheadOfPrint]

  • Cavally virus (CavV) and related viruses in the family Mesoniviridae diverged profoundly from other nidovirus lineages but largely retained the characteristic set of replicative enzymes conserved in the Corona- and Roniviridae. Expression of these enzymes in virus-infected cells requires extensive proteolytic processing of two large replicase polyproteins, pp1a and pp1ab, by the viral 3C-like protease (3CLpro). Here, we show that CavV 3CLpro autoproteolytic cleavage occurs at two N-terminal (N1 and N2) and one C-terminal (C1) processing site(s). The mature form of 3CLpro was revealed to be a 314-residue protein produced by cleavage at FKNK1386|SAAS (N2) and YYNQ1700|SATI (C1). Site-directed mutagenesis data suggest that the mesonivirus 3CLpro employs a catalytic Cys-His dyad comprised of CavV pp1a/pp1ab residues Cys-1539 and His-1434. The study further suggests that mesonivirus 3CLpro substrate specificities differ from those of related nidovirus proteases. The presence of Gln (or Glu) at the P1 position was not required for cleavage although residues that control Gln/Glu specificity in related viral proteases are retained in the CavV 3CLpro sequence. Asn at the P2 position was identified as a key determinant for mesonivirus 3CLpro substrate specificity. Other positions, including P4 and P1', are each occupied by structurally related amino acids, indicating a supportive role in substrate binding. Together, the data identify a new subgroup of nidovirus main proteases and support previous conclusions on phylogenetic relationships between the main nidovirus lineages.

    Importance Mesoniviruses have been suggested to provide an evolutionary link between nidovirus lineages with small (13-16 kb) and large (26-32 kb) RNA genome sizes and it has been proposed that a specific set of enzymes, including a proofreading exoribonuclease and other replicase-gene encoded proteins, play a key role in the major genome expansion leading to the currently known lineages of "large nidoviruses". Despite their smaller genome size (20 kb), mesoniviruses retained most of the replicative domains conserved in large nidoviruses and are thus considered interesting models for studying possible key events in the evolution of RNA genomes of exceptional size and complexity. Our study provides the first characterization of a mesonivirus replicase gene-encoded nonstructural protein. The data confirm and extend previous phylogenetic studies of mesoni- and related viruses and pave the way for studies into the formation of the mesonivirus replication complex and functional and structural studies of its functional subunits.

  • DDX3 DEAD-box RNA Helicase is a Host Factor that Restricts Hepatitis B Virus Replication at the Transcriptional Level [PublishAheadOfPrint]

  • DDX3 is a member of the DEAD-box RNA helicase family involved in mRNA metabolism including transcription, splicing, and translation. We previously identified DDX3 as a HBV Pol binding protein, and by using a transient transfection, we found that DDX3 inhibits HBV replication at post-transcriptional level, perhaps following encapsidation. To determine the exact mechanism of the inhibition, we here employed diverse HBV experimental system. Inconsistently, we found that DDX3-mediated inhibition occurs at the level of transcription. By using tetracycline-inducible HBV-producing cells, we observed that lentivirus-mediated DDX3 expression led to a reduced level of HBV RNAs. Importantly, knockdown of DDX3 by short hairpin RNA resulted in augmentation of HBV RNAs in two distinct HBV replication systems: (i) tetracycline-inducible HBV producing cells and (ii) constitutive HBV-producing HepG2.2.15 cells. Moreover, DDX3 knockdown in HBV-susceptible HepG2-NTCP cells, where covalently closed circular DNA (cccDNA) serves as the template for viral transcription, resulted in increased HBV RNAs, validating that transcription regulation by DDX3 occurs on a physiological template. Overall, our results demonstrate that DDX3 represents an intrinsic host antiviral factor that restricts HBV transcription.

    Importance Upon entry into host cells, viruses encounter host factors that restrict viral infection. During evolution viruses have acquired the ability to subvert cellular factors that adversely affect their replication. Such host factors include TRIM5aalpha; and APOBEC3G, which were discovered in retroviruses. The discovery of host restriction factors provided deeper insight into the innate immune response and viral pathogenesis, leading to better understanding of host-virus interactions. In contrast to retroviruses, little is known about host factors that restrict hepatitis B virus (HBV), a virus distantly related to retroviruses. DDX3 DEAD-box RNA helicase is best characterized as an RNA helicase involved in RNA metabolism such as RNA processing and translation. Here, we show that DDX3 inhibits HBV infection at the level of viral transcription.

  • Identification of Nucleotide-Level Changes Impacting Gene Content and Genome Evolution in Orthopoxviruses. [PublishAheadOfPrint]

  • Poxviruses are composed of large dsDNA genomes coding for several hundred genes whose variation has supported virus adaptation to a wide variety of hosts over their long evolutionary history. Comparative genomics has suggested that the Orthopoxvirus genus in particular has undergone reductive evolution, with the most recent common ancestor likely possessing a gene complement consisting of all genes present in any existing modern-day orthopoxvirus species, similar to the current Cowpox virus species. As orthopoxviruses adapt to new environments, the selection pressure on individual genes may be altered, driving sequence divergence and possible loss of function. This is evidenced by accumulation of mutations and loss of protein-coding open reading frames (ORFs) that progress from individual missense mutations, to gene truncation through the introduction of early stop mutations (ESMs), gene fragmentation, and in some cases, a total loss of the ORF. In this study, we have constructed a whole-genome alignment for representative isolates from each Orthopoxvirus species and used it to identify the nucleotide-level changes that have led to gene content variation. By identifying the changes that have led to ESMs, we were able to determine that short indels were the major cause of gene truncations, and that the genome length is proportional to the number of ESMs present. We also identified the number and types of protein functional motifs still present in truncated genes to assess their functional significance.

    IMPORTANCE This work contributes to our understanding of reductive evolution in poxviruses by identifying genomic remnants such as SNPs and indels left behind by evolutionary processes. Our comprehensive analysis of the genomic changes leading to gene truncation and fragmentation was able to detect some of the remnants of these evolutionary processes still present in orthopoxvirus genomes, and suggest that these viruses are under continual adaptation due to changes in their environment. These results further our understanding of the evolutionary mechanisms that drive virus variation, allowing orthopoxviruses to adapt to particular environmental niches. Understanding the past evolutionary history of these virus pathogens may help predict their future evolutionary potential.

  • Expanded regulatory T cells in chronically Friend retrovirus infected mice suppress immunity to a mCMV superinfection [PublishAheadOfPrint]

  • It is still unclear whether expanded and activated regulatory T cells (Tregs) in chronic viral infections can influence primary immune responses against superinfections with unrelated viruses. Expanded Tregs found in the spleens of chronically FV-infected mice decreased mCMV-specific CD8+ T cell responses during acute mCMV superinfection. This suppression of mCMV-specific T cell immunity was only found in organs with FV-induced Treg expansion. Surprisingly, acute mCMV infection itself did not expand or activate Tregs.

  • Crystal structure of HSV-2 gD bound to nectin-1 reveals a conserved mode of receptor recognition [PublishAheadOfPrint]

  • Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are among the most prevalent human pathogens. Both viruses can recognize, via the surface envelope glycoprotein D (gD), human nectin-1 as a functional receptor. Previous studies have successfully elucidated the molecular basis of the binding between HSV-1 gD and nectin-1 by co-crystallography. Despite a high sequence identity between HSV-1 and -2 gDs, the atomic inter-molecule details for HSV-2-gD/nectin-1 interaction remain elusive. Here, we reported the crystal structures of both the unbound and the nectin-1-bound HSV-2 gD. The free gD structure expectedly comprises an IgV-like core and the surface-exposed terminal extensions as observed in its HSV-1 counterpart, but lacks traceable electron densities for a large portion of the terminal elements. These terminal residues were clearly traced in the complex structure as a definitive loop in the N-terminus and an aalpha;mmdash;helix in the C-terminus, thereby showing a conserved nectin-1-binding mode as reported for HSV-1 gD. The interface residues in nectin-1 were further mutated and tested for the gD-interaction by surface plasmon resonance. The resultant binding patterns were similar between HSV-1 and -2 gDs, further supporting a homologous receptor-binding basis by the two viruses for nectin-1. These data, together with a cell-based fusion assay showing a cross-inhibition of the gD/nectin-1 mediated cell-cell fusion by soluble HSV-1 and -2 gDs, provided solid structural and functional evidence that HSV-1/-2 recognizes nectin-1 via the same binding mode. Finally, we also demonstrated that nectin-1 I80 is an important residue involved in gD interaction.

    Importance Despite intensified studies, a detailed picture of the molecular features in the HSV-2-gD/nectin-1 interaction remains unavailable. Previous work focused on HSV-1 gD, which folds into an IgV-like core with large terminal extensions and utilizes the extension elements to engage nectin-1. Here, we reported the crystal structures of HSV-2 gD in both the free and the nectin-1-bound forms. The atomic inter-molecule details for HSV-2-gD/nectin-1 interaction were clearly presented. The observed binding mode is identical to that reported for its HSV-1 counterpart. This structural observation was further supported by our comparative functional assays showing that nectin-1 mutations similarly affect the ligand/receptor interaction of both virus gDs. Taken together, we provided comprehensive structural and functional data demonstrating a conserved receptor-binding mode between HSV-1 and -2 for nectin-1. Our results also indicate that the tropism-difference between the two viruses likely arises from other aspects rather than the gD/nectin-1 binding-features.

  • Development of Dose-Response Models of Creutzfeldt-Jakob Disease Infection in Nonhuman Primates for Assessing the Risk of Transfusion-Transmitted variant Creutzfeldt-Jakob Disease [PublishAheadOfPrint]

  • Estimates for the risk of transmitting variant Creutzfeldt-Jakob disease (vCJD) via blood transfusion have largely relied on data from rodent experiments, but the relationship between dose (amount of infected blood) and response (vCJD infection) has never been well quantified. The goal of this study was to develop a dose-response model based on nonhuman primate data to better estimate the likelihood of transfusion-transmitted vCJD (TTvCJD) in humans. Our model used dose-response data from nonhuman primates inoculated intracerebrally (IC) with brain tissues of patients with sporadic and familial CJD. We analyzed the data statistically using a beta-Poisson dose-response model. We further adjusted model parameters to account for the differences in infectivity between blood and brain tissue and in transmission efficiency between intravenous (IV) and IC routes to estimate dose-dependent TTvCJD infection. The model estimates a mean infection rate of 76% among recipients who receive one unit of whole blood collected from an infected donor near the end of the incubation period. The nonhuman primate model provides estimates that are more consistent with those derived from a risk analysis of transfused non-leukoreduced red blood cells in United Kingdom compared to prior estimates based on rodent models.

    IMPORTANCE TTvCJD was recently identified as one of three emerging infectious diseases posing the greatest immediate threat to the safety of the blood supply. Cases of TTvCJD were reported in recipients of non-leukoreduced red blood cells and coagulation Factor VIII manufactured from blood of UK donors. As the quantity of abnormal prions (the causative agent of TTvCJD) varies significantly in different blood components and products, it is necessary to quantify the dose-response relationship for a wide range of doses for the vCJD agent in transfused blood and plasma derivatives. In this paper we suggest the first mechanistic dose-response model for TTvCJD infection based on data from experiments with nonhuman primates. This new model may improve estimates of the possible risk to humans.

  • Analysis of the evolution and structure of a complex intra-host viral population in chronic Hepatitis C mapped by ultra-deep pyrosequencing. [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) causes chronic infection in up to 50-80% of infected individuals. Hypervariable region 1 (HVR1) variability is frequently studied to gain an insight into the mechanisms of HCV adaptation during chronic infection, but the changes to and persistence of HCV subpopulations during intra-host evolution are poorly understood. In this study, we have used ultra-deep pyrosequencing (UDPS) to map viral heterogeneity of a single patient over 9.6 years of chronic HCV genotype 4a infection. Informed error correction of the raw UDPS data was performed using a temporally matched clonal data set. The resultant data set reported the detection of low frequency recombinants throughout the study period implying that recombination is an active mechanism through which HCV can explore novel sequence space. The data indicates that poly-virus infection of hepatocytes has occurred but that the fitness quotients of recombinant daughter virions are too low to compete against the parental genomes. The subpopulations of parental genomes contributing to the recombination events highlighted a dynamic virome where subpopulations of variants are in competition. In addition, we provide direct evidence that demonstrates the growth of subdominant populations to dominance in the absence of a detectable humoral response.

    IMPORTANCE Analysis of ultra-deep pyrosequencing data sets derived from virus amplicons frequently relies on software tools that are not optimised for amplicon analysis, assume random incorporation of sequencing errors and are focused on achieving higher specificity at the expense of sensitivity. Such analysis is further complicated by the presence of hypervariable regions. In this study, we make use of a temporally matched reference sequence data set to inform error correction algorithms. Using this methodology we were able to (1) detect multiple instances of Hepatitis C virus intra-subtype recombination at the E1/E2 junction (a phenomenon rarely reported in the literature) and (2) interrogate the longitudinal quasispecies complexity of the virome. Parallel to the UDPS, isolation of IgG-bound virions was found to be co-incident with the collapse of specific viral subpopulations.

  • Comparative Genome Analysis of Four Elephant Endotheliotropic Herpesviruses EEHV3, EEHV4, EEHV5 and EEHV6 from Cases of Hemorrhagic Disease or Viremia. [PublishAheadOfPrint]

  • Summary: The genomes of three types of novel endotheliotropic herpesviruses (EEHV1A, EEHV1B and EEHV2) associated with lethal hemorrhagic disease in Asian elephants have been previously well characterized and assigned to a new Proboscivirus genus. Here we have generated 112-kb of DNA sequence data from segments of four more types of EEHV by direct targeted PCR from blood or necropsy tissue of six viremic elephants. Comparative phylogenetic analysis of nearly 30 protein-encoding genes of EEHV5 and EEHV6 show that they diverge uniformly by nearly 20% from their closest relatives, EEHV2 and EEHV1A respectively, and are likely to have similar overall gene content and genome organization. In contrast, seven EEHV3 and EEHV4 genes analysed differ from those of all other EEHVs by 37% and have a (G-plus-C)-content of 63% compared to just 42% for the others. Three strains of EEHV5 analyzed clustered into two partially chimeric subgroups EEHV5A and EEHV5B that diverge by 19% within three small non-contiguous segments totalling 6.2-kb. We conclude that all six EEHV types should be designated as independent species within a proposed new fourth Deltaherpesvirinae subfamily of mammalian herpesviruses. These likely initially diverged close to 100 million years ago when the ancestors of modern elephants split from all other placental mammals, then evolved into two major branches with high or low (G-plus-C)-content about 35 million years ago. Later additional branching events subsequently generated three paired sister taxon lineages of which EEHV1 plus EEHV6, EEHV5 plus EEHV2 and EEHV4 plus EEHV3 may represent Asian and African elephant versions, respectively.

    Importance: One of the factors threatening the long-term survival of endangered Asian elephants in both wild range countries and in captive breeding populations in zoos is a highly lethal hemorrhagic herpesvirus disease that has killed at least 70 young Asian elephants worldwide. The genomes of the first three types of EEHVs (or Probosciviruses) identified have been partially characterized in the preceding paper. Here we have used PCR DNA sequence analysis from multiple segments of DNA amplified directly from blood or necropsy tissue of six more selected cases of hemorrhagic disease to partially characterize four other types of EEHVs from either Asian or African elephants. We propose that all six types and two chimeric subtypes of EEHV belong to multiple lineages of both AT-rich and GC-rich branches within a new subfamily to be named the Deltaherpesvirinae, which evolved separately from all other mammalian herpesviruses about100 million years ago.

  • Elephant Endotheliotropic Herpesviruses EEHV1A, EEHV1B and EEHV2 from Cases of Hemorrhagic Disease are Highly Diverged from Other Mammalian Herpesviruses and May Form a New Subfamily. [PublishAheadOfPrint]

  • A family of novel endotheliotropic herpesviruses (EEHV) assigned to the genus Proboscivirus have been identified as the cause of fatal hemorrhagic disease in 70 young Asian elephants worldwide. Although EEHV cannot be grown in cell culture, we have determined a total of 378-kb of viral genomic DNA sequence directly from clinical tissue of six lethal cases and two survivors. Overall, the data obtained encompasses 57 genes, including orthologues of 32 core genes common to all herpesviruses, 14 genes found in some other herpesviruses, plus ten novel genes including a single-large putative transcriptional regulatory protein (ORF-L). Based on differences in gene content and organization plus phylogenetic analyses of conserved core proteins that have just 20% to 50% or less identity to orthologues in other herpesviruses, we propose that EEHV1A, EEHV1B and EEHV2 could be considered a new Deltaherpesvirinae subfamily of mammalian herpesviruses that evolved as an intermediate branch between the Betaherpesvirinae and Gammaherpesvirinae. Unlike cytomegaloviruses, EEHV genomes encode RRB, TK and UL9-like OBP proteins and have an alphaherpesvirus-like dyad-symmetry ori-Lyt domain. They also differ from all known betaherpesviruses by having a 40-kb large-scale inversion of core gene blocks I, II and III. EEHV1 and EEHV2 DNA differ uniformly by more than 25%, but EEHV1 clusters into two major sub-groups designated EEHV1A and EEHV1B with ancient partially chimeric features. Whereas large segments are nearly identical, three non-adjacent loci totalling 15-kb diverge by between 21 and 37%. One strain of EEHV1B analyzed is interpreted to be a modern partial recombinant with EEHV1A.

    Importance: Asian elephants are an endangered species whose survival is under extreme pressure in wild range countries and whose captive breeding populations in zoos are not self-sustaining. In 1999, a novel class of herpesviruses called EEHVs was discovered that have caused a rapidly lethal hemorrhagic disease in 20% of all captive Asian elephant calves born in zoos in the USA and Europe since 1980. The disease is increasingly being recognized in Asian range countries as well. These viruses cannot be grown in cell culture, but by direct PCR DNA sequence analysis from segments totalling 15-30% of the genomes from blood or necropsy tissue of eight different cases, we have determined that they not only fall into multiple types and chimeric subtypes of a novel Proboscivirus genus, but propose that they should also be classified as the first examples of a new mammalian herpesvirus subfamily named the Deltaherpesvirinae.

  • Molecular requirement for sterols in herpes simplex virus entry and infectivity [PublishAheadOfPrint]

  • Herpes simplex virus 1 (HSV-1) required cholesterol for virion-induced membrane fusion. HSV successfully entered DHCR24-/- cells, which lack a desmosterol-to-cholesterol conversion enzyme, indicating entry can occur independently of cholesterol. Depletion of desmosterol from these cells resulted in diminished HSV-1 entry, suggesting a general sterol requirement for HSV-1 entry and that desmosterol can operate in virus entry. Cholesterol functioned more effectively than desmosterol, suggesting that the hydrocarbon tail of cholesterol influences viral entry.

  • Amino Acid Changes in the Influenza A Virus PA Protein that Attenuate Avian H5N1 Viruses in Mammals [PublishAheadOfPrint]

  • The influenza viral polymerase complex affects host tropism and pathogenicity. In particular, several amino acids in the PB2 polymerase subunit are essential for the efficient replication of avian influenza viruses in mammals. The PA polymerase subunit also contributes to host range and pathogenicity. Here, we report that the PA proteins of several highly pathogenic avian H5N1 viruses have attenuating properties in mammalian cells, and that the attenuating phenotype is conferred by strain-specific amino acid changes. Specifically, lysine at position 185 of A/duck/Vietnam/TY165/2010 (TY165; H5N1) PA induced strongly attenuating effects in vitro and in vivo. More importantly, the introduction of the arginine residue commonly found at this position in PA significantly increased the viral polymerase activity of TY165 in mammalian cells, and its virulence and pathogenicity in mice. These findings demonstrate that the PA protein plays an important role in influenza virulence and pathogenicity.

    IMPORTANCE Highly pathogenic influenza viruses of the H5N1 subtype cause severe respiratory infections in humans, which have resulted in death in nearly two-thirds of the laboratory-confirmed cases. We found that the viral PA polymerase subunit of several H5N1 viruses possesses amino acid changes that attenuate virus replication in mammalian cells (yet the H5N1 viruses possessing these mutations are highly pathogenic in mice). Specifically, we found that an arginine-to-lysine substitution at position 185 of an H5N1 virus PA protein significantly affected that virus' virulence and pathogenicity in mice. The PA protein thus plays a role in the pathogenicity of highly pathogenic H5N1 influenza viruses.

  • M27 expressed by cytomegalovirus counteracts effective type I IFN induction of myeloid cells, but not of plasmacytoid dendritic cells [PublishAheadOfPrint]

  • In healthy individuals the functional immune system effectively confines human cytomegalovirus (CMV) replication, while viral immune evasion and persistence preclude sterile immunity. Mouse CMV (MCMV) is a well-established model to study the delicate CMV-host balance. Effective control of MCMV infection depends on the induction of protective type I interferon (IFN-I) responses. Nevertheless, it is unclear whether also in professional antigen presenting cell subsets MCMV-encoded evasins inhibit the induction of IFN-I responses. Upon MCMV treatment, enhanced expression of MCMV immediate early and early proteins was detected in bone marrow cultures of macrophages and myeloid dendritic cells when compared with plasmacytoid dendritic cell cultures, whereas plasmacytoid dendritic cells mounted more vigorous IFN-I responses. Experiments with Toll-like receptor (TLR) and/or RIG-I like helicase (RLH) deficient cell subsets revealed that upon MCMV treatment of myeloid cells IFN-I responses were triggered independent of TLR- and RLH-signaling, whereas in plasmacytoid dendritic cells IFN-I induction was strictly TLR-dependent. Macrophages and myeloid dendritic cells treated either with UV-inactivated MCMV or live MCMV that lacked the STAT2 antagonist pM27 mounted significantly higher IFN-I responses than cells treated with live wild-type MCMV. In contrast, plasmacytoid dendritic cells responded similarly to UV-inactivated and live MCMV. These experiments illustrated that pM27 not only inhibited IFN-I mediated receptor signaling but also evaded the induction of IFN responses in myeloid dendritic cells. Furthermore, we found that additional MCMV encoded evasin(s) were needed to efficiently shut off IFN-I responses of macrophages, but not of myeloid dendritic cells, thus further elucidating the subtle adjustment of the host-pathogen balance.

    Importance MCMV may IFN-I responses in fibroblasts and epithelial as well as in antigen presenting cell subsets. We focused on the analysis of IFN-I responses of antigen presenting cell subsets, including plasmacytoid dendritic cells, myeloid dendritic cells, and macrophages, which all are triggered by MCMV to mount IFN-I responses. Interestingly, myeloid dendritic cells and macrophages, but not plasmacytoid dendritic cells, are readily MCMV infected and support viral gene expression. As expected from previous studies, plasmacytoid dendritic cells sense MCMV Toll-like receptor (TLR)9 dependently, whereas in myeloid cells IFN-I induction is entirely TLR and RLH independent. MCMV encoded pM27 does not impair IFN-I induction of plasmacytoid dendritic cells, while in myeloid dendritic cells it reduces IFN-I responses. In macrophages, pM27 plus other not yet identified evasins profoundly inhibit the induction of IFN-I responses. Collectively, these results illustrate that MCMV evolved diverse mechanisms to differentially modulate IFN-I responses in single immune cell subsets.

  • Characterization of a broadly neutralizing monoclonal antibody that targets the fusion domain of group 2 influenza A virus hemagglutinin [PublishAheadOfPrint]

  • Due to continuous changes to its antigenic regions, influenza viruses can evade immune detection and cause a significant amount of morbidity and mortality around the world. Influenza vaccinations can protect against disease, but must be annually reformulated to match the current circulating strains. Towards the development of a broad-spectrum influenza vaccine, the elucidation of conserved epitopes is paramount. To this end, we designed an immunization strategy in mice to boost the humoral response against conserved regions of the hemagglutinin (HA) glycoprotein. Of note, generation and identification of broadly neutralizing antibodies that target group 2 HAs are rare and thus far have yielded only a handful of monoclonal antibodies Here, we demonstrate that mouse mAb, 9H10, has broad and potent in vitro neutralizing activity against H3 and H10 group 2 influenza A subtypes. In the mouse model, mAb 9H10 protects mice against two divergent mouse-adapted H3N2 strains, in both pre- and post-exposure administration regimens. In vitro and cell-free assays suggest that mAb 9H10 inhibits viral replication by blocking HA-dependent fusion of the viral and endosomal membranes early in the replication cycle and by disrupting viral particle egress in the late stage of infection. Interestingly, electron microscopy reconstructions of mAb 9H10 bound to the HA reveal that it binds a similar binding footprint to mAbs CR8020 and CR8043.

    IMPORTANCE The influenza hemagglutinin is the major antigenic target of the humoral immune response. However, due to continuous antigenic changes that occur on the surface of this glycoprotein, influenza viruses can escape the immune system and cause significant disease to the host. Towards the development of broad-spectrum therapeutics and vaccines against influenza virus, elucidation of conserved regions of influenza viruses is crucial. Thus, defining these types of epitopes through the generation and characterization of broadly neutralizing monoclonal antibodies can greatly assist others in highlighting conserved regions of hemagglutinin. Here, we demonstrate that mAb 9H10 that targets the hemagglutinin stalk has broadly neutralizing activity against group 2 influenza A viruses in vitro and in vivo.

  • The HERV-K human endogenous retrovirus envelope protein antagonises Tetherin antiviral activity [PublishAheadOfPrint]

  • Endogenous retroviruses are the remnants of past retroviral infections that are scattered within mammalian genomes. In humans, most of these elements are old degenerate sequences that have lost their coding properties. The HERV-K(HML2) family is an exception: it recently amplified in the human genome and corresponds to the most active proviruses, with some intact open reading frames and the potential to encode viral particles. Here, using a reconstructed consensus element, we show that HERV-K(HML2) proviruses are able to inhibit Tetherin, a cellular restriction factor that is active against most enveloped viruses and acts by keeping the viral particles attached to the cell surface. More precisely, we identify the Envelope protein (Env) as the viral effector active against Tetherin. Through immunoprecipitation experiments, we show that recognition of Tetherin is mediated by the surface subunit of Env. Similar to Ebola glycoprotein, HERV-K(HML2) Env does not mediate Tetherin degradation or cell surface removal, and therefore uses a yet undescribed mechanism to inactivate Tetherin. We also assessed all natural complete "alleles" of endogenous HERV-K(HML2) Env described to date for their ability to inhibit Tetherin, and found that two of them (out of six) can block Tetherin restriction. However, due to their recent amplification, HERV-K(HML2) elements are extremely polymorphic in the human population and it is likely that individuals will not all possess the same "anti-Tetherin" potential. Because of Tetherin role as a restriction factor capable of inducing innate immune responses, this could have functional consequences for individual responses to infection.

    IMPORTANCE Tetherin, a cellular protein initially characterised for its role against HIV-1, has been proven to counteract numerous enveloped viruses. It blocks the release of viral particles from producer cells, keeping them tethered to the cell surface. Several viruses have developed strategies to inhibit Tetherin activity, allowing them to efficiently infect and replicate in their host. Here we show that human HERV-K (HML2) elements, the remnants of an ancient retroviral infection, possess an anti-Tetherin activity which is mediated by the envelope protein. It is likely that this activity was an important factor that contributed to the recent, human-specific amplification of this family of elements. Also, due to their recent amplification, HERV-K(HML2) elements are highly polymorphic in the human population. Since Tetherin is a mediator of innate immunity, inter-individual variations among HERV-K(HML2) Env genes may result in differences in immune responses to infection.

  • Identification of molecular markers associated with alteration of receptor-binding specificity in a novel genotype of highly pathogenic avian influenza A(H5N1) viruses detected in Cambodia in 2013. [PublishAheadOfPrint]

  • Human infections with influenza A(H5N1) virus in Cambodia increased sharply during 2013. Molecular characterization of viruses detected in clinical specimens from human cases revealed the presence of mutations associated with alteration of receptor-binding specificity (K189R, Q222L) and respiratory droplet transmission in ferrets (N220K with Q222L). Discovery of quasispecies at position 222 (Q/L), in addition to absence of the mutations in poultry/environmental samples, suggested the mutations occurred during human infection and did not transmit further.

  • Severity of clinical disease and pathology in ferrets experimentally infected with influenza viruses is influenced by inoculum volume [PublishAheadOfPrint]

  • Ferrets are a valuable model for influenza pathogenesis, virus transmission and antiviral therapy studies. However, the contributions of volume of inoculum administered and the ferret's respiratory tract anatomy to disease outcome have not been explored. We noted variation in clinical disease outcomes and the volume of inoculum administered and investigated these differences by administering two influenza viruses (A/California/07/2009 (H1N1pdm) and A/Minnesota/11/2010 (H3N2v) to ferrets intranasally at a dose of 106 TCID50 in a range of inoculum volumes (0.2, 0.5 or 1.0ml), and followed viral replication, clinical disease and pathology over 6 days. Clinical illness and respiratory tract pathology were most severe and most consistent when the viruses were administered in a volume of 1.0 ml. Using a modified micro-CT imaging method and examining gross specimens, we found that the right main stem bronchus was consistently larger in diameter than the left main stem bronchus though the latter was longer and straighter. These anatomic features likely influence the distribution of inoculum in the lower respiratory tract. A 1.0 ml volume of inoculum is optimal for delivery of virus to the lower respiratory tract of ferrets, particularly when evaluation of clinical disease is desired. Furthermore, we highlight important anatomical features of the ferret lung that influence the kinetics of viral replication, clinical disease severity and lung pathology.

    IMPORTANCE Ferrets are a valuable model for influenza pathogenesis, virus transmission and antiviral therapy studies. Clinical disease in ferrets is an important parameter in evaluating the virulence of novel influenza viruses and findings are extrapolated to virulence in humans. Therefore, the accuracy and reproducibility of data between laboratories is highly desirable. We have found that, even when the same virus was administered at similar doses, a range of clinical disease outcomes were reported by different investigators, from asymptomatic infection to severe weight loss, ocular and nasal discharge, sneezing and lethargy. We found that a wide range of inoculum volumes were used to experimentally infect ferrets and we sought to determine whether the variations in disease outcome were the result of the volume of inoculum administered. These data highlight some less explored features of the model, methods of experimental infection, and clinical disease outcomes in a research setting.

  • Recent Population Expansions of Hepatitis B Virus in the United States [PublishAheadOfPrint]

  • The recent epidemic history of hepatitis B virus (HBV) infections in the United States is complex, as indicated by current disparity in HBV genotype distribution between acute and chronic hepatitis B cases and rapid decline in hepatitis B incidence since the 1990s. We report temporal changes in genetic composition of the HBV population using whole-genome sequences (n=179) from acute hepatitis B cases (n=1206) identified through the Sentinel County Surveillance for Acute Hepatitis (1998-2006). HBV belonged mainly to subtypes A2 (75%) and D3 (18%), with times of their most recent common ancestors being, respectively, 1979 and 1987, respectively. A2 underwent rapid population expansions in ca. 1995 and ca. 2002, coinciding with transient rises in acute hepatitis B notification rates among adults; D3 underwent expansion in ca. 1998. A2 strains from cases identified after 2002, compared to those before 2002, tended to cluster phylogenetically, indicating selective expansion of specific strains, and were significantly reduced in genetic diversity (p = 0.001) and frequency of drug-resistance mutations (p = 0.001). The expansion of genetically close HBV A2 strains was associated with risk of infection among male homosexuals (p = 0.03). Incident HBV strains circulating in the US were recent in origin, and restricted in genetic diversity. Disparate transmission dynamics among phylogenetic lineages affected the genetic composition of HBV populations and their capacity to maintain drug-resistance mutations. The tendency of selectively expanding HBV strains to be transmitted among male homosexuals highlights the need to improve hepatitis B vaccination coverage among at-risk adults.

    IMPORTANCE Hepatitis B virus (HBV) remains an important cause of acute and chronic liver disease globally, and in the United States. Genetic analysis of HBV whole genomes from cases of acute hepatitis B identified from 1998-2006 in the United States showed dominance of genotype A2 (75%), followed by D3 (18%). Strains of both subtypes were recent in origin and underwent rapid population expansions from 1995-2000, indicating increase in transmission rate for certain HBV strains during a period of decline in the reported incidence of acute hepatitis B in the US. HBV A2 strains from a particular cluster that experienced the most recent population expansion were more commonly detected among men who have sex with men. Vaccination needs to be stepped up to protect persons who remain at risk of HBV infection.

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