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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]

  • Guinea Pig Prion Protein Supports Rapid Propagation of Bovine Spongiform Encephalopathy and Variant Creutzfeldt-Jakob Disease Prions [Prions]

  • The biochemical and neuropathological properties of bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) prions are faithfully maintained upon transmission to guinea pigs. However, primary and secondary transmissions of BSE and vCJD in guinea pigs result in long incubation periods of ~450 and ~350 days, respectively. To determine if the incubation periods of BSE and vCJD prions could be shortened, we generated transgenic (Tg) mice expressing guinea pig prion protein (GPPrP). Inoculation of Tg(GPPrP) mice with BSE and vCJD prions resulted in mean incubation periods of 210 and 199 days, respectively, which shortened to 137 and 122 days upon serial transmission. In contrast, three different isolates of sporadic CJD prions failed to transmit disease to Tg(GPPrP) mice. Many of the strain-specified biochemical and neuropathological properties of BSE and vCJD prions, including the presence of type 2 protease-resistant PrPSc, were preserved upon propagation in Tg(GPPrP) mice. Structural modeling revealed that two residues near the N-terminal region of aalpha;-helix 1 in GPPrP might mediate its susceptibility to BSE and vCJD prions. Our results demonstrate that expression of GPPrP in Tg mice supports the rapid propagation of BSE and vCJD prions and suggest that Tg(GPPrP) mice may serve as a useful paradigm for bioassaying these prion isolates.

    IMPORTANCE Variant Creutzfeldt-Jakob disease (vCJD) and bovine spongiform encephalopathy (BSE) prions are two of the prion strains most relevant to human health. However, propagating these strains in mice expressing human or bovine prion protein has been difficult because of prolonged incubation periods or inefficient transmission. Here, we show that transgenic mice expressing guinea pig prion protein are fully susceptible to vCJD and BSE prions but not to sporadic CJD prions. Our results suggest that the guinea pig prion protein is a better, more rapid substrate than either bovine or human prion protein for propagating BSE and vCJD prions.

  • Inhibition of the Membrane Attack Complex by Dengue Virus NS1 through Interaction with Vitronectin and Terminal Complement Proteins [Virus-Cell Interactions]

  • Dengue virus (DENV) infects millions of people worldwide and is a major public health problem. DENV nonstructural protein 1 (NS1) is a conserved glycoprotein that associates with membranes and is also secreted into the plasma in DENV-infected patients. The present study describes a novel mechanism by which NS1 inhibits the terminal complement pathway. We first identified the terminal complement regulator vitronectin (VN) as a novel DENV2 NS1 binding partner by using a yeast two-hybrid system. This interaction was further assessed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) assay. The NS1-VN complex was also detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the DENV2 NS1 protein, either by itself or by interacting with VN, hinders the formation of the membrane attack complex (MAC) and C9 polymerization. Finally, we showed that DENV2, West Nile virus (WNV), and Zika virus (ZIKV) NS1 proteins produced in mammalian cells inhibited C9 polymerization. Taken together, our results points to a role for NS1 as a terminal pathway inhibitor of the complement system.

    IMPORTANCE Dengue is the most important arthropod-borne viral disease nowadays and is caused by dengue virus (DENV). The flavivirus NS1 glycoprotein has been characterized functionally as a complement evasion protein that can attenuate the activation of the classical, lectin, and alternative pathways. The present study describes a novel mechanism by which DENV NS1 inhibits the terminal complement pathway. We identified the terminal complement regulator vitronectin (VN) as a novel DENV NS1 binding partner, and the NS1-VN complex was detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the NS1-VN complex inhibited membrane attack complex (MAC) formation, thus interfering with the complement terminal pathway. Interestingly, NS1 itself also inhibited MAC activity, suggesting a direct role of this protein in the inhibition process. Our findings imply a role for NS1 as a terminal pathway inhibitor of the complement system.

  • Baculovirus FP25K Localization: Role of the Coiled-Coil Domain [Virus-Cell Interactions]

  • Two types of viruses are produced during the baculovirus life cycle: budded virus (BV) and occlusion-derived virus (ODV). A particular baculovirus protein, FP25K, is involved in the switch from BV to ODV production. Previously, FP25K from the model alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was shown to traffic ODV envelope proteins. However, FP25K localization and the domains involved are inconclusive. Here we used a quantitative approach to study FP25K subcellular localization during infection using an AcMNPV bacmid virus that produces a functional AcMNPV FP25K-green fluorescent protein (GFP) fusion protein. During cell infection, FP25K-GFP localized primarily to the cytoplasm, particularly amorphous structures, with a small fraction being localized in the nucleus. To investigate the sequences involved in FP25K localization, an alignment of baculovirus FP25K sequences revealed that the N-terminal putative coiled-coil domain is present in all alphabaculoviruses but absent in betabaculoviruses. Structural prediction indicated a strong relatedness of AcMNPV FP25K to long interspersed element 1 (LINE-1) open reading frame 1 protein (ORF1p), which contains an N-terminal coiled-coil domain responsible for cytoplasmic retention. Point mutations and deletions of this domain lead to a change in AcMNPV FP25K localization from cytoplasmic to nuclear. The coiled-coil and C-terminal deletion viruses increased BV production. Furthermore, a betabaculovirus FP25K protein lacking this N-terminal coiled-coil domain localized predominantly to the nucleus and exhibited increased BV production. These data suggest that the acquisition of this N-terminal coiled-coil domain in FP25K is important for the evolution of alphabaculoviruses. Moreover, with the divergence of preocclusion nuclear membrane breakdown in betabaculoviruses and membrane integrity in alphabaculoviruses, this domain represents an alphabaculovirus adaptation for nuclear trafficking of occlusion-associated proteins.

    IMPORTANCE Baculovirus infection produces two forms of viruses: BV and ODV. Manufacturing of ODV involves trafficking of envelope proteins to the inner nuclear membrane, mediated partly through the FP25K protein. Since FP25K is present in alpha-, beta-, and gammabaculoviruses, it is uncertain if this trafficking function is conserved. In this study, we looked at alpha- and betabaculovirus FP25K trafficking by its localization. Alphabaculovirus FP25K localized primarily to the cytoplasm, whereas betabaculovirus FP25K localized to the nucleus. We found that an N-terminal coiled-coil domain present in all alphabaculovirus FP25K proteins, but absent in betabaculovirus FP25K, was critical for alphabaculovirus FP25K cytoplasmic localization. We believe that this represents an evolutionary process that partly led to the gain of function of this N-terminal coiled-coil domain in alphabaculovirus FP25K to aid in nuclear trafficking of occlusion-associated proteins. Due to betabaculovirus breakdown of the nuclear membrane before occlusion, this function is not needed, and the domain was lost or never acquired.

  • Selection Pressure in the Human Adenovirus Fiber Knob Drives Cell Specificity in Epidemic Keratoconjunctivitis [Genetic Diversity and Evolution]

  • Human adenoviruses (HAdVs) contain seven species (HAdV-A to -G), each associated with specific disease conditions. Among these, HAdV-D includes those viruses associated with epidemic keratoconjunctivitis (EKC), a severe ocular surface infection. The reasons for corneal tropism for some but not all HAdV-Ds are not known. The fiber protein is a major capsid protein; its C-terminal "knob" mediates binding with host cell receptors to facilitate subsequent viral entry. In a comprehensive phylogenetic analysis of HAdV-D capsid genes, fiber knob gene sequences of HAdV-D types associated with EKC formed a unique clade. By proteotyping analysis, EKC virus-associated fiber knobs were uniquely shared. Comparative structural modeling showed no distinct variations in fiber knobs of EKC types but did show variation among HAdV-Ds in a region overlapping with the known CD46 binding site in HAdV-B. We also found signature amino acid positions that distinguish EKC from non-EKC types, and by in vitro studies we showed that corneal epithelial cell tropism can be predicted by the presence of a lysine or alanine at residue 240. This same amino acid residue in EKC viruses shows evidence for positive selection, suggesting that evolutionary pressure enhances fitness in corneal infection, and may be a molecular determinant in EKC pathogenesis.

    IMPORTANCE Viruses adapt various survival strategies to gain entry into target host cells. Human adenovirus (HAdV) types are associated with distinct disease conditions, yet evidence for connections between genotype and cellular tropism is generally lacking. Here, we provide a structural and evolutionary basis for the association between specific genotypes within HAdV species D and epidemic keratoconjunctivitis, a severe ocular surface infection. We find that HAdV-D fiber genes of major EKC pathogens, specifically the fiber knob gene region, share a distinct phylogenetic clade. Deeper analysis of the fiber gene revealed that evolutionary pressure at crucial amino acid sites has a significant impact on its structural conformation, which is likely important in host cell binding and entry. Specific amino acids in hot spot residues provide a link to ocular cell tropism and possibly to corneal pathogenesis.

  • Human Herpesvirus 6B Downregulates Expression of Activating Ligands during Lytic Infection To Escape Elimination by Natural Killer Cells [Virus-Cell Interactions]

  • The Herpesviridae family consists of eight viruses, most of which infect a majority of the human population. One of the less-studied members is human herpesvirus 6 (HHV-6) (Roseolovirus), which causes a mild, well-characterized childhood disease. Primary HHV-6 infection is followed by lifelong latency. Reactivation frequently occurs in immunocompromised patients, such as those suffering from HIV infection or cancer or following transplantation, and causes potentially life-threatening complications. In this study, we investigated the mechanisms that HHV-6 utilizes to remain undetected by natural killer (NK) cells, which are key participants in the innate immune response to infections. We revealed viral mechanisms which downregulate ligands for two powerful activating NK cell receptors: ULBP1, ULBP3, and MICB, which trigger NKG2D, and B7-H6, which activates NKp30. Accordingly, this downregulation impaired the ability of NK cells to recognize HHV-6-infected cells. Thus, we describe for the first time immune evasion mechanisms of HHV-6 that protect lytically infected cells from NK elimination.

    IMPORTANCE Human herpesvirus 6 (HHV-6) latently infects a large portion of the human population and can reactivate in humans lacking a functional immune system, such as cancer or AIDS patients. Under these conditions, it can cause life-threatening diseases. To date, the actions and interplay of immune cells, and particularly cells of the innate immune system, during HHV-6 infection are poorly defined. In this study, we aimed to understand how cells undergoing lytic HHV-6 infection interact with natural killer (NK) cells, innate lymphocytes constituting the first line of defense against viral intruders. We show that HHV-6 suppresses the expression of surface proteins that alert the immune cells by triggering two major receptors on NK cells, NKG2D and NKp30. As a consequence, HHV-6 can replicate undetected by the innate immune system and potentially spread infection throughout the body. This study advances the understanding of HHV-6 biology and the measures it uses to successfully escape immune elimination.

  • Respiratory Syncytial Virus Infection Triggers Epithelial HMGB1 Release as a Damage-Associated Molecular Pattern Promoting a Monocytic Inflammatory Response [Cellular Response to Infection]

  • Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infant and elderly populations worldwide. Currently, there is no efficacious vaccine or therapy available for RSV infection. The molecular mechanisms underlying RSV-induced acute airway disease and associated long-term consequences remain largely unknown; however, experimental evidence suggests that the lung inflammatory response plays a fundamental role in the outcome of RSV infection. High-mobility group box 1 (HMGB1) is a nuclear protein that triggers inflammation when released from activated immune or necrotic cells and drives the pathogenesis of various infectious agents. Although HMGB1 has been implicated in many inflammatory diseases, its role in RSV-induced airway inflammation has not been investigated. This study investigates the molecular mechanism of action of extracellularly released HMGB1 in airway epithelial cells (A549 and small airway epithelial cells) to establish its role in RSV infection. Immunofluorescence microscopy and Western blotting results showed that RSV infection of human airway epithelial cells induced a significant release of HMGB1 as a result of translocation of HMGB1 from the cell nuclei to the cytoplasm and subsequent release into the extracellular space. Treating RSV-infected A549 cells with antioxidants significantly inhibited RSV-induced HMGB1 extracellular release. Studies using recombinant HMGB1 triggered immune responses by activating primary human monocytes. Finally, HMGB1 released by airway epithelial cells due to RSV infection appears to function as a paracrine factor priming epithelial cells and monocytes to inflammatory stimuli in the airways.

    IMPORTANCE RSV is a major cause of serious lower respiratory tract infections in young children and causes severe respiratory morbidity and mortality in the elderly. In addition, to date there is no effective treatment or vaccine available for RSV infection. The mechanisms responsible for RSV-induced acute airway disease and associated long-term consequences remain largely unknown. The oxidative stress response in the airways plays a major role in the pathogenesis of RSV. HMGB1 is a ubiquitous redox-sensitive multifunctional protein that serves as both a DNA regulatory protein and an extracellular cytokine signaling molecule that promotes airway inflammation as a damage-associated molecular pattern. This study investigated the mechanism of action of HMGB1 in RSV infection with the aim of identifying new inflammatory pathways at the molecular level that may be amenable to therapeutic interventions.

  • Neglected but Important Role of Apolipoprotein E Exchange in Hepatitis C Virus Infection [Structure and Assembly]

  • Hepatitis C virus (HCV) is a major cause of chronic liver disease, infecting approximately 170 million people worldwide. HCV assembly is tightly associated with the lipoprotein pathway. Exchangeable apolipoprotein E (apoE) is incorporated on infectious HCV virions and is important for infectious HCV virion morphogenesis and entry. Moreover, the virion apoE level is positively correlated with its ability to escape E2 antibody neutralization. However, the role of apoE exchange in the HCV life cycle is unclear. In this study, the relationship between apoE expression and cell permissiveness to HCV infection was assessed by infecting apoE knockdown and derived apoE rescue cell lines with HCV. Exchange of apoE between lipoproteins and HCV lipoviral particles (LVPs) was evaluated by immunoprecipitation, infectivity testing, and viral genome quantification. Cell and heparin column binding assays were applied to determine the attachment efficiency of LVPs with different levels of incorporated apoE. The results showed that cell permissiveness for HCV infection was determined by exogenous apoE-associated lipoproteins. Furthermore, apoE exchange did occur between HCV LVPs and lipoproteins, which was important to maintain a high apoE level on LVPs. Lipid-free apoE was capable of enhancing HCV infectivity for apoE knockdown cells but not apoE rescue cells. A higher apoE level on LVPs conferred more efficient LVP attachment to both the cell surface and heparin beads. This study revealed that exogenous apoE-incorporating lipoproteins from uninfected hepatocytes safeguarded the apoE level of LVPs for more efficient attachment during HCV infection.

    IMPORTANCE In this study, a neglected but important role of apoE exchange in HCV LVP infectivity after virus assembly and release was identified. The data indicated that apoE expression level in uninfected cells is important for high permissiveness to HCV infection. Secreted apoE-associated lipoprotein specifically enhances infection of HCV LVPs. apoE exchange between HCV LVP and lipoproteins is important to maintain an adequate apoE level on LVPs for their efficient attachment to cell surface. These data defined for the first time an extracellular role of exchangeable apoE in HCV infection and suggested that exchangeable apolipoproteins reach a natural equilibrium between HCV LVPs and lipoprotein particles, which provides a new perspective to the understanding of the heterogeneity of HCV LVPs in composition.

  • DDB1 Stimulates Viral Transcription of Hepatitis B Virus via HBx-Independent Mechanisms [Genome Replication and Regulation of Viral Gene Expression]

  • HBx, a small regulatory protein of hepatitis B virus (HBV), augments viral DNA replication by stimulating viral transcription. Among numerous reported HBx-binding proteins, DDB1 has drawn attention, because DDB1 acts as a substrate receptor of the Cul4-DDB1 ubiquitin E3 ligase. Previous work reported that the DDB1-HBx interaction is indispensable for HBx-stimulated viral DNA replication, suggesting that the Cul4-DDB1 ubiquitin E3 ligase might target cellular restriction factors for ubiquitination and proteasomal degradation. To gain further insight into the DDB1-HBx interaction, we generated HBx mutants deficient for DDB1 binding (i.e., R96A, L98A, and G99A) and examined whether they support HBx-stimulated viral DNA replication. In contrast to data from previous reports, our results showed that the HBx mutants deficient for DDB1 binding supported viral DNA replication to nearly wild-type levels, revealing that the DDB1-HBx interaction is largely dispensable for HBx-stimulated viral DNA replication. Instead, we found that DDB1 directly stimulates viral transcription regardless of HBx expression. Through an HBV infection study, importantly, we demonstrated that DDB1 stimulates viral transcription from covalently closed circular DNA, a physiological template for viral transcription. Overall, we concluded that DDB1 stimulates viral transcription via a mechanism that does not involve an interaction with HBx.

    IMPORTANCE DDB1 constitutes a cullin-based ubiquitin E3 ligase, where DDB1 serves as an adaptor linking the cullin scaffold to the substrate receptor. Previous findings that the DDB1-binding ability of HBx is essential for HBx-stimulated viral DNA replication led to the hypothesis that HBx could downregulate host restriction factors that limit HBV replication through the cullin ubiquitin E3 ligase that requires the DDB1-HBx interaction. Consistent with this hypothesis, recent work identified Smc5/6 as a host restriction factor that is regulated by the viral cullin ubiquitin E3 ligase. In contrast, here we found that the DDB1-HBx interaction is largely dispensable for HBx-stimulated viral DNA replication. Instead, our results clearly showed that DDB1, regardless of HBx expression, enhances viral transcription. Overall, besides its role in the viral cullin ubiquitin E3 ligase, DDB1 itself stimulates viral transcription via HBx-independent mechanisms.

  • High Glucose Induces Reactivation of Latent Kaposi's Sarcoma-Associated Herpesvirus [Virus-Cell Interactions]

  • A high prevalence of Kaposi's sarcoma (KS) is seen in diabetic patients. It is unknown if the physiological conditions of diabetes contribute to KS development. We found elevated levels of viral lytic gene expression when Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells were cultured in high-glucose medium. To demonstrate the association between high glucose levels and KSHV replication, we xenografted telomerase-immortalized human umbilical vein endothelial cells that are infected with KSHV (TIVE-KSHV cells) into hyperglycemic and normal nude mice. The injected cells expressed significantly higher levels of KSHV lytic genes in hyperglycemic mice than in normal mice. We further demonstrated that high glucose levels induced the production of hydrogen peroxide (H2O2), which downregulated silent information regulator 1 (SIRT1), a class III histone deacetylase (HDAC), resulting in the epigenetic transactivation of KSHV lytic genes. These results suggest that high blood glucose levels in diabetic patients contribute to the development of KS by promoting KSHV lytic replication and infection.

    IMPORTANCE Multiple epidemiological studies have reported a higher prevalence of classic KS in diabetic patients. By using both in vitro and in vivo models, we demonstrated an association between high glucose levels and KSHV lytic replication. High glucose levels induce oxidative stress and the production of H2O2, which mediates the reactivation of latent KSHV through multiple mechanisms. Our results provide the first experimental evidence and mechanistic support for the association of classic KS with diabetes.

  • Stimulation of the Replication of ICP0-Null Mutant Herpes Simplex Virus 1 and pp71-Deficient Human Cytomegalovirus by Epstein-Barr Virus Tegument Protein BNRF1 [Genome Replication and Regulation of Viral Gene Expression]

  • It is now well established that several cellular proteins that are components of promyelocytic leukemia nuclear bodies (PML NBs, also known as ND10) have restrictive effects on herpesvirus infections that are countered by viral proteins that are either present in the virion particle or are expressed during the earliest stages of infection. For example, herpes simplex virus 1 (HSV-1) immediate early (IE) protein ICP0 overcomes the restrictive effects of PML-NB components PML, Sp100, hDaxx, and ATRX while human cytomegalovirus (HCMV) IE protein IE1 targets PML and Sp100, and its tegument protein pp71 targets hDaxx and ATRX. The functions of these viral regulatory proteins are in part interchangeable; thus, both IE1 and pp71 stimulate the replication of ICP0-null mutant HSV-1, while ICP0 increases plaque formation by pp71-deficient HCMV. Here, we extend these studies by examining proteins that are expressed by Epstein-Barr virus (EBV). We report that EBV tegument protein BNRF1, discovered by other investigators to target the hDaxx/ATRX complex, increases the replication of both ICP0-null mutant HSV-1 and pp71-deficient HCMV. In addition, EBV protein EBNA-LP, which targets Sp100, also augments ICP0-null mutant HSV-1 replication. The combination of these two EBV regulatory proteins had a greater effect than each one individually. These findings reinforce the concept that disruption of the functions of PML-NB proteins is important for efficient herpesvirus infections.

    IMPORTANCE Whether a herpesvirus initiates a lytic infection in a host cell or establishes quiescence or latency is influenced by events that occur soon after the viral genome has entered the host cell nucleus. Certain cellular proteins respond in a restrictive manner to the invading pathogen's DNA, while viral functions are expressed that counteract the cell-mediated repression. One aspect of cellular restriction of herpesvirus infections is mediated by components of nuclear structures known as PML nuclear bodies (PML NBs), or ND10. Members of the alpha-, beta-, and gammaherpesvirus families all express proteins that interact with, degrade, or otherwise counteract the inhibitory effects of various PML NB components. Previous work has shown that there is the potential for a functional interchange between the viral proteins expressed by alpha- and betaherpesviruses, despite a lack of obvious sequence similarity. Here, this concept is extended to include a member of the gammaherpesviruses.

  • Evidence for the Introduction, Reassortment, and Persistence of Diverse Influenza A Viruses in Antarctica [Genetic Diversity and Evolution]

  • Avian influenza virus (AIV) surveillance in Antarctica during 2013 revealed the prevalence of evolutionarily distinct influenza viruses of the H11N2 subtype in Adeeacute;lie penguins. Here we present results from the continued surveillance of AIV on the Antarctic Peninsula during 2014 and 2015. In addition to the continued detection of H11 subtype viruses in a snowy sheathbill during 2014, we isolated a novel H5N5 subtype virus from a chinstrap penguin during 2015. Gene sequencing and phylogenetic analysis revealed that the H11 virus detected in 2014 had a ggt;99.1% nucleotide similarity to the H11N2 viruses isolated in 2013, suggesting the continued prevalence of this virus in Antarctica over multiple years. However, phylogenetic analysis of the H5N5 virus showed that the genome segments were recently introduced to the continent, except for the NP gene, which was similar to that in the endemic H11N2 viruses. Our analysis indicates geographically diverse origins for the H5N5 virus genes, with the majority of its genome segments derived from North American lineage viruses but the neuraminidase gene derived from a Eurasian lineage virus. In summary, we show the persistence of AIV lineages in Antarctica over multiple years, the recent introduction of gene segments from diverse regions, and reassortment between different AIV lineages in Antarctica, which together significantly increase our understanding of AIV ecology in this fragile and pristine environment.

    IMPORTANCE Analysis of avian influenza viruses (AIVs) detected in Antarctica reveals both the relatively recent introduction of an H5N5 AIV, predominantly of North American-like origin, and the persistence of an evolutionarily divergent H11 AIV. These data demonstrate that the flow of viruses from North America may be more common than initially thought and that, once introduced, these AIVs have the potential to be maintained within Antarctica. The future introduction of AIVs from North America into the Antarctic Peninsula is of particular concern given that highly pathogenic H5Nx viruses have recently been circulating among wild birds in parts of Canada and the Unites States following the movement of these viruses from Eurasia via migratory birds. The introduction of a highly pathogenic influenza virus in penguin colonies within Antarctica might have devastating consequences.

  • Inhibition of Polyamine Biosynthesis Is a Broad-Spectrum Strategy against RNA Viruses [Vaccines and Antiviral Agents]

  • RNA viruses present an extraordinary threat to human health, given their sudden and unpredictable appearance, the potential for rapid spread among the human population, and their ability to evolve resistance to antiviral therapies. The recent emergence of chikungunya virus, Zika virus, and Ebola virus highlights the struggles to contain outbreaks. A significant hurdle is the availability of antivirals to treat the infected or protect at-risk populations. While several compounds show promise in vitro and in vivo, these outbreaks underscore the need to accelerate drug discovery. The replication of several viruses has been described to rely on host polyamines, small and abundant positively charged molecules found in the cell. Here, we describe the antiviral effects of two molecules that alter polyamine levels: difluoromethylornithine (DFMO; also called eflornithine), which is a suicide inhibitor of ornithine decarboxylase 1 (ODC1), and diethylnorspermine (DENSpm), an activator of spermidine/spermine N1-acetyltransferase (SAT1). We show that reducing polyamine levels has a negative effect on diverse RNA viruses, including several viruses involved in recent outbreaks, in vitro and in vivo. These findings highlight the importance of the polyamine biosynthetic pathway to viral replication, as well as its potential as a target in the development of further antivirals or currently available molecules, such as DFMO.

    IMPORTANCE RNA viruses present a significant hazard to human health, and combatting these viruses requires the exploration of new avenues for targeting viral replication. Polyamines, small positively charged molecules within the cell, have been demonstrated to facilitate infection for a few different viruses. Our study demonstrates that diverse RNA viruses rely on the polyamine pathway for replication and highlights polyamine biosynthesis as a promising drug target.

  • NS1 Protein Mutation I64T Affects Interferon Responses and Virulence of Circulating H3N2 Human Influenza A Viruses [Pathogenesis and Immunity]

  • Influenza NS1 protein is the main viral protein counteracting host innate immune responses, allowing the virus to efficiently replicate in interferon (IFN)-competent systems. In this study, we analyzed NS1 protein variability within influenza A (IAV) H3N2 viruses infecting humans during the 2012-2013 season. We also evaluated the impact of the mutations on the ability of NS1 proteins to inhibit host innate immune responses and general gene expression. Surprisingly, a previously unidentified mutation in the double-stranded RNA (dsRNA)-binding domain (I64T) decreased NS1-mediated general inhibition of host protein synthesis by decreasing its interaction with cleavage and polyadenylation specificity factor 30 (CPSF30), leading to increased innate immune responses after viral infection. Notably, a recombinant A/Puerto Rico/8/34 H1N1 virus encoding the H3N2 NS1-T64 protein was highly attenuated in mice, most likely because of its ability to induce higher antiviral IFN responses at early times after infection and because this virus is highly sensitive to the IFN-induced antiviral state. Interestingly, using peripheral blood mononuclear cells (PBMCs) collected at the acute visit (2 to 3 days after infection), we show that the subject infected with the NS1-T64 attenuated virus has diminished responses to interferon and to interferon induction, suggesting why this subject could be infected with this highly IFN-sensitive virus. These data demonstrate the importance of influenza virus surveillance in identifying new mutations in the NS1 protein, affecting its ability to inhibit innate immune responses and, as a consequence, the pathogenicity of the virus.

    IMPORTANCE Influenza A and B viruses are one of the most common causes of respiratory infections in humans, causing 1 billion infections and between 300,000 and 500,000 deaths annually. Influenza virus surveillance to identify new mutations in the NS1 protein affecting innate immune responses and, as a consequence, the pathogenicity of the circulating viruses is highly relevant. Here, we analyzed amino acid variability in the NS1 proteins from human seasonal viruses and the effect of the mutations in innate immune responses and virus pathogenesis. A previously unidentified mutation in the dsRNA-binding domain decreased NS1-mediated general inhibition of host protein synthesis and the interaction of the protein with CPSF30. This mutation led to increased innate immune responses after viral infection, augmented IFN sensitivity, and virus attenuation in mice. Interestingly, using PBMCs, the subject infected with the virus encoding the attenuating mutation induced decreased antiviral responses, suggesting why this subject could be infected with this virus.

  • Chimeric Antigen Receptor T Cells Guided by the Single-Chain Fv of a Broadly Neutralizing Antibody Specifically and Effectively Eradicate Virus Reactivated from Latency in CD4+ T Lymphocytes Isolated from HIV-1-Infected Individuals Receiving Suppressive Combined Antiretroviral Therapy [Pathogenesis and Immunity]

  • Despite the advent of combined antiretroviral therapy (cART), the persistence of viral reservoirs remains a major barrier to curing human immunodeficiency virus type 1 (HIV-1) infection. Recently, the shock and kill strategy, by which such reservoirs are eradicated following reactivation of latent HIV-1 by latency-reversing agents (LRAs), has been extensively practiced. It is important to reestablish virus-specific and reliable immune surveillance to eradicate the reactivated virus-harboring cells. In this report, we attempted to reach this goal by using newly developed chimeric antigen receptor (CAR)-T cell technology. To generate anti-HIV-1 CAR-T cells, we connected the single-chain variable fragment of the broadly neutralizing HIV-1-specific antibody VRC01 to a third-generation CAR moiety as the extracellular and intracellular domains and subsequently transduced this into primary CD8+ T lymphocytes. We demonstrated that the resulting VC-CAR-T cells induced T cell-mediated cytolysis of cells expressing HIV-1 Env proteins and significantly inhibited HIV-1 rebound after removal of antiviral inhibitors in a viral infectivity model in cell culture that mimics the termination of the cART in the clinic. Importantly, the VC-CAR-T cells also effectively induced the cytolysis of LRA-reactivated HIV-1-infected CD4+ T lymphocytes isolated from infected individuals receiving suppressive cART. Our data demonstrate that the special features of genetically engineered CAR-T cells make them a particularly suitable candidate for therapeutic application in efforts to reach a functional HIV cure.

    IMPORTANCE The presence of latently infected cells remains a key obstacle to the development of a functional HIV-1 cure. Reactivation of dormant viruses is possible with latency-reversing agents, but the effectiveness of these compounds and the subsequent immune response require optimization if the eradication of HIV-1-infected cells is to be achieved. Here, we describe the use of a chimeric antigen receptor, comprised of T cell activation domains and a broadly neutralizing antibody, VRC01, targeting HIV-1 to treat the infected cells. T cells expressing this construct exerted specific cytotoxic activity against wild-type HIV-1-infected cells, resulting in a dramatic reduction in viral rebound in vitro, and showed persistent effectiveness against reactivated latently infected T lymphocytes from HIV-1 patients receiving combined antiretroviral therapy. The methods used in this study constitute an improvement over existing CD4-based CAR-T technology and offer a promising approach to HIV-1 immunotherapy.

  • Equine Rhinitis A Virus Mutants with Altered Acid Resistance Unveil a Key Role of VP3 and Intrasubunit Interactions in the Control of the pH Stability of the Aphthovirus Capsid [Virus-Cell Interactions]

  • Equine rhinitis A virus (ERAV) is a picornavirus associated with respiratory disease in horses and is genetically closely related to foot-and-mouth disease virus (FMDV), the prototype aphthovirus. ERAV has recently gained interest as an FMDV alternative for the study of aphthovirus biology, including cell entry and uncoating or antiviral testing. As described for FMDV, current data support that acidic pH inside cellular endosomes triggers ERAV uncoating. In order to provide further insights into aphthovirus uncoating mechanism, we have isolated a panel of ERAV mutants with altered acid sensitivity and that differed on their degree of sensitivity to the inhibition of endosome acidification. These results provide functional evidence of the involvement of acidic pH on ERAV uncoating within endosomes. Remarkably, all amino acid substitutions found in acid-labile or acid-resistant ERAVs were located in the capsid protein VP3, indicating that this protein plays a pivotal role for the control of pH stability of the ERAV capsid. Moreover, all amino acid substitutions mapped at the intraprotomer interface between VP3 and VP2 or between VP3 and the N terminus of VP1. These results expand our knowledge on the regions that regulate the acid stability of aphthovirus capsid and should be taken into account when using ERAV as a surrogate of FMDV.

    IMPORTANCE The viral capsid constitutes a sort of dynamic nanomachine that protects the viral genome against environmental assaults while accomplishing important functions such as receptor attachment for viral entry or genome release. We have explored the molecular determinants of aphthovirus capsid stability by isolating and characterizing a panel of equine rhinitis A virus mutants that differed on their acid sensitivity. All the mutations were located within a specific region of the capsid, the intraprotomer interface among capsid proteins, thus providing new insights into the regions that control the acid stability of aphthovirus capsid. These findings could positively contribute to the development of antiviral approaches targeting aphthovirus uncoating or the refinement of vaccine strategies based on capsid stabilization.

  • Near-Atomic Resolution Structure of a Highly Neutralizing Fab Bound to Canine Parvovirus [Structure and Assembly]

  • Canine parvovirus (CPV) is a highly contagious pathogen that causes severe disease in dogs and wildlife. Previously, a panel of neutralizing monoclonal antibodies (MAb) raised against CPV was characterized. An antibody fragment (Fab) of MAb E was found to neutralize the virus at low molar ratios. Using recent advances in cryo-electron microscopy (cryo-EM), we determined the structure of CPV in complex with Fab E to 4.1 AAring; resolution, which allowed de novo building of the Fab structure. The footprint identified was significantly different from the footprint obtained previously from models fitted into lower-resolution maps. Using single-chain variable fragments, we tested antibody residues that control capsid binding. The near-atomic structure also revealed that Fab binding had caused capsid destabilization in regions containing key residues conferring receptor binding and tropism, which suggests a mechanism for efficient virus neutralization by antibody. Furthermore, a general technical approach to solving the structures of small molecules is demonstrated, as binding the Fab to the capsid allowed us to determine the 50-kDa Fab structure by cryo-EM.

    IMPORTANCE Using cryo-electron microscopy and new direct electron detector technology, we have solved the 4 AAring; resolution structure of a Fab molecule bound to a picornavirus capsid. The Fab induced conformational changes in regions of the virus capsid that control receptor binding. The antibody footprint is markedly different from the previous one identified by using a 12 AAring; structure. This work emphasizes the need for a high-resolution structure to guide mutational analysis and cautions against relying on older low-resolution structures even though they were interpreted with the best methodology available at the time.

  • The Antiviral Alkaloid Berberine Reduces Chikungunya Virus-Induced Mitogen-Activated Protein Kinase Signaling [Vaccines and Antiviral Agents]

  • Chikungunya virus (CHIKV) has infected millions of people in the tropical and subtropical regions since its reemergence in the last decade. We recently identified the nontoxic plant alkaloid berberine as an antiviral substance against CHIKV in a high-throughput screen. Here, we show that berberine is effective in multiple cell types against a variety of CHIKV strains, also at a high multiplicity of infection, consolidating the potential of berberine as an antiviral drug. We excluded any effect of this compound on virus entry or on the activity of the viral replicase. A human phosphokinase array revealed that CHIKV infection specifically activated the major mitogen-activated protein kinase (MAPK) signaling pathways extracellular signal-related kinase (ERK), p38 and c-Jun NH2-terminal kinase (JNK). Upon treatment with berberine, this virus-induced MAPK activation was markedly reduced. Subsequent analyses with specific inhibitors of these kinases indicated that the ERK and JNK signaling cascades are important for the generation of progeny virions. In contrast to specific MAPK inhibitors, berberine lowered virus-induced activation of all major MAPK pathways and resulted in a stronger reduction in viral titers. Further, we assessed the in vivo efficacy of berberine in a mouse model and measured a significant reduction of CHIKV-induced inflammatory disease. In summary, we demonstrate the efficacy of berberine as a drug against CHIKV and highlight the importance of the MAPK signaling pathways in the alphavirus infectious cycle.

    IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne virus that causes severe and persistent muscle and joint pain and has recently spread to the Americas. No licensed drug exists to counter this virus. In this study, we report that the alkaloid berberine is antiviral against different CHIKV strains and in multiple human cell lines. We demonstrate that berberine collectively reduced the virus-induced activation of cellular mitogen-activated protein kinase signaling. The relevance of these signaling cascades in the viral life cycle was emphasized by specific inhibitors of these kinase pathways, which decreased the production of progeny virions. Berberine significantly reduced CHIKV-induced inflammatory disease in a mouse model, demonstrating efficacy of the drug in vivo. Overall, this work makes a strong case for pursuing berberine as a potential anti-CHIKV therapeutic compound and for exploring the MAPK signaling pathways as antiviral targets against alphavirus infections.

  • Functional and Structural Characterization of P[19] Rotavirus VP8* Interaction with Histo-blood Group Antigens [Virus-Cell Interactions]

  • Rotaviruses (RVs) of species A (RVA) are a major causative agent of acute gastroenteritis. Recently, histo-blood group antigens (HBGAs) have been reported to interact with human RVA VP8* proteins. Human P[19] is a rare P genotype of porcine origin that infects humans sporadically. The functional and structural characteristics of P[19] VP8* interaction with HBGAs are unknown. In this study, we expressed and purified the VP8* proteins of human and porcine P[19] RVs. In oligosaccharide and saliva binding assays, P[19] VP8* proteins showed obvious binding to A-, B-, and O-type saliva samples irrespective of the secretor status, implying broad binding patterns. However, they did not display specific binding to any of the oligosaccharides tested. In addition, we solved the structure of human P[19] VP8* at 2.4 AAring;, which revealed a typical galectin-like fold. The structural alignment demonstrated that P[19] VP8* was most similar to that of P[8], which was consistent with the phylogenetic analysis. Structure superimposition revealed the basis for the lack of binding to the oligosaccharides. Our study indicates that P[19] RVs may bind to other oligosaccharides or ligands and may have the potential to spread widely among humans. Thus, it is necessary to place the prevalence and evolution of P[19] RVs under surveillance.

    IMPORTANCE Human P[19] is a rare P genotype of porcine origin. Based on phylogenetic analysis of VP8* sequences, P[19] was classified in the P[II] genogroup, together with P[4], P[6], and P[8], which have been reported to interact with HBGAs in a genotype-dependent manner. In this study, we explored the functional and structural characteristics of P[19] VP8* interaction with HBGAs. P[19] VP8* showed binding to A-, B-, and O-type saliva samples, as well as saliva of nonsecretors. This implies that P[19] has the potential to spread among humans with a broad binding range. Careful attention should be paid to the evolution and prevalence of P[19] RVs. Furthermore, we solved the structure of P[19] VP8*. Structure superimposition indicated that P[19] may bind to other oligosaccharides or ligands using potential binding sites, suggesting that further investigation of the specific cell attachment factors is warranted.

  • Cell Surface THY-1 Contributes to Human Cytomegalovirus Entry via a Macropinocytosis-Like Process [Virus-Cell Interactions]

  • Previously we showed that THY-1 has a critical role in the initial stage of infection of certain cell types with human cytomegalovirus (HCMV) and that THY-1 is important for HCMV-mediated activation of phosphatidylinositol 3-kinase (PI3K)/Akt during virus entry. THY-1 is known to interact with integrins and is a major cargo protein of clathrin-independent endocytic vesicles. Since macropinocytosis involves integrin signaling, is PI3K/Akt dependent, and is a clathrin-independent endocytic process, we determined whether THY-1 has a role in HCMV entry by macropinocytosis. Using electron microscopy in two cell lines that support HCMV infection in a THY-1-dependent manner, we found that HCMV enters these cells by a macropinocytosis-like process. THY-1 associated with HCMV virions on the cell surface and colocalized with virus inside macropinosomes. 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) and soluble THY-1 blocked HCMV infection in the cell lines by gge;80% and 60%, respectively. HCMV entry into the cells triggered increased influx of extracellular fluid, a marker of macropinocytosis, and this increased fluid uptake was inhibited by EIPA and by soluble THY-1. Blocking actin depolymerization, Na+/H+ exchange, PI3K, and Pak1 kinase, which are critical for macropinocytosis, impaired HCMV infection. Neither internalized HCMV virions nor THY-1 in virus-infected cells colocalized with transferrin as determined by confocal microscopy, indicating that clathrin-mediated endocytosis was not involved in THY-1-associated virus entry. These results suggest that HCMV has adapted to utilize THY-1, a cargo protein of clathrin-independent endocytotic vesicles, to facilitate efficient entry into certain cell types by a macropinocytosis-like process.

    IMPORTANCE Human cytomegalovirus (HCMV) infects over half of the population and is the most common infectious cause of birth defects. The virus is the most important infection occurring in transplant recipients. The mechanism of how HCMV enters cells is controversial. In this study, we show that THY-1, a cell surface protein that is critical for the early stage of entry of HCMV into certain cell types, contributes to virus entry by macropinocytosis. Our findings suggest that HCMV has adapted to utilize THY-1 to facilitate entry of HCMV into macropinosomes in certain cell types. Further knowledge about the mechanism of HCMV entry into cells may facilitate the development of novel inhibitors of virus infection.

  • CD95 Signaling Inhibits B Cell Receptor-Mediated Gammaherpesvirus Replication in Apoptosis-Resistant B Lymphoma Cells [Virus-Cell Interactions]

  • While CD95 is an apoptosis-inducing receptor and has emerged as a potential anticancer therapy target, mounting evidence shows that CD95 is also emerging as a tumor promoter by activating nonapoptotic signaling pathways. Gammaherpesviral infection is closely associated with lymphoproliferative diseases, including B cell lymphomas. The nonapoptotic function of CD95 in gammaherpesvirus-associated lymphomas is largely unknown. Here, we show that stimulation of CD95 agonist antibody drives the majority of sensitive gammaherpesvirus-transformed B cells to undergo caspase-dependent apoptosis and promotes the survival and proliferation of a subpopulation of apoptosis-resistant B cells. Surprisingly, CD95-mediated nonapoptotic signaling induced beta interferon (IFN-bbeta;) expression and correlatively inhibited B cell receptor (BCR)-mediated gammaherpesviral replication in the apoptosis-resistant lymphoma cells without influencing BCR signaling. Further analysis showed that IFN-bbeta; alone or synergizing with CD95 blocked the activation of lytic switch proteins and the gene expression of gammaherpesviruses. Our findings indicate that, independent of its apoptotic activity, CD95 signaling activity plays an important role in blocking viral replication in apoptosis-resistant, gammaherpesvirus-associated B lymphoma cells, suggesting a novel mechanism that indicates how host CD95 prototype death receptor controls the life cycle of gammaherpesviruses independent of its apoptotic activity.

    IMPORTANCE Gammaherpesviruses are closely associated with lymphoid malignancies and other cancers. Viral replication and persistence strategies leading to cancer involve the activation of antiapoptotic and proliferation programs, as well as evasion of the host immune response. Here, we provide evidence that the stimulation of CD95 agonist antibody, mimicking one of the major mechanisms of cytotoxic T cell killing, inhibits B cell receptor-mediated gammaherpesviral replication in CD95 apoptosis-resistant lymphoma cells. CD95-induced type I interferon (IFN-bbeta;) contributes to the inhibition of gammaherpesviral replication. This finding sheds new light on the CD95 nonapoptotic function and provides a novel mechanism for gammaherpesviruses that helps them to escape host immune surveillance.

  • Characterization of Clade 7.2 H5 Avian Influenza Viruses That Continue To Circulate in Chickens in China [Genetic Diversity and Evolution]

  • The H5N1 avian influenza viruses emerged in Southeast Asia in the late 20th century and have evolved into multiple phylogenetic clades based on their hemagglutinin (HA)-encoding genes. The clade 7.2 viruses were first detected in chickens in northern China in 2006, and vaccines specifically targeted to the clade were developed and have been used in poultry in China since 2006. During routine surveillance and disease diagnosis, we isolated seven H5 viruses between 2011 and 2014 that bear the clade 7.2 HA genes. Here, we performed extensive studies to understand how the clade 7.2 H5 viruses have evolved in chickens in China. Full genome sequence analysis revealed that the seven viruses formed two subtypes (four H5N1 viruses and three H5N2 viruses) and four genotypes by deriving genes from other influenza viruses. All of the viruses had antigenically drifted from the clade 7.2 viruses that were isolated in 2006. Pathogenicity studies of four viruses, one from each genotype, revealed that all of the viruses are highly pathogenic in chickens, but none of them could replicate in ducks. The four viruses exclusively bound to avian-type receptors and replicated only in the turbinates and/or lungs of mice; none of them were lethal to mice at a dosage of 106 50% egg infective doses (EID50). Our study indicates that although the clade 7.2 viruses have not been eradicated from poultry through vaccination, they have not become more dangerous to other animals (e.g., ducks and mice) and humans.

    IMPORTANCE Animal influenza viruses can acquire the ability to infect and kill humans. The H5N1 viruses have been a concern in recent decades because of their clear pandemic potential. We sorted H5N1 influenza viruses into different phylogenetic clades based on their HA genes. The clade 7.2 viruses were detected in chickens in several provinces of northern China in 2006. Vaccines for these viruses were subsequently developed and have been used ever since to control infection of poultry. Here, we analyzed the genetic and biologic properties of seven clade 7.2 viruses that were isolated from chickens between 2011 and 2014. We found that after nearly 9 years of circulation in chickens, the clade 7.2 viruses still exclusively bind to avian-type receptors and are of low pathogenicity to mice, suggesting that these H5 viruses pose a low risk to human public health.

  • A Single Mutation at Position 190 in Hemagglutinin Enhances Binding Affinity for Human Type Sialic Acid Receptor and Replication of H9N2 Avian Influenza Virus in Mice [Virus-Cell Interactions]

  • H9N2 avian influenza virus (AIV) has an extended host range, but the molecular basis underlying H9N2 AIV transmission to mammals remains unclear. We isolated more than 900 H9N2 AIVs in our 3-year surveillance in live bird markets in China from 2009 to 2012. Thirty-seven representative isolates were selected for further detailed characterization. These isolates were categorized into 8 genotypes (B64 to B71) and formed a distinct antigenic subgroup. Three isolates belonging to genotype B69, which is a predominant genotype circulating in China, replicated efficiently in mice, while the viruses tested in parallel in other genotypes replicated poorly, although they, like the three B69 isolates, have a leucine at position 226 in the hemagglutinin (HA) receptor binding site, which is critical for binding human type sialic acid receptors. Further molecular and single mutation analysis revealed that a valine (V) residue at position 190 in HA is responsible for efficient replication of these H9N2 viruses in mice. The 190V in HA does not affect virus receptor binding specificity but enhances binding affinity to human cells and lung tissues from mouse and humans. All these data indicate that the 190V in HA is one of the important determinants for H9N2 AIVs to cross the species barrier to infect mammals despite multiple genes conferring adaptation and replication of H9N2 viruses in mammals. Our findings provide novel insights on understanding host range expansion of H9N2 AIVs.

    IMPORTANCE Influenza virus hemagglutinin (HA) is responsible for binding to host cell receptors and therefore influences the viral host range and pathogenicity in different species. We showed that the H9N2 avian influenza viruses harboring 190V in the HA exhibit enhanced virus replication in mice. Further studies demonstrate that 190V in the HA does not change virus receptor binding specificity but enhances virus binding affinity of the H9N2 virus to human cells and attachment to lung tissues from humans and mouse. Our findings suggest that more attention should be given to the H9N2 AIVs with HA-190V during surveillance due to their potential threat to mammals, including humans.

  • Antagonism of RNase L Is Required for Murine Coronavirus Replication in Kupffer Cells and Liver Sinusoidal Endothelial Cells but Not in Hepatocytes [Pathogenesis and Immunity]

  • Mouse hepatitis virus strain A59 infection of mice is a useful tool for studying virus-host interaction during hepatitis development. The NS2H126R mutant is attenuated in liver replication due to loss of phosphodiesterase activity, which the wild-type (WT) virus uses to block the 2',5'-oligoadenylate synthetase (OAS)-RNase L (RNase L) antiviral pathway. The activation of RNase L by NS2H126R is cell type dependent and correlates with high basal expression levels of OAS, as found in myeloid cells. We tested the hypothesis that the resident liver macrophages, Kupffer cells (KC), represent the cell type most likely to restrict NS2H126R and prevent hepatitis. As found previously, A59 and NS2H126R replicate similarly in hepatocytes and neither activates RNase L, as assessed by an rRNA degradation assay. In contrast, in KC, A59 exhibited a 100-fold-higher titer than NS2H126R and NS2H126R induced rRNA degradation. Interestingly, in liver sinusoidal endothelial cells (LSEC), the cells that form a barrier between blood and liver parenchymal cells, NS2H126R activates RNase L, which limits viral replication. Similar growth kinetics were observed for the two viruses in KC and LSEC from RNase Lnndash;/nndash; mice, demonstrating that both use RNase L to limit NS2H126R replication. Depletion of KC by gadolinium(III) chloride or of LSEC by cyclophosphamide partially restores liver replication of NS2H126R, leading to hepatitis. Thus, during mouse hepatitis virus (MHV) infection, hepatitis, which damages the parenchyma, is prevented by RNase L activity in both KC and LSEC but not in hepatocytes. This may be explained by the undetectable levels of RNase L as well as by the OASs expressed in hepatocytes.

    IMPORTANCE Mouse hepatitis virus infection of mice provides a useful tool for studying virus-host interactions during hepatitis development. The NS2H126R mutant is attenuated in liver replication due to loss of phosphodiesterase activity, by which the wild-type virus blocks the potent OAS-RNase L antiviral pathway. RNase L activation by NS2H126R is cell type dependent and correlates with high basal expression levels of OAS, as found in myeloid cells. We showed that the hepatocytes that comprise the liver parenchyma do not activate RNase L when infected with NS2H126R or restrict replication. However, both Kupffer cells (KC) (i.e., the liver-resident macrophages) and the liver sinusoidal endothelial cells (LSEC) which line the sinusoids activate RNase L in response to NS2H126R. These data suggest that KC and LSEC prevent viral spread into the parenchyma, preventing hepatitis. Furthermore, hepatocytes express undetectable levels of OASs and RNase L, which likely explains the lack of RNase L activation during NS2H126R infection.

  • Monocytes but Not Lymphocytes Carrying HIV-1 on Their Surface Transmit Infection to Human Tissue Ex Vivo [Virus-Cell Interactions]

  • Unprotected sexual intercourse with HIV-infected men is the major cause of new infections. HIV virions are released into semen by various cells of the male genital tract, as well as by infected monocytes and lymphocytes present in semen. Some of these virions may attach to the surfaces of cells, infected or uninfected. We investigated whether cells carrying attached HIV on their surfaces can transmit infection. We addressed this question in a model system of human tissue exposed ex vivo to monocytes and lymphocytes carrying HIV on their surfaces. We gamma irradiated the cells to prevent their productive infection. In spite of comparable amounts of HIV attached to monocytes and lymphocytes, only monocytes were capable of transmitting infection and triggering productive infection in tissue. This HIV-1 transmission was mediated by cell-cell contacts. Our experiments suggest that in vivo, HIV attached to infected or uninfected monocytes, which far outnumber lymphocytes in HIV-infected semen, may contribute to sexual transmission of HIV from men to their partners.

    IMPORTANCE The vast majority of new HIV infections occur through sexual transmission, in which HIV is transferred from the semen of an infected male to an uninfected partner. In semen, HIV-1 particles may exist as free-floating virions; inside infected cells; or attached to the surfaces of cells, whether they are infected or not. Here, we investigated whether HIV attached to the surfaces of monocytes or lymphocytes could transmit infection to human tissue. Incubation of human tissue with monocyte-attached HIV resulted in productive tissue infection. In contrast, there was no infection of tissues when they were incubated with lymphocyte-attached HIV-1. Our results highlight the important role that seminal monocytes may play in HIV transmission in vivo, especially since monocytes far outnumber lymphocytes in the semen of HIV-infected individuals.

  • LFA-1 Engagement Triggers T Cell Polarization at the HIV-1 Virological Synapse [Virus-Cell Interactions]

  • HIV-1 efficiently disseminates by cell-cell spread at intercellular contacts called virological synapses (VS), where the virus preferentially assembles and buds. Cell-cell contact triggers active polarization of organelles and viral proteins within infected cells to the contact site to support efficient VS formation and HIV-1 spread; critically, however, which cell surface protein triggers contact-induced polarization at the VS remains unclear. Additionally, the mechanism by which the HIV-1 envelope glycoprotein (Env) is recruited to the VS remains ill defined. Here, we use a reductionist bead-coupled antibody assay as a model of the VS and show that cross-linking the integrin LFA-1 alone is sufficient to induce active T cell polarization and recruitment of the microtubule organizing center (MTOC) in HIV-1-infected cells. Mutant cell lines coupled with inhibitors demonstrated that LFA-1-induced polarization was dependent on the T cell kinase ZAP70. Notably, immunofluorescent staining of viral proteins revealed an accumulation of surface Env at sites of LFA-1 engagement, with intracellular Env localized to a Golgi compartment proximal to the polarized MTOC. Furthermore, blocking LFA-1-induced MTOC polarization through ZAP70 inhibition prevented intracellular Env polarization. Taken together, these data reveal that LFA-1 is a key determinant in inducing dynamic T cell remodeling to the VS and suggest a model in which LFA-1 engagement triggers active polarization of the MTOC and the associated Env-containing secretory apparatus to sites of cell-cell contact to support polarized viral assembly and egress for efficient cell-cell spread.

    IMPORTANCE HIV-1 causes AIDS by spreading within immune cells and depletion of CD4 T lymphocytes. Rapid spread between these cells occurs by highly efficient cell-cell transmission that takes place at virological synapses (VS). VS are characterized by striking T cell remodeling that is spatially associated with polarized virus assembly and budding at sites of cell contact. Here, we show that the integrin LFA-1 triggers organelle polarization and viral protein recruitment, facilitating formation of the VS, and that this requires the T cell kinase ZAP70. Taken together, these data suggest a mechanism by which HIV-1-infected T cells sense and respond to cell contact to polarize viral egress and promote cell-cell spread. Understanding how cell-cell spread is regulated may help reveal therapeutic targets to specifically block this mode of HIV-1 dissemination.

  • HIV-1-Neutralizing IgA Detected in Genital Secretions of Highly HIV-1-Exposed Seronegative Women on Oral Preexposure Prophylaxis [Pathogenesis and Immunity]

  • Although nonhuman primate studies have shown that simian immunodeficiency virus/simian-human immunodeficiency virus (SIV/SHIV) exposure during preexposure prophylaxis (PrEP) with oral tenofovir can induce SIV immunity without productive infection, this has not been documented in humans. We evaluated cervicovaginal IgA in Partners PrEP Study participants using a subtype C primary isolate and found that women on PrEP had IgA with higher average human immunodeficiency virus type 1 (HIV-1)-neutralizing magnitude than women on placebo (33% versus 7%; P = 0.008). Using a cutoff of gge;90% HIV-1 neutralization, 19% of women on-PrEP had HIV-1-neutralizing IgA compared to 0% of women on placebo (P = 0.09). We also estimated HIV-1 exposure and found that the proportion of women with HIV-1-neutralizing IgA was associated with the level of HIV-1 exposure (P = 0.04). Taken together, our data suggest that PrEP and high levels of exposure to HIV may each enhance mucosal HIV-1-specific humoral immune responses in sexually exposed but HIV-1-uninfected individuals.

    IMPORTANCE Although there is not yet an effective HIV-1 vaccine, PrEP for at-risk HIV-1-uninfected individuals is a highly efficacious intervention to prevent HIV-1 acquisition and is currently being recommended by the CDC and WHO for all individuals at high risk of HIV-1 acquisition. We previously demonstrated that PrEP use does not enhance peripheral blood HIV-1-specific T-cell responses in HIV-exposed individuals. Here, we evaluate for cervicovaginal HIV-neutralizing IgA responses in genital mucosal secretions of HIV-exposed women, which is likely a more relevant site than peripheral blood for observation of potentially protective immune events occurring in response to sexual HIV-1 exposure for various periods. Furthermore, we assess for host response in the context of longitudinal quantification of HIV-1 exposure. We report that HIV-neutralizing IgA is significantly correlated with higher HIV-1 exposure and, furthermore, that there are more women with HIV-1-neutralizing IgA in the on-PrEP group than in the placebo group.

  • Enterovirus 71 2B Induces Cell Apoptosis by Directly Inducing the Conformational Activation of the Proapoptotic Protein Bax [Virus-Cell Interactions]

  • To survive and replicate within a host, many viruses have evolved strategies that target crucial components within the apoptotic cascade, leading to either inhibition or induction of cell apoptosis. Enterovirus 71 (EV71) infections have been demonstrated to impact the mitochondrial apoptotic pathway and induce apoptosis in many cell lines. However, the detailed mechanism of EV71-induced apoptosis remains to be elucidated. In this study, we report that EV71 2B protein (2B) localized to the mitochondria and induced cell apoptosis by interacting directly with and activating the proapoptotic protein Bax. 2B recruited Bax to the mitochondria and induced Bax conformational activation. In addition, mitochondria isolated from 2B-expressing cells that were treated with a recombinant Bax showed increased Bax interaction and cytochrome c (Cyt c) release. Importantly, apoptosis in cells with either EV71 infection or 2B expression was dramatically reduced in Bax knockdown cells but not in Bak knockdown cells, suggesting that Bax played a pivotal role in EV71- or 2B-induced apoptosis. Further studies indicate that a hydrophobic region of 18 amino acids (aa) in the C-terminal region of 2B (aa 63 to 80) was responsible for the location of 2B in the mitochondria. A hydrophilic region of 14 aa in the N-terminal region of 2B was functional in Bax interaction and its subsequent activation. Moreover, overexpression of the antiapoptotic protein Bcl-XL abrogates 2B-induced release of Cyt c and caspase activation. Therefore, this study provides direct evidence that EV71 2B induces cell apoptosis and impacts the mitochondrial apoptotic pathway by directly modulating the redistribution and activation of proapoptotic protein Bax.

    IMPORTANCE EV71 infections are usually accompanied by severe neurological complications. It has also been postulated that the induction of cell apoptosis resulting from tissue damage is a possible process of EV71-related pathogenesis. In this study, we report that EV71 2B protein (2B) localized to the mitochondria and induced cell apoptosis by interacting directly with and activating the proapoptotic protein Bax. This study provides evidence that EV71 induces cell apoptosis by modulating Bax activation and reveals important clues regarding the mechanism of Cyt c release and mitochondrial permeabilization during EV71 infection.

  • Heparan Sulfate Binding Promotes Accumulation of Intravitreally Delivered Adeno-associated Viral Vectors at the Retina for Enhanced Transduction but Weakly Influences Tropism [Gene Delivery]

  • Many adeno-associated virus (AAV) serotypes efficiently transduce the retina when delivered to the subretinal space but show limited success when delivered to the vitreous due to the inner limiting membrane (ILM). Subretinal delivery of AAV serotype 2 (AAV2) and its heparan sulfate (HS)-binding-deficient capsid led to similar expression, indicating transduction of the outer retina occurred by HS-independent mechanisms. However, intravitreal delivery of HS-ablated recombinant AAV2 (rAAV2) led to a 300-fold decrease in transduction compared to AAV2. Fluorescence in situ hybridization of AAV transgenes was used to identify differences in retinal trafficking and revealed that HS binding was responsible for AAV2 accumulation at the ILM. This mechanism was tested on human ex vivo retinas and showed similar accumulation with HS-binding AAV2 capsid only. To evaluate if HS binding could be applied to other AAV serotypes to enhance their transduction, AAV1 and AAV8 were modified to bind HS with a single-amino-acid mutation and tested in mice. Both HS-binding mutants of AAV1 and AAV8 had higher intravitreal transduction than their non-HS-binding parent capsid due to increased retinal accumulation. To understand the influence that HS binding has on tropism, chimeric AAV2 capsids with dual-glycan usage were tested intravitreally in mice. Compared to HS binding alone, these chimeric capsids displayed enhanced transduction that was correlated with a change in tropism. Taken together, these data indicate that HS binding serves to sequester AAV capsids from the vitreous to the ILM but does not influence retinal tropism. The enhanced retinal transduction of HS-binding capsids provides a rational design strategy for engineering capsids for intravitreal delivery.

    IMPORTANCE Adeno-associated virus (AAV) has become the vector of choice for viral gene transfer and has shown great promise in clinical trials. The need for development of an easy, less invasive injection route for ocular gene therapy is met by intravitreal delivery, but delivery of AAV by this route results in poor transduction outcomes. The inner limiting membrane (ILM) creates a barrier separating the vitreous and the retina. Binding of AAV to heparan sulfate proteoglycan (HSPG) at the ILM may allow the virus to traverse this barrier for better retinal transduction. We show that HSPG binding is correlated with greater accumulation and penetration of AAV in the retina. We demonstrated that this accumulation is conserved across mouse and human retinas and that the addition of HSPG binding to other AAV capsids can increase the number of vectors accumulating at the ILM without dictating tropism.

  • An Essential Role of INI1/hSNF5 Chromatin Remodeling Protein in HIV-1 Posttranscriptional Events and Gag/Gag-Pol Stability [Virus-Cell Interactions]

  • INI1/hSNF5/SMARCB1/BAF47 is an HIV-specific integrase (IN)-binding protein that influences HIV-1 transcription and particle production. INI1 binds to SAP18 (Sin3a-associated protein, 18 kDa), and both INI1 and SAP18 are incorporated into HIV-1 virions. To determine the significance of INI1 and the INI1-SAP18 interaction during HIV-1 replication, we isolated a panel of SAP18-interaction-defective (SID)-INI1 mutants using a yeast reverse two-hybrid screen. The SID-INI1 mutants, which retained the ability to bind to IN, cMYC, and INI1 but were impaired for binding to SAP18, were tested for their effects on HIV-1 particle production. SID-INI1 dramatically reduced the intracellular Gag/Gag-Pol protein levels and, in addition, decreased viral particle production. The SID-INI1-mediated effects were less dramatic in trans complementation assays using IN deletion mutant viruses with Vpr-reverse transcriptase (RT)-IN. SID-INI1 did not inhibit long-terminal-repeat (LTR)-mediated transcription, but it marginally decreased the steady-state gag RNA levels, suggesting a posttranscriptional effect. Pulse-chase analysis indicated that in SID-INI1-expressing cells, the pr55Gag levels decreased rapidly. RNA interference analysis indicated that small hairpin RNA (shRNA)-mediated knockdown of INI1 reduced the intracellular Gag/Gag-Pol levels and further inhibited HIV-1 particle production. These results suggest that SID-INI1 mutants inhibit multiple stages of posttranscriptional events of HIV-1 replication, including intracellular Gag/Gag-Pol RNA and protein levels, which in turn inhibits assembly and particle production. Interfering INI1 leads to a decrease in particle production and Gag/Gag-Pol protein levels. Understanding the role of INI1 and SAP18 in HIV-1 replication is likely to provide novel insight into the stability of Gag/Gag-Pol, which may lead to the development of novel therapeutic strategies to inhibit HIV-1 late events.

    IMPORTANCE Significant gaps exist in our current understanding of the mechanisms and host factors that influence HIV-1 posttranscriptional events, including gag RNA levels, Gag/Gag-Pol protein levels, assembly, and particle production. Our previous studies suggested that the IN-binding host factor INI1 plays a role in HIV-1 assembly. An ectopically expressed minimal IN-binding domain of INI1, S6, potently and selectively inhibited HIV-1 Gag/Gag-Pol trafficking and particle production. However, whether or not endogenous INI1 and its interacting partners, such as SAP18, are required for late events was unknown. Here, we report that endogenous INI1 and its interaction with SAP18 are necessary to maintain intracellular levels of Gag/Gag-Pol and for particle production. Interfering INI1 or the INI1-SAP18 interaction leads to the impairment of these processes, suggesting a novel strategy for inhibiting posttranscriptional events of HIV-1 replication.

  • Primary Structures of the Virion Proteins of Vaccinia Virus at Increased Resolution [Structure and Assembly]

  • Here we examine the protein covalent structure of the vaccinia virus virion. Within two virion preparations, ggt;88% of the theoretical vaccinia virus-encoded proteome was detected with high confidence, including the first detection of products from 27 open reading frames (ORFs) previously designated "predicted," "uncharacterized," "inferred," or "hypothetical" polypeptides containing as few as 39 amino acids (aa) and six proteins whose detection required nontryptic proteolysis. We also detected the expression of four short ORFs, each of which was located within an ORF ("ORF-within-ORF"), including one not previously recognized or known to be expressed. Using quantitative mass spectrometry (MS), between 58 and 74 proteins were determined to be packaged. A total of 63 host proteins were also identified as candidates for packaging. Evidence is provided that some portion of virion proteins are "nicked" via a combination of endoproteolysis and concerted exoproteolysis in a manner, and at sites, independent of virus origin or laboratory procedures. The size of the characterized virion phosphoproteome was doubled from 189 (J. Matson, W. Chou, T. Ngo, and P. D. Gershon, Virology 452-453:310nndash;323, 2014, doi: to 396 confident, unique phosphorylation sites, 268 of which were within the packaged proteome. This included the unambiguous identification of phosphorylation "hot spots" within virion proteins. Using isotopically enriched ATP, 23 sites of intravirion kinase phosphorylation were detected within nine virion proteins, all at sites already partially occupied within the virion preparations. The clear phosphorylation of proteins RAP94 and RP19 was consistent with the roles of these proteins in intravirion early gene transcription. In a blind search for protein modifications, cysteine glutathionylation and O-linked glycosylation featured prominently. We provide evidence for the phosphoglycosylation of vaccinia virus proteins.

    IMPORTANCE Poxviruses are among the most complex and irregular virions, about whose internal structure little is known. To better understand poxvirus virion structure, imaging should be supplemented with other tools. Here, we provide a deep study of the covalent structure of the vaccinia virus virion using the various tools of contemporary mass spectrometry.

  • Exploitation of Interleukin-10 (IL-10) Signaling Pathways: Alternate Roles of Viral and Cellular IL-10 in Rhesus Cytomegalovirus Infection [Vaccines and Antiviral Agents]

  • There is accumulating evidence that the viral interleukin-10 (vIL-10) ortholog of both human and rhesus cytomegalovirus (HCMV and RhCMV, respectively) suppresses the functionality of cell types that are critical to contain virus dissemination and help shape long-term immunity during the earliest virus-host interactions. In particular, exposure of macrophages, peripheral blood mononuclear cells, monocyte-derived dendritic cells, and plasmacytoid dendritic cells to vIL-10 suppresses multiple effector functions including, notably, those that link innate and adaptive immune responses. Further, vaccination of RhCMV-uninfected rhesus macaques with nonfunctional forms of RhCMV vIL-10 greatly restricted parameters of RhCMV infection following RhCMV challenge of the vaccinees. Vaccinees exhibited significantly reduced shedding of RhCMV in saliva and urine following RhCMV challenge compared to shedding in unvaccinated controls. Based on the evidence that vIL-10 is critical during acute infection, the role of vIL-10 during persistent infection was analyzed in rhesus macaques infected long term with RhCMV to determine whether postinfection vaccination against vIL-10 could change the virus-host balance. RhCMV-seropositive macaques, which shed RhCMV in saliva, were vaccinated with nonfunctional RhCMV vIL-10, and shedding levels of RhCMV in saliva were evaluated. Following robust increases in vIL-10-binding and vIL-10-neutralizing antibodies, shedding levels of RhCMV modestly declined, consistent with the interpretation that vIL-10 may play a functional role during persistent infection. However, a more significant association was observed between the levels of cellular IL-10 secreted in peripheral blood mononuclear cells exposed to RhCMV antigens and shedding of RhCMV in saliva. This result implies that RhCMV persistence is associated with the induction of cellular IL-10 receptor-mediated signaling pathways.

    IMPORTANCE Human health is adversely impacted by viruses that establish lifelong infections that are often accompanied with increased morbidity and mortality (e.g., infections with HIV, hepatitis C virus, or human cytomegalovirus). A longstanding but unfulfilled goal has been to develop postinfection vaccine strategies that could "reboot" the immune system of an infected individual in ways that would enable the infected host to develop immune responses that clear reservoirs of persistent virus infection, effectively curing the host of infection. This concept was evaluated in rhesus macaques infected long term with rhesus cytomegalovirus by repeatedly immunizing infected animals with nonfunctional versions of the rhesus cytomegalovirus-encoded viral interleukin-10 immune-modulating protein. Following vaccine-mediated boosting of antibody titers to viral interleukin-10, there was modest evidence for increased immunological control of the virus following vaccination. More significantly, data were also obtained that indicated that rhesus cytomegalovirus is able to persist due to upregulation of the cellular interleukin-10 signaling pathway.

  • Reduction of Neuraminidase Activity Exacerbates Disease in 2009 Pandemic Influenza Virus-Infected Mice [Vaccines and Antiviral Agents]

  • During the first wave of the 2009 pandemic, caused by a H1N1 influenza virus (pH1N1) of swine origin, antivirals were the only form of therapeutic available to control the proliferation of disease until the conventional strain-matched vaccine was produced. Oseltamivir is an antiviral that inhibits the sialidase activity of the viral neuraminidase (NA) protein and was shown to be effective against pH1N1 viruses in ferrets. Furthermore, it was used in humans to treat infections during the pandemic and is still used for current infections without reported complication or exacerbation of illness. However, in an evaluation of the effectiveness of oseltamivir against pH1N1 infection, we unexpectedly observed an exacerbation of disease in virus-infected mice treated with oseltamivir, transforming an otherwise mild illness into one with high morbidity and mortality. In contrast, an identical treatment regime alleviated all signs of illness in mice infected with the pathogenic mouse-adapted virus A/WSN/33 (H1N1). The worsened clinical outcome with pH1N1 viruses occurred over a range of oseltamivir doses and treatment schedules and was directly linked to a reduction in NA enzymatic activity. Our results suggest that the suppression of NA activity with antivirals may exacerbate disease in a host-dependent manner by increasing replicative fitness in viruses that are not optimally adapted for replication in that host.

    IMPORTANCE Here, we report that treatment of pH1N1-infected mice with oseltamivir enhanced disease progression, transforming a mild illness into a lethal infection. This raises a potential pitfall of using the mouse model for evaluation of the therapeutic efficacy of neuraminidase inhibitors. We show that antiviral efficacy determined in a single animal species may not represent treatment in humans and that caution should be used when interpreting the outcome. Furthermore, increased virulence due to oseltamivir treatment was the effect of a shift in the hemagglutinin (HA) and neuraminidase (NA) activity balance. This is the first study that has demonstrated that altering the HA/NA activity balance by reduction in NA activity can result in an increase in virulence in any animal model from nonpathogenic to lethal and the first to demonstrate a situation in which treatment with a NA activity inhibitor has an effect opposite to the intended therapeutic effect of ameliorating the infection.

  • Adoptive Transfer of Engineered Rhesus Simian Immunodeficiency Virus-Specific CD8+ T Cells Reduces the Number of Transmitted/Founder Viruses Established in Rhesus Macaques [Pathogenesis and Immunity]

  • AIDS virus infections are rarely controlled by cell-mediated immunity, in part due to viral immune evasion and immunodeficiency resulting from CD4+ T-cell infection. One likely aspect of this failure is that antiviral cellular immune responses are either absent or present at low levels during the initial establishment of infection. To test whether an extensive, timely, and effective response could reduce the establishment of infection from a high-dose inoculum, we adoptively transferred large numbers of T cells that were molecularly engineered with anti-simian immunodeficiency virus (anti-SIV) activity into rhesus macaques 3 days following an intrarectal SIV inoculation. To measure in vivo antiviral activity, we assessed the number of viruses transmitted using SIVmac239X, a molecularly tagged viral stock containing 10 genotypic variants, at a dose calculated to transmit 12 founder viruses. Single-genome sequencing of plasma virus revealed that the two animals receiving T cells expressing SIV-specific T-cell receptors (TCRs) had significantly fewer viral genotypes than the two control animals receiving non-SIV-specific T cells (means of 4.0 versus 7.5 transmitted viral genotypes; P = 0.044). Accounting for the likelihood of transmission of multiple viruses of a particular genotype, the calculated means of the total number of founder viruses transmitted were 4.5 and 14.5 in the experimental and control groups, respectively (P = 0.021). Thus, a large antiviral T-cell response timed with virus exposure can limit viral transmission. The presence of strong, preexisting T-cell responses, including those induced by vaccines, might help prevent the establishment of infection at the lower-exposure doses in humans that typically transmit only a single virus.

    IMPORTANCE The establishment of AIDS virus infection in an individual is essentially a race between the spreading virus and host immune defenses. Cell-mediated immune responses induced by infection or vaccination are important contributors in limiting viral replication. However, in human immunodeficiency virus (HIV)/SIV infection, the virus usually wins the race, irreversibly crippling the immune system before an effective cellular immune response is developed and active. We found that providing an accelerated response by adoptively transferring large numbers of antiviral T cells shortly after a high-dose mucosal inoculation, while not preventing infection altogether, limited the number of individual viruses transmitted. Thus, the presence of strong, preexisting T-cell responses, including those induced by vaccines, might prevent infection in humans, where the virus exposure is considerably lower.

  • Affinity Purification of the Hepatitis C Virus Replicase Identifies Valosin-Containing Protein, a Member of the ATPases Associated with Diverse Cellular Activities Family, as an Active Virus Replication Modulator [Virus-Cell Interactions]

  • Like almost all of the positive-strand RNA viruses, hepatitis C virus (HCV) induces host intracellular membrane modification to form the membrane-bound viral replication complex (RC), within which viral replicases amplify the viral RNA genome. Despite accumulated information about how HCV co-opts host factors for viral replication, our knowledge of the molecular mechanisms by which viral proteins hijack host factors for replicase assembly has only begun to emerge. Purification of the viral replicase and identification of the replicase-associated host factors to dissect their roles in RC biogenesis will shed light on the molecular mechanisms of RC assembly. To purify the viral replicase in the context of genuine viral replication, we developed an HCV subgenomic replicon system in which two different affinity tags were simultaneously inserted in frame into HCV NS5A and NS5B. After solubilizing the replicon cells, we purified the viral replicase by two-step affinity purification and identified the associated host factors by mass spectrometry. We identified valosin-containing protein (VCP), a member of the ATPases associated with diverse cellular activities (AAA+ATPase) family, as an active viral replication modulator whose ATPase activity is required for viral replication. A transient replication assay indicated that VCP is involved mainly in viral genome amplification. VCP associated with viral replicase and colocalized with a viral RC marker. Further, in an HCV replicase formation surrogate system, abolishing VCP function resulted in aberrant distribution of HCV NS5A. We propose that HCV may co-opt a host AAA+ATPase for its replicase assembly.

    IMPORTANCE Almost all of the positive-strand RNA viruses share a replication strategy in which viral proteins modify host membranes to form the membrane-associated viral replicase. Viruses hijack host factors to facilitate this energy-unfavorable process. Understanding of this fundamental process is hampered by the challenges of purifying the replicase because of the technical difficulties involved. In this study, we developed an HCV subgenomic replicon system in which two different affinity tags were simultaneously inserted in frame into two replicase components. Using this dual-affinity-tagged replicon system, we purified the viral replicase and identified valosin-containing protein (VCP) AAA+ATPase as a pivotal viral replicase-associated host factor that is required for viral genome replication. Abolishing VCP function resulted in aberrant viral protein distribution. We propose that HCV hijacks a host AAA+ATPase for its replicase assembly. Understanding the molecular mechanism of VCP regulates viral replicase assembly may lead to novel antiviral strategies targeting the most conserved viral replication step.

  • Pathogenicity and Transmission of H5 and H7 Highly Pathogenic Avian Influenza Viruses in Mallards [Pathogenesis and Immunity]

  • Wild aquatic birds have been associated with the intercontinental spread of H5 subtype highly pathogenic avian influenza (HPAI) viruses of the A/goose/Guangdong/1/96 (Gs/GD) lineage during 2005, 2010, and 2014, but dispersion by wild waterfowl has not been implicated with spread of other HPAI viruses. To better understand why Gs/GD H5 HPAI viruses infect and transmit more efficiently in waterfowl than other HPAI viruses, groups of mallard ducks were challenged with one of 14 different H5 and H7 HPAI viruses, including a Gs/GD lineage H5N1 (clade 2.2) virus from Mongolia, part of the 2005 dispersion, and the H5N8 and H5N2 index HPAI viruses (clade from the United States, part of the 2014 dispersion. All virus-inoculated ducks and contact exposed ducks became infected and shed moderate to high titers of the viruses, with the exception that mallards were resistant to Ck/Pennsylvania/83 and Ck/Queretaro/95 H5N2 HPAI virus infection. Clinical signs were only observed in ducks challenged with the H5N1 2005 virus, which all died, and with the H5N8 and H5N2 2014 viruses, which had decreased weight gain and fever. These three viruses were also shed in higher titers by the ducks, which could facilitate virus transmission and spread. This study highlights the possible role of wild waterfowl in the spread of HPAI viruses.

    IMPORTANCE The spread of H5 subtype highly pathogenic avian influenza (HPAI) viruses of the Gs/GD lineage by migratory waterfowl is a serious concern for animal and public health. H5 and H7 HPAI viruses are considered to be adapted to gallinaceous species (chickens, turkeys, quail, etc.) and less likely to infect and transmit in wild ducks. In order to understand why this is different with certain Gs/GD lineage H5 HPAI viruses, we compared the pathogenicity and transmission of several H5 and H7 HPAI viruses from previous poultry outbreaks to Gs/GD lineage H5 viruses, including H5N1 (clade 2.2), H5N8 and H5N2 (clade viruses, in mallards as a representative wild duck species. Surprisingly, most HPAI viruses examined in this study replicated well and transmitted among mallards; however, the three Gs/GD lineage H5 HPAI viruses replicated to higher titers, which could explain the transmission of these viruses in susceptible wild duck populations.

  • Glycan Specificity of P[19] Rotavirus and Comparison with Those of Related P Genotypes [Virus-Cell Interactions]

  • The P[19] genotype belongs to the P[II] genogroup of group A rotaviruses (RVs). However, unlike the other P[II] RVs, which mainly infect humans, P[19] RVs commonly infect animals (pigs), making P[19] unique for the study of RV diversity and host ranges. Through in vitro binding assays and saturation transfer difference (STD) nuclear magnetic resonance (NMR), we found that P[19] could bind mucin cores 2, 4, and 6, as well as type 1 histo-blood group antigens (HBGAs). The common sequences of these glycans serve as minimal binding units, while additional residues, such as the A, B, H, and Lewis epitopes of the type 1 HBGAs, can further define the binding outcomes and therefore likely the host ranges for P[19] RVs. This complex binding property of P[19] is shared with the other three P[II] RVs (P[4], P[6], and P[8]) in that all of them recognized the type 1 HBGA precursor, although P[4] and P[8], but not P[6], also bind to mucin cores. Moreover, while essential for P[4] and P[8] binding, the addition of the Lewis epitope blocked P[6] and P[19] binding to type 1 HBGAs. Chemical-shift NMR of P[19] VP8* identified a ligand binding interface that has shifted away from the known RV P-genotype binding sites but is conserved among all P[II] RVs and two P[I] RVs (P[10] and P[12]), suggesting an evolutionary connection among these human and animal RVs. Taken together, these data are important for hypotheses on potential mechanisms for RV diversity, host ranges, and cross-species transmission.

    IMPORTANCE In this study, we found that our P[19] strain and other P[II] RVs recognize mucin cores and the type 1 HBGA precursors as the minimal functional units and that additional saccharides adjacent to these units can alter binding outcomes and thereby possibly host ranges. These data may help to explain why some P[II] RVs, such as P[6] and P[19], commonly infect animals but rarely humans, while others, such as the P[4] and P[8] RVs, mainly infect humans and are predominant over other P genotypes. Elucidation of the molecular bases for strain-specific host ranges and cross-species transmission of these human and animal RVs is important to understand RV epidemiology and disease burden, which may impact development of control and prevention strategies against RV gastroenteritis.

  • Dual-Color Herpesvirus Capsids Discriminate Inoculum from Progeny and Reveal Axonal Transport Dynamics [Virus-Cell Interactions]

  • Alphaherpesviruses such as herpes simplex virus and pseudorabies virus (PRV) are neuroinvasive double-stranded DNA (dsDNA) viruses that establish lifelong latency in peripheral nervous system (PNS) neurons of their native hosts. Following reactivation, infection can spread back to the initial mucosal site of infection or, in rare cases, to the central nervous system, with usually serious outcomes. During entry and egress, viral capsids depend on microtubule-based molecular motors for efficient and fast transport. In axons of PNS neurons, cytoplasmic dynein provides force for retrograde movements toward the soma, and kinesins move cargo in the opposite, anterograde direction. The dynamic properties of virus particles in cells can be imaged by fluorescent protein fusions to the small capsid protein VP26, which are incorporated into capsids. However, single-color fluorescent protein tags fail to distinguish the virus inoculum from progeny. Therefore, we established a dual-color system by growing a recombinant PRV expressing a red fluorescent VP26 fusion (PRV180) on a stable cell line expressing a green VP26 fusion (PK15-mNG-VP26). The resulting dual-color virus preparation (PRV180G) contains capsids tagged with both red and green fluorescent proteins, and 97% of particles contain detectable levels of mNeonGreen (mNG)-tagged VP26. After replication in neuronal cells, all PRV180G progeny exclusively contain monomeric red fluorescent protein (mRFP)-VP26-tagged capsids. We used PRV180G for an analysis of axonal capsid transport dynamics in PNS neurons. Fast dual-color total internal reflection fluorescence (TIRF) microscopy, single-particle tracking, and motility analyses reveal robust, bidirectional capsid motility mediated by cytoplasmic dynein and kinesin during entry, whereas egressing progeny particles are transported exclusively by kinesins.

    IMPORTANCE Alphaherpesviruses are neuroinvasive viruses that infect the peripheral nervous system (PNS) of infected hosts as an integral part of their life cycle. Establishment of a quiescent or latent infection in PNS neurons is a hallmark of most alphaherpesviruses. Spread of infection to the central nervous system is surprisingly rare in natural hosts but can be fatal. Pseudorabies virus (PRV) is a broad-host-range swine alphaherpesvirus that enters neuronal cells and utilizes intracellular transport processes to establish infection and to spread between cells. By using a virus preparation with fluorescent viral capsids that change color depending on the stage of the infectious cycle, we find that during entry, axons of PNS neurons support robust, bidirectional capsid motility, similar to cellular cargo, toward the cell body. In contrast, progeny particles appear to be transported unidirectionally by kinesin motors toward distal egress sites.

  • Intracerebral Inoculation of Mouse-Passaged Saffold Virus Type 3 Affects Cerebellar Development in Neonatal Mice [Pathogenesis and Immunity]

  • Saffold virus (SAFV), a human cardiovirus, is occasionally detected in infants with neurological disorders, including meningitis and cerebellitis. We recently reported that SAFV type 3 isolates infect cerebellar glial cells, but not large neurons, in mice. However, the impact of this infection remained unclear. Here, we determined the neuropathogenesis of SAFV type 3 in the cerebella of neonatal ddY mice by using SAFV passaged in the cerebella of neonatal BALB/c mice. The virus titer in the cerebellum increased following the inoculation of each of five passaged strains. The fifth passaged strain harbored amino acid substitutions in the VP2 (H160R and Q239R) and VP3 (K62M) capsid proteins. Molecular modeling of the capsid proteins suggested that the VP2-H160R and VP3-K62M mutations alter the structural dynamics of the receptor binding surface via the formation of a novel hydrophobic interaction between the VP2 puff B and VP3 knob regions. Compared with the original strain, the passaged strain showed altered growth characteristics in human-derived astroglial cell lines and greater replication in the brains of neonatal mice. In addition, the passaged strain was more neurovirulent than the original strain, while both strains infected astroglial and neural progenitor cells in the mouse brain. Intracerebral inoculation of either the original or the passaged strain affected brain Purkinje cell dendrites, and a high titer of the passaged strain induced cerebellar hypoplasia in neonatal mice. Thus, infection by mouse-passaged SAFV affected cerebellar development in neonatal mice. This animal model contributes to the understanding of the neuropathogenicity of SAFV infections in infants.

    IMPORTANCE Saffold virus (SAFV) is a candidate neuropathogenic agent in infants and children, but the neuropathogenicity of the virus has not been fully elucidated. Recently, we evaluated the pathogenicity of two clinical SAFV isolates in mice. Similar to other neurotropic picornaviruses, these isolates showed mild infectivity of glial and neural progenitor cells, but not of large neurons, in the cerebellum. However, the outcome of this viral infection in the cerebellum has not been clarified. Here, we examined the tropism of SAFV in the cerebellum. We obtained an in vivo-passaged strain from the cerebella of neonatal mice and examined its genome and its neurovirulence in the neonatal mouse brain. The passaged virus showed high infectivity and neurovirulence in the brain, especially the cerebellum, and affected cerebellar development. This unique neonatal mouse model will be helpful for elucidating the neuropathogenesis of SAFV infections occurring early in life.

  • Packaging and Prefusion Stabilization Separately and Additively Increase the Quantity and Quality of Respiratory Syncytial Virus (RSV)-Neutralizing Antibodies Induced by an RSV Fusion Protein Expressed by a Parainfluenza Virus Vector [Vaccines and Antiviral Agents]

  • Human respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are major pediatric respiratory pathogens that lack vaccines. A chimeric bovine/human PIV3 (rB/HPIV3) virus expressing the unmodified, wild-type (wt) RSV fusion (F) protein from an added gene was previously evaluated in seronegative children as a bivalent intranasal RSV/HPIV3 vaccine, and it was well tolerated but insufficiently immunogenic for RSV F. We recently showed that rB/HPIV3 expressing a partially stabilized prefusion form (pre-F) of RSV F efficiently induced "high-quality" RSV-neutralizing antibodies, defined as antibodies that neutralize RSV in vitro without added complement (B. Liang et al., J Virol 89:9499nndash;9510, 2015, doi:10.1128/JVI.01373-15). In the present study, we modified RSV F by replacing its cytoplasmic tail (CT) domain or its CT and transmembrane (TM) domains (TMCT) with counterparts from BPIV3 F, with or without pre-F stabilization. This resulted in RSV F being packaged in the rB/HPIV3 particle with an efficiency similar to that of RSV particles. Enhanced packaging was substantially attenuating in hamsters (10- to 100-fold) and rhesus monkeys (100- to 1,000-fold). Nonetheless, TMCT-directed packaging substantially increased the titers of high-quality RSV-neutralizing serum antibodies in hamsters. In rhesus monkeys, a strongly additive immunogenic effect of packaging and pre-F stabilization was observed, as demonstrated by 8- and 30-fold increases of RSV-neutralizing serum antibody titers in the presence and absence of added complement, respectively, compared to pre-F stabilization alone. Analysis of vaccine-induced F-specific antibodies by binding assays indicated that packaging conferred substantial stabilization of RSV F in the pre-F conformation. This provides an improved version of this well-tolerated RSV/HPIV3 vaccine candidate, with potently improved immunogenicity, which can be returned to clinical trials.

    IMPORTANCE Human respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are major viral agents of acute pediatric bronchiolitis and pneumonia worldwide that lack vaccines. A bivalent intranasal RSV/HPIV3 vaccine candidate consisting of a chimeric bovine/human PIV3 (rB/HPIV3) strain expressing the RSV fusion (F) protein was previously shown to be well tolerated by seronegative children but was insufficiently immunogenic for RSV F. In the present study, the RSV F protein was engineered to be packaged efficiently into vaccine virus particles. This resulted in a significantly enhanced quantity and quality of RSV-neutralizing antibodies in hamsters and nonhuman primates. In nonhuman primates, this effect was strongly additive to the previously described stabilization of the prefusion conformation of the F protein. The improved immunogenicity of RSV F by packaging appeared to involve prefusion stabilization. These findings provide a potently more immunogenic version of this well-tolerated vaccine candidate and should be applicable to other vectored vaccines.

  • Efficiency in Complexity: Composition and Dynamic Nature of Mimivirus Replication Factories [Structure and Assembly]

  • The recent discovery of multiple giant double-stranded DNA (dsDNA) viruses blurred the consensual distinction between viruses and cells due to their size, as well as to their structural and genetic complexity. A dramatic feature revealed by these viruses as well as by many positive-strand RNA viruses is their ability to rapidly form elaborate intracellular organelles, termed "viral factories," where viral progeny are continuously generated. Here we report the first isolation of viral factories at progressive postinfection time points. The isolated factories were subjected to mass spectrometry-based proteomics, bioinformatics, and imaging analyses. These analyses revealed that numerous viral proteins are present in the factories but not in mature virions, thus implying that multiple and diverse proteins are required to promote the efficiency of viral factories as "production lines" of viral progeny. Moreover, our results highlight the dynamic and highly complex nature of viral factories, provide new and general insights into viral infection, and substantiate the intriguing notion that viral factories may represent the living state of viruses.

    IMPORTANCE Large dsDNA viruses such as vaccinia virus and the giant mimivirus, as well as many positive-strand RNA viruses, generate elaborate cytoplasmic organelles in which the multiple and diverse transactions required for viral replication and assembly occur. These organelles, which were termed "viral factories," are attracting much interest due to the increasing realization that the rapid and continuous production of viral progeny is a direct outcome of the elaborate structure and composition of the factories, which act as efficient production lines. To get new insights into the nature and function of viral factories, we devised a method that allows, for the first time, the isolation of these organelles. Analyses of the isolated factories generated at different times postinfection by mass spectrometry-based proteomics provide new perceptions of their role and reveal the highly dynamic nature of these organelles.

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    JVI Accepts: Articles Published Ahead of Print

  • Critical Contribution of Tyr15 in the HIV-1 Integrase (IN) to Facilitate the IN Assembly and Non-enzymatic Function Through the IN Precursor Form with Reverse Transcriptase [PublishAheadOfPrint]

  • Non-enzymatic roles for HIV-1 integrase (IN) at steps prior to the enzymatic integration step have been reported. To obtain structural and functional insights into the non-enzymatic roles of IN, we performed genetic analyses of HIV-1 IN focusing on a highly conserved Tyr15 in the N-terminal domain (NTD), which has previously been shown to regulate an equilibrium state between two NTD dimer conformations. Substitution of Tyr15 with alanine, histidine, or tryptophan prevented HIV-1 infection and caused severe impairment of reverse transcription without apparent defects in reverse transcriptase (RT) or in capsid disassembly kinetics after entry into cells. Cross-link analyses of recombinant IN proteins demonstrated that lethal mutations of Tyr15 severely impaired IN structure for assembly. Notably, substitution of Tyr15 with phenylalanine was tolerated for all IN functions, demonstrating that a benzene ring of the aromatic side chain is a key moiety for IN assembly and functions. Additional mutagenic analyses based on previously proposed tetramer models for IN assembly suggested a key role of Tyr15 in facilitating the hydrophobic interaction among IN subunits, together with other proximal residues within the subunit interface. Rescue experiment of RT-IN-deleted (RTIN) HIV-1 by supplying IN and RT in trans revealed that the non-enzymatic IN function might be exerted through the IN precursor conjugated with RT (RT-IN). Importantly, the lethal mutations of Tyr15 significantly reduced the RT-IN function and assembly. Taken together, Tyr15 seems to play a key role in facilitating the proper assembly of IN and RT on viral RNA through the RT-IN precursor form.

    IMPORTANCE Inhibitors of the IN enzymatic strand-transfer function (INSTI) have been applied in combination antiretroviral therapies to treat HIV-1-infected patients. Recently, allosteric IN inhibitors (ALLINs) that interact with HIV-1 IN residues, the locations of which are distinct from the catalytic sites targeted by INSTI, have been discovered. Importantly, ALLINs affect the non-enzymatic role(s) of HIV-1 IN, providing a rationale for the development of next-generation IN inhibitors with a mechanism that is distinct from that of INSTI. Here, we demonstrate that Tyr15 in the HIV-1 IN NTD plays a critical role during IN assembly by facilitating the hydrophobic interaction of the NTD with the other domains of IN. Importantly, we found that the functional assembly of IN through its fusion form with RT is critical for IN to exert its non-enzymatic function. Our results provide a novel mechanistic insight into the non-enzymatic function of HIV-1 IN and its prevention.

  • JunD/AP-1 antagonizes the induction of DAPK1 to promote the survival of v-Src transformed cells [PublishAheadOfPrint]

  • The increase in AP-1 activity is a hallmark of cell transformation by tyrosine kinases. Previously, we reported that blocking AP-1 using the c-Jun dominant-negative mutant TAM67 induced senescence, adipogenesis or apoptosis in v-Src-transformed chicken embryo fibroblasts (CEF) whereas inhibition of JunD by shRNA specifically induced apoptosis. To investigate the role of AP-1 in Src-mediated transformation, we undertook a gene profiling study to characterize the transcriptomes of v-Src-transformed CEF expressing either TAM67 or the JunD shRNA. Our study revealed a cluster of 18 probe-sets up-regulated exclusively in response to AP-1/JunD impairment and v-Src transformation. Four of these probe-sets correspond to genes involved in the interferon pathway. One gene in particular, death-associated protein kinase 1 (DAPK1), is a C/EBPbbeta;-regulated mediator of apoptosis in IFN-induced cell death. Here we show that inhibition of DAPK1 abrogates cell-death in v-Src-transformed cells expressing the JunD shRNA. Chromatin immunoprecipitation data indicated that C/EBPbbeta; was recruited to the DAPK1 promoter while the expression of a dominant negative mutant of C/EBPbbeta; abrogated the induction of DAPK1 in response to the inhibition of AP-1. In contrast, as determined by ChIP assays, JunD was not detected on the DAPK1 promoter in any conditions, suggesting that JunD promotes survival by indirectly antagonizing the expression of DAPK1 in v-Src transformed cells.

    IMPORTANCE Transformation by the v-Src oncoprotein causes extensive changes in gene expression in primary cells such as chicken embryo fibroblasts. These changes, determining the properties of transformed cells, are controlled in part at the transcriptional level. Much attention has been devoted to transcription factors such as AP-1 and NF-B and the control of genes associated with a more aggressive phenotype. In this report, we describe a novel mechanism of action determined by the JunD component of AP-1, a factor enhancing cell survival in v-Src transformed cells. We show that the loss of JunD results in the aberrant activation of a genetic program leading to cell death. This program requires the activation of the tumour suppressor death-associated protein kinase 1 (DAPK1). Since DAPK1 is phosphorylated and inhibited by v-Src, these results highlight the importance of this kinase and the multiple mechanisms controlled by v-Src to antagonize the tumour suppressor function of DAPK1.

  • Carbon Monoxide Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication by the cGMP/PKG and NF-{kappa}B Signaling Pathway [PublishAheadOfPrint]

  • Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the pork industry worldwide each year. Our previous research had demonstrated that heme oxygenase-1 (HO-1) can suppress PRRSV replication via an unknown molecular mechanism. In this study, inhibition of PRRSV replication was demonstrated to be mediated by carbon monoxide (CO), a downstream metabolite of HO-1. Using several approaches, we demonstrate that CO significantly inhibited PRRSV replication in both PRRSV permissive cell line, MARC-145, and the predominant cell type targeted during in vivo PRRSV infection, porcine alveolar macrophages (PAMs). Our results showed that CO inhibited intercellular spread of PRRSV, however, it did not affect PRRSV entry of host cells. Furthermore, CO was found to suppress PRRSV replication via the activation of the cGMP/PKG signaling pathway. CO significantly inhibits PRRSV-induced NF-B activation, a required step for PRRSV replication. Moreover, CO significantly reduced PRRSV-induced pro-inflammatory cytokine mRNA levels. In conclusion, the present study demonstrates that CO exerts its anti-PRRSV effect by activating the cellular cGMP/PKG signaling pathway and by negatively regulating cellular NF-B signaling. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of PRRSV replication, but also suggest potential new control measures for future PRRSV outbreaks.

    IMPORTANCE PRRSV causes great economic losses each year to the swine industry worldwide. Carbon monoxide (CO), a metabolite of HO-1, has been shown to have antimicrobial and antiviral activities in infected cells. Our previous research demonstrated that HO-1 can suppress PRRSV replication. Here we show that endogenous CO produced through HO-1 catalysis mediates the antiviral effect of HO-1. CO inhibits PRRSV replication by activating the cellular cGMP/PKG signaling pathway and by negatively regulating cellular NF-B signaling. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of PRRSV replication, but also suggest potential new control measures for future PRRSV outbreaks.

  • The cellular DNA helicase ChlR1 regulates chromatin and nuclear matrix attachment of the human papillomavirus type 16 E2 protein and high copy viral genome establishment [PublishAheadOfPrint]

  • In papillomavirus infections, the viral genome is established as a double-stranded DNA episome. To segregate the episomes to daughter cells during mitosis, they are tethered to cellular chromatin by the viral E2 protein. We previously demonstrated that the E2 proteins of diverse papillomavirus types, including bovine papillomavirus (BPV) and human papillomavirus type 16 (HPV16), associate with the cellular DNA helicase ChlR1. This virus-host interaction is important for the tethering of BPV E2 to mitotic chromatin and the stable maintenance of BPV episomes. The role of the association between E2 and ChlR1 in the HPV16 life cycle is unresolved. Here, we show that the HPV16 E2 mutation Y131A has significantly reduced binding to ChlR1, but retains transcriptional activation and viral origin-dependent replication functions. Subcellular fractionation of keratinocytes expressing E2Y131A shows a marked change in the localization of the protein. In comparison to wild type E2, the chromatin-bound pool of E2Y131A is decreased, concomitant with an increase in nuclear matrix-associated protein. Cell cycle synchronization indicates that the shift in subcellular localization of E2Y131A occurs in mid-S phase. A similar alteration between the subcellular pools of E2WT protein occurred upon ChlR1 silencing. Notably, in an HPV16 life cycle model in primary human keratinocytes, mutant E2Y131A genomes were established as episomes, but at a markedly lower copy number than wild type HPV16 genomes, and were not maintained upon cell passage. Our studies indicate that ChlR1 is an important regulator of the chromatin association of E2 and of the establishment and maintenance of HPV16 episomes.

    IMPORTANCE Infection with high-risk human papillomavirus (HPV) types is a major cause of anogenital and oropharyngeal cancers. During infection, the circular DNA HPV genome persists within the nucleus, independently of the host cell chromatin. Persistence of infection is a risk factor for cancer development and is partly achieved by the attachment of viral DNA to cellular chromatin during cell division. The HPV E2 protein plays a critical role in this tethering by simultaneously binding to the viral genome and chromatin during mitosis. We previously showed that the cellular DNA helicase ChlR1 is required for the loading of the bovine papillomavirus E2 protein onto chromatin during DNA synthesis. Here, we have identified a mutation in HPV16 E2 that abrogates interaction with ChlR1 and show that ChlR1 regulates the chromatin association of HPV16 E2 and that this virus-host interaction is essential for viral episome maintenance.

  • A Helical Short-Peptide Fusion Inhibitor with Highly Potent Activities against HIV-1, HIV-2 and Simian Immunodeficiency Virus [PublishAheadOfPrint]

  • HIV-2 has already spread to different regions worldwide and currently about 1-2 million people have been infected, calling for new antiviral agents that are effective on both HIV-1 and HIV-2 isolates. T-20 (Enfuvirtide), a 36-mer peptide derived from the C-terminal heptad repeat region (CHR) of gp41, is the only clinically approved HIV-1 fusion inhibitor, but it easily induces drug-resistance and is not active on HIV-2. In this study, we firstly demonstrated that the M-T hook structure was also a vital strategy to enhance the binding stability and inhibitory activity of diverse CHR-based peptide inhibitors. Then, we designed a novel short-peptide (23-mer) termed 2P23 by introducing the M-T hook structure, HIV-2 sequences and llsquo;salt-bridges'-forming residues. Promisingly, 2P23 was a highly stable helical peptide with high binding to the surrogate targets derived from HIV-1, HIV-2 and simian immunodeficiency virus (SIV). In consistence, 2P23 exhibited potent activity in inhibiting diverse subtypes of HIV-1 isolates, T-20-resistant HIV-1 mutants, and a panel of primary HIV-2 isolates, HIV-2 mutants and SIV isolates. Therefore, we conclude that 2P23 has high potential to be further developed for clinical use and it is also an ideal tool for exploring the mechanisms of HIV-1/2 and SIV-mediated membrane fusion.

    IMPORTANCE The peptide drug T-20 is the only approved HIV-1 fusion inhibitor, but it is not active on HIV-2 isolates which have currently infected 1-2 million people and continuingly spread worldwide. Recent studies have demonstrated that the M-T hook structure can greatly enhance the binding and antiviral activities of gp41 CHR-derived inhibitors, especially for short-peptides otherwise inactive. By combining the hook structure, HIV-2 sequence and llsquo;salt-bridge'-based strategies, the short-peptide 2P23 has been successfully designed. 2P23 exhibits prominent advantages over many other peptide fusion inhibitors, including its potent and broad activity on HIV-1, HIV-2 and even SIV isolates, its stability as a helical, oligomeric peptide, and its high binding to diverse targets. The small size of 2P23 would benefit its synthesis and significantly reduce production cost. Therefore, 2P23 is an ideal candidate for further development and it also provides a novel tool for studying HIV-1/2 and SIV-mediated cell fusion.

  • Naf1 regulates HIV-1 latency by suppressing viral promoter-driven gene expression in primary CD4+ T cells [PublishAheadOfPrint]

  • HIV-1 latency is characterized by reversible silencing of viral transcription driven by the long terminal repeat (LTR) promoter of HIV-1. Cellular and viral factors regulating the LTR activity contribute to HIV-1 latency and certain repressive cellular factors modulate viral transcription silencing. Nef-associated factor 1 (Naf1) is a host nucleo-cytoplasmic shuttling protein and regulates multiple cellular signal pathways and HIV-1 production. We have recently reported that the nucleus-located Naf1 promoted nuclear export of unspliced HIV-1 gag mRNA, leading to the accumulated Gag production. Here, we demonstrate new functions of Naf1 in regulating HIV-1 persistence. We found that Naf1 contributes to the maintenance of HIV-1 latency by inhibiting LTR-driven HIV-1 gene transcription in a nuclear factor kappa B-dependent manner. Interestingly, Naf1 knockdown significantly enhanced viral reactivation in both HIV-1 latently infected Jurkat T cells and primary central memory CD4+ T cells. Furthermore, Naf1 knockdown in resting CD4+ T cells from HIV-1-infected individuals treated with antiretroviral therapy significantly increased viral reactivation upon T-cell activation, suggesting an important role of Naf1 in modulating HIV-1 latency in vivo. Our findings provide new insights into a better understanding of HIV-1 latency, and implicate that the inhibition of Naf1 activity to activate HIV-1 latently infected cells may be a potential therapeutic strategy.

    IMPORTANCE HIV-1 latency is mainly characterized by a reversible silencing of long terminal repeats (LTR) promoter-driven transcription of an integrated provirus. Cellular and viral proteins regulating LTR activity contribute to the modulation of HIV-1 latency. In this study, we found that host protein Naf1 inhibited HIV-1-LTR-driven transcription of HIV genes and contributed to the maintenance of HIV-1 latency. Our findings provide new insights into a better understanding of host-modulation on HIV-1 latency, which may lead to a potential therapeutic strategy for HIV persistence by targeting Naf1 protein.

  • CLEC5A-mediated enhancement of the inflammatory response in myeloid cells contributes to influenza pathogenicity in vivo [PublishAheadOfPrint]

  • Human infections with influenza viruses exhibit mild to severe clinical outcome as a result of complex virus-host interactions. Induction of inflammatory mediators via pattern recognition receptors may dictate subsequent host responses for pathogen clearance and tissue damage. We identified that human C-type lectin member 5A (CLEC5A) interacts with the hemagglutinin protein of influenza viruses expressed on lentiviral pseudoparticles through lectin screening. Silencing CLEC5A gene expression, blocking influenza-CLEC5A interactions with anti-CLEC5A antibodies, or dampening CLEC5A-mediated signaling using a spleen tyrosine kinase inhibitor consistently reduced the levels of pro-inflammatory cytokines produced by human macrophages without affecting the replication of influenza A viruses of different subtypes. Infection of bone marrow-derived macrophages from CLEC5A-deficient mice showed reduced TNF-aalpha; and IP-10 but elevated IFN-aalpha; compared to wild-type mice. The heightened type-I IFN response in the macrophages of CLEC5A-deficient mice was associated with up-regulated TLR3 mRNA after treatment with double stranded RNA. Upon lethal challenges with a recombinant H5N1 virus, CLEC5A-deficient mice showed reduced levels of pro-inflammatory cytokines, decreased immune cell infiltration in the lungs and improved survival compared to the wild-type mice, despite comparable viral loads noted throughout the course of infection. The survival difference was more prominent at a lower dose of inoculum. Our results suggest that CLEC5A-mediated enhancement of the inflammatory response in myeloid cells contributes to influenza pathogenicity in vivo and may be considered as a therapeutic target in combination with effective antivirals. Well-orchestrated host responses together with effective viral clearance are critical for optimal clinical outcome after influenza infections.

    IMPORTANCE Multiple pattern recognition receptors work in synergy to sense viral RNA or proteins synthesized during influenza replication and mediate host responses for viral control. Well-orchestrated host responses may help to maintain the inflammatory response at gate to minimize tissue damage while inducing an effective adaptive immune response for viral clearance. We identified that CLEC5A, a C-type lectin receptor which has previously been reported to mediate flavivirus-induced inflammatory responses, enhanced induction of pro-inflammatory cytokines and chemokines in myeloid cells after influenza infections. CLEC5A-deficient mice infected with influenza virus showed reduced inflammation in the lungs and improved survival than the wild-type mice, despite comparable viral loads. The survival difference was more prominent at a lower dose of inoculum. Collectively, our results suggest that dampening CLEC5A-mediated inflammatory responses in myeloid cells may reduce immunopathogenesis after influenza infections.

  • Efficient transformation of primary human mesenchymal stromal cells by adenovirus early region 1 oncogenes [PublishAheadOfPrint]

  • Previous observations that human amniotic fluid cells (AFC) can be transformed by human adenovirus type 5 (HAdV-5) E1A/E1B oncogenes prompted us to identify the target cells in the AFC population that are susceptible for transformation. Our results demonstrate that one cell type corresponding to mesenchymal stem/stroma cells (hMSC), can be reproducibly transformed by HAdV-5 E1A/E1B oncogenes as efficient as primary rodent cultures. HAdV-5 E1-transformed hMSCs exhibit all properties commonly associated with a high grade of oncogenic transformation, including enhanced cell proliferation, anchorage-independent growth, increased growth rate, high telomerase activity as well as numerical and structural chromosomal aberrations. These data confirm previous work showing that HAdV preferentially transform cells of mesenchymal origin in rodents. More importantly, they demonstrate for the first time that human cells with stem cell characteristics can be completely transformed by HAdV oncogenes in tissue culture at a high efficiency. Our findings strongly support the hypothesis that undifferentiated progenitor cells or cells with stem cell-like properties are highly susceptible targets for HAdV-mediated cell transformation and suggest that virus-associated tumors in humans may originate, at least in part, from infections of these cell types. We expect that primary hMSC will replace the primary rodent cultures in HAdV viral transformation studies and are confident that these investigations will continue to uncover general principles of viral oncogenesis that can be extended to human DNA tumor viruses as well.

    IMPORTANCE It is generally believed that transformation of primary human cells with HAdV-5 E1 oncogenes is very inefficient. However, a few cell lines have been successfully transformed with HAdV-5 E1A and E1B, indicating that there is a certain cell type, which is susceptible for HAdV-mediated transformation. Interestingly, all those cell lines have been derived from human embryonic tissue, albeit the exact cell type, is not known yet. We show for the first time the successful transformation of primary human mesenchymal stromal cells (hMSC) by HAdV-5 E1A and E1B. Further, we show upon HAdV-5 E1A and E1B expression, that these primary progenitor cells exhibit features of tumor cells and can no longer be differentiated into the adipogenic, chondrogenic or osteogenic lineage. Hence, primary hMSC represent a robust and novel model system to elucidate the underlying molecular mechanisms of adenovirus-mediated transformation of multipotent human progenitor cells.

  • Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone [PublishAheadOfPrint]

  • Zika virus (ZIKV; Family Flaviviridae, genus Flavivirus) is a rapidly expanding global pathogen that has been associated with severe clinical manifestations, including devastating neurological disease in infants. There are currently no molecular clones of a New World ZIKV available, hindering progress toward understanding determinants of transmission and pathogenesis. Here we report the development and characterization of a novel ZIKV reverse genetics system based on a 2015 isolate from Puerto Rico (PRVABC59). We generated a two-plasmid infectious clone system from which infectious virus was rescued that replicates in human and mosquito cells with growth kinetics representative of wild-type ZIKV. Infectious clone-derived virus initiated comparable infection and transmission rates in Aedes aegypti mosquitoes compared to the primary isolate and displayed similar pathogenesis in AG129 mice. This infectious clone system provides a valuable resource to the research community to explore ZIKV molecular biology, vaccine development, antiviral development, diagnostics, vector competence, and disease pathogenesis.

    IMPORTANCE ZIKV is a rapidly spreading mosquito-borne pathogen that has been linked to Guillain-Barreeacute; syndrome in adults and congenital microcephaly in developing fetuses and infants. ZIKV can also be sexually transmitted. The viral molecular determinants of any of these phenotypes are not well understood. There is no reverse genetics system available for the current epidemic virus that will allow researchers to study ZIKV immunity, develop novel vaccines, or develop antiviral drugs. Here we provide a novel infectious clone system generated from a recent ZIKV isolated from a patient infected in Puerto Rico. This infectious clone produces virus with similar in vitro and in vivo characteristics to the primary isolate, providing a critical tool to study ZIKV infection and disease.

  • Broadly Neutralising Antibodies Display Superior Potential Over Non-Neutralising Antibodies in Preventing HIV-1 infection of Mucosal Tissue [PublishAheadOfPrint]

  • Definition of the key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue may prove critical to effective vaccine development and the prophylactic use of monoclonal antibodies. Although direct antibody-mediated neutralisation is highly effective against cell-free virus, antibodies targeting different sites of envelope vulnerability may display differential activity against mucosal infection. Non-neutralising antibodies (nnAbs) may also impact on mucosal transmission events through Fc-gamma receptor (FcR)-mediated inhibition. Here, a panel of broadly neutralising (bnAbs) and nnAbs, including those associated with protection in the RV144 vaccine trial, were screened for their ability to block HIV-1 acquisition and replication across a range of cellular and mucosal tissue models. Neutralisation potency, as determined by the TZM-bl infection assay, did not fully predict activity in mucosal tissue. CD4bs-specific bnAbs, and in particular VRC01, were consistent in blocking HIV-1 infection across all cellular and tissue models. MPER (2F5) and outer-domain glycan (2G12) bnAbs were also efficient in preventing infection of mucosal tissues, while the protective efficacy of bnAbs targeting V1-V2 glycans (PG9 and PG16) were more variable. In contrast, nnAb alone and in combinations, while active in a range of cellular assays, were poorly protective against HIV-1 infection of mucosal tissues. These data suggest tissue resident effector cell numbers and low FcR expression may limit the potential of nnAbs to prevent establishment of the initial foci of infection. The solid protection provided by specific bnAbs clearly demonstrates their superior potential over non-neutralising antibodies for preventing HIV-1 infection at the mucosal portals of infection.

    IMPORTANCE Key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue have not been defined. While bnAbs are highly effective against cell-free virus, they are not induced by current vaccine candidates. However, nnAbs, readily induced by vaccines, can trigger antibody-dependent cellular effector functions, through engagement of their Fc-gamma receptors. Fc-mediated antiviral activity has been implicated as a secondary correlate of decreased HIV-1 risk in the RV144 vaccine efficacy trial, suggesting protection might be mediated in the absence of classical neutralisation. To aid vaccine design and selection of antibodies for use in passive protection strategies we assessed a range of bnAbs and nnAbs for their potential to block ex-vivo challenge of mucosal tissues. Our data clearly indicate the superior efficacy of neutralising antibodies in preventing mucosal acquisition of infection. These results underscore the importance of maintaining the central focus of HIV-1 vaccine research on the induction of potently neutralising antibodies.

  • Host cellular protein TRAPPC6A{Delta} interacts with influenza A virus M2 protein and regulates viral propagation by modulating M2 trafficking [PublishAheadOfPrint]

  • Influenza A virus (IAV) matrix protein 2 (M2) plays multiple roles in the early and late phases of viral infection. Once synthesized, M2 is translocated to the endoplasmic reticulum (ER), travels to the Golgi apparatus, and is sorted at the trans-Golgi network (TGN) for transport to the apical plasma membrane, where it functions in virus budding. We hypothesized that M2 trafficking along with its secretory pathway must be finely regulated, and host factors could be involved in this process. However, no studies examining the role of host factors in M2 post-translational transport have been reported. Here, we used a yeast two-hybrid (Y2H) system to screen for host proteins that interact with the M2 protein and identified transport protein particle complex 6A (TRAPPC6A) as a potential binding partner. We found that both TRAPPC6A and its N-terminal internal deletion isoform TRAPPC6A delta (TRAPPC6A) interact with M2. Truncation and mutation analyses showed that the highly conserved leucine residue at position 96 of M2 is critical for mediating this interaction. The role of TRAPPC6A in the viral life cycle was investigated by knockdown of endogenous TRAPPC6A with small interfering RNA (siRNA) and by generating a recombinant virus that was unable to interact with TRAPPC6A/TRAPPC6A. The results indicated that TRAPPC6A, through its interaction with M2, slows down M2 trafficking to the apical plasma membrane, favors viral replication in vitro, and positively modulates virus virulence in mice.

    IMPORTANCE Influenza A virus M2 protein regulates the trafficking of not only other proteins but also itself along the secretory pathway. However, the host factors involved in the regulation of the post-translational transport of M2 are largely unknown. In this study, we identified TRAPPC6A and its N-terminal internal deletion isoform TRAPPC6A as interacting partners of M2. We found that the leucine (L) residue at position 96 of M2 is critical for mediating this interaction, which leads us to propose that the high level of conservation of 96L is a consequence of M2 adaptation to its interacting host factors, TRAPPC6A/TRAPPC6A. Importantly, we discovered that TRAPPC6A can positively regulate viral replication in vitro by modulating M2 trafficking to the plasma membrane.

  • Differential infection patterns and recent evolutionary origins of equine hepaciviruses in donkeys [PublishAheadOfPrint]

  • The hepatitis C virus (HCV) is a major human pathogen. Genetically related viruses in animals suggest a zoonotic origin of HCV. The closest relative of HCV is found in horses (termed equine hepacivirus, EqHV). However, low EqHV genetic diversity implies relatively recent acquisition of EqHV by horses, making a derivation of HCV from EqHV unlikely. To unravel the EqHV evolutionary history within equid sister species, we analyzed 829 donkeys and 53 mules sampled in nine European, Asian, African and American countries by molecular and serologic tools for EqHV infection. Antibodies were found in 278 animals (31.5%), and viral RNA was found in 3 animals (0.3%), all of which were simultaneously seropositive. A low RNA prevalence in spite of high seroprevalence suggests predominance of acute infection, a possible difference from the mostly chronic hepacivirus infection pattern seen in horses and humans. Limitation of transmission due to short courses of infection may explain the existence of entirely seronegative groups of animals. Donkey and horse EqHV strains were paraphyletic and 97.5-98.2% identical in their translated polyprotein sequences, making virus/host co-speciation unlikely. Evolutionary reconstructions supported host switches of EqHV between horses and donkeys without the involvement of adaptive evolution. Global admixture of donkey and horse hepaciviruses was compatible with anthropogenic alterations of EqHV ecology. In summary, our findings do not support EqHV as the origin of the significantly more diversified HCV. Identification of a host system with predominantly acute hepacivirus infection may enable new insights into the chronic infection pattern associated with HCV.

    IMPORTANCE The evolutionary origins of the human hepatitis C virus (HCV) are unclear. The closest animal-associated relative of HCV occurs in horses (equine hepacivirus, EqHV). The low EqHV genetic diversity implies a relatively recent acquisition of EqHV by horses, limiting the time span for potential horse-to-human infections in the past. Horses are genetically related to donkeys and EqHV may have co-speciated with these host species. Here, we investigated a large panel of donkeys from various countries using serologic and molecular tools. We found EqHV to be globally widespread in donkeys and identify potential differences in EqHV infection patterns, with donkeys potentially showing enhanced EqHV clearance compared to horses. We provide strong evidence against EqHV co-speciation and for its capability to switch hosts among equines. Differential hepacivirus infection patterns in horses and donkeys may enable new insights into the chronic infection pattern associated with HCV.

  • The long and complicated relationship between Epstein-Barr virus and epithelial cells [PublishAheadOfPrint]

  • The role of epithelial cells in infection and persistence of Epstein-Barr virus (EBV) has long been difficult to resolve. Recent developments have, however, reinforced both the conclusion that they are a major site of virus replication and raised the possibility that, like papillomaviruses, EBV has evolved to take advantage of epithelial differentiation to ensure both survival persistence and spread.

  • Recombinant receptor-binding domains of multiple MERS-coronaviruses induce cross-neutralizing antibodies against divergent human and camel MERS-coronaviruses and antibody-escape mutants [PublishAheadOfPrint]

  • Middle East respiratory syndrome coronavirus (MERS-CoV) binds to cellular receptor dipeptidyl peptidase 4 (DPP4) via spike (S) protein receptor-binding domain (RBD). The RBD contains critical neutralizing epitopes and serves as an important vaccine target. Since RBD mutations occur in different MERS-CoV isolates and antibody-escape mutants, cross-neutralization of divergent MERS-CoV strains by RBD-induced antibodies remains unknown. Here, we constructed four recombinant RBD (rRBD) proteins with single or multiple mutations detected in representative human MERS-CoV strains from the 2012, 2013, 2014 and 2015 outbreaks, respectively, and one rRBD protein with multiple changes derived from camel MERS-CoV strains. Like the RBD of prototype EMC2012 (EMC-RBD), all five RBDs maintained good antigenicity and functionality, the ability to bind RBD-specific neutralizing mAbs and the DPP4 receptor, and high immunogenicity, able to elicit S-specific antibodies. They induced potent neutralizing antibodies cross-neutralizing 17 MERS pseudoviruses expressing S proteins of representative human and camel MERS-CoV strains identified during the 2012-2015 outbreaks, 5 mAb-escape MERS-CoV mutants, and 2 live human MERS-CoV strains. We then constructed two RBDs mutated in multiple key residues in the receptor-binding motif (RBM) of RBD and demonstrated their strong cross-reactivity with anti-EMC-RBD antibodies. These RBD mutants with diminished DPP4 binding also led to virus attenuation, suggesting that immunoevasion after RBD immunization is accompanied by loss of viral fitness. Therefore, this study demonstrates that MERS-CoV RBD is an important vaccine target able to induce highly potent and broad-spectrum neutralizing antibodies against infection by divergent circulating human and camel MERS-CoV strains.

    IMPORTANCE MERS-CoV was first identified in June 2012 and has since spread in humans and camels. Mutations in its spike (S) protein receptor-binding domain (RBD), a key vaccine target, have been identified, raising concerns over the efficacy of RBD-based MERS vaccines against circulating human and camel MERS-CoV strains. Here, we constructed five vaccine candidates, designated 2012-RBD, 2013-RBD, 2014-RBD, 2015-RBD, and camel-RBD, respectively, containing single or multiple mutations in the RBD of representative human and camel MERS-CoV strains during the 2012-2015 outbreaks. These RBD-based vaccine candidates maintained good functionality, antigenicity and immunogenicity, and they induced strong cross-neutralizing antibodies against infection by divergent pseudotyped and live MERS-CoV strains, as well as antibody-escape MERS-CoV mutants. This study provides impetus for further development of a safe, highly effective, and broad-spectrum RBD-based subunit vaccine to prevent MERS-CoV infection.

  • Potent Inhibition of HCMV by Modulating the Cellular SNARE Syntaxin 5 [PublishAheadOfPrint]

  • Formation of the cytoplasmic viral assembly compartment (cVAC) is an important step for efficient HCMV assembly. To do this, the virus must alter and repurpose the normal cellular balance of membrane and protein flux, a process that is not well understood. Although a recent screen identified three viral proteins essential for cVAC formation, less is known about the contribution of cellular factors. We show that HCMV infection increases the protein level of a cellular trafficking factor, Syntaxin 5 (STX5), a member of the syntaxin family of SNARE proteins. STX5 is recruited to the cVAC in infected cells and is required for the efficient production of infectious virions. We find that STX5 is important for normal cVAC morphology and the proper localization of viral proteins. A previously identified inhibitor of trafficking, Retro94, causes the mislocalization of STX5, an altered cVAC morphology and dispersal of viral proteins. The presence of Retro94 results in the severely impaired production of infectious virions, as great as 5 logs. We show that this inhibition is conserved among different strains of HCMV, the various cell types that support infection, as well as for murine CMV. Thus, our data identify a key cellular trafficking factor important for supporting HCMV infection.

    IMPORTANCE Human cytomegalovirus (HCMV) infection causes severe disease and mortality in immunocompromised individuals, including organ transplant and AIDS patients. In addition, infection of a developing fetus may result in lifelong complications such as deafness and learning disabilities. Understanding in detail the processes involved in HCMV replication is important for developing novel treatments. One of these essential processes, assembly of infectious virions, takes places in the cytoplasmic viral assembly compartment. We identify a cellular protein, syntaxin 5, important for generating this compartment and show that it is required for the efficient production of infectious virions. We also show that a small molecule that disrupts this protein also significantly reduces the amount of infectious virions that are generated. Thus, by pinpointing a cellular protein that is important in the replication cycle of HCMV, we identified a novel target that can be pursued for therapeutic intervention.

  • Native folding of a recombinant gpE1/gpE2 heterodimer vaccine antigen from a precursor protein fused with Fc IgG [PublishAheadOfPrint]

  • A recombinant HCV-1 (1a) gpE1/gpE2 (E1E2) vaccine candidate was previously shown by our group to protect chimpanzees and generate broad cross-neutralizing antibodies in animals and humans. In addition, recent independent studies have highlighted the importance of conserved neutralizing epitopes in HCV vaccine development that map to antigenic clusters in E2 or the E1E2 heterodimer. E1E2 can be purified using Galanthis nivalis lectin agarose (GNA), but this technique is suboptimal for global production. Our goal was to investigate a high affinity and scalable method for isolating E1E2. We generated an Fc-tag-derived (Fc-d) E1E2 that was selectively captured by Protein G Sepharose with the tag being removed subsequently using PreScission protease. Surprisingly, despite the presence of the large Fc tag, Fc-d E1E2 formed heterodimers similar to GNA purified wild type (WT) E1E2 and exhibited near identical binding profiles to HCV monoclonal antibodies that target conserved neutralizing epitopes in E2 (HC33.4, HC84.26 and AR3B) and the E1E2 heterodimer (AR4A and AR5A). Antisera from immunized mice showed that Fc-d E1E2 elicited similar anti-E2 antibody titers and neutralization of HCV pseudotype viruses to WT E1E2. Competition ELISAs showed that antisera from immunized mice inhibited monoclonal antibody binding to neutralizing epitopes. Antisera from Fc-d immunized mice exhibited stronger competition for AR3B and AR5A than WT, whereas competition for HC84.26 and AR4A was similar. We anticipate that Fc-d E1E2 will provide a scalable purification and manufacturing process using Protein A/G-based chromatography.

    IMPORTANCE A prophylactic HCV vaccine is still needed to control this global disease despite the availability of direct-acting antivirals. Previously, we demonstrated that a recombinant envelope glycoprotein (E1E2) vaccine (genotype 1a) elicited cross-neutralizing antibodies from human volunteers. A challenge for isolating the E1E2 antigen is the reliance on GNA, which is unsuitable for large scale-up and global vaccine delivery. We have generated a novel Fc domain tagged E1E2 antigen that forms functional heterodimers similar to the native E1E2. Affinity purification and tag removal of the Fc-tagged E1E2 resulted in an antigen with a near identical profile of cross-neutralizing epitopes. This antigen elicited anti-HCV antibodies that targeted conserved neutralizing epitopes of E1E2. Owing to the high selectivity and cost-effective binding capacity of affinity resins for capture of the Fc-tagged rE1E2, we anticipate our method will provide a means for large scale production of this HCV vaccine candidate.

  • Interleukin-17A Promotes CD8+ T Cell Cytotoxicity to Facilitate West Nile Virus Clearance [PublishAheadOfPrint]

  • CD8+ T cells are crucial components of immunity and play a vital role in recovery from West Nile virus (WNV) infection. Here, we identify a previously unrecognized function of interleukin-17A (IL-17A) in inducing cytotoxic mediator gene expression and promoting CD8+ T cell cytotoxicity against WNV infection in mice. We find that IL-17A deficient mice (Il17a-/-) are more susceptible to WNV infection and develop a higher viral burden compared to wild-type (WT) mice. Interestingly, the CD8+ T cells isolated from Il17a-/- mice are less cytotoxic and express lower levels of cytotoxic mediator genes, which can be restored by supplying recombinant IL-17A in vitro and in vivo. Importantly, treatment of WNV-infected mice with recombinant IL-17A, as late as day 6 post-infection, significantly reduces viral burden and increases survival, suggesting a therapeutic potential of IL-17A. In conclusion, we report a novel function of IL-17A in promoting CD8+ T cell cytotoxicity, which may have broad implications in other microbial infections and cancers.

    IMPORTANCE Interleukin-17A (IL-17A) and CD8+ T cells regulate diverse immune functions during microbial infections, malignancies, and autoimmune diseases. IL-17A is a proinflammatory cytokine produced by diverse cell types, while the CD8+ T cells (known as cytotoxic T cells) are major cells that provide immunity against intracellular pathogens. Previous studies have demonstrated a crucial role of CD8+ T cells in recovery from West Nile virus (WNV) infection. However, the role of IL-17A during WNV infection yet remains unclear. Here, we demonstrate that IL-17A protects mice from lethal WNV infection by promoting CD8+ T cell-mediated clearance of WNV. In addition, treatment of WNV-infected mice with recombinant IL-17A reduces viral burden and increases survival of mice, suggesting a potential therapeutic. This novel IL-17A-CD8+ T cell axis may also have broad implications in immunity to other microbial infections and cancers, where CD8+ T cell functions are crucial.

  • Pivotal role of RIP1 and MLKL in neuronal cell death induced by the human neuroinvasive coronavirus OC43 [PublishAheadOfPrint]

  • Human coronaviruses (HCoV) are respiratory pathogens with neuroinvasive, neurotropic and neurovirulent properties, highlighting the importance to study the potential implication of these viruses in neurological diseases. The OC43 strain (HCoV-OC43) was reported to induce neuronal cell death which may participate in neuropathogenesis. Here, we show that HCoV-OC43 harboring two point mutations in the spike glycoprotein (rOC/Us183-241) was more neurovirulent than the wild-type HCoV-OC43 (rOC/ATCC) in mice and induced more cell death in murine and human neuronal cells. To evaluate the role of regulated cell death (RCD) in HCoV-OC43-mediated neural pathogenesis, we determine if knockdown of Bax, a key regulator of apoptosis, or RIP1, a key regulator of necroptosis, altered the percentage of neuronal cell death following HCoV-OC43 infection. We found that Bax-dependent apoptosis did not play a significant role in RCD following infection, as inhibition of Bax expression mediated by RNA interference did not confer cellular protection against the cell death process. On the other hand, we demonstrated that RIP1 and MLKL were involved in neuronal cell death as RIP1 knockdown and chemical inhibition of MLKL significantly increased cell survival after infection. Taken together, these results indicate that RIP1 and MLKL contribute to necroptotic cell death after HCoV-OC43 infection to limit viral replication. However, this RCD could lead to neuronal loss in the mouse CNS and accentuate the neuroinflammation process reflecting the severity of neuropathogenesis.

    IMPORTANCE OF THE STUDY Because they are naturally neuroinvasive and neurotropic, human coronaviruses are suspected to participate in the development of neurological diseases. Given that the strain OC43 is neurovirulent in mice and induces neuronal cell death, we explored the neuronal response to infection by characterizing the activation of RCD. Our results revealed that classical apoptosis associated with the Bax protein is not playing a significant role in HCoV-OC43-induced neuronal cell death and that RIP1 and MLKL, two cellular proteins usually associated with necroptosis (a RCD back-up system when apoptosis is not adequately induced), both play a pivotal role in the process. As necroptosis disrupts cellular membranes and allows the release of damage-associated molecular patterns (DAMP) and may induce the production of pro-inflammatory cytokines, it may represent a pro-inflammatory cell death mechanism that contributes to excessive neuroinflammation and neurodegeneration and eventually to neurological disorders after a coronavirus infection.

  • An in vitro RNA synthesis assay for rabies virus defines critical ribonucleoprotein interactions for polymerase activity. [PublishAheadOfPrint]

  • We report an in vitro RNA synthesis assay for the RNA dependent RNA polymerase (RdRP) of rabies virus (RABV). We expressed RABV large polymerase protein (L) in insect cells from a recombinant baculovirus vector and the phosphoprotein cofactor (P) in Escherichia coli and purified the resulting proteins by affinity and size exclusion chromatography. Using chemically synthesized short RNA corresponding to the first 19-nt of the rabies virus genome we demonstrate that L alone initiates synthesis on naked RNA, and that P serves to enhance initiation and processivity of the RdRP. The L-P complex lacks full processivity, which we interpret to reflect the lack of the viral nucleocapsid protein (N) on the template. Using this assay, we define the requirements in P for stimulation of RdRP activity as residues 11-50 of P, and formally demonstrate that ribavirin triphosphate (RTP) inhibits the RdRP. By comparing the properties of RABV RdRP with that of the related rhabdovirus, vesicular stomatitis virus (VSV), we demonstrate that both polymerases can copy the heterologous promoter sequence. The requirements for engagement of the N-RNA template of VSV by its polymerase are provided by the C-terminal domain (CTD) of P. A chimeric RABV P in which the oligomerization domain (OD) and the CTD were replaced by that of VSV P stimulated RABV RdRP activity on naked RNA, but was insufficient to permit initiation on the VSV N-RNA template. This result implies that interactions between L and the template N are also required for initiation of RNA synthesis extending our knowledge of ribonucleoprotein interactions that are critical for gene expression.

    IMPORTANCE Current understanding of the structural and functional significance of the components of the rabies virus replication machinery is incomplete. Although structures are available for the nucleocapsid protein in complex with RNA, and also for portions of P information on both the structure and function of the L protein are lacking. This study reports the expression and purification of the full-length L protein of RABV and a characterization of its RdRP activity in vitro. The study provides a new assay that has utility for screening inhibitors and understanding their mechanism of action, as well as defining new interactions that are required for RdRP activity.

  • Vietnamese influenza A viruses of swine between 2010 and 2015: multiple introductions of A(H1N1)pdm09 viruses into the pig population and diversifying genetic constellations of enzootic IAV-S [PublishAheadOfPrint]

  • Active surveillance of influenza A viruses of swine (IAV-S) involving 262 farms and 10 slaughterhouses in seven provinces in northern and southern Vietnam from 2010 to 2015 yielded 388 isolates from 32 farms; these viruses were classified into H1N1, H1N2, and H3N2 subtypes. Whole-genome sequencing followed by phylogenetic analysis revealed that the isolates represented 15 genotypes, according to genetic constellation of the eight segments. All of the H1N1 viruses were entirely A(H1N1)pdm09 viruses, whereas all of the H1N2 and H3N2 viruses were reassortants among 5 distinct ancestral viruses: H1 and H3 triple-reassortant (TR) IAV-S that originated from North America and pre-2009 human seasonal H1, human seasonal H3N2, and A(H1N1)pdm09 viruses. Notably, 93% of the reassortant IAV-S retained M genes that were derived from A(H1N1)pdm09, suggesting some advantage in terms of their host adaptation. Bayesian Markov chain Monte Carlo analysis revealed that multiple introductions of A(H1N1)pdm09 and TR IAV-S into the Vietnamese pig population have driven the genetic diversity of currently circulating Vietnamese IAV-S. In addition, our results indicate that a reassortant IAV-S with human-like H3 and N2 genes and an A(H1N1)pdm09-origin M gene likely caused a human case in Ho Chi Minh City in 2010. Our current findings indicate that human-to-pig transmission as well as co-circulation of different IAV-S have contributed to diversifying the gene constellations of IAV-S in Vietnam.

    IMPORTANCE This comprehensive genetic characterization of 388 influenza A viruses of swine (IAV-S) isolated through active surveillance of Vietnamese pig farms from 2010 through 2015 provides molecular epidemiological insight into the genetic diversification of IAV-S in Vietnam after the emergence of A(H1N1)pdm09 viruses. Multiple reassortments among A(H1N1)pdm09 viruses and enzootic IAV-S yielded 14 genotypes, 9 of which carried novel gene combinations. The reassortants that carried M genes derived from A(H1N1)pdm09 viruses became predominant, replacing those of the IAV-S endemic since 2011. Notably, one of the novel reassortants likely caused a human case in Vietnam. Given that Vietnam is the second-largest pig-producing country in Asia, continued monitoring of IAV-S is highly important from the viewpoints of both the swine industry and human public health.

  • The Potyviral Silencing Suppressor Protein VPg Mediates Degradation of SGS3 via Ubiquitination and Autophagy Pathways [PublishAheadOfPrint]

  • RNA silencing is an innate antiviral immunity of plants and animals. To counteract this host immune response, viruses have evolved an effective strategy to protect themselves by expression of viral suppressors of RNA silencing (VSRs). Most potyviruses encode two VSRs, the helper component-proteinase (HC-Pro) and the viral genome-linked protein (VPg). The molecular biology of the former has been well characterized, whereas how VPg exerts its function in RNA silencing suppression is yet to be understood. In this study, we show that infection by Turnip mosaic virus (TuMV) causes the reduced level of SUPPRESSOR OF GENE SILENCING3 (SGS3), a key component of the RNA silencing pathway that functions in double-stranded RNA synthesis for virus-derived siRNA (vsiRNA) production. We also demonstrate that among 11 TuMV-encoded viral proteins, VPg is the only one that interacts with SGS3. We further present evidence that expression of VPg alone, independent of viral infection, is sufficient to induce the degradation of SGS3 and its intimate partner, RNA-DEPENDENT RNA POLYMERASE6 (RDR6). Moreover, we discover that the VPg-mediated degradation of SGS3 is via both the 20S ubiquitin-proteasome and autophagy pathways. Taken together, our data suggest a role for VPg-mediated degradation of SGS3 in silencing suppression by VPg.

    IMPORTANCE Potyviruses represent the largest group of known plant viruses and cause significant losses in many agriculturally important crops in the world. In order to establish their infections, potyviruses must overcome host antiviral silencing response. A viral protein called VPg has been shown to play a role in this process, but how it works is unclear. In this paper, we found that the VPg protein of Turnip mosaic virus (TuMV), which is a potyvirus, interacts with a host protein named SGS3, a key protein in the RNA silencing pathway. Moreover, this interaction leads to the degradation of SGS3 and its interacting and functional partner RDR6, which is another essential component of the RNA silencing pathway. We also identified the cellular pathways that are recruited for the VPg-mediated degradation of SGS3. Therefore, this work reveals a possible mechanism by which VPg sabotages host antiviral RNA silencing to promote virus infection.

  • Creating an artificial tail-anchor as a novel strategy to enhance the potency of peptide-based HIV fusion inhibitors [PublishAheadOfPrint]

  • T20 (enfuvirtide) and other peptides derived from the human immunodeficiency virus type 1 (HIV-1) gp41 C-terminal heptad repeat (CHR) region inhibit HIV fusion by binding to the hydrophobic grooves on the N-terminal heptad repeat (NHR)-trimer and blocking the six-helix bundle (6-HB) formation. Several strategies focusing on the binding grooves of NHR-trimer have been adopted to increase antiviral activity of the CHR-peptides. Here we developed a novel and simple strategy to greatly enhance the potency of the existing peptide-based HIV fusion inhibitors. Firstly, we identified a shallow pocket adjacent to the groove in the N-terminal region of NHR-trimer as a new drug target, and then designed several short artificial peptides to fit this target. After adding IDL (Ile-Asp-Leu) to the C-terminus of CHR-peptide WQ or MT-WQ, the conjugated peptide WQ-IDL or MT-WQ-IDL showed much more potent activities than WQ and T20, respectively, in inhibiting HIV-1 IIIB infection. WQ-IDL and MT-WQ-IDL were also more effective than WQ in blocking HIV-1 Env-mediated membrane fusion and had higher binding affinity with NHR-peptide N46. We solved the crystal structure of 6-HB formed by MT-WQ-IDL and N46, and found that besides the N-terminal MT hook-tail, the IDL tail-anchor of MT-WQ-IDL also bind with the shallow hydrophobic pocket outside the groove of the NHR-trimer, resulting in the enhanced inhibition of HIV-1 fusion with the target cell. It is expected that this novel approach can be widely used to improve the potency of the peptidic fusion inhibitors against other enveloped viruses with class I fusion protein.

    IMPORTANCE The hydrophobic groove of human immunodeficiency virus type 1 (HIV-1) gp41 NHR-trimer has been known as the classic drug target to develop fusion inhibitors derived from gp41 CHR. Here we developed a novel and simple strategy to improve the existing peptide-based HIV fusion inhibitors. We identified a shallow pocket adjacent to the groove in the NHR-trimer and added a short artificial peptide consisting of three amino acids (IDL) to the C-terminus of a fusion inhibitor to fit this new target. The inhibition activity of this new conjugated peptide was significantly enhanced by 77-fold, much more potent than T20 (enfuvirtide), suggesting that the IDL tail can be adopted for optimizing existing HIV-1 CHR-peptide fusion inhibitors. This new approach by identifying potential binding pocket outside the traditional target and creating an artificial tail-anchor can be widely applied to design novel fusion inhibitors against other class I enveloped viruses, such as MERS-CoV.

  • Identification of novel KSHV Orf50 transcripts: discovery of new RTA isoforms with variable transactivation potential [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that has been associated with primary effusion lymphoma, multicentric Castleman's disease, as well as its namesake Kaposi's sarcoma. As a gammaherpesvirus, KSHV is able to acutely replicate, enter latency, and reactivate from this latent state. A key protein involved in both acute replication and reactivation from latency is the replication and transcriptional activator (RTA) encoded by the gene Orf50. RTA is a known transactivator of multiple viral genes allowing for it to control the switch between latency and virus replication. Here we report the identification of six alternatively spliced Orf50 transcripts that are generated from four distinct promoters. These newly identified promoters are shown to be transcriptionally active in 293T (embryonic kidney), Vero (African-green monkey kidney epithelial cells), 3T12 (mouse fibroblast), and Raw 264.7 (mouse macrophage) cell lines. Notably, the newly identified Orf50 transcripts are predicted to encode four different isoforms of the RTA which differ by 6 -- 10 residues at the amino-terminus of the protein. Here we show the global viral transactivation potential of all four RTA isoforms and demonstrate that all isoforms can transcriptionally activate an array of KSHV promoters to various levels. The pattern of transcriptional activation appears to support a transcriptional interference model within the Orf50 region, where silencing of previously expressed isoforms by transcription initiation from upstream Orf50 promoters has the potential to modulate the pattern of viral gene activation.

    IMPORTANCE Gammaherpesviruses are associated with the development of lymphomas and lymphoproliferative diseases, as well as several other types of cancer. The human gammaherpesvirus, Kaposi's sarcom-associated herpesvirus (KSHV), is tightly associated with the development of Kaposi's sarcoma, multicentric Castleman's disease, as well as a rare form of B cell lymphoma (primary effusion lymphoma) primarily observed in HIV infected individuals. RTA is an essential viral gene product involved in the initiation of gammaherpesvirus replication, ane is conserved among all known gammaherpesviruses. Here we show for KSHV that transcription of the gene encoding RTA is complex and leads to the expression of serveral isoforms of RTA with distinct functions. This observed complexity in KSHV RTA expression and function likely plays a critical role in the regulation of downstream viral and cellular gene expression leading to the efficient production of mature virions.

  • Design and In Vivo Characterization of Immunoconjugates Targeting HIV gp160 [PublishAheadOfPrint]

  • The envelope (Env) glycoprotein of HIV is expressed on the surface of productively-infected cells, and can be used as a target for cytotoxic immunoconjugates (ICs), in which cell-killing moieties including toxins, drugs, or radionuclides are chemically or genetically linked to monoclonal antibodies (MAbs) or other targeting ligands. Such ICs could be used to eliminate persistent reservoirs of HIV infection. We have found that MAbs which bind to the external loop of gp41, e.g. MAb 7B2, make highly effective ICs, particularly when used in combination with soluble CD4. We have evaluated toxicity, immunogenicity, and efficacy of the ICs targeted with 7B2 in mice and in SHIV-infected macaques. In the macaques, we tested immunotoxins (ITs), consisting of protein toxins bound to the targeting agent. ITs were well tolerated, initially efficacious, but ultimately limited by immunogenicity. In an effort to decrease immunogenicity, we tested different toxic moieties, including recombinant toxins, cytotoxic drugs, and tubulin inhibitors. ICs containing deglycosylated ricin A chain prepared from ricin toxin extracted from castor beans were the most effective in killing HIV-infected cells. Having identified immunogenicity as a major concern, we show that conjugation of IT to polyethylene glycol limits immunogenicity. These studies demonstrate that cytotoxic ICs can target virus-infected cells in vivo, but also highlight potential problems to be addressed.

    IMPORTANCE It is not yet possible to cure HIV infection. Even after years of fully effective antiviral therapy, a persistent reservoir of virus-infected cells remains. Here we propose that a targeted conjugate, consisting of an anti-HIV antibody bound to a toxic moiety, could function to kill the HIV-infected cells that constitute this reservoir. We tested this approach in HIV-infected cells grown in the lab, and in animal infections. Our studies demonstrated that these immunoconjugates are effective both in vitro and in test animals. In particular, ITs constructed with the deglycosylated A chain prepared from native ricin were the most effective in killing cells. But their utility was blunted because they provoked immune reactions that interfered with the therapeutic effects. We then demonstrated that coating ITs with polyethylene glycol minimizes immunogenicity, as has been demonstrated with other protein therapies.

  • Importance of highly conserved peptide sites of HCMV gO for the formation of the gH/gL/gO complex [PublishAheadOfPrint]

  • The glycoprotein O (gO) is betaherpesvirus-specific. Together with the viral glycoproteins H and L, gO forms a covalent trimeric complex that is part of the viral envelope. This trimer is crucial for cell-free infectivity of human cytomegalovirus (HCMV) but dispensable for cell-associated spread. We hypothesized that the amino acids (aa) that are conserved among gOs of different cytomegaloviruses are important for the formation of the trimeric complex and hence for efficient virus spread. In a mutational approach, nine peptide sites - containing all 13 highly conserved amino acids - were analyzed in the context of HCMV strain TB40-BAC4 with regard to infection efficiency and formation of the gH/gL/gO complex. Mutation of aa181-186 or aa193-198 resulted in the loss of the trimer and a complete small-plaque phenotype, whereas mutation of aa108 or aa249-254 caused an intermediate phenotype. While individual mutations of the five conserved cysteines had little impact, their relevance was revealed in a combined mutation, which abrogated both complex formation and cell-free infectivity. C343 was particular as it was sufficient and necessary for covalent binding of gO to gH/gL. Remarkably however, C218 together with C167 rescued infectivity in the absence of detectable covalent complex formation. We conclude that all highly conserved amino acids contribute to the function of gO to some extent, but aa181-198 and cysteines 343, 218 and 167 are particularly relevant. Surprisingly, covalent binding of gO to gH/gL is neither required for its incorporation into virions nor for proper function in cell-free infection.

    Importance Like all herpesviruses, the widespread human pathogen HCMV depends on glycoproteins gB, gH and gL for entry into target cells. Additionally, gH and gL have to bind gO in a trimeric complex for efficient cell-free infection. Homologs of gO are shared by all cytomegaloviruses, with 13 amino acids being highly conserved. In a mutational approach we analyzed these amino acids to elucidate their role for the function of gO. All conserved amino acids contributed either to formation of the trimeric complex or to cell-free infection. Notably, these two phenotypes were not inevitably linked, as the mutation of a charged cluster in the center of gO abrogated cell-free infection while trimeric complexes were still being formed. Cysteine 343 was essential for covalent binding of gO to gH/gL, however non-covalent complex formation in the absence of cysteine 343 also allowed for cell-free infectivity.

  • Deep-sequence identification and role in virus replication of a JC virus quasispecies in patients with progressive multifocal leukoencephalopathy [PublishAheadOfPrint]

  • JC virus (JCV) is a DNA virus causing progressive multifocal leukoencephalopathy (PML) in immunodeficient patients. In the present study, 22 genetic quasispecies with more than 1.5% variant frequency were detected in JCV genomes from six clinical samples of PML by next-generation sequencing. A mutation from A to C at nt 3495 in JCV Mad1 resulting in a V to G amino acid substitution at aa position 392 of the large T antigen (TAg) was identified in all six cases of PML at 3%nndash;19% variant frequencies. Transfection of JCV Mad1 DNA having the V392G substitution in TAg into IMR-32 and HEK293 cells resulted in a dramatically decreased production of JCV-encoded proteins. The virus DNA copy number was also reduced in supernatants of the mutant virus-transfected cells. Transfection of the IMR-32 and HEK293 cells with a virus genome containing a revertant mutation recovered viral production and protein expression. Co-transfection with equal amounts of wild type and mutated JCV genome did not reduce the expression of viral proteins nor viral replication, suggesting that the mutation did not have any dominant negative function. Finally, immunohistochemistry demonstrated that TAg was expressed in all six pathological samples in which the quasispecies were detected. In conclusion, the V392G amino acid substitution in TAg identified frequently in PML lesions has a function in suppressing JCV replication, but the frequency of the mutation was restricted and its role in PML lesions was limited.

    IMPORTANCE DNA viruses generally have lower mutation frequency than RNA viruses, and the detection of quasispecies in JCV has been rarely reported. In the present study, a next-generation sequencer identified a JCV quasispecies with an amino acid substitution in the T antigen in patients with PML. In vitro studies showed that the mutation strongly repressed the expression of JC viral proteins and reduced the viral replication. However, because the frequency of the mutation was low in each case, the total expression of virus proteins was sustained in vivo. Thus, JC virus replicates in PML lesions in the presence of a mutant virus which is able to repress virus replication.

  • Structural biology of the arterivirus nsp11 endoribonucleases [PublishAheadOfPrint]

  • Endoribonuclease (NendoU) is unique and conserved as a major genetic marker in nidoviruses that infect vertebrate hosts. Arterivirus non-structural protein (nsp) 11 was shown to have NendoU activity and play essential roles in the viral life cycle. Here, we report three crystal structures of porcine reproductive and respiratory syndrome virus (PRRSV) and equine arteritis virus (EAV) nsp11 mutants. The structures of arterivirus nsp11 contain two conserved compact domains: N-terminal domain (NTD) and C-terminal domain (CTD). The structures of PRRSV and EAV endoribonucleases are similar and conserved in the arterivirus, but they are greatly different from that of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoV) representing important human pathogens in the Nidovirales order. The catalytic center of NendoU activity is located in the CTD, where a positively charged groove is next to the key catalytic residues conserved in nidoviruses. Although the NTD is nearly identical, the catalytic region of the arterivirus nsp11 family proteins is remarkably flexible, and the oligomerization may be concentration-dependent. In summary, our structures provide new insight into this key multifunctional NendoU family of proteins and lay a foundation for better understanding of the molecular mechanism and antiviral drug development.

    IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) and equine arteritis virus are two major members of the arterivirus family. PRRSV, a leading swine pathogen, causes reproductive failure in breeding stock and respiratory tract illness in young pigs. Due to the lack of a suitable vaccine or effective drug treatment and the quick spread of these viruses, infected animals either die quickly or must be culled. PRRSV costs the swine industry around $644 million annually in the USA and almost 1.5b in Europe every year. To find a way to combat these viruses, we focused on the essential viral non-structural protein (nsp) 11. Nsp11 is associated with multiple functions such as RNA-processing and suppressing the infected host innate immunity system. The three solved structures in this study provide new insight into the molecular mechanisms of this crucial protein family and will benefit the development of new treatments against these deadly viruses.

  • The nonstructural protein NSs of Schmallenberg virus is targeted to the nucleolus and induces nucleolar disorganization. [PublishAheadOfPrint]

  • Schmallenberg virus (SBV) was discovered in Germany in late 2011 and then spread rapidly to many European countries. SBV is an orthobunyavirus that causes abortion and congenital abnormalities in ruminants. A virus-encoded non-structural protein, termed NSs, is a major virulence factor of SBV and it is known to promote the degradation of Rpb1, a subunit of the RNA Pol II complex, and therefore hampers global cellular transcription. In this study we found that NSs is mainly localized in the nucleus of infected cells, and, specifically appears to target the nucleolus through a nucleolar localization signal (NoLS) localized between residues 33 and 51 of the protein. NSs co-localizes with nucleolar markers such as B23 (nucleophosmin) and fibrillarin. We observed that in SBV-infected cells, B23 undergoes a nucleolar to nucleoplasm redistribution, evocative of viral-induced nucleolar disruption. In contrast, nucleolar pattern of B23 was unchanged upon infection with a SBV recombinant mutant with NSs lacking the NoLS motif (SBVNoLS). Interestingly, unlike wild type SBV, the inhibitory activity of SBVNoLS towards RNA Pol II transcription is impaired. Overall, our results suggest that a putative link exists between NSs-induced nucleolar disruption and its inhibitory function on cellular transcription, which consequently precludes cellular antiviral response and/or induces cell death.

    IMPORTANCE Schmallenberg virus (SBV) is an emerging arbovirus of ruminants that has spread in Europe between 2011 and 2013. SBV induces fetal abnormalities during gestation with the central nervous system being one of the most affected organs. The viral-encoded NSs protein acts as a virulent factor by impairing host cell transcription. Here, we show that NSs contains a nucleolar localization signal (NoLS) and induces disorganization of the nucleolus. The NoLS motif in the SBV NSs is absolutely necessary for viral induced inhibition of cellular transcription. To our knowledge, this is the first report of nucleolar functions for NSs within the Bunyaviridae family.

  • Intrinsic and innate defenses of neurons: Detente with the herpesviruses. [PublishAheadOfPrint]

  • Neuroinvasive herpesviruses have evolved to efficiently infect and establish latency in neurons. The nervous system has limited capability to regenerate, so immune responses therein are carefully regulated to be non-destructive, with dependence on atypical intrinsic and innate defenses. In this article we review studies of some of these non-canonical defense pathways and how herpesvirus gene products counter them, highlighting the contributions that primary neuronal in vitro models have made to our understanding of this field.

  • The DNA damage signaling is required for replication of the HBoV1 DNA in dividing HEK293 cells [PublishAheadOfPrint]

  • Human bocavirus 1 (HBoV1), an emerging human pathogenic respiratory virus, is a member of the genus Bocaparvovirus of the Parvoviridae family. In human airway epithelia air-liquid interface (HAE-ALI) cultures, HBoV1 infection initiates a DNA damage response (DDR), activating all three phosphatidylinositol 3-kinase-related kinases (PI3KKs): ATM, ATR and DNA-PKcs. In this context, activation of PI3KKs is a requirement for amplification of the HBoV1 genome (PLoS Pathog., 2016; 12:e1005399), and HBoV1 replicates only in terminally differentiated, non-dividing cells. This report builds on the previous discovery that replication of the HBoV1 DNA can also occur in dividing HEK293 cells, demonstrating that such replication is likewise dependent on a DDR. Transfection of HEK293 cells with the duplex DNA genome of HBoV1 induces hallmarks of DDR, including phosphorylation of H2AX and RPA32, as well as activation of all three PI3KKs. The large viral non-structural protein NS1 is sufficient to induce the DDR and the activation of three PI3KKs. Pharmacological inhibition or knockdown of any one of the PI3KKs significantly decreases both replication of the HBoV1 DNA and downstream production of progeny virions. The DDR induced by the HBoV1 NS1 protein does not cause obvious damage to cellular DNA or arrest of the cell cycle. Notably, key DNA-replication factors and major DNA-repair DNA polymerases (Pol and Pol ) are recruited to the viral DNA replication centers and facilitate HBoV1 DNA replication. Our study provides the first evidence of a DDR-dependent parvovirus DNA replication that occurs in dividing cells and is independent of cell cycle arrest.

    IMPORTANCE The human parvovirus bocavirus 1 (HBoV1) is an emerging respiratory virus that causes lower respiratory-tract infections in young children worldwide. HEK293 cells are the only dividing cells tested that fully support replication of the duplex genome of this virus and produce progeny virions. In this study, we demonstrate that the HBoV1 induces a DDR that plays significant roles in replication of the viral DNA and production of progeny virions in HEK293 cells. We also show that both cellular DNA-replication factors and DNA-repair DNA polymerases colocalize within centers of viral DNA replication, and that Pol and Pol play an important role in HBoV1 DNA replication. Whereas the DDR that leads to replication of the DNA of other parvoviruses is facilitated by the cell cycle, the DDR triggered by HBoV1 DNA replication or NS1 is not. HBoV1 is the first parvovirus whose NS1 can activate all three PI3KKs (ATM, ATR, and DNA-PKcs).

  • High EBV load and genomic diversity are associated with generation of gp350 specific neutralizing antibodies following Acute Infectious Mononucleosis [PublishAheadOfPrint]

  • The Epstein Barr virus (EBV) gp350 glycoprotein interacts with the cellular receptor to mediate viral entry and is thought to be the major target for neutralizing antibodies. To better understand the role of EBV-specific antibodies in the control of viral replication and evolution of sequence diversity, we measured EBV gp350-specific antibody responses and sequenced the gp350 gene in samples obtained from individuals experiencing primary EBV infection (acute infectious mononucleosis; AIM) and again 6 months later (convalescence, CONV). EBV gp350-specific IgG was detected in the sera of 17 (71%) of 24 individuals at AIM and all 24 (100%) individuals at CONV; binding antibody titers increased from AIM through CONV, reaching levels equivalent to those in age-matched, chronically-infected individuals. Antibody-dependent cell-mediated phagocytosis (ADCP) was rarely detected in AIM (4 of 24 individuals; 17%) but was commonly detected in CONV (19 of 24 individuals; 79%). The majority (83%) of samples taken during AIM neutralized infection of primary B cells; all samples obtained 6 months post-diagnosis neutralized EBV infection of cultured and primary target cells. Deep sequencing revealed inter-patient gp350 sequence variation, but conservation of the CR2 binding site. Levels of gp350-specific neutralizing activity directly correlated with higher peripheral blood EBV DNA levels in AIM and greater evolution of diversity in gp350 nucleotide sequences from AIM to CONV. In summary, we conclude that viral load and EBV gp350 diversity during early infection are associated with the development of neutralizing antibody responses following AIM.

    IMPORTANCE Antibodies against viral surface proteins can blunt the spread of viral infection by coating viral particles mediating uptake by immune cells or blocking interaction with host cell receptors, making them a desirable component of a sterilizing vaccine. The EBV surface protein gp350 is a major target for antibodies. We report the detection EBV gp350-specific antibodies capable of neutralizing EBV infection in vitro. The majority of gp350-directed vaccines focus on glycoproteins from lab-adapted strains which may poorly reflect primary viral envelop diversity. We report some of the first primary gp350 sequences noting that the gp350 host receptor binding site is remarkably stable across patients and time. However, changes in overall gene diversity were detectable during infection. Patients with higher peripheral blood viral loads in primary infection and greater changes in viral diversity generated more efficient antibodies. Our findings provide insight into the generation of functional antibodies, necessary for vaccine development.

  • In vivo efficacy of measles virus fusion protein-derived peptides is modulated by properties of self-assembly and membrane residence [PublishAheadOfPrint]

  • Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV hemagglutinin (H) and fusion (F) envelope glycoproteins; upon receptor engagement by H, the pre-fusion F undergoes a structural transition, extending and inserting into the target cell membrane and then re-folding into a post-fusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad-repeat (HR) regions of F can inhibit MV infection at the entry stage. In previous work, we have generated potent MV fusion inhibitors by dimerizing the F derived peptides and conjugating them to cholesterol. We have shown that prophylactic intranasal administration of our lead fusion inhibitor efficiently protects from MV infection in vivo. We show here that peptides tagged with lipophilic moieties self-assemble into nanoparticles until they reach the target cells, where they are integrated into cell membranes. The self-assembly feature enhances bio-distribution and half-life of the peptides, while integration into the target cell membrane increases fusion inhibitor potency. These factors together modulate in vivo efficacy. The results suggest a new framework for developing effective fusion inhibitory peptides.

    Importance Measles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that fusion inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. We show here that specific biophysical properties regulate the in vivo efficacy of MV F derived peptides.

  • Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry [PublishAheadOfPrint]

  • Human coronavirus 229E (HCoV-229E), a causative agent of the common cold, enters host cells via two distinct pathways: one is mediated by cell surface proteases, particularly transmembrane protease serine 2 (TMPRSS2), and the other by endosomal cathepsin L. Thus, specific inhibitors of these proteases block virus infection. However, it is unclear which of these pathways is actually utilized by HCoV-229E in the human respiratory tract. Here, we examined the mechanism of cell entry used by a pseudotyped virus bearing the HCoV-229E spike (S) protein in the presence/absence of protease inhibitors. We found that, when compared with a laboratory strain isolated in 1966 and passaged for a half century, clinical isolates of HCoV-229E were less likely to utilize cathepsin L; rather, they showed a preference for TMPRSS2. Two amino acid substitutions (R642M and N714K) in the S protein of HCoV-229E clinical isolates altered their sensitivity to a cathepsin L inhibitor, suggesting that these amino acids were responsible for cathepsin L use. After 20 passages in HeLa cells, the ability of the isolate to use cathepsin increased such that it was equal to that of the laboratory strain; this increase was caused by an amino acid substitution (I577S) in the S protein. The passaged virus showed a reduced ability to replicate in differentiated airway epithelial cells cultured at an air-liquid interface. These results suggest that the endosomal pathway is disadvantageous for HCoV-229E infection of human airway epithelial cells; therefore, clinical isolates are less able to use cathepsin.

    IMPORTANCE Many envelope viruses enter cells through endocytosis. Viral spike proteins drive the fusion of viral and endosomal membranes to facilitate insertion of the viral genome into the cytoplasm. Human coronavirus 229E (HCoV-229E) utilizes endosomal cathepsin L to activate the spike protein after receptor binding. Here, we found that clinical isolates of HCoV-229E preferentially utilize the cell surface protease, TMPRSS2, rather than endosomal cathepsin L. The endosome is a main site of Toll-like receptor recognition, which then triggers an innate immune response; therefore, HCoV-229E presumably evolved to bypass the endosome by entering the cell via TMPRSS2. Thus, the virus uses a simple mechanism to evade the host innate immune system. Therefore, therapeutic agents for coronavirus-mediated diseases such as SARS and MERS should target cell surface TMPRSS2 rather than endosomal cathepsin.

  • Deep Sequencing of Influenza A Virus from a Human Challenge Study Reveals a Selective Bottleneck and Only Limited Intrahost Genetic Diversification [PublishAheadOfPrint]

  • Knowledge of influenza evolution at the point of transmission and at the intra-host level remains limited, particularly for human hosts. Here, we analyze a unique viral dataset of next-generation sequencing (NGS) samples generated from a human influenza challenge study wherein 17 healthy subjects were inoculated with egg-passaged virus. Nasal wash samples collected from 7 of these subjects were successfully deep sequenced. From these, we characterized changes in the subjects' viral populations during infection and identified differences between the virus in these samples and the viral stock used to inoculate the subjects. We first calculated pairwise genetic distances between the subjects' nasal wash samples, the viral stock, and the A/Wisconsin/67/2005 (H3N2) reference strain used to generate the stock virus. These distances revealed that considerable viral evolution occurred at various points in the human challenge study. Further quantitative analyses indicated that: (1) the viral stock contained genetic variants that originated and likely were selected for during the passaging process; (2) direct intranasal inoculation with the viral stock resulted in a selective bottleneck that reduced nonsynonymous genetic diversity in the viral hemagglutinin and nucleoprotein; and (3) intrahost viral evolution continued over the course of infection. These intrahost evolutionary dynamics were dominated by purifying selection. Our findings indicate that rapid viral evolution can occur during acute influenza infection in otherwise healthy human hosts when the founding population size of the virus is large, as is the case with direct intranasal inoculation.

    Importance Influenza viruses circulating among humans are known to rapidly evolve over time. However, little is known about how influenza virus evolves across single transmission events and over the course of a single infection. To address these issues, we analyze influenza sequences from a human challenge experiment that initiated infection with a cell- and egg-passaged viral stock, which appeared to have adapted during its preparation. We find that the subjects' viral populations differ genetically from the viral stock, with subjects' viral populations having lower representation of the amino-acid changing variants that arose during viral preparation. We also find that most of the viral evolution occurring over single infections is characterized by further decreases in the frequencies of these amino-acid changing variants and that only limited intrahost genetic diversification through new mutations is apparent. Our findings indicate that influenza virus populations can undergo rapid genetic changes during acute human infections.

  • TMPRSS12 Is an Activating Protease for Subtype B Avian Metapneumovirus [PublishAheadOfPrint]

  • The entry of avian metapneumovirus (aMPV) into host cells initially requires the fusion of viral and cell membranes, which is exclusively mediated by fusion (F) protein. Proteolysis of aMPV F protein by endogenous proteases of host cells allows F protein to induce membrane fusion; however, these proteases have not been identified. Here, we provide the first evidence that the transmembrane serine protease TMPRSS12 facilitates the cleavage of subtype B aMPV (aMPV/B) F protein. We found that overexpression of TMPRSS12 enhanced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. Subsequently, knockdown of TMPRSS12 with specific small interfering RNAs (siRNAs) reduced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. We also found a cleavage motif in the aMPV/B F protein (amino acids 100 and 101) that was recognized by TMPRSS12. The histidine, aspartic acid, and serine residue (HDS) triad of TMPRSS12 was shown to be essential for the proteolysis of aMPV/B F protein via mutation analysis. Notably, we observed TMPRSS12 mRNA expression in target organs of aMPV/B in chickens. Overall, our results indicate that TMPRSS12 is crucial for aMPV/B F protein proteolysis and aMPV/B infectivity and that TMPRSS12 may serve as a target for novel therapeutics and prophylactics for aMPV.

    IMPORTANCE Proteolysis of the avian metapneumovirus (aMPV) fusion (F) protein is a prerequisite for F protein-mediated membrane fusion of virus-cell and for aMPV infection; however, the proteases used in vitro and vivo are not clear. A combination of analyses, including overexpression, knockdown, and mutation methods, demonstrated that the transmembrane serine protease TMPRSS12 facilitated cleavage of subtype B aMPV (aMPV/B) F protein. Importantly, we located the motif in the aMPV/B F protein recognized by TMPRSS12 and the catalytic triad in TMPRSS12 that facilitated proteolysis of the aMPV/B F protein. This is first report on TMPRSS12 as a protease for proteolysis of viral envelope glycoproteins. Our study will shed light on the mechanism of proteolysis of aMPV F protein and pathogenesis of aMPV.

  • Tetherin antagonism by the Ebola virus glycoprotein requires an intact receptor-binding domain and can be blocked by GP1-specific antibodies [PublishAheadOfPrint]

  • The glycoprotein of Ebola virus (EBOV-GP), a member of the Filoviridae family, facilitates viral entry into target cells. In addition, EBOV-GP antagonizes the antiviral activity of the host cell protein tetherin, which may otherwise restrict EBOV-release from infected cells. However, it is unclear how EBOV-GP antagonizes tetherin and it is unknown whether the GP of Lloviu virus (LLOV), a filovirus found in dead bats in Northern Spain, also counteracts tetherin. Here, we show that LLOV-GP antagonizes tetherin, indicating that tetherin may not impede LLOV spread in human cells. Moreover, we demonstrate that appropriate processing of N-glycans in tetherin/GP-coexpressing cells is required for tetherin counteraction by EBOV-GP. Furthermore, we show that an intact receptor-binding domain (RBD) in the GP1 subunit of EBOV-GP is a prerequisite to tetherin counteraction. In contrast, blockade of Niemann-Pick disease, type C1 (NPC1), a cellular binding partner of the RBD, did not interfere with tetherin antagonism. Finally, we provide evidence that an antibody directed against GP1, which protects mice from a lethal EBOV challenge, may block GP-dependent tetherin antagonism. Our data in conjunction with previous reports indicate that tetherin antagonism is conserved between the GPs of all known filoviruses and demonstrate that the GP1 subunits of EBOV-GP plays a central role in tetherin antagonism.

    IMPORTANCE Filoviruses are re-emerging pathogens that constitute a public health threat. Understanding how Ebola virus (EBOV), a highly pathogenic filovirus responsible for the 2013-2016 Ebola virus disease epidemic in Western Africa, counteracts antiviral effectors of the innate immune system might help to define novel targets for antiviral intervention. Similarly, determining whether Lloviu virus (LLOV), a filovirus detected in bats in Northern Spain, is inhibited by innate antiviral effectors in human cells might help to define whether the virus constitutes a threat to humans. The present study shows that LLOV like EBOV counteracts the antiviral effector protein tetherin via its glycoprotein (GP), suggesting that tetherin does not pose a defense against LLOV spread in humans. Moreover, our work identifies the GP1 subunit of EBOV-GP, in particular an intact receptor-binding domain, as critical for tetherin counteraction and provides evidence that antibodies directed against GP1 can interfere with tetherin counteraction.

  • Identification of IFN-stimulated gene (ISG) proteins that inhibit human parainfluenza virus type 3 [PublishAheadOfPrint]

  • A major arm of cellular innate immunity is type I interferon (IFN), represented by IFN-aalpha; and IFN-bbeta;. Type I IFN transcriptionally induces a large number of cellular genes, collectively known as IFN-Stimulated Genes or ISGs, which act as antivirals. The IFIT (interferon-induced proteins with tetratricopeptide repeats)-family proteins constitute a major subclass of ISG, and are characterized by multiple tetratricopeptide repeats (TPRs). Here, we have interrogated IFIT proteins for their ability to inhibit the growth of human parainfluenza virus type 3 (PIV3), a nonsegmented negative-strand RNA virus of the Paramyxoviridae family and major cause of respiratory disease in children. We found that IFIT1 significantly inhibited PIV3, whereas IFIT2, IFIT3 and IFIT5 were less effective or not at all. In further screening a set of ISGs we discovered that several other ISGs also inhibited PIV3, which included IFITM1, IDO, PKR and viperin/Cig5. The antiviral effect of IDO, the enzyme that catalyzes the first step of tryptophan degradation, could be counteracted by tryptophan. These results advance our knowledge of diverse ISGs functioning as antivirals and may provide novel antiviral approaches against PIV3.

    IMPORTANCE The innate immunity of the host, typified by Interferon (IFN), is a major antiviral defense. IFN inhibits virus growth by inducing a large number of "IFN-stimulated genes" (ISGs), several of which have been shown to have specific antiviral functions. Parainfluenza virus type 3 (PIV3) is major pathogen of children, and no reliable vaccine or specific antiviral against it currently exists. In this article, we report several ISGs that strongly inhibit PIV3 growth, the use of which may allow a better antiviral regimen targeting PIV3.

  • HIV-1 glycan density drives the persistence of the mannose patch within an infected individual [PublishAheadOfPrint]

  • The HIV envelope (Env) is extensively modified with host-derived N-linked glycans. The high density of glycosylation on the viral spike limits enzymatic processing resulting in numerous under-processed oligomannose-type glycans. This extensive glycosylation not only shields conserved regions of the protein from the immune system but also act as targets for HIV broadly neutralizing antibodies (bnAbs). In response to the host immune system, the HIV glycan shield is constantly evolving through mutations affecting both the positions and frequencies of potential N-linked glycans (PNGSs). Here, using longitudinal Env sequences from a clade C infected individual (CAP256), we measure the impact of the shifting glycan shield during HIV infection on the abundance of oligomannose-type glycans. By analyzing the intrinsic mannose patch from a panel of recombinant CAP256 gp120s displaying high protein sequence variability and changes in PNGS frequency and positioning, we show that the intrinsic mannose-patch persists throughout the course of HIV infection and correlates with the number of PNGSs. This effect of glycan density on processing state was also supported by the analysis of a cross-clade panel of recombinant gp120 glycoproteins. Together, these observations underscore the importance of glycan clustering for the generation of carbohydrate epitopes for HIV bnAbs. The persistence of the intrinsic mannose patch over the course of HIV infection further highlights this epitope as an important target for HIV vaccine strategies.

    Importance Development of an HIV vaccine is critical for control of the HIV pandemic, and elicitation of broadly neutralizing antibodies (bnAbs), is likely to be a key component of a successful vaccine response. The HIV Envelope glycoprotein (Env) is covered in an array of host-derived N-linked glycans often referred to as the glycan shield. This glycan shield is a target for many of the recently isolated HIV bnAbs and is therefore under constant pressure from the host immune system leading to changes in both glycan site frequency and location. This study aimed to determine whether these genetic changes impacted the eventual processing of glycans on the HIV Env, and the viruses susceptibility to neutralization. We show that despite this variation in glycan site positioning and frequency over the course of HIV infection, the mannose patch is a conserved feature throughout, making it a stable target for HIV vaccine design.

  • Syncytial Mutations Do not Impair the Specificity of Entry and Spread of a Glycoprotein D Receptor-Retargeted Herpes Simplex Virus [PublishAheadOfPrint]

  • Membrane fusion, which is the key process for both initial cell entry and subsequent lateral spread of herpes simplex virus (HSV), requires the four envelope glycoproteins gB, gD, gH, and gL. Syncytial mutations, predominantly mapped to the gB and gK genes, confer hyperfusogenicity on HSV and cause multinucleated giant cells, termed syncytia. Here, we asked whether interaction of gD with a cognate entry receptor remains indispensable for initiating membrane fusion of syncytial strains. To address this question, we took advantage of mutant viruses for which viral entry into cells relies on the uniquely specific interaction of an engineered gD with the epidermal growth factor receptor (EGFR). We introduced selected syncytial mutations into gB and/or gK of the EGFR-retargeted HSV and found that these mutations, especially when combined, enabled formation of extensive syncytia by human cancer cell lines that express the target receptor; these syncytia were substantially larger than the plaques formed by the parental retargeted HSV. We assessed EGFR dependence for entry and spread separately, using direct-entry and infectious-center assays, respectively, and found that the syncytial mutations did not override the receptor specificity of the retargeted viruses at either stage. We discuss the implications of these results for the development of more effective targeted oncolytic HSV vectors.

    IMPORTANCE Herpes simplex virus (HSV) is not only investigated as a human pathogen, but also as a promising agent for oncolytic virotherapy. We previously showed that both the initial entry and subsequent lateral spread of HSV can be retargeted to cells expressing tumor-associated antigens by single-chain antibodies fused into a receptor-binding-deficient envelope glycoprotein D (gD). Here, we introduced syncytial mutations into the gB and/or gK genes of gD-retargeted HSVs to determine whether viral tropism remained dependent on the interaction of gD with the target receptor. Entry and spread profiles of the recombinant viruses indicated that gD retargeting does not abolish the hyperfusogenic activity of syncytial mutations and that these mutations do not eliminate the dependence of HSV entry and spread on a specific gD-receptor interaction. These observations suggest that syncytial mutations may be valuable for increasing the tumor-specific spreading of retargeted oncolytic HSV vectors.

  • Characterization of human CD8 T cell responses in dengue virus infected patients from India [PublishAheadOfPrint]

  • Epidemiological studies suggest that India has the largest number of dengue virus infected cases world-wide. However, there is minimal information about the immunological responses in these patients. CD8 T cells are important in dengue because they have been implicated in both protection and immunopathology. Here we provide a detailed analysis of HLA-DR+CD38+ and HLA-DR-CD38+ effector CD8 T- cell subsets in dengue-patients from India and Thailand. Both CD8 T cell subsets expanded and expressed markers indicative of antigen-driven proliferation; tissue-homing and cytotoxic effector functions with the HLA-DR+CD38+ subset being the most striking in these effector qualities. The breadth of the dengue specific CD8 T cell response was diverse with NS3-specific cells being the most dominant. Interestingly, only a small fraction of these activated effector CD8 T-cells produced IFN- when stimulated with dengue peptide-pools. Transcriptomics revealed downregulation of key molecules involved in TCR-signaling. Consistent with this, the majority of these CD8 T cells remained IFN- unresponsive even by TCR-dependent polyclonal-stimulation (anti-CD3 plus anti-CD28), but produced IFN- by TCR-independent polyclonal-stimulation (PMA/Ionomycin). Thus, a vast majority of these proliferating highly differentiated effector CD8 T-cells probably acquire TCR refractoriness at the time the patient is experiencing febrile illness that leads to IFN- unresponsiveness. Our studies open novel avenues for understanding the mechanisms that fine-tune the balance between CD8 T-cell mediated protective versus pathological effects in dengue febrile illness.

    Importance Dengue is becoming a global public health concern. Although CD8 T-cells have been implicated in both protection and in cytokine-mediated immunopathology of dengue, how the balance maintained between these opposing functions remains unknown. We comprehensively characterized CD8 T-cell subsets in dengue-patients from India and Thailand; and show that these cells expand massively, express phenotypes indicative of overwhelming antigenic-stimulus, tissue homing/cytotoxic effector functions, but a vast majority of them fail to produce IFN- in vitro. Interestingly, these cells were fully capable of producing the cytokine when stimulated in a TCR-independent manner, but failed to do so in a TCR dependent stimulation. These results, together with transcriptomics, revealed that a vast majority of these CD8 T-cells from dengue patients become cytokine unresponsive due to TCR-signalling insufficiencies. These observations open novel avenues for understanding the mechanisms that fine-tune the balance between CD8-mediated protective versus pathological effects during febrile dengue illness.

  • Rationally Designed Immunogens Targeting HIV-1 gp120 V1V2 Induce Distinct Conformation-Specific Antibody Responses in Rabbits [PublishAheadOfPrint]

  • The V1V2 region of HIV-1 gp120 harbors a major vulnerable site targeted by a group of broadly neutralizing mAbs such as PG9 through strand-strand recognition. However, this epitope region is structurally polymorphic, as it can also form a helical conformation recognized by RV144 vaccine-induced mAb CH58. This structural polymorphism is a potential mechanism for masking the V1V2 vulnerable site. Designing immunogens that can induce conformation-specific Ab responses may lead to vaccines targeting this vulnerable site. We designed a panel of immunogens engrafting the V1V2 domain into trimeric and pentameric scaffolds in structurally constrained conformations. We also fused V1V2 to an Fc fragment to mimic the unconstrained V1V2 conformation. We tested these V1V2-scaffold proteins for immunogenicity in rabbits and assessed the responses by ELISA and competition assays. Our V1V2 immunogens induced distinct conformation-specific Ab responses. Abs induced by structurally unconstrained immunogens reacted preferentially with V1V2-unconstrained antigens, suggesting recognition of the helical configuration, while Abs induced by the structurally constrained immunogens reacted preferentially with V1V2-constrained antigens, suggesting recognition of the bbeta;-strand conformation. The Ab responses induced by the structurally constrained immunogens were more broadly reactive and had higher titers than those induced by the structurally unconstrained immunogens. Our results demonstrate that immunogens presenting the different structural conformations of the gp120 V1V2 vulnerable site can be designed, and that these immunogens induce distinct Ab responses with epitope conformation specificity. Therefore, these structurally constrained V1V2 immunogens are vaccine prototypes targeting the V1V2 domain of the HIV-1 envelope.

    IMPORTANCE Correlates analysis of the RV144 HIV-1 vaccine trial suggested that the presence of antibodies to the V1V2 region of HIV-1 gp120 was responsible for the modest protection observed in the trial. In addition V1V2 harbors one of the key vulnerable sites of HIV-1 Env recognized by a family of broadly neutralizing mAbs such as PG9. Thus V1V2 is a key target for vaccine development. However, this vulnerable site is structurally polymorphic, and designing immunogens that present different conformations is crucial for targeting this site. We show here that such immunogens can be designed and they induced conformation-specific antibody responses in rabbits. Our immunogens are therefore prototypes of vaccine candidates targeting the V1V2 region of HIV-1 Env.

  • SIV-producing cells in follicles are partially suppressed by CD8+ cells in vivo [PublishAheadOfPrint]

  • HIV- and simian immunodeficiency virus (SIV)-specific CD8+ T cells are typically largely excluded from lymphoid B cell follicles where HIV- and SIV-producing cells are most highly concentrated, indicating that B cell follicles are somewhat of an immune privileged site. To gain insights into virus-specific follicular CD8+ T cells, we determined the location and phenotype of follicular SIV-specific CD8+ T cells in situ, the local relationship of these cells to Foxp3+ cells, and effects of CD8 depletion on levels of follicular SIV-producing cells in chronically SIV infected rhesus macaques. We found that follicular SIV-specific CD8+ T cells were able to migrate throughout follicular areas including germinal centers. Many expressed PD-1, indicating they may have been exhausted. A small subset was in direct contact with and likely inhibited by Foxp3+ cells and a few were themselves Foxp3+. In addition, subsets of follicular SIV-specific CD8+ T cells expressed low to medium levels of perforin and subsets were activated and proliferating. Importantly, after CD8 depletion, SIV-producing cells increased in B cell follicles and extrafollicular areas, suggesting that follicular and extrafollicular CD8+ T cells have a suppressive effect on SIV replication. Taken together, these results suggest that during chronic SIV infection, despite high levels of exhaustion and likely inhibition by Foxp3+ cells, a subset of follicular SIV-specific CD8+ T cells are functional and suppress viral replication in vivo. These findings support HIV cure strategies that augment functional follicular virus-specific CD8+ T cells to enhance viral control.

    Importance: HIV- and SIV-specific CD8+ T cells are typically largely excluded from lymphoid B cell follicles where virus-producing cells are most highly concentrated, suggesting that B cell follicles are somewhat of an immune privileged site where virus-specific CD8+ T cells are not able to clear all follicular HIV- and SIV-producing cells. To gain insights into follicular CD8+ T cells function, we characterized follicular virus-specific CD8+ T cells in situ, in an SIV-infected rhesus macaque model of HIV. We found that subsets of follicular SIV-specific CD8+ T cells are able to migrate throughout the follicle, are likely inhibited by Foxp3+ cells, are likely exhausted, and that nonetheless, subsets are likely functional, as they express markers consistent with effector function, and show signs of suppressing viral replication in vivo. These findings support HIV cure strategies that increase frequencies of functional follicular virus-specific CD8+ T cells.

  • Foot-and-mouth disease virus viroporin 2B antagonizes RIG-I mediated antiviral effects by inhibition of its protein expression [PublishAheadOfPrint]

  • The role of retinoic acid-inducible gene I (RIG-I) in foot-and-mouth disease virus (FMDV)-infected cells remains unknown. Herein, we showed that RIG-I inhibits FMDV replication in host cells. FMDV infection increased the transcription of RIG-I, while it decreased RIG-I protein expression. A detailed analysis revealed that FMDV leader proteinase (Lpro), as well as 3C proteinase (3Cpro) and 2B protein, decreased RIG-I protein expression. Lpro and 3Cpro are viral proteinases that can cleave various host proteins and are responsible for several of the viral polyprotein cleavages. However, for the first time, we observed 2B-induced reduction of host protein. Further studies showed that 2B-mediated reduction of RIG-I is specific to FMDV, but not other picornaviruses including encephalomyocarditis virus, enterovirus 71, and coxsackievirus A16. Moreover, we found the decreased protein level of RIG-I is independent of the cleavage of eukaryotic translation initiation factor 4 gamma, the induction of cellular apoptosis, or the association of proteasomes, lysosomes and caspases pathways. A direct interaction was observed between RIG-I and 2B. The carboxyl terminal amino acids 105nndash;114 and 135nndash;144 of 2B were essential for the reduction of RIG-I, while residues 105nndash;114 were required for the interaction. These data suggest the antiviral role of RIG-I against FMDV and a novel antagonistic mechanism of FMDV that is mediated by 2B protein.

    IMPORTANCE This study demonstrated that RIG-I could suppress FMDV replication during virus infection. FMDV infection increased the transcriptional expression of RIG-I, while it decreased RIG-I protein expression. FMDV 2B protein interacted with RIG-I and induced reduction of RIG-I. 2B-induced reduction of RIG-I was independent of the induction of the cleavage of eukaryotic translation initiation factor 4 gamma or cellular apoptosis. In addition, proteasomes, lysosomes and caspases pathways were not involved in this process. This study provides new insight into the immune evasion mediated by FMDV and identifies 2B as an antagonistic factor for FMDV to evade the antiviral response.

  • Constitutively expressed IFITM3 protein in human pulmonary endothelial cells poses an early infection block to human influenza viruses [PublishAheadOfPrint]

  • A role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. Here, we found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion, but failed to uncoat viral nucleocapsid and replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by siRNA could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses an intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans.

    IMPORTANCE Avian influenza viruses, including H5N1 and H7N9 have been associated with severe respiratory disease and fatal outcomes in humans. Although acute respiratory distress syndrome (ARDS) and progressive pulmonary endothelial damage are known to be present during severe human infections, the role of pulmonary endothelial cells in the pathogenesis of avian influenza virus infections is largely unknown. By comparing human seasonal influenza strains to avian influenza viruses we provide greater insight into the interaction of influenza virus with human pulmonary endothelial cells. We show that human influenza virus infection is blocked during the early stages of virus entry, which is likely due to the relatively high expression of the host antiviral factor IFITMs (interferon-induced transmembrane proteins) located in membrane-bound compartments inside cells. Overall, this study provides a mechanism by which human endothelial cells limit replication of human influenza strains whereas avian influenza viruses overcome these restriction factors in this cell type.

  • Phosphorylation requirement of murine leukemia virus p12 [PublishAheadOfPrint]

  • The p12 protein of murine leukemia virus (MLV) Gag is associated with the preintegration complex (PIC), and mutants of p12 (PM14) exhibit defects in nuclear entry/retention. Mutants of the phosphorylated serine-61 also have reported defects in the early life cycle. Here we show that a phosphorylated peptide motif derived from human papillomavirus 8 (HPV 8), the E2 hinge region240-255, can functionally replace the main phosphorylated motif of MLV p12 and can rescue the viral titer of a lethal p12-PM14 mutant. Complementation with the HPV 8 E2 hinge motif generated multiple second site mutations in live viral passage assays. Additional p12 phosphorylation sites were detected, including the late domain of p12 (PPPY), as well as the late domain/protease cleavage site of matrix (LYPAL) by mass spectrometry and Western blots. Chromatin binding of p12-GFP fusion protein and functional complementation of p12-PM14 occurred independent of the E2 hinge region phosphorylation. Substitution of serine-61 to alanine within the minimal tethering domain (61SPMASRLRGRR71) maintained tethering, but in the context of the full-length p12, substitutions in S61 remained untethered and lost infectivity, indicating phosphorylation of p12 serine-61 functions to temporally regulate early and late p12 functions.

    Importance The p12 protein, required for both early and late viral functions, is the predominant phosphorylated viral protein of Moloney MLV and is required for virus viability. Our studies indicate that the N-terminus of p12 represses the early function of the chromatin binding domain, and that deletion of the N-terminus activates chromatin binding in the wild type Moloney MLV p12 protein. Mass spectrometry and mutagenesis studies suggest phosphorylation of both the repression domain and chromatin binding domain acts to temporally regulate this process at the appropriate stages during infection.

  • Protective capacity of the human anamnestic antibody response during acute dengue infection [PublishAheadOfPrint]

  • Half of the world's population is exposed to the risk of dengue virus infection. Although a vaccine for dengue is now available in a few countries, its reported overall efficacy of about 60% is not ideal. Protective immune correlates following natural dengue infection remain undefined, which makes it difficult to predict the efficacy of new vaccines. In this study, we address the protective capacity of dengue-specific antibodies that are produced by plasmablasts a few days after natural secondary infection. Among a panel of 18 dengue reactive human monoclonal antibodies, four groups of antibodies were identified based on their binding properties. While antibodies targeting the fusion loop of the glycoprotein of dengue virus dominated the antibody response, two smaller groups of antibodies bound to previously undescribed epitopes in domain II of the E protein. The latter, largely serotype cross-reactive antibodies, demonstrated increased stability of binding at pH 5. These antibodies possessed weak to moderate neutralization capacity in vitro but were the most efficacious in promoting survival of infected mice. Our data suggest that the cross-reactive anamestic antibody response has a protective capacity despite moderate neutralization in vitro and a moderate decrease of viremia in vivo.

    IMPORTANCE Antibodies can protect from symptomatic dengue infection. However, it is not easy to assess which classes of antibodies provide protection because in vitro assays are not always predictive of in vivo protection. During a repeat infection, dengue-specific immune memory cells are re-activated and large amounts of antibodies are produced. By studying antibodies cloned from patients with heterologous secondary infection we tested the protective value of the serotype cross-reactive "recall" or "anamnestic" response. We found that results from in vitro neutralization assays did not always correlate with the ability of the antibodies to reduce viremia in a mouse model. In addition, a decrease of viremia in mice did not necessarily improve survival. The most protective antibodies were stable at pH5, suggesting that antibody binding in the endosomes, after the antibody-virus complex is internalized, might be important to block virus spread in the organism.

  • Repression of the Chromatin Tethering Domain of Murine Leukemia Virus p12 [PublishAheadOfPrint]

  • Murine Leukemia Virus (MLV) p12, encoded within Gag, binds the viral preintegration complex (PIC) to the mitotic chromatin. This acts to anchor the viral PIC in the nucleus as the nuclear envelope reforms post-mitosis. Mutations within the p12 C-terminus (p12 PM13-15) block early stages in viral replication. Within the p12 PM13 region (p12 60PSPMA65), our studies indicated that chromatin tethering is not detected when the wild type p12 protein (M63) was expressed as a GFP fusion, however, constructs bearing p12-I63 were tethered. N-terminal truncations of the activated p12-I63-GFP indicated that tethering increased further upon deletion of p12 25DLLTEDPPPY34, which includes the late domain required for viral assembly. The p12 PM15 sequence (p12 70RREPP74) is critical for wild type viral viability, however, virus bearing the PM15 mutation (p12 70AAAAA74) with a second M63I mutant were viable, with a titer 18-fold lower than WT. p12 M63I mutation amplified chromatin tethering and compensated for the loss of chromatin binding of p12 PM15. Rescue of the p12-M63-PM15 non-viable mutant with PFV and KSHV tethering sequences confirmed the function of p1270-74 in chromatin binding. Minimally, full strength tethering was seen with only p12 61SPIASRLRGRR71 fused to GFP. These results indicate that the p12 C-terminus alone is sufficient for chromatin binding, and that the presence of the p12 25DLLTEDPPPY34 motif in the N-terminus suppresses the ability to tether.

    Importance This study defines a regulatory mechanism controlling the differential roles of the MLV p12 protein in early and late replication. During viral assembly and egress, the late domain within the p12 N-terminus functions to bind host vesicle release factors. During viral entry, the C-terminus of p12 is required for tethering to host mitotic chromosomes. Our studies indicate that the p12 domain including the PPPY late sequence temporally represses the p12 chromatin tethering motif. Maximal p12 tethering was identified with only an 11 amino acid minimal chromatin tethering motif encoded at p1261-71. Within this region, the p12-M63I substitution switches p12 into a tethering competent state, partially rescuing the p12-PM15 tethering mutant. A model for how this conformational change regulates early versus late functions is presented.

  • The intra-cellular cholesterol transport inhibitor U18666A inhibits the exosome-dependent release of mature hepatitis C virus [PublishAheadOfPrint]

  • Background: HCV particles are described as lipoviroparticles which are released similar to VLDLs. However, the release mechanism is still poorly understood; the canonical ERGIC pathway as well as the endosome-dependent release has been proposed. Recently, also the role of exosomes in the transmission of HCV has been reported. Only a minor fraction of the de novo synthesized lipoviroparticles are finally released by the infected cell.

    Methods: To investigate the relevance of MVBs for viral morphogenesis and release, the MVB inhibitor U18666A was used. Intracellular trafficking was analyzed by confocal microscopy and electron microscopy. Moreover an mcherry-tagged HCV-variant was used.

    Results: Conditions were established that enable an U18666A-dependent inhibition of MVBs without affecting viral replication. Under these conditions a significant inhibition of the HCV release was observed. The assembly of viral particles is not affected. In U18666A-treated cells, intact/infectious viral particles accumulate in CD63-positive exosomal structures and large dysfunctional lysosomal structures (multilamellar bodies). These retained particles possess a lower density reflecting a misloading with lipids.

    Conclusion: Our data indicate that at least a fraction of HCV particles leaves the cell via the endosomal pathway. Endosomes facilitate the sorting of HCV particles for release or degradation.

    Importance There are still a variety of open questions regarding morphogenesis and release of hepatitis C virus. The HCV-infected cell produces significant more viral particles as finally released raising the question about the fate of the non-released particles. Moreover the relevance of the endosomal pathway for the release of HCV is under debate.

    Use of the MVB (multivesicular bodies) inhibitor U18666A enabled a detailed analysis of the impact of MVBs for the viral morphogenesis and release. It was revealed that infectious, fully-assembled HCV particles are either MVB-dependently released or intracellularly degraded by the lysosome. Our data indicate that at least a fraction of HCV particles leaves the cell via the endosomal pathway independent from the constitutive secretory pathway.

    Our study describes a so far unprecedented crosstalk between two pathways regulating on the one hand the release of infectious viral particles and on the other hand the intracellular degradation of non-released particles.

  • Heterodimers as the structural unit of the T=1 capsid of the fungal dsRNA Rosellinia necatrix quadrivirus 1 [PublishAheadOfPrint]

  • Most dsRNA viruses are transcribed and replicated in a specialized icosahedral capsid with a T=1 lattice consisting of 60 asymmetric capsid protein (CP) dimers. These capsids help to organize the viral genome and replicative complex(es). They also act as molecular sieves that isolate the virus genome from host defense mechanisms and allow the passage of nucleotides and viral transcripts. Rosellinia necatrix quadrivirus 1 (RnQV1), the type species of family Quadriviridae, is a dsRNA fungal virus with a multipartite genome consisting of four monocistronic segments (segments 1-4). dsRNA-2 and dsRNA-4 encode two CP (P2 and P4, respectively), which co-assemble into ~450 AAring; diameter capsids. We used three-dimensional cryo-electron microscopy combined with complementary biophysical techniques to determine the structure of RnQV1 virion strains W1075 and W1118. RnQV1 has a quadripartite genome and the capsid is based on a single-shelled, T=1 lattice built of P2-P4 dimers. Whereas the RnQV1-W1118 capsid is built of full-length CP, the P2 and P4 of RnQV1-W1075 are cleaved into several polypeptides, maintaining capsid structural organization. RnQV1 heterodimers have a quaternary organization similar to homodimers of reoviruses and other dsRNA mycoviruses. The RnQV1 capsid is the first T=1 capsid with a heterodimer as an asymmetric unit reported to date, and follows the architectural principle for dsRNA viruses that a 120-subunit capsid is a conserved assembly that supports dsRNA replication and organization.

    Importance Given their importance to health, members of family Reoviridae are the basis of most structural and functional studies and provide much of our knowledge of dsRNA viruses. Analysis of bacterial, protozoal and fungal dsRNA viruses has improved our understanding of their structure, function and evolution as well. Here we studied a dsRNA virus that infects the fungus Rosellinia necatrix, an ascomycete that is pathogenic to a wide range of plants. Using three-dimensional cryo-electron microscopy and analytical ultracentrifugation analysis, we determined the structure and stoichiometry of Rosellinia necatrix quadrivirus 1 (RnQV1). The RnQV1 capsid is a T=1 capsid with 60 heterodimers as the asymmetric units. The large amount of genetic information used by RnQV1 to construct a simple T=1 capsid is probably related to the numerous virus-host and virus-virus interactions that it must face in its life cycle, which lacks an extracellular phase.

  • Reovirus {mu}1 Protein Affects Infection by Altering Virus-Receptor Interactions [PublishAheadOfPrint]

  • Proteins that form the reovirus outer capsid play an active role in the entry of reovirus into host cells. Among these, the 1 protein mediates attachment of reovirus particles to host cells via interaction with cell surface glycans or the proteinaceous receptor, JAM-A. The mmu;1 protein functions to penetrate the host cell membrane to allow delivery of the genome-containing viral core particle into the cytoplasm to initiate viral replication. We demonstrate that a reassortant virus that expresses the M2 gene-encoded mmu;1 protein derived from prototype strain T3D in an otherwise prototype T1L background (T1L/T3DM2) infects cells more efficiently than parental T1L. Unexpectedly, the enhancement in infectivity of T1L/T3DM2 is due to its capacity to attach to cells more efficiently. We present genetic data implicating the central region of mmu;1 in altering the cell attachment property of reovirus. Our data indicate that T3D mmu;1-mediated enhancement in infectivity of T1L is dependent on the function of 1 and requires the expression of JAM-A. We also demonstrate that T1L/T3DM2 utilizes JAM-A more efficiently than T1L. These studies reveal a previously unknown relationship between two non-adjacent reovirus outer capsid proteins, 1 and mmu;1.

    IMPORTANCE How reovirus attaches to host cells has been extensively characterized. Attachment of reovirus to host cells is mediated by the 1 protein and properties of 1 influence the capacity of reovirus to target specific host tissues and produce disease. Here, we present new evidence indicating that the cell attachment properties of 1 are influenced by the nature of mmu;1, a capsid protein that does not physically interact with 1. These studies could explain the previously described role for mmu;1 in influencing reovirus pathogenesis. These studies are also of broader significance because they highlight an example for how genetic reassortment between virus strains could produce phenotypes that are distinct from either parent.

  • Ubiquitination up-regulates influenza virus polymerase function [PublishAheadOfPrint]

  • The influenza A virus polymerase plays an essential role in the virus lifecycle, directing synthesis of viral mRNAs and genomes. It is a trimeric complex composed of subunits PA, PB1, and PB2 and associates with viral RNAs and nucleoprotein (NP) to form higher order ribonucleoprotein (RNP) complexes. The polymerase is regulated temporally over the course of infection to ensure coordinated expression of viral genes as well as replication of the viral genome. Various host factors and processes have been implicated in regulation of the IAV polymerase function, including post-translational modifications, however the mechanisms are not fully understood. Here we demonstrate that ubiquitination plays an important role in stimulating polymerase activity. We show that all protein subunits in the RNP are ubiquitinated, but ubiquitination does not significantly alter protein levels. Instead, ubiquitination and an active proteasome enhance polymerase activity. Expressing ubiquitin up-regulates polymerase function in a dose-dependent fashion causing increased accumulation of vRNA, cRNA and mRNA and enhanced viral gene expression during infection. Ubiquitin expression directly affects polymerase activity independent of NP or RNP assembly. Ubiquitination and the ubiquitin-proteasome pathway play key roles during multiple stages of influenza virus infection, and data presented here now demonstrate that these processes modulate viral polymerase activity independent of protein degradation.

    IMPORTANCE The cellular ubiquitin-proteasome pathway impacts steps during the entire influenza virus life cycle. Ubiquitination suppresses replication by targeting viral proteins for degradation and stimulating innate antiviral signaling pathways. Ubiquitination also enhances replication by facilitating viral entry and virion disassembly. We identify here an addition pro-viral role of the ubiquitin-proteasome system, showing that all of the proteins in the viral replication machinery are subject to ubiquitination and this is crucial for optimal viral polymerase activity. Manipulating the ubiquitin machinery for therapeutic benefit is therefore likely to disrupt the function of multiple viral proteins at stages throughout the course of infection.

  • Specific inhibition of HIV infection by spironolactone in T cells [PublishAheadOfPrint]

  • Tat protein, the HIV transactivator, regulates transcription of the HIV genome by the host transcription machinery. Efficient inhibitors of HIV transcription that target Tat or the cellular cofactor Nuclear Factor-kappa B (NF-B) are well known. However, inhibition of HIV Tat-dependent transcription by targeting the general transcription and DNA-repair factor II human (TFIIH) has not been reported. Here, we show that spironolactone (SP), an aldosterone antagonist approved for clinical use, inhibits HIV-1 and HIV-2 infection of permissive T cells by blocking viral Tat-dependent transcription from the long terminal repeat (LTR). We found that treatment of Jurkat and primary CD4+ T cells with SP induces degradation of the XPB cellular helicase, a component of the TFIIH complex, without affecting cellular mRNA levels, T cell viability or T cell proliferation. We further demonstrate that the effect of SP on HIV infection is independent from its aldosterone antagonist function since the structural analogue, eplerenone, does not induce XPB degradation and does not inhibit HIV infection. Rescue experiments show that the SP-induced block of HIV infection relies, at least partially, on XPB degradation. In addition, we demonstrate that SP specifically inhibits Tat-dependent transcription, since basal transcription from the LTR is not affected. Our results demonstrate that SP is a specific inhibitor of HIV Tat-dependent transcription in T cells, which additionally suggests that XPB is a cofactor required for HIV infection. Targeting a cellular cofactor of HIV transcription constitutes an alternative strategy to inhibit HIV infection together with the existing antiretroviral therapy.

    Importance Transcription from the HIV promoter is regulated by the combined activity of the host transcription machinery and the viral transactivator Tat protein. Here, we report that the drug spironolactone - an antagonist of aldosterone - blocks viral Tat-dependent transcription, thereby inhibiting both HIV-1 and HIV-2 infection of permissive T cells. This inhibition relies on the degradation of the cellular helicase XPB, a component of the TFIIH transcription factor complex. Consequently, XPB appears as a novel HIV cofactor. Our discovery of the HIV inhibitory activity of spironolactone opens the way for the development of novel anti-HIV strategies targeting a cellular cofactor, without the limitations of antiretroviral therapy of drug resistance, and high cost.

  • The Coat Protein and NIa Protease of Two Potyviridae Members Independently Confer Superinfection Exclusion [PublishAheadOfPrint]

  • Superinfection exclusion (SIE) is an antagonistic virus-virus interaction whereby initial infection by one virus prevents subsequent infection by closely related viruses. Although SIE has been described in diverse viruses infecting plants, humans and animals, its mechanisms including involvement of specific viral determinants are just beginning to be elucidated. In this study, SIE determinants encoded by two economically important wheat viruses, Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) and Triticum mosaic virus (TriMV; genus Poacevirus, family Potyviridae), were identified in gain-of-function experiments that used heterologous viruses to express individual virus-encoded proteins in wheat. Wheat plants infected with TriMV expressing WSMV P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg or NIb cistrons permitted efficient superinfection by WSMV expressing GFP (WSMV-GFP). By contrast, wheat infected with TriMV expressing WSMV NIa-Pro or CP substantially excluded superinfection by WSMV-GFP, suggesting that both of these cistrons are SIE effectors encoded by WSMV. Importantly, SIE is due to functional WSMV NIa-Pro or CP proteins rather than their coding RNAs as altering the coded protein products by minimally changing RNA sequences led to abolishment of SIE. Deletion mutagenesis further revealed that elicitation of SIE by NIa-Pro requires the entire protein while CP requires only a 200 amino acid (aa) middle fragment (aa 101-300) of the 349 aa. Strikingly, reciprocal experiments with WSMV-mediated expression of TriMV proteins showed that TriMV CP, and TriMV NIa-Pro to a lesser extent, likewise excluded superinfection by TriMV-GFP. Collectively, these data demonstrate that WSMV- and TriMV-encoded CP and NIa-Pro proteins are effectors of SIE, and these two proteins trigger SIE independent of each other.

    IMPORTANCE Superinfection exclusion (SIE) is an antagonistic virus-virus interaction that prevents secondary invasions by identical or closely related viruses in the same host cells. Although known to occur in diverse viruses, SIE remains an enigma in terms of key molecular determinants and action mechanisms. In this study, we found that Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) encode two independently functioning cistrons that serve as effectors of SIE at the protein but not the RNA level. The coat protein and NIa-Pro encoded by these two viruses, when expressed from a heterologous virus, exerted SIE to the cognate viruses. The identification of virus-encoded effectors of SIE and their transgenic expression could potentially facilitate the development of virus-resistant crop plants. Additionally, functional conservation of SIE in diverse virus groups suggests that a better understanding of the underlying mechanisms of SIE could facilitate the development of novel antiviral therapies against viral diseases.

  • Herpes Simplex Virus 1 Tegument Protein UL41 Counteracts IFIT3 Antiviral Innate Immunity [PublishAheadOfPrint]

  • The interferon-induced protein with tetratricopeptide repeats 3 (IFIT3 or ISG60) is a host intrinsic antiviral factor that restricts many DNA and RNA virus replication. Herpes simplex virus 1 (HSV-1), a DNA virus bearing a large genome, can encode many viral proteins to counteract the host immune responses. However, whether IFIT3 plays a role upon HSV-1 infection is little known. In this study, we show for the first time that HSV-1 tegument protein UL41, a viral endoribonuclease, plays an important role on inhibiting the antiviral activity of IFIT3. Here, we demonstrated that ectopically expressed IFIT3 could restrict the replication of vesicular stomatitis virus (VSV), but had little effect on the replication of wild-type (WT)-HSV-1. Further study showed that WT-HSV-1 infection down-regulated the expression of IFIT3, and ectopic expression of UL41, but not the immediate early protein ICP0, notably reduced the expression of IFIT3. The underlying molecular mechanism was that UL41 diminished the accumulation of IFIT3 mRNA to abrogate its antiviral activity. In addition, our results illustrated that ectopic expression of IFIT3 inhibited the replication of UL41-null mutant virus (R2621), and stably knockdown of IFIT3 facilitated its replication. Taken together, HSV-1 was shown for the first time to evade the antiviral function of IFIT3 via UL41.

    IMPORTANCE The tegument protein UL41 of HSV-1 is an endoribonuclease with the substrate specificity of RNase A, which plays an important role in viral infection. Upon HSV-1 infection, interferons (IFNs) are critical cytokines that regulate immune responses against viral infection. Host antiviral responses are significantly boosted or crippled in the presence or absence of IFIT3, however, whether IFIT3 plays a role during HSV-1 infection is still unknown. Our data shows for the first time that IFIT3 has little effect on HSV-1 replication as UL41 decreases the accumulation of IFIT3 mRNA and subverts its antiviral activity. This study identifies IFIT3 as a novel target of the tegument protein UL41 and provides new insight into HSV-1 mediated immune evasion.

  • Pathogenesis and transmission assessments of two H7N8 influenza A viruses recently isolated from turkey farms in Indiana using mouse and ferret models [PublishAheadOfPrint]

  • Avian influenza A H7 viruses have caused multiple outbreaks in domestic poultry throughout North America, resulting in occasional human infections in close contact. In early 2016, the presence of H7N8 highly pathogenic avian influenza (HPAI) viruses and closely related H7N8 low pathogenic avian influenza (LPAI) viruses were confirmed in commercial turkey farms in Indiana. These H7N8 viruses represent the first isolation of this subtype in domestic poultry in North America, with their virulence in mammalian hosts and the potential risk for human infection largely unknown. In this study, we assessed the ability of H7N8 HPAI and LPAI viruses to replicate in vitro in human airway cells and in vivo in mouse and ferret models. Both H7N8 viruses replicated efficiently in vitro and in vivo, but exhibited substantial differences in disease severity in mammals. In mice, while the H7N8 LPAI virus largely remained avirulent, the H7N8 HPAI virus exhibited greater infectivity, virulence, and lethality. Both H7N8 viruses replicated similarly in ferrets, but only the H7N8 HPAI virus caused moderate weight loss, lethargy, and mortality. The H7N8 LPAI virus displayed limited transmissibility in ferrets placed in direct contact, while no transmission of H7N8 HPAI virus was detected. Our results indicate that the H7N8 avian influenza viruses from Indiana are able to replicate in mammals and cause severe disease, but with limited transmission. The recent appearance of H7N8 viruses in domestic poultry highlights the need for continued influenza surveillance in wild birds and close monitoring of the potential risk to human health.

    Importance: H7 influenza viruses circulate in wild birds in the U.S., but when the virus emerges in domestic poultry populations, the frequency of human exposure and the potential for human infections increases. An H7N8 highly pathogenic avian influenza (HPAI) and an H7N8 low pathogenic avian influenza (LPAI) viruses were recently isolated from commercial turkey farms in Indiana. To determine the risk that these influenza viruses pose to humans, we assessed the pathogenesis and transmission in vitro and in mammalian models. We found that the H7N8 HPAI virus exhibited enhanced virulence and, although transmission was only observed with the H7N8 LPAI virus, the ability of this H7 virus to transmit in a mammalian host and quickly evolve to a more virulent strain is cause for concern. Our findings offer important insight into the potential for emerging H7 avian influenza viruses to acquire the ability to cause disease and transmit among mammals.

  • A Tale of Two PMLs: Elements Regulating the Differential Substrate Recognition by the HSV-1 ICP0 E3 Ubiquitin Ligase [PublishAheadOfPrint]

  • Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an aalpha; gene product required for viral replication at low multiplicity of infection. Upon entry, nuclear domain 10 (ND10) converges at the incoming DNA and represses viral gene expression. ICP0 contains a RING-type E3 ubiquitin ligase, which degrades the ND10 organizer, PML, and disperses ND10 to alleviate the repression. In the present study, we focus on understanding the regulation on ICP0 E3 ligase activity in degrading different ICP0 substrates. We report that: (i) A SIM located at ICP0 residues 362-364 is required for the degradation of PML isoforms II, IV and VI but not isoform I. This differentiation mechanism exists in both HEp-2 and U2OS cells, regardless of the cell permissiveness to the ICP0 null virus. (ii) Physical interaction between SIM362-364 and PML II is necessary but not sufficient for PML II degradation. Both proximal sequences surrounding SIM362-364 and distal sequences located at ICP0 C-terminus enhance the degradation of PML II. (iii) ICP0 C-terminus is dispensable for PML I degradation. Instead, bipartite PML I binding domains located in the N-terminal half of ICP0 coordinate to promote the degradation of PML I. (iv) The stability of ICP0, but not its ND10-fusion ability, affects the rate of PML I degradation. Taken together, ICP0 uses at least two regulatory mechanisms to differentiate its substrates. The disparate recognition of the ICP0 E3 substrates may be related to different roles these substrates may play in the HSV-1 infection.

    IMPORTANCE: Viruses have a limited genetic coding capacity but must encounter a multi-layered comprehensive host defense. To establish a successful infection, viruses usually produce multifunctional proteins to coordinate the counteractions. Here we report that an HSV-1 protein, ICP0, can recognize individual host factors and target them differently for destruction. We identified elements that are important for the ICP0 E3 ubiquitin ligase to differentially recognize two of its substrates, PML I and PML II. This is the first study that has systematically investigated how ICP0 discriminates two similar molecules by very different mechanisms. The work lays the foundation for understanding the role of host defensive factors and the mechanisms viruses use to take advantage of some host proteins while destroying the others.

  • Single-nucleotide-resolution mapping of HBV promoters in infected human livers and hepatocellular carcinoma [PublishAheadOfPrint]

  • Hepatitis B virus (HBV) is a major cause of liver diseases including hepatocellular carcinoma (HCC), and more than 650,000 people die annually due to HBV-associated liver failure. Extensive studies of individual promoters have revealed that heterogeneous RNA 5rrsquo; -ends contribute to the complexity of HBV transcriptome and proteome. Here we provide a comprehensive map of HBV transcription start sites (TSSs) in human liver, HCC and blood, as well as several experimental replication systems, at single nucleotide resolution. Using CAGE analysis of 16 HCC/non-tumor liver pairs, we identify 17 robust TSSs, including a novel promoter for the X gene located in the middle of the gene body, which potentially produces a shorter X protein translated from the conserved second start codon, and two minor anti-sense transcripts that might represent viral ncRNAs. Interestingly, transcription profiles were similar in HCC and non-tumor livers, although quantitative analysis revealed highly variable patterns of TSS usage among clinical samples, reflecting precise regulation of HBV transcription initiation at each promoter. Unlike the variety of TSSs found in liver and HCC, the vast majority of transcripts detected in HBV-positive blood samples are pgRNA, most likely generated and released from liver. Our quantitative TSS mapping using the CAGE technology will allow better understanding of HBV transcriptional responses in further studies aimed at eradicating HBV in chronic carriers.

    IMPORTANCE Despite the availability of a safe and effective vaccine, HBV infection remains a global health problem, and current antiviral protocols are not able to eliminate the virus in chronic carriers. Previous studies of the regulation of HBV transcription have described four major promoters and two enhancers, but little is known about their activity in human livers and HCC. We deeply sequenced the HBV RNA 5rrsquo; ends in clinical human samples and experimental models by using a new, sensitive and quantitative method termed cap analysis of gene expression (CAGE). Our data provide the first comprehensive map of global TSS distribution over the entire HBV genome in the human liver, validating already known promoters and identifying novel locations. Better knowledge of HBV transcriptional activity in the clinical setting has critical implications in the evaluation of therapeutic approaches that target HBV replication.

  • Bipartite network analysis of the archaeal virosphere: evolutionary connections between viruses and capsid-less mobile elements [PublishAheadOfPrint]

  • Archaea and particularly hyperthermophilic crenarchaea are hosts to many unusual viruses with diverse virion shapes and distinct gene compositions. As is typical of viruses in general, there are no universal genes in the archaeal virosphere. Therefore, to obtain a comprehensive picture of the evolutionary relationships between viruses, network analysis methods are more productive than traditional phylogenetic approaches. Here we present a comprehensive comparative analysis of genomes and proteomes from all currently known taxonomically classified and unclassified, cultivated and uncultivated archaeal viruses. We constructed a bipartite network of archaeal viruses that includes two classes of nodes, the genomes and gene families that connect them. Dissection of this network using formal community detection methods reveals strong modularity with 10 distinct modules and 3 putative supermodules. However, compared to the previously analyzed similar networks of eukaryotic and bacterial viruses, the archaeal virus network is sparsely connected. With the exception of the tailed viruses related to the bacteriophages of the order Caudovirales and the families Turriviridae and Sphaerolipoviridae that are linked to a distinct supermodule of eukaryotic viruses, there are few connector genes shared by different archaeal virus modules. In contrast, most of these modules include, in addition to viruses, capsid-less mobile elements, emphasizing tight evolutionary connections between the two types of entities in archaea. The relative contributions of distinct evolutionary origins, in particular from non-viral elements, and insufficient sampling to the sparsity of the archaeal virus network remain to be determined by further exploration of the archaeal virosphere.

    IMPORTANCE Viruses infecting archaea are among the most mysterious denizens of the virosphere. Many of these viruses display no genetic or even morphological relationship to viruses of bacteria and eukaryotes, raising questions regarding their origins and position in the global virosphere. Analysis of 5740 protein sequences from 116 genomes allowed dissection of the archaeal virus network and showed that most groups of the archaeal viruses are evolutionarily connected to capsid-less mobile genetic elements, including various plasmids and transposons. This finding could reflect actual independent origins of the distinct groups of archaeal viruses from different non-viral elements, providing important insights into the emergence and evolution of the archaeal virome.

  • TRIM5{alpha} Resistance Escape Mutations in Capsid are Transferable Between Simian Immunodeficiency Virus Strains [PublishAheadOfPrint]

  • TRIM5aalpha; polymorphism limits and complicates the use of simian immunodeficiency virus (SIV) for evaluation of HIV vaccine strategies in rhesus macaques. We previously reported that the TRIM5aalpha;-sensitive SIVsm clone, SIVsmE543-3, acquired amino acid substitutions in capsid that overcame TRIM5aalpha; restriction when it was passaged in rhesus macaques expressing restrictive TRIM5aalpha; alleles. Here we generated TRIM5aalpha;-resistant clones of the related SIVsmE660 strain without animal passage by introducing the same amino acid capsid substitutions. We evaluated one of the variants in rhesus macaques expressing permissive and restrictive TRIM5aalpha; alleles. The SIVsmE660 variant infected and replicated in macaques with restrictive TRIM5aalpha; genotypes as efficiently as in macaques with permissive TRIM5aalpha; genotypes. These results demonstrated that mutations in SIV capsid can confer SIV resistance to TRIM5aalpha; restriction without animal passage, suggesting an applicable method to generate more diverse SIV strains for HIV vaccine studies.

    IMPORTANCE Many strains of SIV from sooty mangabey monkeys are susceptible to resistance by common rhesus macaque TRIM5aalpha; alleles and result in reduced virus acquisition and replication in macaques that express these restrictive alleles. We previously observed that spontaneous variations in the capsid gene were associated with improved replication in macaques and introduction of two amino acid changes in capsid transfers this improved replication to the parent clone. In the present study we introduced these mutations to a related but distinct strain of SIV that is commonly used for challenge studies for vaccine trials. These mutations also improved replication of this strain in macaques with the restrictive TRIM5aalpha; genotype and thus will eliminate the confounding effects of TRIM5aalpha; in vaccine studies.

  • Structure of Aichi virus 1 and its empty particle: clues towards kobuvirus genome release mechanism [PublishAheadOfPrint]

  • Aichi virus 1 (AiV-1) is a human pathogen from the Kobuvirus genus of the Picornaviridae family. Worldwide, 80-95% of adults have antibodies against the virus. AiV-1 infections are associated with nausea, gastroenteritis, and fever. Unlike most picornaviruses, kobuvirus capsids are composed of only three types of subunits: VP0, VP1, and VP3. Here we present the structure of the AiV-1 virion determined to a resolution of 2.1 AAring; using X-ray crystallography. The surface loops puff of VP0 and knob of VP3 in AiV-1 are shorter than those in other picornaviruses. Instead, the 42-residue-long BC-loop of VP0 forms the most prominent surface feature of the AiV-1 virion. We determined the structure of AiV-1 empty particle to a resolution of 4.2 AAring; using cryo-electron microscopy. The empty capsids are expanded relative to the native virus. The N-terminal arms of capsid proteins VP0, which mediate contacts between the pentamers of capsid protein protomers in the native AiV-1 virion, are disordered in the empty capsid. Nevertheless, the empty particles are stable, at least in vitro, and do not contain pores that might serve as channels for genome release. Therefore, extensive and probably reversible local reorganization of AiV-1 capsid is required for its genome release.

    IMPORTANCE Aichi virus 1 (AiV-1) is a human pathogen that can cause diarrhea, abdominal pain, nausea, vomiting, and fever. AiV-1 is identified in environmental screening studies with higher frequency and greater abundance than other human enteric viruses. Accordingly, 80-95% of adults worldwide have suffered from AiV-1 infections. We determined the structure of the AiV-1 virion. Based on the structure, we show that antiviral compounds that were developed against related enteroviruses are unlikely to be effective against AiV-1. The surface of the AiV-1 virion has a unique topology distinct from other related viruses from the Picornaviridae family. We also determined that AiV-1 capsids form compact shells even after genome release. Therefore, AiV-1 genome release requires large localized and probably reversible reorganization of the capsid.

  • The US3 protein of pseudorabies virus drives viral passage across the basement membrane in porcine respiratory mucosa explants [PublishAheadOfPrint]

  • Passage of the basement membrane (BM), which forms a barrier between epithelium and underlying lamina propria, represents an important step in the early pathogenesis of different alphaherpesviruses. Rho-GTPase signaling plays an important role in transmigration of cells across the BM during physiological and pathological processes. We reported earlier that the US3 protein kinase of the alphaherpesvirus pseudorabies virus (PRV) interferes with Rho GTPase signaling and causes a reorganisation of the host cell cytoskeleton which, as a consequence, enhances viral cell-to-cell spread in epithelial cell cultures.

    Here, using an ex vivo system of porcine nasal respiratory mucosa explants that allows to study PRV invasion through the BM, we found that a PRV strain that lacks US3 expression (US3 PRV) showed a reduced spread in mucosal epithelium and was virtually unable to breach the BM, in contrast to isogenic wild type (WT) or US3-rescue PRV strains. Interestingly, addition of IPA3, an inhibitor of p21-activated kinases that blocks the effects of US3 on the cytoskeleton, suppressed the ability of WT PRV to spread across the BM. In addition, artificial suppression of RhoA signalling using CPC3 (Cell Permeable C3 transferase) to mimic the effects of US3 on Rho-GTPase signaling, significantly increased passage of US3 PRV through the BM whereas it did not significantly affect BM passage of WT or US3-rescue PRV. In conclusion, these data indicate that US3 plays an important role in PRV mucosal invasion across the BM, which involves its interference with Rho-GTPase signaling. This is the first report describing an alphaherpesvirus protein that drives viral BM passage.

    Importance Many viruses, including alphaherpesviruses, primarily replicate in epithelial cells of surface mucosae such as the respiratory mucosa. Some of these viruses breach the basement membrane underlying these epithelial cells to reach underlying connective tissue and blood vessels and invade the host. Hence, epithelial spread and basement membrane passage represent crucial but still poorly understood early steps in (alphaherpes)virus pathogenesis. Here, using ex vivo porcine respiratory mucosa explants, we show that the conserved US3 protein of the porcine alphaherpesvirus pseudorabies virus (PRV) is critical for passage of PRV across the basement membrane and contributes to efficient viral epithelial spread. In addition, we show that US3-mediated viral epithelial spread and passage across the basement membrane depend at least in part on the ability of this viral protein to modulate cellular Rho GTPase signaling. This is the first report that identifies an alphaherpesvirus protein that drives viral basement membrane passage.

  • Altering the anti-inflammatory lipoxin microenvironment: a new insight into KSHV pathogenesis [PublishAheadOfPrint]

  • Lipoxins are host anti-inflammatory molecules that play a vital role in restoring tissue homeostasis. The efficacy of lipoxins and their analog epilipoxins in treating inflammation and its associated diseases has been well documented. Kaposi's Sarcoma (KS) and Primary Effusion Lymphoma (PEL) are two well-known inflammation related diseases caused by Kaposi's Sarcoma-Associated Herpesvirus (KSHV). Controlling inflammation is one of the strategies adopted to treat KS and PEL, a primary motivation for exploring and evaluating the therapeutic potential of using lipoxins. This study documents how KSHV manipulates and downregulates the secretion of the anti-inflammatory lipoxin A4 in host cells and the viral factors involved in this process using in vitro KS and PEL cells as models. The presence of the lipoxin receptor [ALX/Formyl peptidyl receptor (FPR)] in KS patient tissue sections and in vitro KS and PEL cell models offers a novel possibility for treating KS and PEL with lipoxins. Treating de novo KSHV infected endothelial cells with lipoxin and epilipoxin creates an anti-inflammatory environment by decreasing levels of NFB, AKT, ERK1/2, COX-2, and 5-lipoxygenase. Lipoxin treatment on CRISPR/CAS9 technology mediated ALX/FPR gene deletion revealed the importance of the lipoxin receptor ALX for effective lipoxin signaling. A viral miRNA cluster was identified as the primary factor contributing to the downregulation of lipoxin A4 secretion in host cells. The KSHV miRNA cluster probably targets enzyme 15-lipoxygenase, which is involved in lipoxin A4 synthesis. This study provides a new insight into the potential treatment of KS and PEL using nature's own anti-inflammatory molecule, lipoxin.

    IMPORTANCE KSHV infection has been shown to upregulate several host pro-inflammatory factors, which aid in its survival and pathogenesis. Influence of KSHV infection on anti-inflammatory molecules is not well studied. As current treatment methods for KS and PEL are fraught with unwanted side effects and low efficiency, the search for new therapeutics is therefore imperative. The use of nature's own molecule lipoxin as a drug is promising. This study opens up new domains in KSHV research focusing on how the virus modulates lipoxin secretion. Current study warrants further investigation to study the therapeutic potential of lipoxin using in vitro cell models for KS and PEL.

  • Immune and non-immune compartment-specific IFN responses are critical determinants of HSV-induced generalized infections and acute liver failure. [PublishAheadOfPrint]

  • The interferon (IFN) response to viral pathogens is critical for host survival. In humans and mouse models, defects in IFN responses can result in lethal herpes simplex type-1 (HSV-1) infections, usually from encephalitis. Although rare, HSV-1 can also cause fulminant hepatic failure which is often fatal. While herpes simplex encephalitis has been extensively studied, HSV-1 generalized infections and subsequent acute liver failure are less well understood. We previously demonstrated that IFNaalpha;bbeta;R-/- mice are exquisitely susceptible to liver infection following corneal infection with HSV-1. In this study we used bone marrow chimeras of IFNaalpha;bbeta;R-/- (AG129) and WT (129SvEv) mice to probe the underlying IFN-dependent mechanisms that control HSV-1 pathogenesis. Following infection, WT mice with either IFNaalpha;bbeta;R-/- or WT marrow exhibited comparable survival, while IFNaalpha;bbeta;R-/- mice with WT marrow had a significant survival advantage over their counterparts with IFNaalpha;bbeta;R-/- marrow. Furthermore, using bioluminescent imaging (BLI) to maximize data acquisition, we showed that the transfer of IFN-competent hematopoietic cells controlled HSV-1 replication and damage in the livers of IFNaalpha;bbeta;R-/- mice. Consistent with this, the inability of IFNaalpha;bbeta;R-/- immune cells to control liver infection in IFNaalpha;bbeta;R-/-mice manifested as profoundly elevated aspartate transaminase (AST) and alanine transaminase (ALT) levels, indicative of severe liver damage. In contrast, IFNaalpha;bbeta;R-/- mice receiving WT marrow exhibited only modest elevations of AST and ALT levels. These studies indicate that IFN responsiveness of the immune system is a major determinant of viral tropism and damage during visceral HSV infections.

    IMPORTANCE Herpes simplex virus type-1 is an incurable viral infection with the most significant morbidity and mortality occurring in neonates and patients with compromised immune systems. Severe pathologies from HSV include the blindness-inducing herpetic stromal keratitis, highly debilitating and lethal herpes simplex encephalitis, and generalized infections that can lead to herpes simplex-induced acute liver failure. While immune compromise is a known factor, the precise mechanisms that lead to generalized HSV infections are unknown. In this study we used and developed a mouse model system in combination with real-time bioluminescence imaging to demonstrate the relative importance of the immune and non-immune compartments for containing viral spread, and promoting host survival after corneal infection. Our results shed light on the pathogenesis of HSV infections that lead to generalized infection and acute liver failure.

  • Introduction, evolution, and dissemination of influenza A viruses in exhibition swine, USA, 2009-2013 [PublishAheadOfPrint]

  • The swine-human interface created at agricultural fairs, along with the generation of and maintenance of influenza A virus diversity in exhibition swine, presents an ongoing threat to public health. Nucleotide sequences of influenza A virus isolates collected from exhibition swine in Ohio (n=262) and Indiana (n=103) during 2009-2013 were used to investigate viral evolution and movement within this niche sector of the swine industry. Phylogenetic and Bayesian analyses were employed to identify introductions of influenza A virus to exhibition swine and study viral population dynamics. In 2013 alone, we identified ten independent introductions of influenza A virus into Ohio and/or Indiana exhibition swine. Frequently, viruses from the same introduction were identified at multiple fairs within the region, providing evidence of rapid and widespread viral movement within the exhibition swine populations of the two states. While pigs moving fair-to-fair-to-fair is possible in some locations, the concurrent detection of nearly identical strains at several fairs indicates a viral common source was more likely. Importantly, we detected an association between the high number of human H3N2v infections in 2012 and the widespread circulation of influenza A viruses of the same genotype in exhibition swine in Ohio fairs sampled that year. The extent of viral diversity observed in exhibition swine, and the rapidity with which it disseminated across long distances, indicates that novel strains of influenza A virus will continue to emerge and spread within exhibition swine populations, presenting an ongoing threat to humans.

    Importance Understanding the underlying population dynamics of influenza A viruses in commercial and exhibition swine is central to assessing the risk for human infections with variant viruses, including H3N2v. We used viral genomic sequences from isolates collected from exhibition swine during 2009-2013 to understand how the peak of H3N2v cases in 2012 relates to long-term trends in the population dynamics of pandemic viruses recently introduced into commercial and exhibition swine in the United States. Our spatial analysis underscores the key role of rapid viral dispersal in spreading multiple genetic lineages throughout a multi-state network of agricultural fairs, providing opportunities for divergent lineages to co-infect, reassort, and generate new viral genotypes. The higher genetic diversity of genotypes co-circulating in exhibition swine since 2013 could facilitate the evolution of new reassortants, potentially with even greater ability to cause severe infections in humans or transmit human-to-human, highlighting the need for continued vigilance.

  • Antagonism of BST-2/tetherin is a conserved function of the Env glycoprotein of primary HIV-2 isolates [PublishAheadOfPrint]

  • Although HIV-2 does not encode a vpu gene, the ability to antagonize BST-2 is conserved in some HIV-2 isolates where it is controlled by the Env glycoprotein. We previously reported that a single amino acid difference between the lab-adapted ROD10 and ROD14 Envs controlled the enhancement of virus release (referred to here as Vpu-like) activity. Here we investigated how conserved the Vpu-like activity is in primary HIV-2 isolates. We found that almost half of the 35 tested primary HIV-2 Env isolates obtained from 7 different patients enhanced virus release. Interestingly, most HIV-2 patients harbored a mixed population of viruses containing or lacking Vpu-like activity. Vpu-like activity and Envelope functionality varied significantly among Env isolates; however, there was no direct correlation between these two functions suggesting they evolved independently. In comparing the Env sequences from one HIV-2 patient, we found that similar to the ROD10/ROD14 Envs, a single amino acid change (T568I) in the ectodomain of the TM subunit was sufficient to confer Vpu-like activity to an inactive Env variant. Surprisingly, however, absence of Vpu-like activity was not correlated with absence of BST-2 interaction. Taken together, our data suggest that maintaining the ability to antagonize BST-2 is of functional relevance not only to HIV-1 but to HIV-2 as well. Our data show that as with Vpu, binding of HIV-2 Env to BST-2 is important but not sufficient for antagonism. Finally, as observed previously, the Vpu-like activity in HIV-2 Env can be controlled by single residue changes in the TM subunit.

    IMPORTANCE Lentiviruses such as HIV-1 and HIV-2 encode accessory proteins whose function is to overcome host restriction mechanisms. Vpu is a well-studied HIV-1 accessory protein that enhances virus release by antagonizing the host restriction factor BST-2. HIV-2 does not encode a vpu gene. Instead, the HIV-2 Env glycoprotein was found to antagonize BST-2 in some isolates. Here, we cloned multiple Env sequences from 7 HIV-2-infected patients and found that about half were able to antagonize BST-2. Importantly, most HIV-2 patients harbored a mixed population of viruses containing or lacking the ability to antagonize BST-2. In fact, in comparing Env sequences from one patient combined with site-directed mutagenesis, we were able to restore BST-2 antagonism to an inactive Env protein by a single amino acid change. Our data suggest that targeting BST-2 by HIV-2 Env is a dynamic process that can be regulated by simple changes in the Env sequence.

  • Absence of evidence for a causal link between L-BSE and known forms of sporadic CJD in human PrP transgenic mice [PublishAheadOfPrint]

  • Prions are proteinaceous pathogens responsible for subacute spongiform encephalopathies in animals and humans. The prions responsible for bovine spongiform encephalopathy (BSE) are zoonotic agents, causing variant Creutzfeldt-Jakob disease (CJD) in humans. The transfer of prions between species is limited by a species barrier, which is thought to reflect structural incompatibilities between host cellular prion protein (PrPC) and the infecting pathological PrP assemblies (PrPSc) constituting the prion. A BSE strain variant, designated L-BSE and responsible for atypical, supposedly spontaneous forms of prion diseases in aged cattle, demonstrates zoonotic potential, as evidenced by its capacity to propagate more easily than classical BSE in transgenic mice expressing human PrPC and in non-human primates. In humanized mice, L-BSE propagates without any apparent species barrier and shares similar biochemical PrPSc signatures with the CJD subtype designated MM2-cortical, thus opening the possibility that certain CJD cases classified as sporadic may actually originate from L-type BSE cross-transmission. To address this issue, we compared the biological properties of L-BSE and those of a panel of CJD subtypes representative of the human prion strain diversity using standard strain-typing criteria in human PrP transgenic mice. We found no evidence that L-BSE causes a known form of sporadic CJD.

    IMPORTANCE Since the quasi-extinction of classical BSE, atypical BSE forms are the sole BSE variants circulating in cattle worldwide. They are observed in rare cases of old cattle, making them difficult to detect. Extrapolation of our results suggests that L-BSE may propagate in humans as an unrecognized form of CJD and we urge both the continued utilization of precautionary measures to eliminate these agents from the human food chain and active surveillance for CJD phenotypes in the general population.

  • Suppression of Host Innate Immune Response by Hepatitis C Virus via the Induction of Autophagic Degradation of TRAF6 [PublishAheadOfPrint]

  • Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an important adapter molecule that mediates the TNFR family and interleukin-1/Toll-like receptor (IL-1/TLR) signaling cascades. These pathways are important for the host to control viral infections. In this report, we demonstrated that hepatitis C virus (HCV) depleted TRAF6 from its host cells through a post-translational mechanism. This depletion was independent of proteasomes, as it was not affected by the proteasome inhibitor MG132, but it was suppressed by bafilomycin A1, which led to the association of TRAF6 with autophagosomes. As bafilomycin A1 is a vacuolar ATPase inhibitor that inhibits the autophagic protein degradation, these results suggested that HCV depleted TRAF6 via autophagy. The degradation of TRAF6 was apparently mediated by the p62 sequestosome protein, which is a factor important for selective autophagy, as it could bind to TRAF6 and its silencing stabilized TRAF6. The depletion of TRAF6 suppressed the activation of NF-B and the induction of pro-inflammatory cytokines and enhanced HCV replication. In contrast, the over-expression of TRAF6 suppressed HCV replication. These results revealed a novel mechanism that was used by HCV to disrupt the host innate immune responses for viral replication and persistence.

    IMPORTANCE HCV can cause severe liver diseases and is one of the most important human pathogens. It establishes chronic infection in the great majority of patient that it infects, indicating that it has evolved sophisticated mechanisms to evade host immunity. TRAF6 is an important signaling molecule that mediates the activation of NF-B and the expression of pro-inflammatory cytokines and interferons. In this study, we found that HCV infection suppressed the host innate immune response through the induction of autophagic degradation of TRAF6. This finding provided important information for further understanding how HCV evades host immunity to establish persistence.

  • The splicing history of an mRNA affects its level of translation and sensitivity to cleavage by the virion host shutoff (VHS) endonuclease during Herpes Simplex Virus infections [PublishAheadOfPrint]

  • During lytic herpes simplex virus (HSV) infections, the virion host shutoff (Vhs) (UL41) endoribonuclease degrades many cellular and viral mRNAs. In uninfected cells, spliced mRNAs emerge into the cytoplasm bound by exon junction complexes (EJCs), and are translated several times more efficiently than unspliced mRNAs that have the same sequence, but lack EJCs. Notably, most cellular mRNAs are spliced, whereas most HSV mRNAs are not. To examine the effect of splicing on gene expression during an HSV infection, cells were transfected with plasmids encoding an unspliced renilla luciferase (RLuc) reporter mRNA, or RLuc constructs with introns near the 5rrsquo; or 3rrsquo; ends of the gene. After splicing of intron-containing transcripts, all three RLuc mRNAs had the same primary sequence. Upon infection in the presence of actinomycin D, spliced mRNAs were much less sensitive to degradation by copies of Vhs from infecting virions than were unspliced mRNAs. During productive infections (in the absence of drugs) RLuc was expressed at substantially higher levels from spliced than unspliced mRNAs. Interestingly, the stimulatory effect of splicing on RLuc expression was significantly greater in infected than in uninfected cells. The translational stimulatory effect of an intron during HSV-1 infections could be replicated by artificially tethering various EJC components to an unspliced RLuc transcript. Thus, the splicing history of an mRNA, and the consequent presence or absence of EJCs, affects its level of translation and sensitivity to Vhs cleavage during lytic HSV infections.

    IMPORTANCE Most mammalian mRNAs are spliced. In contrast, of the more than 80 mRNAs encoded by herpes simplex virus type 1 (HSV-1), only five are spliced. In addition, synthesis of the immediate-early protein ICP27 causes partial inhibition of pre-mRNA splicing, with the resultant accumulation of both spliced and unspliced versions of some mRNAs in the cytoplasm. A common perception is that HSV-1 infection necessarily inhibits expression of spliced mRNAs. In contrast, this study demonstrates two instances in which pre-mRNA splicing actually enhances the synthesis of proteins from mRNAs during HSV-1 infections. Specifically, splicing stabilized an mRNA against degradation by copies of the Vhs endoribonuclease from infecting virions, and greatly enhanced the amount of protein synthesized from spliced mRNAs at late times after infection. The data suggest that splicing, and the resultant presence of exon junction complexes on an mRNA, may play an important role in gene expression during HSV-1 infections.

  • Acute simian varicella infection causes robust and sustained changes in gene expression in the sensory ganglia [PublishAheadOfPrint]

  • Primary infection with varicella zoster virus (VZV), a neurotropic alpha herpesvirus, results in varicella. VZV establishes latency in the sensory ganglia and can reactivate later in life to cause herpes zoster. The relationship between VZV and its host during acute infection in the sensory ganglia is not well understood due to limited access to clinical specimens. Intrabronchial inoculation of rhesus macaques with simian varicella virus (SVV), recapitulates the hallmarks of VZV infection in humans. We leveraged this animal model to characterize the host-pathogen interactions in the ganglia during both acute and latent infection by measuring both viral and host transcriptomes on days 3, 7, 10, 14 and 100-post infection (DPI). SVV DNA and transcripts were detected in sensory ganglia 3 DPI, before the appearance of rash. CD4 and CD8 T cells were also detected in the sensory ganglia 3 DPI. Moreover, lung-resident T-cells isolated from the same animals 3 DPI also harbored SVV DNA and transcripts, suggesting that T-cells may be responsible for trafficking SVV to the ganglia. RNA-Seq analysis showed that cessation of viral transcription 7 DPI coincides with a robust antiviral innate immune response in the ganglia. Interestingly, a significant number of genes that play a critical role in nervous system development and function remained down-regulated into latency. These studies provide novel insights into host-pathogen interactions in the sensory ganglia during acute varicella, and demonstrate that SVV infection results in profound and sustained changes in neuronal gene expression.

    IMPORTANCE Many aspects of VZV infection of sensory ganglia remain poorly understood, due to limited access to human specimens and the fact that VZV is strictly a human virus. Infection of rhesus macaques with simian varicella virus (SVV), a homolog of VZV, provides a robust model of the human disease. Using this model, we show that SVV reaches the ganglia early after infection, most likely by T cells and that the induction of a robust innate immune response correlates with cessation of virus transcription. We also report significant changes in the expression of genes that play an important role in neuronal function. Importantly, these changes persist long after viral replication ceases. Given the homology between SVV and VZV, and the genetic and physiological similarities between rhesus macaques and humans, our results provide novel insight into the interactions between VZV and its human host and explain some of the neurological consequences of VZV infection.

  • Antagonism of the SERINC5 Particle Infectivity Restriction by HIV-1 Nef Involves Counteraction of Virion-associated Pools of the Restriction Factor [PublishAheadOfPrint]

  • SERINC 3 and 5 are recently identified host cell inhibitors of HIV-1 particle infectivity that are counteracted by the viral pathogenesis factor Nef. Here we confirm that HIV-1 Nef, but not HIV-1 Vpu, antagonizes the particle infectivity restriction of SERINC5. SERINC5 antagonism occurred in parallel to other Nef activities including cell surface receptor downregulation, trans-Golgi-network targeting of Lck and inhibition of host cell actin dynamics. Interaction motifs with host cell endocytic machinery, Nef-associated kinase complex as well as CD4 cytoplasmic tail/HIV-1 protease were identified as essential Nef determinants for SERINC5 antagonism. Characterizing antagonism-deficient Nef mutants revealed that counteraction of SERINC5 occurs in the absence of retargeting of the restriction factor to intracellular compartments and reduction of SERINC5 cell surface density is insufficient for antagonism. Consistent with virion incorporation of SERINC5 being a prerequisite for its antiviral activity, the infectivity of HIV-1 particles produced in the absence of a SERINC5 antagonist decreased with increasing amounts of virion SERINC5. At low levels of SERINC5 expression, enhancement of virion infectivity by Nef was associated with reduced virion incorporation of SERINC5 and antagonism-defective Nef mutants failed to exclude SERINC5 from virions. However, at elevated levels of SERINC5 expression, Nef maintained HIV particles infectious despite significant virion incorporation of the restriction factor. These results suggest that in addition to virion exclusion, Nef employs a cryptic mechanism to antagonize virion-associated SERINC5. The involvement of common determinants suggests that SERINC5 antagonism and downregulation of cell surface CD4 by Nef involve related molecular mechanisms.

    Importance HIV-1 Nef critically determines virus spread and disease progression in infected individuals by incompletely defined mechanisms. SERINC3 and SERINC5 were recently identified as potent inhibitors of HIV particle infectivity whose antiviral activity is antagonized by HIV-1 Nef. To address the mechanism of SERINC5 antagonism we identified four molecular determinants of Nef antagonism that are all linked to the mechanism by which Nef downregulates cell surface CD4. Functional characterization of these mutants revealed that endosomal targeting and cell surface downregulation of SERINC5 is dispensable and insufficient for antagonism, respectively. In contrast, virion exclusion and antagonism of SERINC5 were correlated, however Nef was also able to enhance the infectivity of virions that incorporated robust levels of SERINC5. These results suggest that antagonism of the SERINC5 restriction to virion infectivity by HIV-1 Nef is mediated by a dual mechanism that is related to CD4 downregulation.

  • Attenuated Infectious Hematopoietic Necrosis Virus (IHNV) with rearranged gene order as potential vaccine [PublishAheadOfPrint]

  • The genome of the Infectious Hematopoietic Necrosis Virus (IHNV), a salmonid Novirhabdovirus has been engineered to modify the gene order and to evaluate the impact on a possible attenuation of the virus in vitro and in vivo. By reverse genetics, eight recombinant rIHNV, termed NxGy according to the respective position of the nucleoprotein (N) and glycoprotein (G) genes along the genome have been recovered. All rIHNV have been fully characterized in vitro for their cytopathic effect, kinetics of replication and profile of viral gene transcription. These rIHNV are stable through up to ten passages in cell culture. Following bath immersion administration of the various rIHNV to juvenile trout, some of the rIHNV were clearly attenuated (N2G3, N2G4, N3G4, and N4G1). Position of the N gene seems to be one of the most critical features correlated to the level of viral attenuation. The induced-immune response potential in fish was evaluated by ELISPOT and seroneutralization assays. The recombinant virus N2G3 induced a strong antibody response in immunized fish and conferred 86% of protection against wild-type IHNV challenge in trout, thus representing a promising starting point for the development of live-attenuated vaccine candidate.

    IMPORTANCE In Europe, no vaccines are available against Infectious Hematopoietic Necrosis Virus (IHNV), one of the major economic threats in fish aquaculture. Live-attenuated vaccines are conditioned by a sensible balance between attenuation and pathogenicity. Moreover, non-segmented negative strain RNA viruses (NNSV) are subject to a transcription gradient dictated by the order of their genes in their genome. In the perspective of developing a vaccine against IHNV, we engineered various recombinant IHNV with reordered genome in order to artificially attenuate the virus. Our results validate the gene rearrangement approach as a potent and stable attenuation strategy for fish Novirhabdovirus and open new perspective for other NNSV vaccine design.

  • Rhinovirus 16 2A protease affects nuclear localization of 3CD during infection [PublishAheadOfPrint]

  • The human rhinovirus (HRV) 3C and 2A proteases (3Cpro and 2Apro, respectively) are critical in HRV infection as they are required for viral polyprotein processing as well as proteolysing key host factors to facilitate virus replication. Early in infection, 3Cpro is present as its precursor 3CD, which, although the mechanism of subcellular targeting is unknown, is found in the nucleus as well as in the cytoplasm. In this study, we use transfected and infected cell systems to show that 2Apro activity is required for 3CD nuclear localization. Using GFP-tagged forms of 3Cpro, 3D and mutant derivatives thereof, we show that 3Cpro is located in the cytoplasm and the nucleus, whereas 3CD and 3D are localized predominantly in the cytoplasm, implying that 3D lacks nuclear targeting ability, and that 3Cpro activity within 3CD is not sufficient to allow the larger protein into the nucleus. Importantly, by co-expressing mCherry-2Apro fusion proteins, we demonstrate formally that 2Apro activity is required to allow HRV 3CD access to the nucleus. In contrast, mCherry-3Cpro is insufficient to allow 3CD access to the nucleus. Finally, we confirm the relevance of these results to HRV infection by demonstrating that nuclear localization of 3CD correlates with 2Apro activity and not 3Cpro activity, which is only observed later in infection. The results thus define the temporal activities of 2Apro and 3CD/3Cpro activities in HRV16 infection.

    IMPORTANCE The human rhinovirus genome encodes two proteases, 2A and 3C, as well as a precursor protease, 3CD. These proteases are essential for efficient virus replication. The 3CD protein is found in the nucleus early during infection, though the mechanism of subcellular localization is unknown. Here we show that 2A protease is required for this localization, the 3C protease activity of 3CD is not sufficient to allow 3CD entry into the nucleus, and 3D lacks nuclear targeting ability. This study demonstrates that both 2A and 3C proteases are required for the correct localization of proteins during infection, as well as defining the temporal regulation of 2A and 3CD/3C protease activities during HRV16 infection.

  • Resistance to mutant group-2 influenza neuraminidases of an oseltamivir-zanamivir hybrid inhibitor [PublishAheadOfPrint]

  • Influenza neuraminidase (NA) drug-resistance is one of the challenges to preparedness against epidemic and pandemic influenza infections. NA N1- and N2-containing influenza viruses are the primary cause of seasonal epidemics and past pandemics. The structural and functional basis underlying drug-resistance of the influenza N1 NA is well characterized. Yet, drug-resistance of the N2 strain is not well understood. Here, we confirm that substitution of N2 E119 or I222 results in multi-drug resistance, and when occurring together the sensitivity to NA inhibitors (NAI) is reduced severely. Using crystallographic studies, we show that E119 substitution results in a loss of hydrogen bonding to oseltamivir and zanamivir whereas I222 substitution results in a change in the hydrophobic environment that is critical for oseltamivir binding. Moreover, we find that MS-257, a zanamivir/oseltamivir hybrid inhibitor, is less susceptible to drug-resistance. The binding mode of MS-257 shows that increased hydrogen bonding interactions between the inhibitor and NA active site anchor the inhibitor within the active site and allow adjustments in response to active-site modifications. Such stability is likely responsible for the observed reduced susceptibility to drug-resistant .MS-257 serves as a next-generation anti-influenza drug candidate and serves also as a scaffold for further design of NAIs.

    Importance Oseltamivir and zanamivir are the two major antiviral drugs available for the treatment of influenza virus infections. However, multi-drug resistant viruses have emerged in clinical cases, which pose a challenge for the development of new drugs. N1 and N2 subtypes exist in the viruses which cause seasonal epidemics and past pandemics. Although N1 drug resistance is well characterized, the molecular mechanisms underlying N2 drug resistance are unknown. A previous report showed that an N2 E119V/I222L dual mutant conferred drug resistance to seasonal influenza virus. Here, we confirm that these substitutions result in multi-drug resistance and dramatically reduced sensitivity to NAI. We further elucidate the molecular mechanism underlying N2 drug resistance by solving crystal structures of N2 E119V, I222L, and the dual mutant. Most importantly, we found that a novel oseltamivir/zanamivir hybrid inhibitor, MS-257, remains more effective against drug-resistant N2 and is a promising candidate as a next generation anti-influenza drug.

  • Hepaciviral NS3/4A proteases interfere with MAVS signalling of their cognate animal hosts and also with human MAVS: implications for zoonotic transmission [PublishAheadOfPrint]

  • Multiple novel members of the genus Hepacivirus have recently been discovered in diverse mammalian species. However, to date, their replication mechanisms and zoonotic potential have not been explored in detail. The NS3/4A serine protease of HCV is critical for cleavage of the viral polyprotein. It also cleaves the cellular innate immune adaptor MAVS, thus decreasing IFN production and contributing to HCV persistence in the human host.

    To investigate conservation of fundamental aspects of the hepaciviral life-cycle, we explored if MAVS cleavage and suppression of innate immune signaling represents a common mechanism employed across different clades of the genus Hepacivirus to enhance viral replication. To estimate the zoonotic potential of these non-human hepaciviruses, we assessed if their NS3/4A proteases were capable of cleaving human MAVS.

    NS3/4A proteases of viruses infecting Colobus monkeys, rodents, horses, and cows cleaved the MAVS protein of their cognate hosts and interfered with its ability to induce the IFN-bbeta; promoter. All NS3/4A proteases from non-human viruses readily cleaved human MAVS. Thus, NS3/4A-dependent cleavage of MAVS is a conserved replication strategy across multiple clades within the genus Hepacivirus. Human MAVS is susceptible to cleavage by these non-human viral proteases indicating that it does not pose a barrier for zoonotic transmission of these viruses to humans.

    IMPORTANCE Virus infection is recognized by cellular sensor proteins triggering innate immune signaling and antiviral defenses. While viruses have evolved strategies to thwart these antiviral programs in their cognate host species, these evasion mechanisms are often ineffective in a novel host, thus limiting viral transmission across species. HCV, the best characterized member of the genus Hepacivirus within the family Flaviviridae, uses its NS3/4A protease to disrupt innate immune signaling by cleaving the cellular adaptor protein MAVS. Recently, a large number of HCV-related viruses were discovered in various animal species including wild, live-stock and companion animals. We show that the NS3/4A proteases of these hepaciviruses from different animals and representing various clades of the genus cleave their cognate host MAVS proteins, in addition to human MAVS. Therefore, cleavage of MAVS is a common strategy of hepaciviruses and human MAVS is likely unable to limit replication of these non-human viruses upon zoonotic exposure.

  • HIV-1 escape from a peptidic anchor inhibitor by envelope glycoprotein spike stabilization [PublishAheadOfPrint]

  • The trimeric HIV-1 envelope glycoprotein spike (Env) mediates viral entry into cells using a spring-loaded mechanism that allows for the controlled insertion of the Env fusion peptide into the target membrane, followed by membrane fusion. Env is the focus of vaccine research aimed at inducing protective immunity by antibodies as well as efforts to develop drugs that inhibit the viral entry process. The molecular factors contributing to Env stability and decay need to be better understood in order to optimally design vaccines and therapeutics. We generated escape viruses against VIR165, a peptidic inhibitor that binds the fusion peptide of the gp41 subunit and prevents its insertion into the target membrane. Interestingly, a number of escape viruses acquired substitutions in the C1 domain of the gp120 subunit (A60E, E64K and H66R) that rendered these viruses dependent on the inhibitor. These viruses could only infect target cells when VIR165 was present after CD4 binding. Furthermore, the VIR165-dependent viruses were resistant to soluble CD4-induced Env destabilization and decay. These data suggest that VIR165-dependent Env proteins are kinetically trapped in the unliganded state and require the drug to negotiate CD4-induced conformational changes. These studies provide mechanistic insight into the action of the gp41 fusion peptide and its inhibitors, and provide new ways to stabilize Env trimer vaccines.

    IMPORTANCE Because of the rapid development of HIV-1 drug resistance new drug targets need to be continuously explored. The fusion peptide of the envelope glycoprotein can be targeted by anchor inhibitors. Here we describe virus escape from the anchor inhibitor VIR165. Interestingly, some escape viruses became dependent on the inhibitor for cell entry. We show that the identified escape mutations stabilize the ground state of the envelope glycoprotein and should thus be useful in the design of stabilized envelope-based HIV vaccines.

  • PrPC governs prion strain susceptibility in bank vole while others host factors modulate strain features [PublishAheadOfPrint]

  • Bank vole is a rodent species that shows a differential susceptibility to the experimental transmission of different prion strains. In this work, the transmission features of a panel of diverse prions with distinct origin have been assayed in both bank vole expressing methionine at codon 109 (Bv109M) or in transgenic mice expressing physiological levels of bank vole PrPC (BvPrP-Tg407 mouse line). This work compiles the first systematic comparison of the transmission features of a collection of prion isolates, representing a panel of diverse prion strains, in a transgenic mouse model and in its natural counterpart. The results showed very similar transmission properties in both natural species and transgenic mouse model demonstrating the key role of PrP amino acid sequence in prion transmission susceptibility. However, differences in the PrpSc type propagated by Bv109M and BvPrP-Tg407 suggest that host factors other than PrPC would modulate prion strain features.

    IMPORTANCE The differential susceptibility of bank vole to prion strains can be modeled in transgenic mice, suggesting that this selective susceptibility is only controlled by the vole PrP sequence rather than by other species specific factors. Differences in the phenotypes observed after prion transmissions in bank voles and in the transgenic mice suggest that other host factors than PrPC sequence may affect the selection of the substrain replicating in the animal model.

  • Glycosyl Phosphatidylinositol-Anchored Variable Region of Llama Heavy Chain Only Antibody JM4 Efficiently Blocks Both Cell-Free and T Cell-T Cell Transmission of Human Immunodeficiency Virus Type 1 [PublishAheadOfPrint]

  • JM2 and JM4 are two recently isolated variable regions (VHH) of heavy chain only antibodies from llamas that have been immunized with a trimeric gp140 bound to a CD4 mimic. JM2 binds the CD4-binding site of gp120 and neutralizes HIV-1 strains from subtypes B, C and G. JM4 binds gp120 and neutralizes HIV-1 strains from subtypes A, B, C, A/E and G in a CD4-dependent manner. In the present study, we constructed glycosyl-phosphatidylinositol (GPI)-anchored VHH JM2 and JM4 along with a E4 control and transduced them into human CD4+ cell lines and primary CD4 T cells. We report that by genetically linking the VHHs with a GPI attachment signal, VHHs are targeted to the lipid rafts of the plasma membranes. Expression of GPI-VHH JM4, but not GPI-VHH E4 and JM2, on the surface of transduced cells potently neutralizes multiple subtypes of HIV-1 isolates including tier 2 or 3 strains, transmitted/founders, quasispecies and soluble single domain antibody (sdAb) JM4-resistant viruses. Moreover, transduction of CEMss-CCR5 cells with GPI-VHH JM4, but not with GPI-VHH E4, confers resistance to both cell-free and T cell-T cell transmission of HIV-1 and HIV-1 envelope-mediated fusion. Finally, GPI-VHH JM4-transduced human primary CD4 T cells efficiently resist both cell-free and T cell-T cell transmission of HIV-1. Thus, we conclude that the VHH JM4, when targeted to the lipid rafts of the plasma membrane, efficiently neutralizes HIV-1 infection via both cell-free and T cell-T cell transmission. Our findings should have important implications for GPI-anchored antibody-based therapy against HIV-1.

    Importance Lipid rafts are specialized dynamic microdomains of the plasma membrane and have been shown to be gateways for HIV-1 budding as well as entry into T cells and macrophages. In nature, many glycosyl-phosphatidylinositol (GPI)-anchored proteins localize in the lipid rafts. In the present study, we developed GPI-anchored variable region (VHH) of two heavy chain only antibodies JM2 and JM4 from immunized llamas. We show that by genetically linking the VHHs with a GPI attachment signal, VHHs are targeted to the lipid rafts of the plasma membranes. GPI-VHH JM4, but not GPI-VHH JM2, in transduced CD4+ cell lines and human primary CD4 T cells not only efficiently blocks diverse HIV-1 strains including tier 2 or 3 strains, transmitted founders, quasispecies and soluble sdAb JM4-resistant strains, but also efficiently interferes T cell-T cell transmissions of HIV-1 and HIV-1 envelope-mediated fusion. Our findings should have important implications in GPI-anchored antibody-based therapy against HIV-1.

  • Extragenic Suppression of a Mutation in Herpes Simplex Virus Type 1 (HSV-1) UL34 That Affects Lamina Disruption and Nuclear Egress [PublishAheadOfPrint]

  • Nuclear egress of herpesviruses is accompanied by changes in the architecture of the nuclear membrane and nuclear lamina that are thought to facilitate capsid access to the INM and curvature of patches of the INM around the capsid during budding. Here we report the properties of a point mutant of pUL34 (Q163A) that fails to induce gross changes in nuclear architecture or redistribution of lamin A/C. UL34(Q163A) shows a roughly 100-fold defect in single-step growth, and forms small plaques. This mutant has a defect in nuclear egress and furthermore fails to disrupt nuclear shape or cause observable displacement of lamin A/C despite retaining the ability to recruit pUS3 and PKC protein kinases, and to mediate phosphorylation of emerin. Extragenic suppressors of the UL34(Q163A) phenotype were isolated, all of which carry a single mutation of arginine 229 to leucine in UL31. Surprisingly, although this UL31 mutation largely restores virus replication, it does not correct the lamina disruption defect suggesting that, in Vero cells, changes in nuclear shape and gross displacements of lamin A/C may facilitate, but are unnecessary for nuclear egress.

    Importance Herpesvirus nuclear egress is an essential and conserved process that requires close association of the virus capsid with the inner nuclear membrane and budding of the capsid into that membrane. Access to the nuclear membrane and tight curvature of that membrane is thought to require disruption of the nuclear lamina that underlies the inner nuclear membrane and, consistent with this idea, herpesvirus infection induces biochemical and architectural changes at the nuclear membrane. The significance of the nuclear membrane architectural changes is poorly characterized. The results presented here address that deficiency in our understanding and show that a combination of mutations in two of the viral nuclear egress factors results in failure to accomplish at least two components of lamina disruption while still allowing relatively efficient viral replication, suggesting that changes in nuclear shape and displacement of lamins are not necessary for HSV-1 nuclear egress.

  • Multiple Strategies Reveal a Bi-dentate Interaction Between the Nipah Virus Attachment and Fusion Glycoproteins [PublishAheadOfPrint]

  • The paramyxoviral family contains many medically important viruses including measles virus, mumps virus, parainfluenza viruses, respiratory syncytial virus, human metapneumovirus, and the deadly zoonotic henipaviruses Hendra and Nipah virus (NiV). To both enter host cells and spread from cell to cell within infected hosts, the vast majority of paramyxoviruses utilize two viral envelope glycoproteins: the attachment glycoprotein (G, H, or HN) and the fusion glycoprotein (F). Binding of G/H/HN to a host cell receptor triggers structural changes in G/H/HN that in turn trigger F to undergo a series of conformational changes that result in viral-cell (viral entry) or cell-cell (syncytia) membrane fusion. The actual regions of G/H/HN and F that interact during the membrane fusion process remain relatively unknown, though it is generally thought that the paramyxoviral G/H/HN stalk region interacts with the F head region. Studies to determine such interactive regions have relied heavily on co-immunoprecipitation approaches, whose limitations include the use of detergents and the micelle-mediated association of proteins. Here we developed a flow cytometric strategy capable of detecting membrane protein-protein interactions by interchangeably using the full-length form of G and a soluble form of F, or vice versa. Using both co-immunoprecipitation and flow cytometric strategies, we found a bi-dentate interaction between NiV G and F, where both the stalk and head regions of NiV G interact with F. This is a new structural-biological finding for the paramyxoviruses. Additionally, our studies disclosed regions of the NiV G and F glycoproteins dispensable for the G and F interactions.

    IMPORTANCE Nipah virus (NiV) is a zoonotic paramyxovirus that causes high mortality rates in humans, with no approved treatment or vaccine available for human use. Viral entry into host cells relies on two viral envelope glycoproteins: the attachment (G) and fusion (F) glycoproteins. Binding of G to the ephrinB2 or ephrinB3 cell receptors triggers conformational changes in G that in turn cause F to undergo conformational changes that result in viral/cell membrane fusion and viral entry. It is currently unknown, however, which specific regions of G and F interact during membrane fusion. Past efforts to determine the interacting regions have relied mainly on co-immunoprecipitation, a technique with some pitfalls. We developed a flow cytometric assay to study membrane protein-protein interactions, and using this assay we report a bi-dentate interaction whereby both the head and stalk regions of NiV G interact with NiV F, a new finding for the paramyxovirus family.

  • Tetherin Antagonism by HIV-1 Group M Nef Proteins [PublishAheadOfPrint]

  • Although Nef is the viral gene product used by most simian immunodeficiency viruses to overcome restriction by tetherin, this activity was acquired by the Vpu protein of HIV-1 group M viruses due to the absence of sequences in human tetherin that confer susceptibility to Nef. Thus, it is widely accepted that HIV-1 group M uses Vpu instead of Nef to counteract tetherin. Challenging this paradigm, we identified Nef alleles of HIV-1 group M isolates with significant activity against human tetherin. These Nef proteins promoted virus release and tetherin downmodulation from the cell surface, and in the context of vpu-deleted HIV-1 recombinants, enhanced virus replication and resistance to antibody-dependent cell-mediated cytotoxicity (ADCC). Further analysis revealed that the Vpu proteins from several of these viruses lack anti-tetherin activity, suggesting that under certain circumstances, HIV-1 group M Nef may acquire the ability to counteract tetherin to compensate for the loss of this function by Vpu. These observations illustrate the remarkable plasticity of HIV-1 in overcoming restriction by tetherin and challenge the prevailing view that all HIV-1 group M isolates use Vpu to counteract tetherin.

    IMPORTANCE Most HIV-1 group M viruses, the main group of HIV-1 responsible for the global AIDS pandemic, use their Vpu proteins to overcome restriction by tetherin (BST-2 or CD317), which is a transmembrane protein that inhibits virus release from infected cells. Here we show that the Nef proteins of certain HIV-1 group M isolates can acquire the ability to counteract tetherin. These results challenge the current paradigm that HIV-1 group M exclusively uses Vpu to counteract tetherin and underscore the importance of tetherin antagonism for efficient viral replication.

  • The P Protein of Spring Viremia of Carp Virus Negatively Regulates Fish IFN Response by Inhibiting the Kinase Activity of TANK-binding kinase 1 [PublishAheadOfPrint]

  • Spring viremia of carp virus (SVCV) is an efficient pathogen causing high mortality in the common carp. Fish interferon (IFN) is a powerful cytokine enabling host cells to establish an antiviral response; therefore, the strategies that SVCV uses to avoid the cellular IFN response were investigated. Here, we report that the SVCV P protein is phosphorylated by cellular TANK-binding kinase 1 (TBK1), which decreases IFN regulatory factor 3 (IRF3) phosphorylation and suppresses IFN production. First, overexpression of P protein inhibited the IFN promoter activation induced by SVCV and the IFN activity activated by mitochondrial antiviral signaling protein (MAVS), although TBK1 activity was not blocked by P protein. Second, P protein co-localized and interacted with TBK1. Dominant-negative experiments suggested that the TBK1 N terminal kinase domain interacted with P protein and was essential for P protein and IRF3 phosphorylation. Finally, P protein overexpression reduced the IRF3 phosphorylation activated by TBK1 and reduced host cellular ifn transcription. Collectively, our data demonstrated that the SVCV P protein is a decoy substrate for the host phosphokinase TBK1, preventing IFN production and facilitating SVCV replication.

    IMPORTANCE TBK1 is a pivotal phosphokinase that activates host IFN production to defend against viral infection, thus, it is a potential target for viruses to negatively regulate IFN response and facilitate viral evasion. We report that the SVCV P protein functions as a decoy substrate for cellular TBK1, leading to the reduction of IRF3 phosphorylation and suppression of IFN expression. These findings reveal a novel immune evasion mechanism of SVCV.

  • A Novel Benzodiazepine Compound Inhibits Yellow Fever Virus Infection by Specifically Targeting NS4B Protein [PublishAheadOfPrint]

  • Although a highly effective vaccine is available, the number of yellow fever cases has increased over the past two decades, which highlights the pressing need for antiviral therapeutics. In a high throughput screening campaign, we identified an acetic acid benzodiazepine (BDAA) compound, which potently inhibits yellow fever virus (YFV). Interestingly, while treatment of YFV infected cultures with 2 mmu;M of BDAA reduced the virion production by greater than 2 logs, the compound is not active against 21 other viruses from 14 different viral families. Selection and genetic analysis of drug resistant viruses revealed that substitution of proline at amino acid 219 (P219) of the nonstructural protein 4B (NS4B) with serine, threonine or alanine confers YFV resistance to BDAA without apparent loss of replication fitness in cultured mammalian cells. However, substitution of P219 with glycine confers BDAA resistance with significant loss of replication ability. Bioinformatics analysis predicts that the P219 localizes at the endoplasmic reticulum lumen side of the fifth putative trans-membrane domain of NS4B and the mutation may render the viral protein incapable of interacting with BDAA. Our studies thus revealed important role and structural basis for NS4B protein in supporting YFV replication. Moreover, in YFV-infected hamsters, oral administration of BDAA protected 90% of the animals from death, significantly reduced viral load by greater than 2 logs and attenuated viral infection-induced liver injury and body weight loss. The encouraging preclinical results thus warrant further development of BDAA or its derivatives as antiviral agents to treat yellow fever.

    Importance Yellow fever is an acute viral hemorrhagic disease which threatens approximately one billion people living in tropical areas of Africa and Latin America. Although a highly effective yellow fever vaccine has been available for more than seven decades, the low vaccination rate fails to prevent outbreaks in at-risk regions. It has been estimated that up to 1.7 million YFV infections occur in Africa each year, resulting in 29,000 to 60,000 death. Thus far, there is no specific antiviral treatment for yellow fever. To cope with this medical challenge, we identified a benzodiazepine compound that selectively inhibits YFV by targeting the viral NS4B protein. To our knowledge, this is the first report demonstrating in vivo safety and antiviral efficacy of an YFV NS4B inhibitor in an animal model. We have thus reached a critical milestone toward the development of specific antiviral therapeutics for clinical management of yellow fever.

  • The cytoskeletal adaptor obscurin-like 1 interacts with the HPV16 capsid protein L2 and is required for HPV16 endocytosis [PublishAheadOfPrint]

  • The human papillomavirus (HPV) capsid protein L2 is essential for viral entry. To gain a deeper understanding of the role of L2, we searched for novel cellular L2-interacting proteins. A yeast two-hybrid analysis uncovered the actin depolymerizing factor gelsolin, the membrane glycoprotein dysadherin, the centrosomal protein 68 (Cep68), and the cytoskeletal adaptor protein obscurin-like 1 protein (OBSL1) as putative L2 binding molecules. Pseudovirus (PsV) infection assays identified OBSL1 as a host factor required for gene transduction by three oncogenic human papillomavirus types, HPV16, HPV18, and HPV31. In addition, we detected OBSL1 expression in cervical tissue sections and noted the involvement of OBSL1 during gene transduction of primary keratinocytes by HPV16 PsV. Complex formation of HPV16 L2 with OBSL1 was demonstrated in co-immunofluorescence and co-immunoprecipitation studies after overexpression of L2 or after PsV exposure. We observed a strong co-localization of OBSL1 with HPV16 PsV and CD151 at the plasma membrane suggesting a role of OBSL1 in viral endocytosis. Indeed, viral entry assays exhibited a reduction of viral endocytosis in OBSL1 depleted cells. Our results suggest OBSL1 as a novel L2-interacting protein and endocytosis factor in HPV infection.

    Importance Human papillomaviruses infect mucosal and cutaneous epithelium and the high risk HPV types account for 5% of cancer cases worldwide. As recently discovered, HPV entry occurs by a clathrin-, caveolin- and dynamin-independent endocytosis via tetraspanin-enriched microdomains. At present, the cellular proteins involved in the underlying mechanism of this type of endocytosis are under investigation. In this study, the cytoskeletal adaptor OBSL1 was discovered as a previously unrecognized interaction partner of the minor capsid protein L2 and was identified as a proviral host factor required for HPV16 endocytosis into target cells. The findings of this study advance the understanding of a so far less well characterized endocytic pathway that is used by oncogenic HPV subtypes.

  • Influenza virus susceptibility of wild-derived CAST/EiJ mice results from two amino acid changes in the MX1 restriction factor [PublishAheadOfPrint]

  • The interferon-regulated Mx1 gene of the A2G mouse strain confers a high degree of resistance against influenza A and Thogoto viruses. Most other laboratory inbred mouse strains carry truncated non-functional Mx1 alleles and, consequently, exhibit high virus susceptibility. Interestingly, CAST/EiJ mice derived from wild Mus musculus castaneus possess a seemingly intact Mx1 gene, but are highly susceptible to influenza A virus challenge. To determine whether enhanced influenza virus susceptibility is due to intrinsically reduced antiviral activity of the CAST-derived Mx1 allele, we generated a congenic C57BL/6J mouse line that carries the Mx locus of CAST/EiJ. Adults animals of this line were almost as susceptible to influenza virus challenge as standard C57BL/6J mice lacking functional Mx1 alleles but exhibited far more pronounced resistance to Thogoto virus. Sequencing revealed that CAST-derived MX1 differs from A2G-derived MX1 by two amino acids (G83R and A222V) in the GTPase domain. Especially the A222V mutation reduced GTPase activity of purified MX1 and diminished the inhibitory effect of MX1 in influenza A virus polymerase activity assays. Further, MX1 protein was substantially less abundant in organs of interferon-treated mice carrying the CAST Mx1 allele in comparison to mice carrying the A2G Mx1 allele. We found that the CAST-specific mutations reduced the metabolic stability of the MX1 protein although Mx1 mRNA levels were unchanged. Thus, enhanced influenza virus susceptibility of CAST/EiJ mice can be explained by minor alterations in the MX1 restriction factor that negatively affect its enzymatic activity and reduce its half-life.

    IMPORTANCE Although the crystal structures of the prototypic human MXA protein is known, the importance of specific protein domains for antiviral activity is still incompletely understood. Novel insights might come from studying naturally occuring MX protein variants with altered antiviral activity. Here we identified two seemingly minor amino acid changes in the GTPase domain that negatively affect enzymatic activity and metabolic stability of murine MX1 and thus dramatically reduce influenza virus resistance of the respective mouse inbred strain. These observations highlight our current inability to predict the biological consequences of previously uncharacterized MX mutations in mice. Since this is probably also true for naturally occurring mutations in Mx genes of humans, careful experimental analysis of any natural MXA variants for altered activity is necessary in order to assess possible consequences of such mutations on innate antiviral immunity.

  • The Respiratory Syncytial Virus Phosphoprotein, Matrix Protein, and Fusion Protein Carboxy-Terminal Domain Drive Efficient Filamentous Virus-Like Particle Formation. [PublishAheadOfPrint]

  • Virus-like particles (VLP) are attractive as a vaccine concept. For human respiratory syncytial virus (hRSV), VLP assembly is poorly understood and appears inefficient. Hence hRSV antigens are often incorporated into foreign VLP systems to generate anti-RSV vaccine candidates. To better understand assembly and utimately enable efficient production of authentic hRSV VLPs, we examined the associated requirements and mechanisms. In a previous analysis in HEp-2 cells, the nucleoprotein (N), phosphoprotein (P), matrix protein (M) and fusion protein (F) were required for formation of filamentous VLPs, which, similar to wildtype virus, were associated with the cell surface. Using fluorescence and electron microscopy combined with immuno-gold labeling we examined the surface of transfected HEp-2 cells and further dissected the process of filamentous VLP formation. Our results show that N was not required. Co-expression of P+M+F, but not P+M, M+F, or P+F, induced both viral protein coalescence and formation of filamentous VLPs which resembled wildtype virions. Despite sub-optimal coalescence in absence of P, the M and F proteins when co-expressed formed cell surface -associated filaments of abnormal morphology, appearing longer and thinner than wildtype virions. For F, only the carboxy-terminus (Fstem) was required, and addition of foreign protein sequences to Fstem allowed incorporation into VLPs. Together, the data show that P, M, and the F carboxy-terminus are sufficient for robust viral protein coalescence and filamentous VLP formation, and suggest that M-F interaction drives viral filament formation with P acting as a type of co-factor facilitating the process and exerting control over particle morphology.

    IMPORTANCE hRSV is responsible for ggt;100,000 deaths in children worldwide, and a vaccine is not available. Among the potential anti-hRSV approaches are virus-like particle (VLP) vaccines, which, based on resemblance to virus or viral components, can induce protective immunity. For hRSV, few reports are available concerning authentic VLP production or testing, in large part because VLP production is inefficient and the mechanisms underlying particle assembly are poorly understood. Here, we took advantage of the cell-associated nature of RSV particles, and used high-resolution microscopy analyses to examine the viral proteins required for formation of wildtype virus-resembling VLPs, the contribution of these proteins to morphology, and the domains involved in incorporation of the antigenically important viral F protein. The results provide new insights that will facilitate future production of hRSV VLPs of defined shape and composition and may translate into improved manufacture of live-attenuated hRSV vaccines.

  • Comprehensive Genome-Scale Phylogenetic Study Provides New Insights on the Global Expansion of Chikungunya Virus [PublishAheadOfPrint]

  • Since the India and Indian Ocean outbreaks of 2005-2006, the global distribution of chikungunya virus (CHIKV) and the locations of epidemics have dramatically shifted. First, the Indian Ocean lineage (IOL) caused sustained epidemics in India and has radiated to many other countries. Second, the Asian lineage has caused frequent outbreaks in the Pacific islands and in 2013 was introduced to the Caribbean, followed by rapid spread to nearly all of the neotropics. Further, CHIKV epidemics as well as exported cases have been reported in central Africa after a long period of perceived silence. To understand these changes and anticipate the future of this virus, the exact distribution, genetic diversity, transmission routes, and future epidemic potential of CHIKV require further assessment. To do so, we conducted the most comprehensive phylogenetic analysis to date, examined CHIKV evolution and transmission, and explored distinct genetic factors associated with the emergence of the East/Central/South African (ECSA), IOL, and Asian lineages. Our results reveal contrasting evolutionary patterns among lineages with growing genetic diversities observed in each, and suggest that CHIKV will continue to be a major public health threat with the potential for further emergence and spread.

    IMPORTANCE Chikungunya fever is a re-emerging infectious disease that is transmitted by Aedes mosquitoes and causes severe health and economic burden in affected populations. Since the unprecedented Indian Ocean and Indian Subcontinent outbreaks of 2005-2006, CHIKV has further expanded its geographic range including to the Americas in 2013. Its evolution and transmission during and following these epidemics, as well as the recent evolution and spread of other lineages require optimal assessment. Using newly obtained genome sequences, we provide a comprehensive update of the global distribution of CHIKV genetic diversity and analyze factors associated with recent outbreaks. These results provide a solid foundation for future evolutionary studies of CHIKV that not can only elucidate emergence mechanisms, and also may help to predict future epidemics.

  • Assessment of the PrPc amino-terminal domain in prion species barriers [PublishAheadOfPrint]

  • Chronic wasting disease (CWD) in cervids and bovine spongiform encephalopathy (BSE) in cattle are prion diseases that are caused by the same protein-misfolding mechanism, but appear to pose different risks to humans. We are interested in understanding the differences between the species barriers of CWD and BSE. We used real-time, quaking-induced conversion (RT-QuIC) to model the central molecular event in prion disease, the templated misfolding of the normal prion protein, PrPc, to a pathogenic, amyloid isoform, PrPSc. We examined the role of the PrPc amino-terminal domain (NTD, aa23-90) in cross-species conversion by comparing the conversion efficiency of various prion seeds in either full-length (aa23-231) or truncated (aa90-231) PrPc. We demonstrate that the presence of white-tailed deer and bovine NTDs hindered seeded conversion of PrPc, but human and bank vole NTDs did the opposite. Additionally, full-length human and bank vole PrPc were more likely to be converted to amyloid by CWD prions than were their truncated forms. A chimera with replacement of the human NTD by the bovine NTD resembled human PrPc. The requirement for an NTD, but not for the specific human sequence, suggests that the NTD interacts with other regions of the human PrPc to increase promiscuity. These data contribute to the evidence that, in addition to primary sequence, prion species barriers are controlled by interactions of the substrate NTD with the rest of the substrate PrPc molecule.

    Importance We demonstrate that the amino-terminal domain of the normal prion protein, PrPc, hinders seeded conversion of bovine and white-tailed deer PrPc to the prion form, but it facilitates conversion of the human and bank vole PrPc to the prion form. Additionally, we demonstrate that the amino-terminal domain of human and bank vole PrPc requires interaction with the rest of the molecule to facilitate conversion by CWD prions. These data suggest that interactions of the amino-terminal domain with the rest of the PrPc molecule play an important role in the susceptibility of humans to CWD prions.

  • Convallatoxin-induced reduction of methionine import effectively inhibits human cytomegalovirus infection and replication [PublishAheadOfPrint]

  • Cytomegalovirus (CMV) is a ubiquitous human pathogen that increases morbidity and mortality of immunocompromised individuals. The current FDA approved treatments for CMV infection are intended to be virus specific, yet they have significant adverse side effects including nephrotoxicity and hematological toxicity. Thus, there is a medical need for safer and more effective CMV therapeutics. Using a high-content screen, we have identified the cardiac glycoside convallatoxin as an effective compound that inhibits CMV infection. Using a panel of cardiac glycoside variants, we assessed the structural elements critical for anti-CMV activity by both experimental and in silico methods. Analysis of the antiviral effect, toxicity, and pharmacodynamics of different variants of cardiac glycosides identified the mechanism of inhibition as reduction of methionine import leading to decreased immediate early gene translation without significant toxicity. Also, convallatoxin was found to dramatically reduce viral proliferation of clinical CMV strains implying its mechanism of action as an effective strategy to block CMV dissemination. Our study has uncovered the mechanism and structural elements of convallatoxin that are important for effectively inhibiting CMV infection by targeting the expression of immediate early genes.

    Importance: Cytomegalovirus is a highly prevalent virus capable of causing severe disease in certain populations. The current FDA approved therapeutics all target the same stage of the viral life cycle and induce toxicity and viral resistance. We identified convallatoxin, a novel, cell-targeting antiviral that inhibits CMV infection by decreasing the synthesis of viral proteins. At doses low enough for cells to tolerate, convallatoxin was able to inhibit primary isolates of CMV, including those resistant to the anti-CMV drug ganciclovir. In addition to identifying convallatoxin as a novel antiviral, limiting mRNA translation has a dramatic impact on CMV infection and proliferation.

  • Multiple antibody lineages in one donor target the glycan-V3 supersite of the HIV-1 envelope glycoprotein and display a preference for quaternary binding [PublishAheadOfPrint]

  • One of the goals of HIV-1 vaccine development is the elicitation of neutralizing antibodies against vulnerable regions on the envelope glycoproteins (Env) viral spike. Broadly neutralizing antibodies targeting the Env glycan-V3 region (also called the N332-glycan supersite) have previously been described, with several single lineages each derived from individual donors. We used a high-throughput B-cell culture method to isolate neutralizing antibodies from an HIV-1 infected donor with high serum neutralization breadth. Clonal relatives from three distinct antibody lineages were isolated. Each of these antibody lineages displayed modest breadth and potency, but shared several characteristics with the well-characterized glycan-V3 antibodies including dependence on glycans N332 and N301, VH4 family gene utilization, a CDRH2 insertion, and a longer than average CDRH3. In contrast to previously described glycan-V3 antibodies, the most potent of these antibodies preferentially recognized the native Env trimer compared to monomeric gp120. These data indicate the diversity of antibody specificities that target the glycan-V3 site. The quaternary binding preference of these antibodies suggests that that their elicitation likely requires presentation of a native-like trimeric Env immunogen.

    Importance Broadly neutralizing antibodies targeting the HIV-1 glycan-V3 region have previously been described with single lineages from individual donors. Here we describe three lineages from a single donor that each target glycan-V3. Unlike the previously described glycan-V3 antibodies, these mature antibodies bind preferentially to the native Env trimer and weakly to gp120 monomer. These data extend our knowledge of immune response recognition of the N332 supersite region and suggest that the mode of epitope recognition is more complex than previously anticipated.

  • SUMO Modification Stabilizes Enterovirus 71 Polymerase 3D to Facilitate Viral Replication [PublishAheadOfPrint]

  • Accumulating evidences suggest that viruses hijack cellular proteins to circumvent the host immune system. Ubiquitination and SUMOylation are extensively studied post-translational modifications (PTMs) that play critical roles in diverse biological processes. Crosstalk between ubiquitination and SUMOylation of both host and viral proteins has been reported to result in distinct functional conses1quences. Enterovirus 71 (EV71), an RNA virus belonging to Picornaviridae family, is a common cause of hand, foot and mouth disease. Little is known concerning how host PTM systems interact with enteroviruses. Here, we demonstrated that the 3D protein, an RNA-dependent RNA polymerase (RdRp) of EV71, is modified by small ubiquitin-like modifier-1 both during infection and in vitro. Residues K159 and L150/D151/L152 were responsible for 3D SUMOylation determined by bioinformatic prediction combined with site-directed mutagenesis. And primer-dependent polymerase assays indicated that mutation of SUMOylation sites impaired 3D polymerase activity and virus replication. Moreover, 3D is ubiquitinated in a SUMO-dependent manner, and SUMOylation is crucial for 3D stability which may be due to the interplay between the two PTMs. Of importance, increasing the level of SUMO-1 in EV71-infected cells augmented the SUMOylation and ubiquitination level of 3D, leading to enhanced replication of EV71. These results together suggested that SUMO and ubiquitin cooperatively regulated EV71 infection either by SUMO-ubiquitin hybrid chains or by ubiquitin conjugating to the exposed lysine residue through SUMOylation. Our study provides a new insight into how a virus utilizes cellular pathways to facilitate its replication.

    Importance Infection with Enterovirus 71(EV71) often causes neurological diseases in children and EV71 is responsible for the majority of fatalities. Based on a better understanding of interplay between virus and host cell, antiviral drugs against enteroviruses may be developed. As a dynamic cellular process of post-translational modification, SUMOylation regulates global cellular protein localization, interaction, stability, and enzymatic activity. However, little is known concerning the SUMOylation directly influence virus replication by targeting viral polymerase. Herein, we found that EV71 polymerase 3D was SUMOylated during EV71 infection and in vitro. Moreover, the SUMOylation sites were determined. And in vitro polymerase assays indicated that mutations at SUMOylation sites could impair polymerase synthesis. Importantly, 3D is ubiquitinated in a SUMOylation-dependent manner which enhances the stability of the viral polymerase. Our findings indicate that the two modifications likely cooperatively enhance virus replication. Our study may offer a new therapeutic strategy against virus replication.

  • Immunodominant T-cell epitopes in the VP1 capsid protein of rhinovirus species A and C. [PublishAheadOfPrint]

  • Rhinovirus (RV) species A and C are the most frequent cause of respiratory viral illness worldwide and RV-C has been linked to more severe exacerbations of asthma in young children. Little is known about the immune response against the different RV species, although studies comparing IgG1 antibody titers found impaired antibody responses to RV-C. Therefore, the aim of this study was to assess whether T-cell immunity to RV-C is similarly impaired. We measured T-cell proliferation to overlapping synthetic peptides covering the entire VP1 capsid protein of an RV-A and RV-C genotype for 20 healthy adult donors. Human leukocyte antigen (HLA) was typed in all donors in order to investigate possible associations between HLA type and RV peptide recognition. Total and specific IgG1 antibody titers to VP1 of both RV-A and RV-C were also measured to examine associations between the antibody and T-cell responses. We identified T-cell epitopes that are specific of, and representative for each RV-A and RV-C species. These epitopes stimulated CD4+ specific T-cell proliferation with a similar magnitude of response for both RV species. All donors, independent of their HLA-DR or DQ type, were able to recognize the immunodominant RV-A and C regions of VP1. Furthermore, the presence or absence of specific antibody titers was not related with changes in T-cell recognition. Our results indicate a dissociation between the antibody and T-cell response against rhinoviruses. The species-representative T-cell epitopes identified in this study are valuable tools for future studies investigating T-cell responses to the different RV species.

    Importance Rhinoviruses (RV) are mostly associated with the common cold and asthma exacerbations, although their contribution in most upper and lower respiratory tract diseases have increasingly been reported. Species C (RV-C) has been associated with more frequent and severe asthma exacerbations in young children, and with RV-A are the most clinically relevant species. Little is known about how our immune system responds to rhinoviruses and there are limited tools to study specific adaptive immunity against each rhinovirus species.

    In this study, we identified immunodominant T-cell epitopes of the VP1 protein of RV-A and RV-C, which are representative of each species. The study found T-cell responses to RV-A and RV-C were of a similar magnitude, in contrast with previous findings showing RV-C specific antibody responses were low. These findings will provide the basis for future studies on immune response against rhinoviruses and can help elucidate the mechanisms of severity of rhinovirus-induced infections.

  • Hepatitis C virus is released via a non-canonical secretory route. [PublishAheadOfPrint]

  • We analyzed HCV morphogenesis using viral genomes encoding for a mCherry-tagged E1 glycoprotein. HCV-E1-mCherry polyprotein expression, intracellular localization and replication kinetics were comparable to untagged HCV and E1-mCherry tagged viral particles were assembled and released into cell culture supernatants. Expression and localization of structural E1 and non-structural NS5A followed a tempo-spatial pattern with succinct decrease in replication complexes and the appearance of E1-mCherry punctae. Interaction of the structural proteins E1, Core and E2 increased at E1-mCherry punctae in a time-dependent manner, indicating that E1-mCherry punctae represent assembled or assembling virions. E1-mCherry did not colocalize with Golgi markers. Furthermore, the bulk of viral glycoproteins within released particles revealed an EndoH-sensitive glycosylation pattern, indicating absence of viral glycoprotein processing by the Golgi. In contrast, HCV-E1-mCherry trafficked with Rab9-positive compartments and inhibition of endosomes specifically suppressed HCV release. Our data suggests that assembled HCV particles are released via a non-canonical secretory route involving the endosomal compartment.

    IMPORTANCE STATEMENT The goal of this study was to shed light on the poorly understood trafficking and release routes of hepatitis C virus (HCV). For this, we generated novel HCV genomes which result in the production of fluorescently labeled viral particles. We used live cell microscopy and other imaging techniques to follow up on the temporal dynamics of virus particle formation and trafficking in HCV-expressing liver cells. While viral particles and viral structural protein were found in endosomal compartments, no overlap with Golgi structures could be observed. Furthermore, biochemical and inhibitor-based experiments support a HCV release route which is distinguishable from canonical Golgi-mediated secretion. Since viruses hijack cellular pathways to generate viral progeny, our results point towards the possible existence of a not yet described cellular secretion route.

  • FIV Vif N-Terminal Residues Selectively Counteract Feline APOBEC3s [PublishAheadOfPrint]

  • Feline immunodeficiency virus (FIV) Vif protein counteracts feline APOBEC3s (FcaA3s) restriction factors by inducing their proteasomal degradation. The functional domains in FIV Vif for interaction with FcaA3s are poorly understood. Here, we have identified several motifs in FIV Vif that are important for selective degradation of different FcaA3s. Cats (Felis catus) express three types of A3s: single-domain A3Z2, A3Z3 and double-domain A3Z2Z3. We proposed that FIV Vif would selectively interact with the Z2 and the Z3 A3s. Indeed, we identified two N-terminal Vif motifs (12LF13 and 18GG19) that specifically interacted with the FcaA3Z2 protein but not with A3Z3. In contrast, the exclusive degradation of FcaA3Z3 was regulated by a region of three residues (M24, L25 and I27). Only a FIV Vif carrying a combination of mutations from both interaction sites lost the capacity to degrade and counteract FcaA3Z2Z3. However, alterations in the specific A3s interaction sites did not affect the cellular localization of the FIV Vif protein and binding to feline A3s. Pull-down experiments demonstrated that the A3 binding region localized to FIV Vif residues 50 to 80, outside the specific A3 interaction domain. Finally, we found that the Vif sites specific to individual A3s are conserved in several FIV lineages of domestic cat and non-domestic cats, while being absent in the FIV Vif of pumas. Our data support a complex model of multiple Vif-A3 interactions in which the specific region for selective A3 counteraction is discrete from a general A3 binding domain.

    IMPORTANCE Both human immunodeficiency virus (HIV) and feline immunodeficiency virus (FIV) Vif proteins counteract their host's APOBEC3 restriction factors. However, these two Vif proteins have limited sequence homology. The molecular interaction between FIV Vif and feline APOBEC3s are not well understood. Here we have identified N-terminal FIV Vif sites that regulate the selective interaction of Vif with either feline APOBEC3Z2 or APOBEC3Z3. These specific Vif sites are conserved in several FIV lineages of domestic cat and non-domestic cats, while being absent in FIV Vif from puma. Our findings provide important insights for future experiments describing the FIV Vif interaction with feline APOBEC3s and also indicate that the conserved feline APOBEC3s interaction sites of FIV Vif allow FIV transmissions in Felidae.

  • HSV1 Enters Human Keratinocytes by a Nectin-1-Dependent, Rapid Plasma Membrane Fusion Pathway that Functions at Low Temperature [PublishAheadOfPrint]

  • Herpes simplex virus 1 (HSV1) infects humans through stratified epithelia that are composed primarily of keratinocytes. The route of HSV1 entry into keratinocytes has been the subject of limited investigation, but is proposed to involve pH-dependent endocytosis, requiring the gD-binding receptor, nectin-1. Here, we have utilized the nTERT human keratinocyte cell line as a new model for dissecting the mechanism of HSV1 entry in to the host. Although immortalised, these cells nonetheless retain normal growth and differentiation properties of primary cells. Using siRNA depletion studies, we confirm that, despite nTERT cells expressing high levels of the alternative gD receptor HVEM, HSV1 requires nectin-1, not HVEM, to enter these cells. Strikingly, virus entry into nTERT cells occured with unusual rapidity, such that maximum penetration was achieved within 5 minutes. Moreover, HSV1 was able to enter keratinocytes but not other cell types at temperatures as low as 7ddeg;C, conditions where endocytosis was shown to be completely inhibited. Transmission electron microscopy of early entry events at both 37ddeg;C and 7ddeg;C identified numerous examples of naked virus capsids located immediately beneath the plasma membrane, with no evidence of virions in cytoplasmic vesicles. Taken together, these results imply that HSV1 uses the nectin-1 receptor to enter human keratinocyte cells via a previously uncharacterised rapid plasma membrane fusion pathway that functions at low temperature. These studies have important implications for current understanding of the relationship between HSV1 and its relevant in vivo target cell.

    IMPORTANCE The gold standard of antiviral treatment for any human virus infection is the prevention of virus entry into the host cell. In the case of HSV1, primary infection in the human begins in the epidermis of the skin or the oral mucosa, where the virus infects keratinocytes, and it is therefore important to understand the molecular events involved in HSV1 entry into this cell type. Nonetheless, few studies have looked specifically at entry into these relevant human cells. Our results reveal a new route for virus entry that is specific to keratinocytes, involves rapid entry, and functions at low temperatures. This may reflect the environmental conditions encountered by HSV1 when entering its host through the skin and emphasizes the importance of studying virus-host interactions in physiologically relevant cells.

  • Varicella Zoster Virus Downregulates Programmed Death Ligand 1 and Major Histocompatibility Complex 1 in Human Brain Vascular Adventitial Fibroblasts, Perineurial Cells and Lung Fibroblasts [PublishAheadOfPrint]

  • Varicella zoster virus (VZV) vasculopathy produces stroke, giant cell arteritis and granulomatous aortitis and develops after virus reactivates from ganglia and spreads transaxonally to arterial adventitia, resulting in persistent inflammation and pathological vascular remodeling. The mechanism(s) by which inflammatory cells persist in VZV-infected arteries is unknown; however, virus-induced dysregulation of programmed death ligand 1 (PD-L1) may play a role. Specifically, PD-L1 is expressed on virtually all nucleated cells and suppresses the immune system by interacting with the programmed cell death protein receptor 1 found exclusively on immune cells; thus, downregulation of PD-L1 may promote inflammation as seen in some autoimmune diseases. Both flow cytometry and immunofluorescence analyses to test whether VZV infection of adventitial cells downregulates PD-L1 showed decreased PD-L1 expression in VZV-infected compared to mock-infected human brain vascular adventitial fibroblasts (HBVAFs), perineurial cells (HPNCs) and fetal lung fibroblasts (HFLs) at 72 hours post-infection. Quantitative RT-PCR analyses showed no change in PD-L1 transcript levels between mock- and VZV-infected cells, indicating a post-transcriptional mechanism for VZV-mediated downregulation of PD-L1. Flow cytometry analyses showed decreased major histocompatibility complex 1 (MHC-1) expression in VZV-infected cells and adjacent uninfected cells compared to mock-infected cells. These data suggest that reduced PD-L1 expression in VZV-infected adventitial cells may potentially contribute to persistent vascular inflammation observed in virus-infected arteries from patients with VZV vasculopathy, while downregulation of MHC-1 prevents viral clearance.

    IMPORTANCE Here, we provide the first demonstration that VZV downregulates PD-L1 expression in infected HBVAFs, HPNCs and HFLs, which together with the noted VZV-mediated downregulation of MHC-1, might foster persistent inflammation in vessels leading to pathological vascular remodeling during VZV vasculopathy, as well as persistent inflammation in infected lungs to promote subsequent infection of T cells and hematogenous virus spread. Identification of a potential mechanism by which persistent inflammation in the absence of effective viral clearance occurs in VZV vasculopathy and VZV infection of the lung is a step toward targeted therapy of VZV-induced disease.

  • HIV envelope gp120 alters T-cell receptor mobilization in the immunological synapse of uninfected CD4 T cells and augments the T cell activation [PublishAheadOfPrint]

  • HIV is transmitted most efficiently from cell to cell and productive infection occurs mainly in activated CD4 T cells. It is postulated that HIV exploits immunological synapses formed between CD4 T cells and antigen-presenting cells to facilitate the targeting and infection of activated CD4 T cells. This study sought to evaluate how the presence of the HIV envelope (Env) in the CD4 T cell immunological synapse affects synapse formation and intracellular signaling to impact the downstream T cell activation events. CD4 T cells were applied onto supported lipid bilayers (SLBs) that were reconstituted with HIV Env gp120, anti-TCR antibody OKT3 and ICAM-1 to represent the surface of HIV Env-bearing antigen-presenting cells. The results showed that the HIV Env did not disrupt immunological synapse formation. Instead, the HIV Env accumulated with TCR at the center of the synapse, altered the kinetics of TCR recruitment to the synapse, and affected the synapse morphology over time. The HIV Env also prolonged Lck phosphorylation at the synapse and enhanced TCR-induced CD69 upregulation, IL-2 secretion, and proliferation to promote virus infection. These results suggest that HIV uses the immunological synapse as a conduit not only for selective virus transmission to activated CD4 T cells, but also for boosting the T cell activation state, thereby increasing its likelihood for undergoing productive replication in the targeted CD4 T cells.

    IMPORTANCE There are about two million new HIV infections every year. A better understanding on how HIV is transmitted to the susceptible cells is critical to devise effective strategies to prevent HIV infection. Activated CD4 T cells are preferentially infected by HIV, although how this is accomplished is not fully understood. This study examined whether HIV co-opts the normal T-cell activation process through the so-called immunological synapse. We found that the HIV envelope is recruited to the center of the immunological synapse together with the T-cell receptor and enhances the T cell receptor-induced activation of the CD4 T cells. The heightened cellular activation promotes the capacity of the CD4 T cells to support productive HIV replication. This study provides evidence for the exploitation of the normal immunological synapse and T-cell activation process by HIV to boost the activation state of the targeted CD4 T cells and promote infection of these cells.

  • Regulation of HSV glycoprotein induced cascade of events governing cell-cell fusion [PublishAheadOfPrint]

  • Receptor dependent HSV-induced fusion requires glycoproteins gD, gH/gL, and gB. Our current model posits that during fusion receptor-activated conformational changes in gD activate gH/gL, which subsequently triggers transformation of the pre-fusion form of gB into a fusogenic state. To examine the role of each glycoprotein in receptor dependent cell-cell fusion we took advantage of our discovery that fusion by wild type HSV-2 glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we established that fusion speed was governed by gH/gL, with gH being the main contributor. While the mutant forms of gB fuse at distinct rates that are dictated by their molecular structure, these restrictions can be overcome by gH2/gL2, thereby enhancing their activity. We also found that deregulated forms of gD1 and gH2/gL2 can alter the fusogenic potential of gB, promoting cell fusion in the absence of a cellular receptor and that deregulated forms of gB can drive the fusion machinery to even higher levels. Low pH enhanced fusion by affecting the structure of both gB and gH/gL mutants. Together, our data highlight the complexity of the fusion machinery, the impact of the activation state of each glycoprotein on the fusion process and the critical role of gH/gL in regulating HSV induced fusion.

    IMPORTANCE Cell-cell fusion mediated by HSV glycoproteins requires gD, gH/gL, gB and a gD receptor. Here, we show that fusion by wild type HSV-2 glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we found that the fusion process was controlled by gH/gL. Restrictions imposed on the gB structure by mutations could be overcome by gH2/gL2, enhancing their activity. Using low pH or deregulated forms of gD1 and gH2/gL2 the fusogenic potential of gB could only be increased in the absence of receptor, underlining the exquisite regulation that occurs in the presence of receptor. Our data highlight the complexity of the fusion machinery, the impact of the activation state of each glycoprotein on the fusion process and the critical role of gH/gL in regulating HSV induced fusion.

  • Dynamics and Correlates of CD8 T-cell Counts in Africans with Primary Human Immunodeficient Virus Type I (HIV-1) Infection [PublishAheadOfPrint]

  • In individuals with HIV-1 infection, depletion of CD4+ T-cells is often accompanied by a malfunction of CD8+ T-cells that are persistently activated and/or exhausted. While the dynamics and correlates of CD4 counts have been well-documented, the same does not apply to CD8 counts. Here, we examined CD8 counts in a cohort of 497 Africans with primary HIV-1 infection and monthly to quarterly follow-up visits for up to three years in the absence of antiretroviral therapy. Statistical models revealed that i) CD8 counts were relatively steady in the 3-36 months period of infection and similar between men and women; ii) neither geography nor heterogeneity in HIV-1 set-point viral load could account for the roughly 10-fold range of CD8 counts in the cohort (P ggt;0.25 in all tests); and iii) factors independently associated with relatively high CD8 counts included demographics (age lle;40 years, adjusted P = 0.010) and several human leukocyte antigen class I (HLA-I) alleles, including HLA-A*03:01 (P = 0.013), B*15:10 (P = 0.007), and B*58:02 (P llt;0.001). Several sensitivity analyses provided supporting evidence for these novel relationships. Overall, these findings suggest that factors associated with CD8 count have little overlap with those previously reported for other HIV-1-related outcome measures, including viral load, CD4 count, and CD4:CD8 ratio.

    Importance Longitudinal data from 497 HIV-1 seroconverters have allowed us to systematically evaluate the dynamics and correlates of CD8+ T-cell counts during untreated primary HIV-1 infection in eastern and southern Africans. Our findings suggest that individuals with certain HLA-I alleles, including A*03 (exclusively A*03:01), persistently maintain a relatively high CD8 counts following HIV-1 infection, which may offer an intriguing explanation for the recently reported, negative association of A*03 with HIV-1-specific, broadly neutralizing antibody responses. In future studies, attention to HLA-I genotyping data may benefit in-depth understanding of both cellular and humoral immunity, as well as their intrinsic balances, especially in settings where there is emerging evidence of antagonism between the two arms of adaptive immunity.

  • Altered glycosylation patterns increase immunogenicity of a subunit HCV vaccine inducing neutralizing antibodies which confer protection in mice [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) infection is a global health problem for which no vaccine is available. HCV has a highly heterogeneous RNA genome and can be classified into seven genotypes. Due to the high genetic and resultant antigenic variation among genotypes, inducing antibodies capable of neutralizing most of the HCV genotypes by experimental vaccination has been challenging. Previous efforts focused on priming humoral immune responses with recombinant HCV envelope E2 protein produced in mammalian cells. Here, we report that a soluble form of HCV E2 (sE2) produced in insect cells possess different glycosylation patterns and is more immunogenic as evidenced by the induction of higher titers of broadly neutralizing antibodies (bNAbs) against cell culture-derived HCV (HCVcc) harboring structural proteins from a diverse array of HCV genotypes. We affirm that continuous and discontinuous epitopes of well-characterized bNAbs are conserved, thus suggesting that sE2 produced in insect cells is properly folded. In a genetically humanized mouse model, active immunization with sE2 efficiently protected against challenge with a heterologous HCV genotype. These data not only demonstrate that sE2 is a promising HCV vaccine candidate but also highlight the importance of glycosylation patterns in developing subunit viral vaccines.

    Importance: A prophylactic vaccine with high efficacy and low cost is greatly needed for global control of HCV infection. Induction of broadly neutralizing antibodies against most HCV genotypes has been challenging due to the antigenic diversity of the HCV genome. Herein, we refined a high-yield subunit HCV vaccine that elicited broadly neutralizing antibody responses in preclinical trials. We found that soluble HCV E2 protein (sE2) produced in insect cells is distinctly glycosylated and is more immunogenic than sE2 produced in mammalian cells, suggesting that glycosylation patterns should be taken into consideration in efforts to generate antibody-based, recombinant vaccines against HCV. We further showed that sE2 vaccination confers protection against HCV infection in a genetically humanized mouse model. Thus our work identified a promising broadly protective HCV vaccine candidate, which should be considered of further pre-clinical and clinical development.

  • Fc receptor mediated activities by Env-specific human mAbs generated from volunteers receiving the DNA prime-protein boost HIV vaccine DP6-001 [PublishAheadOfPrint]

  • HIV-1 is able to elicit broadly potent neutralizing antibodies in a very small subset of individuals only after several years' infection and, therefore, vaccines that elicit these types of antibodies have been difficult to design. The RV144 trial showed that a moderate protection is possible, which may correlate with ADCC activity. Our previous studies demonstrated that in an HIV vaccine phase I trial, DP6-001, a polyvalent Env DNA prime-protein boost formulation, could elicit potent and broadly reactive, gp120-specific antibodies with positive neutralization activities. Here we report the production and analysis of HIV-1 Env-specific human monoclonal antibodies (mAbs) isolated from DP6-001 vaccinees. For this initial report, 13 mAbs from four DP6-001 vaccinees showed broad binding activities to gp120 proteins of diverse subtypes, both autologous and heterologous to vaccine immunogens. Equally cross-reactive Fc-mediated functional activities, including ADCC and ADCP, were present with both immune sera and isolated mAbs, confirming the induction of non-neutralizing functional mAbs by the DNA prime-protein boost vaccination. Elicitation of broadly reactive mAbs by vaccination in healthy human volunteers confirms the value of the polyvalent formulation in this HIV vaccine design.

    Importance The roles of FcR mediated protective antibody responses are gaining more attention due their potential contribution to the low level protection against HIV-1 infection in RV144 trail. At the same time, the information from other HIV vaccine studies from humans is very limited. In the current report, both immune sera and monoclonal antibodies from vaccinated humans showed not only high level but also cross-subtype ADCC and ADCP activities using a polyvalent DNA prime-protein boost vaccine formulation.


  • Strong antibody (Ab) responses against V1V2 epitopes of the HIV-1 gp120 envelope (Env) correlated with reduced infection rates in studies of HIV, SHIV, and SIV. In order to focus the Ab response on V1V2, we used six V1V2 sequences and nine scaffold proteins to construct immunogens which were tested using various immunization regimens for their ability to induce cross-reactive and biologically active V2 Abs in rabbits. A prime/boost immunization strategy was employed using gp120 DNA and various V1V2-scaffold proteins. The rabbit polyclonal Ab responses (a) were successfully focused on the V1V2 region, with weak or only transient responses to other Env epitopes, (b) displayed broad cross-reactive binding activity with gp120s and the V1V2 regions of diverse strains from clades B, C, and E, (c) included V2 Abs with specificities similar to those found in HIV-infected individuals, and (d) were detectable gge;1 year after the last boosting dose. Importantly, sera from rabbits receiving V1V2-scaffold immunogens displayed Ab-dependent cellular phagocytosis whereas sera from rabbits receiving only gp120 did not. The results represent the first fully successful example of reverse vaccinology in the HIV vaccine field with rationally-designed epitope-scaffold immunogens inducing Abs that recapitulate the epitope specificity and biologic activity of the human monoclonal Abs from which the immunogens were designed. Moreover, this is the first immunogenicity study using epitope-targeting rationally designed vaccine constructs that induced an Fc-mediated activity associated with protection from infection with HIV, SIV and SHIV.

    IMPORTANCE Novel immunogens were designed to focus the antibody response of rabbits on the V1V2 epitopes of HIV-1 gp120 since such antibodies were associated with reduced infection rates of HIV, SIV, and SHIV. The vaccine-induced antibodies were broadly cross-reactive with the V1V2 regions of HIV subtypes B, C and E, and, importantly, facilitated Fc-mediated phagocytosis, an activity not induced upon immunization of rabbits with gp120. This is the first immunogenicity study of vaccine constructs that focuses the antibody response on V1V2 and induces V2-specific antibodies with the ability to mediate phagocytosis, an activity that has been associated with protection from infection with HIV, SIV and SHIV.

  • Herpes Simplex Virus 1 Induces Phosphorylation and Reorganization of lamin A/C Through the {gamma}134.5 Protein That Facilitates Nuclear Egress [PublishAheadOfPrint]

  • Herpes simplex virus 1 (HSV-1) remodels nuclear membranes during virus egress. Although the UL31 and UL34 proteins control nucleocapsid transit in infected cells, the molecular interactions required for their function are unclear. Here we report that the 134.5 gene product of HSV-1 facilitates nucleocapsid release to the cytoplasm through bridging the UL31/UL34 complex, cellular p32 and protein kinase C. Unlike wild type virus, an HSV mutant devoid of 134.5 or its amino-terminus is crippled for viral growth and release. This is attributable to a defect in virus nuclear egress. In infected cells, wild type virus recruits protein kinase C to the nuclear membrane and triggers its activation whereas the 134.5 mutants fail to exert such effect. Accordingly, the 134.5 mutants are unable to induce phosphorylation and reorganization of lamin A/C. When expressed in host cells 134.5 targets p32 and protein kinase C. Meanwhile, it communicates with the UL31/UL34 complex through UL31. Deletion of the amino-terminus from 134.5 disrupts its activity. These results suggest that disintegration of the nuclear lamina mediated by 134.5 promotes HSV replication.

    IMPORTANCE HSV nuclear egress is a key step that determines the outcome of viral infection. While the nuclear egress complex mediates capsid transit across the nuclear membrane, the regulatory components are not clearly defined in virus infected cells. We report that the 134.5 gene product, a virulence factor of HSV-1, facilitates nuclear egress cooperatively with cellular p32, protein kinase C and the nuclear egress complex. This work highlights a viral mechanism that may contribute to the pathogenesis of HSV infection.

  • The presumed polyomavirus viroporin VP4 of SV40 or BKPyV is not required for viral progeny release [PublishAheadOfPrint]

  • The minor capsid protein of human BK polyomavirus (BKPyV), VP2, and its N-terminally truncated form VP3, are both important for viral entry. The closely related Simian Virus 40 (SV40) reportedly produces an additional truncated form of VP2/3, denoted VP4, apparently functioning as a viroporin promoting progeny release. The VP4 open reading frame is conserved in some polyomaviruses including BKPyV. In this study, we investigated the role of VP4 in BKPyV replication. By transfecting viral genomes into primary human renal proximal tubule epithelial cells we demonstrated that unaltered BKPyV and mutants with start codon substitutions in VP4 (VP2M229I and VP2M229A) abolishing putative VP4 production, were released at the same level to supernatants. However, during infection studies, VP2M229I and VP2M229A exhibited 90% and 65% reduced infectivity, respectively, indicating that isoleucine substitution inadvertently disrupted VP2/3 function to the detriment of viral entry, while inhibition of VP4 production during late infection was well tolerated. Unexpectedly, and similarly to BKPyV, SV40 WT and the corresponding VP4 start codon mutants (VP2M228I and VP2M228A) transfected into monkey kidney cell lines, were also released at equal levels. Upon infection, only the VP2M228I mutant exhibited reduced infectivity, a 43% reduction, which also subsequently lead to delayed host cell lysis. Mass spectrometry analysis of nuclear extracts from SV40 infected cells failed to identify VP4. Our results suggest that neither BKPyV nor SV40 require VP4 for progeny release. Moreover, our results reveal an important role in viral entry for the amino acid in VP2/VP3 unavoidably changed by VP4 start codon mutagenesis.

    IMPORTANCE Almost a decade ago, SV40 was reported to produce a late non-structural protein, VP4, which forms pores in the nuclear membrane, facilitating progeny release. By performing transfection studies with unaltered BKPyV and SV40 and their respective VP4-deficient mutants, we found that VP4 is dispensable for progeny release contrary to the original findings. However, infection studies demonstrated a counterintuitive reduction of infectivity of certain VP4-deficient mutants. In addition to the isoleucine-substituted SV40 mutant of the original study, we included alanine-substituted VP4-deficient mutants of BKPyV (VP2M229A) and SV40 (VP2M228A). These revealed that the reduction in infectivity was not caused by a lack of VP4 but rather depended on the identity of the single amino acid substituted within VP2/3 for VP4 start codon mutagenesis. Hopefully, our results will correct the longstanding misconception of VP4's role during infection, and stimulate continued work on unravelling the mechanism for release of polyomavirus progeny.

  • Human monoclonal antibody 81.39a effectively neutralizes emerging influenza A viruses of group 1 and 2 hemagglutinins [PublishAheadOfPrint]

  • The pandemic threat posed by emerging zoonotic influenza A viruses necessitate development of antiviral agents effective against various antigenic subtypes. Human monoclonal antibody (hmAb) targeting the hemagglutinin (HA) stalk offers a promising approach to control influenza virus infections. Here we investigated the ability of hmAb 81.39a to inhibit in vitro replication of human and zoonotic viruses, representing 16 HA subtypes. The majority of viruses were effectively neutralized by 81.39a, EC50 llt;0.01nndash;4.9mmu;g/ml. Among group 2 HA viruses tested, a single A(H7N9) virus was not neutralized at 50mmu;g/ml; it contained HA2-Asp19Gly, an amino acid position previously associated with resistance to neutralization by the group 2 HA-neutralizing mAb CR8020. Notably, among group 1 HA viruses, H11nndash;H13, and H16 subtypes were not neutralized at 50mmu;g/ml; they shared a substitution HA2-Asp19Asn/Ala. Conversely, H9 viruses harboring HA2-Asp19Ala were fully susceptible to neutralization. Therefore, amino acid variance at HA2-Asp19 has subtype-specific adverse effects on in vitro neutralization. Mice given a single injection (15 or 45 mg/kg) at 24 or 48 hours after infection with recently emerged A(H5N2), A(H5N8), A(H6N1) or A(H7N9) viruses were protected from mortality and showed drastically reduced lung viral titers. Furthermore, 81.39a protected mice infected with A(H7N9) harboring HA2-Asp19Gly, although the antiviral effect was lessened. A(H1N1)pdm09-infected ferrets receiving a single dose (25 mg/kg) had reduced viral titers and showed less lung tissue injury, despite 24nndash;72 hours delayed treatment. Taken together, this study provides experimental evidence for the therapeutic potential of 81.39a against diverse influenza A viruses.

    Importance Zoonotic influenza viruses, such as A(H5N1) and A(H7N9) subtypes, have caused severe disease and deaths in humans raising public health concerns. Development of novel anti-influenza therapeutics with a broad spectrum of activity against various subtypes is necessary to mitigate disease severity. Here we demonstrate that the hemagglutinin (HA) stalk-targeting human monoclonal antibody 81.39a effectively neutralized the majority of influenza A viruses tested, representing 16 HA subtypes. Furthermore, 81.39a delayed treatment significantly suppressed virus replication in the lungs, prevented dramatic body weight loss and increased survival rates of mice infected with A(H5Nx), A(H6N1) or A(H7N9) viruses. When tested in ferrets, 81.39a delayed treatment reduced viral titers, particularly in the lower respiratory tract, and substantially alleviated disease symptoms associated with severe A(H1N1)pdm09 influenza. Collectively, our data demonstrated the effectiveness of 81.39a against both seasonal and emerging influenza A viruses.

  • Herpes Simplex Virus Type 1 UL37 Protein Tyrosine Residues Conserved among all Alphaherpesviruses are Required for Interactions with Glycoprotein K (gK), Cytoplasmic Virion Envelopment, and Infectious Virus Production [PublishAheadOfPrint]

  • The HSV-1 UL37 protein functions in virion envelopment at trans-Golgi membranes, as well as in retrograde and anterograde transport of virion capsids. Recently, we reported that UL37 interacts with glycoprotein K(gK) and its interacting partner protein UL20 (Jambunathan, et. al, J. Virol. 88:5927-5935) facilitating cytoplasmic virion envelopment. Alignment of UL37 homologues encoded by alphaherpesviruses revealed the presence of highly conserved residues in the central portion of the UL37 protein. A cadre of nine UL37 site-specific mutations were produced and tested for their ability to inhibit virion envelopment and infectious virus production. Complementation analysis revealed that replacement of tyrosines 474 and 480 with alanine failed to complement the UL37-null virus, while all other mutated UL37 genes complemented efficiently. The recombinant virus DC474-480 constructed with tyrosines 474, 476, 477 and 480 mutated to alanine residues produced a gK-null-like phenotype characterized by the production of very small plaques and accumulation of capsids in the cytoplasm of infected cells. Recombinant viruses having either tyrosine 476 or 477 replaced with alanine residues produced a wild-type phenotype. Immunoprecipitation assays revealed that replacement of all four tyrosines to alanines substantially reduced the ability of gK to interact with UL37. Alignment of herpes simplex virus (HSV) UL37 to the human cytomegalovirus and Epstein Barr Virus UL37 homologues revealed that Y480 was conserved only for alphaherpesviruses. Collectively, these results suggest that UL37 conserved tyrosine residue Y480 plays crucial role in interactions with gK to facilitate cytoplasmic virion envelopment and infectious virus production.

    Importance: The HSV-1 UL37 protein is conserved among all herpesviruses, functions in both retrograde and anterograde transport of virion capsids, and plays critical roles in cytoplasmic virion envelopment by interacting with glycoprotein K (gK). We show here that UL37 tyrosine residues conserved among all alphaherpesviruses serve critical roles in cytoplasmic virion envelopment and interactions with gK.

  • {gamma}134.5 neurovirulence gene of herpes simplex virus type 1 modifies the exosome secretion profile in epithelial cells [PublishAheadOfPrint]

  • Recently, it has been demonstrated that herpes simplex virus type 1 (HSV-1) -infected cells secrete exosomes that deliver to uninfected cells the innate immune sensor, STING, and viral RNAs (1, 2)....

  • Characterization of the glycoprotein stable signal peptide in mediating Pichinde viral replication and virulence [PublishAheadOfPrint]

  • Arenaviruses can cause lethal hemorrhagic fevers in humans with limited preventative and therapeutic measures. The arenaviral glycoprotein stable signal peptide (SSP) is unique among signal peptides in that it is an integral component of the mature glycoprotein complex (GPC) and plays important roles not only in the GPC expression and processing but also in the membrane fusion process during viral entry. Using the Pichinde virus (PICV) reverse genetics system, we analyzed the effects of alanine substitutions at many conserved residues within SSP on viral replication in cell culture and in a guinea-pig infection model. Our data showed that the K33A, F49A, and C57A mutations abolished GPC-mediated cell entry and, therefore, could not allow for the generation of viable recombinant viruses, demonstrating that these residues are essential for the PICV life cycle. The G2A mutation caused a marked reduction of cell entry at the membrane fusion step, and while this mutant virus was viable, it was significantly attenuated in vitro and in vivo. The N20A mutation also reduced membrane fusion activity and viral virulence in guinea pigs, but did not significantly affect cell entry or viral growth in cell culture. Two other mutations (N37A and R55A) did not affect membrane fusion or viral growth in vitro but significantly reduced viral virulence in vivo. Taken together, our data suggest that GPC SSP plays an essential role in mediating viral entry and also contributes to viral virulence in vivo.

    Authors' summary Several arenaviruses, such as Lassa fever virus, can cause severe and lethal hemorrhagic fever diseases with high mortality and morbidity, for which no FDA-approved vaccines or therapies are currently available. Viral entry into cells is mediated by arenavirus glycoprotein complex (GPC) that consists of a stable signal peptide (SSP), the receptor-binding GP1, and transmembrane GP2 protein subunits. Using a reverse genetics system of a prototypic arenavirus, Pichinde virus (PICV), we have shown for the first time in the context of infectious virus infections of cell culture and of guinea pigs that the SSP plays an essential role in mediating the membrane fusion step as well as at other yet-to-be-determined processes during viral infection. Our study provides important insights into the biological roles of GPC SSP and implicates it as a potentially good target for the development of antivirals against deadly human arenavirus pathogens.

  • Magnitude and quality of cytokine and chemokine storm during acute infection distinguish non-progressive and progressive simian immunodeficiency virus infections of nonhuman primates [PublishAheadOfPrint]

  • Acute HIV infection represents a period of intense immune perturbation and activation of the host immune system. Study of the eclipse and viral expansion phases of infection is difficult in humans, but studies in non-progressive and progressive nonhuman primate infection models can provide significant insight into critical events occurring during this time. Cytokines, chemokines and other soluble immune factors were measured in longitudinal samples from rhesus macaques infected with either SIVmac251 (progressive infection) or SIVmac239nef (attenuated/non-progressive infection), and from African green monkeys infected with SIVsab9315BR (non-pathogenic infection). Levels of acute-phase peak viral replication were highest in SIVmac251 infection, but correlated positively with viremia at three months post-infection in all three infection models. SIVmac251 infection was associated with stronger corresponding acute-phase cytokine/chemokine responses than the non-progressive infections. Production of IL-15, IL-18, IFN-, G-CSF, MCP-1, MIP-1bbeta; and SAA during acute SIVmac251 infection, but not SIVmac239nef or SIVsab9315BR infection, correlated positively with chronic viremia at three months post infection. Acute-phase production of MCP-1 correlated with viremia at three months post infection in both non-progressive infections. Finally, a positive correlation between the acute-phase area under the curve (AUC) IL-6 and sCD40L and chronic viremia was only observed in the non-progressive infections models. While we observed dynamic acute inflammatory immune responses in both progressive and non-progressive SIV infections, the responses in the non-progressive infections were not only lower in magnitude but also qualitatively different biomarkers of disease progression.

    IMPORTANCE NHP models of HIV infection constitute a powerful tool to study viral pathogenesis to gain critical information for a better understanding of HIV infection in humans. Here, we studied progressive and non-progressive SIV infection models in both natural and non-natural host NHP species. Regardless of the pathogenicity of the virus infection or NHP species studied, the magnitude of viremia, as measured by area under the curve during the first 4 weeks of infection, positively correlated with viremia in chronic infection. The magnitude of cytokine and chemokine responses during primary infection also correlated positively with both acute-phase and chronic viremia. However, the pattern and levels of specific cytokines and chemokines produced differed between non-progressive and progressive SIV infection models. The qualitative differences in the early immune response in progressive and non-progressive infections identified here correlate with and may provide insights into the basis of differences in the subsequent course of disease.

  • An E2 gene infectious HCV library reveals growth adaptive mutations that modulate interactions with the scavenger receptor class B type I [PublishAheadOfPrint]

  • While natural hepatitis C virus (HCV) infection results in highly diverse quasispecies of related viruses over time, mutations accumulate more slowly in tissue culture, in part because of the inefficiency of replication in cells. To create a highly diverse population of HCV particles in cell culture and identify novel growth-enhancing mutations, we engineered a library of infectious HCV with all codons represented at most positions in the ectodomain of the E2 gene. We identified many putative growth adaptive mutations and selected nine highly represented E2 mutants for further study: Q412R, T416R, S449P, T563V, A579R, L619T, V626S, K632T, and L644I. We evaluated these mutants for changes in particle to infectious unit ratio, sensitivity to neutralizing antibody or CD81 large extracellular loop (CD81-LEL) inhibition, entry factor usage, and buoyant density profiles. Q412R, T416R, S449P, T563V and L619T were neutralized more efficiently by anti-E2 antibodies and T416R, T563V and L619T by CD81-LEL. Remarkably, all nine variants showed reduced dependence on scavenger receptor class B type I (SR-BI) for infection. This shift from SR-BI usage did not correlate with a change in the buoyant density profiles of the variants, suggesting an altered E2-SR-BI interaction rather than changes in the virus-associated lipoprotein-E2 interaction. Our results demonstrate that residues influencing SR-BI usage are distributed across E2 and support the development of large scale mutagenesis studies to identify viral variants with unique functional properties.

    IMPORTANCE Characterizing variant viruses can reveal new information about the lifecycle of Hepatitis C virus (HCV) and the roles played by different viral genes. However, it is difficult to recapitulate high levels of diversity in the laboratory because of limitations in the HCV culture system. To overcome this limitation, we engineered a library of mutations into the E2 gene in the context of an infectious clone of the virus. We used this library of viruses to identify nine mutations that enhance the growth rate of HCV. These growth enhancing mutations reduced the dependence on a key entry receptor, scavenger receptor class B type I (SR-BI). By generating a highly diverse library of infectious HCV, we mapped regions of the E2 protein that influence a key virus-host interaction and provide proof-of-principle for the generation of large-scale mutant libraries for the study of pathogens with great sequence variability.

  • Monitoring integration over time supports a role for CTL and ongoing replication as determinants of reservoir size [PublishAheadOfPrint]

  • The dynamics of HIV reservoir accumulation off antiretroviral therapy (ART) is underexplored. Levels of integrated HIV DNA in peripheral blood mononuclear cells (PBMCs) were longitudinally monitored before and after antiviral therapy. HIV integration increased over time in both Elite Controllers (ECs, n=8) and Non-Controllers (NCs, n=6) before ART, whereas integration remained stable in patients on ART (n=4). The median annual fold-change was higher in NCs compared to ECs and negatively correlated with CD4/CD8 T-cell ratio. CTL function as assessed by infected CD4 T-cell elimination (ICE) and granzyme B activity did not significantly change over time in ECs, suggesting the gradual increase in integrated HIV DNA observed in ECs was not a result of progressive loss of immune-mediated control. Also, acutely infected (n=7) but not chronically infected (n=6) patients exhibited a significant drop in integrated HIV DNA 12 months after ART initiation. In conclusion, in the absence of ART, integrated HIV accumulates over time both in NCs and in ECs, at variable individual rates. Starting ART early in infection leads to a greater drop in integrated HIV DNA compared to initiating treatment after years of infection. The increase in integrated HIV DNA over time suggests early treatment may be of benefit in limiting HIV reservoirs.

    Importance The establishment of a latent reservoir represents a barrier to cure among HIV-infected individuals. The dynamics of HIV reservoir accumulation over time in patients before antiviral therapy is underexplored, in large part because it is difficult to accurately and reproducibly measure the size of HIV reservoir in this setting. In our study, we compared the dynamics of integrated HIV DNA over time in ECs and NCs before and after ART is initiated. We found that integrated HIV DNA levels progressively increase over time in the absence of ART, but with a faster, albeit variable, rate in NCs compared to ECs. In addition, integrated HIV DNA declines more dramatically when ART is initiated in acute rather than chronic HIV infection, suggesting important differences between acute and chronic infection. Our study highlights the role of HIV replication and CTL control in reservoir accumulation in sanctuary sites and why ART appears to be more effective in acute infection.

  • Characterization of VSV pseudotypes bearing essential entry glycoproteins gB, gD, gH, and gL of Herpes Simplex virus Type 1 [PublishAheadOfPrint]

  • Herpes Simplex viruses (HSV) are unusual in that unlike most enveloped viruses, they require at least four entry glycoproteins, gB, gD, gH, and gL, for entry into target cells in addition to a cellular receptor for gD. The dissection of the HSV-1 entry mechanism is complicated by the presence of over a dozen proteins on the viral envelope. To investigate HSV-1 entry requirements in a simplified system, we generated VSV virions pseudotyped with HSV-1 essential entry glycoproteins gB, gD, gH, and gL but lacking the native VSV fusogen G. These virions, referred to here as VSVG-BHLD virions, infected a cell line expressing a gD receptor, demonstrating for the first time that the four essential entry glycoproteins of HSV-1 are not only required but also sufficient for cell entry. To our knowledge, this is the first time the VSV pseudotyping system has been successfully extended beyond two proteins. Entry of pseudotyped virions required a gD receptor and was inhibited by HSV-1 specific anti-gB or anti-gH/gL neutralizing antibodies, which suggests that membrane fusion during the entry of the pseudotyped virions shares common requirements with the membrane fusion involved in HSV-1 entry and HSV-1-mediated syncytia formation. The HSV pseudotyping system established here presents a novel tool for systematic exploration of the HSV entry and membrane fusion mechanisms.

    IMPORTANCE Herpes simplex viruses (HSVs) are human pathogens that can cause cold sores, genital herpes, and blindness. No vaccines or preventatives are available. HSV entry into cells nndash; a prerequisite for a successful infection nndash; is a complex process that involves multiple viral and host proteins and occurs by different routes. The detailed mechanistic knowledge of the HSV entry is important for understanding its pathogenesis and would benefit antiviral and vaccine development; yet, the presence of over a dozen proteins on the viral envelope complicates the dissection of the HSV entry mechanisms. Here, we generated heterologous virions displaying the four essential entry proteins of HSV-1 and showed that they are capable of cell entry and, like HSV-1, require all four entry glycoproteins along with a gD receptor. This HSV pseudotyping system pioneered here opens doors for future systematic exploration of the herpesvirus entry mechanisms.

  • Cell-cell sensing of viral infection by pDCs [PublishAheadOfPrint]

  • All cells possess signaling pathways designed to trigger antiviral responses, notably characterized by type I interferon (IFN) production, upon recognition of invading viruses. Especially, host sensors recognize viral nucleic acids. Nonetheless, virtually all viruses have evolved potent strategies that preclude host responses within the infected cells. The plasmacytoid dendritic cell (pDC) is an immune cell type known as a robust type I IFN producer in response to viral infection. Evidence suggests that such functionality of the pDCs participates to viral clearance. Nonetheless, their contribution, which is likely complex and varies depending on the pathogen, is still enigmatic for many viruses. pDCs are not permissive to most viral infections, and consistently recent examples suggest that pDCs respond to immuno-stimulatory viral RNA transferred via non-infectious and/or non-canonical viral/cellular carriers. Therefore, the pDC response likely bypasses innate signaling blockage induced by virus within infected cells. Importantly, the requirement for cell-cell contact is increasingly recognized as a hallmark of the pDC-mediated antiviral state, triggered by evolutionarily divergent RNA viruses.

  • The role of LAMP1 binding and pH sensing by the spike complex of Lassa virus [PublishAheadOfPrint]

  • To effectively infect cells, Lassa virus needs to switch in endosomal compartment from its primary receptor aalpha;-dystroglycan to a protein termed LAMP1. A unique histidine triad on the surface of the receptor-binding domain from the glycoprotein spike complex of Lassa virus is important for LAMP1 binding. Here we investigate mutated spikes that have impaired ability to interact with LAMP1 and show that although LAMP1 is important for efficient infectivity it is not required for spike-mediated membrane fusion per se. Our studies reveal an important regulatory role for histidines from the triad in sensing acidic pH and preventing premature spike triggering. We further show that LAMP1 requires a positively charged His230 to engage with the spike complex and that LAMP1 binding promotes membrane fusion. These results elucidate the molecular role for LAMP1 binding during Lassa virus cell-entry and provide new insights for how pH is sensed by the spike.

    IMPORTANCE Lassa virus is a devastating disease-causing agent in West Africa with a significant yearly death toll and severe long-term complications associated with its infection in survivors. In recent years we learned that Lassa virus needs to switch receptors in a pH dependent manner to efficiently infect cells, but neither the molecular mechanisms that allow switching nor the actual effect of switching were known. Here we investigate the activity of the viral spike complex after abrogating its ability to switch receptors. These studies informed us about the role of switching receptors and provided new insights for how the spike senses acidic pH.

  • Alteration of a second putative fusion peptide of structural glycoprotein E2 of classical swine fever virus alters virus replication and virulence in swine [PublishAheadOfPrint]

  • E2, the major envelope glycoprotein of classical swine fever virus (CSFV), is involved in several critical virus functions including cell attachment, host range susceptibility, and virulence in natural hosts. Functional structural analysis of E2 based on Wimley-White interfacial hydrophobicity distribution predicted the involvement of a loop (residues 864-881) stabilized by a disulfide bond (869CKWGGNWTCV878, named FPII) in establishing interactions with the host cell membrane. This loop further contains an 872GG873 dipeptide, and two aromatic residues (871W and 875W) accessible to solvent. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how amino acid substitutions within FPII may affect replication of BICv in vitro and virus virulence in swine. Recombinant CSFVs containing mutations in different residues of FPII were constructed. A particular construct, harboring amino acid residue substitutions W871T, W875D, and V878T (FPII.2), demonstrated a significantly decreased ability to replicate in a swine cell line (SK6) and swine macrophage primary cell cultures. Interestingly, mutated virus FPII.2 was completely attenuated in pigs. Also, animals infected with FPII.2 virus were protected against virulent challenge with Brescia virus at 21 days post-vaccination. Supporting a role for the E2 864-881 loop in membrane fusion, only synthetic peptides that were based on the native E2 functional sequence were competent for inserting into model membranes and perturbing their integrity and this functionality is lost in synthetic peptides harboring substitutions at FPII.2 amino acid residues W871T, W875D, and V878T.

    Importance This report constitutes the identification and characterization of a putative fusion peptide (FP) in the major structural protein E2 of classical swine fever virus (CSFV). The FP identified was performed by functional structural analysis of E2. We characterized the functional significance of this FP by using artificial membranes. Substitution of critical amino acid residues within the FP radically alters how it interacts with the artificial membranes. When we introduced these same mutations into the viral sequence there was a reduction in replication in cell cultures, and when we infected domestic swine, the natural host of CSFV host, we observed that the virus was now completely attenuated in swine. In addition, the virus mutant that was attenuated in vivo efficiently protects pigs against wild-type virus. These results provide the proof-of-principle to support as a strategy for vaccine development the discovery and manipulation of FPs.

  • Identification of essential genes in the Salmonella phage SPN3US reveals novel insights into giant phage head structure and assembly. [PublishAheadOfPrint]

  • Giant tailed bacterial viruses or phages, such as Pseudomonas aeruginosa phage KZ, have long genomes packaged into large, atypical virions. Many aspects of KZ and related phage biology are poorly understood mostly due to the fact that the functions of the majority of their proteins are unknown. We hypothesized that the Salmonella enterica phage SPN3US could be a useful model phage to address this gap in knowledge. The 240 kb SPN3US genome shares a core set of 91 genes with KZ and related phages, of which ~61 are virion genes, consistent with the expectation that virion complexity is an ancient, conserved feature. Nucleotide sequencing of eighteen mutants enabled assignment of thirteen genes as essential, information which could not have been determined by sequence-based searches for eleven genes. Proteome analyses of two SPN3US virion protein mutants with knockouts in 64 and 241 revealed new insight into the composition and assembly of giant phage heads. The 64 mutant analyses revealed all the genetic determinants required for assembly of the SPN3US head and a likely head-tail joining role for gp64, and its homologs in related phages, due to the tailless-particle phenotype produced. Analyses of the mutation in 241, which encodes a RNA polymerase bbeta; subunit, revealed that without this subunit, no other subunits are assembled into the head, and enabled identification of a "missing" bbeta;' subunit domain. These findings support SPN3US as an excellent model for giant phage research, laying the groundwork for future analyses of their highly unusual virions, host-interactions and evolution.

    Importance In recent years there has been a paradigm shift in virology with the realization that extremely large viruses infecting prokaryotes (giant phages) can be found in many environments. A group of phages related to the prototype giant phage KZ are of great interest due to their virions being amongst the most complex of prokaryotic viruses and their potential for biocontrol and phage therapy applications. Our understanding of the biology of these phages is limited as a large proportion of their proteins have either not been characterized and/or deemed putative without any experimental verification. In this study, we analyzed Salmonella phage SPN3US using a combination of genomics, genetics and proteomics and in doing so revealed new information regarding giant phage head structure and assembly, and virion RNA polymerase composition. Our findings demonstrate the suitability of SPN3US as model phage for the growing group of phages related to KZ.

  • Mitogen-activated Protein Kinase Kinase 2 (MEK2), a Novel E2-interacting Protein, Promotes the Growth of Classical Swine Fever Virus via Attenuation of the JAK-STAT Signaling Pathway [PublishAheadOfPrint]

  • Mitogen-activated protein kinase kinase/extracellular regulated kinase (MEK1/2/ERK1/2) cascade is involved in the replication of several members of the Flaviviridae family including hepatitis C virus and dengue virus. The effects of the cascade on the replication of classical swine fever virus (CSFV), a fatal pestivirus of pigs, remain unknown. In this study, MEK2 was identified as a novel binding partner of the E2 protein of CSFV using yeast two-hybrid screening. The E2-MEK2 interaction was confirmed by glutathione S-transferase pulldown, coimmunoprecipitation, and laser confocal microscopy assays. The C-termini of E2 [amino acids (aa) 890nndash;1053] and MEK2 (aa 266nndash;400) were mapped to be crucial for the interaction. Overexpression of MEK2 significantly promoted the replication of CSFV, whereas knockdown of MEK2 by lentivirus-mediated small hairpin RNAs dramatically inhibited CSFV replication. In addition, CSFV infection induced a biphasic activation of ERK1/2, the downstream signaling molecules of MEK2. Furthermore, the replication of CSFV was markedly inhibited in PK-15 cells treated with U0126, a specific inhibitor for MEK1/2/ERK1/2, whereas MEK2 did not affect CSFV replication after blocking the interferon-induced Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway by ruxolitinib, a JAK-STAT-specific inhibitor. Taken together, our results indicate that MEK2 positively regulates the replication of CSFV through inhibiting the JAK-STAT signaling pathway.

    IMPORTANCE Mitogen-activated protein kinase kinase 2 (MEK2) is a kinase that operates immediately upstream of extracellular regulated kinase 1/2 (ERK1/2) and links to Raf and ERK via phosphorylation. Currently, little is known about the role of MEK2 in the replication of classical swine fever virus (CSFV), a devastating porcine pestivirus. Here, we investigate the roles of MEK2 and the MEK2/ERK1/2 cascade in the growth of CSFV for the first time. We show that MEK2 positively regulates CSFV replication. Notably, we demonstrate that MEK2 promotes CSFV replication through inhibiting the interferon-induced JAK-STAT signaling pathway, a key antiviral pathway involved in the innate immunity. Our work reveals a novel role of MEK2 in CSFV infection and sheds light on the molecular basis by which pestiviruses interplay with the host cell.

  • Activation of the RLR/MAVS signaling pathway by the L protein of Mopeia virus [PublishAheadOfPrint]

  • The Arenaviridae family includes several important human pathogens that can cause severe hemorrhagic fever and greatly threaten public health. As a major component of the innate immune system, the RLR/MAVS signaling pathway is involved in recognizing viral components and initiating antiviral activity. It has been reported that arenavirus infection can suppress the innate immune response, and arenavirus NP and Z proteins of pathogenic arenaviruses can disrupt RLR/MAVS signaling, thus inhibiting production of IFN-I. However, recent studies have shown elevated IFN-I levels in certain arenavirus-infected cells. The mechanism by which arenavirus infection induces IFN-I responses remains unclear. In this study, we determined that the Lp of Mopeia virus (MOPV), an Old World (OW) arenavirus, can activate the RLR/MAVS pathway and thus induce the production of IFN-I. This activation is associated with the RNA-dependent RNA polymerase activity of Lp. This study provides a foundation for further studies of interactions between arenaviruses and the innate immune system and the elucidation of arenavirus pathogenesis.

    Importance Distinct innate immune responses are observed when hosts are infected with different arenaviruses. It has been widely accepted that NP and certain Z proteins of arenaviruses inhibit the RLR/MAVS signaling pathway. The viral components responsible for the activation of RLR/MAVS signaling pathway remain to be determined. In the current study, we demonstrate for the first time that the Lp of MOPV, an OW arenavirus, can activate the RLR/MAVS signaling pathway and thus induce the production of IFN-I. Based on our results, we proposed that dynamic interactions exist among Lp-produced RNA, NP and the RLR/MAVS signaling pathway, and the outcome of these interactions may determine the final IFN-I response pattern: elevated or declined. Our study here provided a possible explanation for how IFN-I can become activated during arenavirus infection and may help gain insights into the interactions that form between different arenavirus components and the innate immune system.

  • Canonical and variant forms of histone H3 are deposited onto the human cytomegalovirus genome during lytic and latent infections [PublishAheadOfPrint]

  • Chromatin is the nucleoprotein complex that protects and compacts eukaryotic genomes. It is responsible for a large part of the epigenetic control of transcription. The genomes of DNA viruses such as human cytomegalovirus (HCMV) are devoid of histones within virions but are chromatinized and epigenetically regulated following delivery to the host cell nucleus. How chromatin is initially assembled on viral genomes, and which variant forms of the core histone proteins are deposited is incompletely understood. We monitored the deposition of both ectopically expressed and endogenous histone H3.1/2 and H3.3 during lytic and latent HCMV infections. Here we show both are deposited on HCMV genomes during lytic and latent infections suggesting similar mechanisms of viral chromatin assembly during the different infection types and indicating both canonical and variant core histones may be important modulators of infecting viral genomes. We further show that association of both H3.1/2 and H3.3 occurs independent of viral DNA synthesis or de novo viral gene expression, implicating cellular factors and/or virion components in the formation of chromatin on virion-delivered genomes during both lytic and latent infections.

    Importance It is well established that infecting herpesvirus genomes are chromatinized upon entry into the host cell nucleus. Why or how this occurs is a mystery. It is important to know why they are chromatinized in order to better understand cellular pathogen recognition (DNA sensing) pathways, viral fate determinations (lytic or latent), and to anticipate how artificially modulating chromatinization may impact viral infections. It is important to know how they are chromatinized in order to potentially modulate the process for therapeutic effect. Our work showing HCMV genomes are loaded with canonical and variant H3 histones during both lytic and latent infections strengthens the hypothesis that chromatinization pathways are similar between the two infection types, implicates virion or cellular factors in this process, and exposes the possibility that histone variants, in addition to post-translational modification, may impact viral gene expression. These revelations are important to understanding and intelligently intervening in herpesvirus infections.

  • Increased Protein Degradation Improves Influenza Virus Nucleoprotein-specific CD8+ T cell Activation in vitro but not in C57BL/6 mice [PublishAheadOfPrint]

  • Due to antigenic drift of influenza viruses, seasonal influenza vaccines need to be updated annually. These vaccines are based on predictions of strains likely to circulate in the next season. However, vaccine efficacy is greatly reduced in case of a mismatch between circulating and vaccine strains. Furthermore, novel antigenically distinct influenza viruses are introduced into the human population from animal reservoirs occasionally and may cause pandemic outbreaks. To dampen the impact of seasonal and pandemic influenza, vaccines that induce broadly protective and long lasting immunity are preferred. Because influenza virus-specific CD8+ T cells are mainly directed against relatively conserved internal proteins, like nucleoprotein (NP), they are highly cross-reactive and afford protection against infection with antigenically distinct influenza virus strains, so-called heterosubtypic immunity. Here, we used modified vaccinia virus Ankara (MVA) as a vaccine vector for the induction of influenza virus NP-specific CD8+ T cells. To optimize induction of CD8+ T cell responses, we made several modifications to NP, which aimed at retaining the protein in the cytosol or targeting it to the proteasome. We hypothesized that these strategies would increase antigen processing and presentation and thus improve induction of CD8+ T cell responses. We showed that NP proteins with increased degradation rates improved CD8+ T cell activation in vitro, if the amount of antigen was limited or if CD8+ T cells were of low functional avidity. However, after immunization of C57BL/6 mice, no differences were detected between modified NP and NPwt, since NPwt already induced optimal CD8+ T cell responses.

    IMPORTANCE: Due to the continuous antigenic drift of seasonal influenza viruses and the threat of a novel pandemic, there is a great need for the development of novel influenza vaccines that offer broadly protective immunity against multiple subtypes. CD8+ T cells can provide immunity against multiple subtypes of influenza viruses by the recognition of relatively conserved internal antigens. In this study, we aimed at optimizing the CD8+ T cell response to influenza A virus by making modifications to influenza A virus nucleoprotein (NP) expressed from the vaccine vector modified vaccinia virus Ankara (MVA). These modifications resulted in increased antigen degradation, thereby producing elevated levels of peptides that can be presented on MHC class I molecules to CD8+ T cells. Although we were unable to increase the NP-specific immune response in the mouse strain used, this approach may have benefits for vaccine development using less immunogenic proteins.

  • Vif Proteins from Diverse HIV/SIV Lineages have Distinct Binding Sites in A3C [PublishAheadOfPrint]

  • Lentiviruses have evolved the Vif protein to counteract APOBEC3 (A3) restriction factors by targeting them for proteasomal degradation. Previous studies have identified important residues in the interface of HIV-1 Vif and human APOBEC3C (hA3C) or human APOBEC3F (hA3F). However, the interaction between primate A3C proteins with HIV-1 Vif or natural HIV-1 Vif variants is still poorly understood. Here, we report that HIV-1 Vif is inactive against A3Cs of rhesus macaques (rhA3C), sooty mangabey monkeys (smmA3C) and African green monkeys, while HIV-2, SIVagm and SIVmac Vif proteins efficiently mediate the depletion of all tested A3Cs. We identified that residues N/H130 and Q133 in rhA3C and smmA3C are determinants for this HIV-1 Vif triggered counteraction. We also found that the HIV-1 Vif interaction sites in helix 4 of hA3C and hA3F differ. Vif alleles from diverse HIV-1 subtypes were tested for degradation activities to hA3C. The subtype F-1 Vif was identified to be inactive to degrade hA3C and hA3F. The residues that determined F-1 Vif inactivity to A3C/A3F were located at the C-terminal region (K167 and D182). Structural analysis of F-1 Vif revealed that impairing the internal salt bridge of E171-K167 restored its reduction capacities to A3C/A3F. Furthermore, we found that D101 could also form an internal interaction with K167. Replacing D101 by glycine and R167 by lysine in NL4-3 Vif impaired its counteractivity to A3F and A3C. This finding indicates that internal interactions outside the A3 binding region in HIV-1 Vif influence the capacity to induce degradation of A3C/A3F.

    IMPORTANCE The APOBEC3 restriction factors can serve as potential barriers to lentiviral cross-species transmissions. Vif proteins from lentiviruses counteract APOBEC3 by proteasomal degradation. In this study, we found that monkey-derived APOBEC3C (A3C), rhA3C and smmA3C were resistant to HIV-1 Vif. This was determined by A3C residues N/H130 and Q133. However, HIV-2, SIVagm and SIVmac Vif proteins were found to be able to mediate the depletion of all tested primate A3C proteins. In addition, we identified a natural HIV-1 Vif (F-1 Vif) that was inactive to hA3C/hA3F. Here we provide for the first time a model that explains how an internal salt bridge of E171-K167-D101 influences Vif-mediated degradation of hA3C/hA3F. This finding provides a novel way to develop HIV-1 inhibitors by targeting the internal interactions of the Vif protein.

  • Sindbis virus can exploit a host antiviral protein to evade immune surveillance [PublishAheadOfPrint]

  • Viral infection induces production of type I interferons (IFNs), which stimulate the expression of a variety of antiviral factors to inhibit viral replication. To establish effective infection, viruses need to develop strategies to evade the immune responses. A neurovirulent Sindbis virus strain with neuroinvasive properties (SVNI) causes lethal encephalitis in mice, and its replication in cultured cells is inhibited by the zinc-finger antiviral protein (ZAP), a host factor that specifically inhibits the replication of certain viruses by binding to the viral mRNAs and repressing the translation and promoting the degradation of target mRNA. We report here that MEF cells from ZAP knockout mice supported more efficient SVNI replication than wildtype cells. SVNI infection of 10 day-old suckling mice led to reduced survival in the knockout mice. Unexpectedly, however, SVNI infection of 23 day-old weanling mice, whose immune system is more developed than the suckling mice, resulted in significantly improved survival in ZAP knockout mice. Further analyses revealed that in the weanling knockout mice, SVNI replicated more efficiently in lymphoid tissues at early times postinfection, and induced higher levels of IFN production, which restricted viral spread to the central nervous system. Blocking IFN activity through the use of receptor neutralizing antibodies rendered knockout mice more sensitive to SVNI infection than wildtype mice. These results uncover a mechanism by which SVNI exploits a host antiviral factor to evade innate immune surveillance.

    Importance Sindbis virus, a prototypic member of the Alphavirus genus, has been used to study the pathogenesis of acute viral encephalitis in mice for many years. How the virus evades immune surveillance to establish effective infection is largely unknown. ZAP is a host antiviral factor that potently inhibits Sindbis virus replication in cell culture. Here we show that infection of ZAP knockout suckling mice with a neuroinvasive Sindbis strain (SVNI) led to faster disease progression. However, SVNI infection of weanling mice led to slower disease progression in knockout mice. Further analyses revealed that in weanling knockout mice, SVNI replicated more efficiently in lymphoid tissues at early times postinfection, and induced higher levels of interferon production, which restricted viral spread to the central nervous system. These results uncover a mechanism by which SVNI exploits a host antiviral factor to evade innate immune surveillance and allow enhanced neuroinvasion.

  • Vaccinia virus uses retromer-independent cellular retrograde transport pathways to facilitate the wrapping of intracellular mature virions during viral morphogenesis [PublishAheadOfPrint]

  • Poxviruses such as Vaccinia virus (VACV) undertake a complex cytoplasmic replication cycle which involves morphogenesis through four distinct virion forms, and includes a crucial "wrapping" step whereby intracellular mature virions (IMVs) are wrapped in two additional membranes to form intracellular enveloped virions (IEVs). To determine if cellular retrograde transport pathways were required for this wrapping step we examined VACV morphogenesis in cells with reduced expression of the tetrameric tethering factor complex GARP (Golgi-associated retrograde pathway complex), a central component of retrograde transport. VACV multi-step replication was significantly impaired in cells transfected with siRNA targeting the GARP complex or in cells with a mutated GARP complex. Detailed analysis revealed that depletion of the GARP complex resulted in a reduction in the number of IEVs, thereby linking retrograde transport with the wrapping of IMVs. In addition foci of viral wrapping membrane proteins without an associated internal core accumulated in cells with a mutated GARP complex, suggesting that impaired retrograde transport uncouples nascent IMVs from the IEV membranes at the site of wrapping. Finally, small molecule inhibitors of retrograde transport strongly suppressed VACV multi-step growth in vitro and reduced weight loss and clinical signs in an in vivo murine model of systemic poxviral disease. This work links cellular retrograde transport pathways with morphogenesis of poxviruses and identifies a panel of novel inhibitors of poxvirus replication.

    Importance: Cellular retrograde transport pathways traffic cargo from endosomes to the trans-Golgi network and are a key part of the intracellular membrane network. This work reveals the prototypic poxvirus Vaccinia virus (VACV) exploits cellular retrograde transport pathways to facilitate the wrapping of intracellular mature virions and therefore promote the production of extracellular virus. Inhibition of retrograde transport by small molecule inhibitors reduced replication of VACV in cell culture and alleviated disease in mice experimentally infected with VACV. This research provides fundamental new knowledge about the "wrapping" step of poxvirus morphogenesis, furthers our knowledge of the complex cellular retrograde pathways, and identifies a new group of anti-poxvirus drugs.

  • Visualizing herpesvirus procapsids in living cells [PublishAheadOfPrint]

  • A complete understanding of herpesvirus morphogenesis requires studies of capsid assembly dynamics in living cells. Although fluorescent tags fused to the VP26 and pUL25 capsid proteins are available, neither of these components are present on the initial capsid assembly: the procapsid. To make procapsids accessible to live-cell imaging, we made a series of recombinant pseudorabies viruses that encoded GFP fused in-frame to the internal capsid scaffold and maturation protease. One recombinant, a GFP-VP24 fusion, maintained wild-type propagation kinetics in vitro and approximated wild-type virulence in vivo. The fusion also proved well tolerated in herpes simplex virus. Viruses encoding GFP-VP24 along with a traditional capsid reporter fusion (pUL25/mCherry) demonstrated that GFP-VP24 was a reliable capsid marker, and revealed that the protein remained capsid associated following entry into cells and upon nuclear docking. These dual-fluorescent viruses made possible the discrimination of procapsids during infection, and monitoring capsid shell maturation kinetics. The results demonstrate the feasibility of imaging herpesvirus procapsids and their morphogenesis in living cells, and indicate that the encapsidation machinery does not substantially help coordinate capsid shell maturation.

    IMPORTANCE The Herpesviridae family consists of human and veterinary pathogens that cause a wide range of diseases in their respective hosts. These viruses share a structurally-related icosahedral capsid that encases the dsDNA viral genome. The dynamics of capsid assembly and maturation has been inaccessible to examination in living cells. This study has overcome this technical hurdle and provides new insights into this fundamental stage of herpesvirus infection.

  • Releasing the genomic RNA sequestered in the mumps virus nucleocapsid [PublishAheadOfPrint]

  • In a negative strand RNA virus, the genomic RNA is sequestered inside the nucleocapsid when the viral RNA-dependent RNA polymerase uses it as the template for viral RNA synthesis. It must require a conformational change in the nucleocapsid protein (NP) to make the RNA accessible by the viral polymerase during this process. The structure of an empty mumps virus nucleocapsid-like particle is determined to 10.4 AAring; resolution by cryoEM image reconstruction. By modeling the crystal structure of parainfluenza virus 5 into the density, it is shown that the aalpha;-helix close to the RNA became flexible when RNA was removed. Point mutations in this helix resulted in loss of polymerase activities. Since the core of NP is rigid in the nucleocapsid, we suggest that interactions between this region of the mumps virus NP and its polymerase leads to exposure of the sequestered genomic RNA, instead of large NP domain rotations.

    Importance Mumps virus (MuV) infection may cause serious diseases including hearing loss, orchitis, oophoritis, mastitis, and pancreatitis. MuV is a negative strand RNA virus, similar to rabies virus or Ebola virus, that has a unique mechanism of viral RNA synthesis. They all make their own RNA-dependent RNA polymerase (RdRp). The viral RdRp uses the genomic RNA inside the viral nucleocapsid as the template to synthesize viral RNAs. Since the template RNA is always sequestered in the nucleocapsid, the viral RdRp must find a way to open it up in order to gain access to the covered template. Our work reported here shows that a helix structural element in the MuV nucleocapsid protein becomes open when the sequestered RNA is released. The amino acids related to this helix are required for RdRp to synthesize viral RNA. We propose that the viral RdRp pulls this helix open to release the genomic RNA.

  • Multiple Roles of the Cytoplasmic Domain of Herpes Simplex Virus 1 Envelope Glycoprotein D in Infected Cells [PublishAheadOfPrint]

  • Herpes simplex virus 1 (HSV-1) envelope glycoprotein D (gD) plays an essential role in viral entry. The functional regions of gD responsible for viral entry have been mapped to its extracellular domain, whereas the gD cytoplasmic domain plays no obvious role in viral entry. Thus far, the role(s) of the gD cytoplasmic domain in HSV-1 replication remained to be elucidated. In this study, we showed that ectopic expression of gD induced microvilli-like tubular structures at the plasma membrane, which resembled the reported projection structures of the plasma membrane induced in HSV-1-infected cells. Mutations in the arginine cluster (residues 365 to 367) in the gD cytoplasmic domain greatly reduced gD-induced plasma membrane remodeling. In agreement with this, the mutations in the arginine cluster in the gD cytoplasmic domain reduced the number of microvilli-like tubular structures at the plasma membrane in HSV-1-infected cells. In addition, the mutations produced an accumulation of unenveloped nucleocapsids in the cytoplasm, and reduced viral replication and cell-cell spread. These results suggested that the arginine cluster in the gD cytoplasmic domain was required for the efficient induction of plasma membrane projections and viral final envelopment, and these functions of the gD domain may lead to efficient viral replication and cell-cell spread.

    IMPORTANCE The cytoplasmic domain of HSV-1 gD, an essential envelope glycoprotein for viral entry, was reported to promote viral replication and cell-cell spread, but the role(s) of the domain during HSV-1 infection remained unknown. In this study, we clarified two functions of the arginine cluster in the HSV-1 gD cytoplasmic domain, both of which require host cell membrane remodeling; i.e., formation of microvilli-like projections at the plasma membrane and viral final envelopment in HSV-1-infected cells. We also showed that the gD arginine cluster was required for efficient HSV-1 replication and cell-cell spread. This is the first report clarifying not only the functions of the gD cytoplasmic domain but also identifying the gD arginine cluster as the HSV-1 factor responsible for the induction of plasma membrane projections in HSV-1-infected cells. Our results elucidate some of the functions of this multifunctional envelope glycoprotein during HSV-1 infection.

  • Structure of an N276-dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole adjacent to the CD4 Binding Site [PublishAheadOfPrint]

  • All HIV-1 infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap with conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We have previously described an HIV-1 clade C infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier-2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single B cell sorting. We report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody, representative of the early CD4 binding site plasma response in CAP257. A cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similar to HJ16 and 179NC75. This antibody was derived from IGHV3-33 and IGLV3-10 genes, and neutralized RHPA but not the CAP257 transmitted/founder virus. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops, present in almost all HIV-1 strains including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops likely resulted in unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B cell lineage.

    Importance The conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of different antibody lineages to this site remain elusive. Studies have shown that strain-specific antibodies can evolve into broadly neutralizing antibodies, or in some cases act as helper lineages. Therefore characterizing the epitopes of strain-specific antibodies will help to inform the design of HIV-1 immunogens to elicit broadly neutralizing antibodies. In this study we isolate a narrowly neutralizing N276 glycan-dependent antibody, and use x-ray crystallography and viral deep sequencing to describe how gp120 lacking glycans in V5 might have elicited these early glycan-dependent CD4 binding site antibodies. These data highlight the importance of glycan holes in the elicitation of B cell lineages targeting the CD4 binding site.

  • Residues K465 and G467 Within The Cytoplasmic Domain of GP2 Critically Contribute To LCMV Persistence In Mice [PublishAheadOfPrint]

  • Several arenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever disease in humans and pose serious public health concerns in their endemic regions. Moreover, mounting evidence indicates that the worldwide-distributed, prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), is a neglected human pathogen of clinical significance. We have documented that a recombinant LCMV containing the glycoprotein gene (GPC) of LASV within the backbone of the immunosuppressive Clone 13 (Cl-13) variant of Armstrong strain of LCMV (rCl-13/LASV-GPC) exhibited Cl-13 like growth properties in cultured cells but in contrast to Cl-13, rCl-13/LASV-GPC was unable to establish persistence in immune competent adult mice, which prevented its use for some in vivo experiments. Recently, V459K and K461G mutations within the GP2 cytoplasmic domain (CD) of rCl-13/LASV-GPC were shown to increase rCl-13/LASV-GPC infectivity in mice. Here we generated rCl-13(GPC/VGKS) by introducing the corresponding revertant mutations K465V and G467K within GP2 of rCl-13 and show that rCl-13(GPC/VGKS) was unable to persist in mice. K465V and G467K mutations did not affect GPC processing, virus RNA replication or gene expression. In addition, rCl-13(GPC/VGKS) grew to high titer in cultured cell lines and in immunodeficient mice. Further analysis revealed that rCl-13(GPC/VGKS) infected fewer splenic plasmacytoid dendritic cells when compared to rCl-13, yet both viruses there induced a similar type I interferon response in mice. Our findings have identified novel viral determinants of Cl-13 persistence and also revealed that virus GPC-host interactions yet to be elucidated critically contribute to Cl-13 persistence.

    IMPORTANCE The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV) provides investigators with a superb experimental model system to investigate virus-host interactions. Armstrong strain (ARM) of LCMV causes an acute infection whereas its' derivative, Clone 13 (Cl-13), causes a persistent infection. Mutations F260L and K1079Q within GP1 and L polymerase, respectively, have been shown to play a critical role in Cl-13's ability to persist in mice. However, there is an overall lack of knowledge about other viral determinants required for Cl-13's persistence. Here we report that mutations K465V and G467K within the cytoplasmic domain of Cl-13 GP2 resulted in a virus, rCl-13(GPC/VGKS), that failed to persist in mice despite exhibiting Cl-13 wild type-like fitness in cultured cells and immune compromised mice. This finding has uncovered novel viral determinants of viral persistence, and a detailed characterization of rCl-13(GPC/VGKS) can provide novel insights about the mechanisms underlying virus persistent infection.

  • Effect of host species on the topography of fitness landscape for a plant RNA virus [PublishAheadOfPrint]

  • Adaptive fitness landscapes are a fundamental concept in evolutionary biology that relate the genotype of individuals with their fitness. At the end, the evolutionary fate of evolving populations depends on the topography of the landscape, that is, the number of accessible mutational pathways and of possible fitness peaks (i.e, adaptive solutions). For long time, fitness landscapes were only theoretical constructions due to a lack of precise information on the mapping between genotypes and phenotypes. In recent years, however, efforts have been devoted to characterize the properties of empirical fitness landscapes for individual proteins or for microbes adapting to artificial environments. In a previous study, we had characterized the properties of the empirical fitness landscape defined by the first five mutations fixed during adaptation of tobacco etch potyvirus (TEV) to a new experimental host, Arabidopsis thaliana. Here we evaluate the topography of this landscape in the ancestral host Nicotiana tabacum. Comparing the topographies of the landscape in the two hosts, we found that some features remain similar, such as the existence of fitness holes and the prevalence of epistasis, including cases of sign and of reciprocal sign that create rugged, uncorrelated and highly random topographies. However, we also observed significant differences in the fine-grained details among both landscapes due to changes in the fitness and epistatic interactions of some genotypes. Our results support the idea that not only fitness tradeoffs between hosts but also topographical incongruences among fitness landscapes in alternative hosts may contribute to virus specialization.

    IMPORTANCE Despite its importance for understanding virus' evolutionary dynamics, very little is known about the topography of virus adaptive fitness landscapes and even less is known about the effect that different host species and environmental conditions may have of this topography. To bring this gap, we have evaluated the topography of a small fitness landscape formed by all genotypes that result from every possible combination of the five mutations fixed during adaptation of TEV to its novel host A. thaliana. To assess the effect that host species may have on this topography, we evaluated the fitness of every genotype both in the ancestral and novel hosts. We found both landscapes share some macroscopic properties such as the existence of holes and being highly rugged and uncorrelated, yet they differ in microscopic details due to changes in the magnitude and sign of fitness and epistatic effects.

  • Virologic differences do not fully explain the diversification of swine influenza viruses in the United States [PublishAheadOfPrint]

  • Influenza A(H1N1) viruses entered the US swine population following the 1918 pandemic and remained genetically stable for roughly 80 years. In 1998, there was an outbreak of influenza-like illness among swine caused by A(H3N2) viruses containing the triple reassortant internal gene (TRIG) cassette. Following the TRIG cassette emergence, numerous reassortant viruses were isolated in nature suggesting the TRIG virus had an enhanced ability to reassort compared to the classical swine virus. This study was designed to quantify the relative reassortment capacity of classical and TRIG swine viruses. Reverse genetic viruses were generated from the classical H1N1 virus A/swine/MN/37866/1999 (MN/99), the TRIG virus A/swine/NC/18161/2002 (NC/02), and a seasonal human H3N2 virus A/TX/6/1996 (TX/96) to measure in vitro reassortment and growth potentials. After co-infection with NC/02 or MN/99 and TX/96, H1/H3 double positive cells were identified. Delayed TX/96 infection was fully excluded by both swine viruses equally. We then analyzed reassortant H3 viruses. 77 of 81 (95.1%) TX/96-NC/02 reassortants contained at least one polymerase gene segment from NC/02 whereas only 34 of 61 (55.7%) MN/99-TX/96 reassortants contained at least one polymerase gene segment from MN/99. Additionally, 38 of 81 (46.9%) NC/02-TX/96 reassortants contained all NC/02 polymerase gene segments while none of the MN/99-TX/96 reassortants contained all MN/99 polymerase genes. There were 21 H3 reassortants between MN/99-TX/96 compared to only 17 H3 reassortants between NC/02-TX/96. Overall, the results indicated that there are no distinct differences in the ability of the TRIG to reassort with a human virus compared to the classical swine virus.

    IMPORTANCE There appear to be no differences between the ability of classical swine and TRIG swine viruses to exclude a second virus suggesting that under the right circumstances both viruses have similar opportunities to reassort. The increased percentage of TRIG polymerase gene segments in reassortant H3 viruses indicate that they may be more compatible with gene segments from other viruses, however this needs to be further investigated. Nevertheless, the classical swine virus also showed the ability to reassort suggesting that factors other than reassortment capacity alone is responsible for the different epidemiology's of TRIG and classical swine viruses. The post-TRIG diversity was likely driven by increased intensive farming practices rather than virologic properties. Our results indicate that host ecology can be a significant factor in viral evolution.

  • Differential Control of BST2 restriction and Plasmacytoid Dendritic Cell Antiviral Response by Antagonists encoded by HIV-1 Group M and O Strains [PublishAheadOfPrint]

  • BST2/Tetherin is a type-I interferon (IFN-I) stimulated host factor that restricts the release of HIV-1 by entrapping budding virions at the cell surface. This membrane-associated protein can also engage and activate the plasmacytoid dendritic cell (pDC)-specific ILT7 inhibitory receptor to downregulate the IFN-I response by pDCs. Pandemic HIV-1 group M uses Vpu (M-Vpu) to counteract the two BST2 isoforms (long and short) that are expressed in human cells. M-Vpu efficiently downregulates surface long BST2, while it displaces short BST2 molecules away from viral assembly sites. We recently found that this attribute is used by M-Vpu to activate the BST2/ILT7-dependent negative-feedback pathway and to suppress pDC IFN-I responses during sensing of infected cells. However, whether this property is conserved with the endemic HIV-1 group O, which has evolved Nef (O-Nef) to counteract specifically the long BST2 isoform, remains unknown. In the present study, we validated that O-Nefs have the capacity to downregulate surface BST2 and enhance HIV-1 particle release, although less efficiently than M-Vpu. In contrast to M-Vpu, O-Nef did not enhance viral spread in T cell culture nor displace short BST2 from viral assembly sites to prevent its occlusion by tethered HIV-1 particles. Consequently, O-Nef impairs the ability of BST2 to activate negative ILT7 signaling to suppress the IFN-I response by pDC-containing PBMCs during sensing of infected cells. These distinctive features of BST2 counteraction by O-Nefs may in part explain the limited spread of the HIV-1 group O in the human population.

    Importance: Geographical distribution and prevalence of different HIV-1 groups show large variation. Understanding drivers of distinctive viral spread may aid the development of therapeutic strategies for controlling HIV-1 pandemic strains spread. Differential spread of HIV-1 groups appears linked to their capacities to antagonize the long and short isoforms of the BST2 restriction factor. We found that the endemic HIV-1 group O-encoded BST2 antagonist, Nef, is unable to counteract the restriction mediated by short BST2, a condition that impairs its ability to activate ILT7 and suppress pDC antiviral responses. This is in contrast to the pandemic HIV-1 group M-specified BST2 countermeasure, Vpu, which displays a diverse array of mechanisms to counteract short and long BST2, an attribute that allows an effective control of pDC antiviral responses. These findings may help explain the limited spread of HIV-1 group O as well as the continued predominance of HIV-1 group M throughout the world.

  • Imperfect symmetry of Sp1 and core promoter elements regulates early and late viral gene expression of the bidirectional BK polyomavirus non-coding control region [PublishAheadOfPrint]

  • Rearrangements or point mutations in the non-coding control region (NCCR) of BK polyomavirus (BKPyV) have been associated with higher viral loads and more pronounced organ pathology in immunocompromised patients. The respective mutations affect a multitude of transcription factor binding sites (TFBS), but consistently cause increased expression of the early viral gene region (EVGR) at the expense of late viral gene region (LVGR) expression. By mutating TFBS, we identified three phenotypic groups leading to strong, intermediate, or impaired EVGR expression and corresponding BKPyV replication. Unexpectedly, Sp1 TFBS mutants either activated or inhibited EVGR expression, when located proximal to the LVGR (SP1-4) or the EVGR (SP1-2), respectively. We now demonstrate that the bidirectional balance of EVGR and LVGR expression is dependent on affinity, strand orientation, and number of Sp1 sites. Swapping the LVGR-proximal high-affinity SP1-4 with the EVGR-proximal low-affinity SP1-2, in-site strand flipping, or inserting an additional SP1-2 site caused a rearranged NCCR phenotype of increased EVGR expression and faster BKPyV replication. 5rrsquo; -RACE revealed an imperfect symmetry between the EVGR- and LVGR-proximal parts of the NCCR, consisting of TATA- and TATA-like elements, initiator elements, and downstream promoter elements. Mutation or deletion of the archetypal LVGR promoter, as found in activated NCCR variants, abrogated LVGR expression, which could be restored by providing large T-antigen in trans. Thus, whereas Sp1 sites control the initial EVGR-LVGR expression balance, LTag expression can override inactivation of the LVGR promoter and acts as a key driver of LVGR expression, independent of the Sp1 sites and core promoter elements.

    IMPORTANCE Polyomaviridae currently comprise more than 70 members including 13 human PyVs, all of which share the bidirectional genome organization mediated by the NCCR that determines species and host cell specificity, persistence, replication, and virulence. Here, we demonstrate that the BKPyV NCCR is fine-tuned by an imperfect symmetry of core promoter elements centering around TATA- and TATA-like sequences close to the EVGR and LVGR, respectively, which are governed by directionality and affinity of two Sp1 sites. The data indicated that the BKPyV NCCR is poised towards EVGR expression that can be readily unlatched by a simple switch affecting Sp1 binding. The resulting LTag as the major EVGR protein drives viral genome replication, renders subsequent LVGR expression independent of archetypal promoter elements, and can overcome enhancer/promoter mutations and deletions. The data are pivotal for understanding how human PyV NCCRs mediate secondary host cell specificity, reactivation and virulence in their natural hosts.

  • Non-coding subgenomic flavivirus RNA is processed by the mosquito RNAi machinery and determines West Nile virus transmission by Culex pipiens mosquitoes [PublishAheadOfPrint]

  • Flaviviruses, such as Zika, Yellow fever, dengue and West Nile (WNV) are a serious concern for human health. Flaviviruses produce an abundant non-coding subgenomic flavivirus RNA (sfRNA) in infected cells. SfRNA results from stalling of the host 5rrsquo; -3rrsquo; exoribonuclease XRN1/Pacman on conserved RNA structures in the 3rrsquo; UTR of the viral genomic RNA. SfRNA production is conserved in insect-specific, mosquito-borne, tick-borne and no-known-vector flaviviruses, suggesting a pivotal role for sfRNA in the flavivirus life cycle. Here we investigated the function of sfRNA during WNV infection of Culex pipiens mosquitoes and evaluated its role in determining vector competence. An sfRNA1-deficient WNV was generated that displayed similar growth kinetics as wildtype WNV in both RNAi-competent and -compromised mosquito cell lines. Small RNA deepsequencing of WNV-infected mosquitoes indicated an active siRNA-based antiviral response for both the wildtype and sfRNA1-deficient virus. Additionally, we provide the first evidence that sfRNA is an RNAi substrate in vivo. Two reproducible small RNA hotspots within the 3rrsquo; UTR/sfRNA of the wildtype virus mapped to RNA stem loops SL-III and 3'SL, which stick out of the 3D sfRNA structure model. Importantly, we demonstrate that sfRNA-deficient WNV displays significantly decreased infection and transmission rates in vivo when administrated via the blood meal. Finally, we show that transmission and infection rates are not affected by sfRNA after intrathoracic injection, thereby identifying sfRNA as a key driver to overcome the mosquito midgut infection barrier. This is the first report to describe a key biological function of sfRNA for flavivirus infection of the arthropod vector, providing an explanation for the strict conservation of sfRNA production.

    IMPORTANCE Understanding the flavivirus transmission cycle is important to identify novel targets to interfere with disease and to aid development of virus control strategies. Flaviviruses produce an abundant, non-coding viral RNA called sfRNA in both arthropod and mammalian cells. To evaluate the role of sfRNA in flavivirus transmission, we infected mosquitoes with the flavivirus West Nile and an sfRNA-deficient mutant West Nile virus. We demonstrate that sfRNA determines the infection and transmission rates of West Nile virus in Culex pipiens mosquitoes. Comparison of infection via the blood meal versus intrathoracic injection, which bypasses the midgut, revealed that sfRNA is important to overcome the mosquito midgut barrier. We also show that sfRNA is processed by the antiviral RNA interference machinery in mosquitoes. This is the first report to describe a pivotal biological function of sfRNA in arthropods. The results explain why sfRNA production is evolutionary conserved.

  • Production of cytomegalovirus dense bodies by scalable bioprocess methods maintains immunogenicity and improves neutralizing antibody titers. [PublishAheadOfPrint]

  • With the goal of developing a virus-like particle-based vaccine based on dense bodies (DB) produced by human cytomegalovirus (HCMV) infections, we evaluated scalable culture, isolation, and inactivation methods, and applied technically advanced assays to determine relative purity, composition, and immunogenicity of DB particles. Our results increase our understanding on the benefits and disadvantages of methods to recover immunogenic DB and inactivate contaminating viral particles. Our results indicate: 1) HCMV strain Towne replicates in MRC-5 fibroblasts grown on microcarriers, 2) DB particles recovered from 2-bromo-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole riboside (BDCRB)-treated cultures and purified by tangential flow filtration (TFF-DB) or glycerol tartrate gradient sedimentation (GT-DB) constitute 92% or 98%, respectively, of all particles in the final product, 3) epithelial cell trophic DB particles are recovered from a single round of coinfection by AD169 and Towne strain viruses, consistent with complementation between UL130 and UL131A expressed by these strains and restoration of gH/gL/UL128-UL131A (gH-pentamer), 4) equivalent neutralizing antibody titers are induced in mice following immunization with epithelial cell tropic DB or gH-pentamer-deficient DB preparations, 5) UV-inactivation of residual virus in GT-DB or TFF-DB preparations retained immunogenicity and induction of neutralizing antibody preventing epithelial cell entry, 6) GT-DB and TFF-DB induced cellular immune responses to multiple HCMV peptides. Collectively, this work provides a foundation for future development of DB as an HCMV-based particle vaccine.

    Importance Development of a vaccine to prevent congenital HCMV infection remains a high priority. Human cytomegalovirus-derived noninfectious particles, or dense bodies, may constitute a safe vaccine strategy that mimics natural infection. The standard approach for purification of virus particles has been a multiple-step, complex gradient that presents a potential barrier to production scale-up and commercialization. Here we employ an approach that combines treatment with an anti-viral terminase inhibitor and purification by a simplified process to produce a vaccine candidate providing broad antiviral humoral and cellular immunity as a foundation for future development.

  • Lineage-specific differences in the gp120 Inner Domain Layer 3 of Human and Simian Immunodeficiency Viruses [PublishAheadOfPrint]

  • Binding of HIV-1 and SIV gp120 exterior envelope glycoprotein to CD4 triggers conformational changes in gp120 that promote its interaction with one of the chemokine receptors, usually CCR5, ultimately leading to gp41-mediated virus-cell membrane fusion and entry. We previously described that topological Layers (Layer 1, Layer 2 and Layer 3) in the gp120 inner domain contribute to gp120-trimer association in the unliganded state but also help secure CD4 binding. Relative to Layer 1 of HIV-1 gp120, the SIVmac239 gp120 Layer 1 plays a more prominent role in maintaining gp120-trimer association but is minimally involved in promoting CD4 binding, which could be explained by the existence of a well-conserved Tryptophan 375 (Trp 375) in HIV-2/SIVsmm. Here we investigated the role of SIV Layer 3 on viral entry, cell-to-cell fusion and CD4 binding. We observed that a network of interactions involving some residues of the bbeta;8-aalpha;5 region in SIVmac239 Layer 3 may contribute to CD4 binding by helping shape the nearby Phe 43 cavity which directly contacts CD4. In summary, our results suggest that SIV Layer 3 has a greater impact on CD4 binding than in HIV-1. This work defines lineage-specific differences in Layer 3 from HIV-1 and SIV.

    IMPORTANCE CD4-induced conformational changes in the gp120 inner domain involve rearrangements between three topological layers. While the role of Layers 1-3 for HIV-1 and 1-2 for SIV on gp120 transition to the CD4-bound conformation has been reported, the role of SIV Layer 3 remains unknown. Here we report that SIV Layer 3 has a greater impact on CD4 binding than in HIV-1 gp120. This work defines lineage specific differences in Layer 3 from HIV-1 and SIV.

  • In vitro studies show that sequence variability contributes to marked variation in Hepatitis B virus replication, protein expression and function observed across genotypes. [PublishAheadOfPrint]

  • The hepatitis B virus (HBV) exists as 9 major genotypes (A to I), one minor strain (designated J) and multiple subtypes. Marked differences in HBV natural history, disease progression and treatment response are exhibited by many of these genotypes and subtypes. For example, HBV genotype C is associated with later HBeAg seroconversion and high rates of liver cancer compared to other HBV genotypes, whereas genotype A2 is rarely associated with HBeAg negative disease or liver cancer. The reasons for these and other differences in HBV natural history are yet to be determined but could in part be due to sequence differences in the HBV genome that alter replicative capacity and / or gene expression. Direct comparative studies on HBV replication and protein expression have been limited to date, due largely to the absence of infectious HBV cDNA clones for each of the HBV genotypes present in the same genetic arrangement. We have produced replication competent infectious cDNA clones of the most common subtypes of genotypes A to D, namely A2, B2, C2, D3 and the minor strain J and compared their HBV replication phenotype using transient transfection models. We identified striking differences in HBV replicative capacity as well as hepatitis B e antigen (HBeAg) and surface (HBsAg) protein expression across genotypes, which may in part be due to sequence variability in regulatory regions of the HBV genome. Functional analysis showed that sequence differences in the major upstream regulatory region across genotypes impacted promoter activity.

    Importance There have been very few studies directly comparing the replication phenotype of different HBV genotypes, for which there are marked differences in natural history and disease progression worldwide. We have generated replication competent 1.3 mer cDNA clones of the major genotypes A2, B2, C2, D3, as well as a recently identified strain J, and identified striking differences in replicative capacity and protein expression that may contribute to some of the observed differences in HBV natural history observed globally.

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