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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]

  • MAVS Coordination of Antiviral Innate Immunity [Gem]

  • RNA virus infection is sensed in the cytoplasm by the retinoic acid-inducible gene I (RIG-I)-like receptors. These proteins signal through the host adaptor protein MAVS to trigger the antiviral innate immune response. Here, we describe how MAVS subcellular localization impacts its function and the regulation underlying MAVS signaling. We propose a model to describe how the coordination of MAVS functions at the interface between the mitochondria and the mitochondrion-associated endoplasmic reticulum (ER) membrane programs antiviral signaling.

  • Ecological Drivers of Virus Evolution: Astrovirus as a Case Study [Gem]

  • Although RNA viruses exhibit a high frequency of host jumps, major differences exist among the different virus families. Astroviruses infect a wide range of hosts, affecting both public health systems and economic production chains. Here we delineate the ecological and adaptive processes that drive the cross-species transmission of astroviruses. We observe that distinct transmission zones determine the prevailing astrovirus host and virus diversity, which in turn suggests that no single host group (e.g., bats) can be the natural reservoir, as illustrated through our phylogenetic analysis.

  • Fluorescence-Activated Cell Sorting-Based Analysis Reveals an Asymmetric Induction of Interferon-Stimulated Genes in Response to Seasonal Influenza A Virus [Cellular Response to Infection]

  • Influenza A virus (IAV) infection provokes an antiviral response involving the expression of type I and III interferons (IFN) and IFN-stimulated genes (ISGs) in infected cell cultures. However, the spatiotemporal dynamics of the IFN reaction are incompletely understood, as previous studies investigated mainly the population responses of virus-infected cultures, although substantial cell-to-cell variability has been documented. We devised a fluorescence-activated cell sorting-based assay to simultaneously quantify expression of viral antigens and ISGs, such as ISG15, MxA, and IFIT1, in IAV-infected cell cultures at the single-cell level. This approach revealed that seasonal IAV triggers an unexpected asymmetric response, as the major cell populations expressed either viral antigen or ISG, but rarely both. Further investigations identified a role of the viral NS1 protein in blocking ISG expression in infected cells, which surprisingly did not reduce paracrine IFN signaling to noninfected cells. Interestingly, viral ISG control was impaired in cultures infected with avian-origin IAV, including the H7N9 virus from eastern China. This phenotype was traced back to polymorphic NS1 amino acids known to be important for stable binding of the polyadenylation factor CPSF30 and concomitant suppression of host cell gene expression. Most significantly, mutation of two amino acids within the CPSF30 attachment site of NS1 from seasonal IAV diminished the strict control of ISG expression in infected cells and substantially attenuated virus replication. In conclusion, our approach revealed an asymmetric, NS1-dependent ISG induction in cultures infected with seasonal IAV, which appears to be essential for efficient virus propagation.

    IMPORTANCE Interferons are expressed by infected cells in response to IAV infection and play important roles in the antiviral immune response by inducing hundreds of interferon-stimulated genes (ISGs). Unlike many previous studies, we investigated the ISG response at the single-cell level, enabling novel insights into this virus-host interaction. Hence, cell cultures infected with seasonal IAV displayed an asymmetric ISG induction that was confined almost exclusively to noninfected cells. In comparison, ISG expression was observed in larger cell populations infected with avian-origin IAV, suggesting a more resolute antiviral response to these strains. Strict control of ISG expression by seasonal IAV was explained by the binding of the viral NS1 protein to the polyadenylation factor CPSF30, which reduces host cell gene expression. Mutational disruption of CPSF30 binding within NS1 concomitantly attenuated ISG control and replication of seasonal IAV, illustrating the importance of maintaining an asymmetric ISG response for efficient virus propagation.

  • SAMHD1 Limits HIV-1 Antigen Presentation by Monocyte-Derived Dendritic Cells [Spotlight]

  • Monocyte-derived dendritic cells (MDDC) stimulate CD8+ cytotoxic T lymphocytes (CTL) by presenting endogenous and exogenous viral peptides via major histocompatibility complex class I (MHC-I) molecules. MDDC are poorly susceptible to HIV-1, in part due to the presence of SAMHD1, a cellular enzyme that depletes intracellular deoxynucleoside triphosphates (dNTPs) and degrades viral RNA. Vpx, an HIV-2/SIVsm protein absent from HIV-1, antagonizes SAMHD1 by inducing its degradation. The impact of SAMHD1 on the adaptive cellular immune response remains poorly characterized. Here, we asked whether SAMHD1 modulates MHC-I-restricted HIV-1 antigen presentation. Untreated MDDC or MDDC pretreated with Vpx were exposed to HIV-1, and antigen presentation was examined by monitoring the activation of an HIV-1 Gag-specific CTL clone. SAMHD1 depletion strongly enhanced productive infection of MDDC as well as endogenous HIV-1 antigen presentation. Time-lapse microscopy analysis demonstrated that in the absence of SAMHD1, the CTL rapidly killed infected MDDC. We also report that various transmitted/founder (T/F) HIV-1 strains poorly infected MDDC and, as a consequence, did not stimulate CTL. Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyping of T/F alleviated a block in viral entry and induced antigen presentation only in the absence of SAMHD1. Furthermore, by using another CTL clone that mostly recognizes incoming HIV-1 antigens, we demonstrate that SAMHD1 does not influence exogenous viral antigen presentation. Altogether, our results demonstrate that the antiviral activity of SAMHD1 impacts antigen presentation by DC, highlighting the link that exists between restriction factors and adaptive immune responses.

    IMPORTANCE Upon viral infection, DC may present antigens derived from incoming viral material in the absence of productive infection of DC or from newly synthesized viral proteins. In the case of HIV, productive infection of DC is blocked at an early postentry step. This is due to the presence of SAMHD1, a cellular enzyme that depletes intracellular levels of dNTPs and inhibits viral reverse transcription. We show that the depletion of SAMHD1 in DCs strongly stimulates the presentation of viral antigens derived from newly produced viral proteins, leading to the activation of HIV-1-specific cytotoxic T lymphocytes (CTL). We further show in real time that the enhanced activation of CTL leads to killing of infected DCs. Our results indicate that the antiviral activity of SAMHD1 not only impacts HIV replication but also impacts antigen presentation by DC. They highlight the link that exists between restriction factors and adaptive immune responses.

  • Identification of a Novel Hepacivirus in Domestic Cattle from Germany [Genetic Diversity and Evolution]

  • Hepatitis C virus (HCV) continues to represent one of the most significant threats to human health. In recent years, HCV-related sequences have been found in bats, rodents, horses, and dogs, indicating a widespread distribution of hepaciviruses among animals. By applying unbiased high-throughput sequencing, a novel virus of the genus Hepacivirus was discovered in a bovine serum sample. De novo assembly yielded a nearly full-length genome coding for a polyprotein of 2,779 amino acids. Phylogenetic analysis confirmed that the virus represents a novel species within the genus Hepacivirus. Viral RNA screening determined that 1.6% (n = 5) of 320 individual animals and 3.2% (n = 5) of 158 investigated cattle herds in Germany were positive for bovine hepacivirus. Repeated reverse transcription-PCR (RT-PCR) analyses of animals from one dairy herd proved that a substantial percentage of cows were infected, with some of them being viremic for over 6 months. Clinical and postmortem examination revealed no signs of disease, including liver damage. Interestingly, quantitative RT-PCR from different organs and tissues, together with the presence of an miR-122 binding site in the viral genome, strongly suggests a liver tropism for bovine hepacivirus, making this novel virus a promising animal model for HCV infections in humans.

    IMPORTANCE Livestock animals act as important sources for emerging pathogens. In particular, their large herd size and the existence of multiple ways of direct and food-borne infection routes emphasize their role as virus reservoirs. Apart from the search for novel viruses, detailed characterization of these pathogens is indispensable in the context of risk analysis. Here, we describe the identification of a novel HCV-like virus in cattle. In addition, determination of the prevalence and of the course of infection in cattle herds provides valuable insights into the biology of this novel virus. The results presented here form a basis for future studies targeting viral pathogenesis of bovine hepaciviruses and their potential to establish zoonotic infections.

  • Differential Expression of CX3CL1 in Hepatitis B Virus-Replicating Hepatoma Cells Can Affect the Migration Activity of CX3CR1+ Immune Cells [Pathogenesis and Immunity]

  • In addition to stellate cells and immune cells, inflamed hepatocytes and hepatoma cells express various kinds of chemokines that attract various kinds of immune cells. Previously, we reported that hepatitis B virus (HBV) replication can induce physiological stress. The aim of this study was to analyze the effect of chemokines produced by HBV-infected hepatocytes and hepatoma cells. A real-time PCR array targeting genes related to chemokines and enzyme-linked immunosorbent assay (ELISA) were carried out to detect the specific chemokines produced by Huh7 cells and HepG2 cells infected with various HBV genotypes. A migration assay, flow cytometry analysis, and immunohistochemistry were carried out to analyze the candidate immune cells that can affect the immunopathogenesis of HBV infection. The expressions of CX3CL1 mRNA and protein were significantly different among HBV genotypes A, B, and C and control cells (mock) (P llt; 0.05). CD56+ NK cells and CD8+ T cells migrated to the hepatoma cells with HBV replication. Moreover, the migration activity of both immune cells was partially cancelled after the treatment of CX3CL1 neutralizing antibody. The expression level of NKG2D on CX3CR1+ NK cells in HCC with HBV infection was significantly lower than that in hepatocellular carcinoma (HCC) with HCV infection and chronic hepatitis B and C patients (P llt; 0.05). On the other hand, the frequency of PD-1high CX3CR1+ CD8+ T cells in HCC with HBV infection was significantly higher than that in HCC with HCV infection and chronic hepatitis B and C (P llt; 0.05). The expression of CX3CL1 in HBV-replicating hepatocytes and hepatoma cells could contribute to the immunopathogenesis of HBV infection.

    IMPORTANCE The progressions of the disease are significantly different among HBV genotypes. However, it has not been clear that how different HBV genotypes could induce different inflammatory responses. Here, we first report that the levels of expression of CX3CL1 mRNA and protein were significantly different among HBV genotypes A, B, and C and mock. Not only the differential expression of CX3CL1 among the genotypes but also the phenotype of CX3CR1+ NK cells and T cells were gradually changed during the progression of the disease status. In addition to in vitro study, the analysis of immunohistochemistry with human samples and NOG mice with human lymphocytes and hepatoma cells supports this phenomenon. The quantification of CX3CL1 could contribute to better understanding of the disease status of HBV infection. Moreover, modifying CX3CL1 might induce an immune response appropriate to the disease status of HBV infection.

  • Enterovirus 71 Proteins 2A and 3D Antagonize the Antiviral Activity of Gamma Interferon via Signaling Attenuation [Pathogenesis and Immunity]

  • Enterovirus 71 (EV71) infection causes severe mortality involving multiple possible mechanisms, including cytokine storm, brain stem encephalitis, and fulminant pulmonary edema. Gamma interferon (IFN-) may confer anti-EV71 activity; however, the claim that disease severity is highly correlated to an increase in IFN- is controversial and would indicate an immune escape initiated by EV71. This study, investigating the role of IFN- in EV71 infection using a murine model, showed that IFN- was elevated. Moreover, IFN- receptor-deficient mice showed higher mortality rates and more severe disease progression with slower viral clearance than wild-type mice. In vitro results showed that IFN- pretreatment reduced EV71 yield, whereas EV71 infection caused IFN- resistance with attenuated IFN- signaling in IFN regulatory factor 1 (IRF1) gene transactivation. To study the immunoediting ability of EV71 proteins in IFN- signaling, 11 viral proteins were stably expressed in cells without cytotoxicity; however, viral proteins 2A and 3D blocked IFN--induced IRF1 transactivation following a loss of signal transducer and activator of transcription 1 (STAT1) nuclear translocation. Viral 3D attenuated IFN- signaling accompanied by a STAT1 decrease without interfering with IFN- receptor expression. Restoration of STAT1 or blocking 3D activity was able to rescue IFN- signaling. Interestingly, viral 2A attenuated IFN- signaling using another mechanism by reducing the serine phosphorylation of STAT1 following the inactivation of extracellular signal-regulated kinase without affecting STAT1 expression. These results demonstrate the anti-EV71 ability of IFN- and the immunoediting ability by EV71 2A and 3D, which attenuate IFN- signaling through different mechanisms.

    IMPORTANCE Immunosurveillance by gamma interferon (IFN-) may confer anti-enterovirus 71 (anti-EV71) activity; however, the claim that disease severity is highly correlated to an increase in IFN- is controversial and would indicate an immune escape initiated by EV71. IFN- receptor-deficient mice showed higher mortality and more severe disease progression, indicating the anti-EV71 property of IFN-. However, EV71 infection caused cellular insusceptibility in response to IFN- stimulation. We used an in vitro system with viral protein expression to explore the novel IFN- inhibitory properties of the EV71 2A and 3D proteins through the different mechanisms. According to this study, targeting either 2A or 3D pharmacologically and/or genetically may sustain a cellular susceptibility in response to IFN-, particularly for IFN--mediated anti-EV71 activity.

  • Kallikrein-8 Proteolytically Processes Human Papillomaviruses in the Extracellular Space To Facilitate Entry into Host Cells [Virus-Cell Interactions]

  • The entry of human papillomaviruses into host cells is a complex process. It involves conformational changes at the cell surface, receptor switching, internalization by a novel endocytic mechanism, uncoating in endosomes, trafficking of a subviral complex to the Golgi complex, and nuclear entry during mitosis. Here, we addressed how the stabilizing contacts in the capsid of human papillomavirus 16 (HPV16) may be reversed to allow uncoating of the viral genome. Using biochemical and cell-biological analyses, we determined that the major capsid protein L1 underwent proteolytic cleavage during entry. In addition to a dispensable cathepsin-mediated proteolysis that occurred likely after removal of capsomers from the subviral complex in endosomes, at least two further proteolytic cleavages of L1 were observed, one of which was independent of the low-pH environment of endosomes. This cleavage occurred extracellularly. Further analysis showed that the responsible protease was the secreted trypsin-like serine protease kallikrein-8 (KLK8) involved in epidermal homeostasis and wound healing. Required for infection, the cleavage was facilitated by prior interaction of viral particles with heparan sulfate proteoglycans. KLK8-mediated cleavage was crucial for further conformational changes exposing an important epitope of the minor capsid protein L2. Occurring independently of cyclophilins and of furin that mediate L2 exposure, KLK8-mediated cleavage of L1 likely facilitated access to L2, located in the capsid lumen, and potentially uncoating. Since HPV6 and HPV18 also required KLK8 for entry, we propose that the KLK8-dependent entry step is conserved.

    IMPORTANCE Our analysis of the proteolytic processing of incoming HPV16, an etiological agent of cervical cancer, demonstrated that the capsid is cleaved extracellularly by a serine protease active during wound healing and that this cleavage was crucial for infection. The cleavage of L1 is one of at least four structural alterations that prime the virus extracellularly for receptor switching, internalization, and possibly uncoating. This step was also important for HPV6 and HPV18, which may suggest that it is conserved among the papillomaviruses. This study advances the understanding of how HPV16 initially infects cells, strengthens the notion that wounding facilitates infection of epidermal tissue, and may help the development of antiviral measures.

  • The Human Adenovirus Type 5 L4 Promoter Is Negatively Regulated by TFII-I and L4-33K [Genome Replication and Regulation of Viral Gene Expression]

  • The late phase of adenovirus gene expression is controlled by proteins made in the intermediate phase, including L4 proteins of 22,000- and 33,000-Da apparent molecular mass (L4-22K and -33K proteins) that are expressed initially from the L4 promoter (L4P). The L4P is activated by a combination of viral proteins and cellular p53 and is ultimately inhibited again by its own products. Here, we have examined the L4P of human adenovirus type 5 in detail and have defined its transcription start site, which our data suggest is positioned by a weak TATA box. Rather than contributing positively to promoter activity, a putative initiator element at the transcription start site acts as a target for negative regulation imposed on the L4P by cellular TFII-I. We show that this TFII-I inhibition is relieved by one of the previously defined viral activators of the L4P, the E4 Orf3 protein, which alters the pool of TFII-I in the cell. We also explore further the negative regulation of the L4P by its products and show that the L4-33K protein is more significant in this process than L4-22K. It is the combined actions of positive and negative factors that lead to the transient activation of the L4P at the onset of the late phase of adenovirus gene expression.

    IMPORTANCE The adenovirus replication cycle proceeds through multiple phases of gene expression in which a key step is the activation of late-phase gene expression to produce proteins from which progeny particles can be formed. Working with human adenovirus type 5, we showed previously that two proteins expressed from the L4 region of the viral genome perform essential roles in moving the infection on into the late phase; these two proteins are produced by the action of a dedicated promoter, the L4P, and without them the infection does not proceed successfully to progeny generation. In this new work, we delineate further aspects of L4P activity and regulation. Understanding how the L4P works, and how it contributes to activation of the late phase of infection, is important to our understanding of natural infections by the virus, in which late gene expression can fail to occur, allowing the virus to persist.

  • Heterogeneous Nuclear Ribonucleoprotein M Facilitates Enterovirus Infection [Spotlight]

  • Picornavirus infection involves a dynamic interplay of host and viral protein interactions that modulates cellular processes to facilitate virus infection and evade host antiviral defenses. Here, using a proteomics-based approach known as TAILS to identify protease-generated neo-N-terminal peptides, we identify a novel target of the poliovirus 3C proteinase, the heterogeneous nuclear ribonucleoprotein M (hnRNP M), a nucleocytoplasmic shuttling RNA-binding protein that is primarily known for its role in pre-mRNA splicing. hnRNP M is cleaved in vitro by poliovirus and coxsackievirus B3 (CVB3) 3C proteinases and is targeted in poliovirus- and CVB3-infected HeLa cells and in the hearts of CVB3-infected mice. hnRNP M relocalizes from the nucleus to the cytoplasm during poliovirus infection. Finally, depletion of hnRNP M using small interfering RNA knockdown approaches decreases poliovirus and CVB3 infections in HeLa cells and does not affect poliovirus internal ribosome entry site translation and viral RNA stability. We propose that cleavage of and subverting the function of hnRNP M is a general strategy utilized by picornaviruses to facilitate viral infection.

    IMPORTANCE Enteroviruses, a member of the picornavirus family, are RNA viruses that cause a range of diseases, including respiratory ailments, dilated cardiomyopathy, and paralysis. Although enteroviruses have been studied for several decades, the molecular basis of infection and the pathogenic mechanisms leading to disease are still poorly understood. Here, we identify hnRNP M as a novel target of a viral proteinase. We demonstrate that the virus subverts the function of hnRNP M and redirects it to a step in the viral life cycle. We propose that cleavage of hnRNP M is a general strategy that picornaviruses use to facilitate infection.

  • Highly Pathogenic New World and Old World Human Arenaviruses Induce Distinct Interferon Responses in Human Cells [Pathogenesis and Immunity]

  • The arenavirus family includes several important pathogens that cause severe and sometimes fatal diseases in humans. The highly pathogenic Old World (OW) arenavirus Lassa fever virus (LASV) is the causative agent of Lassa fever (LF) disease in humans. LASV infections in severe cases are generally immunosuppressive without stimulating interferon (IFN) induction, a proinflammatory response, or T cell activation. However, the host innate immune responses to highly pathogenic New World (NW) arenaviruses are not well understood. We have previously shown that the highly pathogenic NW arenavirus, Junin virus (JUNV), induced an IFN response in human A549 cells. Here, we report that Machupo virus (MACV), another highly pathogenic NW arenavirus, also induces an IFN response. Importantly, both pathogenic NW arenaviruses, in contrast to the OW highly pathogenic arenavirus LASV, readily elicited an IFN response in human primary dendritic cells and A549 cells. Coinfection experiments revealed that LASV could potently inhibit MACV-activated IFN responses even at 6 h after MACV infection, while the replication levels of MACV and LASV were not affected by virus coinfection. Our results clearly demonstrated that although all viruses studied herein are highly pathogenic to humans, the host IFN responses toward infections with the NW arenaviruses JUNV and MACV are quite different from responses to infections with the OW arenavirus LASV, a discovery that needs to be further investigated in relevant animal models. This finding might help us better understand various interplays between the host immune system and highly pathogenic arenaviruses as well as distinct mechanisms underlying viral pathogenesis.

    IMPORTANCE Infections of humans with the highly pathogenic OW LASV are accompanied by potent suppression of interferon or proinflammatory cytokine production. In contrast, infections with the highly pathogenic NW arenavirus JUNV are associated with high levels of IFNs and cytokines in severe and fatal cases. Arenaviruses initially target macrophages and dendritic cells, which are potent IFN/cytokine-producers. In human macrophages, JUNV reportedly does not trigger IFN responses. We here demonstrated that JUNV activated IFN responses in human dendritic cells. MACV, another highly pathogenic NW arenavirus, also activated IFN responses. LASV did not induce detectable IFN responses, in spite of higher replication levels, and blocked the MACV-triggered IFN response in a coinfection assay. Although these viruses are highly pathogenic to humans, our study highlights distinct innate immune responses to infections with the NW arenaviruses JUNV and MACV and to infection with the OW arenavirus LASV and provides important insights into the virus-host interaction and pathogenesis.

  • The Nectin-4/Afadin Protein Complex and Intercellular Membrane Pores Contribute to Rapid Spread of Measles Virus in Primary Human Airway Epithelia [Virus-Cell Interactions]

  • The discovery that measles virus (MV) uses the adherens junction protein nectin-4 as its epithelial receptor provides a new vantage point from which to characterize its rapid spread in the airway epithelium. We show here that in well-differentiated primary cultures of airway epithelial cells from human donors (HAE), MV infectious centers form rapidly and become larger than those of other respiratory pathogens: human respiratory syncytial virus, parainfluenza virus 5, and Sendai virus. While visible syncytia do not form after MV infection of HAE, the cytoplasm of an infected cell suddenly flows into an adjacent cell, as visualized through wild-type MV-expressed cytoplasmic green fluorescent protein (GFP). High-resolution video microscopy documents that GFP flows through openings that form on the lateral surfaces between columnar epithelial cells. To assess the relevance of the protein afadin, which connects nectin-4 to the actin cytoskeleton, we knocked down its mRNA. This resulted in more-limited infectious-center formation. We also generated a nectin-4 mutant without the afadin-binding site in its cytoplasmic tail. This mutant was less effective than wild-type human nectin-4 at promoting MV infection in primary cultures of porcine airway epithelia. Thus, in airway epithelial cells, MV spread requires the nectin-4/afadin complex and is based on cytoplasm transfer between columnar cells. Since the viral membrane fusion apparatus may open the passages that allow cytoplasm transfer, we refer to them as intercellular membrane pores. Virus-induced intercellular pores may contribute to extremely efficient measles contagion by promoting the rapid spread of the virus through the upper respiratory epithelium.

    IMPORTANCE Measles virus (MV), while targeted for eradication, still causes about 120,000 deaths per year worldwide. The recent reemergence of measles in insufficiently vaccinated populations in Europe and North America reminds us that measles is extremely contagious, but the processes favoring its spread in the respiratory epithelium remain poorly defined. Here we characterize wild-type MV spread in well-differentiated primary cultures of human airway epithelial cells. We observed that viral infection promotes the flow of cytoplasmic contents from infected to proximal uninfected columnar epithelial cells. Cytoplasm flows through openings that form on the lateral surfaces. Infectious-center growth is facilitated by afadin, a protein connecting the adherens junction and the actin cytoskeleton. The viral fusion apparatus may open intercellular pores, and the cytoskeleton may stabilize them. Rapid homogenization of cytoplasmic contents in epithelial infectious centers may favor rapid spread and contribute to the extremely contagious nature of measles.

  • Inefficient Codon Usage Impairs mRNA Accumulation: the Case of the v-FLIP Gene of Kaposi's Sarcoma-Associated Herpesvirus [Genome Replication and Regulation of Viral Gene Expression]

  • Latent Kaposi's sarcoma-associated herpesvirus (KSHV) genomes encode a homolog of cellular FLICE-inhibitory proteins (termed v-FLIP) that activates NF-B and can trigger important proinflammatory and antiapoptotic changes in latently infected cells. The protein is present at very low levels in infection and has generally been difficult to efficiently express in recombinant vectors. Here we show that codon usage in the v-FLIP gene is strikingly suboptimal. Optimization of codon use in expression vectors, as expected, restores efficient protein expression. Surprisingly, however, it also dramatically increases the steady-state level of v-FLIP mRNA, at least in part by increasing mRNA stability. When codon-optimized v-FLIP sequences are reintroduced into intact KSHV genomes, the resulting virus expresses readily detectable monocistronic v-FLIP mRNAs that are undetectable in wild-type (WT) infection by blot hybridization, suggesting that such RNAs are in fact transcribed in WT infection but fail to accumulate. The overexpression of v-FLIP by codon-optimized latent genomes results in a 5- to 7-fold decrement in virus production following lytic induction, indicating that maximizing NF-B signaling is deleterious to induction. These studies provide a clear explanation for the evolution of inefficient codon usage in this gene and point to a strong connection between translational efficiency and RNA accumulation in mammalian cells.

    IMPORTANCE This study reports that inefficient codon usage in a herpesviral gene is strikingly correlated with the inability of its mRNA to accumulate in cells; correction of efficient translatability restores RNA abundance. A similar correlation has been reported in yeast species, but the mechanisms operating in mammalian cells appear substantially different.

  • Human Cytokinome Analysis for Interferon Response [Cellular Response to Infection]

  • Cytokines are a group of small secreted proteins that mediate a diverse range of immune and nonimmune responses to inflammatory and microbial stimuli. Only a few of these cytokines mount an antiviral response, including type I, II, and III interferons (IFNs). During viral infections and under inflammatory conditions, a number of cytokines and chemokines are coproduced with IFN; however, no systematic study exists on the interactions of the cytokine repertoire with the IFN response. Here, we performed the largest cytokine and chemokine screen (the human cytokinome, with ggt;240 members) to investigate their modulation of type I and type II IFN responses in a cell line model. We evaluated the cytokine activities in both IFN-stimulated response element (ISRE) and IFN- activation sequence (GAS) reporter systems. Several cytokine clusters that augment either or both ISRE- and GAS-mediated responses to IFNs were derived from the screen. We identified novel modulators of IFN responsemmdash;betacellulin (BTC), interleukin 11 (IL-11), and IL-17Fmmdash;that caused time-dependent induction of the IFN response. The ability to induce endogenous IFN-bbeta; and IFN-stimulated genes varies among these cytokines and was largely dependent on Stat1, as assessed by Stat1 mutant fibroblasts. Certain cytokines appear to augment the IFN-bbeta; response through the NF-B pathway. The novel IFN-like cytokines augmented the antiviral activity of IFN-aalpha; against several RNA viruses, including encephalomyocarditis virus, vesicular stomatitis virus, and influenza virus, in susceptible cell lines. Overall, the study represents a large-scale analysis of cytokines for enhancing the IFN response and identified cytokines capable of enhancing Stat1, IFN-induced gene expression, and antiviral activities.

    IMPORTANCE Innate immunity to viruses is an early defense system to ward off viruses. One mediator is interferon (IFN), which activates a cascade of biochemical events that aim to control the virus life cycle. In our work, we examined more than 200 cytokines, soluble mediators produced within the body as a result of infection, for the ability to enhance IFN action. We identified enhanced interactions with specific IFNs and cytokines. We also revealed that betacellulin, IL-17, and IL-11 cytokines have the novel property of enhancing the antiviral action of IFN against several viruses. These results demonstrate that the human genome codes for previously unknown proteins with unrelated functions that can augment the innate immunity to viruses. Knowing these interactions not only helps our understanding of immunity to viruses and emerging diseases, but can also lead to devising possible new therapeutics by enhancing the mediator of antiviral action itself, IFN.

  • New Noncoding Lytic Transcripts Derived from the Epstein-Barr Virus Latency Origin of Replication, oriP, Are Hyperedited, Bind the Paraspeckle Protein, NONO/p54nrb, and Support Viral Lytic Transcription [Genome Replication and Regulation of Viral Gene Expression]

  • We have previously shown that the Epstein-Barr virus (EBV) likely encodes hundreds of viral long noncoding RNAs (vlncRNAs) that are expressed during reactivation. Here we show that the EBV latency origin of replication (oriP) is transcribed bi-directionally during reactivation and that both leftward (oriPtLs) and rightward (oriPtRs) transcripts are largely localized in the nucleus. While the oriPtLs are most likely noncoding, at least some of the oriPtRs contain the BCRF1/vIL10 open reading frame. Nonetheless, oriPtR transcripts with long 5' untranslated regions may partially serve noncoding functions. Both oriPtL and oriPtR transcripts are expressed with late kinetics, and their expression is inhibited by phosphonoacetic acid. RNA sequencing (RNA-seq) analysis showed that oriPtLs and oriPtRs exhibited extensive "hyperediting" at their Family of Repeat (FR) regions. RNA secondary structure prediction revealed that the FR region of both oriPtLs and oriPtRs may form large evolutionarily conserved and thermodynamically stable hairpins. The double-stranded RNA-binding protein and RNA-editing enzyme ADAR was found to bind to oriPtLs, likely facilitating editing of the FR hairpin. Further, the multifunctional paraspeckle protein, NONO, was found to bind to oriPt transcripts, suggesting that oriPts interact with the paraspeckle-based innate antiviral immune pathway. Knockdown and ectopic expression of oriPtLs showed that it contributes to global viral lytic gene expression and viral DNA replication. Together, these results show that these new vlncRNAs interact with cellular innate immune pathways and that they help facilitate progression of the viral lytic cascade.

    IMPORTANCE Recent studies have revealed that the complexity of lytic herpesviral transcriptomes is significantly greater than previously appreciated with hundreds of viral long noncoding RNAs (vlncRNAs) being recently discovered. Work on cellular lncRNAs over the past several years has just begun to give us an initial appreciation for the array of functions they play in complex formation and regulatory processes in the cell. The newly identified herpesvirus lncRNAs are similarly likely to play a variety of different functions, although these functions are likely tailored to specific needs of the viral infection cycles. Here we describe novel transcripts derived from the EBV latency origin of replication. We show that they are hyperedited, that they interact with a relatively newly appreciated antiviral pathway, and that they play a role in facilitating viral lytic gene expression. These investigations are a starting point to unraveling the complex arena of vlncRNA function in herpesvirus lytic replication.

  • Recombination of Globally Circulating Varicella-Zoster Virus [Spotlight]

  • Varicella-zoster virus (VZV) is a human herpesvirus, which during primary infection typically causes varicella (chicken pox) and establishes lifelong latency in sensory and autonomic ganglia. Later in life, the virus may reactivate to cause herpes zoster (HZ; also known as shingles). To prevent these diseases, a live-attenuated heterogeneous vaccine preparation, vOka, is used routinely in many countries worldwide. Recent studies of another alphaherpesvirus, infectious laryngotracheitis virus, demonstrate that live-attenuated vaccine strains can recombine in vivo, creating virulent progeny. These findings raised concerns about using attenuated herpesvirus vaccines under conditions that favor recombination. To investigate whether VZV may undergo recombination, which is a prerequisite for VZV vaccination to create such conditions, we here analyzed 115 complete VZV genomes. Our results demonstrate that recombination occurs frequently for VZV. It thus seems that VZV is fully capable of recombination if given the opportunity, which may have important implications for continued VZV vaccination. Although no interclade vaccine-wild-type recombinant strains were found, intraclade recombinants were frequently detected in clade 2, which harbors the vaccine strains, suggesting that the vaccine strains have already been involved in recombination events, either in vivo or in vitro during passages in cell culture. Finally, previous partial and complete genomic studies have described strains that do not cluster phylogenetically to any of the five established clades. The additional VZV strains sequenced here, in combination with those previously published, have enabled us to formally define a novel sixth VZV clade.

    IMPORTANCE Although genetic recombination has been demonstrated to frequently occur for other human alphaherpesviruses, herpes simplex viruses 1 and 2, only a few ancient and isolated recent recombination events have hitherto been demonstrated for VZV. In the present study, we demonstrate that VZV also frequently undergoes genetic recombination, including strains belonging to the clade containing the vOKA strain.

  • Lymph Node Macrophages Restrict Murine Cytomegalovirus Dissemination [Pathogenesis and Immunity]

  • Cytomegaloviruses (CMVs) establish chronic infections that spread from a primary entry site to secondary vascular sites, such as the spleen, and then to tertiary shedding sites, such as the salivary glands. Human CMV (HCMV) is difficult to analyze, because its spread precedes clinical presentation. Murine CMV (MCMV) offers a tractable model. It is hypothesized to spread from peripheral sites via vascular endothelial cells and associated monocytes. However, viral luciferase imaging showed footpad-inoculated MCMV first reaching the popliteal lymph nodes (PLN). PLN colonization was rapid and further spread was slow, implying that LN infection can be a significant bottleneck. Most acutely infected PLN cells were CD169+ subcapsular sinus macrophages (SSM). Replication-deficient MCMV also reached them, indicating direct infection. Many SSM expressed viral reporter genes, but few expressed lytic genes. SSM expressed CD11c, and MCMV with a cre-sensitive fluorochrome switch showed switched infected cells in PLN of CD11c-cre mice but yielded little switched virus. SSM depletion with liposomal clodronate or via a CD169-diphtheria toxin receptor transgene shifted infection to ER-TR7+ stromal cells, increased virus production, and accelerated its spread to the spleen. Therefore, MCMV disseminated via LN, and SSM slowed this spread by shielding permissive fibroblasts and poorly supporting viral lytic replication.

    IMPORTANCE HCMV chronically infects most people, and it can cause congenital disability and harm the immunocompromised. A major goal of vaccination is to prevent systemic infection. How this is established is unclear. Restriction to humans makes HCMV difficult to analyze. We show that peripheral MCMV infection spreads via lymph nodes. Here, MCMV infected filtering macrophages, which supported virus replication poorly. When these macrophages were depleted, MCMV infected susceptible fibroblasts and spread faster. The capacity of filtering macrophages to limit MCMV spread argued that their infection is an important bottleneck in host colonization and might be a good vaccine target.

  • A Functional Interaction between Herpes Simplex Virus 1 Glycoprotein gH/gL Domains I and II and gD Is Defined by Using Alphaherpesvirus gH and gL Chimeras [Genome Replication and Regulation of Viral Gene Expression]

  • Whereas most viruses require only a single protein to bind to and fuse with cells, herpesviruses use multiple glycoproteins to mediate virus entry, and thus communication among these proteins is required. For most alphaherpesviruses, the minimal set of viral proteins required for fusion with the host cell includes glycoproteins gD, gB, and a gH/gL heterodimer. In the current model of entry, gD binds to a cellular receptor and transmits a signal to gH/gL. This signal then triggers gB, the conserved fusion protein, to insert into the target membrane and refold to merge the viral and cellular membranes. We previously demonstrated that gB homologs from two alphaherpesviruses, herpes simplex virus 1 (HSV-1) and saimiriine herpesvirus 1 (SaHV-1), were interchangeable. In contrast, neither gD nor gH/gL functioned with heterotypic entry glycoproteins, indicating that gD and gH/gL exhibit an essential type-specific functional interaction. To map this homotypic interaction site on gH/gL, we generated HSV-1/SaHV-1 gH and gL chimeras. The functional interaction with HSV-1 gD mapped to the N-terminal domains I and II of the HSV-1 gH ectodomain. The core of HSV-1 gL that interacts with gH also was required for functional homotypic interaction. The N-terminal gH/gL domains I and II are the least conserved and may have evolved to support species-specific glycoprotein interactions.

    IMPORTANCE The first step of the herpesvirus life cycle is entry into a host cell. A coordinated interaction among multiple viral glycoproteins is required to mediate fusion of the viral envelope with the cell membrane. The details of how these glycoproteins interact to trigger fusion are unclear. By swapping the entry glycoproteins of two alphaherpesviruses (HSV-1 and SaHV-1), we previously demonstrated a functional homotypic interaction between gD and gH/gL. To define the gH and gL requirements for homotypic interaction, we evaluated the function of a panel of HSV-1/SaHV-1 gH and gL chimeras. We demonstrate that domains I and II of HSV-1 gH are sufficient to promote a functional, albeit reduced, interaction with HSV-1 gD. These findings contribute to our model of how the entry glycoproteins cooperate to mediate herpesvirus entry into the cell.

  • A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus NS4B Critical for Interaction with NS3 and Viral Replication [Genome Replication and Regulation of Viral Gene Expression]

  • Dengue virus (DENV) infection causes the most prevalent arthropod-borne viral disease worldwide. Approved vaccines are not available, and targets suitable for the development of antiviral drugs are lacking. One possible drug target is nonstructural protein 4B (NS4B), because it is absolutely required for virus replication; however, its exact role in the DENV replication cycle is largely unknown. With the aim of mapping NS4B determinants critical for DENV replication, we performed a reverse genetic screening of 33 NS4B mutants in the context of an infectious DENV genome. While the majority of these mutations were lethal, for several of them, we were able to select for second-site pseudoreversions, most often residing in NS4B and restoring replication competence. To identify all viral NS4B interaction partners, we engineered a fully viable DENV genome encoding an affinity-tagged NS4B. Mass spectrometry-based analysis of the NS4B complex isolated from infected cells identified the NS3 protease/helicase as a major interaction partner of NS4B. By combining the genetic complementation map of NS4B with a replication-independent expression system, we identified the NS4B cytosolic loopmmdash;more precisely, amino acid residue Q134mmdash;as a critical determinant for NS4B-NS3 interaction. An alanine substitution at this site completely abrogated the interaction and DENV RNA replication, and both were restored by pseudoreversions A69S and A137V. This strict correlation between the degree of NS4B-NS3 interaction and DENV replication provides strong evidence that this viral protein complex plays a pivotal role during the DENV replication cycle, hence representing a promising target for novel antiviral strategies.

    IMPORTANCE With no approved therapy or vaccine against dengue virus infection, the viral nonstructural protein 4B (NS4B) represents a possible drug target, because it is indispensable for virus replication. However, little is known about its precise structure and function. Here, we established the first comprehensive genetic interaction map of NS4B, identifying amino acid residues that are essential for virus replication, as well as second-site mutations compensating for their defects. Additionally, we determined the NS4B viral interactome in infected cells and identified the NS3 protease/helicase as a major interaction partner of NS4B. We mapped residues in the cytosolic loop of NS4B as critical determinants for interaction with NS3, as well as RNA replication. The strong correlation between NS3-NS4B interaction and RNA replication provides strong evidence that this complex plays a pivotal role in the viral replication cycle, hence representing a promising antiviral drug target.

  • Human Endogenous Retrovirus Type K (HERV-K) Particles Package and Transmit HERV-K-Related Sequences [Genome Replication and Regulation of Viral Gene Expression]

  • Human endogenous retroviruses (HERV) make up 8% of the human genome. While the youngest of these retroviruses, HERV-K(HML-2), termed HK2, is able to code for all viral proteins and produce virus-like particles, it is not known if these virus particles package and transmit HK2-related sequences. Here, we analyzed the capacity of HK2 for packaging and transmitting HK2 sequences. We created an HK2 probe, termed Bogota, which can be packaged into HK2 viruses, and transfected it into cells that make HK2 particles. Supernatants of the transfected cells, which contained HK2 viral particles, then were added to target cells, and the transmissibility of the HK2 Bogota reporter was tracked by G418 resistance. Our studies revealed that contemporary HK2 virions produced by some teratocarcinoma and breast cancer cell lines, as well as by peripheral blood lymphocytes from lymphoma patients, can package HK2 Bogota probes, and these viruses transmitted these probes to other cells. After transmission, HK2 Bogota transcripts undergo reverse transcription, a step impaired by antiretroviral agents or by introduction of mutations into the probe sequences required for reverse transcription. HK2 viruses were more efficiently transmitted in the presence of HK2 Rec or HIV-1 Tat and Vif. Transmitted Bogota probes formed episomes but did not integrate into the cellular genome. Resistance to integration might explain the relatively low number of HK2 insertions that were acquired during the last 25 million years of evolution. Whether transient transmission of modern HK2 sequences, which encode two putative oncoproteins, can lead to disease remains to be studied.

    IMPORTANCE Retroviruses invaded the genome of human ancestors over the course of millions of years, yet these viruses generally have been inactivated during evolution, with only remnants of these infectious sequences remaining in the human genome. One of these viruses, termed HK2, still is capable of producing virus particles, although these particles have been regarded as being noninfectious. Using a genetic probe derived from HK2, we have discovered that HK2 viruses produced in modern humans can package HK2 sequences and transmit them to various other cells. Furthermore, the genetic sequences packaged in HK2 undergo reverse transcription. The transmitted probe circularized in the cell and failed to integrate into the cellular genome. These findings suggest that modern HK2 viruses can package viral RNA and transmit it to other cells. Contrary to previous views, we provide evidence of an extracellular viral phase of modern HK2 viruses. We have no evidence of sustained, spreading infection.

  • Human Coronavirus HKU1 Spike Protein Uses O-Acetylated Sialic Acid as an Attachment Receptor Determinant and Employs Hemagglutinin-Esterase Protein as a Receptor-Destroying Enzyme [Virus-Cell Interactions]

  • Human coronavirus (hCoV) HKU1 is one of six hCoVs identified to date and the only one with an unidentified cellular receptor. hCoV-HKU1 encodes a hemagglutinin-esterase (HE) protein that is unique to the group a betacoronaviruses (group 2a). The function of HKU1-HE remains largely undetermined. In this study, we examined binding of the S1 domain of hCoV-HKU1 spike to a panel of cells and found that the S1 could specifically bind on the cell surface of a human rhabdomyosarcoma cell line, RD. Pretreatment of RD cells with neuraminidase (NA) and trypsin greatly reduced the binding, suggesting that the binding was mediated by sialic acids on glycoproteins. However, unlike other group 2a CoVs, e.g., hCoV-OC43, for which 9-O-acetylated sialic acid (9-O-Ac-Sia) serves as a receptor determinant, HKU1-S1 bound with neither 9-O-Ac-Sia-containing glycoprotein(s) nor rat and mouse erythrocytes. Nonetheless, the HKU1-HE was similar to OC43-HE, also possessed sialate-O-acetylesterase activity, and acted as a receptor-destroying enzyme (RDE) capable of eliminating the binding of HKU1-S1 to RD cells, whereas the O-acetylesterase-inactive HKU1-HE mutant lost this capacity. Using primary human ciliated airway epithelial (HAE) cell cultures, the only in vitro replication model for hCoV-HKU1 infection, we confirmed that pretreatment of HAE cells with HE but not the enzymatically inactive mutant blocked hCoV-HKU1 infection. These results demonstrate that hCoV-HKU1 exploits O-Ac-Sia as a cellular attachment receptor determinant to initiate the infection of host cells and that its HE protein possesses the corresponding sialate-O-acetylesterase RDE activity.

    IMPORTANCE Human coronaviruses (hCoV) are important human respiratory pathogens. Among the six hCoVs identified to date, only hCoV-HKU1 has no defined cellular receptor. It is also unclear whether hemagglutinin-esterase (HE) protein plays a role in viral entry. In this study, we found that, similarly to other members of the group 2a CoVs, sialic acid moieties on glycoproteins are critical receptor determinants for the hCoV-HKU1 infection. Interestingly, the virus seems to employ a type of sialic acid different from those employed by other group 2a CoVs. In addition, we determined that the HKU1-HE protein is an O-acetylesterase and acts as a receptor-destroying enzyme (RDE) for hCoV-HKU1. This is the first study to demonstrate that hCoV-HKU1 uses certain types of O-acetylated sialic acid residues on glycoproteins to initiate the infection of host cells and that the HKU1-HE protein possesses sialate-O-acetylesterase RDE activity.

  • Recombination Analysis of Herpes Simplex Virus 1 Reveals a Bias toward GC Content and the Inverted Repeat Regions [Genome Replication and Regulation of Viral Gene Expression]

  • Herpes simplex virus 1 (HSV-1) causes recurrent mucocutaneous ulcers and is the leading cause of infectious blindness and sporadic encephalitis in the United States. HSV-1 has been shown to be highly recombinogenic; however, to date, there has been no genome-wide analysis of recombination. To address this, we generated 40 HSV-1 recombinants derived from two parental strains, OD4 and CJ994. The 40 OD4-CJ994 HSV-1 recombinants were sequenced using the Illumina sequencing system, and recombination breakpoints were determined for each of the recombinants using the Bootscan program. Breakpoints occurring in the terminal inverted repeats were excluded from analysis to prevent double counting, resulting in a total of 272 breakpoints in the data set. By placing windows around the 272 breakpoints followed by Monte Carlo analysis comparing actual data to simulated data, we identified a recombination bias toward both high GC content and intergenic regions. A Monte Carlo analysis also suggested that recombination did not appear to be responsible for the generation of the spontaneous nucleotide mutations detected following sequencing. Additionally, kernel density estimation analysis across the genome found that the large, inverted repeats comprise a recombination hot spot.

    IMPORTANCE Herpes simplex virus 1 (HSV-1) virus is the leading cause of sporadic encephalitis and blinding keratitis in developed countries. HSV-1 has been shown to be highly recombinogenic, and recombination itself appears to be a significant component of genome replication. To date, there has been no genome-wide analysis of recombination. Here we present the findings of the first genome-wide study of recombination performed by generating and sequencing 40 HSV-1 recombinants derived from the OD4 and CJ994 parental strains, followed by bioinformatics analysis. Recombination breakpoints were determined, yielding 272 breakpoints in the full data set. Kernel density analysis determined that the large inverted repeats constitute a recombination hot spot. Additionally, Monte Carlo analyses found biases toward high GC content and intergenic and repetitive regions.

  • Cross-Reactive Neuraminidase-Inhibiting Antibodies Elicited by Immunization with Recombinant Neuraminidase Proteins of H5N1 and Pandemic H1N1 Influenza A Viruses [Vaccines and Antiviral Agents]

  • Neuraminidase (NA), an influenza virus envelope glycoprotein, removes sialic acid from receptors for virus release from infected cells. For this study, we used a baculovirus-insect cell expression system to construct and purify recombinant NA (rNA) proteins of H5N1 (A/Vietnam/1203/2004) and pandemic H1N1 (pH1N1) (A/Texas/05/2009) influenza viruses. BALB/c mice immunized with these proteins had high titers of NA-specific IgG and NA-inhibiting (NI) antibodies against H5N1, pH1N1, H3N2, and H7N9 viruses. H5N1 rNA immunization resulted in higher quantities of NA-specific antibody-secreting B cells against H5N1 and heterologous pH1N1 viruses in the spleen. H5N1 rNA and pH1N1 rNA immunizations both provided complete protection against homologous virus challenges, with H5N1 rNA immunization providing better protection against pH1N1 virus challenges. Cross-reactive NI antibodies were further dissected via pH1N1 rNA protein immunizations with I149V (NA with a change of Ile to Val at position 149), N344Y, and I365T/S366N NA mutations. The I365T/S366N mutation of pH1N1 rNA enhanced cross-reactive NI antibodies against H5N1, H3N2, and H7N9 viruses. It is our hope that these findings provide useful information for the development of an NA-based universal influenza vaccine.

    IMPORTANCE Neuraminidase (NA) is an influenza virus enzymatic protein that cleaves sialic acid linkages on infected cell surfaces, thus facilitating viral release and contributing to viral transmission and mucus infection. In currently available inactivated or live, attenuated influenza vaccines based on the antigenic content of hemagglutinin proteins, vaccine efficacy can be contributed partly through NA-elicited immune responses. We investigated the NA immunity of different recombinant NA (rNA) proteins associated with pH1N1 and H5N1 viruses. Our results indicate that H5N1 rNA immunization induced more potent cross-protective immunity than pH1N1 rNA immunization, and three mutated residues, I149V, I365T, and S366N, near the NA enzyme active site(s) are linked to enhanced cross-reactive NA-inhibiting antibodies against heterologous and heterosubtypic influenza A viruses. These findings provide useful information for the development of an NA-based universal influenza vaccine.

  • Novel Functions of Hendra Virus G N-Glycans and Comparisons to Nipah Virus [Structure and Assembly]

  • Hendra virus (HeV) and Nipah virus (NiV) are reportedly the most deadly pathogens within the Paramyxoviridae family. These two viruses bind the cellular entry receptors ephrin B2 and/or ephrin B3 via the viral attachment glycoprotein G, and the concerted efforts of G and the viral fusion glycoprotein F result in membrane fusion. Membrane fusion is essential for viral entry into host cells and for cell-cell fusion, a hallmark of the disease pathobiology. HeV G is heavily N-glycosylated, but the functions of the N-glycans remain unknown. We disrupted eight predicted N-glycosylation sites in HeV G by conservative mutations (Asn to Gln) and found that six out of eight sites were actually glycosylated (G2 to G7); one in the stalk (G2) and five in the globular head domain (G3 to G7). We then tested the roles of individual and combined HeV G N-glycan mutants and found functions in the modulation of shielding against neutralizing antibodies, intracellular transport, G-F interactions, cell-cell fusion, and viral entry. Between the highly conserved HeV and NiV G glycoproteins, similar trends in the effects of N-glycans on protein functions were observed, with differences in the levels at which some N-glycan mutants affected such functions. While the N-glycan in the stalk domain (G2) had roles that were highly conserved between HeV and NiV G, individual N-glycans in the head affected the levels of several protein functions differently. Our findings are discussed in the context of their contributions to our understanding of HeV and NiV pathogenesis and immune responses.

    IMPORTANCE Viral envelope glycoproteins are important for viral pathogenicity and immune evasion. N-glycan shielding is one mechanism by which immune evasion can be achieved. In paramyxoviruses, viral attachment and membrane fusion are governed by the close interaction of the attachment proteins H/HN/G and the fusion protein F. In this study, we show that the attachment glycoprotein G of Hendra virus (HeV), a deadly paramyxovirus, is N-glycosylated at six sites (G2 to G7) and that most of these sites have important roles in viral entry, cell-cell fusion, G-F interactions, G oligomerization, and immune evasion. Overall, we found that the N-glycan in the stalk domain (G2) had roles that were very conserved between HeV G and the closely related Nipah virus G, whereas individual N-glycans in the head quantitatively modulated several protein functions differently between the two viruses.

  • K1 and K15 of Kaposi's Sarcoma-Associated Herpesvirus Are Partial Functional Homologues of Latent Membrane Protein 2A of Epstein-Barr Virus [Transformation and Oncogenesis]

  • The human herpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are associated with Hodgkin's lymphoma (HL) and Primary effusion lymphomas (PEL), respectively, which are B cell malignancies that originate from germinal center B cells. PEL cells but also a quarter of EBV-positive HL tumor cells do not express the genuine B cell receptor (BCR), a situation incompatible with survival of normal B cells. EBV encodes LMP2A, one of EBV's viral latent membrane proteins, which likely replaces the BCR's survival signaling in HL. Whether KSHV encodes a viral BCR mimic that contributes to oncogenesis is not known because an experimental model of KSHV-mediated B cell transformation is lacking. We addressed this uncertainty with mutant EBVs encoding the KSHV genes K1 or K15 in lieu of LMP2A and infected primary BCR-negative (BCRnndash;) human B cells with them. We confirmed that the survival of BCRnndash; B cells and their proliferation depended on an active LMP2A signal. Like LMP2A, the expression of K1 and K15 led to the survival of BCRnndash; B cells prone to apoptosis, supported their proliferation, and regulated a similar set of cellular target genes. K1 and K15 encoded proteins appear to have noncomplementing, redundant functions in this model, but our findings suggest that both KSHV proteins can replace LMP2A's key activities contributing to the survival, activation and proliferation of BCRnndash; PEL cells in vivo.

    IMPORTANCE Several herpesviruses encode oncogenes that are receptor-like proteins. Often, they are constitutively active providing important functions to the latently infected cells. LMP2A of Epstein-Barr virus (EBV) is such a receptor that mimics an activated B cell receptor, BCR. K1 and K15, related receptors of Kaposi's sarcoma-associated herpesvirus (KSHV) expressed in virus-associated tumors, have less obvious functions. We found in infection experiments that both viral receptors of KSHV can replace LMP2A and deliver functions similar to the endogenous BCR. K1, K15, and LMP2A also control the expression of a related set of cellular genes in primary human B cells, the target cells of EBV and KSHV. The observed phenotypes, as well as the known characteristics of these genes, argue for their contributions to cellular survival, B cell activation, and proliferation. Our findings provide one possible explanation for the tumorigenicity of KSHV, which poses a severe problem in immunocompromised patients.

  • Rift Valley Fever Virus MP-12 Vaccine Is Fully Attenuated by a Combination of Partial Attenuations in the S, M, and L Segments [Vaccines and Antiviral Agents]

  • Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and characterized by a high rate of abortion in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. RVF is caused by Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus), which has a tripartite negative-stranded RNA genome (consisting of the S, M, and L segments). Further spread of RVF into countries where the disease is not endemic may affect the economy and public health, and vaccination is an effective approach to prevent the spread of RVFV. A live-attenuated MP-12 vaccine is one of the best-characterized RVF vaccines for safety and efficacy and is currently conditionally licensed for use for veterinary purposes in the United States. Meanwhile, as of 2015, no other RVF vaccine has been conditionally or fully licensed for use in the United States. The MP-12 strain is derived from wild-type pathogenic strain ZH548, and its genome encodes 23 mutations in the three genome segments. However, the mechanism of MP-12 attenuation remains unknown. We characterized the attenuation of wild-type pathogenic strain ZH501 carrying a mutation(s) of the MP-12 S, M, or L segment in a mouse model. Our results indicated that MP-12 is attenuated by the mutations in the S, M, and L segments, while the mutations in the M and L segments confer stronger attenuation than those in the S segment. We identified a combination of 3 amino acid changes, Y259H (Gn), R1182G (Gc), and R1029K (L), that was sufficient to attenuate ZH501. However, strain MP-12 with reversion mutations at those 3 sites was still highly attenuated. Our results indicate that MP-12 attenuation is supported by a combination of multiple partial attenuation mutations and a single reversion mutation is less likely to cause a reversion to virulence of the MP-12 vaccine.

    IMPORTANCE Rift Valley fever (RVF) is a mosquito-transmitted viral disease that is endemic to Africa and that has the potential to spread into other countries. Vaccination is considered an effective way to prevent the disease, and the only available veterinary RVF vaccine in the United States is a live-attenuated MP-12 vaccine, which is conditionally licensed. Strain MP-12 is different from its parental pathogenic RVFV strain, strain ZH548, because of the presence of 23 mutations. This study determined the role of individual mutations in the attenuation of the MP-12 strain. We found that full attenuation of MP-12 occurs by a combination of multiple mutations. Our findings indicate that a single reversion mutation will less likely cause a major reversion to virulence of the MP-12 vaccine.

  • Regulation of Latent Membrane Protein 1 Signaling through Interaction with Cytoskeletal Proteins [Transformation and Oncogenesis]

  • Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) induces constitutive signaling in EBV-infected cells to ensure the survival of the latently infected cells. LMP1 is localized to lipid raft domains to induce signaling. In the present study, a genome-wide screen based on bimolecular fluorescence complementation (BiFC) was performed to identify LMP1-binding proteins. Several actin cytoskeleton-associated proteins were identified in the screen. Overexpression of these proteins affected LMP1-induced signaling. BiFC between the identified proteins and LMP1 was localized to lipid raft domains and was dependent on LMP1-induced signaling. Proximity biotinylation assays with LMP1 induced biotinylation of the actin-associated proteins, which were shifted in molecular mass. Together, the findings of this study suggest that the association of LMP1 with lipid rafts is mediated at least in part through interactions with the actin cytoskeleton.

    IMPORTANCE LMP1 signaling requires oligomerization, lipid raft partitioning, and binding to cellular adaptors. The current study utilized a genome-wide screen to identify several actin-associated proteins as candidate LMP1-binding proteins. The interaction between LMP1 and these proteins was localized to lipid rafts and dependent on LMP1 signaling. This suggests that the association of LMP1 with lipid rafts is mediated through interactions with actin-associated proteins.

  • Inclusion of Flagellin during Vaccination against Influenza Enhances Recall Responses in Nonhuman Primate Neonates [Vaccines and Antiviral Agents]

  • Influenza virus can cause life-threatening infections in neonates and young infants. Although vaccination is a major countermeasure against influenza, current vaccines are not approved for use in infants less than 6 months of age, in part due to the weak immune response following vaccination. Thus, there is a strong need to develop new vaccines with improved efficacy for this vulnerable population. To address this issue, we established a neonatal African green monkey (AGM) nonhuman primate model that could be used to identify effective influenza vaccine approaches for use in young infants. We assessed the ability of flagellin, a Toll-like receptor 5 (TLR5) agonist, to serve as an effective adjuvant in this at-risk population. Four- to 6-day-old AGMs were primed and boosted with inactivated PR8 influenza virus (IPR8) adjuvanted with either wild-type flagellin or inactive flagellin with a mutation at position 229 (m229), the latter of which is incapable of signaling through TLR5. Increased IgG responses were observed following a boost, as well as at early times after challenge, in infants vaccinated with flagellin-adjuvanted IPR8. Inclusion of flagellin during vaccination also resulted in a significantly increased number of influenza virus-specific T cells following challenge compared to the number in infants vaccinated with the m229 adjuvant. Finally, following challenge infants vaccinated with IPR8 plus flagellin exhibited a reduced pathology in the lungs compared to that in infants that received IPR8 plus m229. This study provides the first evidence of flagellin-mediated enhancement of vaccine responses in nonhuman primate neonates.

    IMPORTANCE Young infants are particularly susceptible to severe disease as a result of influenza virus infection. Compounding this is the lack of effective vaccines for use in this vulnerable population. Here we describe a vaccine approach that results in improved immune responses and protection in young infants. Incorporation of flagellin during vaccination resulted in increased antibody and T cell responses together with reduced disease following virus infection. These results suggest that flagellin may serve as an effective adjuvant for vaccines targeted to this vulnerable population.

  • Characterization of the Human Papillomavirus 16 E8 Promoter [Genome Replication and Regulation of Viral Gene Expression]

  • Persistent infections with certain human papillomaviruses (HPV) such as HPV16 are a necessary risk factor for the development of anogenital and oropharyngeal cancers. HPV16 genomes replicate as low-copy-number plasmids in the nucleus of undifferentiated keratinocytes, which requires the viral E1 and E2 replication proteins. The HPV16 E8^E2C (or E8^E2) protein limits genome replication by repressing both viral transcription and the E1/E2-dependent DNA replication. How E8^E2C expression is regulated is not understood. Previous transcript analyses indicated that the spliced E8^E2C RNA is initiated at a promoter located in the E1 region upstream of the E8 gene. Deletion and mutational analyses of the E8 promoter region identify two conserved elements that are required for basal promoter activity in HPV-negative keratinocytes. In contrast, the transcriptional enhancer in the upstream regulatory region of HPV16 does not modulate basal E8 promoter activity. Cotransfection studies indicate that E8^E2C inhibits, whereas E2 weakly activates, the E8 promoter. Interestingly, the cotransfection of E1 and E2 induces the E8 promoter much more strongly than the major early promoter, and this is partially dependent upon binding of E2 to Brd4. Mutation of E8 promoter elements in the context of HPV16 genomes results in an increased genome copy number and elevated levels of viral early and late transcripts. In summary, the promoter responsible for the expression of E8^E2C is both positively and negatively regulated by viral and cellular factors, and this regulatory circuit may be crucial to maintain a low but constant copy number of HPV16 genomes in undifferentiated cells.

    IMPORTANCE HPV16 replicates in differentiating epithelia and can cause cancer. How HPV16 maintains its genome in undifferentiated cells at a low but constant level is not well understood but may be relevant for the immunological escape of HPV16 in the basal layers of the infected epithelium. This study demonstrates that the expression of the viral E8^E2C protein, which is a potent inhibitor of viral replication in undifferentiated cells, is driven by a separate promoter. The E8 promoter is both positively and negatively regulated by viral proteins and thus most likely acts as a sensor and modulator of viral copy number.

  • Human Cytomegalovirus pUL47 Modulates Tegumentation and Capsid Accumulation at the Viral Assembly Complex [Structure and Assembly]

  • Human cytomegalovirus (HCMV) tegument protein pUL47 is an interaction partner of pUL48 and highly conserved among herpesviruses. It is closely associated with the capsid and has an important function early in infection. Here, we report a specific role of pUL47 in the tegumentation of capsids in the cytoplasm. A newly generated mutant virus (TB-47stop), in which expression of pUL47 is blocked, exhibited a severe impairment in cell-to-cell spread and release of infectivity from infected cells. Ultrastructural analysis of TB-47stop-infected cells clearly showed cytoplasmic accumulations of nonenveloped capsids that were only partially tegumented, indicating that these capsids failed to complete tegumentation. Nevertheless, these accumulations were positive for HCMV inner tegument proteins pp150 and pUL48, suggesting that their attachment to capsids occurs independently of pUL47. Despite these morphological alterations, fully enveloped virus particles were found in the extracellular space and at the viral assembly complex (vAC) of TB-47stop-infected cells, indicating that pUL47 is not essential for the generation of virions. We confirmed findings that incorporation of pUL48 into virions is impaired in the absence of pUL47. Interestingly, pUL47 exhibited a strong nuclear localization in transfected cells, whereas it was found exclusively at the vAC in the context of virus infection. Colocalization of pUL47 and pUL48 at the vAC is consistent with their interaction. We also found a shift to a more nuclear localization of pUL47 when the expression of pUL48 was reduced. Summarizing our results, we hypothesize that pUL48 directs pUL47 to the vAC to promote tegumentation and secondary envelopment of capsids.

    IMPORTANCE Generation of infectious HCMV particles requires an organized and multistep process involving the action of several viral and cellular proteins as well as protein-protein interactions. A better understanding of these processes is important for understanding the biology of HCMV and may help to identify targets for antiviral intervention. Here, we identified tegument protein pUL47 to function in tegumentation and proper trafficking of capsids during late phases of infection. Although pUL47 is not essential for the generation and release of infectious virions, its absence led to massive accumulations of partially tegumented capsids at the cell periphery. Detection of pUL48 at these accumulations indicated a pUL47-independent attachment of pUL48 to the capsid. On the other hand, localization of pUL47 to the vAC during infection appeared to be dependent on tegument protein pUL48, which suggests an intricate interplay of these proteins for normal generation of infectious virus progeny.

  • Adaptive Mutations That Occurred during Circulation in Humans of H1N1 Influenza Virus in the 2009 Pandemic Enhance Virulence in Mice [Pathogenesis and Immunity]

  • During the 2009 H1N1 influenza pandemic, infection attack rates were particularly high among young individuals who suffered from pneumonia with occasional death. Moreover, previously reported determinants of mammalian adaptation and pathogenicity were not present in 2009 pandemic H1N1 influenza A viruses. Thus, it was proposed that unknown viral factors might have contributed to disease severity in humans. In this study, we performed a comparative analysis of two clinical 2009 pandemic H1N1 strains that belong to the very early and later phases of the pandemic. We identified mutations in the viral hemagglutinin (HA) and the nucleoprotein (NP) that occurred during pandemic progression and mediate increased virulence in mice. Lethal disease outcome correlated with elevated viral replication in the alveolar epithelium, increased proinflammatory cytokine and chemokine responses, pneumonia, and lymphopenia in mice. These findings show that viral mutations that have occurred during pandemic circulation among humans are associated with severe disease in mice.

    IMPORTANCE In this study, novel determinants of 2009 pandemic H1N1 influenza pathogenicity were identified in the viral hemagglutinin (HA) and the nucleoprotein (NP) genes. In contrast to highly pathogenic avian influenza viruses, increased virulence in mice did not correlate with enhanced polymerase activity but with reduced activity. Lethal 2009 pandemic H1N1 infection in mice correlated with lymphopenia and severe pneumonia. These studies suggest that molecular mechanisms that mediate 2009 pandemic H1N1 influenza pathogenicity are distinct from those that mediate avian influenza virus pathogenicity in mice.

  • Roles of Phosphorylation of the Nucleocapsid Protein of Mumps Virus in Regulating Viral RNA Transcription and Replication [Genome Replication and Regulation of Viral Gene Expression]

  • Mumps virus (MuV) is a paramyxovirus with a negative-sense nonsegmented RNA genome. The viral RNA genome is encapsidated by the nucleocapsid protein (NP) to form the ribonucleoprotein (RNP), which serves as a template for transcription and replication. In this study, we investigated the roles of phosphorylation sites of NP in MuV RNA synthesis. Using radioactive labeling, we first demonstrated that NP was phosphorylated in MuV-infected cells. Using both liquid chromatography-mass spectrometry (LC-MS) and in silico modeling, we identified nine putative phosphorylated residues within NP. We mutated these nine residues to alanine. Mutation of the serine residue at position 439 to alanine (S439A) was found to reduce the phosphorylation of NP in transfected cells by over 90%. The effects of these mutations on the MuV minigenome system were examined. The S439A mutant was found to have higher activity, four mutants had lower activity, and four mutants had similar activity compared to wild-type NP. MuV containing the S439A mutation had 90% reduced phosphorylation of NP and enhanced viral RNA synthesis and viral protein expression at early time points after infection, indicating that S439 is the major phosphorylation site of NP and its phosphorylation plays an important role in downregulating viral RNA synthesis.

    IMPORTANCE Mumps virus (MuV), a paramyxovirus, is an important human pathogen that is reemerging in human populations. Nucleocapsid protein (NP) of MuV is essential for viral RNA synthesis. We have identified the major phosphorylation site of NP. We have found that phosphorylation of NP plays a critical role in regulating viral RNA synthesis. The work will lead to a better understanding of viral RNA synthesis and possible novel targets for antiviral drug development.

  • Complexity of Neutralizing Antibodies against Multiple Dengue Virus Serotypes after Heterotypic Immunization and Secondary Infection Revealed by In-Depth Analysis of Cross-Reactive Antibodies [Spotlight]

  • The four serotypes of dengue virus (DENV) cause the most important and rapidly emerging arboviral diseases in humans. The recent phase 2b and 3 studies of a tetravalent dengue vaccine reported a moderate efficacy despite the presence of neutralizing antibodies, highlighting the need for a better understanding of neutralizing antibodies in polyclonal human sera. Certain type-specific (TS) antibodies were recently discovered to account for the monotypic neutralizing activity and protection after primary DENV infection. The nature of neutralizing antibodies after secondary DENV infection remains largely unknown. In this study, we examined sera from 10 vaccinees with well-documented exposure to first and second DENV serotypes through heterotypic immunization with live-attenuated vaccines. Higher serum IgG avidities to both exposed and nonexposed serotypes were found after secondary immunization than after primary immunization. Using a two-step depletion protocol to remove different anti-envelope antibodies, including group-reactive (GR) and complex-reactive (CR) antibodies separately, we found GR and CR antibodies together contributed to more than 50% of neutralizing activities against multiple serotypes after secondary immunization. Similar findings were demonstrated in patients after secondary infection. Anti-envelope antibodies recognizing previously exposed serotypes consisted of a large proportion of GR antibodies, CR antibodies, and a small proportion of TS antibodies, whereas those recognizing nonexposed serotypes consisted of GR and CR antibodies. These findings have implications for sequential heterotypic immunization or primary immunization of DENV-primed individuals as alternative strategies for DENV vaccination. The complexity of neutralizing antibodies after secondary infection provides new insights into the difficulty of their application as surrogates of protection.

    IMPORTANCE The four serotypes of dengue virus (DENV) are the leading cause of arboviral diseases in humans. Despite the presence of neutralizing antibodies, a moderate efficacy was recently reported in phase 2b and 3 trials of a dengue vaccine; a better understanding of neutralizing antibodies in polyclonal human sera is urgently needed. We studied vaccinees who received heterotypic immunization of live-attenuated vaccines, as they were known to have received the first and second DENV serotype exposures. We found anti-envelope antibodies consist of group-reactive (GR), complex-reactive (CR), and type-specific (TS) antibodies, and that both GR and CR antibodies contribute significantly to multitypic neutralizing activities after secondary DENV immunization. These findings have implications for alternative strategies for DENV vaccination. Certain TS antibodies were recently discovered to contribute to the monotypic neutralizing activity and protection after primary DENV infection; our findings of the complexity of neutralizing activities after secondary immunization/infection provide new insights for neutralizing antibodies as surrogates of protection.

  • Different Effects of Nonnucleoside Reverse Transcriptase Inhibitor Resistance Mutations on Cytotoxic T Lymphocyte Recognition between HIV-1 Subtype B and Subtype A/E Infections [Cellular Response to Infection]

  • The effect of antiretroviral drug resistance mutations on cytotoxic T lymphocyte (CTL) recognition has been analyzed in HIV-1 subtype B infections, but it remains unclear in infections by other HIV-1 subtypes that are epidemic in countries where antiretroviral drugs are not effectively used. We investigated the effect of nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI)-resistance mutations (Y181C, Y181I, and Y181V) on epitope recognition by CTLs specific for 3 different HIV-1 epitopes (HLA-A*02:01-restricted IV10, HLA-B*35:01-restricted NY9, and HLA-C*12:02-restricted KY9) in subtype B and subtype A/E infections and the accumulation of these mutations in treatment-naive Japanese and Vietnamese. These NNRTI-resistance mutations critically affected NY9-specific and KY9-specific T cell responses in the subtype B infections, whereas they showed a different effect on IV10-specific T cell responses among the subtype B-infected individuals. These mutations affected IV10-specific T cell responses but weakly affected NY9-specific T cell responses in the subtype A/E infections. The substitution at position 3 of NY9 epitope which was found in the subtype A/E virus differently influenced the peptide binding to HLA-B*35:01, suggesting that the differences in peptide binding may result in the differences in T cell recognition between the subtype B virus and A/E virus infections. The Y181C mutation was found to be accumulating in treatment-naive Vietnamese infected with the subtype A/E virus. The present study demonstrated different effects of NNRTI-resistance RT181 mutations on CTL responses between the 2 subtype infections. The Y181C mutation may influence HIV-1 control by the CTLs in Vietnam, since this mutation has been accumulating in treatment-naive Vietnamese.

    IMPORTANCE Antiretroviral therapy leads to the emergence of drug-resistant HIV-1, resulting in virological and clinical failures. Though HIV-1-specific CTLs play a critical role in HIV-1 infection, some of drug resistance mutations located in CTL epitopes are known to affect HIV-1-specific CTL responses. Nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistance RT181 mutations are frequently observed in patients treated with NNRTIs. Such drug resistance mutations may have an influence on immune control by HIV-1-specific CTLs, especially in countries where antiretroviral drugs are not effectively used. We here investigated the effect of three NNRTI-resistance RT181 mutations on immune responses by HIV-1-specific CTLs and the recent accumulation of these mutations in treatment-naive Vietnamese infected with HIV-1 subtype A/E virus. RT181 mutations affected CTL recognition in both subtype A/E and B infections, while the RT Y181C mutation has been accumulating in treatment-naive Vietnamese. The results suggest that the Y181C mutation may influence HIV-1 control by CTLs in Vietnam.

  • Arenavirus Genome Rearrangement for the Development of Live Attenuated Vaccines [Vaccines and Antiviral Agents]

  • Several members of the Arenaviridae family cause hemorrhagic fever disease in humans and pose serious public health problems in their geographic regions of endemicity as well as a credible biodefense threat. To date, there have been no FDA-approved arenavirus vaccines, and current antiarenaviral therapy is limited to an off-label use of ribavirin that is only partially effective. Arenaviruses are enveloped viruses with a bisegmented negative-stranded RNA genome. Each genome segment uses an ambisense coding strategy to direct the synthesis of two viral polypeptides in opposite orientations, separated by a noncoding intergenic region. Here we have used minigenome-based approaches to evaluate expression levels of reporter genes from the nucleoprotein (NP) and glycoprotein precursor (GPC) loci within the S segment of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). We found that reporter genes are expressed to higher levels from the NP than from the GPC locus. Differences in reporter gene expression levels from the NP and GPC loci were confirmed with recombinant trisegmented LCM viruses. We then used reverse genetics to rescue a recombinant LCMV (rLCMV) containing a translocated viral S segment (rLCMV/TransS), where the viral NP and GPC open reading frames replaced one another. The rLCMV/TransS showed slower growth kinetics in cultured cells and was highly attenuated in vivo in a mouse model of lethal LCMV infection, but immunization with rLCMV/TransS conferred complete protection against a lethal challenge with wild-type LCMV. Attenuation of rLCMV/TransS was associated with reduced NP expression levels. These results open a new avenue for the development of arenavirus live attenuated vaccines based on rearrangement of their viral genome.

    IMPORTANCE Several arenaviruses cause severe hemorrhagic fever in humans and also pose a credible bioterrorism threat. Currently, no FDA-licensed vaccines are available to combat arenavirus infections and antiarenaviral therapy is limited to the off-label use of ribavirin, which is only partially effective and associated with side effects. Here we describe, for the first time, the generation of a recombinant LCMV where the viral protein products encoded by the S RNA segment (NP and GPC) were swapped to generate rLCMV/TransS. rLCMV/TransS exhibited reduced viral multiplication in cultured cells and was highly attenuated in vivo while conferring protection, upon a single immunization dose, against a lethal challenge with wild-type LCMV. Our studies provide a proof of concept for the rational development of safe and protective live attenuated vaccine candidates based on genome reorganization for the treatment of pathogenic arenavirus infections in humans.

  • Vinexin {beta} Interacts with Hepatitis C Virus NS5A, Modulating Its Hyperphosphorylation To Regulate Viral Propagation [Virus-Cell Interactions]

  • Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is essential for HCV genome replication and virion production and is involved in the regulation of multiple host signaling pathways. As a proline-rich protein, NS5A is capable of interacting with various host proteins containing Src homology 3 (SH3) domains. Previous studies have suggested that vinexin, a member of the sorbin homology (SoHo) adaptor family, might be a potential binding partner of NS5A by yeast two-hybrid screening. However, firm evidence for this interaction is lacking, and the significance of vinexin in the HCV life cycle remains unclear. In this study, we demonstrated that endogenously and exogenously expressed vinexin bbeta; coimmunoprecipitated with NS5A derived from different HCV genotypes. Two residues, tryptophan (W307) and tyrosine (Y325), in the third SH3 domain of vinexin bbeta; and conserved Pro-X-X-Pro-X-Arg motifs at the C terminus of NS5A were indispensable for the vinexin-NS5A interaction. Furthermore, downregulation of endogenous vinexin bbeta; significantly suppressed NS5A hyperphosphorylation and decreased HCV replication, which could be rescued by expressing a vinexin bbeta; short hairpin RNA-resistant mutant. We also found that vinexin bbeta; modulated the hyperphosphorylation of NS5A in a casein kinase 1aalpha;-dependent on manner. Taken together, our findings suggest that vinexin bbeta; modulates NS5A phosphorylation via its interaction with NS5A, thereby regulating HCV replication, implicating vinexin bbeta; in the viral life cycle.

    IMPORTANCE Hepatitis C virus (HCV) nonstructural protein NS5A is a phosphoprotein, and its phosphorylation states are usually modulated by host kinases and other viral nonstructural elements. Additionally, cellular factors containing Src homology 3 (SH3) domains have been reported to interact with proline-rich regions of NS5A. However, it is unclear whether there are any relationships between NS5A phosphorylation and the NS5A-SH3 interaction, and little is known about the significance of this interaction in the HCV life cycle. In this work, we demonstrate that vinexin bbeta; modulates NS5A hyperphosphorylation through the NS5A-vinexin bbeta; interaction. Hyperphosphorylated NS5A induced by vinexin bbeta; is casein kinase 1aalpha; dependent and is also crucial for HCV propagation. Overall, our findings not only elucidate the relationships between NS5A phosphorylation and the NS5A-SH3 interaction but also shed new mechanistic insight on Flaviviridae NS5A (NS5) phosphorylation. We believe that our results may afford the potential to offer an antiviral therapeutic strategy.

  • Newcastle Disease Virus-Vectored H7 and H5 Live Vaccines Protect Chickens from Challenge with H7N9 or H5N1 Avian Influenza Viruses [Vaccines and Antiviral Agents]

  • Sporadic human infections by a novel H7N9 virus occurred over a large geographic region in China. In this study, we show that Newcastle disease virus (NDV)-vectored H7 (NDV-H7) and NDV-H5 vaccines are able to induce antibodies with high hemagglutination inhibition (HI) titers and completely protect chickens from challenge with the novel H7N9 or highly pathogenic H5N1 viruses, respectively. Notably, a baculovirus-expressed H7 protein failed to protect chickens from H7N9 virus infection.

  • Human Plasmacytoid Dendritic Cells Elicited Different Responses after Infection with Pathogenic and Nonpathogenic Junin Virus Strains [Cellular Response to Infection]

  • The arenavirus Junin virus (JUNV) is the etiologic agent of Argentine hemorrhagic fever. We characterized the JUNV infection of human peripheral blood-derived plasmacytoid dendritic cells (hpDC), demonstrating that hpDC are susceptible to infection with the C#1 strain (attenuated) and even more susceptible to infection with the P (virulent) JUNV strain. However, hpDC elicited different responses in terms of viability, activation, maturation, and cytokine expression after infection with both JUNV strains.

  • A Seminomadic Population in Bangladesh with Extensive Exposure to Macaques Does Not Exhibit High Levels of Zoonotic Simian Foamy Virus Infection [Pathogenesis and Immunity]

  • Simian foamy viruses (SVF) are ubiquitous in nonhuman primates (NHP). SFV can be zoonotically transmitted to humans who either work with or live commensally with NHP. We analyzed the blood of 45 Bangladeshi performing monkey owners (an ethnic group called the Bedey) for SFV infection. Surprisingly, a PCR assay failed to detect SFV infection in any of these participants. This is in contrast to our previously reported infection rate of about 5% among Bangladeshi villagers.

  • Design and Structure of an Engineered Disulfide-Stabilized Influenza Virus Hemagglutinin Trimer [Structure and Assembly]

  • We engineered a disulfide-stabilized influenza virus hemagglutinin (HA) trimer, termed HA3-SS, by introducing cysteine residues into the HA stem to covalently bridge the three protomers. HA3-SS has increased thermostability compared to wild-type HA, and binding of head- and stem-targeted antibodies (Abs) is preserved; only minor structural changes are found in the vicinity of the additional disulfide. This platform has been applied to H1 and H3 HAs and provides prospects for design of intact, stabilized influenza virus HA immunogens.

  • Immediate and Ongoing Detection of Prions in the Blood of Hamsters and Deer following Oral, Nasal, or Blood Inoculations [Spotlight]

  • Infectious prions traverse epithelial barriers to gain access to the circulatory system, yet the temporal parameters of transepithelial transport and persistence in the blood over time remain unknown. We used whole-blood real-time quaking-induced conversion (wbRT-QuIC) to analyze whole blood collected from transmissible spongiform encephalopathy (TSE)-inoculated deer and hamsters throughout the incubation period for the presence of common prion protein-conversion competent amyloid (PrPC-CCA). We observed PrPC-CCA in the blood of TSE-inoculated hosts throughout the disease course from minutes postexposure to terminal disease.

  • Interferon Gamma Prolongs Survival of Varicella-Zoster Virus-Infected Human Neurons In Vitro [Virus-Cell Interactions]

  • Infection of human neurons in vitro with varicella-zoster virus (VZV) at a low multiplicity of infection does not result in a cytopathic effect (CPE) within 14 days postinfection (dpi), despite production of infectious virus. We showed that by 28 dpi a CPE ultimately developed in infected neurons and that interferon gamma inhibited not only the CPE but also VZV DNA accumulation, transcription, and virus production, thereby prolonging the life of VZV-infected neurons.

  • Presence of a trs-Like Motif Promotes Rep-Mediated Wild-Type Adeno-Associated Virus Type 2 Integration [Virus-Cell Interactions]

  • High-throughput integration site (IS) analysis of wild-type adeno-associated virus type 2 (wtAAV2) in human dermal fibroblasts (HDFs) and HeLa cells revealed that juxtaposition of a Rep binding site (RBS) and terminal resolution site (trs)-like motif leads to a 4-fold-increased probability of wtAAV integration. Electrophoretic mobility shift assays (EMSAs) confirmed binding of Rep to off-target RBSs. For the first time, we show Rep protein off-target nicking activity, highlighting the importance of the nicking substrate for Rep-mediated integration.

  • Hepatitis B Virus-Infected HepG2hNTCP Cells Serve as a Novel Immunological Tool To Analyze the Antiviral Efficacy of CD8+ T Cells In Vitro [Pathogenesis and Immunity]

  • CD8+ T cells are the main effector lymphocytes in the control of hepatitis B virus (HBV) infection. However, limitations of model systems, such as low infection rates, restrict mechanistic studies of HBV-specific CD8+ T cells. Here, we established a novel immunological cell culture model based on HBV-infected HepG2hNTCP cells that endogenously processed viral antigens and presented them to HBV-specific CD8+ T cells. This induced cytolytic and noncytolytic CD8+ T-cell effector functions and reduction of viral loads.

  • New Zealand Stresses that It Is High Plains Virus Free, and the Virus Struggles with an Identity Crisis [Letters to the Editor]

  • Reply to "New Zealand Stresses that It Is High Plains Virus Free, and the Virus Struggles with an Identity Crisis" [Letters to the Editor]

  • Erratum for O'Connor et al., Peptide-Dependent Recognition of HLA-B*57:01 by KIR3DS1 [Errata]

  • Correction for Tatineni et al., An Eriophyid Mite-Transmitted Plant Virus Contains Eight Genomic RNA Segments with Unusual Heterogeneity in the Nucleocapsid Protein [Author Correction]

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    JVI Accepts: Articles Published Ahead of Print

  • The Impact of Nucleoporin Mediated Chromatin localization and Nuclear Architecture on HIV Integration Site Selection [PublishAheadOfPrint]

  • It has been known for a number of years that integration sites of human immunodeficiency virus 1 (HIV-1) DNA show a preference for actively expressed chromosomal locations. A number of viral and cellular proteins are implicated in this process, but the underlying mechanism is not clear. Two recent breakthrough publications advance our understanding of HIV integration site selection by focusing on the localization of the preferred target genes of integration. These studies reveal that knockdown of certain nucleoporins and components of nucleocytoplasmic trafficking alter integration site preference; not by altering the trafficking of the viral genome, but by altering the chromatin subtype localization relative to the structure of the nucleus. Here we describe the link between the nuclear basket nucleoporins (Tpr and Nup153) and chromatin organization and how altering the host environment by manipulating nuclear structure could have important implications for the preferential integration of HIV into actively transcribed genes facilitating efficient viral replication.

  • Blockage of CD59 function restores activities of neutralizing and non-neutralizing antibodies in triggering antibody-dependent complement-mediated lysis of HIV-1 virions and provirus-activated latently infected cells [PublishAheadOfPrint]

  • Both HIV-1 virions and infected cells use their surface regulators of complement activation (RCA) to resist antibody-dependent complement-mediated lysis (ADCML). Blockage of the biological function of RCA members, particularly CD59 (a key RCA member that controls formation of the membrane attack complex at the terminal stage of the complement activation cascades via all three activation pathways), has rendered both HIV-1 virions and infected cells sensitive to ADCML mediated by anti-Env antibodies (Abs) or sera/plasma from patients at different stages of viral infection. In the current study, we used the well-characterized anti-HIV-1 neutralizing Abs (nAbs) including 2G12, 2F5, and 4E10 and non-nAbs including 2.2C, A32, N5-i5, and N12-i15 to investigate whether the enhancement of ADCML by blockage of CD59 function is mediated by nAbs, non-nAbs, or both. We found that all nAbs and two non-nAbs (N5-i5 and A32) strongly reacted to three HIV-1 laboratory strains (R5, X4, and R5/X4), six primary isolates, and provirus-activated ACH-2 cells examined. In contrast, two non-nAbs 2.2C and N12-i15 reacted weakly and did not react to these targets, respectively. After blockage of CD59 function, the reactive Abs, regardless of their neutralizing activities, significantly enhanced specific ADCML of HIV-1 virions (both laboratory strains and primary isolates) and provirus-activated latently infected cells. The ADMCL efficacy positively correlated with the ELISA-reactive intensity of those Abs with their targets. Thus, blockage of RCA function represents a novel approach to restore activities of both nAbs and non-nAbs in triggering ADCML of HIV-1 virions and provirus-activated latently infected cells.

    Importance There is a renewed interest in the potential role of non-nAbs in the control of HIV-1 infection. Our data, for the first time, demonstrated that blockage of the biological function of RCA members rendered both HIV-1 virions and infected cells sensitive to ADCML mediated by not only nAbs, but also non-nAbs. Our results are significant in developing novel immune-based approaches to restore the functions of nAbs and non-nAbs in the circulation of HIV-1-infected individuals to specifically target and clear HIV-1 virions and infected cells. Our data also provide new insights into the mechanisms by which HIV-1 virions and infected cells escape Ab-mediated immunity and could aid in the design and/or development of therapeutic HIV-1 vaccines. In addition, a combination of antiretroviral therapy (ART) with RCA blockage, provirus activators, and therapeutic vaccines may represent a novel approach to eliminate HIV-1 reservoirs, i.e. the infected cells harboring replication-competent proviruses and residual viremia.

  • The intracellular domain of the coxsackievirus and adenovirus receptor differentially influences adenovirus entry [PublishAheadOfPrint]

  • The coxsackievirus and adenovirus receptor (CAR) is a cell adhesion molecule used as a docking molecule by some adenoviruses (AdVs) and group B coxsackieviruses. We previously proposed that the preferential transduction of neurons by canine adenovirus type 2 (CAV-2) is due to CAR-mediated internalization. Our proposed pathway of CAV-2 entry is in contrast to that of human AdV type 5 (HAdV-C5) in non-neuronal cells, where internalization is mediated by auxiliary receptors such as integrins. We therefore asked if in fibroblast-like cells the intracellular domain (ICD) of CAR plays a role in the internalization of the CAV-2 fiber knob (FKCAV), CAV-2, or HAdV-C5 when the capsid cannot engage integrins. Here, we show that in fibroblast-like cells, the CAR ICD is needed for FKCAV entry and efficient CAV-2 transduction, but dispensable for HAdV-C5 and a HAdV-C5 capsid lacking the RGD sequence (an integrin-interacting motif) in the penton. Moreover, the deletion of CAR ICD further impacts CAV-2 intracellular trafficking, highlighting the crucial role of CAR in CAV-2 intracellular dynamics. These data demonstrate that CAR ICD contains sequences important for the recruitment of the endocytic machinery that differentially influences AdV cell entry.

    Importance Understanding how viruses interact with the host cell surface and reach the intracellular space is of crucial importance for applied and fundamental virology. Here, we compare the role of a cell adhesion molecule (CAR), in the internalization of adenoviruses that naturally infect humans and Canidae. We show that the intracellular domain of CAR differentially regulate AdV entry and trafficking. Our study highlights the mechanistic differences that a receptor can have for two viruses from the same family.

  • Role of nectin-1 and HVEM as cellular receptors for Herpes Simplex Virus Type 1 on primary murine dermal fibroblasts [PublishAheadOfPrint]

  • The cellular proteins nectin-1 and HVEM can both mediate the entry of herpes simplex virus type 1 (HSV-1). We have recently shown how these receptors contribute to infection of skin by investigating HSV-1 entry into murine epidermis. Ex vivo infection studies reveal nectin-1 as the primary receptor in epidermis whereas HVEM has a more limited role. While the epidermis represents the outermost layer of skin, the contribution of nectin-1 and HVEM in the underlying dermis is still open. Here we analyzed the role of each receptor during HSV-1 entry in murine dermal fibroblasts that were deficient in expression of either nectin-1 or HVEM or both receptors. Because infection was not prevented by the absence of either nectin-1 or HVEM, we conclude that they can act as alternative receptors. Although HVEM was found to be highly expressed on fibroblasts, entry was delayed in nectin-1-deficient cells suggesting that nectin-1 acts as the more efficient receptor. In the absence of both receptors, entry was strongly delayed leading to a much reduced viral spread and virus production. These results suggest an unidentified cellular component that acts as alternate but inefficient receptor for HSV-1 on dermal fibroblasts. Characterization of the cellular entry mechanism sugeests that HSV-1 can enter dermal fibroblasts both by direct fusion with the plasma membrane and via endocytic vesicles and that this is not dependent on the presence or absence of nectin-1. Entry was also shown to require dynamin and cholesterol, suggesting comparable entry pathways in keratinocytes and dermal fibroblasts.

    Importance Herpes simplex virus (HSV) is a human pathogen which infects its host via mucosal surfaces or abraded skin. To understand how HSV-1 overcomes the protective barrier of mucosa or skin and reaches its receptors in tissue, it is essential to know which receptors contribute to the entry into individual skin cells. Previously, we have explored the contribution of nectin-1 and HVEM as receptors for HSV-1 entry into murine epidermis where keratinocytes form the major cell type. Since the underlying dermis consists primarily of fibroblasts, we have now extended our study of HSV-1 entry to dermal fibroblasts isolated from nectin-1- or HVEM-deficient mice or from mice deficient in both receptors. Our results demonstrate a role for both nectin-1 and HVEM as receptors and suggest a further receptor which appears much less efficient.

  • Double-strand RNA is detected by immunofluorescence analysis in RNA and DNA virus infections including those by negative-strand RNA viruses [PublishAheadOfPrint]

  • Early biochemical studies of viral replication suggested that most viruses produce dsRNA that is essential for the induction of the host immune response. However, it was reported in 2006 that dsRNA could be detected by immunofluorescence antibody staining in dsDNA and positive-strand RNA but not in negative-strand RNA virus infections. Other reports in the literature seemed to support these observations. This suggested that negative-strand RNA viruses produce little, if any dsRNA, or that more efficient viral countermeasures to mask dsRNA are mounted. Because of our interest in use of dsRNA antibodies for virus discovery, particularly in pathologic specimens, we wanted to determine how universal immunostaining for dsRNA might be in animal virus infections. We have detected in situ formation of dsRNA in cells infected with vesicular stomatitis virus, measles virus, influenza A virus and Nyamanini virus infections that represent different negative-strand RNA virus families. dsRNA was also detected in cells infected with lymphocytic choriomeningitis virus, an ambisense RNA virus, and minute virus of mice, a ssDNA parvovirus, but not hepatitis B virus. Although dsRNA staining was primarily observed in the cytoplasm it was also seen in the nucleus of cells infected with influenza A virus Nyamanini virus and minute virus of mice (MVM). Thus, it is likely that most animal virus infections produce dsRNA species that can be detected by immunofluorescence staining. Apoptosis induced in several uninfected cell lines failed to up-regulate dsRNA formation.

    IMPORTANCE An effective antiviral host immune response depends on recognition of viral invasion and an intact innate immune system as a first line of defense. Double-stranded RNA (dsRNA) is an essential viral product in the induction of innate immunity, leading to the production of type I interferons (IFN) and activation of hundreds of IFN-stimulated genes. The present study demonstrates that infections, including those by ssDNA and positive- and negative-strand RNA viruses, produce dsRNAs detectable by standard immunofluorescence staining. While dsRNA staining was primarily observed in the cytoplasm, nuclear staining was also present in some RNA and DNA virus infections. The nucleus is unlikely to have pathogen associated molecular pattern (PAMP) receptors for dsRNA because of the presence of host dsRNA molecules. Thus, it is likely that most animal virus infections produce dsRNA species detectable by immunofluorescence staining that may prove useful in viral discovery as well.

  • A Coronavirus E Protein is Present in Two Distinct Pools with Different Effects on Assembly and the Secretory Pathway [PublishAheadOfPrint]

  • Coronaviruses (CoVs) assemble by budding into the lumen of the early Golgi prior to exocytosis. The small CoV envelope (E) protein plays roles in assembly, virion release, and pathogenesis. CoV E has a single hydrophobic domain (HD), is targeted to Golgi membranes, and has cation channel activity in vitro. However, the precise functions of the CoV E protein during infection are still enigmatic. Structural data for the severe acute respiratory syndrome (SARS)-CoV E protein suggests that it assembles into a homo-pentamer. Specific residues in the HD regulate the ion-conducting pore formed by SARS-CoV E in artificial bilayers and the pathogenicity of the virus during infection. The E protein from the avian infectious bronchitis virus (IBV) has dramatic effects on the secretory system, which requires residues in the HD. Here, we use the known structural data from SARS-CoV E to infer residues important for ion channel activity and oligomerization of IBV E. We present biochemical data for the formation of two distinct oligomeric pools of IBV E in transfected and infected cells, and residues required for their formation. A high-order oligomer of IBV E is required for the production of virus-like particles (VLPs), implicating this form of the protein in virion assembly. Additionally, disruption of the secretory pathway by IBV E correlates with a form that is likely monomeric, suggesting that the effects on the secretory pathway are independent of E ion channel activity.

    IMPORTANCE CoVs are important human pathogens with significant zoonotic potential as demonstrated by the emergence of SARS-CoV and Middle East respiratory syndrome (MERS)-CoV. Progress has been made toward identifying potential vaccine candidates in mouse models of CoV infection, including use of attenuated viruses that lack the CoV E protein or express E mutants. However, no approved vaccines and anti-viral therapeutics exist. We previously reported that the hydrophobic domain of the IBV E protein, a putative viroporin, causes disruption of the mammalian secretory pathway when exogenously expressed in cells. Understanding the mechanism of this disruption could lead to the identification of novel anti-viral therapeutics. Here, we present biochemical evidence for two distinct oligomeric forms of IBV E, one essential for assembly and the other with a role in disruption of the secretory pathway. Discovery of two forms of CoV E protein will provide additional targets for anti-viral therapeutics.

  • Myd88 initiates early innate responses and promotes CD4 T cells during coronavirus encephalomyelitis [PublishAheadOfPrint]

  • Myd88 signaling is critical to control numerous central nervous system (CNS) infections by promoting both innate and adaptive immune responses. Nevertheless, the extent to which Myd88 regulates type I IFN versus proinflammatory factors and T cell function, as well as anatomical site of action varies extensively with the pathogen. CNS infection by neurotropic coronavirus with confined replication in brain and spinal cord induces protective IFNaalpha;/bbeta; via Myd88 independent activation of melanoma differentiation-associated gene 5 (MDA5). However, a contribution of Myd88 dependent signals to CNS pathogenesis has not been assessed. Infected Myd88-/- mice failed to control virus, exhibited enhanced clinical disease coincident with increased demyelination, and succumbed to infection within three weeks. Induction of IFNaalpha;/bbeta;, as well as proinflammatory cytokines and chemokines was impaired early during infection. However, defects in both IFNaalpha;/bbeta; and select proinflammatory factors were rapidly overcome prior to T cell recruitment. Myd88 deficiency also specifically blunted myeloid and CD4 T cell recruitment into the CNS without affecting CD8 T cells. Moreover, CD4 T cells, but not CD8 T cells, were impaired in IFN production. Ineffective virus control indeed correlated most prominently with reduced anti-viral IFN in the CNS of Myd88-/- mice. The results demonstrate a crucial role for Myd88 both in early induction of innate responses during coronavirus induced encephalomyelitis and in specifically promoting protective CD4 T cell activation. In the absence of these responses, functional CD8 T cells are insufficient to control viral spread within the CNS resulting in severe demyelination.

    Importance During central nervous system (CNS) infections signaling through the adaptor protein Myd88 promotes both innate and adaptive immune responses. The extent to which Myd88 regulates antiviral type I IFN, pro inflammatory factors, adaptive immunity and pathology is pathogen dependent. These results reveal that Myd88 protects from lethal neurotropic coronavirus induced encephalomyelitis by accelerating, but not enhancing, IFNaalpha;/bbeta; as well as promoting peripheral activation and CNS accumulation of virus specific CD4 T cells secreting IFN. By controlling both early innate responses and CD4 T cell mediated antiviral IFN, Myd88 signaling limits initial viral dissemination and is vital for T cell mediated control of viral load. Uncontrolled viral replication in the absence of Myd88 leads to severe demyelination and pathology despite overall reduced inflammatory responses. These data support a vital role of Myd88 signaling in protective antimicrobial function in the CNS by promoting proinflammatory mediators and T cell mediated IFN production.

  • P2X1 receptor antagonists inhibit HIV-1 fusion by blocking virus-coreceptor interactions [PublishAheadOfPrint]

  • HIV-1 Env glycoprotein-mediated fusion is initiated upon sequential binding of Env to CD4 and coreceptors, CXCR4 or CCR5. Whereas these interactions are thought to be necessary and sufficient to promote HIV-1 fusion, other host factors can modulate this process. Previous studies have reported potent inhibition of HIV-1 fusion by selective P2X1 receptor antagonists, including NF279, and suggested that these receptors play a role in HIV-1 entry. Here we investigated the mechanism of anti-viral activity of NF279 and found that this compound does not inhibit HIV-1 fusion by preventing the activation of P2X1 channels, but effectively blocks the virus binding to CXCR4 or CCR5. The notion of an off-target effect of NF279 on HIV-1 fusion is supported by the lack of detectable expression of P2X1 receptors in cells used in fusion experiments and by the fact that addition of ATP or enzymatic depletion of ATP in a culture medium does not modulate viral fusion. Importantly, NF279 fails to inhibit HIV-1 fusion with cell lines and primary macrophages when added at an intermediate stage downstream of Env-CD4-coreceptor engagement. Conversely, in the presence of NF279, HIV-1 fusion is arrested downstream of CD4 binding but prior to coreceptor engagement. NF279 also antagonizes the signaling function of CCR5, CXCR4 and another chemokine receptor, as evidenced by the suppression of calcium responses elicited by specific ligands and by recombinant gp120. Collectively, our results demonstrate that NF279 is a dual HIV-1 coreceptor inhibitor that interferes with the functional engagement of CCR5 and CXCR4 by Env.

    Importance The well-documented inhibition of HIV-1 fusion and replication by purinergic receptor antagonists implicated these proteins in viral entry/fusion and suggested new targets for anti-viral therapy. Mechanistic studies of the role of P2X1 receptor in HIV-1 fusion performed in this study strongly imply that this channel is not expressed in target cells or involved in viral fusion. Instead, we found that inhibition of HIV-1 fusion by a specific P2X1 receptor antagonist NF279 is due to blocking the virus interactions with both CXCR4 and CCR5 coreceptors. The ability of NF279 to abrogate cellular calcium signaling induced by respective chemokines showed that this compound acts as a dual-coreceptor antagonist. P2X1 receptor antagonists could thus represent a new class of dual-coreceptor inhibitors with a structure and mechanism of action that are distinct from known HIV-1 coreceptor antagonists.

  • Salmon gill poxvirus, the deepest representative of the Chordopoxvirinae [PublishAheadOfPrint]

  • Poxviruses are large DNA viruses of vertebrates and insects causing disease in many animal species including reptiles, birds and mammals. Although poxvirus-like particles were detected in diseased farmed koi carp, ayu and Atlantic salmon, their genetic relationships to poxviruses were not established. Here, we provide the first genome sequence of a fish poxvirus, which was isolated from farmed Atlantic salmon, and use qPCR and immunohistochemistry to describe aspects of salmon gill poxvirus disease. The gill was the main target organ where immature and mature poxvirus particles were detected in detaching, apoptotic respiratory epithelial cells, preceding clinical disease in the form of lethargy, respiratory distress and mortality. In moribund salmon blocking of gas exchange would likely be caused by adherence of respiratory lamellae and epithelial proliferation obstructing respiratory surfaces. The virus was not found in healthy salmon or in controls of gill disease without apoptotic cells, although transmission remains to be demonstrated. PCR on archival tissue confirmed virus infection in 14 cases with gill apoptosis in Norway starting from 1995. Phylogenomic analyses showed that the fish poxvirus is the deepest available branch of chordopoxviruses. The virus encompasses most key chordopoxvirus genes that are required for genome replication and expression although the gene order is substantially different. Nevertheless, many highly conserved chordopoxvirus genes involved in viral membranes biogenesis or virus-host interactions are missing. Instead, the salmon poxvirus carries numerous genes encoding unknown proteins many of which have low sequence complexity and contain simple repeats suggestive of intrinsic disorder or distinct protein structures.

    IMPORTANCE Aquaculture is an increasingly important global source of high quality food. To sustain this growth, disease control in fish farming is essential. Moreover, the spread of disease from farmed fish to wild life is a concern. Serious poxviral diseases are emerging in aquaculture, but very little is known about the viruses and the diseases they cause. There is a possibility that viruses may spread to new species with enhanced virulence as has occurred with the myxoma poxvirus in rabbits. Provision of the first fish poxvirus genome sequence and specific diagnostics for the salmon gill poxvirus in Atlantic salmon may help curb this disease and provide comparative knowledge. Furthermore, because salmon gill poxvirus represents the deepest branch of chordopoxvirus so far discovered, the genome analysis provided substantial insight into the evolution of different functional modules in this important group of viruses.

  • Exploring the balance between DNA pressure and capsid stability in Herpes and phage. [PublishAheadOfPrint]

  • We have recently shown in both herpesviruses and phages that packaged viral DNA creates a pressure of tens of atmospheres pushing against the interior capsid wall. For the first time, using differential scanning microcalorimetry, we directly measure the energy powering the release of pressurized DNA from the capsid. Furthermore, using a new calorimetric assay to accurately determine the temperature inducing DNA release, we found a direct influence of internal DNA pressure on the stability of the viral particle. We show that the balance of forces between the DNA pressure and capsid strength, required for DNA retention between rounds of infection, is conserved between evolutionarily diverse bacterial viruses (phage and P22), as well as a eukaryotic virus, human Herpes Simplex 1 (HSV-1). Our data also suggest that the portal vertex in these viruses is the weakest point in the overall capsid structure and presents the "Achilles' heel" of virus's stability. Comparison between these viral systems shows that viruses with higher DNA packing density (resulting in higher capsid pressure) have inherently stronger capsid structures preventing spontaneous genome release prior to infection. This force balance is of key importance for viral survival and replication. Investigating the ways to disrupt this balance can lead to development of new mutation resistant anti-virals.

    IMPORTANCE A virus can generally be described as a nucleic acid genome contained within a protective protein shell, called the capsid. For many double-stranded DNA viruses, confinement of the large DNA molecule within the small protein capsid results in an energetically stressed DNA state exerting tens of atmospheres of pressures of on the inner capsid wall. We show that stability of viral particles (which directly relates to infectivity) is strongly influenced by the state of the packaged genome. Using scanning calorimetry on a bacterial virus (phage ), as an experimental model system, we investigate the thermodynamics of genome release associated with destabilizing the viral particle. Furthermore, we compare the influence of tight genome confinement on the relative stability for diverse bacterial and eukaryotic viruses. These comparisons reveal an evolutionarily conserved force balance between the capsid stability and the density of the packaged genome.

  • Longitudinal Detection of Prion Shedding in Saliva and Urine by CWD-Infected Deer by RT-QuIC [PublishAheadOfPrint]

  • Chronic Wasting Disease (CWD) is an emergent, rapidly spreading prion disease of cervids. Shedding of infectious prions in saliva and urine is thought to be an important factor in CWD transmission. To help elucidate this issue, we applied an in vitro amplification assay to determine the onset, duration, and magnitude of prion shedding in longitudinally collected saliva and urine samples from CWD-exposed white-tailed deer. We detected prion shedding as early as 3 months after CWD exposure and sustained shedding throughout the disease course. We estimated that a 50% lethal dose (LD50) for cervidized transgenic mice would be contained in 1 ml of infected deer saliva or 10 ml or urine. Given the average course of infection and daily production of these body fluids, an infected deer would shed thousands of prion infectious dosesover the course of CWD infection. The direct and indirect environmental impact of this magnitude of prion shedding for cervid and non-cervid species is surely significant.

    Importance: Chronic wasting disease (CWD) is an emerging and uniformly fatal prion disease affecting free ranging deer and elk and now recognized in 22 United States and 2 C anadian Provinces. It is unique among prion diseases in that it is transmitted naturally though wild populations. A major hypothesis for CWD's florid spread is that prions are shed in excreta and transmitted via direct or indirect environmental contact. Here we use a rapid in vitro assay to show that infectious doses of CWD prions are in fact shed throughout the multi-year disease course in deer. This finding is an important advance in assessing the risks posed by shed CWD prions to animals as well as humans.

  • Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of the Ebola Virus [PublishAheadOfPrint]

  • Lipid enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. The Ebola virus, which buds from the plasma membrane of the host cell causes viral hemorrhagic fever and has a high fatality rate. To date little is known about how budding and egress of the Ebola virus is mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of the Ebola virus matrix protein VP40. VP40 binds PS containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress; whereas PS is typically only on the inner leaflet. Together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus like particles.

    IMPORTANCE The lipid-enveloped Ebola virus causes severe infection, has a high mortality rate, and currently lacks FDA approved therapeutics or vaccines. The Ebola virus harbors just seven genes in its genome and during the replication process there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell it infects. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This study, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry.

  • Characterization of T-antigens including Middle T and ALTO expressed by the human polyomavirus associated with trichodysplasia spinulosa [PublishAheadOfPrint]

  • The polyomavirus tumor (T) antigens play crucial roles in viral replication, transcription and cellular transformation. They are encoded by partially overlapping open reading frames located in the early region through alternative mRNA splicing. The T expression pattern of the Trichodysplasia Spinulosa-associated Polyomavirus (TSPyV) has not been established yet, hampering further study of its pathogenic mechanisms and taxonomic relationship.

    Here we characterized TSPyV T-expression in human cell lines transfected with the TSPyV early region. Sequencing of T-encoded RT-PCR products revealed three splice donor and acceptor sites creating six mRNA splice products that potentially encode Small T (ST), Middle T (MT), Large T (LT), TinyT, 21kT and alternative T (ALTO). Except for 21kT, these splice products were also detected in skin of TSPyV-infected patients. At least three splice products were confirmed by Northern blotting likely encoding LT, MT, ST, 21kT and ALTO. Protein expression was demonstrated for LT, ALTO and possibly MT, of which LT was detected in the nucleus and ALTO in the cytoplasm of transfected cells. Splice site and start codon mutations indicated that ALTO is encoded by the same splice product that encodes LT and uses internal start codons for initiation. Genuineness of ALTO was indicated by the identification of acetylated N-terminal ALTO peptides by mass spectrometry.

    Summarizing, TSPyV exhibits an expression pattern characterized by both MT and ALTO expression, combining features of rodent and human polyomaviruses. This unique expression pattern provides important leads to further study of polyomavirus-related disease and to understand polyomavirus evolution.

    IMPORTANCE The human Trichodysplasia Spinulosa-associated Polyomavirus (TSPyV) is distinguished among polyomaviruses for combining productive infection with cell-transforming properties. In the research presented here, we further substantiate this unique position by indicating expression of both Middle (MT) and alternative T-(ALTO) antigens in TSPyV. So far, none of the human polyomaviruses was shown to express MT, which is considered the most important viral oncoprotein of rodent polyomaviruses. Co-expression of ALTO and MT, which involves a conserved, recently recognized overlapping ORF subject to positive selection, has not been observed before for any polyomavirus. Altogether, our study provides valuable new insights in polyomavirus T gene use and expression. Obviously these insights will be instrumental in further study and understanding of TSPyV pathogenicity. More importantly, however, they provide important leads with regard to interrelationship, functionality and evolution of polyomaviruses as a whole, pushing TSPyV forward as a suitable model virus to further study these entities.

  • Evolution of influenza B virus in Kuala Lumpur, Malaysia between 1995 and 2008 [PublishAheadOfPrint]

  • Influenza B virus causes significant disease but remains understudied in tropical regions. We sequenced 72 influenza B viruses collected in Kuala Lumpur, Malaysia from 1995nndash;2008. The predominant circulating lineage (Victoria or Yamagata) changed every 1nndash;3 years, and these shifts were associated with increased incidence of influenza B. We also found poor lineage matches with recommended influenza vaccine strains. While most influenza B lineages in Malaysia were short-lived, one circulated for 3nndash;4 years.

  • Phylodynamics of Enterovirus A71-Associated Hand, Foot and Mouth Disease in Viet Nam [PublishAheadOfPrint]

  • Enterovirus A71 (EV-A71) is a major cause of hand, foot and mouth disease (HFMD) and is particularly prevalent in parts of Southeast Asia, affecting thousands of children and infants each year. Revealing the evolutionary and epidemiological dynamics of EV-A71 through time and space is central to understanding its outbreak potential. We generated the full genome sequences of 200 EV-A71 strains sampled from various locations in Viet Nam between 2011-2013, and used these sequence data to determine the evolutionary history and phylodynamics of EV-A71 in Viet Nam, providing estimates of the effective reproduction number (Re) of the infection through time. In addition, we described the phylogeography of EV-A71 throughout Southeast Asia, documenting patterns of viral gene flow. Accordingly, our analysis reveals that a rapid genogroup switch from C4 to B5 likely took place during 2012 in Viet Nam. We show that the Re of subgenogroup C4 decreased during the time-frame of sampling, while that of B5 increased and remained ggt;1 at the end of 2013, corresponding to a rise in B5 prevalence. Our study reveals that the subgenogroup B5 virus that emerged into Viet Nam is closely related to variants that were responsible for large epidemics in Malaysia and Taiwan and therefore extends our knowledge regarding its associated endemic area. Subgenogroup B5 evidently has the potential to cause more widespread outbreaks across Southeast Asia.

    Importance EV-A71 is one of many viruses that cause HFMD, a common syndrome that largely affects infants and children. HFMD usually causes only mild illness with no long-term consequences. Occasionally, however, severe infection may arise, especially in very young children, causing neurological complications and even death. EV-A71 is highly contagious and is associated with the most severe HFMD cases, with large and frequent epidemics of the virus recorded worldwide. Although major advances have been made in the development of a potential EV-A71 vaccine, there is no current prevention and little is known about the patterns and dynamics of EV-A71 spread. In this study we utilize full-length genome sequence data obtained from HFMD patients in Viet Nam, a geographical region where the disease has been endemic since 2003, to characterize the phylodynamics of this important emerging virus.

  • HIV-1 cell-free and cell-to-cell infection are differentially regulated by distinct determinants in the Env gp41 cytoplasmic tail [PublishAheadOfPrint]

  • The HIV-1 envelope (Env) glycoprotein mediates viral entry during both cell-free and cell-to-cell infection of CD4+ T cells. The highly conserved long cytoplasmic tail (CT) of Env is required in a cell type-dependent manner for optimal infectivity of cell-free virus. To probe the role of the CT in cell-to-cell infection, we tested a panel of mutants in the CT region that maintain or attenuate cell-free infection to ask whether the functions of the CT are conserved during cell-free and cell-to-cell infection. The mutants tested included truncations of structural motifs in the gp41 CT, and two point mutants in lentiviral lytic peptide 3 (LLP-3) previously described as disrupting the infectivity of cell-free virus. We found that small truncations of 28 to 43 amino acids (aa) or two LLP-3 point mutations YW_SL and LL_RQ severely impaired single round cell-free infectivity by 10-fold or more relative to wild type full-length CT. These mutants showed a modest 2-fold reduction in cell-to-cell infection assays. Conversely, large truncations of 93 to 124 aa severely impaired cell-to-cell infectivity by 20-fold or more, while displaying a 50% increase in infectivity of cell-free viral particles when produced in 293T cells. Intermediate truncations of 46 to 90 aa showed profound impairment of both modes of infection. Our results show that the ability of Env to support cell-free and cell-to-cell infection are genetically distinct. These differences are cell-type dependent for large CT truncation mutants. Additionally, point mutants in LLP-3 can maintain multi-round propagation from cell-to-cell in primary CD4+ T cells.

    Importance The functions of HIV Env gp41 CT remain poorly understood despite being widely studied in the context of cell-free infection. We have identified domains of the gp41 CT responsible for striking selective deficiencies in either cell-free or cell-to-cell infectivity. These differences may reflect a different intrinsic regulatory influence of the CT on cell-associated versus particle-associated Env, or differential interaction with host or viral proteins. Our findings provide novel insight into the key regulatory potential of the gp41 CT in cell-free and cell-to-cell HIV-1 infection, particularly for short truncation mutants lle;43 amino acids, or point mutations in the LLP-3 helical domain of the CT which are able to propagate via cell-to-cell in the absence of infectious cell-free virus production. These mutants may also serve as tools to further define the contributions of cell-free and cell-to-cell infection in vitro and in vivo.

  • An aptamer against the matrix binding domain on the hepatitis B virus capsid impairs virion formation [PublishAheadOfPrint]

  • The hepatitis B virus (HBV) particle is an icosahedral nucleocapsid surrounded by a lipid envelope containing viral surface proteins. A small domain (matrix domain, MD) in the large surface protein L and a narrow region (matrix binding domain, MBD) including isoleucine 126 on the capsid surface have been mapped where point mutations like core-I126A specifically blocked nucleocapsid envelopment. Possibly, both domains interact with each other during virion morphogenesis. By the SELEX method we evolved DNA aptamers from an oligonucleotide library binding to purified recombinant capsids but not binding to the corresponding I126A mutant capsids. Aptamers bound to capsids were separated from unbound molecules by filtration. After 13 rounds of selections and amplifications 16 different aptamers were found among 73 clones. The four most frequent aptamers represented more than 50 % of the clones. The main aptamer AO-01 (13 clones, 18 %) showed the lowest dissociation constant (Kd) of 180 +/- 82 nM for capsid binding among the four molecules. Its Kd value for I126A capsids was 1306 +/- 503 nM. Cotransfection of Huh7 cells with AO-01 and an HBV genomic construct resulted in 47 % inhibition of virion production 3 days post transfection but showed no inhibition by cotransfection of an aptamer with random sequence. The half-life of AO-01 in cells was 2 hours which might explain the incomplete inhibition. The results support the importance of the MBD for nucleocapsid envelopment. Inhibiting the MD-MBD interaction by a low molecular weight substance might represent a new approach for an antiviral therapy.

    Importance Approximately 240 million people are persistently infected with HBV. To date, antiviral therapies depend on a single target, the viral reverse transcriptase. Future additional targets could be viral protein-protein interactions. We selected a 55 base long single stranded DNA molecule (aptamer) which binds with relatively high affinity to a region on the HBV capsid interacting with viral envelope proteins during budding. This aptamer inhibits virion formation in cell culture. The result substantiates the current model for HBV morphogenesis and shows that the capsid envelope interaction is a potential antiviral target.

  • The great (nuclear) escape: new insights into the role of the nuclear egress complex of herpesviruses [PublishAheadOfPrint]

  • Herpesviruses are unusual among enveloped viruses because they bud twice yet acquire a single envelope. They are also the only known viruses that bud into the nuclear envelope. We discovered that the herpesvirus nuclear egress complex (NEC) could bud membranes without the help of other proteins by forming a coat-like hexagonal scaffold inside the budding membrane. This finding raises a possibility that a phenotypically similar nuclear export of large RNAs is cargo-driven.

  • Impaired EBV-specific neutralizing antibody response during acute infectious mononucleosis is coincident with global B-cell dysfunction [PublishAheadOfPrint]

  • Here we present evidence for previously unappreciated B-cell immune dysregulation during acute EBV-associated infectious mononucleosis (IM). Longitudinal analyses revealed that patients with acute IM have undetectable EBV-specific neutralizing antibodies and gp350-specific B-cell responses, which was associated with a significant reduction in memory B-cells and no evidence of circulating antibody-secreting cells. These observations correlate with dysregulation of tumour necrosis factor (TNF)-family members BAFF and APRIL and increased expression of FAS on circulating B-cells.

  • Functional and structural characterization of human V3-specific monoclonal antibody 2424 with neutralizing activity against HIV-1 JRFL [PublishAheadOfPrint]

  • The V3 region of HIV-1 gp120 is important for virus-coreceptor interaction and highly immunogenic. Although most anti-V3 antibodies neutralize only the sensitive Tier 1 viruses, anti-V3 antibodies effective against the more resistant viruses exist, and a better understanding about these antibodies and their epitopes would be beneficial for the development of novel vaccine immunogens against HIV. The HIV-1 isolate JRFL with its cryptic V3 is resistant to most V3-specific monoclonal antibodies (mAbs). However, the V3 mAb 2424 achieves 100% neutralization against JRFL. 2424 is encoded by IgH V3-53 and IgL V2-28 genes, a pairing rarely used by the other V3 mAbs.. 2424 also has distinct binding and neutralization profiles. Studies of 2424-mediated neutralization of JRFL produced with a mannosidase inhibitor further revealed that its neutralizing activity is unaffected by the glycan composition of the virus envelope. To understand the distinct activity of 2424, we determined the crystal structure of 2424 Fab in complex with a JRFL V3 peptide, and showed that the 2424 epitope is located at the tip of the V3 crown (307IHIGPGRAFY318), dominated by interactions with HisP308, ProP313, and ArgP315. The binding mode of 2424 is similar to that of the well-characterized 447-52D, although 2424 is more side-chain dependent. The 2424 epitope is focused on the very apex of V3, away from nearby glycans, facilitating antibody access. This feature distinguishes the 2424 epitope from the other V3 crown epitopes and indicates that the tip of V3 is a potential site to target and incorporate into HIV vaccine immunogens.

    IMPORTANCE HIV/AIDS vaccines are crucial for controlling the HIV epidemics that continue to afflict millions of people worldwide. However, HIV vaccine development has been hampered by significant scientific challenges, one of which is the inability of HIV vaccine candidates evaluated thus far to elicit production of potent and broadly neutralizing antibodies. The V3 loop is one of the few immunogenic targets on the virus envelope glycoprotein that can induce neutralizing antibodies, but in many viruses, parts of V3 are inaccessible for antibody recognition. This study examined a V3-specific monoclonal antibody that can completely neutralize HIV-1 JRFL, a virus isolate resistant to most V3 antibodies. Our data reveal that this antibody recognizes the most distal tip of V3 that is not as occluded as other parts of V3. Hence, the epitope of 2424 is in one of the vulnerable sites on the virus that may be exploited in designing HIV vaccine immunogens.

  • Dual-Tropic CXCR6/CCR5 Simian Immunodeficiency Virus Infection of Sooty Mangabey Primary Lymphocytes: Distinct Coreceptor Use in SIV Natural versus Pathogenic Hosts [PublishAheadOfPrint]

  • Natural host sooty mangabeys (SM) infected with SIV exhibit high viral loads but do not develop disease, whereas infection of rhesus macaques (RM) causes CD4+ T cell loss and AIDS. Several mechanisms have been proposed to explain these divergent outcomes, including differences in cell targeting, which has been linked to low expression of the canonical SIV entry receptor CCR5 on CD4+ T cells of SM and other natural hosts. We previously showed that infection and high-level viremia occur even in a subset of SM that genetically lack functional CCR5, which indicates that alternative entry coreceptors are used by in SM in vivo in these animals. We also showed that SM CXCR6 is a robust coreceptor for SIVsmm in vitro. Here we identify CXCR6 as a principal entry pathway for SIV in SM primary lymphocytes. We show that ex vivo SIVinfection of lymphocytes from CCR5 wild-type SM is mediated by both CXCR6 and CCR5. In contrast, infection of RM lymphocytes is fully dependent on CCR5. These data raise the possibility that CXCR6-directed tropism in CCR5-low natural hosts may alter CD4+ T cell subset targeting compared with non-natural hosts, enabling SIV to maintain high level replication without leading to widespread CD4+ T cell loss.

    IMPORTANCE Natural hosts of SIV such as sooty mangabeys sustain high viral load but do not develop disease, while non-natural hosts like rhesus macaques develop AIDS. Understanding this difference may help elucidate mechanisms of pathogenesis. Natural hosts have very low levels of the SIV entry coreceptor CCR5 suggesting that restricted entry may limit infection of certain target cells, although it is unclear how the virus replicates so robustly. Here we show that, in sooty mangabey lymphocytes, infection is mediated by the alternative entry coreceptor CXCR6, as well as CCR5. In rhesus macaque lymphocytes, however, infection occurs entirely through CCR5. The use of CXCR6 for entry, combined with very low CCR5 levels, may re-direct the virus to different cell targets in natural hosts. It is possible that differential targeting may favor infection of non-essential cells and limit infection of critical cells in natural hosts, and thus contribute to benign outcome of infection.

  • Patient-specific neutralizing antibody responses to herpes simplex virus are attributed to epitopes on either gD, gB, or both and can be type-specific [PublishAheadOfPrint]

  • Herpes simplex virus (HSV) types 1 and 2 infect many humans and establish a latent infection in sensory ganglia. While some infected people suffer periodic recurrences, others do not. Infected people mount both cell-mediated and humoral responses, including the production of virus-neutralizing antibodies (Abs) directed at viral entry glycoproteins. Previously, we examined IgGs from 10 HSV-seropositive individuals; all neutralized virus and were directed primarily against gD or gD+gB. Here, we expand our studies and examine 32 additional sera from HSV-infected individuals, 23 of whom had no recurrent disease. Using an Octet RED96rreg; system, we screened all 32 serum samples directly for both glycoprotein binding and competition with known neutralizing anti-gD and -gB monoclonal Abs (MAbs). On average, the recurrent cohort exhibited higher binding to gD and gB and had higher neutralization titers. There were similar trends in the blocking of MAbs to critical gD and gB epitopes. When we depleted 6 sera of Abs to specific glycoproteins, we found different types of responses, but always directed primarily at gD and/or gB. Interestingly, in one dual-infected person, the neutralizing response to HSV-2 was due to gD2 and gB2 while HSV-1 neutralization was due to gD1 and gB1. In another case, virus neutralization was HSV-1 specific with the Ab response directed entirely at gB1, despite this serum blocking type-common anti-gD and -gB neutralizing MAbs. These data are pertinent in the design of future HSV vaccines as they demonstrate the importance of both serotypes of gD and gB as immunogens.

    IMPORTANCE We previously showed that people infected with HSV produce neutralizing Abs directed against gD or a combination of gD+gB (and in one case, gD+gB+gC, which was type-1 specific). In this more extensive study, we again found that gD or gD+gB can account for the virus neutralizing response and critical epitopes of one or both of these proteins are represented in sera of naturally-infected humans. However, we also found that some individuals produced a strong response against gB alone. In addition, we identified type-specific contributions to HSV neutralization from both gD and gB. Contributions from the other entry glycoproteins, gC and gH/gL, were minimal and limited to HSV-1 neutralization. Knowing the variations in how humans see and mount a response to HSV will be important to vaccine development.

  • A dual role for the non-receptor tyrosine kinase Pyk2 during the intracellular trafficking of human papillomavirus 16 [PublishAheadOfPrint]

  • The infectious process of human papillomaviruses (HPV) has been studied considerably and many cellular components required for viral entry and trafficking continue to be revealed. In this study we investigated the role of the non-receptor tyrosine kinase Pyk2 during HPV16 pseudovirion infection of human keratinocytes. We found that Pyk2 is necessary for infection and appears to be involved in the intracellular trafficking of the virus. siRNA-mediated reduction of Pyk2 resulted in a significant decrease in infection, but did not prevent viral entry at the plasma membrane. Pyk2 depletion resulted in altered endo-lysosomal trafficking of HPV16 and accelerated unfolding of the viral capsid. Furthermore, we observed retention of the HPV16 pseudogenome in the trans-Golgi network (TGN) in Pyk2-depleted cells, suggesting that the kinase could be required for the viral DNA to exit the TGN. While Pyk2 has previously been shown to function during entry of enveloped viruses at the plasma membrane, the kinase has not yet been implicated during the intracellular trafficking of a non-enveloped virus, such as HPV. Additionally, these data enrich the current literature on Pyk2's function in human keratinocytes.

    IMPORTANCE In this study we investigate the role of the non-receptor tyrosine kinase, Pyk2, during human papillomavirus (HPV) infection of human skin cells. Infections with high-risk types of HPV, such as HPV16, are the leading cause of cervical cancer and major cause of genital and oropharyngeal cancers. As a non-enveloped virus, HPV enters cells by interacting with cellular receptors and established cellular trafficking routes to ensure that the viral DNA reaches the nucleus for productive infection. This study identifies Pyk2 as a required cellular component for the intracellular trafficking of HPV16 during infection. Understanding the infectious pathways of HPVs is critical for developing additional preventative therapies. Furthermore, this study advances knowledge on intracellular trafficking processes in keratinocytes.

  • Screening of human kinome identifies MSK1/2-CREB1 as an essential pathway mediating KSHV lytic replication during primary infection [PublishAheadOfPrint]

  • Viruses often hijack cellular pathways to facilitate infection and replication. Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus etiologically associated with Kaposi's sarcoma, a vascular tumor of endothelial cells. Despite intensive studies, cellular pathways mediating KSHV infection and replication are still not well defined. Using an antibody array approach, we examined cellular proteins phosphorylated during KSHV primary infection of primary human umbilical vein endothelial cells. Enrichment analysis identified integrin/MAPK, insulin/EGFR and JAK/STAT as the activated networks during KSHV primary infection. Transcriptional factor CREB1 (cAMP responsive element-binding protein 1) had the strongest increase in phosphorylation. While knock down of CREB1 had no effect on KSHV entry and trafficking, it drastically reduced the expression of lytic transcripts and proteins, and production of infectious virions. Chemical activation of CREB1 significantly enhanced viral lytic replication. In contrast, CREB1 neither influenced the expression of latent gene LANA nor affected KSHV infectivity. Mechanistically, CREB1 was neither activated through the classic cAMP/PKA pathway, nor via the AKT, MK2 and RSK pathways. Rather, CREB1 was activated by the mitogen- and stress-activated protein kinases-1 and -2 (MSK1/2). Consequently, chemical inhibition or knock down of MSKs significantly inhibited KSHV lytic replication program; however, it had minimal effect on LANA expression and KSHV infectivity. Together, these results identify MSK1/2-CREB1 as novel essential effectors for KSHV lytic replication during primary infection. The differential effect of the MSK1/2-CREB1 pathway on the expression of viral latent and lytic genes might control the robustness of viral lytic replication, and therefore the KSHV replication program during primary infection.

    Importance Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus associated with several cancers. Through genome-wide kinase screening, we have found that KSHV activates the mitogen- and stress-activated protein kinases-1 and -2 (MSK1/2) and cAMP responsive element-binding protein 1 (CREB1) pathway during primary infection, and depends on this pathway for viral lytic replication. Inhibition of this pathway blocks KSHV lytic replication. These results illustrate a mechanism by which KSHV hijacks a cellular pathway for its replication and identify a potential therapeutic target.

  • A Screen for ERK Primed GSK-3 Substrates Identifies the p53 Inhibitor iASPP [PublishAheadOfPrint]

  • The Kaposi sarcoma associated herpesvirus (KSHV) LANA protein is essential for the replication and maintenance of virus genomes in latently KSHV infected cells. LANA also drives dysregulated cell growth through a multiplicity of mechanisms that include altering the activity of the cellular kinases ERK (extracellular signal-regulated kinase) and GSK-3 (glycogen synthase kinase -3). To investigate the potential impact of these changes in enzyme activity, we used protein microarrays to identify cell proteins that were phosphorylated by the combination of ERK and GSK-3. The assays identified 58 potential ERK primed GSK-3 substrates of which 23 had evidence for in vivo phosphorylation in mass spectrometry databases. Two of these, SMAD4 and iASPP, were selected for further analysis and were confirmed as ERK primed GSK-3 substrates. Co-transfection experiments revealed that iASPP, but not SMAD4, was targeted for degradation in the presence of GSK-3. iASPP interferes with apoptosis induced by p53 family members. To determine the importance of iASPP to KSHV infected cell growth, PEL cells were treated with an iASPP inhibitor, in the presence or absence of the MDM2 inhibitor Nutlin-3. Drug inhibition of iASPP activity induced apoptosis in BC3 and BCBL1 PEL cells but did not induce PARP cleavage in virus negative BJAB cells. The effect of iASPP inhibition was additive with Nutlin-3. Interfering with iASPP function is therefore another mechanism that can sensitize KSHV positive PEL cells to cell death.

    IMPORTANCE: KSHV is associated with several malignancies including primary effusion lymphoma (PEL). The KSHV encoded LANA protein is multi-functional and promotes both cell growth and resistance to cell death. LANA is known to activate the ERK kinase and limit the activity of another kinase, GSK -3. To discover ways in which LANA manipulation of these two kinases might impact on the PEL cell survival, we screened a human protein microarray for ERK primed, GSK-3 substrates. One of the proteins identified, iASPP, showed reduced levels in the presence of GSK-3. Further, blocking iASPP activity increased cell death, particularly in p53 wild-type BC3 PEL cells.

  • HIV-1 Reservoir Dynamics after Vaccination and Antiretroviral Therapy Interruption Are Associated with Dendritic Cell-Vaccine Induced T-Cell Responses [PublishAheadOfPrint]

  • HIV-1 specific immune responses induced by a dendritic cells (DCs) therapeutic vaccine might have some effect on viral reservoir. We measured total and integrated HIV-1 DNA in isolated CD4 T cells in patients on cART randomized to receive DC pulsed with autologous HIV-1 (n=24) (DC-HIV-1) or with non-pulsed DCs (n=12) (DC-control) at 6 time-points: before any cART, before STOP1 (first cART interruption 56 weeks before the first immunization to isolate virus for pulsing DCs), before and after vaccinations (VAC1 and VAC2) and at weeks 12 and 48 after second cART interruption. Vaccinations did not influence HIV-1 DNA levels in vaccinated subjects. After cART interruption post-vaccination (week 12), while total HIV-1 DNA significantly increased in both arms, integrated HIV-1 DNA did not change in vaccinees (1.8 to 1.9, p=0.22) and increased in controls (1.8 to 2.1, p=0.02) (p=0.03 for the difference between groups). However, this lack of increase of integrated HIV-1 DNA observed in DC-HIV-1 group was transient and at week 48 after cART interruption no differences were observed between groups. HIV-1 specific T cells responses at VAC2 time-point were inversely correlated with total and integrated HIV-1 after cART interruption in vaccinees (r=-0.69, p=0.002 and r=-0.82, pllt;0.0001, respectively). No correlations were found in controls. HIV-1-specific T-cell immune responses elicited by DC therapeutic vaccines drive changes in HIV-1 DNA after vaccination and cART interruption.

    IMPORTANCE There is an intense interest in developing strategies to target HIV-1 reservoirs that create barriers to cure. The development of therapeutic vaccines aimed at enhancing immune mediated clearance of virus producing cells is of high priority. Few therapeutic vaccine clinical trials have investigate the role of therapeutic vaccines as a strategy to safely eliminate or control viral reservoirs. We recently reported that a dendritic cell based therapeutic vaccine was able to decrease significantly viral set-point in vaccinated patients with a concomitant increase in HIV-1--specific T cell responses. HIV-1 specific T cell immune responses elicited by this therapeutic dendritic cell vaccine drove changes of viral reservoir after vaccinations and significantly delayed the replenishment of integrated HIV-1 DNA after cART interruption. These data help to understand how an immunization could shift the virus/host balance and are instrumental to better design strategies to reach the functional cure of HIV-1 infection.

  • Superoxide-generating Nox5{alpha} is functionally required for HTLV-1-induced cell transformation phenotype [PublishAheadOfPrint]

  • Human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and transforms T-cells in vitro. To our knowledge, the functional role of reactive oxygen species (ROS)-generating NADPH oxidase (Nox) 5 in HTLV-1 transformation remains undefined. Here, we found that the Nox5aalpha; expression was upregulated in 88% of 17 ATL patient samples but not in normal peripheral blood T-cells. Upregulation of Nox5aalpha; variant was transcriptionally sustained by the constitutive Jak-STAT5 signaling pathway in IL-2-independent HTLV-1-transformed cell lines including MT1 and MT2, whereas it was transiently induced by the IL-2-triggered Jak-STAT5 axis in uninfected T-cells. A Nox inhibitor, diphenylene iodonium and anti-oxidants such as N-acetyl cysteine blocked proliferation of MT1 and MT2 cells. Ablation of Nox5aalpha; by small interfering RNAs abrogated ROS production, inhibited cellular activities including proliferation, migration, and survival, and suppressed tumorigenicity in immunodeficient NOG mice. The findings suggest that Nox5aalpha; is a key molecule for redox-signal-mediated maintenance of HTLV-1 transformation phenotype and could be a potential molecular target for therapeutic intervention of cancer development.

    IMPORTANCE HTLV-1 is the first human oncogenic retrovirus associated with ATL. Despite the extensive study over the years, the mechanism underlying HTLV-1-induced cell transformation is not fully understood. In this study, we addressed the expression and function of ROS-generating Nox family genes in HTLV-1-transformed cells. Our study provides the first evidence that the upregulated expression of Nox5aalpha; is associated with the pathological state of ATL peripheral blood mononuclear cells, and that Nox5aalpha; is an integral component of the Jak-STAT5 signaling pathway in HTLV-1-transformed T-cells. Nox5aalpha;-derived ROS are critically involved in the regulation of cellular activities in HTLV-1-transformed cells including proliferation, migration, survival, and tumorigenicity. These results indicate that Nox5aalpha;-derived ROS are functionally required for maintenance of HTLV-1 transformation phenotype. The finding provides new insight into the redox-dependent mechanism of HTLV-1 transformation and raises an intriguing possibility that Nox5aalpha; serves as a potential molecular target to treat HTLV-1-related leukemia.

  • Interferon-inducible Protein Tetherin Inhibits Hepatitis B Virus Virion Secretion [PublishAheadOfPrint]

  • Interferon alpha (IFN-aalpha;) is an approved medication for chronic hepatitis B therapy. Besides acting as an immunomodulator, IFN-aalpha; elicits a pleiotropic antiviral state in HBV infected hepatocytes, but whether or not IFN-aalpha; impedes the late steps of HBV life cycle, such as HBV secretion, remains elusive. Herein we report that IFN-aalpha; treatment of HepAD38 cells with established HBV replication selectively reduced HBV virion release without altering the intracellular viral replication or the secretion of HBV subviral particles and nonenveloped capsids. In search of the interferon stimulated gene(s) that is responsible for HBV virion reduction, we found that tetherin, a broad spectrum antiviral transmembrane protein that inhibits the egress of a variety of enveloped viruses, was highly induced by IFN-aalpha; in HepAD38 cells and in primary human hepatocytes. We further demonstrated that the expression of full-length tetherin, but not the C-terminal glycosylphosphatidylinositol (GPI) anchor-truncated form, inhibited HBV virion egress from HepAD38 cells. In addition, the GPI anchor-truncated tetherin exhibited a dominant negative effect and was incorporated into the liberated virions. We also found the colocalization of tetherin and HBV L protein at intracellular multivesicular body, where the budding of HBV virion takes place. In line with this, electron microscopy demonstrated the HBV virion was tethered in the lumen of the cisterna membrane under tetherin expression. Finally, knock down of tetherin or overexpression of the dominant-negative tetherin attenuated IFN-aalpha;-mediated reduction of HBV virion release. Taken together, our study suggests that IFN-aalpha; inhibits HBV virion egress from hepatocytes through the induction of tetherin.

    IMPORTANCE Tetherin is a host restriction factor that blocks the egress of a variety of enveloped viruses through tethering the budding virions on the cell surface by its membrane anchor domains. Herein we report that interferon directly and selectively inhibits the secretion of HBV virion, but not subviral particles or nonenveloped capsids, through the induction of tetherin in hepatocyte-derived cells. The antiviral function of tetherin requires the carboxyl-terminal GPI anchor, while the GPI anchor deletion mutant exhibits dominant negative activity and attaches to the liberated HBV virion. Consistent with the fact that HBV is an intracellular budding virus, microscopy analyses demonstrated that the tethering of HBV virion occurs in the intracellular cisterna, and that tetherin colocalizes with HBV virion on multivesicular body, where is the HBV virion budding site. Our study not only expands the antiviral spectrum of tetherin, but also sheds light on the mechanisms of interferon-elicited anti-HBV responses.

  • The expected effect of deleterious mutations on within-host adaptation of pathogens [PublishAheadOfPrint]

  • Adaptation is a common theme in both pathogen emergence, for example in zoonotic cross-species transmission, and pathogen control, where adaptation might limit the effect of the immune response and antiviral treatment. When such evolution requires deleterious intermediate mutations, fitness ridges and valleys arise in the pathogen's fitness landscape. The effect of deleterious intermediate mutations on within-host pathogen adaptation is examined with deterministic calculations, appropriate for pathogens replicating in large populations with high error rates. The effect of deleterious intermediates on pathogen adaptation is smaller than their name might suggest: when two mutations are required, and each individual single mutation is fully deleterious, the pathogen can jump across the fitness valley by obtaining two mutations at once, leading to a proportion of adapted mutant that is 20-fold lower than for the situation where all mutants are neutral. The negative effects of deleterious intermediates are typically substantially smaller, and outweighed, by fitness advantages of the adapted mutant. Moreover, requiring a specific mutation order has a substantially smaller effect on pathogen adaptation than the effect of all intermediates being deleterious. These results can be rationalized when calculating the number of routes of mutation available to the pathogen, providing a simple approach to estimate the effect of deleterious mutations. The calculations discussed here are applicable when assessing the effect of deleterious mutations on the within-host adaptation of pathogens, for example in the context of zoonotic emergence, antigenic escape, and drug resistance.

    IMPORTANCE Adaptation is critical for pathogens after zoonotic transmission into a new host species, or to achieve antigenic immune escape and drug resistance. Using a deterministic approach, the effects of deleterious intermediate mutations on pathogen adaptation are calculated whilst avoiding commonly made simplifications that do not apply to large pathogen populations replicating with high mutations rates. Perhaps unexpectedly, pathogen adaptation does not halt when the intermediate mutations are fully deleterious. Negative effects of deleterious mutations are substantially outweighed by fitness gains of adaptation. To gain an understanding of the effect of deleterious mutations on pathogen adaptation, a simple approach is introduced that counts the number of routes available to the pathogen with and without deleterious intermediate mutations. This methodology enables a straightforward calculation of the proportion of the pathogen population that will cross a fitness valley or traverse a fitness ridge, without reverting to more complicated mathematical models.

  • Serotonin Receptor Agonist 5-nonyloxytryptamine Alters the Kinetics of Reovirus Cell Entry [PublishAheadOfPrint]

  • Mammalian orthoreoviruses (reoviruses) are nonenveloped double-stranded RNA viruses that infect most mammalian species including humans. Reovirus binds to cell-surface glycans, junctional adhesion molecule-A (JAM-A), and the Nogo-1 receptor (depending on the cell type) and enters cells by receptor-mediated endocytosis. Within the endocytic compartment, reovirus undergoes stepwise disassembly, which is followed by release of the transcriptionally active viral core into the cytoplasm. In a small-molecule screen to identify host mediators of reovirus infection, we found that treatment of cells with 5-nonyloxytryptamine (5-NT), a prototype serotonin receptor agonist, diminished reovirus cytotoxicity. 5-NT also blocked reovirus infection. In contrast, treatment of cells with methiothepin mesylate, a serotonin antagonist, enhanced infection by reovirus. 5-NT did not alter cell-surface expression of JAM-A or attachment of reovirus to cells. However, 5-NT altered the distribution of early endosomes with a concomitant impairment of reovirus transit to late endosomes and a delay in reovirus disassembly. Consistent with an inhibition of viral disassembly, 5-NT treatment did not alter infection by in vitro-generated infectious subvirion particles, which bind to JAM-A but bypass a requirement for proteolytic uncoating in endosomes to infect cells. We also found that treatment of cells with 5-NT decreased infectivity of alphavirus chikungunya virus and coronavirus mouse hepatitis virus. These data suggest that serotonin receptor signaling influences cellular activities that regulate entry of diverse virus families and provide a new, potentially broad-spectrum target for antiviral drug development.

    IMPORTANCE Identification of well-characterized small molecules that modulate viral infection can accelerate development of antiviral therapeutics while also providing new tools to increase our understanding of the cellular processes that underlie virus-mediated cell injury. We conducted a small-molecule screen to identify compounds capable of inhibiting cytotoxicity caused by reovirus, a prototype double-stranded RNA virus. We found that 5-NT impairs reovirus infection by altering viral transport during cell entry. Remarkably, 5-NT also inhibits infection by an alphavirus and a coronavirus. The antiviral properties of 5-NT suggest that serotonin receptor signaling is an important regulator of infection by diverse virus families and illuminate a potential new drug target.

  • Tetherin sensitivity of influenza A viruses is strain specific: Role of hemagglutinin and neuraminidase [PublishAheadOfPrint]

  • The expression of the antiviral host cell factor tetherin is induced by interferon and can inhibit the release of enveloped viruses from infected cells. The Vpu protein of HIV-1 antagonizes the antiviral activity of tetherin and tetherin antagonists with Vpu-like activity have been identified in other viruses. In contrast, it is incompletely understood whether tetherin inhibits influenza A virus (FLUAV) release and whether FLUAV encodes tetherin antagonists. Here, we show that release of several laboratory-adapted and a seasonal FLUAV strain is inhibited by tetherin while pandemic FLUAV A/Hamburg/4/2009 is resistant. Studies with a virus-like particle system and analysis of reassortant viruses provided evidence that the viral hemagglutinin (HA) is an important determinant of tetherin antagonism but requires the presence of its cognate neuraminidase (NA) to inhibit tetherin. Finally, tetherin antagonism by FLUAV was dependent on the virion context, since retrovirus release from tetherin-positive cells was not rescued, and correlated with a HA, NA-dependent reduction in tetherin expression. In sum, our study identifies HA and NA proteins of certain pandemic FLUAV as tetherin antagonists, which has important implications for understanding FLUAV pathogenesis.

    IMPORTANCE Influenza A virus (FLUAV) infection is responsible for substantial global morbidity and mortality and understanding how the virus evades immune defenses of the host may uncover novel targets for antiviral intervention. Tetherin is an antiviral effector molecule of the innate immune system which can contribute to control of viral invasion. However, it has been unclear whether FLUAV are inhibited by tetherin and whether these viruses encode tetherin antagonizing proteins. Our observation that several pandemic FLUAV can counteract tetherin via their HA and NA proteins identifies these proteins as novel tetherin antagonists and indicates that HA/NA-dependent inactivation of innate defenses may contribute to the efficient spread of pandemic FLUAV.

  • Alfalfa leaf curl virus: An aphid-transmitted geminivirus [PublishAheadOfPrint]

  • The family Geminiviridae comprises seven genera differentiated by genome organisation, sequence similarity and insect vector. Capulavirus, an eighth genus, has been proposed so as to accommodate two newly discovered highly divergent geminiviruses that presently have no known vector. Alfalfa leaf curl virus, identified here as a third capulavirus is shown to be transmitted by Aphis craccivora: This is the first report of an aphid-transmitted geminivirus.

  • HIV-1 Resistance to the Capsid-Targeting Inhibitor PF74 Results in Altered Dependence on Host Factors Required for Virus Nuclear Entry [PublishAheadOfPrint]

  • During HIV-1 infection of cells, the viral capsid plays critical roles in reverse transcription and nuclear entry of the virus. The capsid-targeting small molecule PF74 inhibits HIV-1 at early stages of infection. HIV-1 resistance to PF74 is complex, requiring multiple amino acid substitutions in the viral CA protein. Here we report the identification and analysis of a novel PF74-resistant mutant encoding amino acid changes in both domains of CA, three of which are near the pocket where PF74 binds. Interestingly, the mutant virus retained partial PF74 binding, and its replication was stimulated by the compound. The mutant capsid structure was not significantly perturbed by binding of PF74; rather, the mutations inhibited capsid interactions with CPSF6 and Nup153 and altered HIV-1 dependence on these host factors and on TNPO3. Moreover, the replication of the mutant virus was markedly impaired in activated primary CD4+ T cells and macrophages. Our results suggest that HIV-1 escapes a capsid-targeting small molecule inhibitor by altering the virus's dependence on host factors normally required for entry into the nucleus. They further imply that clinical resistance to inhibitors targeting the PF74 binding pocket is likely to be strongly limited by functional constraints on HIV-1 evolution.

    IMPORTANCE The HIV-1 capsid plays critical roles in early steps of infection and is an attractive target for therapy. Here we show that selection for resistance to a capsid-targeting small molecule inhibitor can result in viral dependence on the compound. The mutant virus was debilitated in primary T cells and macrophages---cellular targets of infection in vivo. The mutations also altered the virus's dependence on cellular factors that are normally required for HIV-1 entry into the nucleus. This work provides new information regarding mechanisms of HIV-1 resistance that should be useful in efforts to develop clinically useful drugs targeting the HIV-1 capsid.

  • Cataloguing of Potential HIV Susceptibility Factors during the Menstrual Cycle of Pigtail Macaques Using a Systems Biology Approach [PublishAheadOfPrint]

  • Our earlier studies in pig-tailed macaques demonstrated varying SHIV susceptibility during the menstrual cycle, likely caused by cyclic variations in immune responses in the female genital tract. There is concern that high-dose, long-lasting, injectable progestin-based contraception could mimic the high-progesterone luteal phase and predispose women to HIV-1 acquisition and transmission. In this study, we adopted a systems biology approach employing proteomics (tandem mass spectrometry), transcriptomics (RNA microarray hybridization), and other specific protein assays (enzyme-linked immunosorbent assays and multiplex chemokine-cytokine measurements) to characterize the effects of hormonal changes on the expression of innate factors and secreted proteins in the macaque vagina. Several antiviral factors and pathways (including acute phase response signaling and complement system) were overexpressed in the follicular phase. Conversely, during the luteal phase there were factors overexpressed (including moesins, syndecans, integrins, among others) that could play direct or indirect roles in enhancing HIV-1 infection. Thus, our study showed that specific pathways and proteins/genes might be working in tandem to regulate innate immunity, thus fostering further investigation and future design of approaches to help counter HIV-1 acquisition in the female genital tract.

    IMPORTANCE HIV infection in women is poorly understood. High levels of the hormone progesterone may make women more vulnerable to infection. This could be the case during the menstrual cycle, when using hormone-based birth control, or during pregnancy. The biological basis for increased HIV vulnerability is not known. We used an animal model with high risk for infection during periods of high progesterone. Genital secretions and tissues were studied during the menstrual cycle. Our goal was to identify biological factors upregulated at high progesterone levels, and we indeed show an upregulation of genes and proteins which enhance the ability of HIV to infect when progesterone is high. In contrast, during low progesterone periods, we find more HIV inhibitory factors. This basic research study contributes to our understanding of mechanisms that may regulate HIV infection in females under hormonal influences. Such knowledge is needed for the development of novel prevention strategies.

  • Rotavirus NSP3 is a translational surrogate of the PABP-poly(A) complex [PublishAheadOfPrint]

  • Through its interaction with the 5rrsquo; translation initiation factor eIF4G, poly(A) binding protein (PABP) facilitates the translation of 5rrsquo; -capped and 3rrsquo; -poly(A)-tailed mRNAs. Rotavirus mRNAs are capped but not polyadenylated, instead terminating in a 3rrsquo; GACC motif that is recognized by the viral protein NSP3, which competes with PABP for eIF4G binding. Upon rotavirus infection, viral, GACC-tailed mRNAs are efficiently translated while host poly(A)-tailed mRNA translation is, in contrast, severely impaired. To explore the roles of NSP3 in these two opposing events, the translational capabilities of three capped mRNAs, distinguished by either a GACC, a poly(A) or a non-GACC and non-poly(A) 3rrsquo; end, have been monitored after electroporation of cells expressing all rotavirus proteins (infected cells) or only NSP3 (stably or transiently transfected cells). In infected cells, we found that the magnitudes of translation induction (GACC-tailed mRNA) and translation reduction (poly(A)-tailed mRNA) both depended on the rotavirus strain used but were not genetically linked to NSP3. In transfected cells, even a small amount of NSP3 was sufficient to dramatically enhance GACC-tailed mRNA translation and, surprisingly, to slightly favor the translation of both poly(A)- and non-poly(A)-tailed mRNAs, likely through by stabilizing the eIF4E-eIF4G interaction. These data suggest that NSP3 is a translational surrogate of the PABP-poly(A) complex and therefore cannot by itself be responsible for inhibiting the translation of host poly(A)-tailed mRNAs upon rotavirus infection.

    IMPORTANCE To control host cell physiology and to circumvent innate immunity, many viruses have evolved powerful mechanisms aimed at inhibiting host mRNA translation while stimulating translation of their own mRNAs. How rotavirus tackles this challenge is still a matter of debate. Using rotavirus-infected cells, we show that the magnitude of cellular poly(A) mRNA translation differs with respect to rotavirus strains but is not genetically linked to NSP3. Using cells expressing rotavirus NSP3, we show that NSP3 alone not only dramatically enhances rotavirus-like mRNA translation but also enhances poly(A) mRNA translation rather than inhibiting it, likely by stabilizing the eIF4E-eIF4G complex. Thus, the inhibition of cellular polyadenylated mRNA translation during rotavirus infection cannot be solely attributed to NSP3 and is more likely the result of global competition between viral and host mRNAs for the cellular translation machinery.

  • Identification of the Receptor Binding Domain of the Spike Glycoprotein of Human Betacoronavirus HKU1. [PublishAheadOfPrint]

  • Coronavirus spike (S) glycoproteins mediate receptor binding, membrane fusion, and virus entry and determine host range. Murine betacoronavirus (bbeta;-CoV) in group A uses the N-terminal domain (NTD) of S protein to bind to its receptor, whereas bbeta;-CoVs SARS-CoV in group B and MERS-CoV in group C, respectively, and several aalpha;-CoVs use the downstream C-domain in their S proteins to recognize their receptor proteins. To identify the receptor-binding domain in the spike of human bbeta;-CoV HKU1 in group A, we generated and mapped a panel of monoclonal antibodies (mAbs) to the ectodomain of HKU1 spike. They did not cross-react with S proteins of any other CoV tested. Most of the HKU1 spike mAbs recognized epitopes in the C-domain, between amino acids 535 to 673, indicating that this region is immunodominant. Two of the mAbs blocked HKU1 virus infection of primary human tracheal-bronchial epithelial (HTBE) cells. Pre-incubation of HTBE cells with a truncated HKU1 S protein that includes the C-domain blocked infection with HKU1 virus, but pre-incubation of cells with truncated S protein containing only the NTD did not block infection. These data suggest that the receptor-binding domain (RBD) of HKU1 spike protein is located in the C-domain, where the spike proteins of aalpha;-CoVs and bbeta;-CoVs in groups B and C bind to their specific receptor proteins. Thus, two bbeta;-CoVs in group A, HKU1 and murine CoV, have evolved to use different regions of their spike glycoproteins to recognize their respective receptor proteins.

    IMPORTANCE Mouse hepatitis virus, a bbeta;-CoV in group A, uses the galectin-like NTD in its spike protein to bind its receptor protein, while HCoV-OC43, another bbeta;-CoV in group A, uses the NTD to bind to its sialic acid containing receptor. In marked contrast, the NTD of the spike glycoprotein of human respiratory bbeta;-CoV HKU1, which is also in group A, does not bind sugar. In this study, we showed that for the spike protein of HKU1, the purified C-domain, downstream of the NTD, could block HKU1 virus infection of human respiratory epithelial cells, and that several monoclonal antibodies that mapped to the C-domain neutralized virus infectivity. Thus the receptor-binding domain of HKU1 spike glycoprotein is located in the C-domain. Surprisingly, two bbeta;-CoVs in group A, MHV and HKU1, have evolved to use different regions of their spike glycoproteins to recognize their respective receptors.

  • The human adenovirus type 5 E4orf4 protein targets two phosphatase regulators of the Hippo signaling pathway [PublishAheadOfPrint]

  • When expressed alone at high levels, the human adenovirus E4orf4 protein exhibits tumor cell-specific p53-independent toxicity. A major E4orf4 target is the B55 class of PP2A regulatory subunits and we have shown recently that binding of E4orf4 inhibits PP2AB55 phosphatase activity in a dose-dependent fashion by preventing access of substrates. While interaction with B55 subunits is essential for toxicity, E4orf4 mutants exist that, despite binding B55 at high levels, are defective in cell killing, suggesting that other essential targets may exist. In an attempt to identify additional targets we undertook a proteomics approach to characterize E4orf4-interacting proteins. Our findings indicated that, in addition to PP2AB55 subunits, ASPP-PP1 complex subunits were found among the major E4orf4-binding species. Both the PP2A and ASPP-PP1 phosphatases are known to positively regulate effectors of the Hippo signaling pathway that controls expression of cell growth/survival genes by dephosphorylating the YAP transcriptional co-activator. We find here that expression of E4orf4 results in hyperphosphorylation of YAP, suggesting that Hippo signaling may be affected by E4orf4 interactions with PP2AB55 and/or ASPP-PP1 phosphatases. Furthermore, knockdown YAP1 expression was seen to enhance E4orf4 killing, again consistent with a link between E4orf4 toxicity and inhibition of the Hippo pathway. This effect may in fact contribute to the cancer cell specificity of E4orf4 toxicity as many human cancer cells rely heavily on the Hippo pathway for their enhanced proliferation.

    Importance The human adenovirus E4orf4 protein has been known for some time to induce tumor cell-specific death when expressed at high levels, and thus knowledge of its mode of action could be of importance for development of new cancer therapies. Although the B55 form of the phosphatase PP2A has long been known as an essential E4orf4 target, genetic analyses indicated that others must exist. To identify additional E4orf4 targets we performed, for the first time, a large scale affinity purification/mass spectrometry analysis of E4orf4 binding partners. Several additional candidates were detected, including key regulators of the Hippo signaling pathway that enhances cell viability in many cancers, and results of preliminary studies suggested a possible link between inhibition of Hippo signaling and E4orf4 toxicity.

  • The Nucleocapsid Protein of Coronaviruses Acts as a Viral Suppressor of RNA Silencing in Mammalian Cells [PublishAheadOfPrint]

  • RNA interference (RNAi) is a process of eukaryotic posttranscriptional gene silencing that functions in antiviral immunity in plants, nematodes, and insects. However, recent studies provided strong supports that RNAi also plays a role in antiviral mechanism in mammalian cells. To combat RNAi-mediated antiviral responses, many viruses encode viral suppressors of RNA silencing (VSR) to facilitate their replication. VSRs have been widely studied for plant and insect viruses but only few have been defined for mammalian viruses currently. Here, we identified a novel VSR from coronaviruses, a group of medically important mammalian viruses including Severe acute respiratory syndrome coronavirus (SARS-CoV), and showed that the nucleocapsid protein (N protein) of coronaviruses suppresses RNAi triggered by either short hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs) in mammalian cells. Mouse hepatitis virus (MHV) is closely related to SARS-CoV in the family Coronaviridae and was used as a coronavirus replication model. The replication of MHV increased when the N proteins were expressed in trans, while knockdown of Dicer1 or Ago2 transcripts facilitated the MHV replication in mammalian cells. These results support the hypothesis that RNAi is a part of the antiviral immunity responses in mammalian cells.

    Importance RNAi has been well known to play important antiviral roles from plants to invertebrates. However, recent studies provided strong supports that RNAi is also involved in antiviral response in mammalian cells. An important indication for RNAi-mediated antiviral activity in mammals is the fact that a number of mammalian viruses encode potent suppressor of RNA silencing (VSR). Our current results demonstrate that coronaviruses N protein could function as a VSR through its dsRNA binding activity. Mutational analysis of N protein allowed us to find out the critical residues for the VSR activity. Using the MHV-A59 as the coronavirus replication model, we showed that ectopic expression of SARS-CoV N protein could promote MHV replication in RNAi-active cells but did not in RNAi depleted cells. These results indicate that coronaviruses encode a VSR that functions in the replication cycle, and provide further evidences to support that RNAi-mediated antiviral response exists in mammalian cells.

  • Human cytomegalovirus gH/gL/gO promotes the fusion step of entry into all cell types whereas gH/gL/UL128-131 broadens virus tropism through a distinct mechanism. [PublishAheadOfPrint]

  • Interaction between gH/gL and the fusion protein gB is likely a conserved feature of the entry mechanism for all herpesviruses. Human cytomegalovirus (HCMV) gH/gL can be bound by gO, or by the set of proteins UL128, UL130, and UL131, forming gH/gL/gO and gH/gL/UL128-131. The mechanisms by which these complexes facilitate entry are poorly understood. Mutants lacking UL128-131 replicate well on fibroblasts, but fail to enter epithelial/endothelial cells, and this has lead to the general assumption that gH/gL/UL128-131 promotes gB-mediated fusion on epithelial/endothelial cells whereas gH/gL/gO provides this function on fibroblasts. This was challenged by observations that gO-null mutants were defective on all of these cell types, suggesting that entry into epithelial/endothelial cells requires both of the gH/gL complexes, but the severe replication defect of the gO mutants precluded detailed analysis. We previously reported that the ratio of gH/gL/gO and gH/gL/UL128-131 in the virion envelope varied dramatically among HCMV strains. Here, we show that stains not only differ in the ratio, but also vary in the total amount of gH/gL in the virion. Cell-type specific particle-to-PFU ratios of HCMV strains that contained different amounts of gH/gL/gO and gH/gL/UL128-131 were determined. Infection of both fibroblasts and epithelial cells was generally correlated with the abundance of gH/gL/gO, but not with gH/gL/UL128-131. The low infectivity of virions rich in gH/gL/UL128-131, but low in gH/gL/gO could be overcome by treatment with the chemical fusogen PEG, strongly arguing that gH/gL/gO provides the conserved herpesvirus "gH/gL entry function" of promoting gB-mediated fusion for entry into all cell types, whereas gH/gL/UL128-131 acts through a distinct mechanism to allow infection of select cell types.

    IMPORTANCE The functions of HCMV gH/gL complexes in entry are unclear. Unlike the well-studied Epstein-Barr virus (EBV), where gH/gL and gH/gL/gp42 complexes both seem capable of promoting gB fusion during entry into different cell types, our studies here suggest that for HCMV, gH/gL/gO promotes gB fusion on all cell types, whereas gH/gL/UL128-131 broadens virus tropism through a distinct, as yet unknown mechanism. To our knowledge, this is the first suggestion of a herpesvirus gH/gL that does not act by promoting gB fusion, which might make HCMV a useful model to study the fundamental mechanisms by which herpesvirus gH/gL regulates gB fusion. Moreover, gH/gL/UL128-131 is a candidate vaccine target. Our findings help to explain the cell type-dependent virus neutralization exhibited by anti-gH/gL/UL128-131 antibodies, and underscore the importance of gH/gL/gO as another important part of vaccine or therapeutic strategies.

  • Structural Determinants for the Selective Anti-HIV-1 Activity of the All-{beta} Alternative Conformer of XCL1 [PublishAheadOfPrint]

  • HIV-1 replication is regulated in vivo by a complex network of cytokines and chemokines. XCL1/lymphotactin, a unique metamorphic chemokine, was recently identified as a broad-spectrum endogenous HIV-1 inhibitor that blocks viral entry via direct interaction with the gp120 envelope glycoprotein. HIV-1 inhibition by XCL1 requires access to the alternative all-bbeta; conformation, which interacts with glycosaminoglycans (GAG) but not with the specific XCL1 receptor, XCR1. To investigate the structural determinants of the HIV-inhibitory function of XCL1, we performed a detailed structure-function analysis of a stabilized all-bbeta; variant, XCL1 W55D. Individual alanine substitutions of two basic residues within the 40s' loop, K42 and R43, abrogated the ability of XCL1 to bind to the viral envelope and block HIV-1 infection; moreover, a loss of HIV-inhibitory function, albeit less marked, was seen upon individual mutation of three additional basic residues, R18, R35 and K46. In contrast, mutation of K42 to arginine did not cause any loss of function, suggesting that the interaction with gp120 is primarily electrostatic in nature. Strikingly, four of these five residues cluster to form a large (~350AAring;2) positively-charged surface in the all-bbeta; XCL1 conformation, while they are dissociated in the classic chemokine fold, which is inactive against HIV-1, providing a structural basis for the selective antiviral activity of the alternatively-folded XCL1. Furthermore, we observed that changes to the N-terminal domain, which is proximal to the cluster of putative HIV-1 gp120-interacting residues, also affect the antiviral activity of XCL1. Interestingly, the complement of residues involved in HIV-1 blockade is partially overlapping, but distinct from those involved in the GAG-binding function of XCL1. These data identify key structural determinants of anti-HIV activity in XCL1, providing new templates for the development of HIV-1 entry inhibitors.

    Importance The host immune system controls HIV-1 infection through a wide array of inhibitory responses, including the induction of cytotoxic effector cells and the secretion of non-cytolytic soluble antiviral factors such as cytokines and chemokines. We recently identified XCL1/lymphotactin, a chemokine primarily produced by CD8+ T cells, as a novel endogenous factor with broad anti-HIV activity. Strikingly, only one of the two conformations that XCL1 can adopt in solution, the alternative all-bbeta; fold, mediates antiviral activity. At variance with the classic HIV-inhibitory chemokines such as CCL5/RANTES, XCL1 acts via direct interaction with the external viral envelope glycoprotein, gp120. Here, we identify the interactive surface of XCL1 that is implicated in binding to the HIV-1 envelope and HIV-1 inhibition, providing a structural basis to explain why only the all-bbeta; XCL1 conformer is effective against HIV-1. Our findings may be useful in guiding the rational design of new inhibitors of HIV-1 entry.

  • Hepatitis B Virus Covalently Closed Circular DNA Formation in Immortalized Mouse Hepatocytes Associated with Nucleocapsid Destabilization [PublishAheadOfPrint]

  • Hepatitis B virus (HBV) infects hundreds of millions of people worldwide and causes acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. HBV is an enveloped virus with a relaxed circular (RC) DNA genome. In the nuclei of infected human hepatocytes, conversion of RC DNA from the incoming virion or cytoplasmic mature nucleocapsid (NC) to the covalently closed circular (CCC) DNA, which serves as the template for producing all viral transcripts, is essential to establish and sustain viral replication. For reasons yet to be understood, HBV is apparently unable to make CCC DNA in normal mouse hepatocytes in the liver. We report here that HBV CCC DNA was formed efficiently in an immortalized mouse hepatocyte cell line, AML12HBV10, and this is associated with destabilization of mature NCs in these cells. These results suggest that destabilization of mature HBV NCs in AML12HBV10 cells facilitates efficient NC uncoating and subsequent CCC DNA formation. They further implicate NC uncoating as an important step in CCC DNA formation that is subject to host regulation and potentially a critical determinant of host range and/or cell tropism of HBV.

    Importance Persistent infection by hepatitis B virus (HBV), afflicting hundreds of millions worldwide, is sustained by the episomal viral covalently closed circular (CCC) DNA in the nuclei of infected hepatocytes. CCC DNA is converted from the viral genomic (precursor) DNA contained in cytoplasmic viral nucleocapsids. The conversion process remains ill-defined but host cell factors are thought to play an essential role. In particular, HBV fails to make CCC DNA in normal mouse hepatocytes despite the presence of large amounts of nucleocapsids containing the precursor viral DNA. We have found that in an immortalized mouse hepatocyte cell line, HBV is able to make abundant amounts of CCC DNA. This ability correlates with increased instability of viral nucleocapsids in these cells, which likely facilitates nucleocapsid disassembly (uncoating) to release the genomic DNA for conversion to CCC DNA. Our studies have thus revealed a novel mechanism of controlling viral persistence via regulating nucleocapsid disassembly.

  • Host Range Restriction of Epstein-Barr Virus and Related Lymphocryptoviruses [PublishAheadOfPrint]

  • Epstein-Barr related herpesviruses, or lymphocryptoviruses (LCV) naturally infect humans and nonhuman primates (NHP), but their host range is not well characterized. Using LCV and B cells from multiple species of Hominidae and Cercopithicidae, we show LCV can immortalize B cells from some non-native species, but growth transformation is restricted to B cells from their own family of hominoids or Old World NHP suggesting a high degree of LCV adaptation to their natural primate host.

  • Co-crystal structures of antibody N60-i3 and antibody JR4 in complex with gp120 define more Cluster A epitopes involved in effective antibody-dependent effector function against HIV-1 [PublishAheadOfPrint]

  • Accumulating evidence indicates a role for Fc receptor (FcR)-mediated effector functions of antibodies, including antibody-dependent cell-mediated cytotoxicity (ADCC), in prevention of HIV-1 acquisition and in post-infection control of viremia. Consequently, an understanding of the molecular basis for Env epitopes that constitute effective ADCC targets is of fundamental interest for humoral anti-HIV-1 immunity and for HIV-1 vaccine design. A substantial portion of FcR-effector function of potentially protective anti-HIV-1 antibodies is directed toward non-neutralizing, transitional, CD4-induceable (CD4i) epitopes associated with the gp41 reactive region of gp120 (Cluster A epitopes). Our previous studies defined the A32-like epitope within the Cluster A region and mapped it to the highly conserved and mobile layers 1 and 2 of the gp120 inner domain within C1-C2 regions of gp120. Here we elucidate additional Cluster A epitope structures, including an A32-like epitope, recognized by human mAb N60-i3 and a hybrid A32-C11-like epitope, recognized by rhesus macaque mAb JR4. These studies define for the first time a hybrid A32-C11-like epitope and map it to elements of both the A32-like sub-region and the 7 layered bbeta;-sheet of the gp41-interactive region of gp120. These studies provide additional evidence that effective antibody-dependent effector function to the Cluster A region depends on precise epitope targeting nndash; a combination of epitope footprint and mode of antibody attachment. All together these findings help in understanding how Cluster A epitopes are targeted by humoral responses

    Importance HIV/AIDS has claimed the lives of over 30 million people. Although antiretroviral drugs can control viral replication no vaccine has yet been developed to prevent the spread of the disease. Studies of natural HIV-1 infection, SIV or SHIV infected non-human primates (NHPs) and HIV-1-infected humanized mice models, passive transfer studies in infants born to HIV-infected mothers and the RV144 clinical trial have linked FcR-mediated effector functions of anti-HIV-1 antibodies with post-infection control of viremia and/or blocking viral acquisition. With this report we provide additional definition of the molecular determinants for Env antigen engagement which lead to effective antibody-dependent effector function directed to the non-neutralizing CD4-dependent epitopes in the gp41 reactive region of gp120. These findings have important implications for the development of an effective HIV-1 vaccine.

  • Role of Host Cell p32 in Herpes Simplex Virus 1 De-envelopment During Viral Nuclear Egress [PublishAheadOfPrint]

  • To clarify the function(s) of the herpes simplex virus 1 (HSV-1) major virion structural protein UL47 (also designated VP13/14), we screened cells overexpressing UL47 for UL47-binding cellular proteins. Tandem affinity purification of transiently expressed UL47 coupled with mass spectrometry-based proteomics technology and subsequent analyses showed that UL47 interacted with cell protein p32 in HSV-1-infected cells. Unlike in mock-infected cells, p32 accumulated at the nuclear rim in HSV-1-infected cells and this p32 recruitment to the nuclear rim required UL47. p32 formed a complex(es) with HSV-1 proteins UL31, UL34, Us3, UL47 and/or ICP22 in HSV-1-infected cells. All these HSV-1 proteins were previously reported to be important for HSV-1 nuclear egress, in which nucleocapsids bud through the inner nuclear membrane (primary envelopment) and the enveloped nucleocapsids then fuse with the outer nuclear membrane (de-envelopment). Like viral proteins UL31, UL34, Us3 and UL47, p32 was detected in primary enveloped virions. p32 knock-down reduced viral replication and induced membranous invaginations adjacent to the nuclear rim containing primary enveloped virions and aberrant localization of UL31 and UL34 in punctate structures at the nuclear rim. These effects of p32 knock-down were reduced in the absence of UL47. Therefore, the effects of p32 knock-down in HSV-1 nuclear egress were similar to those of the previously reported mutation(s) in HSV-1 regulatory proteins for HSV-1 de-envelopment during viral nuclear egress. Collectively, these results suggested that p32 regulated HSV-1 de-envelopment and replication, in a UL47-dependent manner.

    IMPORTANCE In this study, we have presented data suggesting that: (i) the HSV-1 major virion structural protein UL47 interacted with host cell protein p32 and mediated the recruitment of p32 to the nuclear rim in HSV-1-infected cells; (ii) p32 was a component of the HSV-1 nuclear egress complex (NEC), whose core components were UL31 and UL34; and (iii) p32 regulated HSV-1 de-envelopment during viral nuclear egress. It has been reported that p32 was a component of human cytomegalovirus NEC and was required for efficient disintegration of nuclear lamina, which has been thought to facilitate HSV-1 primary envelopment during viral nuclear egress. Thus, p32 appeared to be a core component of herpesvirus NECs, like UL31 and UL34 homologues in other herpesviruses, and to play multiple roles in herpesvirus nuclear egress.

  • Immunoglobulin with High-Titer In-vitro Cross Neutralizing Hepatitis C Virus Antibody Passively Protects Chimpanzee from Homologous, but not Heterologous Challenge [PublishAheadOfPrint]

  • The importance of neutralizing antibodies (NtAb) in protection against hepatitis C virus (HCV) remains controversial. We infused a chimpanzee with H06-immunoglobulin from a genotype 1a HCV-infected patient, and challenged with genotype strains efficiently neutralized by H06 in vitro. 1a-NtAb afforded no protection against 4a or 5a. Protection against homologous 1a lasted 18 weeks, but infection emerged when NtAb-titers waned. However, 6a-infection was prevented. The differential in vivo neutralization patterns have implications for HCV vaccine development.

  • Cellular and protein-directed glycosylation of native cleaved HIV-1 envelope [PublishAheadOfPrint]

  • The gp120/gp41 HIV-1 envelope glycoprotein (Env) is highly glycosylated with up to 50% of its mass consisting of N-linked glycans. This dense carbohydrate coat has emerged as a promising vaccine target, with its glycans acting as epitopes for a number of potent and broadly neutralizing antibodies (bnAbs). Characterizing the glycan structures present on native HIV-1 Env is thus a critical goal for the design of Env immunogens. Here, we use a complementary, multistep approach involving ion-mobility mass spectrometry and high performance liquid chromatography to comprehensively characterize the glycan structures present on HIV-1 gp120 produced in peripheral blood mononuclear cells (PBMCs). The capacity of different expression systems, including pseudoviral particles and recombinant cell surface trimers, to reproduce native-like glycosylation was then assessed. A population of oligomannose glycans on gp120 was reproduced across all expression systems, supporting this as an intrinsic property of Env that can be targeted for vaccine design. In contrast, Env produced in HEK 293T cells failed to accurately reproduce the highly processed complex-type glycan structures observed on PBMC-derived gp120, and in particular the precise linkage of sialic acid residues that cap these glycans. Finally we show that, unlike gp120, the glycans decorating gp41 are mostly complex-type sugars, consistent with the glycan specificity of bnAbs that target this region. These findings provide insights into the glycosylation of native and recombinant HIV-1 Env and can be used to inform strategies for immunogen design and preparation.

    Importance: Development of an HIV vaccine is desperately needed to control new infections and elicitation of HIV bnAbs will likely be an important component of an effective vaccine. Increasingly, HIV bnAbs are being identified that bind to the N-linked glycans coating the HIV envelope glycoproteins gp120 and gp41, highlighting them as important targets for vaccine design. It is therefore important to characterize the glycan structures present on native, virion-associated gp120 and gp41 for development of vaccines that accurately mimic native-Env glycosylation. Here we use a number of analytical techniques to precisely study the structures of both the oligomannose and complex-type glycans present on native Env to provide a reference for determining the ability of potential HIV immunogens to accurately replicate the glycosylation pattern on these native structures.

  • The ORF61 protein encoded by Simian Varicella Virus and Varicella Zoster Virus inhibits NF{kappa}B signaling by interfering with I{kappa}B{alpha} degradation [PublishAheadOfPrint]

  • Varicella Zoster Virus (VZV) causes chickenpox upon primary infection and establishes latency in ganglia. Reactivation from latency causes herpes zoster, which may be complicated by post-herpetic neuralgia. Innate immunity mediated by interferon and pro-inflammatory cytokines represent the first line of immune defense upon infection and reactivation. VZV is known to interfere with multiple innate immune signaling pathways including the central transcription factor NFB. However the role of these inhibitory mechanisms in vivo is unknown. Simian varicella virus (SVV)-infection of rhesus macaques recapitulates key aspects of VZV pathogenesis and this model thus permits examining the role of immune evasion mechanisms in vivo. Here we compare SVV and VZV with respect to interference of NFB activation. We demonstrate that both viruses prevent ubiquitination of the NFB inhibitor IĸBaalpha;, whereas SVV additionally prevents IĸBaalpha; phosphorylation. We show that the ORF61 proteins of VZV and SVV are sufficient to prevent IĸBaalpha; ubiquitination upon ectopic expression. We further demonstrate that SVV ORF61 interacts with bbeta;-TrCP, a subunit of the SCF ubiquitin ligase complex that mediates the degradation of IĸBaalpha;. This interaction seems to inactivate SCF-mediated protein degradation in general since the unrelated bbeta;-TrCP-target Snail is also stabilized by ORF61. In addition to ORF61, SVV seems to encode additional inhibitors of the NFB pathway since ORF61-deleted SVV still prevented IĸBaalpha; phosphorylation and degradation. Taken together, our data demonstrate that SVV interferes with TNFaalpha;-induced NFB activation at multiple levels which is consistent with the importance of these counter mechanisms for Varicella Virus infection.

    Importance The role of innate immunity during the establishment of primary infection, latency and reactivation by Varicella Zoster Virus (VZV) is incompletely understood. Since infection of rhesus macaques by Simian Varicella Virus (SVV) is being used as an animal model of VZV infection we characterized the molecular mechanism by which SVV interferes with innate immune activation. Specifically, we studied how SVV prevents activation of the transcription factor NFB, a central factor in eliciting pro-inflammatory responses. The identification of molecular mechanisms that counteract innate immunity might ultimately lead to better vaccines and treatments of VZV since overcoming these mechanisms either by small molecule inhibition of by genetic modification of vaccine strains is expected to reduce the pathogenic potential of VZV. Moreover, using SVV-infection of rhesus macaques it will be possible to study increasing the vulnerability of varicella viruses to innate immunity will impact viral pathogenesis.

  • Assembly of Epstein-Barr virus capsid in promyelocytic leukemia nuclear bodies [PublishAheadOfPrint]

  • The Epstein-Barr virus (EBV) capsid contains a major capsid protein, VCA; two minor capsid proteins, BDLF1 and BORF1; and a small capsid protein, BFRF3. During the lytic cycle, these capsid proteins are synthesized and imported into the host nucleus for capsid assembly. This study finds that EBV capsid proteins colocalize with promyelocytic leukemia nuclear bodies (PML-NBs) in P3HR1 cells during the viral lytic cycle, appearing as nuclear speckles under a confocal laser-scanning microscope. In a GST-pulldown study, we show that BORF1 interacts with PML-NBs in vitro. BORF1 also colocalizes with PML-NBs in EBV-negative Akata cells after transfection, and is responsible for bringing VCA and the VCA-BFRF3 complex from the cytoplasm to PML-NBs in the nucleus. Furthermore, BDLF1 is dispersed throughout the cell when expressed alone, but colocalizes with PML-NBs when BORF1 is also present in the cell. In addition, this study finds that knockdown of PML expression by shRNA does not influence intracellular levels of capsid proteins, but reduces the number of viral particles produced by P3HR1 cells. Together, these results demonstrate that BORF1 plays a critical role in bringing capsid proteins to PML-NBs, which may likely be the assembly sites of EBV capsids. The mechanisms elucidated in this study are critical to understanding the process of EBV capsid assembly.

    IMPORTANCE Capsid assembly is an important event during the Epstein-Barr virus (EBV) lytic cycle, as this process is required for the production of virions. In this study, confocal microscopy revealed that the EBV capsid protein BORF1 interacts with promyelocytic leukemia nuclear bodies (PML-NBs) in the host nucleus, and is responsible for transporting the other EBV capsid proteins, including VCA, BDLF1, and BFRF3, to these subnuclear locations prior to initiation of capsid assembly. This study also found that knockdown of PML expression by shRNA significantly reduces EBV capsid assembly capabilities. This enhanced understanding of capsid assembly offers potential for the development of novel antiviral strategies and therapies that can prevent the propagation and spread of EBV.

  • Preliminary Evaluation of a Bunyavirus Vector for Cancer Immunotherapy [PublishAheadOfPrint]

  • Replicon particles of Rift Valley fever virus, referred to as nonspreading RVFV (NSR), are intrinsically safe and highly immunogenic. Here, we demonstrate that NSR-infected human dendritic cells can activate CD8+ T-cells in vitro, and that prophylactic and therapeutic vaccination of mice with NSR encoding a tumor associated CD8 peptide can control outgrowth of lymphoma cells in vivo. These results suggest that the NSR system holds promise for cancer immunotherapy.

  • The partial attenuation of Small Hydrophobic (SH) gene deleted RSV is associated with elevated IL-1{beta} responses. [PublishAheadOfPrint]

  • The small hydrophobic (SH) gene of respiratory syncytial virus (RSV), a major cause of infant hospitalisation, encodes a viroporin of unknown function. SH gene knockout virus (RSV SH) is partially attenuated in vivo but not in vitro, suggesting that the SH protein may have an immunomodulatory role. RSV SH has been tested as a live attenuated vaccine in humans and cattle and here we demonstrate that it protected against viral re-challenge in mice. We compared the immune response to infection with wild type and SH RSV, in vivo using BALB/c mice and in vitro using epithelial cells, neutrophils and macrophages. Strikingly, the IL-1bbeta; response to RSV SH infection was greater than wild type RSV, in spite of decreased viral load and when IL-1bbeta; was blocked in vivo, viral load returned to wild type levels. A significantly higher IL-1bbeta; response to RSV SH was also detected in vitro, with greater magnitude responses in neutrophils and macrophages than epithelial cells. Depleting macrophages (with clodronate liposome) and neutrophils (with anti-Ly6G/1A8) demonstrated the contribution of these cells to the IL-1bbeta; response in vivo, the first demonstration of neutrophilic IL-1bbeta; production in response to viral lung infection. In this study we describe an increased IL-1bbeta; response to RSV SH, which may explain the attenuation in vivo and supports targeting the SH gene in live attenuated vaccines.

    Importance There is a pressing need for a vaccine for Respiratory Syncytial Virus (RSV). A number of live attenuated RSV vaccine strains have been developed in which the small hydrophobic (SH) gene has been deleted, even though the function of the SH protein is unknown. The structure of the SH protein has recently been solved showing it is a pore forming protein (viroporin). Here we demonstrate that the IL-1bbeta; response to RSV SH is greater in spite of lower viral load, which contributes to the attenuation in vivo. This potentially suggests a novel method by which viruses can evade the host response. As all Pneumovirinae and some Paramyxovirinae encode similar SH genes this new understanding may also enable the development of live attenuated vaccines for both RSV and other members of the Paramyxoviridae.

  • Genomic and Proteomic Analyses Indicate that Banchine and Campoplegine Polydnaviruses Have Similar, if not Identical, Viral Ancestors [PublishAheadOfPrint]

  • Polydnaviruses form a group of unconventional dsDNA viruses transmitted by endoparasitic wasps during egg laying into caterpillar hosts, where viral gene expression is essential to immature wasp survival. A copy of the viral genome is present in wasp chromosomes, thus ensuring vertical transmission. Polydnaviruses comprise two taxa, Bracovirus and Ichnovirus, shown to have distinct viral ancestors whose genomes were "captured" by ancestral wasps. While evidence indicates bracoviruses derive from a nudivirus ancestor, the identity of the ichnovirus progenitor remains unknown. In addition, ichnoviruses are found in two ichneumonid wasp subfamilies, Campopleginae and Banchinae, where they constitute morphologically and genomically different virus types. To address the question of whether these two ichnovirus subgroups have distinct ancestors, we used genomic, proteomic and transcriptomic analyses to characterize particle proteins of the banchine Glypta fumiferanae ichnovirus and the genes encoding them. Several proteins were found to be homologous to those identified earlier for campoplegine ichnoviruses while the corresponding genes were located in clusters of the wasp genome similar of those observed previously in a campoplegine wasp. However, for the first time in a polydnavirus system, these clusters also revealed sequences encoding enzymes presumed to form the replicative machinery of the progenitor virus and observed to be overexpressed in the virogenic tissue. Homology searches pointed to nucleocytoplasmic large DNA viruses as the likely source of these genes. These data, along with an analysis of the chromosomal form of five viral genome segments, provide clear evidence for the relatedness of the banchine and campoplegine ichnovirus ancestors.

    Importance Recent work indicates that the two recognized polydnavirus taxa, bracovirus and ichnovirus, are derived from distinct viruses whose genomes integrated into the genomes of ancestral wasps. However, the identity of the ichnovirus ancestor is unknown and questions remain regarding the possibility that the two described ichnovirus subgroups, banchine and campoplegine ichnoviruses, have distinct origins. Our study provides unequivocal evidence that these two ichnovirus types are derived from related viral progenitors. This suggests that morphological and genomic differences observed between the ichnovirus lineages, including features unique to banchine ichnovirus genome segments, result from evolutionary divergence either before or after their endogenization. Strikingly, analysis of selected wasp genomic regions revealed genes presumed to be part of the replicative machinery of the progenitor virus, shedding new light on the likely identity of this virus. Finally, these genes could well play a role in ichnovirus replication as they were overexpressed in the virogenic tissue.

  • The ERdj5 reductase cooperates with PDI to promote SV40 ER membrane translocation [PublishAheadOfPrint]

  • The non-enveloped polyomavirus (PyV) SV40 traffics from the cell surface to the endoplasmic reticulum (ER) where it penetrates the ER membrane to reach the cytosol before mobilizing into the nucleus to cause infection. Prior to ER membrane penetration, ER lumenal factors impart structural rearrangements to the virus, generating a translocation-competent virion capable of crossing the ER membrane. Here we identify ERdj5 as an ER enzyme that reduces SV40's disulfide bonds, a reaction important for its ER membrane transport and infection. ERdj5 also mediates human BK PyV infection. This enzyme cooperates with protein disulfide isomerase (PDI), a redox chaperone previously implicated in unfolding SV40, to fully stimulate membrane penetration. Negative stain electron microscopy of ER-localized SV40 suggests that ERdj5/PDI impart structural rearrangements to the virus. These conformational changes enable SV40 to engage BAP31, an ER membrane protein essential for supporting membrane penetration of the virus. Uncoupling SV40 from BAP31 traps the virus in an ER subdomain called foci that likely serve as depots from where SV40 gains access to the cytosol. Our study thus pinpoints two ER lumenal factors that coordinately prime SV40 for ER membrane translocation, and establishes a functional connection between lumenal and membrane events driving this process.

    Importance PyVs are established etiologic agents of many debilitating human diseases, especially in immunocompromised individuals. To infect cells at the cellular level, this virus family must penetrate the host ER membrane to reach the cytosol, a critical entry step. In this report, we identify two ER lumenal factors that prepare the virus for ER membrane translocation, and connect these lumenal events with events on the ER membrane. Pinpointing cellular components necessary for supporting PyV infection should lead to rational therapeutic strategies in preventing and treating PyV-related diseases.

  • Lentiviral protein transfer vectors are an efficient vaccine-platform inducing strong antigen-specific cytotoxic T cell response [PublishAheadOfPrint]

  • To induce and trigger innate and adaptive immune responses, antigen presenting cells (APCs) take up and process antigens. Retroviral particles are capable of transferring not only genetic information, but also foreign cargo proteins when genetically fused to viral structural proteins. Here, we demonstrate the capacity of lentiviral protein transfer vectors (PTV) for targeted antigen transfer directly into APCs and thereby induction of cytotoxic T cell responses. Targeting of lentiviral PTVs to APCs can be achieved analogously to gene transfer vectors by pseudotyping the particles with truncated wild-type measles virus (MV) glycoproteins (GPs), which use human SLAM as main entry receptor. SLAM is expressed on stimulated lymphocytes and APCs including dendritic cells. SLAM-targeted PTVs transferred the reporter proteins GFP or Cre recombinase with strict receptor specificity into SLAM-expressing CHO and B cell lines, in contrast to broadly transducing VSV-G pseudotyped PTVs. Primary myeloid dendritic cells (mDCs) incubated with targeted or non-targeted, ovalbumin (Ova) transferring PTVs stimulated Ova-specific T lymphocytes, especially CD8+ T cells. Administration of Ova-PTVs into SLAM-transgenic and control mice confirmed the observed predominant induction of antigen-specific CD8+ T cells and demonstrated capacity of protein transfer vectors as suitable vaccines for the induction of antigen-specific immune responses.

    Importance This study demonstrates specificity and efficacy of antigen transfer by SLAM-targeted and non-targeted lentiviral protein transfer vectors into antigen presenting cells to trigger antigen-specific immune responses in vitro and in vivo. The observed predominant activation of antigen-specific CD8+ T cells indicates the suitability of SLAM-targeted, but also non-targeted PTVs as vaccine for the induction of cytotoxic immune responses. Since cytotoxic CD8+ T lymphocytes are a mainstay of anti-tumoral immune responses, PTVs could be engineered for the transfer of specific tumor antigens provoking tailored anti-tumoral immunity. Therefore, PTVs can be used as safe and efficient alternative to gene transfer vectors or life-attenuated replicating vector platforms avoiding genotoxicity or general toxicity in highly immunocompromised patients, respectively. Thereby, the potential for easy envelope exchange allows to circumvent neutralizing antibodies e.g. during repeated boost immunizations.

  • Possible HPV38 contamination of endometrial cancer RNA-Seq samples in The Cancer Genome Atlas database [PublishAheadOfPrint]

  • Viruses are causally associated with a number of human malignancies. In this study, we sought to identify new viral-cancer associations by searching RNA-Sequencing datasets from ggt;2000 patients, encompassing 21 cancers from The Cancer Genome Atlas (TCGA), for the presence of viral sequences. In agreement with previous studies, we found human papillomavirus type 16 (HPV16) and HPV18 in oropharyngeal cancer and hepatitis B and C viruses in liver cancer. Unexpectedly, however, we found HPV38, a cutaneous form of HPV associated with skin cancer, in 32 of 168 samples with endometrial cancer. In 12 of the HPV38+ samples, we observed at least one paired read that mapped to both human and HPV38 genomes, indicative of viral integration into host DNA, something not previously demonstrated for HPV38. The expression levels of HPV38 transcripts were relatively low, and all 32 HPV38+ samples belonged to the same experimental batch of 40 samples, whereas none of the other 128 endometrial carcinoma samples were HPV38+, raising doubts about the significance of the HPV38 association. Moreover, the HPV38+ samples contained the same 10 novel single nucleotide variations (SNVs), leading us to hypothesize that one patient was infected with this new isolate of HPV38, which was integrated into his/her genome and may have cross-contaminated other TCGA samples within batch #228. Based on our analysis, we propose guidelines to examine batch effect, virus expression level, and SNVs as part of NGS data analysis for evaluating the significance of viral/pathogen sequences in clinical samples.

    Importance High-throughput RNA-Sequencing followed by computational analysis has vastly accelerated the identification of viral and other pathogenic sequences in clinical samples, but cross-contamination during the processing of the samples remain a major problem that can lead to erroneous conclusions. We found HPV38 sequences specifically present in RNA-Seq samples of endometrial cancer patients from TCGA, a virus not previously associated with this type of cancer. However, multiple lines of evidence suggest possible cross-contamination in these samples, which were processed together in the same batch. Despite this potential cross-contamination, our data indicate that we have detected a new isolate of HPV38 that appears to be integrated into the human genome. We also herein provide a general guideline for computational detection and interpretation of pathogen-disease associations.

  • Molecular signatures of immune activation and epithelial barrier remodeling are enhanced during the luteal phase of the menstrual cycle: implications for HIV susceptibility [PublishAheadOfPrint]

  • Variable infectivity and transmissibility of HIV/SHIV has been recently associated with the menstrual cycle, with particular susceptibility observed during the luteal phase in non-human primate models and ex vivo human explant cultures, but the mechanism is poorly understood. Here, we performed an unbiased, mass spectrometry-based proteomic analysis to better understand the mucosal immunological processes underpinning this observed susceptibility to HIV infection. Cervicovaginal lavage samples (n=19) were collected, characterized as follicular or luteal phase using days since last menstrual period, and analyzed by tandem-mass spectrometry. Biological insights from these data were gained using a spectrum of computational methods including hierarchical clustering, pathway analysis, gene set enrichment analysis, and partial least-squares discriminant analysis with LASSO feature selection. Of the 384 proteins identified, 43 were differentially abundant between phases (pllt;0.05, gge;2 fold change). Cell-cell adhesion proteins and antiproteases were reduced, and leukocyte recruitment (IL-8 pathway, p=1.41E-5) and extravasation proteins (p=5.62E-4) were elevated during the luteal phase. LASSO/PLSDA identified a minimal profile of 18 proteins that best distinguished the luteal phase. This profile included cytoskeletal elements and proteases known to be involved in cellular movement. Gene set enrichment analysis associated CD4+ T cell and neutrophil gene set signatures with the luteal phase (pllt;0.05). Taken all together, our findings indicate a strong association between proteins involved in tissue remodeling and leukocyte infiltration with the luteal phase, which may represent potential hormone-associated mechanisms of increased susceptibility to HIV.

    Importance Recent studies have discovered an enhanced susceptibility to HIV infection during the progesterone-dominant luteal phase of the menstrual cycle. Yet, the mechanism responsible for this enhanced susceptibility has yet to be determined. Understanding the source of this vulnerability will be important for designing efficacious HIV prevention technologies for women. Furthermore, these findings may also be extrapolated to better understand the impact of exogenous hormone application, such as the use of hormonal contraceptives, on HIV acquisition risk. Hormonal contraceptives are the most widely used contraceptive method in sub-Saharan Africa, the most HIV burdened area of the world. For this reason, research conducted to better understand how hormones impact host immunity and susceptibility factors important for HIV infection is a global health priority.

  • Newly-emergent highly pathogenic H5N9 subtype avian influenza A virus [PublishAheadOfPrint]

  • The Novel H7N9 avian influenza virus (AIV), was demonstrated to cause severe human respiratory infections in China. Here, we examined poultry specimens from live bird markets linked to human H7N9 infection in Hangzhou, China. Metagenomic sequencing revealed mixed subtypes (H5, H7, H9, N1, N2 and N9). Subsequently AIV subtypes H5N9, H7N9 and H9N2 were isolated. Evolutionary analysis showed that the hemagglutination and neuraminidase genes of the novel H5N9 virus originated from A/Muscovy duck/Vietnam/LBM227/2012 (H5N1) belonging to Clade 2.3.2.1 and human-infective A/Hangzhou/1/2013 (H7N9), six internal genes were similar to those of the H5N1, H7N9 and H9N2 viruses. The virus harbored the PQRERRRKR/GL motif characteristic of highly pathogenic AIVs at the HA cleavage site. Receptor-binding experiments demonstrated that the virus binds aalpha;-2,3 sialic acid, but not aalpha;-2,6 sialic acid. Identically, pathogenicity experiment also showed that the virus caused low mortality rates in mice. This newly isolated H5N9 virus is a highly pathogenic reassortant virus originating from H5N1, H7N9 and H9N2 subtypes. Live bird markets represent a potential transmission risk to public health and the poultry industry.

    IMPORTANCE This investigation confirm that the novel H5N9 subtype avian influenza A virus is a reassortant strain originating from H5N1, H7N9 and H9N2 subtypes, which is totally different from those H5N9 viruses reported before. The novel H5N9 virus got a highly pathogenic H5 gene and an N9 gene from human-infecting H7N9, but caused low mortality rates in mice. Whether this novel H5N9 virus will cause human infections from its avian host and become a pandemic subtype, is not known yet. So it is interesting to assess the risk of the emergence of novel reassortant virus with potential transmissibility to public health.

  • An enhanced synthetic multi-clade DNA prime induces improved cross-clade reactive functional antibodies when combined with an adjuvanted protein boost in non-human primates [PublishAheadOfPrint]

  • The search for an efficacious human immunodeficiency virus (HIV-1) vaccine remains a pressing need. The moderate success of the RV144 Thai clinical vaccine trial suggested that vaccine-induced HIV-1 specific antibodies can reduce the risk of HIV-1 infection. We have made several improvements to the DNA platform and have previously shown that improved DNA vaccines alone are capable of inducing both binding and neutralizing antibodies in small animal models. In this study, we explored how an improved DNA prime and recombinant protein boost would impact HIV-specific vaccine immunogenicity in rhesus macaques (RhM). After DNA immunization with either a single HIV Env consensus sequence or multiple constructs expressing HIV subtype-specific Env consensus sequences, we detected both CD4+ and CD8+ T-cell responses to all vaccine immunogens. These T-cell responses were further increased after protein boosting to levels exceeding those of DNA only or protein only immunization. In addition, we observed antibodies that exhibited robust cross-clade binding, neutralizing and ADCC activity after immunization with the DNA prime-protein boost regimen with the multiple Env formulation inducing a more robust and broad response compared to the single Env formulation. The magnitude and functionality of these responses emphasize the strong priming effect improved DNA immunogens can induce which are further expanded upon protein boost. These results support further study of an improved synthetic DNA prime together with a protein boost for enhancing anti-HIV immune responses.

    Importance Even with effective anti-retroviral drugs, HIV remains an enormous global health burden. Vaccine development has been problematic in part due to the high degree of diversity and poor immunogenicity of the HIV Env protein. Studies suggest that a relevant HIV vaccine will likely need to induce broad cellular and humoral responses from a simple vaccine regimen due to the resource limited setting in which the HIV pandemic is most rampant. DNA vaccination lends itself well to increasing the amount of diversity included in a vaccine due to the ease of manufacturing multiple plasmids and formulating them as a single immunization. By increasing the number of Envs within a formulation, we were able to show increased breadth of responses as well as improved functionality induced in a non-human primate model. This increased breadth could be built upon, leading to better coverage against circulating strains with broader vaccine-induced protection.

  • Large tailed spindle viruses of Archaea: A new way of doing viral business [PublishAheadOfPrint]

  • Viruses of Archaea continue to surprise us. Archaeal viruses have revealed new morphologies, protein folds, and gene content. This is especially true for large spindle viruses, which only infect Archaea. We present a comparison of particle morphology, major coat protein structure, and gene content among the five characterized large spindle viruses to elucidate defining characteristics. Structural similarities and a core set of genes support the grouping of the large spindle viruses into a new superfamily.

  • Structural analysis of the roles of influenza A virus membrane-associated proteins in assembly and morphology. [PublishAheadOfPrint]

  • The assembly of influenza A virus at the plasma membrane of infected cells leads to release of enveloped virions that are typically round in tissue-culture adapted strains, but filamentous in strains isolated from patients. The viral proteins hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1) and the M2 ion channel all contribute to virus assembly. When expressed individually or in combination in the cells, they can all, under certain conditions, mediate release of membrane-enveloped particles, but their relative roles in virus assembly, release and morphology remain unclear. To investigate this, we have produced membrane-enveloped particles by plasmid-derived expression of combinations of HA, NA and M proteins (M1 and M2), or by infection with influenza A virus. We have monitored particle release, particle morphology, and plasma membrane morphology using biochemical methods, electron microscopy, electron tomography and cryo-electron tomography. Our data suggest that HA, NA or HANA expression leads to particle release through non-specific induction of membrane curvature. In contrast, co-expression with the M proteins clusters the glycoproteins into filamentous membrane protrusions, which by forming a constricted neck at the base, can be released as particles. HA and NA preferentially distribute to differently curved membranes within these particles. Both the budding intermediates and the released particles are morphologically similar to those produced during infection with influenza A virus. Together, our data provide new insights into influenza virus assembly and show that the M segment together with either of the glycoproteins is the minimal requirement to assemble and release membrane-enveloped particles that are truly virus-like.

    IMPORTANCE Influenza A virus is a major respiratory pathogen. It assembles membrane-enveloped virus particles whose shapes vary from spherical to filamentous. Here we have studied the roles of individual viral proteins in mediating virus assembly and in determining virus shape. To do this, we used a range of electron microscopy techniques to obtain and compare 2D and 3D images of virus particles and virus-like particles during and after assembly. The virus-like particles were produced using different combinations of viral proteins. Among our results, we found that co-expression of one or both of the viral surface proteins (hemagglutinin and neuraminidase), together with the viral membrane associated proteins encoded in the M segment, results in assembly and release of filamentous virus-like particles in a manner very similar to the budding and release of influenza virions. These data provide novel insights into the roles played by individual viral proteins in influenza A virus assembly.

  • Targeting importin-{alpha}7 as a therapeutic approach against pandemic influenza viruses [PublishAheadOfPrint]

  • Viral drug resistance is believed to occur less likely if compounds are directed against cellular rather than viral proteins. In this study, we analyzed the feasibility of a crucial viral replication factor, namely importin-aalpha;7, as a potential cellular drug target to combat pandemic influenza. Surprisingly, only five viral lung-to-lung passages were required to achieve 100% lethality in importin-aalpha;7-/- mice that are otherwise resistant. Viral escape from importin-aalpha;7 requirement was mediated by five mutations in the viral ribonucleoprotein complex and the surface glycoproteins. Moreover, the importin-aalpha;7-/- mouse-adapted strain became even more virulent for wild-type mice compared to the parental strain. These studies show that targeting host proteins may still result in viral escape by alternative pathways eventually giving rise to even more virulent virus strains. Thus, therapeutic intervention strategies should consider a multi-target approach to reduce viral drug resistance.

    Importance Here, we investigated the longstanding hypothesis based on in vitro studies that viral drug resistance occurs less likely if compounds are directed against cellular rather than viral proteins. Here, we challenged this hypothesis by analyzing in an in vivo animal model the feasibility of targeting the cellular factor importin-aalpha;7 - that is crucial for human influenza virus replication and pathogenesis - as an efficient antiviral strategy against pandemic influenza viruses. In summary, our studies suggest that resistance against cellular factors is possible in vivo and the emergence of even more virulent viral escape variants calls for particular caution. Thus, therapeutic intervention strategies should consider a multi-target approach using compounds against viral as well as cellular factors to reduce the risk of viral drug resistance and potentially increased virulence.

  • Structure of C-Terminal Domain of the Multifunctional ICP27 Protein from Herpes Simplex Virus-1 [PublishAheadOfPrint]

  • Herpes viruses are nuclear-replicating viruses that have successfully evolved to evade the immune system of humans, establishing life-long infections. ICP27 from herpes simplex virus (HSV) is a multifunctional regulatory protein that is functionally conserved in all known human herpes viruses. It has the potential to interact with an array of cellular proteins as well as intronless viral RNAs. ICP27 plays an essential role in viral transcription, nuclear export of intronless RNAs, translation of viral transcripts and virion host shut-off function. It has also been implicated in several signaling pathways and prevention of apoptosis. Although much is known about its central role in viral replication and infection, very little is known about the structure and mechanistic properties of ICP27 and its homologs. We present the first crystal structure of ICP27 C-terminal domain at 2.0 AAring; resolution. The structure reveals the C-terminal half of ICP27 to have a novel fold consisting of aalpha;-helices and long loops, along with a unique CHCC-type of zinc-binding motif. The two termini of this domain extend out from the central core and hint to possibilities of making interactions. ICP27 essential domain is capable of forming self-dimers as seen in the structure, which is confirmed by analytical ultracentrifugation study. Preliminary in vitro phosphorylation assays reveal that this domain may be regulated by cellular kinases.

    IMPORTANCE ICP27 is a key regulatory protein of the Herpes Simplex Virus and has functional homologs in all known human herpes viruses. Understanding the structure of this protein is a step ahead in deciphering the mechanism by which the virus thrives. In this study, we present the first structure of the C-terminal domain of ICP27 and describe its novel features. We critically analyze the structure and compare our results to the information available form earlier studies. This structure can act as a guide in future experimental designs, and can add to a better understanding of mechanism of ICP27 as well as that of its homologs.

  • BIOLOGY OF ZIKA VIRUS INFECTION IN HUMAN SKIN CELLS [PublishAheadOfPrint]

  • Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family that includes Dengue, West Nile, Yellow Fever and Japanese encephalitis viruses, causing a mosquito-borne disease transmitted by the Aedes genus, with recent outbreaks in the South Pacific. Here, we determine the importance of the human skin in the entry of ZIKV and its contribution to the induction of anti-viral immune responses. We show that human dermal fibroblasts, epidermal keratinocytes and immature dendritic cells are permissive to the most recent ZIKV isolate, responsible for the epidemic in French Polynesia. Several entry and/or adhesion factors, among which DC-SIGN, AXL, TYRO3, and to a lesser extent, TIM-1, permitted ZIKV entry with a major role for the TAM receptor AXL. ZIKV permissiveness of human skin fibroblasts was confirmed by the use of a neutralizing Ab and specific RNA silencing. ZIKV induced the transcription of TLR-3, RIG-I and MDA5, as well as several interferon-stimulated genes, including OAS2, ISG15 and MX1, characterized by a strongly enhanced interferon-bbeta; gene expression. ZIKV was found to be sensitive to the antiviral effect of both type I and type II interferons. Finally, infection of skin fibroblasts resulted in the formation of autophagosomes whose presence was associated with enhanced viral replication, as shown by the use of Torin 1, a chemical inducer of autophagy or the specific autophagy inhibitor 3-Methyladenine. The results presented herein permit to gain better insight in the biology of ZIKV and to devise strategies aiming to interfere with the pathology caused by this emerging Flavivirus.

    IMPORTANCE Zika virus (ZIKV) is an arbovirus belonging to Flaviviridae family. Vector-mediated transmission of ZIKV is initiated when a blood-feeding female Aedes mosquito injects the virus into the skin of its mammalian host, followed by infection of permissive cells via specific receptors. Indeed, skin immune cells, including dermal fibroblasts, epidermal keratinocytes and immature dendritic cells, were all found to be permissive to ZIKV infection. The results also show a major role for the phosphatidylserine receptor AXL as a ZIKV entry receptor, and cellular autophagy in enhancing ZIKV replication in permissive cells. ZIKV replication leads to activation of an antiviral innate immune response and the production of type I interferons in infected cells. Taken together, these results provide for the first time a general insight into the interaction between ZIKV and its mammalian host.

  • Mutation of a Single Envelope N-linked Glycosylation Site Enhances the Pathogenicity of Bovine Leukemia Virus [PublishAheadOfPrint]

  • Viruses have co-evolved with their host to ensure efficient replication and transmission without inducing excessive pathogenicity that would indirectly impair their persistence. This is exemplified by the bovine leukemia virus (BLV) system in which lymphoproliferative disorders develop in ruminants after latency periods of several years. In principle, the equilibrium reached between the virus and its host could be disrupted by emergence of more pathogenic strains. Intriguingly but fortunately, such a hyperpathogenic BLV strain was never observed in the field nor designed in vitro. In this study, we aimed at understanding the role of envelope N-linked glycosylation with the hypothesis that this posttranslational modification could either favor BLV infection by allowing viral entry or allow immune escape by using glycans as a shield. Using reverse genetics of an infectious molecular provirus, we have identified a N-linked envelope glycosylation site (N230) that limits viral replication and pathogenicity. Indeed, mutation N230E unexpectedly leads to enhanced fusogenicity and protein stability.

    IMPORTANCE Infection by retroviruses requires the interaction of the viral envelope protein (SU) with a membrane-associated receptor allowing fusion and release of the viral genomic RNA into the cell. Here, we have shown that N-linked glycosylation of the Bovine Leukemia Virus (BLV) SU protein is, as expected, essential for cell infection in vitro. Consistently, mutation of all glycosylation sites of a BLV provirus destroys infectivity in vivo. However, single mutations do not significantly modify replication in vivo. Instead, a particular mutation at SU codon 230 increases replication and accelerates pathogenesis. This unexpected observation has important consequences in terms of disease control and managing.

  • Characterization of Two Human Monoclonal Antibodies Neutralizing Influenza A H7N9 Viruses [PublishAheadOfPrint]

  • H7N9 caused a significant global health concern, due to its severe infection and approximately 35% mortality in humans. By screening a Fab antibody phage library derived from patients who recovered from H7N9 infections, we characterized two human monoclonal antibodies (HuMAbs), HNIgGD5 and HNIgGH8. The epitope of these two antibodies was dependent on two residues in the receptor binding site at positions V186 and L226 of the hemagglutinin glycoprotein. Both antibodies possessed high neutralizing activity.

  • Two mutations were critical for bat-to-human transmission of MERS coronavirus [PublishAheadOfPrint]

  • To understand how MERS coronavirus (MERS-CoV) transmitted from bats to humans, we compared the virus-surface spikes of MERS-CoV and a related bat coronavirus HKU4. Although HKU4 spike cannot mediate viral entry into human cells, two mutations enabled it to do so by allowing it to be activated by human proteases. These mutations are present in MERS-CoV spike, explaining why MERS-CoV infects human cells. These mutations therefore played critical roles in the bat-to-human transmission of MERS-CoV.

  • Context-dependent cleavage of the capsid protein by the West Nile virus protease modulates the efficiency of virus assembly [PublishAheadOfPrint]

  • Molecular mechanisms that define the specificity of flavivirus RNA encapsulation are poorly understood. Virions composed of the structural proteins of one flavivirus and the genomic RNA of a heterologous strain can be assembled and have been developed as live-attenuated vaccine candidates for several flaviviruses. In this study, we discovered that not all combinations of flavivirus components are possible. While a West Nile virus (WNV) sub-genomic RNA could readily be packaged by structural proteins of the DENV2 strain 16681, production of infectious virions with DENV2 strain NGC structural proteins was not possible, despite the very high amino acid identity between these viruses. Mutagenesis studies identified a single residue (position 101) of the DENV capsid (C) protein as the determinant for heterologous virus production. C101 is located at the P1rrsquo; position of the NS2B/3 protease cleavage site at the carboxy-terminus of the C protein. WNV NS2B/3 cleavage of the DENV structural polyprotein was possible when a threonine (Thr101, 16681) but not a serine (Ser101, NGC) occupied the P1rrsquo; position, a finding not predicted by in vitro protease specificity studies. Critically, both serine and threonine were tolerated at the P1rrsquo; position of WNV capsid. More extensive mutagenesis revealed the importance of flanking residues within the polyprotein in defining the cleavage specificity of the WNV protease. A more detailed understanding of the context-dependence of viral protease specificity may aid the development of new protease inhibitors and provide insight into associated patterns of drug resistance.

    IMPORTANCE West Nile virus (WNV) and dengue virus (DENV) are mosquito-borne flaviviruses that cause considerable morbidity and mortality in humans. No specific anti-flavivirus therapeutics are available for treatment of infection. Proteolytic processing of the flavivirus polyprotein is an essential step in the replication cycle, and is an attractive target for antiviral development. The design of protease inhibitors has been informed by insights into the molecular details of the interactions of proteases and their substrates. In this manuscript, studies of the processing of WNV and DENV capsid proteins by the WNV protease identified an unexpected contribution of the sequence surrounding critical residues within the cleavage site on protease specificity. This demonstration of context-dependent protease cleavage has implications for the design of chimeric flaviviruses, new therapeutics, and the interpretation of flavivirus protease substrate specificity studies.

  • The Canonical Immediate-Early 3 Gene Product pIE611 of Mouse Cytomegalovirus Is Dispensable for Viral Replication, but Mediates Transcriptional and Post-transcriptional Regulation of Viral Gene Products [PublishAheadOfPrint]

  • Transcription of mouse cytomegalovirus (MCMV) immediate-early ie1 and ie3 is controlled by the major immediate-early promoter/enhancer (MIEP) and requires differential splicing. Based on complete loss of genome replication of an MCMV mutant carrying a deletion of the ie3-specific exon 5, the multifunctional IE3 protein (611 amino acids, pIE611) is considered essential for viral replication. Our analysis of ie3 transcription resulted in the identification of novel ie3 isoforms derived from alternatively spliced ie3 transcripts. Construction of an IE3-HA virus by insertion of an in-frame HA-epitope sequence allowed detection of the IE3 isoforms in infected cells, verifying that the newly identified transcripts are coding for proteins. This prompted the construction of an MCMV mutant lacking ie611, but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using ie611 MCMV, we demonstrate the dispensability of the canonical ie3 gene product pIE611 for viral replication. To determine the role of pIE611 for viral gene expression during MCMV infection in an unbiased global approach, we used label-free quantitative mass-spectrometry to delineate pIE611-dependent changes of the MCMV proteome. Interestingly, further analysis revealed transcriptional as well as post-transcriptional regulation of MCMV gene products by pIE611.

    IMPORTANCE Cytomegaloviruses are pathogenic bbeta;-herpesviruses persisting in a lifelong latency from which reactivation can occur under conditions of immuno-suppression, immuno-immaturity or inflammation. The switch from latency to reactivation requires expression of immediate-early genes. Therefore, understanding of immediate-early gene regulation might add insights into viral pathogenesis. The mouse cytomegalovirus (MCMV) immediate-early (ie) 3 protein (611 amino acids, pIE611) is considered essential for viral replication. The identification of novel protein isoforms derived from alternatively spliced ie3 transcripts prompted the construction of an MCMV mutant lacking ie611, but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using ie611 MCMV, we demonstrate the dispensability of the canonical ie3 gene product pIE611 for viral replication and delineated pIE611-dependent changes of the MCMV proteome. Our findings have fundamental implications for the interpretation of earlier studies on llsquo;pIE3rrsquo; functions and highlight the complex orchestration of MCMV gene regulation.

  • SIRT1 suppresses human T-cell leukemia virus type 1 transcription [PublishAheadOfPrint]

  • Human T-cell leukemia virus type 1 (HTLV-1)-associated diseases are poorly treatable and HTLV-1 vaccines are not available. High proviral load is one major risk factor for disease development. HTLV-1 encodes Tax oncoprotein that activates transcription from viral long terminal repeats (LTR) and various types of cellular promoters. Counteracting Tax function might have prophylactic and therapeutic benefits. In this work, we report on the suppression of Tax activation of HTLV-1 LTR by SIRT1 deacetylase. The transcriptional activity of Tax on the LTR was largely ablated when SIRT1 was overexpressed, but Tax activation of NF-B was unaffected. On the contrary, the activation of the LTR by Tax was boosted when SIRT1 was depleted. Treatment of cells with resveratrol shunted Tax activity in a SIRT1-dependent manner. The activation of SIRT1 in HTLV-1-transformed T cells by resveratrol potently inhibited HTLV-1 proviral transcription and Tax expression, whereas compromising SIRT1 by specific inhibitors augmented HTLV-1 mRNA expression. The administration of resveratrol also decreased the production of cell-free HTLV-1 virion from MT2 cells and the transmission of HTLV-1 from MT2 cells to uninfected Jurkat cells in co-culture. SIRT1 associated with Tax in HTLV-1-transformed T cells. Treatment with resveratrol prevented the interaction of Tax with CREB and the recruitment of CREB, CRTC1 and p300 to Tax-responsive elements in the LTR. Our work demonstrates the negative regulatory function of SIRT1 in Tax activation of HTLV-1 transcription. Small molecule activators of SIRT1 such as resveratrol might be considered as new prophylactic and therapeutic agents in HTLV-1-associated diseases.

    Importance Human T-cell leukemia virus type 1 (HTLV-1) causes a highly lethal blood cancer or a chronic debilitating disease of the spinal cord. Treatments are unsatisfactory and vaccines are not available. Disease progression is associated with robust expression of HTLV-1 genes. Suppressing HTLV-1 gene expression might have preventive and therapeutic benefits. It is therefore critical that host factors controlling HTLV-1 gene expression be identified and characterized. This work reveals a new host factor that suppresses HTLV-1 gene expression and a natural compound that activates this suppression. Our findings not only provide new knowledge of the host control of HTLV-1 gene expression, but also suggest a new strategy of using natural compounds for prevention and treatment of HTLV-1-associated diseases.

  • VP2 exchange and NS3/NS3a deletion in African horsesickness virus (AHSV) towards AHS Disabled Infectious Single Animal (DISA) vaccine candidates [PublishAheadOfPrint]

  • ABSTRACT

    African horsesickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae. There are nine serotypes of AHSV showing different levels of cross neutralization. AHSV is transmitted by species of Culicoides biting midges and causes African Horsesickness (AHS) in equids with a mortality rate of up to 95% in naiiuml;ve horses. AHS has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climate conditions appear to be competent vectors for the related bluetongue virus (BTV). To control AHS, live-attenuated vaccines (LAVs) are used in Africa. We used reverse genetics to generate llsquo;synthetic' reassortants of AHSV for all nine serotypes by exchange of genome segment 2 (Seg-2). This segment encodes VP2 which is the serotype determining protein and the dominant target for neutralizing antibodies. Single Seg-2 AHSV reassortants showed similar cytopathogenic effect in mammalian cells, but displayed different growth kinetics. Reverse genetics for AHSV was also used to study Seg-10 expressing NS3/NS3a proteins. We demonstrated that NS3/NS3a proteins are not essential for AHSV replication in vitro. NS3/NS3a of AHSV is however involved in cytopathogenic effect in mammalian cells, and is very important for virus release from cultured insect cells in particular. Similar to the concept of BT Disabled Infectious Single Animal (DISA) vaccine platform, an AHS DISA vaccine platform lacking NS3/NS3a expression was developed. Using Seg-2[lsqb]VP2[rsqb] exchange we will be able to develop AHS DISA vaccine candidates for all current AHSV serotypes.

    IMPORTANCE African horsesickness virus is transmitted by species of Culicoides biting midges and causes African Horsesickness in equids with a mortality rate of up to 95% in naiiuml;ve horses. African Horsesickness has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climate conditions are supposed to be competent vectors. Using reverse genetics, viruses for all nine serotypes were constructed by exchange of Seg-2 expressing serotype determining VP2 protein. Further, we demonstrated that non-structural protein NS3/NS3a is not essential for virus replication in vitro. However, potential spread of virus by biting midges is supposed to be blocked, since in vitro release of virus is strongly reduced due to this deletion. VP2 exchange and NS3/NS3a deletion in African horsesickness virus were combined in the concept of Disabled Infectious Single Animal vaccine for all nine serotypes.

  • Characterization of ribosomal frameshifting in Theiler's murine encephalomyelitis virus [PublishAheadOfPrint]

  • Theiler's murine encephalomyelitis virus (TMEV) is a member of the genus Cardiovirus in the Picornaviridae family of positive-sense single-stranded RNA viruses. Previously we demonstrated that in the related cardiovirus, Encephalomyocarditis virus, a programmed -1 ribosomal frameshift (-1 PRF) occurs at a conserved G_GUU_UUU sequence within the 2B-encoding region of the polyprotein ORF. Here we show that -1 PRF occurs at a similar site during translation of the TMEV genome. In addition we demonstrate that a predicted 3rrsquo; RNA stem-loop structure at a non-canonical spacing downstream of the shift site is required for efficient frameshifting in TMEV, and that frameshifting also requires virus infection. Mutating the G_GUU_UUU shift site to inhibit frameshifting results in an attenuated virus with reduced growth kinetics and a small plaque phenotype. Frameshifting in the virus context was found to be extremely efficient at 74-82%, which, to our knowledge, is the highest frameshifting efficiency recorded to date for any virus. We propose that highly efficient -1 PRF in TMEV provides a mechanism to escape the confines of equimolar expression normally inherent in the single-polyprotein expression strategy of picornaviruses.

    Importance Many viruses utilize programmed -1 ribosomal frameshifting (-1 PRF) to produce different protein products at a defined ratio, or to translate overlapping ORFs to increase coding capacity. With few exceptions, -1 PRF occurs on specific llsquo;slipperyrrsquo; heptanucleotide sequences and is stimulated by RNA structure beginning 5-9 nt downstream of the slippery site. Here we describe an unusual case of -1 PRF in Theiler's murine encephalomyelitis virus (TMEV) that is extraordinarily efficient (74-82% of ribosomes shift into the alternative reading frame) and, in stark contrast to other examples of -1 PRF, is dependent upon a stem-loop structure beginning 14 nt downstream of the slippery site. Furthermore, in TMEV-based reporter constructs in transfected cells, efficient frameshifting is critically dependent upon virus infection. We suggest that TMEV evolved frameshifting as a novel mechanism for removing ribosomes from the message (a llsquo;ribosome sinkrrsquo;) to downregulate synthesis of the 3rrsquo; -encoded replication proteins.

  • The epidemiology, evolution and recent outbreaks of avian influenza viruses in China: A Review [PublishAheadOfPrint]

  • Novel reassortants of H7N9, H10N8, and H5N6 avian influenza viruses (AIVs) are currently circulating in China's poultry flocks, occasionally infecting humans and other mammals. Combined with the sometimes enzootic H5N1 and H9N2 strains, this cauldron of genetically diverse AIVs pose significant risks to public health. Here, we review the epidemiology, evolution and recent outbreaks of AIVs in China, discuss reasons behind the recent increase in the emergence of novel AIVs, and identify warning signs which may point to the emergence of a potentially virulent and highly transmissible AIV to humans. This review will be useful to authorities who consider options for the detection and control of AIV transmission in animals and humans, with the goal of preventing future epidemics and pandemics.

  • Infection patterns induced in naive adult woodchucks by virions of woodchuck hepatitis virus collected during either acute or chronic phases of infection. [PublishAheadOfPrint]

  • The infectivity of hepadnavirus virions produced during either acute or chronic stages of infection was compared by testing the ability of the virions of woodchuck hepatitis virus (WHV) to induce productive acute infection in naiiuml;ve adult woodchucks. Serum WHV collected during acute infection was compared to virions harvested from WHV-infected woodchucks during either (i) early chronic infection, when WHV-induced hepatocelluar carcinoma (HCC) was not yet developed; or (ii) late chronic infection, when established HCC was terminal. All tested types of WHV inoculum were related, because they were collected from woodchucks that originally were infected with standardized WHV7 inoculum. Despite the individual differences between animals, the kinetics of accumulation of serum relaxed circular DNA of WHV demonstrated that the virions produced during early or late chronic infection are fully capable of inducing productive acute infection with long lasting high viremia. These findings were further supported by the analysis of such intrahepatic markers of WHV infection, as replicative intermediate DNA, covalently-closed circular DNA, pre-genomic RNA and percentage of WHV core antigen-positive hepatocytes measured at several time points over the course of seventeen and a half weeks after the inoculation. In addition, the observed relationship between the production of antibodies against WHV surface antigens and parameters of WHV infection appears to be complex. Taken together, the generated data suggest that in vivo hepadnavirus virions produced during different phases of chronic infection did not demonstrate any considerable deficiencies in infectivity, when compared to that of virions generated during the acute phase of infection.

    IMPORTANCE The generated data suggest that infectivity of virions produced during the early or late stages of chronic hepadnavirus infection is not compromised. Our novel results provided several lines of further evidence supporting the idea that during the state of chronic infection in vivo, the limitations of hepadnavirus cell-to-cell spread/superinfection (observed recently in woodchuck model) are not due to diminished infectivity of the virions circulating in the blood; and are likely (i) related to the properties of hepatocytes (i.e., their capacity to support hepadnavirus infection/replication) and (ii) influenced by the immune system. The obtained results further extend the understanding of the mechanisms regulating the persistence of hepadnavirus infection. The follow-up studies that will further investigate hepadnavirus cell-to-cell spread as a potential regulator of the chronic state of the infection are warranted.

  • Deletion of African Swine Fever Virus Georgia 2007 virulence-associated gene 9GL (B119L) leads to virus attenuation in swine at low doses while inducing an effective protection against homologous challenge [PublishAheadOfPrint]

  • African swine fever virus (ASFV) is the etiological agent of an often lethal disease of domestic pigs. Disease control strategies have been hampered by the unavailability of vaccines against ASFV. Since its introduction in the Republic of Georgia, a highly virulent virus, ASFV Georgia 2007 (ASFV-G) has caused an epizootic that spread rapidly into Eastern Europeans countries. Currently, no vaccines are available or under development to control ASFV-G. In the past, genetically modified ASFVs harboring deletions of virulence-associated genes have proven attenuated in swine inducing protective immunity against challenge with homologous parental viruses. Deletion of 9GL (B119L) gene in highly virulent ASFV Malawi-Lil-20/1 produced an attenuated phenotype even when administered to pigs at a dose of 106 HAD50. Here we report the construction of a genetically modified ASFV-G harboring a deletion of 9GL (B119L) gene (ASFV-G-9GLv). Like Malawi-Lil-20/1-9GL, ASFV-G-9GL showed limited replication in primary swine macrophages. However, intramuscular inoculation of swine with 104 HAD50 of ASFV-G-9GL produced a virulent phenotype that unlike Malawi-Lil-20/1-9GL induced a lethal disease in swine as parental ASFV-G. Interestingly, lower doses (102-103 HAD50) of ASFV-G-9GL did not induce a virulent phenotype in swine and when challenged protected pigs against disease. A dose of 102 HAD50 of ASFV-G-9GLv conferred partial protection when pigs were challenged either at 21 or 28 days post-infection (dpi). A dose of 103 HAD50 of ASFV-G-9GL conferred partial and complete protection at 21 and 28 dpi respectively. Information here adds to our recent report as the first attempts towards experimental vaccines against ASFV-G.

    Importance The main problem for controlling ASF is the lack of vaccines. Studies on ASFV virulence lead to the production of genetically modified attenuated viruses that induce protection in pigs but only against homologous virus challenges. Here we produced a recombinant ASFV lacking virulence-associated gene 9GL in an attempt to produce a vaccine against virulent ASFV-G, a highly virulent virus isolate detected in Caucasus region in 2007 and now spreading though the Caucasus region and Eastern Europe. Deletion of 9GL, unlike with other ASFV isolates, did not attenuate completely ASFV-G. However, when delivered once at low dosages recombinant ASFV-G-9GL induces protection in swine against parental ASFV-G. The protection against ASFV-G is highly effective after 28 days post -vaccination, whereas at 21 days post-vaccination animals survived the lethal challenge but showing signs of ASF. Here we report the design and development of an experimental vaccine that induces protection against virulent ASFV-G.

  • Identification of owl monkey CD4 receptors broadly compatible with early-stage HIV-1 isolates [PublishAheadOfPrint]

  • Most HIV-1 variants isolated from early-stage human infections do not use nonhuman primate versions of the CD4 receptor for cellular entry, or do so poorly. We and others have previously shown that CD4 has experienced strong natural selection over the course of primate speciation, but it is unclear whether this selection has influenced the functional characteristics of CD4 as an HIV-1 receptor. Surprisingly we find that selection on CD4 has been most intense in the New World monkeys, animals that have never been found to harbor lentiviruses related to HIV-1. Based on this, we sampled CD4 genetic diversity within populations of individuals from seven different species, including five species of New World Monkeys. We found that some, but not all, CD4 alleles found in Spix's owl monkeys (Aotus vociferans) encode functional receptors for early-stage human HIV-1 isolates representing all of the major group M clades (A, B, C, and D). However, only some isolates of HIV-1 subtype C can use the CD4 receptor encoded by permissive Spix's owl monkey alleles. We characterize the prevalence of functional CD4 alleles in a colony of captive Spix's owl monkeys and find that 88% of surveyed individuals are homozygous for permissive CD4 alleles, which encode an asparagine at position 39 of the receptor. We find that the CD4 receptors encoded by two other species of owl monkeys (Aotus azarae and Aotus nancymaae) also serve as functional entry receptors for early-stage isolates of HIV-1.

    Importance. Nonhuman primates, particularly macaques, are used for preclinical evaluation of HIV-1 vaccine candidates. However, a significant limitation of the macaque model is the fact that most circulating HIV-1 variants cannot use the macaque CD4 receptor to enter cells and have to be adapted to these species. This is particularly true for viral variants from early stages of infection, which represent the most relevant vaccine targets. Here, we find that some individuals from captive owl monkey populations harbor CD4 alleles that are compatible with a broad collection of HIV-1 isolates, including those isolated from early in infection in highly affected populations and representing diverse subtypes.

  • Differential impact of in vivo CD8+ T-lymphocyte depletion in controller versus progressor SIV-infected macaques. [PublishAheadOfPrint]

  • Numerous studies have demonstrated that CD8+ T-lymphocytes suppress virus replication during HIV/SIV infection. However, the mechanisms underlying this activity of T-cells remain incompletely understood. Here we conducted CD8+ T-lymphocyte depletion in 15 rhesus macaques (RMs) infected i.v. with SIVmac239. At day 70 post-infection the animals (10 "progressors" with high viremia and 5 "controllers" with low viremia) were CD8-depleted by s.c. administration of the antibody M-T807R1. As expected, CD8 depletion resulted in increased virus replication, more prominently in controllers as compared to progressors, which correlated inversely with pre-depletion viremia. Of note, the feature of CD8+ T-lymphocytes pre-depletion that correlated best with the increase in viremia post-depletion was the level of CD8+T-bet+ lymphocytes. We next found that CD8 depletion resulted in a homogenous increase of SIV-RNA in superficial and mesenteric lymph nodes, spleen, and the gastro-intestinal tract of both controllers and progressors. Interestingly, the level of SIV-DNA increased post-depletion in both central-memory (TCM) and effector-memory (TEM) CD4+ T-lymphocytes in progressor RMs, but decreased in the CD4+ TCM of 4 out of 5 controllers. Finally, we found that CD8 depletion is associated with a greater increase in CD4+ T-lymphocyte activation (measured by Ki-67 expression) in controllers as compared to progressors. Overall, these data reveal a differential impact of CD8+ T-lymphocyte depletion between controller and progressor SIV-infected RMs, thus emphasizing the complexity of the in vivo antiviral role of CD8+ T-lymphocytes.

    Importance In this study we further dissect the impact of CD8+ T-lymphocytes on HIV/SIV replication during SIV infection. CD8+ T-lymphocyte depletion leads to a relatively homogenous increase in viral replication in peripheral blood and tissues. CD8+ T-lymphocytes depletion resulted in a more prominent increase in viral loads and CD4+ T-lymphocyte activation in controllers relative to progressors. Interestingly, we found T-bet expression on CD8+ T-lymphocytes to be the best predictor of viral load increase following depletion. The levels of SIV-DNA increase post-depletion in both central-memory (TCM) and effector-memory (TEM) CD4+ T-lymphocytes in progressors RMs, but decrease in the CD4+ TCM of controllers. The findings described in this study provide key insights into the differential function of CD8+ T-lymphocytes in controller and progressor RMs.

  • Lower Respiratory Tract Infection of the Ferret by 2009 H1N1 Pandemic Influenza A Virus Triggers Biphasic, Systemic and Local Neutrophil Recruitment [PublishAheadOfPrint]

  • Infection of the lower respiratory tract by influenza A viruses results in an increase in inflammation and immune cell infiltration in the lung. The dynamic relationships among the lung microenvironments, the lung and systemic host responses during infection remain poorly understood. Herein, we used an extensive systematic histologic analysis coupled with live imaging to gain access to these relationships in ferrets infected with the pandemic A(H1N1)2009 virus [H1N1pdm]. Neutrophil levels rose in lungs of H1N1pdm-infected ferrets 6 hours post-infection and became concentrated at areas of H1N1pdm-infected bronchiolar epithelium by 1 dpi (days post-infection). In addition, neutrophils were increased throughout the alveolar spaces during the first 3 dpi, and returned to baseline density by 6 dpi. Histochemical staining revealed neutrophil infiltration in the lungs occurred in two waves, at 1 and 3 dpi, and gene expression within microenvironments suggested two types of neutrophils. Specifically, CCL3, but not CXCL8/IL-8, levels were greater within discrete lung microenvironments, and coincided with increased infiltration of neutrophils in the lung. We used live imaging of ferrets to monitor host responses within the lung over time with 18fluorodeoxyglucose (FDG). Sites within the H1N1pdm-infected ferret lung with high FDG had high levels of proliferative epithelium. In summary, neutrophils invaded the H1N1pdm-infected ferret lung, globally, and focally, at sites of infection. The microenvironments with increased neutrophils, did not correlate with FDG, and hence, FDG-uptake may reflect prior infection and inflammation that has experienced damage as reflected by bronchial regeneration of tissues in the lungs at sites of high FDG.

    IMPORTANCE Severe influenza disease is characterized by an acute infection of the lower airways that may rapidly progress to organ failure and death. Well-developed animal models that mimic human disease are essential to understanding the complex relationships of the microenvironment, organ and system in controlling virus replication, inflammation, and disease progression. Employing the ferret model of H1N1pdm infection, we used live imaging and comprehensive histological analyses to address specific hypothesis regarding spatial and temporal relationships that occur over the progression of infection and inflammation. We show the general invasion of neutrophils at the organ level (lung), but a distinct pattern of localized accumulation within the local microenvironment at the site of infection. Moreover, we show that these responses were biphasic within the lung. Finally, live imaging revealed an early and sustained host metabolic response at sites of infection that may reflect damage and repair of tissues in the lungs.

  • Restriction of HIV-1 Requires the N-terminal Region of MxB/Mx2 as a Capsid-Binding Motif but not as a Nuclear Localization Signal [PublishAheadOfPrint]

  • The IFN-aalpha;-inducible restriction factor MxB blocks HIV-1 infection after reverse transcription but prior to integration. Fate of the capsid experiments have correlated the ability of MxB to block HIV-1 infection with stabilization of viral cores during infection. We have previously demonstrated that HIV-1 restriction by MxB requires capsid binding and oligomerization. Deletion and gain of function experiments have mapped the HIV-1 restriction ability of MxB to its N-terminal 25 amino acids. This report reveals that the N-terminal 25 amino acids of MxB exhibit two separate functions: 1) the ability of MxB to bind to HIV-1 capsid, and 2) the nuclear localization signal of MxB, which is important for the ability of MxB to shuttle into the nucleus. To understand whether MxB restriction of HIV-1 requires capsid binding and/or nuclear localization, we genetically separated these two functions and evaluated their contribution to restriction. Our experiments demonstrated that the 11RRR13 motif is important for the ability of MxB to bind capsid and to restrict HIV-1 infection. These experiments suggested that capsid binding is necessary for the ability of MxB to block HIV-1 infection. Separately from the capsid binding function of MxB, we found that residues 20KY21 regulate the ability of the N-terminal 25 amino acids of MxB to function as a nuclear localization signal; however, the ability of the N-terminal 25 amino acids to function as a nuclear localization signal was not required for restriction.

    IMPORTANCE MxB/Mx2 blocks HIV-1 infection in cells from the immune system. MxB blocks infection by preventing the uncoating process of HIV-1. The ability of MxB to block HIV-1 infection requires that MxB binds to the HIV-1 core by using its N-terminal domain. The present study shows that MxB uses residues 11RRR13 to bind to the HIV-1 core during infection and are required for the ability of MxB to block HIV-1 infection. We also found that residues 20KY21 constitute a nuclear localization signal that is not required for the ability of MxB to block HIV-1 infection.

  • Novel receptor specificity of avian gammacoronaviruses causing enteritis [PublishAheadOfPrint]

  • ABSTRACT

    Viruses exploit molecules on target membrane as receptors for attachment and entry into the host cells. Thus, receptor expression patterns can define viral tissue tropism and might to some extent predict the susceptibility of a host for a particular virus. Previously, others and we have shown that respiratory pathogens of the genus gammacoronavirus, including chicken infectious bronchitis virus (IBV), require specific aalpha;2-3-linked sialylated glycans for attachment and entry. Here, we studied determinants for binding of enterotropic avian gammacoronaviruses, including those of turkey (TCoV-US), guineafowl (GfCoV) and quail (QCoV), which are evolutionary distant from respiratory avian coronaviruses based on the viral attachment protein spike (S1). We profiled the binding of recombinantly expressed S1 proteins of TCoV, GfCoV and QCoV to tissues of their respective hosts. Protein histochemistry showed that the tissue binding specificity of S1 of turkey, quail, and guineafowl CoVs was limited to intestinal tissues of each particular host, in accordance with the reported pathogenicity of these viruses in vivo. Glycan array analyses revealed that, in contrast to IBV, S1 of enteric gammacoronaviruses recognize a unique set of non-sialylated type 2 poly-N-acetyl lactosamines. Lectin histochemistry as well as tissue binding patterns of TCoV-S1 further indicated that these complex N-glycans are prominently expressed on the intestinal tract of various avian species. In conclusion, our data demonstrates not only that enteric gammacoronaviruses recognize a novel glycan receptor, but also that enterotropism may be correlated with the high specificity of spike proteins for such glycans expressed in the intestines of the avian host.

    IMPORTANCE Avian coronaviruses are economically important viruses for the poultry industry. While infectious bronchitis virus (IBV), a respiratory pathogen of chickens, is rather well known, other viruses of the genus gammacoronavirus, including those causing enteric disease, are hardly studied. In turkey, guineafowl and quail, coronaviruses have been reported to be the major causative agent of enteric diseases. Specifically, turkey coronavirus outbreaks have been reported in North America, Europe and Australia for several decades. Recently a gammacoronavirus was isolated from guineafowl with fulminating disease. To date, it is not clear why these avian coronaviruses are enteropathogenic, whereas other closely related avian coronaviruses like IBV cause respiratory disease. A comprehensive understanding of the virus' tropism and pathogenicity explained by their receptor specificity, and the receptor expression on tissues was therefore needed. Here we identify a novel glycan receptor for enteric avian coronaviruses, which will further support the development of vaccines.

  • Human Papillomavirus Infectious Entry and Trafficking is a Rapid Process [PublishAheadOfPrint]

  • ABSTRACT

    Previous studies have indicated that Human Papillomavirus (HPV) infectious entry is slow requiring many hours after initial infection for the virus to gain entry into the nucleus. However intracellular transport pathways are typically very rapid and in the context of a natural HPV infection in a wounded epithelium, such slow intracellular transport would seem at odds with a normal viral infection. Using synchronised cell populations we show that HPV trafficking can however be a rapid process. In cells that are infected in the late S-early G2/M phase of the cell cycle, HPV16 pseudovirion (PsV) reporter DNA gene expression is detectable by 8hrs post-infection. Likewise reporter DNA can be visualised within the nucleus in conjunction with PML nuclear bodies 1-2hrs post-infection in cells that are infected with PsVs just prior to mitotic entry. This demonstrates that endosomal trafficking of HPV is rapid, with mitosis being the main restriction on nuclear entry.

    IMPORTANCE HPV infectious entry appears to be slow and requires mitosis to occur before the incoming viral DNA can access the nucleus. In this study we show that HPV trafficking in the cell is actually very rapid. This demonstrates that in the context of a normal virus infection, the cell cycle state will have a major influence on the time it takes for an incoming virus to enter the nucleus and initiate viral gene expression.

  • Lung CD8+ T cell impairment occurs during human metapneumovirus infection despite virus-like particle (VLP) induction of functional CD8+ T cells [PublishAheadOfPrint]

  • Human metapneumovirus (HMPV) is a major cause of respiratory disease in infants, the elderly, and immunocompromised individuals worldwide. There is currently no licensed HMPV vaccine. Virus-like particles (VLPs) are an attractive vaccine candidate because they are non-infectious and elicit a neutralizing antibody response. However, studies show that serum neutralizing antibodies are insufficient for complete protection against reinfection, and that adaptive T cell immunity is important for viral clearance. HMPV and other respiratory viruses induce lung CD8+ T cell (TCD8) impairment, mediated by programmed death-1 (PD-1). In this study, we generated HMPV VLPs by expressing the fusion and matrix proteins in mammalian cells and tested whether VLP immunization induces functional HMPV-specific TCD8 responses in mice. C57BL/6 mice vaccinated twice with VLPs and subsequently challenged with HMPV were protected from lung viral replication for at least 20 weeks post-immunization. A single VLP dose elicited F- and M-specific lung TCD8 with higher function and lower expression of PD-1 and other inhibitory receptors compared to TCD8 from HMPV-infected mice. However, after HMPV challenge, lung TCD8 from VLP-vaccinated mice exhibited inhibitory receptor expression and functional impairment similar to those of mice experiencing secondary infection. HMPV challenge of VLP-immunized mmu;MT mice also elicited a large percentage of impaired lung TCD8, similar to mice experiencing secondary infection. Together, these results indicate that VLPs are a promising vaccine candidate but do not prevent lung TCD8 impairment upon HMPV challenge.

    IMPORTANCE Human metapneumovirus (HMPV) is a leading cause of acute respiratory disease for which there is no licensed vaccine. Virus-like particles (VLPs) are an attractive vaccine candidate and induce antibodies, but T cell responses are less defined. Moreover, HMPV and other respiratory viruses induce lung CD8+ T cell (TCD8) impairment mediated by programmed death-1 (PD-1). In this study, HMPV VLPs containing viral fusion and matrix proteins elicited epitope-specific TCD8 that were functional and low in PD-1 expression. Two VLP doses conferred sterilizing immunity in C57BL/6 mice and facilitated HMPV clearance in antibody-deficient mmu;MT mice without enhancing lung pathology. However, regardless of whether responding lung TCD8 had previously encountered HMPV antigens in the context of VLPs or virus, a similar proportion were impaired and expressed comparable levels of PD-1 upon viral challenge. These results suggest that VLPs are a promising vaccine candidate but do not prevent lung TCD8 impairment upon HMPV challenge.

  • Marine snails and slugs: a great place to look for antiviral drugs [PublishAheadOfPrint]

  • Molluscs, one of the most successful phyla, lack clear evidence of adaptive immunity, yet thrive in the oceans, which are rich in viruses. There are thought to be nearly 120,000 species of Mollusca, most living in marine habitats. Despite the extraordinary abundance of viruses in oceans, molluscs often have very long lifespans (10-100s yrs). Thus, their innate immunity must be highly effective at countering viral infections. Antiviral compounds are a crucial component of molluscan defenses against viruses, and have diverse mechanisms of action against a wide variety of viruses, including many that are human pathogens. Antiviral compounds found in abalone, oyster, mussels and other cultured molluscs are available in large supply, providing good opportunities for future research and development. However, most members of the phylum Mollusca have not been examined for the presence of antiviral compounds. The enormous diversity and adaptations of molluscs implies a potential source of novel antiviral compounds for future drug discovery.

  • Characterization of a novel megabirnavirus from Sclerotinia sclerotiorum reveals horizontal gene transfer from ssRNA virus to dsRNA virus [PublishAheadOfPrint]

  • Mycoviruses have been detected in all major groups of filamentous fungi and their study represents an important branch of virology. Here, we characterized a novel double-stranded (ds) RNA mycovirus, Sclerotinia sclerotiorum megabirnavirus 1 (SsMBV1), in an apparently hypovirulent strain (SX466) of Sclerotinia sclerotiorum. Two similarly sized dsRNA segments (L1- and L2-dsRNA), the genome of SsMBV1, are packaged in rigid spherical particles purified from strain SX466. The full-length cDNA sequence of L1-dsRNA/SsMBV1 comprises two large open reading frames (ORF1 and ORF2), which encode a putative coat protein and an RNA-dependent RNA polymerases (RdRp), respectively. Phylogenetic analysis of the RdRp domain clearly indicates that SsMBV1 is related to Rosellinia necatrix megabirnavirus 1. L2-dsRNA/SsMBV1 comprises two non-overlapping ORFs (ORFA and ORFB) encoding two hypothetical proteins with unknown functions. The 5rrsquo; -terminal regions of L1 and L2-dsRNA/SsMBV1 share strictly conserved sequences and form stable stem-loop structures. Although L2-dsRNA/SsMBV1 is dispensable for replication, genome packaging, and pathogenicity of SsMBV1, it enhances transcript accumulation of L1-dsRNA/SsMBV1 and stability of virus-like particles (VLPs). Interestingly, a conserved papain-like protease domain similar to a multifunctional protein (p29) of Cryphonectria hypovirus 1, was detected in ORFA-encoded protein of L2-dsRNA/SsMBV1. Phylogenetic analysis based on protease domain suggests that horizontal gene transfer may have occurred from an ssRNA virus (hypovirus) to a dsRNA virus, SsMBV1. Our results reveal SsMBV1 has a slight impact on fundamental biological characteristics of its host regardless of the presence or absence of L2-dsRNA/SsMBV1.

    Importance Mycoviruses are widespread in all major fungal groups and they possess diverse genomes of mostly ssRNA and dsRNA and recently circular ssDNA. Here, we have characterized a novel dsRNA virus (Sclerotinia sclerotiorum megabirnavirus 1, SsMBV1) that was isolated from an apparently hypovirulent strain SX466 of Sclerotinia sclerotiorum. Although SsMBV1 is phylogenetically related to RnMBV1, SsMBV1 is markedly distinct from other reported megabirnaviruses with two features of VLP and conserved domains. Our results convincingly showed that SsMBV1 is viable in the absence of L2-dsRNA/SsMBV1 (a potential large satellite-like RNA or genuine genomic virus component). More interestingly, we detected a conserved papain-like protease domain that commonly exists in ssRNA viruses including members of families Potyviridae and Hypoviridae. Phylogenetic analysis based on protease domain suggests that horizontal gene transfer might have occurred from ssRNA virus to dsRNA virus, which may provide new insights into the evolutionary history of dsRNA and ssRNA viruses.

  • Careers in virology: Science writing and journalism [PublishAheadOfPrint]

  • This article condenses some highlights from a presentation I've now given at several universities about the bench-to-newsroom career path. For readers who simply want a short explanation of how to parlay their hard-earned critical thinking skills from graduate school into a lucrative job in a growing industry: go to law school.

  • Head-to-head comparison of poxvirus NYVAC and ALVAC vectors expressing identical HIV-1 clade C immunogens in prime/boost combination with Env protein in non-human primates [PublishAheadOfPrint]

  • We have compared the HIV-1-specific cellular and humoral immune responses elicited in rhesus macaques immunized with two poxvirus vectors (NYVAC and ALVAC) expressing the same HIV-1 antigens from clade C, Env gp140 as a trimeric cell released protein and Gag-Pol-Nef as Gag-induced virus-like particles (VLPs) (referred as NYVAC-C and ALVAC-C). The immunization protocol consisted of two doses of the corresponding poxvirus vector plus two doses of a combination of the poxvirus vector and a purified HIV-1 gp120 protein from clade C. This immunogenicity profile was also compared to that elicited by vaccine regimens consisting of two doses of the ALVAC vector expressing HIV-1 antigens from clades B/E (ALVAC-vCP1521) plus two doses of a combination of ALVAC-vCP1521 and HIV-1 gp120 protein from clades B/E (similar to the RV144 trial regimen) or clade C. The results showed that immunization of macaques with NYVAC-C stimulated at different times more potent HIV-1-specific CD4+ T-cell responses and induced a trend toward higher magnitude of HIV-1-specific CD8+ T-cell immune responses than ALVAC-C. Furthermore, NYVAC-C induced a trend toward higher levels of binding IgG antibodies against clade C HIV-1 gp140, gp120 or MuLV gp70-scaffolded V1/V2 and toward best cross-clade binding IgG responses against HIV-1 gp140 from clades A, B and group M consensus, compared to ALVAC-C. Of the linear binding IgG responses most were directed against the V3 loop in all immunization groups. Additionally, NYVAC-C and ALVAC-C also induced similar levels of HIV-1 neutralizing antibodies and antibody-dependent cellular cytotoxicity (ADCC) responses. Interestingly, binding IgA antibodies against HIV-1 gp120 or MuLV gp70-scaffolded V1/V2 were absent or very low in all immunization groups. Overall, these results provide a comprehensive survey of the immunogenicity of NYVAC versus ALVAC expressing HIV-1 antigens in non-human primates and indicate that NYVAC may represent an alternative candidate to ALVAC in the development of a future HIV-1 vaccine.

    IMPORTANCE The finding of a safe and effective HIV/AIDS vaccine immunogen is one of the main research priorities. Here, we have generated two poxvirus-based HIV vaccine candidates (NYVAC and ALVAC vectors) expressing the same clade C HIV-1 antigens in separate vectors and tested in non-human primates their immunogenicity profile. The results showed that immunization with NYVAC-C induced a trend toward higher HIV-1-specific cellular and humoral immune responses than those elicited by ALVAC-C, indicating that this new NYVAC vector could be considered a novel optimized HIV/AIDS vaccine candidate for human clinical trials.

  • Influenza A virus co-infection through transmission can support high levels of reassortment [PublishAheadOfPrint]

  • The reassortment of gene segments between influenza viruses increases genomic diversity and plays an important role in viral evolution. We have shown previously that this process is highly efficient within a co-infected cell and, given synchronous co-infection at moderate or high doses, can give rise to ~60-70% of progeny shed from an animal host. Conversely, reassortment in vivo can be rendered undetectable by lowering viral doses or extending the time between infections. One might also predict that seeding of transmitted viruses into different sites within the target tissue could limit subsequent reassortment. Given the potential for stochastic factors to restrict reassortment during natural infection, we sought to determine its efficiency in a host co-infected through transmission. Two scenarios were tested in a guinea pig model, using influenza A/Panama/2007/99 (H3N2) virus (wt) and a silently mutated variant (var) thereof as parental virus strains. In the first, co-infection was achieved by exposing a naiiuml;ve guinea pig to two cagemates, one infected with wt and the other with var virus. When such exposure led to co-infection, robust reassortment was typically seen, with 50-100% of isolates carrying reassortant genomes at one or more time points. In the second scenario, naiiuml;ve guinea pigs were exposed to a cagemate that had been co-inoculated with wt and var viruses. Here, reassortment occurred in the co-inoculated donor host, multiple variants were transmitted, and reassortants were prevalent in the recipient host. Together, these results demonstrate the immense potential for reassortment to generate viral diversity in nature.

    Importance Influenza viruses evolve rapidly under selection due to the generation of viral diversity through two mechanisms. The first is the introduction of random errors into the genome by the viral polymerase, which occurs with a frequency of approximately 10-5 errors/nucleotide replicated. The second is reassortment, or the exchange of gene segments between viruses. Reassortment is known to occur readily under well-controlled laboratory conditions, but its frequency in nature is not clear. Here we tested the hypothesis that reassortment efficiency following co-infection through transmission would be reduced compared to that seen with co-inoculation. Contrary to this hypothesis, our results indicate that co-infection achieved through transmission supports high levels of reassortment. These results suggest that reassortment is not exquisitely sensitive to stochastic effects associated with transmission and likely occurs in nature whenever a host is infected productively with more than one influenza A virus.

  • The Torsin activator LULL1 is required for efficient growth of HSV-1 [PublishAheadOfPrint]

  • TorsinA is a membrane-tethered AAA+ ATPase implicated in nuclear envelope dynamics as well as the nuclear egress of herpes simplex virus-1 (HSV-1). The activity of TorsinA and the related ATPase TorsinB strictly depends on LAP1 and LULL1, type II transmembrane proteins that are integral parts of the Torsin/cofactor AAA ring, forming a composite, membrane-spanning assembly. Here we use CRISPR/Cas9-mediated genome engineering to create single- and double-knockout (KO) cell lines of TorA and TorB as well as their activators LAP1 and LULL1 to investigate the effect on HSV-1 production. Consistent with LULL1 being the more potent Torsin activator, a LULL1 KO reduces HSV-1 growth by one order of magnitude, while the deletion of other components of the Torsin system in combination causes subtle defects. Notably, LULL1 deficiency leads to a 10-fold decrease in the number of viral genomes per host cell without affecting viral protein production, allowing us to tentatively assign LULL1 to an unexpected role that precedes HSV-1 nuclear egress.

    Importance In this study we conduct the first comprehensive genetic and phenotypic analysis of the Torsin/cofactor system in the context of HSV-1 infection, establishing LULL1 as the most important component of the Torsin system with respect to viral production.

  • Mapping of a region of the PA-X protein of influenza A virus that is important for its shut-off activity. [PublishAheadOfPrint]

  • The influenza A virus PA-X comprises an N-terminal PA endonuclease domain and a C-terminal PA-X-specific domain. PA-X reduces host and viral mRNA accumulation via its endonuclease function. Here, we found that the N-terminal 15 amino acids, particularly 6 basic amino acids, in the C-terminal PA-X-specific region are important for PA-X shut-off activity. These 6 basic amino acids enabled a PA deletion mutant to suppress protein expression at a level comparable to that of wild-type PA-X.

  • Arenavirus Co-Infections are Common in Snakes with Boid Inclusion Body Disease [PublishAheadOfPrint]

  • Recently, novel arenaviruses were found in snakes with Boid Inclusion Body Disease (BIBD); these form a new genus Reptarenavirus within the family Arenaviridae. We used NGS and de novo sequence assembly to investigate reptarenavirus isolates from our previous study. Four of the six isolates and all samples from snakes with BIBD contained at least two reptarenaviruses species. The sequenced viruses comprise four novel reptarenavirus species and a representative of a new arenavirus genus.

  • A dual modality HSV-2 vaccine for preventing genital herpes using glycoproteins C and D subunit antigens to induce potent antibody responses and adenovirus vectors containing capsid and tegument proteins as T cell immunogens [PublishAheadOfPrint]

  • We evaluated a genital herpes prophylactic vaccine containing HSV-2 glycoproteins C (gC2) and D (gD2) to stimulate humoral immunity and UL19 (capsid protein VP5) and UL47 (tegument protein VP13/14) as T-cell immunogens. HSV-2 gC2 and gD2 proteins were expressed in baculovirus, while UL19 and UL47 gene were expressed in replication defective adenovirus vectors. The adenovirus vectors containing UL19/UL47 stimulated CD4+ and CD8+ human and murine T-cell responses. Guinea pigs were either i) mock immunized; ii) immunized with gC2/gD2 with CpG/alum as adjuvants; iii) immunized with adenovirus vectors UL19/UL47; or iv) immunized with combined gC2/gD2-CpG/alum and adenovirus vectors UL19/UL47. Immunization with gC2/gD2 produced potent neutralizing antibodies, while UL19 and UL47 also stimulated antibody responses. After intravaginal HSV-2 challenge, the mock and adenovirus UL19/UL47 groups developed severe acute disease, while 2/8 animals in the gC2/gD2 alone and none in the combined group developed acute disease. No animals in the gC2/gD2 or combined group developed recurrent disease; however, 5/8 animals in each group had subclinical shedding of HSV-2 DNA on 15/168 days in the gC2/gD2 group and 13/168 days in the combined group. Lumbosacral dorsal root ganglia were positive for HSV-2 DNA and Latency Associated Transcripts in 5/8 animals in the gC2/gD2 and 2/8 in the combined group. None of the differences comparing gD2/gD2 alone with the combined group was statistically significant. Therefore, adding T-cell immunogens UL19/UL47 to gC2/gD2 did not significantly reduce genital disease and vaginal HSV-2 DNA shedding compared with the excellent protection provided by gC2/gD2 in the guinea pig model.

    Importance HSV-2 infection is a common cause of genital ulcer disease and a significant public health concern. Genital herpes increases the risk of transmission and acquisition of HIV-1 infection by 3-to-4 fold. A herpes vaccine that prevents genital lesions and asymptomatic genital shedding will have a substantial impact on two epidemics, HSV-2 and HIV-1. Previously we reported that a vaccine containing HSV-2 glycoprotein C (gC2) and glycoprotein D (gD2) reduced genital lesions and asymptomatic HSV-2 genital shedding in guinea pigs, yet the protection was not complete. We evaluated whether adding T cell immunogens UL19 (capsid protein VP5) and UL47 (tegument protein VP13/14) will enhance protection provided by gC2/gD2, which produces potent antibody responses. Here we report the efficacy of a combination vaccine containing gC2/gD2 and UL19/UL47 for prevention of genital disease, vaginal shedding of HSV-2 DNA and latent infection of dorsal root ganglia in guinea pigs.

  • A kinome-wide siRNA screen identifies proviral and antiviral host factors in SARS-coronavirus replication, including PKR and early secretory pathway proteins [PublishAheadOfPrint]

  • To identify host factors relevant for SARS-coronavirus (SARS-CoV) replication, we performed an siRNA library screen targeting the human kinome. Protein kinases are key regulators of many cellular functions and the systematic knockdown of their expression should provide a broad perspective on factors and pathways promoting or antagonizing coronavirus replication. In addition to 40 proteins that promote SARS-CoV replication, our study identified 90 factors exhibiting an antiviral effect. Pathway analysis grouped subsets of these factors in specific cellular processes, including the innate immune response and the metabolism of complex lipids, which thus appear to play a role in SARS-CoV infection. Several factors were selected for in-depth validation in follow-up experiments. In cells depleted for the bbeta;2 subunit of the coatomer protein complex (COPB2), the strongest proviral hit, we observed reduced SARS-CoV protein expression and a ggt;2-log reduction in virus yield. Knockdown of the COPB2-related proteins COPB1 and Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) also suggested that COPI-coated vesicles and/or the early secretory pathway are important for SARS-CoV replication. Depletion of the antiviral double-stranded RNA-activated protein kinase (PKR) enhanced virus replication in the primary screen, and validation experiments confirmed increased SARS-CoV protein expression and virus production upon PKR depletion. In addition, cyclin dependent kinase 6 (CDK6) was identified as a novel antiviral host factor in SARS-CoV replication. The inventory of pro- and antiviral host factors and pathways described here substantiates and expands our understanding of SARS-CoV replication and may contribute to the identification of novel targets for antiviral therapy.

    Importance Replication of all viruses including SARS-coronavirus (SARS-CoV) depends on and is influenced by cellular pathways. Although substantial progress has been made in dissecting the coronavirus replicative cycle, our understanding of the host factors that stimulate (proviral factors) or restrict (antiviral factors) infection remains far from complete. To study the role of host proteins in SARS-CoV infection, we set out to systematically identify kinase-regulated processes that influence virus replication. Protein kinases are key regulators in signal transduction, control a wide variety of cellular processes, and many of them are targets of approved drugs and other compounds. Our screen identified a variety of hits and will form the basis for more detailed follow-up studies that should contribute to a better understanding of SARS-CoV replication and coronavirus-host interactions in general. The identified factors could be interesting targets for the development of host-directed antiviral therapy to treat infections with SARS-CoV or other pathogenic coronaviruses.

  • Tracking the emergence of host-specific SIV env and nef populations reveals nef early adaptation and convergent evolution in brain of naturally progressing Rhesus macaques [PublishAheadOfPrint]

  • While a clear understanding of the events leading to successful establishment of host-specific viral populations and productive infection in the central nervous system (CNS) has not yet been reached, the simian immunodeficiency virus (SIV)-infected Rhesus Macaque provides a powerful model for the study of human immunodeficiency virus (HIV) intra-host evolution and neuropathogenesis. The evolution of gp120 and nef genes, which encode two key proteins required for the establishment and maintenance of infection, was assessed in macaques that were intravenously inoculated with the same viral swarm and allowed to naturally progress to simian AIDS and potential SIV-encephalitis (SIVE). Longitudinal plasma samples and immune markers were monitored until terminal illness. Single genome-sequencing was employed to amplify full-length env through nef transcripts from plasma over time and brain tissues at necropsy. Nef sequences diverged from the founder virus at a faster than gp120 diverged. Host-specific sequence populations were detected in nef (~92 days) before they were detected in gp120 (~182 days). At necropsy, similar brain nef sequences were found in different macaques, indicating convergent evolution, while gp120 brain sequences remained largely host-specific. Molecular clock and selection analysis showed weaker clock-like behavior and stronger selection pressure in nef than in gp120 with the strongest nef selection in the macaque with SIVE. Rapid nef diversification, occurring prior to gp120 diversification, indicates that early adaptation of nef in the new host is essential for successful infection. Moreover, the convergent evolution of nef sequences in the CNS suggests a significant role for nef in establishing neurotropic strains.

    IMPORTANCE The SIV-infected rhesus macaque model closely resembles HIV-1 immunopathogenesis, neuropathogenesis and disease progression in humans. Macaques were intravenously infected with an identical viral swarm to investigate evolutionary patterns in the gp120 and nef genes leading to the emergence of host-specific viral populations, and potentially linked to disease progression. Although each macaque exhibited unique immune profiles, macaque-specific nef sequences evolving under selection were consistently detected in plasma samples at three months post infection, significantly earlier than in gp120 macaque-specific sequences. On the other hand, nef sequences in brain tissues, collected at necropsy of two animals with detectable infection in the central nervous system (CNS), revealed convergent evolution. Results not only indicate that early adaptation of nef in the new host may be essential for successful infection, but also suggest that specific nef variants may be required for SIV to efficiently invade CNS macrophages and/or enhance macrophage migration resulting in HIV neuropathology.

  • Inhibition of O-linked N-acetylglucosamine Transferase Reduces Replication of Herpes Simplex Virus and Human Cytomegalovirus [PublishAheadOfPrint]

  • O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential cellular enzyme that posttranslationally modifies nuclear and cytoplasmic proteins via O-linked addition of a single N-acetylglucosamine (GlcNAc) moiety. Among the many targets of OGT is host cell factor 1 (HCF-1), a transcriptional regulator that is required for transactivation of the immediate-early genes of herpes simplex virus (HSV). HCF-1 is synthesized as a large precursor that is proteolytically cleaved by OGT, which may regulate its biological function. In this study, we tested whether inhibition of the enzymatic activity of OGT with a small molecule inhibitor OSMI-1 affects initiation of HSV immediate-early gene expression and viral replication. We found that inhibiting OGT's enzymatic activity significantly decreased HSV replication. The major effect of the inhibitor occurred late in the viral replication cycle, when it reduced the levels of late proteins and inhibited capsid formation. However, depleting OGT levels with siRNA reduced the expression of HSV immediate-early genes, in addition to reducing viral yields. In this study, we identify OGT as a novel cellular factor involved in HSV replication. Our results using a small molecule inhibitor and siRNA depletion suggest that OGT's glycosylation and scaffolding functions play distinct roles in the replication cycle of HSV.

    Importance Antiviral agents can target viral or host gene products essential for viral replication. O-GlcNAc transferase (OGT) is an important cellular enzyme that catalyzes the posttranslational addition of GlcNAc sugar residues to hundreds of nuclear and cytoplasmic proteins, and this modification regulates their activity and function. Some of the known OGT targets are cellular proteins that are critical for the expression of herpes simplex virus (HSV) genes, suggesting a role for OGT in the replication cycle of HSV. In this study, we found that OGT is required for the efficient expression of viral genes, and for assembly of new virions. Thus, we identify OGT as a novel host factor involved in the replication of HSV and a potential target for antiviral therapy.

  • Novel arenavirus entry inhibitors discovered using a minigenome rescue system for high-throughput drug screening [PublishAheadOfPrint]

  • Certain members of the Arenaviridae family are Category A agents, capable of causing severe hemorrhagic fevers in humans. Specific antiviral treatments do not exist, and the only commonly used drug, Ribavirin, has limited efficacy and can cause severe side effects. The discovery and development of new antivirals is inhibited by the biohazardous nature of the viruses, making them a relatively poorly understood group of human pathogens. We therefore adapted a reverse genetics minigenome (MG) rescue system based on Junin virus, the causative agent of Argentine hemorrhagic fever, for high throughput screening (HTS). The MG rescue system recapitulates all stages of the virus life-cycle, and enables screening of small molecule libraries under BSL2 conditions. The HTS resulted in the identification of 4 candidate compounds with potent activity against a broad panel of arenaviruses, three of which were completely novel. The target for all 4 compounds was the stage of viral entry, which positions the compounds as potentially important leads for future development.

    Importance The arenavirus family includes several members that are highly pathogenic, causing acute viral hemorrhagic fevers with high mortality rates. No specific effective treatments exist, and although a vaccine is available for Junin virus, the causative agent of Argentine hemorrhagic fever (AHF), it is only licensed for use in the endemic area of AHF. For these reasons it is important to identify specific compounds that could be developed as antivirals against these deadly viruses.

  • Coronavirus nsp10/nsp16 methyltransferase can be targeted by nsp10-derived peptide in vitro and in vivo to reduce replication and pathogenesis [PublishAheadOfPrint]

  • The 5rrsquo; cap structures of eukaryotic mRNAs are important for RNA stability and protein translation. Many viruses that replicate in the cytoplasm of eukaryotes have evolved 2rrsquo; -O-methyltransferases (2rrsquo; -O-MTase) to autonomously modify their mRNAs and thus carry a cap-1 structure (m7Gppp-Nm) at the 5rrsquo; -end, thereby facilitating viral replication and escaping innate immune recognition in host cells. Previous studies showed that the 2rrsquo; -O-MTase activity of severe acute respiratory syndrome coronavirus (SARS-CoV) non-structural protein 16 (nsp16) needs to be activated by nsp10 whereas nsp16 of feline coronavirus (FCoV) alone possesses 2rrsquo; -O-MTase activity. In this study, we demonstrate that stimulation of nsp16 2rrsquo; -O-MTase activity by nsp10 is a universal and conserved mechanism in coronaviruses including FCoV and that nsp10 is functionally interchangeable in stimulation of nsp16 of different coronaviruses. Based on our current and previous studies, we designed a peptide (TP29) from the sequence of the interaction interface of mouse hepatitis virus (MHV) nsp10 and demonstrated that the peptide inhibits the 2rrsquo; -O-MTase activity of different coronaviruses in biochemical assays and the viral replication in MHV infection and SARS-CoV replicon models. Interestingly, the peptide TP29 exerted robust inhibitory effects in vivo in MHV infected mice by impairing the MHV virulence and pathogenesis through suppressing virus replication and enhancing type I interferon production at an early stage of infection. Therefore, as a proof-of-principle, the current results indicate that coronavirus 2rrsquo; -O-MTase activity can be targeted in vitro and in vivo.

    Importance Coronaviruses (CoVs) are important pathogens of animals and human with high zoonotic potential. SARS-CoV encodes the 2rrsquo; -O-methyltransferase (2rrsquo; -O-MTase) that is composed of the catalytic subunit nsp16 and the stimulatory subunit nsp10, and plays an important role in virus genome replication and evasion from innate immunity. Our current results demonstrate that stimulation of nsp16 2rrsquo; -O-MTase activity by nsp10 is a common mechanism for coronaviruses. And nsp10 is functionally interchangeable in the stimulation of nsp16 among different coronaviruses, which underlies the rationale for developing inhibitory peptides. We demonstrate that a peptide derived from the nsp16-interacting domain of mouse hepatitis virus (MHV) nsp10 could inhibit 2rrsquo; -O-MTase activity of different coronaviruses in vitro and viral replication of MHV and SARS-CoV replicon in cell culture. And it could strongly inhibit virus replication and pathogenesis in MHV-infected mice. The work makes it possible to develop broad-spectrum peptide inhibitors by targeting the nsp16/nsp10 2rrsquo; -O-MTase of coronaviruses.

  • Broadly neutralizing human immunodeficiency virus type 1 antibody gene transfer protects non-human primates from mucosal simian-human immunodeficiency virus infection. [PublishAheadOfPrint]

  • Broadly neutralizing antibodies (bnAbs) can prevent lentiviral infection in non-human primates and may slow the spread of human immunodeficiency virus type 1 (HIV-1). Although protection by passive transfer of human bnAbs has been demonstrated in monkeys, durable expression is essential for its broader use in humans. Gene-based expression of bnAbs provides a potential solution to this problem, though immune responses to the viral vector or to the antibody may limit its durability and efficacy. Here, we delivered an adeno-associated viral vector encoding a simianized form of a CD4bs bnAb, VRC07, and evaluated its immunogenicity and protective efficacy. The expressed antibody circulated in macaques for 16 weeks at levels up to 66 mmu;g/ml, though immune suppression with cyclosporine A (CsA) was needed to sustain expression. Gene-delivered simian VRC07 protected against SHIV infection in monkeys 5.5 weeks after treatment. Gene transfer of an anti-HIV antibody can therefore protect against infection by viruses that cause AIDS in primates when the host immune responses are controlled.

    IMPORTANCE Sustained interventions that can prevent HIV-1 infection are needed to halt the spread of the HIV-1 pandemic. The protective capacity of anti-HIV antibody gene therapy has been established in mouse models of HIV-1 infection, but has not been established for primates. We show here proof-of-concept that gene transfer of anti-HIV antibody genes can protect against infection by viruses that cause AIDS in primates when host immune responses are controlled.

  • Poxvirus protein MC132 from Molluscum Contagiosum Virus inhibits NF{kappa}B activation by targeting p65 for degradation [PublishAheadOfPrint]

  • Molluscum Contagiosum Virus (MCV) is unique in being the only known extant, human-adapted poxvirus, yet to date is very poorly characterized in terms of host-pathogen interactions. MCV causes persistent skin lesions filled with live virus yet these generally appear immunologically silent suggesting the presence of potent inhibitors of human anti-viral immunity and inflammation. However, less than five MCV immunomodulatory genes have been characterized in detail yet it is likely that many more remain to be discovered given the density of such sequences in all well characterized poxviruses. Following virus infection, NFB activation occurs in response to both pattern recognition receptor (PRR) signaling and cellular activation by virus-elicited proinflammatory cytokines such as TNF. As such NFB activation is required for virus detection, anti-viral signaling, inflammation and clearance of viral infection. Hence we screened a library of MCV genes for effects on TNF-stimulated NFB activation. This revealed MC132, a unique protein with no orthologs in other poxviral genomes, as a novel inhibitor of NFB. Interestingly, MC132 also inhibited PRR- and virus-activated NFB, since MC132 interacted with the NFB subunit p65, and caused p65 degradation. Unbiased affinity purification to identify host targets of MC132 revealed that MC132 acted by targeting NFB p65 for ubiquitin-dependent proteasomal degradation by recruiting p65 to a host Cullin-5/Elongin B/Elongin C complex. These data reveal a novel mechanism for poxviral inhibition of human innate immunity and further clarify how the human-adapted poxvirus MCV can so effectively evade anti-viral immunity to persist in skin lesions.

    Importance How human cells detect and respond to viruses is incompletely understood, but great leaps in our understanding have been made by studying both the early innate immune response and the ways viruses evade it. Poxviruses adapt to specific hosts over time evolving elegantly precise inhibitors targeting the rate-limiting steps of immunity. These inhibitors reveal new features of the anti-viral response whilst also offering potent new tools for approaching therapeutic intervention in autoimmunity. Molluscum contagiosum virus (MCV) is the only known extant poxvirus specifically adapted to human infection yet remains poorly characterized. We report the identification of MCV protein MC132 as a potent inhibitor of NFB, an essential regulatory crux of innate immunity. Further, identification of the mechanism of inhibition of NFB by MC132 reveals an elegant example of convergent evolution with human herpesviruses. This discovery greatly expands our understanding of how MCV so effectively evades human immunity.

  • Robust Protection against Highly Virulent Foot-and-Mouth Disease Virus in Swine by Combination Treatment with Recombinant Adenoviruses Expressing Porcine Interferon-{alpha}{gamma} and Multiple Small Interfering RNAs [PublishAheadOfPrint]

  • Because the currently available vaccines against foot-and-mouth disease (FMD) provide no protection until 4-7 days post-vaccination, the only alternative method to halt the spread of the FMD virus (FMDV) during outbreaks is the application of antiviral agents. Combinatorial treatment strategies have been used to enhance the efficacy of antiviral agents, which may be advantageous in overcoming viral resistance mechanisms against antiviral treatments. We have developed recombinant adenoviruses for simultaneous expression of porcine interferon aalpha; and (Ad-porcine IFN-aalpha;), as well as 3 small interfering RNAs (Ad-3siRNA) targeting FMDV mRNAs encoding non-structural proteins. The antiviral effects of Ad-porcine IFN-aalpha; and Ad-3siRNA expression were tested in combination in porcine cells, suckling mice, and swine. We observed enhanced antiviral effects in porcine cells and mice, as well as robust protection against the highly pathogenic strain O/Andong/SKR/2010 and increased expression of cytokines in swine following combination treatment. In addition, we showed that combination treatment was effective against all serotypes of FMDV. Therefore, we suggest that the combined treatment with Ad-porcine IFN-aalpha; and Ad-3siRNA may offer fast-acting antiviral protection and be used with a vaccine during the period that the vaccine does not provide protection against FMD.

    IMPORTANCE The use of current foot-and-mouth disease (FMD) vaccines to induce rapid protection provides limited effectiveness because the protection does not become effective until a minimum of 4 days after vaccination. Therefore, antiviral agents remain the only available treatment to confer rapid protection and reduce the spread of foot-and-mouth disease virus (FMDV) in livestock during outbreaks until vaccine-induced protective immunity can become effective. Interferons and small interfering RNAs (siRNAs) have been reported to be effective antiviral agents against FMDV, although the virus has associated resistance mechanisms for type I interferons and siRNAs. We have developed recombinant adenoviruses for simultaneous expression of porcine interferon aalpha; and (Ad-porcine IFN-aalpha;), as well as multiple siRNAs (Ad-3siRNA) to enhance the inhibitory effects of these antiviral agents observed in previous studies. Here, we show enhanced antiviral effects against FMDV by combination treatment with Ad-porcine IFN-aalpha; and Ad-3siRNA to overcome resistant mechanisms of FMDV in swine.

  • mRNA capping by Venezuelan Equine Encephalitis Virus nsP1: Functional characterization and implication for antiviral research. [PublishAheadOfPrint]

  • Alphaviruses are known to possess a unique viral mRNA capping mechanism involving the viral non structural protein nsP1. This enzyme harbours methyltransferase (MTase) and nsP1 guanylylation (GT) activities catalysing the transfer of the methyl group from S-Adenosylmethionine (AdoMet) to the N7 position of a GTP molecule followed by the formation of a m7GMP-nsP1 adduct. Subsequent transfer m7GMP onto the 5rrsquo; -end of the viral mRNA has not been demonstrated in vitro yet. In this study we report the biochemical characterization of Venezuelan equine encephalitis virus (VEEV) nsP1. We have developed enzymatic assays uncoupling the different reactions steps catalysed by nsP1. The MTase and GT reaction activities were followed using a non-hydrolysable GTP (GIDP) substrate and an original Western Blot assay using anti-m3G/m7G-cap monoclonal antibody, respectively. The GT reaction is stimulated by S-Adenosyl-L-homocysteine (Ado-Hcy), product of the preceding MTase reaction, and metallic ions. The covalent linking between nsP1 and m7GMP involves a phosphamide bond between the nucleotide a histidine residue. Final guanylyltransfer onto RNA was observed for the first time with an alphavirus nsP1 using a 5rrsquo; -diphosphate RNA oligonucleotide whose sequence corresponds to the 5rrsquo; end of the viral genome. Alanine scanning mutagenesis of residues H37, H45, D63, E118, Y285, D354, R365, N369, N375 reveals their respective role in MT and GT reactions. Finally, the inhibitory effect of Sinefungin, Aurintricarboxylic acid (ATA) and ribavirin-triphosphate on MTase and capping reactions was investigated, providing possible avenues for antiviral research.

    Importance Emergence or re-emergence of alphaviruses represents a serious health concern and the elucidation of their replication mechanisms is a prerequisite for the development of specific inhibitors targeting viral enzymes. In particular, alphaviruses are able, through an original reactions sequence, to add to their mRNA a cap required for their protection against cellular nucleases and initiation of viral proteins translation. In this study, the capping of a 5rrsquo; diphosphate synthetic RNA mimicking the 5rrsquo; end of an alphavirus mRNA was reconstituted in vitro for the first time. The different steps for this capping are performed by the non structural protein 1 (nsP1). Reference compounds known to target the viral capping inhibited nsP1 enzymatic functions, highlighting the value of this enzyme in the antiviral development.

  • Early step of JSRV-mediated cell transformation involves the interaction between Env and the RALBP1 cellular protein [PublishAheadOfPrint]

  • Ovine pulmonary adenocarcinoma is a naturally occurring lung cancer in sheep induced by the Jaagsiekte Sheep RetroVirus (JSRV). Its envelope glycoprotein (Env) carries oncogenic properties, and its expression is sufficient to induce in vitro cell transformation and in vivo lung adenocarcinoma. The identification of cellular partners of the JSRV envelope remains crucial for deciphering mechanisms leading to cell transformation. We initially identified RALBP1 (Ral A Binding Protein also known as RLIP76 or RIP), a cellular protein implicated in the ras pathway, as a partner of JSRV Env by yeast double-hybrid screening and confirmed formation of RALBP1/Env complexes in mammalian cells. Expression of the RALBP1 protein was repressed in tumoral lungs as well as in tumor-derived alveolar type II cells. Through its inhibition using specific siRNA, we showed that RALBP1 was involved in envelope-induced cell transformation and in modulation of the mTOR/p70S6K pathway by the retroviral envelope.

    IMPORTANCE JSRV-induced lung adenocarcinoma is of importance for the sheep industry. While the envelope has been reported as the oncogenic determinant of the virus, the cellular proteins directly interacting with Env are still not known. Our report on the formation of RALBP/Env complexes and the role of this interaction in cell transformation opens new hypothesis for the dysregulation observed upon virus infection in sheep.

  • H3K27 demethylation at the proviral promoter sensitizes latent HIV to the effects of vorinostat in ex-vivo cultures of resting CD4+ T cells [PublishAheadOfPrint]

  • Histone methyltransferase inhibitors (HMTi) and histone deacetylase inhibitors (HDACi) are reported to synergistically induce the expression of latent human immunodeficiency virus type 1 (HIV), but studies have largely been performed in cell lines. As specific and potent HMTis directed at EZH1/2 are now in human testing, we wished to rigorously test such an inhibitor in a primary resting T-cell model of HIV latency. We found that GSK343, a potent and selective EZH2/EZH1 (enhancer of zeste 2 Polycomb repressive complex 2 subunit 2 or 1) inhibitor, reduced histone 3 trimethylation at lysine 27 (H3K27) at the HIV provirus in resting cells. Remarkably, this epigenetic change was not associated with increased proviral expression in latently infected resting cells.

    However, following the reduction in H3K27 at the HIV long terminal repeat (LTR), subsequent exposure to the HDACi suberoylanilide hydroxamic acid or vorinostat (VOR) resulted in an increase in HIV gag RNA and HIV p24 antigen production up to 2.5-fold greater than that induced by VOR alone.

    Therefore, in primary, resting CD4+ T cells true mechanistic synergy in the reversal of HIV latency may be achieved by the combination of HMTis and HDACis. Although other cellular effects of EZH2 inhibition may contribute to the sensitization of the HIV LTR to subsequent exposure to VOR, and to increase viral antigen production, this synergistic effect is directly associated with H3K27 demethylation at Nuc-1. Based upon our findings the combination of HMTis and HDACis should be considered for testing in animal models or clinical trials.

    Importance Demethylation of H3K27 mediated by the histone methyltransferase inhibitor GSK343 in primary resting T cells is slow; occurring over 96 hours, but by itself does not result in a significant upregulation of cell-associated HIV RNA expression or viral antigen production. However, following H3K27 demethylation, latent viral expression within infected primary resting CD4+ T cells in synergistically increased upon exposure to the histone deacetylase inhibitor vorinostat. Demethylation at H3K27 sensitizes the HIV promoter to the effects an HDAC inhibitor, and provides proof of concept for the testing of combination epigenetic approaches to disrupt latent HIV infection, a necessary step towards the eradication of HIV infection.

  • Frequency, private specificity and cross-reactivity of pre-existing HCV-specific CD8+ T cells in HCV seronegative individuals: implication for vaccine responses [PublishAheadOfPrint]

  • T cell responses play a critical role in controlling or clearing viruses. Therefore, strategies to prevent or treat infections include boosting T cell responses. T cells specific for various pathogens have been reported in unexposed individuals and an influence of such cells on the response towards vaccines is conceivable. However, little is known about their frequency, repertoire and impact on vaccination. We performed a detailed characterization of CD8+ T cells specific to a Hepatitis C Virus (HCV) epitope (NS3-1073) in 121 HCV seronegative individuals. We show that in vitro HCV NS3-1073 specific CD8+ T cell responses were rather abundantly detectable in one third of HCV seronegative individuals irrespective of risk factors for HCV exposure. Ex vivo, these NS3-1073 specific CD8+ T cells were found to be both naiiuml;ve- and memory-like cells. Importantly, recognition of various peptides derived from unrelated viruses by NS3-1073 specific CD8+ T cells showed a considerable degree of T cell cross-reactivity, suggesting that they might in part originate from previous heterologous infections. Finally, we further provide evidence that pre-existing NS3-1073 specific CD8+ T cells can impact the T cell response towards peptide vaccination. Healthy vaccinated individuals who showed an in vitro response towards NS3-1073 already before vaccination displayed a more vigorous and earlier response towards the vaccine.

    IMPORTANCE Preventive and therapeutic vaccines are being developed for many viral infections and often aim on inducing T cell responses. Despite effective antiviral drugs against HCV, there is still a need for a preventive vaccine. However, the responses to vaccines can be highly variable among different individuals. Pre-existing T cells in unexposed individuals could be one reason that helps to explain the variable T cell responses to vaccines. Based on our findings, we suggest that HCV CD8+ T cells are abundant in HCV seronegative individuals but their repertoire is highly diverse due to both involvement of naiiuml;ve precursors and cross-reactive memory cells of different specificities which can influence the response to vaccines. The data may emphasize the need to personalize immune based therapies based on the individuals T cell repertoire that is present before the immune intervention.

  • Primer ID Validates Template Sampling Depth and Greatly Reduces the Error Rate of Next Generation Sequencing of HIV-1 Genomic RNA Populations [PublishAheadOfPrint]

  • Validating the sampling depth and reducing sequencing errors are critical for studies of viral populations using next generation sequencing (NGS). We previously described the use of Primer ID to tag each viral RNA template with a block of degenerate nucleotides in the cDNA primer. We now show that low abundance Primer IDs (offspring Primer IDs) are generated due to PCR/sequencing errors. These artifactual Primer IDs can be removed using a cut-off model for the number of reads required to make a template consensus sequence. We have modeled the fraction of sequences lost due to Primer ID resampling. For a typical sequencing run less than 10% of raw reads are lost to offspring Primer ID filtering and resampling. The remaining raw reads are used to correct for PCR resampling and sequencing errors. We also demonstrate that Primer ID reveals bias intrinsic to PCR, especially at low template input or utilization. cDNA synthesis and PCR convert around 20% of RNA templates into recoverable sequences, and 30X sequence coverage recovers most of these template sequences. We have directly measured the residual error rate to be around 1 in 10,000 nucleotides. We use this error rate and the Poisson distribution to define the cut-off to identify pre-existing drug resistance mutations at low abundance in an HIV-infected subject. Collectively these studies show that greater than 90% of the raw sequence reads can be used to validate template sampling depth and to dramatically reduce the error rate in assessing a genetically diverse viral population using NGS.

    Importance: While NGS has revolutionized sequencing strategies it suffers from serious limitations in defining sequence heterogeneity in a genetically diverse population such as HIV-1 due to PCR resampling and PCR/sequencing errors. The Primer ID approach reveals the true sampling depth and greatly reduces errors. Knowing sampling depth allows the construction of a model of how to maximize the recovery of sequences from input templates and reduce resampling of the Primer ID so that appropriate multiplexing can be included in the experimental design. With defined sampling depth and measured error rate we are able to assign cut-offs for the accurate detection of minority variants in viral populations. This approach allows the power of NGS to be realized without having to guess about sampling depth and ignoring the problem of PCR resampling while also being able to correct most of the errors in the dataset.

  • HSV-1 Reactivates from Autonomic Ciliary Ganglia Independently from Sensory Trigeminal Ganglia to Cause Recurrent Ocular Disease [PublishAheadOfPrint]

  • Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) establish latency in sensory and autonomic neurons after ocular or genital infection, but their recurrence patterns differ. HSV-1 reactivates from latency to cause recurrent orofacial disease and while HSV-1 also causes genital lesions, HSV-2 recurs more efficiently in the genital region and rarely causes ocular disease. Mechanisms regulating these anatomical preferences are unclear. To determine whether differences in latent infection and reactivation in autonomic ganglia contribute to differences in HSV-1 and HSV-2 anatomical preferences for recurrent disease, we compared HSV-1 and HSV-2 clinical disease, acute and latent viral loads, and viral gene expression, in sensory trigeminal and autonomic superior cervical and ciliary ganglia in a guinea pig ocular infection model. HSV-2 produced more severe acute disease, correlating with higher viral DNA loads in sensory and autonomic ganglia, as well as higher levels of thymidine kinase expression, a marker of productive infection, in autonomic ganglia. HSV-1 reactivated in ciliary ganglia, independently from trigeminal ganglia, to cause more frequent recurrent symptoms, while HSV-2 replicated simultaneously in autonomic and sensory ganglia to cause more persistent disease. While both HSV-1 and HSV-2 expressed the latency-associated transcript (LAT) in the trigeminal and superior cervical ganglia, only HSV-1 expressed LAT in ciliary ganglia, suggesting that HSV-2 is not reactivation competent or does not fully establish latency in ciliary ganglia. Thus, differences in replication and viral gene expression in autonomic ganglia may contribute to differences in HSV-1 and HSV-2 acute and recurrent clinical disease.

    IMPORTANCE Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) establish latent infections, from which the viruses reactivate to cause recurrent disease throughout the life of the host. However, the viruses exhibit different manifestations and frequencies of recurrent disease. HSV-1 and HSV-2 establish latency in sensory and autonomic ganglia. However, autonomic ganglia are more responsive to stimuli associated with recurrent disease in humans, such as stress and hormone fluctuations, suggesting that autonomic ganglia may play an important role in recurrent disease. We show that HSV-1 can reactivate from autonomic ganglia, independently from sensory ganglia, to cause recurrent ocular disease symptoms. We found no evidence that HSV-2 could reactivate from autonomic independently from sensory ganglia after ocular infection, but replicated in both ganglia simultaneously to cause persistent disease. Thus, viral replication and reactivation in autonomic ganglia contributes to different clinical disease manifestations of HSV-1 and HSV-2 after ocular infection.

  • Thioredoxin 2 is a Novel E2-Interacting Protein that Inhibits the Replication of Classical Swine Fever Virus [PublishAheadOfPrint]

  • The E2 protein of classical swine fever virus (CSFV) is an envelope glycoprotein that is involved in virus attachment and entry. To date, the E2-interacting cellular proteins and their involvement in viral replication have been poorly documented. In this study, thioredoxin 2 (Trx2) was identified to be a novel E2-interacting partner using yeast two-hybrid screening from a porcine macrophage cDNA library. Trx2 is a mitochondria-associated protein that participates in diverse cellular events. The Trx2-E2 interaction was further confirmed by GST pulldown, in situ proximity ligation and laser confocal assays. The thioredoxin domain of Trx2 and the asparagine at position 37 (N37) in the E2 protein were shown to be critical for the interaction. Silencing of the Trx2 expression in PK-15 cells by small interfering RNAs significantly promotes CSFV replication, and conversely, overexpression of Trx2 markedly inhibits viral replication of the wild-type (wt) CSFV and to a greater extent that of the CSFV N37D mutant that is defective in binding Trx2. The wt CSFV but not the CSFV N37D mutant was shown to reduce the Trx2 protein expression in PK-15 cells. Furthermore, we demonstrated that Trx2 increases nuclear factor-kappaB (NF-B) promoter activity by promoting the nuclear translocation of the p65 subunit of NF-B. Notably, activation of the NF-B signaling pathway induced by tumor necrosis factor (TNF)-aalpha; significantly inhibits CSFV replication in PK-15 cells, whereas blocking the NF-B activation in Trx2-overexpressing cells no longer suppresses CSFV replication. Taken together, our findings reveal that Trx2 inhibits CSFV replication via the NF-B signaling pathway.

    IMPORTANCE Thioredoxin 2 (Trx2) is a mitochondria-associated protein that participates in diverse cellular events, such as antioxidative and antiapoptotic processes and the modulation of transcription factors. However, little is known about the involvement of Trx2 in viral replication. Here, we investigated, for the first time, the role of Trx2 in the replication of classical swine fever virus (CSFV), a devastating pestivirus of pigs. By knockdown and overexpression, we showed that Trx2 negatively regulates CSFV replication. Notably, we demonstrated that Trx2 inhibits CSFV replication by promoting the nuclear translocation of the p65 subunit of NF-B, a key regulator of the host's innate immunity and inflammatory response. Our findings reveal a novel role of Trx2 in the host's antiviral response and provide new insights into the complex mechanisms by which CSFV interacts with the host cell.

  • Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling molecules Rab11 and Rab22 [PublishAheadOfPrint]

  • Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. Here we used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASHnndash;VPEF/FAM21nndash;retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm.

    IMPORTANCE Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into PI3P-positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. Here we dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We showed that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There the complex executes endosomal membrane fission and cargo sorting to the Rab11-positive and Rab22-positive recycling pathway, resulting in membrane fusion and virus core uncoating in the cytoplasm.

  • New insights into the understanding of hepatitis C virus entry and cell-to-cell transmission by using the ionophore Monensin A [PublishAheadOfPrint]

  • In our study, we characterized the effect on the Hepatitis C Virus (HCV) life cycle of Monensin, an ionophore that is known to raise the intracellular pH. We show that Monensin inhibits HCV entry in a pangenotypic and dose-dependent manner. Monensin induces an alkalization of intracellular organelles, leading to an inhibition of the fusion step between viral and cellular membranes. Interestingly, we demonstrated that HCV cell-to-cell transmission is dependent on the vesicular pH. Under the selective pressure of Monensin, we selected a Monensin-resistant virus which has evolved to use a new entry route that is partially pH- and clathrin-independent. Characterization of this mutant led to the identification of two mutations in envelope proteins, Y297H in E1 and I399T in the hypervariable region 1 (HVR1) of E2, which confer resistance to Monensin and thus allow HCV to use a pH-independent entry route. Interestingly, the I399T mutation introduces a N-glycosylation site within HVR1, increases the density of virions and their sensitivity to neutralization with anti-apolipoprotein E (ApoE) antibodies, suggesting that this mutation likely induces conformational changes in HVR1 that in turn modulate the association with ApoE. Strikingly, the I399T mutation dramatically reduces HCV cell-to-cell spread. In conclusion, we identified a mutation in HVR1 that overcomes the vesicular pH-dependence, modifies the biophysical properties of particles and drastically reduces cell-to-cell transmission, indicating that the regulation by HVR1 of particle association with ApoE might control the pH-dependence of cell-free and cell-to-cell transmission. Thus, HVR1 and ApoE are critical regulators of HCV propagation.

    IMPORTANCE Although several cell surface proteins have been identified as entry factors for Hepatitis C Virus (HCV), precise mechanisms regulating its transmission into hepatic cells are still unclear. In our study, we used Monensin A, an ionophore that is known to raise the intracellular pH, and demonstrated that cell-free and cell-to-cell transmission are both pH-dependent processes. We generated Monensin-resistant viruses that displayed different entry routes and biophysical properties. Thanks to these mutants, we highlighted the importance of the hypervariable region 1 (HVR1) in E2 envelope protein for the association of particles with apolipoprotein E, which in turn might control the pH-dependency of cell-free and cell-to-cell transmission.

  • Distinct immune responses in resistant and susceptible strains of mice during neurovirulent alphavirus encephalomyelitis [PublishAheadOfPrint]

  • Susceptibility to alphavirus encephalomyelitis is dependent on a variety of factors including the genetic background of the host. Neuroadapted Sindbis virus (NSV) causes uniformly fatal disease in adult C57BL/6 (B6) mice, but adult BALB/c (Bc) mice recover from infection. In B6 mice fatal encephalomyelitis is immune-mediated rather than a direct result of virus infection. To identify the immunological determinants of host susceptibility to fatal NSV-induced encephalomyelitis, we compared virus titers and immune responses in adult B6 and Bc mice infected intranasally with NSV. B6 mice had higher levels of virus replication higher levels of type I interferon and slower virus clearance compared to Bc mice. B6 mice had more neuronal apoptosis, more severe neurologic disease and higher mortality than Bc mice. B6 mice had more infiltration of inflammatory cells and higher brain levels of IL1b, IL-6, TNFa, Csf2 and CCL2 mRNAs and IL-6, TNFaalpha;, IFN- and CCL2 protein than Bc mice. However, Bc mice had more brain antibody at day 7 and a higher percentage of CD4+ T cells. CD4+ T cells in the brains of Bc mice included fewer Th17 cells and more Tregs producing IL-10 than B6 mice accompanied by higher levels of Il2 and Cxcl10 mRNAs. In the absence of IL-10, resistant Bc mice became susceptible to fatal encephalomyelitis after NSV infection. These studies demonstrate the importance of the immune response and its regulation in determining host survival during alphavirus encephalomyelitis.

    IMPORTANCE Mosquito-borne alphavirus infections are an important cause of encephalomyelitis in humans. The severity of disease is dependent both on the strain of the virus and the age and genetic background of the host. A neurovirulent strain of Sindbis virus causes immune-mediated fatal encephalomyelitis in adult C57BL/6 mice, but not in BALB/c mice. To determine the host-dependent immunological mechanisms underlying the differences in susceptibility between these two strains of mice, we compared their immune responses to infection. Resistance to fatal disease in BALB/c mice was associated with better antibody responses, more rapid virus clearance, fewer Th17 cells and more potent regulatory T cell responses than occurred in susceptible C57BL/6 mice. In the absence of interleukin-10, a component of the regulatory immune response, resistant mice became susceptible to lethal disease. This study demonstrates the importance of the immune response and its regulation for host survival during alphavirus encephalomyelitis.

  • Vaccine Induced Epitope Specific Antibodies to SIVmac239 Envelope Are Distinct from Those Induced to the HIV-1 Envelope in Non-Human Primates [PublishAheadOfPrint]

  • To evaluate antibody specificities induced by SIV versus HIV-1 envelope antigens in non-human primate (NHP), we profiled binding antibody responses to linear epitopes in NHP studies with HIV-1 or SIV immunogens. We found that overall HIV-1 Env IgG responses were dominated by V3, notably with the exception of responses to the RV144 vaccine strain immunogen (A244 Env) that was dominated by V2, whereas the anti-SIVmac239 Env responses were dominated by V2 regardless of vaccine regimens.

  • Innovations in Undergraduate Science Education: Going Viral [PublishAheadOfPrint]

  • Bacteriophage discovery and genomics provides a powerful and effective platform for integrating missions in research and education. Implementation as the Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program facilitates a broad impact by including a diverse array of schools, faculty, and students. The program generates new insights into the diversity and evolution of the bacteriophage population and presents a model for introducing first-year undergraduate students to discovery-based research experiences.

  • Dark matter of the biosphere: The amazing world of bacteriophage diversity [PublishAheadOfPrint]

  • Bacteriophages are the most abundant biological entities in the biosphere, and this dynamic and old population is not surprisingly highly diverse genetically. Relative to bacterial genomics, phage genomics has advanced slowly, and a higher resolution picture of the phagosphere is only just emerging. This view reveals substantial diversity even among phages known to infect a common host strain, but the relationships are complex with mosaic genomic architectures generated by illegitimate recombination over a long period of evolutionary history.

  • Genome Sequencing and Analysis of Geographically Diverse Clinical Isolates of Herpes Simplex Virus 2 [PublishAheadOfPrint]

  • Herpes simplex virus 2 (HSV-2), the principal causative agent of recurrent genital herpes, is a highly prevalent viral infection worldwide. Limited information is available on the amount of genomic DNA variation between HSV-2 strains because there have been only two genomes determined, the HG52 laboratory strain and the newly sequenced SD90e low-passage clinical isolate strain, each from a different geographical area. In this study we report the near-complete genome sequences of 34 HSV-2 low passage and laboratory strains, of which 14 were collected in Uganda, 1 in South Africa, 11 in the USA and 8 in Japan. Our analyses of these genomes demonstrated remarkable sequence conservation, regardless of geographic origin, with maximum nucleotide divergence between strains being 0.4% across the genome. In contrast, prior studies indicated that HSV-1 genomes exhibit more sequence diversity as well as geographical clustering. Additionally, unlike HSV-1, little viral recombination between HSV-2 strains could be substantiated. These results are interpreted in light of HSV-2 evolution, epidemiology, and pathogenesis. Finally, the newly generated sequences more closely resemble the low passage SD90e than HG52, supporting the use of the former as the new reference genome of HSV-2.

    IMPORTANCE Herpes simplex virus 2 (HSV-2) is a causative agent of genital and neonatal herpes. Therefore, knowledge of its DNA genome and genetic variability are central to preventing and treating genital herpes. However, only two full-length HSV-2 genomes have been reported. In his study we have sequenced 34 additional HSV-2 low passage and laboratory viral genomes and have initiated analysis of the genetic diversity of HSV-2 strains from around the world. The analysis of these genomes will facilitate research aimed at vaccine development, diagnosis, and the evaluation of clinical manifestations and transmission of HSV-2. This information will also contribute to our understanding of HSV evolution.

  • Global Diversity Within and Between Human Herpes Virus 1 and 2 Glycoproteins [PublishAheadOfPrint]

  • Human herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) are large-genome DNA viruses that establish a persistent infection in sensory neurons and commonly manifest with recurring oral or genital erosions that transmit virus. HSV encodes twelve predicted glycoproteins that serve various functions including cellular attachment, entry, and egress. Glycoprotein G is currently the target of an antibody test to differentiate HSV-1 from HSV-2; however, this test has shown reduced capacity to differentiate HSV strains in East Africa. Until the recent availability of 26 full-length HSV-1 and 36 full-length HSV-2 sequences, minimal comparative information was available for these viruses. In this study we use a variety of sequence analysis methods to compare all available sequence data for HSV-1 and HSV-2 glycoproteins, using viruses isolated in Europe, Asia, North America, the Republic of South Africa and East Africa. We find numerous differences in diversity, nonsynonymous/synonymous substitution rates and recombination rates between HSV-1 glycoproteins and their HSV-2 counterparts. Phylogenetic analysis revealed that while most global HSV-2 glycoprotein G sequences did not form cluster within or between continents, one clade (supported at 60.5%) contained 37% of the African sequences analyzed. Accordingly, sequences from this African subset contained unique amino acid signatures, not only in glycoprotein G, but also in glycoproteins I and E, which may account for the failure of sensitive antibody tests to distinguish HSV-1 from HSV-2 in some African individuals. Consensus sequences generated in the study can be used to improve diagnostic assays that differentiate HSV-1 from HSV-2 in global populations.

    Importance Human herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) are large DNA viruses associated with recurring oral or genital erosions that transmit virus. Up to twelve HSV-1 and HSV -2 glycoproteins are involved in HSV cell entry or are required for viral spread in animals, albeit some are dispensable for replication in vitro. The recent availability of a comparable number of full-length HSV-1 and HSV-2 sequences enabled comparative analysis of gene diversity of glycoproteins within and between HSV types. Overall, we found less glycoprotein sequence diversity within HSV-2 than within the HSV-1 strains studied, while at the same time, several HSV-2 glycoproteins were evolving under less selective pressure. Because HSV glycoproteins are the focus of antibody tests to detect and differentiate between infections with the two strains and are constituents of vaccines in clinical stage development, these findings will aid in refining the targets for diagnostic tests and vaccines.

  • Characterization and Implementation of a Diverse SIVsm Envelope Panel in the Assessment of Neutralizing Antibody Breadth Elicited in Rhesus Macaques by Multi-Modal Vaccines Expressing the SIVmac239 Envelope. [PublishAheadOfPrint]

  • Antibodies that can neutralize diverse viral strains are likely to be an important component of a protective HIV-1 vaccine. To this end, pre-clinical SIV-based non-human primate immunization regimens have been designed to evaluate and enhance antibody-mediated protection. However, these trials often rely on a limited selection of SIV strains with extreme neutralization phenotypes to assess vaccine-elicited antibody activity. To mirror the viral panels used to assess HIV-1 antibody breadth, we created and characterized a novel panel of 14 genetically and phenotypically diverse SIVsm Envs. To assess the utility of this panel, we characterized the neutralizing activity elicited by four SIVmac239 envelope-expressing DNA/MVA and protein-based vaccination regimens that included the immunomodulatory adjuvants GM-CSF, Toll-like receptor (TLR) ligands, and CD40 ligand. The SIVsm Env panel exhibited a spectrum of neutralization sensitivity to SIV-infected plasma pools and monoclonal antibodies, allowing categorization into three tiers. Pooled sera from 91 rhesus macaques immunized in the four trials consistently neutralized only the highly sensitive tier 1a SIVsm Envs, regardless of the immunization regimen. The inability of vaccine-mediated antibodies to neutralize the moderately resistant tier 1b and tier 2 SIVsm Envs defined here suggests that those antibodies were directed towards epitopes that are not accessible on most SIVsm Envs. To achieve a broader and more effective neutralization profile in pre-clinical vaccine studies that is relevant to known features of HIV-1 neutralization, more emphasis should be placed on optimizing the Env immunogen, as adjuvants do not appear to supersede the neutralizing antibody profile determined by the immunogen.

    Importance Many in the HIV/AIDS vaccine field believe that the ability to elicit broadly neutralizing antibodies capable of blocking genetically diverse HIV-1 variants is a critical component of a protective vaccine. Various SIV-based non-human primate vaccine studies have investigated ways to improve antibody-mediated protection against a heterologous SIV challenge, including administering adjuvants that might stimulate greater neutralization breadth. Using a novel SIV neutralization panel and samples from four rhesus macaque vaccine trials designed for cross-comparison, we show that different regimens expressing the same SIV envelope immunogen consistently elicit antibodies that neutralize only the very sensitive tier 1a SIV variants. The results argue that the neutralizing antibody profile elicited by a vaccine is primarily determined by the envelope immunogen and is not substantially broadened by including adjuvants, resulting in the conclusion that the envelope immunogen itself should be the primary consideration in efforts to elicit antibodies with greater neutralization breadth.

  • Functional Analysis of the 60 Nucleotide Duplication in the Respiratory Syncytial Virus Buenos Aires Strain Attachment Glycoprotein [PublishAheadOfPrint]

  • There are two subgroups of respiratory syncytial virus (RSV), A and B, and within each subgroup isolates are further divided into clades. Several years ago, multiple subgroup B isolates were described which contained a duplication of 60 nucleotides in the G gene. These isolates were given a new clade designation of BA based on the site of isolation, Buenos Aires, Argentina. BA RSV strains have since become the predominant circulating clade of RSV B viruses. We hypothesized that the duplicated region in G serves to enhance the function of G in the virus life cycle. We generated recombinant viruses that express a consensus BA G gene, or a consensus BA G gene lacking the duplication (Gdup). We determined that the duplicated region functions during virus attachment to cells. Additionally, we showed that in vitro, the virus containing the duplication has a fitness advantage compared to the virus without the duplication. Our data demonstrate that the duplicated region in the BA strain G protein augments virus attachment and fitness.

    Importance Respiratory syncytial virus (RSV) is an important pathogen for infants, for which there is no vaccine. Different strains of RSV circulate from year to year, and the predominating strains change over time. Subgroup B RSV strains with a duplication in the attachment glycoprotein (G) emerged and then became the dominant B genotype. We found that a recombinant virus harboring the duplication bound more efficiently to cells and was more fit than a recombinant strain lacking the duplication. Our work advances a mechanism for an important natural RSV mutation.

  • Human blood circulating basophils capture HIV-1 and mediate viral trans-infection of CD4+ T cells [PublishAheadOfPrint]

  • Cell-associated HIV-1 infection has been proposed to play a pivotal role in the spread of HIV-1 infection. Granulocytes are a category of white blood cells, comprising mainly basophils, neutrophils and eosinophils, and participate in various inflammatory reactions and defense against pathogens. Here, we investigated the role of human blood granulocytes in the dissemination of HIV-1. These cells were found to express a variety of HIV-1 attachment factors (HAFs). Basophils expressed HAFs DC-SIGN, DCIR, HSPG and aalpha;4bbeta;7 integrin, and mediated the most efficient capture of HIV-1 on the cell surface. Neutrophils were found to express DCIR, and demonstrated limited efficiency of viral capture. Eosinophils expressed aalpha;4bbeta;7 integrin, but exhibited little or no virus-binding capacity. . Intriguingly, following direct contact with CD4+ T cells, viruses harbored on the surface of basophils-were transferred to T cells. The contact between basophils and CD4+ T cells and formation of infectious synapses appeared necessary for efficient HIV-1 spread. In HIV-1 infected individuals, the frequency of basophils remained fairly stable over the course of disease, regardless of CD4+ T depletion or the emergence of AIDS-associated opportunistic infections. . Collectively, our results provide novel insights into the roles of granulocytes, particularly basophils, in HIV-1 dissemination. Thus, strategies designed to prevent basophil-mediated viral capture and transfer may be developed into a new form of therapy.

    IMPORTANCE Cell-associated HIV-1 infection has been proposed to play a pivotal role in the spread of HIV-1 infection. Here, we demonstrate that human blood circulating granulocytes, particularly basophils, can capture HIV-1 and mediate viral trans-infection of CD4+ T cells. The expression of a variety of HIV-1 attachment factors, such as the C-type lectins, etc., facilitates viral capture and transfer. Intriguingly, the frequency of basophils remains fairly stable during the course of disease, in patients with different levels of CD4+ T count. Our results provide novel insights into the roles of granulocytes, particularly basophils, in HIV-1 dissemination. We suggest that strategies designed to prevent basophils-mediated viral capture and transfer may be a new direction for the development of anti-HIV therapy.

  • Immunization with Immune Complexes Modulates the Fine-Specificity of Antibody Responses to a Flavivirus Antigen [PublishAheadOfPrint]

  • The antibody response to proteins may be modulated by the presence of pre-existing antigen-specific antibodies and the formation of immune complexes (ICs). Effects such as a general increase or decrease of the response as well as epitope-specific phenomena have been described. In this study, we investigated influences of IC-immunization on the fine-specificity of antibody responses in a structurally well-defined system, using the envelope protein (E) of tick-borne encephalitis (TBE) virus as an immunogen. TBE virus occurs in Europe and Asia and nndash; together with yellow fever, dengue, West Nile and Japanese encephalitis viruses nndash; represents one of the major human-pathogenic flaviviruses. Mice were immunized with a dimeric soluble form of E (sE) alone or in complex with monoclonal antibodies specific for each of the three domains of E, and the antibody response induced by these ICs was compared to that after immunization with sE alone. Immunoassays using recombinant domains and domain combinations of TBE virus sE as well as the distantly related West Nile virus sE allowed the dissection and quantification of antibody subsets present in post-immunization sera, thus generating fine-specificity patterns of the polyclonal responses. There were substantially different responses with two of the ICs and could be mechanistically related to (i) epitope-shielding and (ii) antibody-mediated structural changes leading to dissociation of the sE dimer. The phenomena described may also be relevant for polyclonal responses upon secondary infections and/or booster immunizations and affect antibody responses in an individual-specific way.

    IMPORTANCE Infections with flaviviruses such as yellow fever, dengue, Japanese encephalitis, West Nile and tick-borne encephalitis (TBE) viruses pose substantial public health problems in different parts of the world. Antibodies to the viral envelope protein E induced by natural infection or vaccination were shown to confer protection from disease. Such antibodies can target different epitopes in E and the fine-specificities of polyclonal responses can vary between individuals. We conducted a mouse immunization study with TBE E protein alone or complexed to monoclonal antibodies specific for each of the three protein domains. We demonstrate that phenomena such as epitope shielding and antibody-induced structural changes can profoundly influence the fine-specificity of antibody responses to the same immunogen. The study thus provides important new information on the potential immunomodulatory role of pre-existing antibodies in a flavivirus system that can be relevant for understanding individual-specific factors influencing antibody responses in sequential flavivirus infections and/or immunizations.

  • Population Bottlenecks and Pathogen Extinction: "Make this Everyone's Mission to Mars, Including Yours" [PublishAheadOfPrint]

  • Kapusinsky et al. report that host population bottlenecks may result in pathogen extinction, which provides a compelling argument for an alternative approach to vaccination for the control of virus spread. By comparing the prevalence levels of three viral pathogens in two populations of African green monkeys (AGMs) of sabaeus species from Africa and Caribbean Islands, they convincingly show that a major host bottleneck resulted in the eradication of select pathogens from a given host.

  • Identification of influenza A virus PB2 residues involved in enhanced polymerase activity and virus growth in mammalian cells at low temperature [PublishAheadOfPrint]

  • Mutations in the polymerase genes are known to play a major role in avian influenza virus adaptation to mammalian hosts. Despite having avian origin PA and PB2, the 2009 pandemic H1N1 virus (pH1N1) can replicate well in mammalian respiratory tracts, suggesting that these proteins have acquired mutations for efficient growth in humans. We have previously shown that PA from the pH1N1 virus A/California/04/09 (Cal) strongly enhances activity of an otherwise avian polymerase complex derived from A/chicken/Nanchang/3-120/01 (Nan) in mammalian cells. However, this enhancement was observed at 37ddeg;C but not at the lower temperature of 34ddeg;C. An additional introduction of Cal PB2 enhanced activity at 34ddeg;C, suggesting the presence of unidentified residues in Cal PB2 that are required for efficient growth at low temperature. Here, we sought to determine the key PB2 residues which confer enhanced polymerase activity and virus growth in human cells at low temperature. Using a reporter gene assay, we identified novel mutations, PB2 V661A and V683T/A684S, which are involved in enhanced Cal polymerase activity at low temperature. The PB2 T271A mutation, which we previously reported, also contributed to enhanced activity. Growth of recombinant Cal containing PB2 with Nan residues 271T/661V/683V/684A was strongly reduced in human cells compared to wild type virus at low temperature. Among the four residues, 271A and 684S are conserved in human and pH1N1 viruses, but not in avian viruses, suggesting an important role in mammalian adaptation of pH1N1 virus.

    Importance The PB2 protein plays a key role in host adaptation, cold sensitivity and pathogenesis of influenza A virus. Despite containing an avian origin PB2 lacking the mammalian adaptive mutations 627K or 701N, pH1N1 influenza strains can replicate efficiently in the low temperature upper respiratory tract of mammals, suggesting the presence of unknown mutations in the pH1N1 PB2 protein responsible for its low temperature adaptation. Here, in addition to PB2 271A, which has been shown to increase polymerase activity, we identified novel PB2 residues 661A and 683T/684S in pH1N1 which confer enhanced polymerase activity and virus growth in mammalian cells especially at low temperature. Our findings suggest that the presence of these PB2 residues contributes to efficient replication of the pH1N1 virus in the upper respiratory tract, which resulted in efficient human-to-human transmission of this virus.

  • Discovery of dengue virus NS4B inhibitors [PublishAheadOfPrint]

  • The four serotypes of dengue virus (DENV-1 to -4) represent the most prevalent mosquito-borne viral pathogen in humans. No clinically approved vaccine or antiviral is currently available for DENV. Here we report a spiropyrazolopyridone compound that potently inhibits DENV both in vitro and in vivo. The inhibitor was identified through screening a 1.8-million compound library using a DENV-2 replicon assay. The compound selectively inhibits DENV-2 and -3 (EC5010-80 nM), but not DENV-1 and -4 (EC50 ggt;20 mmu;M). Resistance analysis showed that a mutation at amino acid 63 of DENV-2 NS4B (a non-enzymatic transmembrane protein and a component of viral replication complex) could confer resistance to compound inhibition. Genetic studies demonstrate that variations at amino acid 63 of viral NS4B are responsible for the selective inhibition of DENV-2 and -3. Medicinal chemistry improved the physicochemical properties of the initial "hit" (compound-1), leading to compound-14a that has good in vivo pharmacokinetics. Treatment of DENV-2-infected AG129 mice with compound-14a suppressed viremia, even when the treatment started after viral infection. The results have proved the concept that inhibitors of NS4B could potentially be developed for clinical treatment of DENV infection. Compound-14a represents a potential preclinical candidate for treatment of DENV-2 and -3 infected patients.

    IMPORTANCE Dengue virus (DENV) threatens up to 2.5 billion people and is now spreading in many regions in the world where it was not previously endemic. While there are several promising vaccine candidates in clinical trials, there are yet approved vaccines or antivirals available. Here, we describe the identification and characterization of spiropyrazolopyridone as a novel inhibitor of DENV by targeting viral NS4B protein. The compound potently inhibits two of the four serotypes of DENV (DENV-2 and -3) both in vitro and in vivo. Our results validate, for the first time, that NS4B inhibitors could potentially be developed for antiviral therapy for treatment of DENV infection in humans.

  • Sequence-specific modifications enhance the broad spectrum antiviral response activated by RIG-I agonists [PublishAheadOfPrint]

  • The cytosolic RIG-I receptor plays a pivotal role in the initiation of the immune response against RNA virus infection by recognizing short 5rrsquo; -triphosphate (5rrsquo; ppp) containing viral RNA and activating the host antiviral innate response. In the present study, we generated novel 5rrsquo; ppp RIG-I agonists of varied length, structure, and sequence and evaluated the generation of the antiviral and inflammatory response in human epithelial A549 cells, human innate immune primary cells, and murine models of influenza and chikunguna viral pathogenesis. A 99 nucleotide, uridine-rich, hairpin 5rrsquo; -pppRNA termed M8 stimulated an extensive and robust interferon response compared to other modified 5rrsquo; pppRNA structures, RIG-I aptamers, or poly (I:C). Interestingly, manipulation of the primary RNA sequence alone was sufficient to modulate antiviral activity and inflammatory response, in a manner dependent exclusively on RIG-I, and independent of MDA5 and TLR3. Both prophylactic and therapeutic administration of M8 effectively inhibited influenza and dengue viral replication in vitro. Furthermore, multiple strains of influenza virus that were resistant to oseltamivir, an FDA-approved therapeutic treatment for influenza, were highly sensitive to inhibition by M8. Finally, prophylactic M8 treatment in vivo prolonged survival and reduced lung viral titers of mice challenged with influenza, as well as reduced chikungunya-associated foot swelling and viral load. Altogether, these results demonstrate that 5rrsquo; pppRNA can be rationally designed to achieve a maximal RIG-I-mediated protective antiviral response against human pathogenic RNA viruses.

    IMPORTANCE The development of novel therapeutics to treat human pathogenic RNA viral infections is an important goal that seeks to reduce spread of infection and to improve human health and safety. This study investigated the design of an RNA agonist with enhanced antiviral and inflammatory properties against influenza, dengue, and chikungunya viruses. A novel, sequence-dependent, uridine-rich RIG-I agonist generated a protective antiviral response in vitro and in vivo and was effective at concentrations 100-fold lower than prototype sequences or other RNA agonists, highlighting the robust activity and potential clinical use of the 5rrsquo; pppRNA against RNA virus infection. Altogether, the results identify a novel, sequence-specific RIG-I agonist as an attractive therapeutic candidate for the treatment of a broad range of RNA viruses, a pressing issue in which a need for new and more effective options persists.

  • A multi-antigenic DNA vaccine that induces broad HCV-specific T-cell responses in mice [PublishAheadOfPrint]

  • There are 3-4 million new hepatitis C virus (HCV) infections annually around the world, but no vaccine is available. Robust T-cell mediated responses are necessary for effective clearance of the virus and DNA vaccines result in a cell-mediated bias. Adjuvants are often required for effective vaccination, but during natural lytic viral infections damage associated molecular patterns (DAMPs) are released, which act as natural adjuvants. Hence, a vaccine that induces cell necrosis and releases DAMPs will result in cell mediated immunity (CMI), similar to that resulting from natural lytic viral infection. We have generated a DNA vaccine with the ability to elicit strong CMI against the HCV non-structural (NS) proteins (3, 4A, 4B, and 5B) by encoding a cytolytic protein, perforin (PRF), and the antigens on a single plasmid. We examined the efficacy of the vaccines in C57BL/6 mice, as determined by IFN- EliSpot, cell proliferation studies, and intracellular cytokine production. Initially, we showed that encoding the NS4A protein in a vaccine which encoded only NS3 reduced the immunogenicity of NS3, whereas including PRF increased NS3 immunogenicity. In contrast, the inclusion of NS4A increased the immunogenicity of the NS3, NS4B andNS5B proteins, when encoded in a DNA vaccine that also encoded PRF. Finally, vaccines that also encoded PRF elicited similar levels of CMI against each protein after vaccination with DNA encoding NS3, NS4A, NS4B and NS5B compared with mice vaccinated with DNA encoding only NS3 or NS4B/5B. Thus, we have developed a promising "multi-antigen" vaccine that elicits robust CMI.

    Importance Since their development, vaccines have reduced the global burden of disease. One strategy for vaccine development is to use commercially viable DNA technology, which has the potential to generate robust immune responses. Hepatitis C virus causes chronic liver infection and is a leading cause of liver cancer. To date, no vaccine is currently available, treatment is costly and often results in side effects, limiting the number of patients who are treated. Despite recent advances in treatment, prevention remains the key to efficient control and elimination of this virus. Here, we describe a novel DNA vaccine against hepatitis C virus that is capable of inducing robust cell mediated immune responses in mice and is a promising vaccine candidate for humans.

  • The baculovirus anti-apoptotic p35 protein functions an inhibitor of the host RNAi antiviral response [PublishAheadOfPrint]

  • RNA interference (RNAi) is considered as an ancient antiviral-defense in diverse organisms including insects. Virus infections generate double-strand RNAs (dsRNA) that trigger the RNAi machinery to process dsRNAs into virus-derived siRNAs (vsiRNAs) which target virus genomes, mRNAs or replication intermediates. Viruses, in turn, have evolved suppressors of RNAi (VSR) to counter hosts antiviral RNAi. Following recent discoveries that insects mount the RNAi response against DNA viruses, in this study, we found that Autographa californica MNPV (AcMNPV) infection similarly induces an RNAi response in Spodoptera frugiperda cells by generating a large number of vsiRNAs post-infection. Interestingly, we found that AcMNPV expresses a potent VSR to counter the RNAi. The viral p35 gene, which is well known as an inhibitor of apoptosis, was found responsible for suppression of RNAi in diverse insect and mammalian cells. The VSR activity of p35 was further confirmed by a p35-null AcMNPV that did not suppress the response. In addition, our results showed that the VSR activity is not due to inhibition of dsRNA cleavage by Dicer-2, but acts downstream in the RNAi pathway. Furthermore, we found that the VSR activity is not linked to the anti-apoptotic activity of the protein. Overall, our results provide evidence for the existence of VSR activity from a double-stranded DNA virus, and identify the responsible gene, which is involved in inhibition of RNAi as well as apoptosis.

    Importance Our findings demonstrate the occurrence of an insect RNAi response against a baculovirus (AcMNPV) that is highly utilized in microbial control, biological and biomedical research, and protein expression. Moreover, our investigations led to the identification of a viral suppressor of RNAi activity and the gene responsible for the activity. Notably, the gene is also a potent inhibitor of apoptosis. The outcomes signify the dual role of a virus-encoded protein to nullify two key antiviral responses, apoptosis and RNAi.

  • Dengue virus inhibition of autophagic flux and dependency of viral replication on proteasomal degradation of the autophagy receptor p62 [PublishAheadOfPrint]

  • Autophagic flux involves formation of autophagosomes and their degradation by lysosomes. Autophagy can either promote or restrict viral replication. In the case of Dengue virus (DENV) several studies report that autophagy supports the viral replication cycle, and describe an increase of autophagic vesicles (AVs) following infection. However, it is unknown how autophagic flux is altered to result in increased AVs. To address this question, and gain insight into the role of autophagy during DENV infection, we established an unbiased, image-based flow cytometry approach to quantify autophagic flux under normal growth conditions and in response to activation by nutrient deprivation or the mTOR inhibitor Torin1. We found that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Early after infection, basal and activated autophagic flux was enhanced. However, during established replication, basal and Torin1-activated autophagic flux was blocked, while autophagic flux activated by nutrient deprivation was reduced, indicating a block to AV formation and reduced AV degradation capacity. During late infection AV levels increased as a result of inefficient fusion of autophagosomes with lysosomes. Additionally, endo-lysosomal trafficking was suppressed, while lysosomal activities were increased. We further determined that DENV infection progressively reduced levels of the autophagy receptor SQSTM1/p62 via proteasomal degradation. Importantly, stable over-expression of p62 significantly suppressed DENV replication suggesting a novel role for p62 as viral restriction factor. Overall our findings indicate that in the course of DENV infection, autophagy shifts from a supporting to an anti-viral role, which is countered by DENV.

    IMPORTANCE Autophagic flux is a dynamic process starting with the formation of autophagosomes and ending with their degradation after fusion with lysosomes. Autophagy impacts the replication cycle of many viruses. However, thus far the dynamics of autophagy in case of Dengue virus (DENV) infections has not been systematically quantified. Therefore, we employed high-content, imaging-based flow cytometry to quantify autophagic flux and endo-lysosomal trafficking in response to DENV infection. We report that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Further, lysosomal activity was increased, but endo-lysosomal trafficking was suppressed confirming the block of autophagic flux. Importantly, we provide evidence that p62, an autophagy receptor, restrict DENV replication and was specifically depleted in DENV-infected cells via increased proteasomal degradation. These results suggest that during DENV infection autophagy shifts from a pro- to an antiviral cellular process, which is counteracted by the virus.

  • The V1V2 Region of HIV-1 gp120 Forms a Five-Stranded Beta Barrel [PublishAheadOfPrint]

  • The first and second variable regions (V1V2) of gp120 play vital roles in the functioning of the HIV-1 envelope (Env). V1V2, which harbors multiple glycans and is highly sequence diverse, is located at the Env apex and stabilizes the trimeric gp120 spike on the virion surface. It shields V3 and the co-receptor binding sites in the pre-fusion state and exposes them upon CD4 binding. Data from the RV144 human HIV-1 vaccine trial suggested that antibody responses targeting V1V2 region inversely correlated with the risk of infection; thus, understanding the antigenic structure of V1V2 can contribute to vaccine design. We have determined a crystal structure of a V1V2-scaffold molecule (V1V2ZM109-1FD6) in complex with 830A, a human monoclonal antibody that recognizes a V1V2 epitope overlaping the integrin-binding motif in V2. The structure revealed that V1V2 assumes a five-stranded beta barrel structure with the region of the integrin-binding site (AAs 179-181) included in a "kink" followed by an extra beta strand. The complete barrel structure naturally presents the glycans on its outer surface and packs into its core conserved hydrophobic residues, including the Ile at position 181 which was highly correlated with vaccine efficacy in RV144. The epitope of monoclonal antibody 830A is discontinuous and composed of three segments: 1) Thr175, Tyr177, Leu179 and Asp180 at the kink overlapping the integrin-binding site; 2) Arg153 and Val154 in V1, and 3) Ile194 at the C-terminus of V2. This study thus provides the atomic details of the immunogenic "V2i epitope".

    IMPORTANCE Data from the RV144 Phase III clinical trial suggested that the presence of antibodies to the first and second variable regions (V1V2) of gp120 were associated with the modest protection afforded by the vaccine. V1V2 is a highly variable and immunogenic region of the HIV-1 surface glycoprotein gp120, and structural information about this region and its antigenic landscape will be crucial in the design of an effective HIV-1 vaccine. We have determined a crystal structure of V1V2 in complex with human mAb 830A and showed that mAb 830A recognizes a region overlapping the aalpha;4bbeta;7 integrin-binding site. We also show that V1V2 forms a 5-stranded beta barrel, an elegant structure allowing for sequence variations in the strand-connecting loops while preserving a conserved core.

  • Local virus extinctions following a host population bottleneck [PublishAheadOfPrint]

  • A small number of African green monkeys (AGM) were introduced from West Africa to the Caribbean in the 1600s. To determine the impact of this population bottleneck on the AGM virome, we used metagenomics to compare the viral nucleic acids in the plasma of 43 wild AGMs from West Africa (Gambia) to those in 44 AGMs from the Caribbean (St. Kitts and Nevis). Three viruses were detected in the blood of Gambian primates: Simian immunodeficiency virus (SIVagm in 42% of animals), a novel simian pegivirus (SPgVagm, 7%), and numerous novel simian anelloviruses (100%). Only anelloviruses were detected in the Caribbean AGMs with a prevalence and level of viral genetic diversity similar to that in the Gambian animals. A host population bottleneck therefore resulted in the exclusion of adult-acquired SIV and pegivirus from the Caribbean AGMs. The successful importation of AGM anelloviruses into the Caribbean may be the result of their early transmission to infants, very high prevalence in African AGMs, and frequent co-infections with highly distinct variants.

    IMPORTANCE The extent to which viruses can persist in small isolated populations depends on multiple hosts, viral, and environmental factors. The absence of prior infections may put an immunologically naive population at risk for disease outbreaks. Small or isolated populations originating from a small number of founder individuals are therefore considered at increased risk following contact with those with a greater variety of viruses. Here, we compared the plasma virome of West African green monkeys to that in their descendants after importation of a small number of animals to the Caribbean. A lentivirus and a pegivirus were found in the West African population but not in the Caribbean. Highly diverse anelloviruses were found on both continents. A small founder population, limited to infants and young juvenile monkeys, may have eliminated the sexually transmitted viruses from the Caribbean AGMs, while anelloviruses, acquired at an earlier age, persisted through the host population bottleneck.

  • Comparative Analysis of the Glycosylation Profiles of Membrane-anchored HIV-1 Envelope Glycoprotein Trimers and Soluble gp140 [PublishAheadOfPrint]

  • The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer, which consists of gp120 and gp41 subunits, is the focus of multiple strategies for vaccine development. Extensive Env glycosylation provides the HIV-1 virus with protection from the immune system, yet the glycans are also essential components of binding epitopes for numerous broadly neutralizing antibodies. Recent studies have shown that when Env is isolated from virions, its glycosylation profile differs significantly from that of soluble forms of Env (gp120 or gp140) predominantly used in vaccine discovery research. Here, we show that exogenous membrane-anchored Envs, which can be produced in large quantities in mammalian cells, also display a virion-like glycan profile, where the glycoprotein is extensively decorated with high-mannose glycans. Additionally, because we characterized the glycosylation with a high-fidelity profiling method, glycopeptide analysis, an unprecedented level of molecular detail regarding membrane Env glycosylation and its heterogeneity is presented. Each glycosylation site was characterized individually, with about 500 glycoforms characterized per Env protein. While many of the sites exclusively contain high-mannose glycans, others retain complex glycans, resulting in a glycan profile that cannot be currently mimicked on soluble gp120 or gp140 preparations. These site-level studies are important for understanding antibody/glycan interactions on native Env trimers. Additionally, we report a newly observed O-linked glycosylation site, T606, and we show that the full O-linked glycosylation profile of membrane-associated Env is similar to that of soluble gp140. These findings provide new insight into Env glycosylation and clarify key molecular-level differences between membrane-anchored Env and soluble gp140.

    Importance A vaccine that protects against human immunodeficiency virus (HIV-1) infection should elicit antibodies that bind to the surface envelope glycoproteins on the membrane of the virus. The envelope glycoproteins have an extensive coat of carbohydrates (glycans), some of which are recognized by virus-neutralizing antibodies and some of which protect the virus from neutralizing antibodies. We found that the HIV-1 membrane envelope glycoproteins have a unique pattern of carbohydrates, with many high-mannose glycans and also, in some places, complex glycans. This pattern was very different from the carbohydrate profile seen for a more easily produced, soluble version of the envelope glycoprotein. Our results provide a detailed characterization of the glycans on the natural membrane envelope glycoproteins of HIV-1, a carbohydrate profile that would be desirable to mimic with a vaccine.

  • Protective efficacy of recombinant Modified Vaccinia virus Ankara (MVA) delivering Middle East Respiratory Syndrome coronavirus spike glycoprotein [PublishAheadOfPrint]

  • Middle East Respiratory Syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans. We tested a recombinant MVA vaccine expressing full-length MERS-CoV spike glycoprotein (S) by immunizing BALB/c mice using either intramuscular or subcutaneous regimens. In all cases MVA-MERS-S induced MERS-CoV-specific CD8+ T-cells and virus-neutralizing antibodies. Vaccinated mice were protected against MERS-CoV challenge infection after transduction with the human dipeptidyl peptidase 4 receptor. This MERS-CoV infection model demonstrates the safety and efficacy of the candidate vaccine.

  • Involvement of the Rac1-IRSp53-Wave2-Arp2/3 signaling pathway in HIV-1 Gag particle release in CD4 T cells. [PublishAheadOfPrint]

  • During HIV-1 assembly, the Gag viral proteins are targeted and assemble at the inner leaflet of the cell plasma membrane. This process could modulate the cortical actin cytoskeleton, located underneath the plasma membrane, since actin dynamic is able to promote localized membrane reorganization. In addition, activated small RhoGTPases are known for regulating actin dynamics and membrane remodeling. Therefore, the modulation of such RhoGTPase activity and of F-actin by the Gag protein during viral particle formation was to be considered. Here, we thus studied the implication of the main Rac1, Cdc42 and RhoA small GTPases, and some of their effectors, in this process. The effect of siRNA-mediated Rho GTPases and their effectors silencing was analyzed on Gag localization, Gag membrane attachment and virus-like particle production by immunofluorescence coupled to confocal microscopy, membrane flotation assays and immunoblot assays, respectively. In parallel, the effect of Gag expression on Rac1 activation level and intracellular F-actin content was monitored in T-cells, respectively, by G-LISA, flow cytometry and fluorescence microscopy. Our results revealed the involvement of an activated Rac1 and of the IRSp53-Wave2-Arp2/3 signalling pathway in HIV-1 Gag membrane localization and particle release in T cells, as well as a role for actin branching and polymerization. And this was solely dependent on the Gag viral protein. In conclusion, our results highlight a new role for the Rac1-IRSp53-Wave2-Arp2/3 signalling pathway in the late steps of HIV-1 replication in CD4 T lymphocytes.

    Importance During HIV-1 assembly, the Gag proteins are targeted and assembled at the inner leaflet of the host cell plasma membrane. Gag interacts with specific membrane phospholipids that can also modulate the regulation of underneath cortical actin cytoskeleton dynamics. Actin dynamic can promote localized membrane reorganization and thus can be involved in facilitating Gag assembly and particle formation. Activated small RhoGTPases and effectors are regulators of actin dynamics and membrane remodeling. We thus studied the implication of the Rac1, Cdc42 and RhoA GTPases and their specific effectors on HIV-1 Gag membrane localization and viral particle release in T cells. Our results show that an activated Rac1 and the IRSp53-Wave2-Arp2/3 signalling pathway are involved in Gag plasma membrane localization and viral particle production. This current work uncovers a role for cortical actin through the activation of Rac1 and IRSp53/Wave2 signalling pathway in HIV-1 particle formation in CD4 T lymphocytes.

  • Promotion of Endoplasmic Reticulum-Associated Degradation of Pro-Cathepsin D by Human Herpesvirus 8-Encoded Viral Interleukin-6 [PublishAheadOfPrint]

  • The interleukin-6 homologue (vIL-6) of human herpesvirus 8 is implicated in viral pathogenesis due to its pro-proliferative, inflammatory, and angiogenic properties, effected through gp130 receptor signaling. In primary effusion lymphoma (PEL) cells, vIL-6 is latently expressed and essential for normal cell growth and viability. This is mediated partly via suppression of proapoptotic cathepsin D (CatD) via co-complexing of the ER-localized CatD precursor, pro-CatD (pCatD), and vIL-6 with the previously uncharacterized ER membrane protein vitamin K epoxide reductase complex subunit 1 variant-2 (VKORC1v2). vIL-6 suppression of CatD occurs also during reactivated productive replication in PEL cells and is likely to contribute to pro-replication functions of vIL-6. Here we report that vIL-6 suppresses CatD through vIL-6, VKORC1v2 and pCatD association with components of the ER-associated degradation (ERAD) machinery. In transfected cells, expression of vIL-6 along with CatD led to proteasome-dependent (inhibitor-sensitive) decreases in CatD levels and the promotion of pCatD polyubiquitination. Depletion of particular ERAD-associated isomerases, lectins, and translocon components, including ERAD E3 ubiquitin ligase HRD1, diminished suppression of CatD by vIL-6. Coprecipitation assays identified direct or indirect interactions of VKORC1v2, vIL-6 and pCatD with translocon proteins (SEL1L and/or HRD1) and ERAD-associated lectins OS9 and XTP3-B. Endogenous CatD expression in PEL cells was increased by depletion of ERAD components and suppression of CatD by vIL-6 overexpression in PEL cells was dependent on HRD1. Our data reveal a new mechanism of ER-localized vIL-6 activity and further characterize VKORC1v2 function.

    IMPORTANCE Human herpesvirus 8 (HHV-8) viral interleukin-6 (vIL-6), unlike cellular IL-6 proteins, is secreted inefficiently and sequestered mainly in the endoplasmic reticulum (ER), from where it can signal through the gp130 receptor. We have recently reported that vIL-6 also associates with a novel membrane protein termed vitamin K epoxide reductase complex subunit 1 variant-2 (VKORC1v2) and mediates suppression of VKORC1v2-cointeracting cathepsin D, a stress-released pro-apoptotic protein negatively impacting HHV-8 latently-infected primary effusion lymphoma (PEL) cell viability and reactivated virus productive replication. Here, we have examined the mechanistic basis of the VKORC1v2-vIL-6 interaction-dependent suppression of cathepsin D and have found that this novel activity of vIL-6 is mediated through co-association of VKORC1v2, pro-cathepsin D and vIL-6 with components of the ER-associated degradation (ERAD) machinery. Our findings provide information of significance for potential antiviral and therapeutic targeting of VKORC1v2-mediated vIL-6 activities and also indicate the nature of VKORC1v2 function in normal cell biology.

  • Pentosan polysulfate: a novel glycosaminoglycan-like molecule for the effective treatment of alphavirus-induced cartilage destruction and inflammatory disease [PublishAheadOfPrint]

  • Arthritogenic alphaviruses such as Ross River virus (RRV) and chikungunya virus (CHIKV) cause large-scale epidemics of severe musculoskeletal disease and have been progressively expanding their global distribution. Since its introduction in July 2014, CHIKV now circulates in the USA . The hallmark of alphavirus disease is crippling pain and inflammation of the joints, a similar immunopathology to rheumatoid arthritis. The use of glycans as novel therapeutics is an area of research that has increased in recent years. Here we describe the promising therapeutic potential of the glycosaminoglycan (GAG)-like molecule pentosan polysulfate (PPS) to alleviate viral-induced arthritis. Mouse models of RRV and CHIKV disease were used to characterise the extent of cartilage damage in infection and investigate the potential of PPS to treat disease. This was assessed using histological analysis, real-time PCR and FACS. Alphaviral-infection resulted in cartilage destruction, the severity of which was alleviated by PPS therapy during RRV and CHIKV clinical disease. Reduction in cartilage damage corresponded with a significant reduction in immune infiltrates. Using multiplex analysis, PPS treatment significantly increased the anti-inflammatory cytokine interleukin-10 and reduced pro-inflammatory cytokines, typically correlated with disease severity. Furthermore, we reveal that the severe RRV-induced joint pathology, including thinning of articular cartilage and loss of proteoglycans in the cartilage matrix, was diminished with treatment. PPS is a promising new therapy for alphavirus induced arthritis acting to preserve the cartilage matrix, which is damaged during alphavirus infection. Overall the data demonstrate the potential of glycotherapeutics as a new class of treatment for infectious arthritis.

    Importance The hallmark of alphavirus disease is crippling pain and joint arthritis, which often has an extended duration. In the past year, CHIKV has expanded into the Americas with approximately 1-million cases reported to date, whereas RRV continues to circulate in the South Pacific. Currently there is no licenced specific treatment for alphavirus disease and the increasing spread of infection highlights an urgent need for therapeutic intervention strategies. Pentosan polysulfate (PPS) is a glycan derivative that is orally bioavailable, has few toxic side effects and is currently licensed under the name Elmironrreg; for the treatment of cystitis in the USA. Our findings show RRV infection damages the articular cartilage including a loss of proteoglycans within joint. Furthermore, treatment with PPS reduced the severity of both RRV- and CHIKV-induced musculoskeletal disease, including a reduction in inflammation and joint swelling, suggesting PPS is a promising candidate for drug-repurposing for the treatment of alphavirus-induced arthritis.

  • Non-glycosylated G protein vaccine protects against homologous and heterologous RSV challenge while glycosylated G enhances RSV lung pathology and cytokine levels [PublishAheadOfPrint]

  • New efforts are under way to develop a vaccine against RSV that will provide protective immunity without the potential for vaccine-associated disease enhancement as observed in infants following vaccination with formalin-inactivated RSV vaccine. In addition to the F fusion protein, the G attachment surface protein represents a target for neutralizing antibodies and thus represents an important vaccine candidate. However, glycosylated G protein expressed in mammalian cells was shown to induce pulmonary eosinophilia upon RSV infection in mouse model. In the current study, we evaluated in parallel the safety and protective efficacy of RSV A2 recombinant unglycosylated G protein ectodomain (amino acids 67-298) expressed in E. coli (REG) vs. glycosylated G produced in mammalian cells (RMG) in a mouse RSV challenge model. Vaccination with REG generated neutralizing antibodies against RSV A2 in 7/11 BALB/c mice, while RMG did not elicit neutralizing antibodies. Total serum binding antibodies against the recombinant proteins (both REG and RMG) were measured by Surface Plasmon Resonance (SPR) and found to be ggt;10-fold higher for REG compared with RMG-vaccinated animals. Complete reduction of lung viral loads after homologous (RSV-A2) and heterologous (RSV-B1) viral challenge was observed in 7/8 animals vaccinated with REG but not in RMG vaccinated animals. Furthermore, enhanced lung pathology and elevated Th2 cytokines/chemokines were observed exclusively in animals vaccinated with RMG (but not with REG or PBS) after homologous or heterologous RSV challenge. This study suggests that bacterially produced unglycosylated G protein could be developed alone or as a component of a protective vaccine against RSV disease.

    Importance New efforts are under way to develop vaccines against RSV that will provide protective immunity without the potential for disease enhancement. The G attachment protein represents an important candidate for inclusion in an effective RSV vaccine. In the current study, we evaluated the safety and protective efficacy of RSV A2 recombinant unglycosylated G protein ectodomain produced in E. coli (REG) vs. glycosylated G produced in mammalian cells (RMG) in a mouse RSV challenge model (A2 and B1 strains). The unglycosylated G generated high protective immunity and no lung pathology even in animals that lacked anti-RSV neutralizing antibodies prior to RSV challenge. Control of viral loads correlated with antibody binding to the G protein. In contrast, the glycosylated G protein provided poor protection and enhanced lung pathology after RSV challenge. Therefore, bacterially produced unglycosylated G protein presents an economical approach as a protective vaccine against RSV.

  • Rethinking the Response to Emerging Microbes: Vaccines and Therapeutics in the Ebola Era - A Conference at Harvard Medical School [PublishAheadOfPrint]

  • Harvard Medical School convened a meeting of biomedical and clinical experts on March 5, 2015 on the topic of "Rethinking the Response to Emerging Microbes: Vaccines and Therapeutics in the Ebola Era," with the goals of discussing the lessons from the recent Ebola outbreak and using those lessons as a case study to aid preparations for future emerging infections. The speakers and audience discussed the special challenges in combatting an infectious agent that causes sporadic outbreaks in resource-poor countries. The meeting led to a call for improved basic medical care for all and continued support of basic discovery research to provide the foundation for preparedness for future outbreaks in addition to the targeted emergency response to outbreaks and targeted research programs against Ebola and other specific emerging pathogens.

  • Tick saliva enhances Powassan virus transmission to the host influencing its dissemination and the course of disease [PublishAheadOfPrint]

  • Powassan virus (POWV) is an encephalitic tick-borne flavivirus which can result in serious neuroinvasive disease with up to 10% case fatality rate. The study objective was to determine whether the salivary gland extract (SGE) from I. scapularis ticks facilitates the transmission and dissemination of POWV in a process known as saliva-activated transmission. Groups of BALB/c mice were footpad inoculated with one of the following: high dose of POWV with and without SGE, or low dose of POWV with and without SGE. Mice from each group were sacrificed daily. Organ viral loads and gene expression profiles were evaluated by quantitative real-time PCR. Both groups of mice infected with POWV high dose showed severe neurological signs of disease preceding death. The presence of SGE did not affect POWV transmission or disease outcome for mice infected with the high dose of POWV. Neuroinvasion, paralysis, and death occurred for all mice infected with the low dose of POWV plus SGE; however, for mice infected with the low dose of POWV in the absence of SGE, there were no clinical signs of infection nor did any mice succumb to disease. Although this group displayed low-level viremias, all mice were completely healthy and it was the only group in which POWV was cleared from the lymph nodes. We conclude that saliva-activated transmission occurs in mice infected with a low dose of POWV. Our study is the first to demonstrate virus dose-dependent saliva-activated transmission, warranting further investigation of the specific salivary factors responsible for enhancing POWV transmission.

    IMPORTANCE. Powassan virus (POWV) is a tick-borne flavivirus that continues to emerge in the United States, as is evident by the surge in number and expanding geographic range of confirmed cases in the past decade. This neuroinvasive virus is transmitted to humans by infected tick bites. Successful tick feeding is facilitated by a collection of pharmacologically active factors in tick saliva. In a process known as saliva-activated transmission, tick bioactive salivary molecules are thought to modulate the host environment making it more favorable for the transmission and establishment of a pathogen. This phenomenon has been demonstrated for several tick-borne pathogens; however, a systematic investigation of the role of tick saliva on dissemination and pathogenesis of a tick-borne viral disease has never been attempted before. This study will fill that gap by systematically examining whether the presence of tick saliva contributes to the transmission and dissemination of POWV in mice.

  • HIV-1 is Restricted Prior to Integration in Primary Cord-Derived Human CD34+ Cells [PublishAheadOfPrint]

  • Certain cells have the ability to block retroviral infection at specific stages of the viral cycle by the activities of well-characterized factors and transcriptional silencing machinery. Infection of murine stem cells (MSCs) by the murine leukemia viruses (MLVs) is profoundly blocked post-integration by transcriptional silencing. Here we show that a dominant point of restriction of HIV-1 in human CD34+ cells is prior to integration, and that HIV-1 restriction by human CD34+ cells is fundamentally different.

  • Expanding regulatory T cells alleviates chikungunya virus-induced pathology in mice [PublishAheadOfPrint]

  • Chikungunya virus (CHIKV) infection is a re-emerging pandemic human arboviral disease. CD4+ T cells were previously shown to contribute to joint inflammation in the course of CHIKV infection in mice. The JES6-1 anti-IL-2 antibody selectively expands mouse regulatory T cells (Tregs) by forming a complex with IL-2. In this study, we show that the IL-2 JES6-1-mediated expansion of Tregs ameliorates CHIKV-induced joint pathology. It does so by inhibiting the infiltration of CD4+ T cells due to the induction of anergy in CHIKV-specific CD4+ effector T cells. These findings suggest that activation of Tregs could also become an alternative approach to control CHIKV-mediated disease.

    Importance Chikungunya virus (CHIKV) has re-emerged as a pathogen of global significance. Patients infected with CHIKV suffer from incapacitating joint pain that severely affects their daily functioning. Despite the best efforts, effective treatment is still inadequate. While T cells-mediated immunopathology in CHIKV infections has been reported, the role of regulatory T cells (Tregs) has not been explored. The JES6-1 anti-IL-2 antibody has been demonstrated to selectively expand mouse Tregs by forming a complex with IL-2. We reveal here that IL-2 JES6-1-mediated expansion of Tregs ameliorates the CHIKV-induced joint pathology in mice by neutralizing virus-specific CD4+ effector T (Teff) cells. We show that this treatment abrogates the infiltration of pathogenic CD4+ T cells through induction of anergy in CHIKV-specific CD4+ Teff cells. This is the first evidence where the role of Tregs is demonstrated in CHIKV pathogenesis and its expansion could control virus-mediated immunopathology.

  • Repeated Low-dose Influenza Virus Infection Causes Severe Disease in Mice: a Model for Vaccine Evaluation [PublishAheadOfPrint]

  • Influenza infection causes severe disease and death in humans. In traditional vaccine research and development, a single high-dose virus challenge of animals is used to evaluate vaccine efficacy. This type of challenge model may have limitations. In the present study, we developed a novel challenge model by infecting mice repeatedly in short intervals with low-doses of influenza A virus. Our results show that compared to a single high-dose infection, mice that received repeated low-dose challenges showed earlier morbidity and mortality and more severe disease. They developed higher vial loads, more severe lung pathology, greater inflammatory responses, and generated only limited influenza A virus-specific B and T cell responses. A commercial trivalent influenza vaccine protected mice against a single high and lethal dose of influenza A virus but was ineffective against repeated low-dose virus challenges. Overall our data show that the repeated low-dose influenza A virus infection mouse model is more stringent and may thus be more suitable to select for highly efficacious influenza vaccines.

    Importance Influenza epidemics and pandemics pose serious threats to public health. Animal models are crucial for evaluating the efficacy of influenza vaccines. Traditional models based on a single high-dose virus challenge may have limitations. Here, we describe a new mouse model based on repeated low dose influenza A virus challenges given within a short period. Repeated low-dose challenges caused more severe disease in mice associated with higher viral loads and increased lung inflammation and reduced influenza A virus-specific B and T cell responses. A commercial influenza vaccine that was shown to protect mice from high-dose challenge was ineffective against repeated low-dose challenges. Overall, our results show that the low-dose repeated challenge model is more stringent and may therefore be better suited for pre-clinical vaccine efficacy studies.

  • A transgenic Drosophila melanogaster model to study Human T-Lymphotropic Virus oncoprotein Tax-1-driven transformation in vivo [PublishAheadOfPrint]

  • HTLV-1-induced adult T-cell leukemia/lymphoma is an aggressive malignancy. HTLV-2 is genetically related to HTLV-1 but does not cause any malignant disease. HTLV-1 Tax transactivator (Tax-1) contributes to leukemogenesis via NF-B. We describe transgenic Drosophila models expressing Tax in the compound eye and plasmatocytes. We demonstrate that Tax-1 but not Tax-2 induces ommatidia perturbation and increased plasmatocyte proliferation and that the eye phenotype is dependent on Kenny (IKK/NEMO), thus validating this new in vivo model.

  • Caribbean and La Reunion Chikungunya virus isolates differ in their capacity to induce pro-inflammatory Th1 and NK cell responses and acute joint pathology [PublishAheadOfPrint]

  • Chikungunya virus (CHIKV) is a mosquito-borne arthralgic alphavirus that has garnered international attention as an important emerging pathogen since 2005. More recently, it invaded the Caribbean islands and the western hemisphere. Intriguingly, the current CHIKV outbreak in the Caribbean is caused by the Asian CHIKV genotype, which differs from the La Reeacute;union LR2006 OPY1 isolate belonging to the Indian Ocean lineage. Here, we adopted a systematic and comparative approach against LR2006 OPY1 to characterize the pathogenicity of the Caribbean CNR20235 isolate and consequential host immune responses in mice. Ex vivo infection using primary mouse-tail fibroblasts revealed weaker replication efficiency by CNR20235 isolate. In the CHIKV mouse model, CNR20235 infection induced an enervated joint pathology characterized by moderate edema and swelling, independent of mononuclear cell infiltration. Based on systemic cytokine analysis, localized immune-phenotyping and gene expression profiles in the popliteal lymph node and inflamed joints, two pathogenic phases were defined for CHIKV infection: early acute (2-3 dpi) and late acute (6-8 dpi). Reduced joint pathology during early acute phase of CNR20235 infection was associated with a weaker pro-inflammatory Th1 response and natural killer (NK) cells activity. The pathological role of NK cells was further demonstrated as depletion of NK cells reduced joint pathology in LR2006 OPY1. Taken together, this study provides evidence that the Caribbean CNR20235 isolate has an enfeebled replication and induces a less pathogenic response in the mammalian host.

    IMPORTANCE The introduction of CHIKV in the Americas has heightened the risk of large-scale outbreaks due to the close proximity between the United States and the Caribbean. Immune-pathogenicity of the circulating Caribbean CHIKV isolate was explored, where it was demonstrated to exhibit reduced infectivity resulting in a weakened joint pathology. Analysis of serum cytokine levels, localized immune-phenotyping, and gene expression profile in the organs revealed that a limited Th1 response and reduced NK cells activity could underlie the reduced pathology in the host. Interestingly, higher asymptomatic infections were observed in the Caribbean compared to the La Reeacute;union outbreaks in 2005-2006. This is the first study that showed an association between key pro-inflammatory factors and pathology-mediating leukocytes with a less severe pathological outcome in Caribbean CHIKV infection. Given the limited information regarding the sequela of Caribbean CHIKV infection, our study is timely and will aid the understanding of this increasingly important disease.

  • Activated Nrf2 Interacts with Kaposi's Sarcoma-Associated Herpesvirus Latency Protein LANA-1 and Host Protein KAP1 to Mediate Global Lytic Gene Repression [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman's disease. We have previously shown that KSHV utilizes the host transcription factor Nrf2 to aid in infection of endothelial cells and oncogenesis. Here, we investigate the role of Nrf2 in PEL and PEL-derived cell lines and show that KSHV latency induces Nrf2 protein levels and transcriptional activity through the COX-2/PGE2/EP4/PKC axis. Next-generation sequencing of KSHV transcripts in the PEL-derived BCBL-1 cell line reveals that knockdown of this activated Nrf2 results in global elevation of lytic genes. Nrf2 inhibition by the chemical Brusatol also induces lytic gene expression. Both Nrf2 knockdown and Brusatol-mediated inhibition induced KSHV lytic reactivation in BCBL-1 cells. In a series of follow-up experiments, we characterized the mechanism of Nrf2-mediated regulation of KSHV lytic repression during latency. Biochemical assays show that Nrf2 interacted with the KSHV latency-associated nuclear antigen (LANA-1) and the host transcriptional repressor KAP1, which together have been shown to repress lytic gene expression. Promoter studies show that although Nrf2 alone induces the ORF50 promoter, its association with LANA-1 and KAP1 abrogates this effect. Interestingly, LANA-1 is crucial for efficient KAP1/Nrf2 association, while Nrf2 is essential for LANA-1 and KAP1 recruitment to the ORF50 promoter and its repression. Overall, these results suggest that activated Nrf2, LANA-1 and KAP1 assemble on the ORF50 promoter in a temporal fashion. Initially, Nrf2 binds to and activates the ORF50 promoter during early de novo infection, an effect that is exploited during latency by LANA-1-mediated recruitment of the host transcriptional repressor KAP1 on Nrf2. Cell death assays further show that Nrf2 and KAP1 knockdown induce significant cell death in PEL cell lines. Our studies suggest that Nrf2 modulation through available oral agents is a promising therapeutic approach in the treatment of KSHV-associated malignancies.

    Importance KS and PEL are aggressive KSHV-associated malignancies with moderately effective, highly toxic chemotherapies. Besides Ganciclovir and IFN-aalpha; prophylaxis, no KSHV-associated chemotherapy targets the underlying infection, a major oncogenic force. Hence, drugs that selectively target KSHV infection are necessary to eradicate the malignancy while sparing healthy cells. We recently showed that KSHV infection of endothelial cells activates the transcription factor Nrf2 to promote an environment conducive to infection and oncogenesis. Nrf2 is modulated through several well-tolerated oral agents, and may be an important target in KSHV biology. Here, we investigate the role of Nrf2 in PEL, and demonstrate that Nrf2 plays an important role in KSHV gene expression, lytic reactivation and cell survival by interacting with the host transcriptional repressor KAP1 and the viral latency-associated protein LANA-1 to mediate global lytic gene repression and thus cell survival. Hence, targeting Nrf2 with available therapies is a viable approach in the treatment of KSHV malignancies.

  • Naturally-occurring capsid protein variants of human papillomavirus genotype 31 represent a single L1 serotype. [PublishAheadOfPrint]

  • We investigated naturally-occurring variation within the major (L1) and minor (L2) capsid proteins of oncogenic human papillomavirus (HPV) genotype HPV31 to determine the impact on capsid antigenicity. L1L2 pseudoviruses (PsV) representing the three HPV31 variant lineages A, B and C exhibited comparable particle to infectivity ratios and morphology. Lineage-specific L1L2 PsV demonstrated subtle differences in susceptibility to neutralization by antibodies elicited following vaccination, pre-clinical L1 VLP immunization or by monoclonal antibodies; however, these differences were generally of a low magnitude. These data indicate diagnostic lineage-specific single nucleotide polymorphisms within the HPV31 capsid genes have a limited effect on L1 antibody mediated neutralization and that the three HPV31 variant lineages belong to a single L1 serotype. These data contribute to our understanding of HPV L1 variant antigenicity.

    Importance The virus coat (capsid) of the human papillomavirus contains major (L1) and minor (L2) capsid proteins. These proteins facilitate host cell attachment and viral infectivity and are the targets for antibodies which interfere with these events. In this study we investigated the impact of naturally-occurring variation within these proteins upon susceptibility to viral neutralization by antibodies induced by L1 VLP immunization. We demonstrate that HPV31 L1 and L2 variants exhibit similar susceptibility to antibody-mediated neutralization and that for the purposes of L1 VLP-based vaccines these variant lineages represent a single serotype.

  • A novel Leu92 mutant of HIV-1 reverse transcriptase with a selective deficiency in strand transfer causes a loss of viral replication [PublishAheadOfPrint]

  • The process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between the two functions and that they are both direct and unique functions the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting the in vitro data. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectivily blocking this RT activity and consequently HIV-1 infectivity and pathogenesis.

    Importance Reverse transcription in retroviruses is essential for the viral life cycle. This multi-step process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its ribonuclease H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost, due to an impaired intracellular strand transfer, thus supporting the in vitro data. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity.

  • PI(4,5)P2 acyl chains differentiate membrane binding of HIV-1 Gag from that of the phospholipase C{delta}1 PH domain [PublishAheadOfPrint]

  • HIV-1 Gag, which drives virion assembly, interacts with a plasma-membrane(PM)-specific phosphoinositide, phosphatidylinositol- (4, 5)-bisphosphate [PI(4,5)P2]. While cellular acidic-phospholipid-binding proteins/domains, such as the PI(4,5)P2-specific pleckstrin homology domain of phospholipase C1 (PHPLC1), mediate headgroup-specific interactions with corresponding phospholipids, the exact nature of the Gag-PI(4,5)P2 interaction remains undetermined. In this study, we used giant unilamellar vesicles (GUVs) to examine how PI(4,5)P2 with unsaturated or saturated acyl chains affect membrane binding of PHPLC1 and Gag. Both unsaturated dioleoyl-PI(4,5)P2 [DO-PI(4,5)P2] and saturated dipalmitoyl-PI(4,5)P2 [DP-PI(4,5)P2] successfully recruited PHPLC1 to membranes of single-phase GUVs. In contrast, DO-PI(4,5)P2 but not DP-PI(4,5)P2 recruited Gag to GUVs, indicating that PI(4,5)P2 acyl chains contribute to stable membrane binding of Gag. GUVs containing PI(4,5)P2, cholesterol, and dipalmitoyl phosphatidylserine separated into two coexisting phases: one was a liquid phase, and the other appeared to be a phosphatidylserine-enriched gel phase. In these vesicles, the liquid phase recruited PHPLC1 regardless of PI(4,5)P2 acyl chains. Likewise, Gag bound to the liquid phase when PI(4,5)P2 has DO-acyl chains. DP-PI(4,5)P2-containing GUVs showed no detectable Gag binding to the liquid phase. Unexpectedly, however, DP-PI(4,5)P2 still promoted recruitment of Gag, but not PHPLC1, to the dipalmitoyl-phosphatidylserine-enriched gel phase of these GUVs. Altogether, these results revealed different roles for PI(4,5)P2 acyl chains in membrane binding of two PI(4,5)P2-binding proteins, Gag and PHPLC1. Notably, we observed that non-myristylated Gag retains the preference for PI(4,5)P2 containing an unsaturated acyl chain over DP-PI(4,5)P2, suggesting that Gag sensitivity to PI(4,5)P2-acyl-chain saturation is determined directly by the MA-PI(4,5)P2 interaction, rather than indirectly by a myristate-dependent mechanism.

    Importance Binding of HIV-1 Gag to the plasma membrane is promoted by its interaction with a plasma-membrane-localized phospholipid PI(4,5)P2. Many cellular proteins are also recruited to the plasma membrane via PI(4,5)P2-interacting domains represented by PHPLC1. However, differences and/or similarities between these host proteins and viral Gag protein in the nature of their PI(4,5)P2 interactions, especially in the context of membrane binding, remain to be determined. Using a novel giant-unilamellar-vesicle-based system, we found that PI(4,5)P2 with an unsaturated acyl chain recruits PHPLC1 and Gag similarly, whereas PI(4,5)P2 with saturated acyl chains either recruits PHPLC1 but not Gag or sorts these proteins to different phases of vesicles. To our knowledge, this is the first study to show that PI(4,5)P2 acyl chains differentially modulate membrane binding of PI(4,5)P2-binding proteins. Since Gag membrane binding is essential for progeny virion production, the PI(4,5)P2 acyl chain property may serve as a potential target for anti-HIV therapeutic strategies.

  • Prolonged antiretroviral therapy preserves HIV-1-specific CD8 T cells with stem cell-like properties [PublishAheadOfPrint]

  • HIV-1-specific CD8 T cells can influence HIV-1 disease progression during untreated HIV-1 infection, but the functional and phenotypic properties of HIV-1-specific CD8 T cells in individuals treated with suppressive antiretroviral therapy remain less well understood. Here we show that a subgroup of HIV-1-specific CD8 T cells with stem cell-like properties (CD8 TSCM) is enriched in patients receiving suppressive antiretroviral therapy, as opposed to untreated progressors or controllers. In addition, prolonged duration of antiretroviral therapy was associated with a progressive increase in the relative proportions of these stem cell-like HIV-1-specific CD8 T cells. Interestingly, proportions of HIV-1-specific CD8 TSCM and total HIV-1-specific CD8 TSCM cells were associated with CD4 T cell counts during treatment with antiretroviral therapy, but not with CD4 T cell counts, viral loads or immune activation parameters in untreated patients, including controllers. HIV-1-specific CD8 TSCM cells seemed to preferentially secrete IL-2 in response to viral stimulation, while secretion of IFN- was more limited; however, only proportions of IFN--secreting HIV-1-specific CD8 TSCM were associated with CD4 T cell counts during antiretroviral therapy. Together, these data suggest that HIV-1-specific CD8 TSCM cells represent a long-lasting component of the cellular immune response to HIV-1 that persists in an antigen-independent fashion during antiretroviral therapy, but seems unable to survive and expand under conditions of ongoing viral replication during untreated infection.

    Importance Memory CD8 T cells that imitate functional properties of stem cells to maintain life-long cellular immunity have been hypothesized for many years, but only recently have such cells, termed T memory stem cells (TSCM), been physically identified and isolated in humans, mice and non-human primates. Here, we investigated whether cellular immune responses against HIV-1 include such T memory stem cells. Our data show that HIV-1-specific CD8 T memory stem cells are detectable during all stages of HIV-1 infection, but occur most visibly at times of prolonged viral antigen suppression by antiretroviral combination therapy. These cells may therefore be particularly relevant for designing antiviral immune defense strategies against the residual reservoir of HIV-1 infected cells that persists despite treatment and leads to viral rebound upon treatment discontinuation.

  • Inhibitory effect of individual or combinations of broadly neutralizing antibodies and antiviral reagents against cell-free and cell-to-cell HIV-1 transmission [PublishAheadOfPrint]

  • To date, most therapeutic and vaccine candidates for HIV-1 are evaluated pre-clinically for efficacy against cell-free viral challenges. However, cell-associated HIV-1 is suggested to be a major contributor to sexual transmission by mucosal routes. To determine if neutralizing antibodies or inhibitors block cell-free and cell-associated virus transmission of diverse HIV-1 strains with different efficiencies, we tested twelve different antibodies and five inhibitors against four GFP-labeled HIV-1 envelope (Env) variants from transmitted/founder or chronic isolates. We evaluated antibody/inhibitor-mediated virus neutralization using either TZM-bl target cells, in which infectivity was determined by virus-driven luciferase expression, or A3R5 lymphoblastoid target cells, in which infectivity was evaluated by GFP expression. In both the TZM-bl and A3R5 assay, cell-free virus or infected CD4+ lymphocytes were used as targets for neutralization. We further hypothesized that the combined use of specific neutralizing antibodies targeting HIV-1 Env would more effectively prevent cell-associated virus transmission than the use of individual antibodies. The tested antibody combinations included two gp120-directed antibodies, VRC01 and PG9, or VRC01 with the gp41-directed antibody, 10E8. Our results demonstrated that cell-associated virus was less sensitive to neutralizing antibodies and inhibitors, particularly using the A3R5 neutralization assay, and potencies of these neutralizing agents differed among Env variants. A combination of different neutralizing antibodies that target specific sites on gp120 led to a significant reduction in cell-associated virus transmission. These assays will help identify ideal combinations of broadly neutralizing antibodies to use for passive preventive antibody administration and further characterize targets for the most effective neutralizing antibodies/inhibitors.

    Importance: Preventing the transmission of human immunodeficiency virus-1 (HIV-1) remains a prominent goal of HIV research. The relative contribution of HIV-1 within an infected cell versus cell-free HIV-1 to virus transmission remains debated. It has been suggested that cell-associated virus is more efficient at transmitting HIV-1 and more difficult to neutralize than cell-free virus. Several broadly neutralizing antibodies and retroviral inhibitors are currently being studied as potential therapies against HIV-1 transmission. The present study demonstrates a decrease in neutralizing antibody and inhibitor efficiencies against cell-associated compared to cell-free HIV-1 transmission among different strains of HIV-1. We also observed a significant reduction in virus transmission using a combination of two different neutralizing antibodies that target specific sites on the outermost region of HIV-1, the virus envelope. Therefore, our findings support the use of antibody combinations against both cell-free and cell-associated virus in future candidate therapy regimens.

  • Pseudorabies virus fast-axonal transport occurs by a pUS9-independent mechanism [PublishAheadOfPrint]

  • Reactivation from latency results in transmission of neurotropic herpesviruses from the nervous system to body surfaces, referred to as anterograde axonal trafficking. The virus-encoded protein, pUS9, promotes axonal dissemination by sorting virus particles into axons, but whether it is also an effector of fast axonal transport within axons is unknown. To determine the role of pUS9 in anterograde trafficking, we analyzed the axonal transport of pseudorabies virus in the presence and absence of pUS9.

  • IFN{beta} and IFN{alpha}2 differentially protect head and neck cancer cells from VSV-induced oncolysis [PublishAheadOfPrint]

  • Oncolytic viruses (OV) preferentially kill cancer cells due in part to defects in their antiviral responses upon exposure to type I interferons (IFNs). However, IFN responsiveness of some tumor cells confers resistance to OV treatment. The human type I IFNs include one IFNbbeta; and multiple IFNaalpha; subtypes that share the same receptor but are capable of differentially inducing biological responses. The role of individual IFN subtypes in promoting tumor cell resistance to OV is addressed here. Two human IFNs which have been produced for clinical use, IFNaalpha;2a and IFNbbeta;, were compared for activity in protecting human head and neck squamous cell carcinoma (HNSCC) lines from oncolysis by vesicular stomatitis virus (VSV). Susceptibility of HNSCC lines to killing by VSV varied. VSV infection induced increased production of IFNbbeta; in resistant HNSCC cells. When added exogenously, IFNbbeta; was significantly more effective at protecting HNSCC cells from VSV oncolysis than was IFNaalpha;2a. In contrast, normal keratinocytes and endothelial cells were equivalently protected by both IFN subtypes. Differential responsiveness of tumor cells to IFNs aalpha; and bbeta; was further supported by the finding that autocrine IFNbbeta; but not IFNaalpha; promoted survival of HNSCC cells during persistent VSV infection. Therefore, IFNs aalpha; and bbeta; differentially affect VSV oncolysis, justifying the evaluation and comparison of IFN subtypes for use in combination with VSV therapy. Pairing VSV with IFNaalpha;2a may enhance selectivity of oncolytic VSV therapy for HNSCC by inhibiting VSV replication in normal cells without a corresponding inhibition in cancer cells.

    Importance There has been a great deal of progress in the development of oncolytic viruses. However, a major problem is that individual cancers vary in their sensitivity to oncolytic viruses. In many cases this is due to differences in their production and response to interferons (IFNs). The experiments described here compared the responses of head and neck squamous cell carcinoma cell lines to two IFN subtypes, IFNaalpha;2a and IFNbbeta;, in protection from oncolytic vesicular stomatitis virus. We found that IFNaalpha;2a was significantly less protective for cancer cells than was IFNbbeta;, whereas normal cells were equivalently protected by both IFNs. These results suggest that from a therapeutic standpoint, selectivity for cancer versus normal cells may be enhanced by pairing VSV with IFNaalpha;2a.

  • Herpes simplex virus 1 recruits CD98 heavy chain and {beta}1 integrin to the nuclear membrane for viral de-envelopment [PublishAheadOfPrint]

  • Herpesviruses have evolved a unique mechanism for nucleocytoplasmic transport of nascent nucleocapsids: the nucleocapsids bud through the inner nuclear membrane (INM) (primary envelopment), and the enveloped nucleocapsids then fuse with the outer nuclear membrane (de-envelopment). Little is known about the molecular mechanism of herpesviral de-envelopment. Here we showed that knockdown of both CD98 heavy chain (CD98hc) and its binding partner bbeta;1 integrin induced membranous structures containing enveloped herpes simplex virus 1 (HSV-1) virions that are invaginations of the INM into the nucleoplasm, and induced aberrant accumulation of enveloped virions in the perinuclear space and in the invagination structures. These effects were similar to those of the previously reported mutation(s) in HSV-1 proteins gB, gH, UL31 and/or Us3, which were shown here to form a complex(es) with CD98hc in HSV-1-infected cells. These results suggested that cellular proteins CD98hc and bbeta;1 integrin synergistically or independently regulated HSV-1 de-envelopment, probably by interacting directly and/or indirectly with these HSV-1 proteins.

    IMPORTANCE Certain cellular and viral macromolecular complexes, such as Drosophila large ribonucleoprotein complexes and herpesvirus nucleocapsids, utilize a unique vesicle-mediated nucleocytoplasmic transport: the complexes acquire primary envelopes by budding through the inner nuclear membrane into the space between the inner and outer nuclear membranes (primary envelopment), and the enveloped complexes then fuse with the outer nuclear membrane to release de-enveloped complexes into the cytoplasm (de-envelopment). However, there is a lack of information on the molecular mechanism of de-envelopment fusion. We report here that HSV-1 recruited cellular fusion regulatory proteins CD98hc and bbeta;1 integrin to the nuclear membrane for viral de-envelopment fusion. This is the first report of cellular proteins required for efficient de-envelopment of macromolecular complexes during their nuclear egress.

  • FUSE Binding Protein1 Facilitates Persistent Hepatitis C Virus Replication in Hepatoma Cells by Regulating Tumor Suppressor p53 [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) is a leading cause of chronic hepatitis C (CHC), liver cirrhosis, and hepatocellular carcinoma (HCC). Immunohistochemistry of archived HCC tumors showed abundant FBP1 expression in HCC tumors with CHC background. Oncomine data analysis of normal versus HCC tumors with CHC background indicated a 4-fold increase in FBP1 expression with a concomitant 2.5-fold decrease in the expression of p53. We found that FBP1 promotes HCV replication by inhibiting p53, and regulating BCCIP and TCTP, which, respectively, are positive and negative regulators of p53. The severe inhibition of HCV replication in FBP1-knockdown Huh7.5 cells was restored to a normal level by downregulation of either p53 or BCCIP. Although p53 in Huh7.5 cells is transcriptionally inactive as a result of Y220C mutation, we found that activation and DNA binding ability of Y220C p53 were strongly suppressed by FBP1, but significantly activated upon knockdown of FBP1. Transient expression of FBP1 in FBP1-knockdown cells fully restored the control phenotype in which the DNA binding ability of p53 was strongly suppressed. Using EMSA and ITC, we found no significant difference in in-vitro target DNA binding affinity of recombinant wild-type p53 and its Y220C mutant p53. However, in the presence of recombinant FBP1, DNA binding ability of p53 is strongly inhibited. We confirmed that FBP1 downregulates BCCIP, p21, and p53, and upregulates TCTP under radiation-induced stress. Since FBP1 is overexpressed in most HCC tumors with HCV background, it may have a role in promoting persistent virus infection and tumorigenesis.

    Importance It is our novel finding that Fuse binding protein1 (FBP1) strongly inhibits the function of tumor suppressor p53 and is an essential host-cell factor required for HCV replication. Oncomine data analysis on a large number of samples has revealed that overexpression of FBP1 in most HCC tumors with chronic hepatitis C is significantly linked with the decreased expression level of p53. The most significant finding is that FBP1 not only physically interacts with p53 and interferes with its binding to the target DNA but also functions as a negative regulator of p53 under cellular stress. FBP1 is barely detectable in normal differentiated cells; its overexpression in HCC tumors with CHC background suggests that FBP1 may have an important role in promoting HCV infection and HCC tumors by suppressing p53.

  • RESPIRATORY SYNCYTIAL VIRUS INHIBITOR AZ-27 DIFFERENTIALLY INHIBITS DIFFERENT POLYMERASE ACTIVITIES AT THE PROMOTER [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) is the leading cause of pediatric respiratory disease. RSV has an RNA dependent RNA polymerase that transcribes and replicates the viral negative sense RNA genome. The large polymerase subunit (L) has multiple enzymatic activities, having the capability to synthesize RNA, and add and methylate a cap on each of the viral mRNAs. Previous studies had identified a small molecule inhibitor, AZ-27, that targets the L protein. In this study, we examined the effect of AZ-27 on different aspects of RSV polymerase activity. AZ-27 was found to equally inhibit both mRNA transcription and genome replication in cell-based minigenome assays, indicating that it inhibits a step common to both these RNA synthesis processes. Analysis in an in vitro transcription run-on assay, containing RSV nucleocapsids, showed that AZ-27 inhibits synthesis of transcripts from the 3rrsquo; end of the genome to a greater extent that those from the 5rrsquo; end, indicating that it inhibits transcription initiation. Consistent with this finding, experiments that assayed polymerase activity on the promoter showed that AZ-27 inhibited transcription and replication initiation. The RSV polymerase can also utilize the promoter sequence to perform a back-priming reaction. Interestingly, addition of AZ-27 had no effect on addition of up to three nucleotides by back-priming, but inhibited further extension of the back-primed RNA. These data provide new information regarding the mechanism of inhibition by AZ-27. They also suggest that the RSV polymerase adopts different conformations to perform its different activities at the promoter.

    IMPORTANCE Currently, there are no effective antiviral drugs to treat RSV infection. The RSV polymerase is an attractive target for drug development, but this large enzymatic complex is poorly characterized, hampering drug development efforts. AZ-27 is a small molecule inhibitor previously shown to target the RSV large polymerase subunit, but its inhibitory mechanism was unknown. Understanding this would be valuable both for characterizing the polymerase and for further development of inhibitors. Here we show that AZ-27 inhibits an early stage in mRNA transcription, as well as genome replication, by inhibiting initiation of RNA synthesis from the promoter. However, the compound does not inhibit back-priming, another RNA synthesis activity of the RSV polymerase. These findings provide insight into the different activities of the RSV polymerase and will aid further development of antiviral agents against RSV.

  • CONSERVATION OF G PROTEIN EPITOPES IN RESPIRATORY SYNCYTIAL VIRUS (GROUP A) DESPITE BROAD GENETIC DIVERSITY: IS ANTIBODY SELECTION INVOLVED IN VIRUS EVOLUTION? [PublishAheadOfPrint]

  • Worldwide G glycoprotein phylogeny of human respiratory syncytial virus (hRSV) group A sequences revealed diversification in major clades and genotypes over more than fifty years of recorded history. Multiple genotypes co-circulated during prolonged periods of time but recent dominance of the GA2 genotype was noticed in several studies and it is highlighted here with sequences from viruses circulating recently in Spain and Panama. Reactivity of group A viruses with MAbs that recognize strain-variable epitopes of the G glycoprotein failed to correlate genotype diversification with antibody reactivity. Additionally, no clear correlation was found between changes in strain-variable epitopes and predicted sites of positive selection, despite both traits being associated to the C-terminal third of the G glycoprotein. Hence, our data do not lend support to the proposed antibody-driven selection of variants as major determinant of hRSV evolution. Other alternative mechanisms are considered to account for the high degree of hRSV_G variability.

    IMPORTANCE An unusual characteristic of the G glycoprotein of human respiratory syncytial virus (hRSV) is the accumulation of non-synonymous (N) changes at higher rates than synonymous (S) changes, reaching dN/dS values at certain sites predictive of positive selection. Since these sites cluster preferentially in the C-terminal third of the G protein, as certain epitopes recognized by murine antibodies, it was proposed that immune-(antibody) selection might be driving the apparent positive selection, analogous to the antigenic drift observed in the influenza virus hemagglutinin (HA). However, careful antigenic and genetic comparison of the G glycoprotein does not provide evidence of antigenic drift in the G molecule, in agreement with recent published data which did not sense antigenic drift in the G protein with human sera. Alternative explanations to the immune driven selection hypothesis are offered to account for the high level of G protein genetic diversity highlighted in this study.

  • Varicella Zoster Virus and Herpes Simplex Virus-1 Differentially Modulate NKG2D Ligand Expression During Productive Infection [PublishAheadOfPrint]

  • Natural killer (NK) cell deficient patients are particularly susceptible to severe infection with herpesviruses, especially varicella zoster virus (VZV) and herpes simplex virus (HSV-1). The critical role that NK cells play in controlling these infections denotes an intricate struggle for dominance between virus and NK cell antiviral immunity; however, research in this area has remained surprisingly limited. Our study addressed this absence of knowledge and found that infection with VZV was not associated with enhanced NK cell activation, suggesting that the virus uses specific mechanisms to limit NK cell activity. Analysis of viral regulation of ligands for NKG2D, a potent activating receptor ubiquitously expressed on NK cells, revealed that VZV differentially modulates expression of the NKG2D ligands, MICA, ULBP2 and ULBP3, by upregulating MICA expression while reducing ULBP2 and ULBP3 expression on the surface of infected cells. Despite being closely related to VZV, infection with HSV-1 produced a remarkably different effect on NKG2D ligand expression. A significant decrease in MICA, ULBP2 and ULBP3 was observed with HSV-1 infection at a total cellular protein level, as well as on the cell surface. We also demonstrate that HSV-1 differentially regulates expression of an additional NKG2D ligand, ULBP1, by reducing cell-surface expression while total protein levels are unchanged. Our findings illustrate both a striking point of difference between two closely related alphaherpesviruses, as well as suggest a powerful capacity for VZV and HSV-1 to evade antiviral NK cell activity through novel modulation of NKG2D ligand expression.

    IMPORTANCE Patients with deficiencies in NK cell function experience an extreme susceptibility to infection with herpesviruses, in particular, VZV and HSV-1. Despite this striking correlation, research into understanding how these two alphaherpesviruses interact with NK cells is surprisingly limited. Through examination of viral regulation of ligands to the activating NK cell receptor, NKG2D, we reveal patterns of modulation by VZV, which were unexpectedly varied to regulation by HSV-1 infection. Our study begins to unravel the undoubtedly complex interactions that occur between NK cells and alphaherpesvirus infection by providing novel insights into how VZV and HSV-1 manipulate NKG2D ligand expression to modulate NK cell activity, while also illuminating a distinct variation between two closely related alphaherpesviruses.

  • Cytokine-mediated activation of NK cells during viral infection [PublishAheadOfPrint]

  • Natural killer cells provide a first line of defense against infection via the production of antiviral cytokines and direct lysis of target cells. Cytokines such as IL-12 and IL-18 are critical regulators of NK cell activation, but much remains to be learned about how cytokines interact to regulate NK cell function. Here, we have examined cytokine-mediated activation of NK cells during infection with two natural mouse pathogens: lymphocytic choriomenengitis virus (LCMV) and murine cytomegalovirus (MCMV). Using a systematic screen of 1,849 cytokine pairs, we identified the most potent combinations capable of eliciting IFN production in NK cells. We observed that NK cell responses to cytokine stimulation were reduced at 8 days after acute LCMV infection, but recovered to pre-infection levels by 60 days post-infection. In contrast, during MCMV infection NK cell responses to cytokines remained robust at all time points examined. Ly49H+ NK cells recognizing the viral ligand, m157, showed preferential proliferation during early MCMV infection. A population of these cells remained detectable beyond 60 days post-infection, but these divided cells did not demonstrate enhanced IFN production in response to innate cytokine stimulation. Instead, the maturation state of the NK cells (as determined by CD11b/CD27 surface phenotype) was predictive of responsiveness to cytokines, regardless of Ly49H expression. These results help define cytokine interactions that regulate NK cell activation, and highlight variations in NK cell function during two unrelated viral infections.

    Importance Natural killer cells play an important role in immunity to many viral infections. From an initial screen of 1,849 cytokine pairs, we identified the most stimulatory cytokine combinations capable of inducing IFN production by NK cells. Ly49H+ NK cells, which can be directly activated by the MCMV protein, m157, preferentially proliferated during MCMV infection but did not show enhanced IFN production following direct ex vivo cytokine stimulation. Instead, mature CD11b+ and/or CD27+ NK cells responded similarly to innate cytokine stimulation regardless of Ly49H expression. Collectively, our data provide a better foundation for understanding cytokine-mediated NK cell activation during viral infection.

  • Adaptive mutations enhance assembly and cell-to-cell transmission of a high-titer hepatitis C virus genotype 5a Core-NS2 JFH1-based recombinant [PublishAheadOfPrint]

  • Recombinant hepatitis C virus (HCV) clones propagated in human hepatoma cell cultures yield relatively low infectivity titers. Here we adapted the JFH1-based Core-NS2 recombinant SA13/JFH1C3405G,A3696G (termed SA13/JFH1orig), of the poorly characterized genotype 5a, to Huh7.5 cells, yielding a virus with greatly improved spread kinetics and an infectivity titer of 6.7 log10 focus forming units (FFU)/mL. We identified several putative adaptive amino acid changes. In head-to-head infections at fixed multiplicities of infection, one SA13/JFH1orig mutant termed SA13/JFH1Core-NS5B, containing 13 amino acid changes (R114W, V187A [Core]; V235L [E1]; T385P [E2]; L782V [p7]; Y900C [NS2]; N2034D, E2238G, V2252A, L2266P, I2340T [NS5A]; A2500S, V2841A [NS5B]), displayed fitness comparable to the polyclonal high-titer adapted virus. Single-cycle virus production assays in CD81-deficient Huh7-derived cells demonstrated that these changes did not affect replication, but increased HCV assembly and specific infectivity as early as 24 hours post-transfection. Infectious co-culture assays in Huh7.5 cells showed a significant increase in cell-to-cell transmission for SA13/JFH1Core-NS5B viruses as well as viruses with only p7 and nonstructural protein mutations. Interestingly, the E2 HVR1 mutation T385P caused (i) increased sensitivity to neutralizing patient IgG and human monoclonal antibodies AR3A and AR4A and (ii) increased accessibility to the CD81 binding site without affecting the usage of CD81 and SR-BI. We finally demonstrated that SA13/JFH1orig and SA13/JFH1Core-NS5B, with and without the E2 mutation T385P, displayed similar biophysical properties following iodixanol gradient ultracentrifugation. This study has implications for investigations requiring high virus concentrations, such as studies of HCV particle composition and development of whole virus vaccine antigens.

    Importance Hepatitis C virus (HCV) is a major global healthcare burden, affecting more than 150 million people worldwide. These individuals are at high risk of developing severe end-stage liver diseases. No vaccine exists. While it is possible to produce HCV particles resembling isolates of all HCV genotypes in human hepatoma cells (HCVcc), production efficacy varies. Thus, for several important studies, including vaccine development, in vitro systems enabling high titer production of diverse HCV strains would be advantageous. Our study offers important functional data on how cell culture adaptive mutations identified in a genotype 5a JFH1-based HCVcc permit high-titer culture by affecting HCV genesis through increasing virus assembly and HCV fitness by enhancing the virus specific infectivity and cell-to-cell transmission ability, without influencing the biophysical particle properties. High-titer HCVcc like the one described in this study may be pivotal in future vaccine-related studies where large quantities of infectious HCV particles are necessary.

  • Different temporal effects of Ebola virus VP35 and VP24 proteins on the global gene expression in human dendritic cells [PublishAheadOfPrint]

  • Ebola virus (EBOV) causes a severe hemorrhagic fever with a deficient immune response, lymphopenia and lymphocyte apoptosis. Dendritic cells (DC), which trigger the adaptive response, do not mature despite EBOV infection. We recently demonstrated that DC maturation is unblocked by disabling the innate response antagonizing domains (IRADs) in EBOV VP35 and VP24 by mutations R312A and K142A, respectively. Here we analyzed the effects of VP35 and VP24 with the IRADs disabled on global gene expression in human DC. Human monocyte-derived DC were infected by wild-type EBOV (wt EBOV), or EBOVs carrying the mutation in VP35 (EBOV/VP35m), VP24 (EBOV/VP24m), or both (EBOV/VP35m/VP24m). Global gene expression at 8 and 24 hrs was analyzed by deep sequencing, and the expression of interferon (IFN) subtypes up to 5 days post-infection was analyzed by quantitative RT-PCR (qRT-PCR). Wt EBOV induced a weak global gene expression response, including markers of DC maturation, cytokines, chemokines, chemokine receptors, and multiple IFN. The VP35 mutation unblocked the expression resulting in a dramatic increase in expression of these transcripts at 8 and 24 hrs. Surprisingly, DC infected with EBOV/VP24m expressed lower levels of many of these transcripts at 8 hrs after infection, compared to wt EBOV. In contrast, at 24 hrs, expression of the transcripts increased in DC infected with any of the three mutants, as compared to wt EBOV. Moreover, sets of genes affected by the two mutations only partially overlapped. Pathway analysis demonstrated that the VP35 mutation unblocked pathways involved in antigen processing and presentation, and IFN signaling. These data suggest EBOV IRADs have profound effects on the host adaptive immune response through massive transcriptional downregulation of DC.

    IMPORTANCE. The study shows that infection of DC with EBOV, but not its mutant forms with VP35 and/or VP24 IRADs disabled, causes the global block in expression of host genes. The temporal effects of mutations disrupting the two IRADs differ, and the lists of affected genes only partially overlap such that VP35 and VP24 IRADs each have profound effects on antigen presentation by exposed DC. The global modulation of DC gene expression and the resulting lack of their maturation represent a major mechanism by which EBOV disables the T cell response, and suggests that these suppressive pathways are a therapeutic target that may unleash the T cell responses during EBOV infection.

  • The ability of the encephalitic arbovirus Semliki Forest virus to cross the blood brain barrier is determined by the charge of the E2 glycoprotein [PublishAheadOfPrint]

  • Semliki Forest virus (SFV) provides a well-characterized model system to study the pathogenesis of virus encephalitis. Several studies have used virus derived from the molecular clone SFV4. SFV4 virus does not have the same phenotype as the closely related L10 or the prototype virus from which its molecular clone was derived. In mice, L10 generates a high titer plasma viremia, is efficiently neuroinvasive and produces a fatal panencephalitis. Whereas, low dose SFV4 produces a low titer viremia, rarely enters the brain and is generally avirulent. To determine the genetic differences responsible, the consensus sequence of L10 was determined and compared to SFV4. There were twelve nucleotide differences, six were non-synonymous; these were engineered into a new molecular clone, termed SFV6. The derived virus, SFV6, generated a high titer viremia and was efficiently neuroinvasive and virulent. The phenotypic difference mapped to a single aa residue at position 162 in the E2 envelope glycoprotein; lysine in SFV4, glutamic acid in SFV6. Analysis of the L10 virus showed it contained different plaque phenotypes which differed in virulence. A lysine at E2 247 conferred a small plaque avirulent phenotype and glutamic acid a large plaque virulent phenotype. Viruses with a positively charged lysine at E2 162 or 247 were more reliant on glycosaminoglycans (GAGs) to enter cells and were selected for by passage in BHK-21 cells. Interestingly, viruses with the greatest reliance on binding to GAGs replicated to higher titers in the brain and more efficiently crossed an in vitro blood-brain barrier (BBB).

    Importance Virus encephalitis is a major disease and alphaviruses, as highlighted by the recent epidemic of chikungunya virus (CHIKV), are medically important pathogens. In addition, alphaviruses provide well-studied experimental systems with extensive literature, many tools and easy genetic modification. In this study we elucidate the genetic basis for the difference in phenotype between SFV4 and the virus stocks from which it was derived and correct this by engineering a new molecular clone. We then use this in one comprehensive study to demonstrate that positively charged aa residues on the surface of the E2 glycoprotein, mediated by binding to GAGs, determine selective advantage and plaque size in BHK-21 cells, level of viremia in mice, ability to cross an artificial BBB, efficiency of replication in the brain and virulence. Together with studies on Sindbis virus (SINV), this study provides an important advance in understanding alphavirus, and probably other virus, encephalitis.

  • A Rhesus Rhadinovirus Viral Interferon Regulatory Factor is Virion-associated and Inhibits the Early IFN Antiviral Response [PublishAheadOfPrint]

  • The interferon (IFN) response is the earliest host immune response dedicated to combating viral infection. As such, viruses have evolved strategies to subvert this potent antiviral response. Two closely related gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) and rhesus macaque rhadinovirus (RRV), are unique in that they express viral homologues to cellular interferon regulatory factors (IRFs), deemed viral IRFs (vIRFs). Cellular IRFs are a family of transcription factors that are particularly important for the transcription of type I IFNs. Here, we demonstrate a strategy employed by RRV to ensure rapid inhibition of virus-induced type I IFN induction. We found that RRV vIRF R6, when expressed ectopically, interacts with transcriptional coactivator, CREB-binding protein (CBP), in the nucleus. As a result, phosphorylated IRF3, an important transcriptional regulator in IFNbbeta; transcription, fails to effectively bind to the IFNbbeta; promoter, thus inhibiting the activation of IFNbbeta; genes. In addition, we found R6 within RRV virion particles via immunoelectron microscopy and furthermore, that, virion-associated R6 is capable of inhibiting the type I IFN response by preventing efficient binding of IRF3/CBP complexes to the IFNbbeta; promoter in the context of infection. The work shown here is the first example of a vIRF being associated with either the KSHV or RRV virion. The presence of this immunomodulatory protein in the RRV virion provides the virus with an immediate mechanism to evade the host IFN response, thus enabling the virus to effectively establish an infection within the host.

    IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related rhesus macaque rhadinovirus (RRV) are the only known viruses to encode viral homologues to cellular interferon regulatory factors (IRFs) known as vIRFs. In KSHV, these proteins have been shown to play major roles in a variety of cellular processes and are particularly important in the evasion of the host type I interferon (IFN) response. In this study, we delineate the immunomodulatory mechanism of an RRV vIRF and its ability to assist the virus in rapid immune evasion by being prepackaged within the virion, thus providing evidence, for the first time, of a virion-associated vIRF. This work further contributes to our understanding of the mechanisms behind the immunomodulation by the RRV vIRFs during infection.

  • Bile acids reduce prion conversion, reduce neuronal loss, and prolong male survival in models of prion disease. [PublishAheadOfPrint]

  • Prion diseases are fatal neurodegenerative disorders associated with the conversion of cellular prion protein (PrPC) into its aberrant infectious form (PrPSc). There is no treatment available for these diseases. The bile acids tauroursodeoxycholic acid (TUDCA) and ursodeoxycholic acid (UDCA) have been recently shown to be neuroprotective in other protein misfolding disease models including Parkinson's, Huntington's and Alzheimer's diseases, and also in humans with amyotrophic lateral sclerosis (ALS). Here we studied the therapeutic efficacy of these compounds in prion disease. We demonstrated that TUDCA and UDCA substantially reduced PrP conversion in cell-free aggregation assays as well as in chronically and acutely infected cell cultures. This effect was mediated through reduction of PrPSc seeding ability, rather than an effect on PrPC. We also demonstrated the ability of TUDCA and UDCA to reduce neuronal loss in prion infected cerebellar slice cultures. UDCA treatment reduced astrocytosis and prolonged survival in RML prion-infected mice. Interestingly, these effects were limited to the males, implying a gender-specific difference in drug metabolism. Beyond effects on PrPSc, we found that levels of phosphorylated eIF2aalpha; were increased at early time points, with correlated reductions in PSD-95. As demonstrated for other neurodegenerative diseases, we now show that TUDCA and UDCA may have a therapeutic role in prion diseases, with effects on both prion conversion and neuroprotection. Our findings, together with the fact that these natural compounds are orally bioavailable, permeable to the blood-brain barrier and FDA-approved for use in humans, make these compounds promising alternatives for the treatment of prion diseases.

    IMPORTANCE Prion diseases are fatal neurodegenerative diseases that are transmissible to humans and other mammals. There are no disease-modifying therapies available, despite decades of research. Treatment targets have included inhibition of protein accumulation, clearance of toxic aggregates, and prevention of downstream neurodegeneration. No one target may be sufficient; rather, compounds which have a multi-modal mechanism, acting on different targets, would be ideal. TUDCA and UDCA are bile acids that may fulfill this dual role; previous studies have demonstrated their neuroprotective effects in several neurodegenerative disease models, and we now demonstrate that this effect occurs in prion disease, with an added mechanistic target of upstream prion seeding. Importantly, these are natural compounds which are orally bioavailable, permeable to the blood-brain barrier and FDA-approved for use in humans with primary biliary cirrhosis. They have recently been proven efficacious in human ALS. Therefore, these compounds are promising options for the treatment of prion diseases.

  • Isolation and analysis of rare norovirus recombinants from co-infected mice using drop-based microfluidics [PublishAheadOfPrint]

  • Human noroviruses (HuNoVs) are positive-sense RNA viruses that can cause severe, highly infectious gastroenteritis. HuNoV outbreaks are frequently associated with recombination between circulating strains. Strain genotyping and phylogenetic analyses show that noroviruses often recombine in a highly conserved region near the junction of the viral polyprotein (ORF1) and capsid (ORF2) genes and occasionally within the RNA-dependent RNA polymerase (RdRP) gene. Although genotyping methods are useful for tracking changes in circulating viral populations, they only report the dominant recombinant strains and do not elucidate the frequency or range of recombination events. Furthermore, the relatively low frequency of recombination in RNA viruses has limited studies to cell culture or in vitro systems that do not reflect the complexities and selective pressures present in an infected organism. Using two murine norovirus (MNV) strains to model co-infection, we developed a microfluidic platform to amplify, detect, and recover individual recombinants following in vitro and in vivo co-infection. One-step RT-PCR was performed in picoliter drops with primers that identified the wild-type and recombinant progenies and scanned for recombination breakpoints at approximately 1-kb intervals. We detected recombination between MNV strains at multiple loci spanning the viral protease, RdRP, and capsid ORFs and isolated individual recombinant RNA genomes that were present at a frequency of 1/300,000 or greater. This study is the first to examine norovirus recombination following co-infection of an animal and suggests that the exchange of RNA among viral genomes in the infected host occurs in multiple locations and is an important driver of genetic diversity.

    IMPORTANCE RNA viruses increase diversity and escape host immune barriers by genomic recombination. Studies from a number of viral systems indicate that recombination occurs via template switching by the virus-encoded RNA-dependent RNA polymerase (RdRP). However, factors that govern the frequency and positions of recombination in an infected organism remain largely unknown. This work leverages advances in the applied physics of drop-based microfluidics to isolate and sequence rare recombinants arising from the co-infection of mice with two distinct strains of murine norovirus. This report is the first to detect and analyze norovirus recombination in an animal model.

  • Conservation of the STING-mediated cytosolic DNA sensing pathway in zebrafish [PublishAheadOfPrint]

  • Zebrafish (Danio rerio) is a unique and potential model animal for dissecting innate immune signalings. Here, we demonstrate that herpes simplex virus type -1(HSV-1) could infect zebrafish at its different developmental stages, and trigger the expression of type I interferons (IFNs) as well as ISGs in zebrafish larva. Silencing of zSTING, but not zMAVS, markedly attenuates the DNA-virus induced antiviral responses. Notably, a conserved serine residue (S373) is essential for the action of zSTING. Unexpectedly, zebrafish cGAS is dispensable for the STING signaling, whereas zDHX9 and zDDX41 are potential sensors for HSV-1 infection in vivo. Taken together, this proof-of-concept study establishes the zebrafish larva as a feasible model for investigating the cytosolic DNA sensing mechanism, shedding light on the conservation of the STING antiviral signaling pathway.

    Importance The zebrafish larva provides technical advantages for understanding host-pathogen interactions. In this study, we establish the zebrafish larva as a useful model for studying HSV-1 infection. HSV-1 infection triggers strong type I interferon production, which depends on STING expression. In addition, STING-mediated antiviral signaling is conserved in zebrafish. Interestingly, zDHX9 and zDDX41 are indispensable for detecting HSV-1 while cGAS is dispensable. This proof-of-concept study indicates that the zebrafish represents an amenable model for the investigation of cytosolic DNA sensing mechanisms.

  • Historical Outbreaks of Simian Hemorrhagic Fever in Captive Macaques Were Caused by Distinct Arteriviruses [PublishAheadOfPrint]

  • Simian hemorrhagic fever (SHF) is lethal for macaques. Based on clinical presentation and serological diagnosis, all reported SHF outbreaks were thought to be caused by different strains of the same virus, simian hemorrhagic fever virus (SHFV; Arteriviridae). Here we show that SHF outbreaks in Sukhumi in 1964 and in Alamogordo in 1989 were not caused by SHFV, but by two novel, divergent arteriviruses. Our results indicate that multiple, divergent simian arteriviruses can cause SHF.

  • Tuberous Sclerosis Complex Protein 2 (TSC2)-Independent Activation of mTORC1 by Human Cytomegalovirus pUL38 [PublishAheadOfPrint]

  • The mammalian target of rapamycin complex 1 (mTORC1) controls cell growth and anabolic metabolism and is a critical host factor activated by human cytomegalovirus (HCMV) for successful infection. The multi-functional HCMV protein pUL38 has previously been reported to activate mTORC1 by binding to and antagonizing tuberous sclerosis complex protein 2 (TSC2). pUL38 also plays a role in blocking endoplasmic reticulum stress-induced cell death during HCMV infection. In this study, we showed that a mutant pUL38 lacking the N-terminal 24 amino acids (pHA-UL3825-331) was fully functional in suppressing cell death during infection. Interestingly, pHA-UL3825-331 lost the ability to interact with TSC2 but it retained the ability to activate mTORC1, although to a lesser extent than full-length pHA-UL38. Recombinant virus expressing pHA-UL3825-331 replicated with ~10 fold less efficiency than the wild type virus at a low multiplicity of infection (MOI), but it grew similarly well at a high MOI, suggesting an MOI dependent importance of pUL38-TSC2 interaction in supporting virus propagation. Site-directed mutational analysis identified a TQ motif at amino acid residues 23-24 as critical for pUL38 interaction with TSC2. Importantly, when expressed in isolation, the TQ/AA substitution mutant pHA-UL38 TQ/AA was capable of activating mTORC1 just like pHA-UL3825-331. We also created TSC2-null U373-MG cell lines by CRISPR genome editing and showed that pUL38 was capable of further increasing mTORC1 activity in TSC2-null cells. This study therefore identified the residues important for pUL38-TSC2 interaction and demonstrated that pUL38 can activate mTORC1 in both TSC2-dependent and -independent manners.

    Importance Human cytomegalovirus (HCMV), like other viruses, depends exclusively on its host cell to propagate. It has therefore developed methods to protect against host stress responses and to usurp cellular processes to complete its life cycle. Mammalian target of rapamycin complex 1 (mTORC1) is believed to be important for virus replication, and HCMV maintains high mTORC1 activity despite the stressful cellular environment associated with infection. mTORC1 inhibitors suppressed HCMV replication in vitro and reduced the incidence of HCMV reactivation in transplant recipients. We demonstrated that mTORC1 was activated by HCMV protein pUL38 in both tuberous sclerosis complex protein 2 (TSC2)-dependent and TSC2-independent manners. pUL38-independent mode of mTORC1 activation has also been reported. These novel findings suggest the evolution of sophisticated approaches whereby HCMV activates mTORC1, indicating its importance in the biology and pathogenesis of HCMV.

  • Measles Virus Defective-interfering RNAs are Generated Frequently and Early in the Absence of C Protein and can be Destabilized by Adenosine Deaminase Acting on RNA 1-like Hypermutations [PublishAheadOfPrint]

  • Defective-interfering viral genome RNAs (DI-RNAs) can form during infections of negative strand RNA viruses and outgrow full-length viral genomes, thereby modulating the severity and duration of infection. Here we document frequent de novo generation of copyback DI-RNAs from independent rescue events both for vaccine (vac2) and wild-type (IC323) measles viruses as early as passage 1 after virus rescue. Moreover, vaccine and wild-type C protein-deficient (CKO) measles viruses generated about ten times more DI-RNAs than parental virus, suggesting that C enhances processivity of the viral polymerase. We obtained nucleotide sequences of 65 individual DI-RNAs, identified breakpoints and re-initiation sites and predicted their structural features. Several DI-RNAs possessed clusters of A-to-G or U-to-C transitions. Sequences flanking these mutation sites were characteristic of those favored by adenosine deaminase acting on RNA 1 (ADAR1), which catalyzes in double-stranded RNA the C6 deamination of adenosine to produce inosine that is recognized as guanosine, a process known as A-to-I RNA editing. In individual DI-RNAs the transitions were of the same type and occurred on both sides of the breakpoint. These patterns of mutations suggest that ADAR1 edits unencapsidated DI-RNAs that formed double strand RNA structures. Encapsidated DI-RNAs were incorporated into virus particles, which reduced infectivity of virus stocks. The CKO-phenotype was dominant: DI-RNAs derived from vac2-CKO suppressed replication of vac2, as shown by co-infections of interferon-incompetent lymphatic cells with viruses expressing different fluorescent reporter proteins. In contrast, co-infection with a C protein-expressing virus did not counteract the suppressive phenotype of DI-RNAs.

    Importance Recombinant measles viruses (MV) are in clinical trials as cancer therapeutics and as vectored vaccines for HIV-AIDS and other infectious diseases. Efficacy of MV-based vectors depends on their replication proficiency and immune activation capacity. Here we document that copyback defective-interfering RNAs (DI-RNAs) are generated by recombinant vaccine and wild-type MVs immediately after rescue. The MV C protein interferes with DI-RNA generation and may enhance processiviry of the viral polymerase. We frequently detected clusters of A-to-G or U-to-C transitions and noted that sequences flanking individual mutations contain motifs favoring recognition by the adenosine deaminase acting on RNA 1 (ADAR1). The consistent type of transitions on the DI-RNAs indicates that these were direct substrates for editing by ADAR1. The ADAR1-mediated biased hypermutation events are consistent with the protein kinase R (PKR)-ADAR1 balancing model of innate immunity activation. We show by co-infection that the C-defective phenotype is dominant.

  • Regulation of DNA Damage Signaling and Cell Death Responses by Epstein-Barr virus Latent Membrane Proteins (LMP) 1 and LMP2A in Nasopharyngeal Carcinoma Cells [PublishAheadOfPrint]

  • Nasopharyngeal carcinoma (NPC) is closely associated with latent Epstein-Barr virus (EBV) infection. Although EBV infection of pre-neoplastic epithelial cells is not immortalizing, EBV can modulate oncogenic and cell death mechanisms. The viral latent membrane proteins (LMP) 1 and LMP2A are consistently expressed in NPC, and can co-operate in bi-transgenic mice expressed from the keratin-14 promoter to enhance carcinoma development in an epithelial chemical carcinogenesis model. In this study, LMP1 and LMP2A were co-expressed in the EBV-negative NPC cell line HK1, and examined for combined effects in response to genotoxic treatments. In response to DNA damage activation, LMP1 and LMP2A co-expression reduced H2AX (S139) phosphorylation and caspase cleavage induced by a lower dose (5mmu;M) of the topoisomerase II inhibitor etoposide. Regulation of H2AX occurred before the onset of caspase activation, without modulation of other DNA damage signaling mediators including ATM, Chk1 or Chk2, and was additionally suppressed by inducers of DNA single strand breaks (SSBs) and replication stress. Despite reduced DNA damage repair signaling, LMP1-2A co-expressing cells recovered from cytotoxic doses of etoposide, however LMP1 expression was sufficient for this effect. LMP1 and LMP2A co-expression did not enhance cell growth, with moderate increase of cell motility to fibronectin. This study supports that LMP1 and LMP2A jointly regulate DNA repair signaling and cell death activation with no further enhancement in the growth properties of neoplastic cells.

    Importance NPC is characterized by clonal EBV infection and accounts for ggt;78,000 annual cancer cases with increased incidence in endemic regions such as Southeast Asia. The latent proteins LMP1 and LMP2A co-expressed in NPC can individually enhance growth or survival properties in epithelial cells, but their combined effects and potential regulation of DNA repair and checkpoint mechanisms are relatively undetermined. In this study, LMP1-2A co-expression suppressed activation of the DNA damage response (DDR) protein H2AX induced by selective genotoxins that promote DNA replication stress or SSBs. Expression of LMP1 was sufficient to recover cells, resulting in outgrowth of LMP1 and LMP1-2A co-expressing cells and indicating distinct LMP1-dependent effects in restoration of replicative potential. These findings demonstrate novel properties for LMP1 and LMP2A in the co-operative modulation of DDR and apoptotic signaling pathways, further implicating both proteins in the progression of NPC and epithelial malignancies.

  • HIV-1 Env and Nef cooperatively contribute to pDCs activation via CD4-dependent mechanisms [PublishAheadOfPrint]

  • Plasmacytoid dendritic cells (pDCs) are the major source of type I IFN (IFN-I) in response to Human Immunodeficiency Virus type-1 (HIV-1) infection. pDCs are rapidly activated during HIV-1 infection and are implicated in both reducing early viral load as well as contributing to HIV-1-induced pathogenesis. However, most cell-free HIV-1 isolates are inefficient in activating human pDCs, and the mechanisms of HIV-1 recognition by pDCs and pDC activation are not clearly defined. In this study we report that two genetically similar HIV-1 variants (R3A and R3B) isolated from a rapid progressor differentially activated pDCs to produce IFNaalpha;. The highly pathogenic R3A efficiently activated pDCs to induce robust IFNaalpha; production, while the less pathogenic R3B did not. The viral determinant for efficient pDC activation was mapped to the V1V2 region of R3A Env, which also correlated with enhanced CD4 binding activity. Furthermore, we showed that the Nef protein was also required for the activation of pDCs by R3A. Analysis of a panel of R3A Nef functional mutants demonstrated that Nef domains involved in CD4 down-regulation were necessary for R3A to activate pDCs. Our data indicate that R3A-induced pDC activation depends on (1) the high affinity of R3A Env to bind CD4 receptor and (2) Nef activity, which is involved in CD4 down-regulation. Our findings provide new insights into the mechanism by which HIV-1 induces IFNaalpha; in pDCs, which contributes to pathogenesis.

    Importance Plasmacytoid dendritic cells (pDCs) are the major type I interferon (IFN-I) producing cells, and IFN-Is actually contribute to pathogenesis during chronic viral infections. How HIV-1 activates pDCs and the role of pDC/IFN-I in HIV-1 pathogenesis remain unclear. We report here the highly pathogenic HIV-R3A efficiently activated pDCs to induce IFNaalpha; production, while most HIV-1 isolates are inefficient in activating pDCs. We have discovered that R3A-induced pDC activation depends on (1) the high affinity of R3A Env to bind CD4 receptor and (2) Nef activity, which is involved in CD4 down-regulation. Our findings thus provide new insights into the mechanism by which HIV-1 induces IFNaalpha; in pDCs and contributes to HIV-1 pathogenesis. These novel findings will be of great interest to those working on the role of IFN and pDCs in HIV-1 pathogenesis in general, and on the interaction of HIV-1 with pDCs in particular.

  • Molecular mechanism for LAMP1 recognition by Lassa Virus [PublishAheadOfPrint]

  • Lassa virus is a notorious human pathogen that infects many thousands of people each year in West Africa, causing severe viral hemorrhagic fevers and significant mortality. The surface glycoprotein of Lassa virus mediates receptor recognition through its GP1 subunit. Here we report the crystal structure of GP1 from Lassa virus, which is the first representative GP1 structure for Old World arenaviruses. We identify a unique triad of histidines that forms a binding site for LAMP1, a known lysosomal protein recently discovered to be a critical receptor for internalized Lassa virus at acidic pH. We demonstrate that mutation of this histidine-triad, which is highly conserved among Old World arenaviruses, impairs LAMP1 recognition. Our biochemical and structural data further suggest that GP1 from Lassa may undergo irreversible conformational changes that could serve as an immunological decoy mechanism. Together with a variable region that we identify on the surface of GP1, those could be two distinct mechanisms that Lassa virus utilizes to avoid antibody-based immune response.

    IMPORTANCE Structural data at atomic resolution for viral proteins is key for understanding their function at the molecular level and can facilitate novel avenues for combating viral infections. Here we use X-ray protein crystallography to decipher the crystal structure of the receptor-binding domain (GP1) from Lassa virus. This is a pathogenic virus that causes significant illness and mortality in West Africa. This structure reveals the overall architecture of GP1 domains from the group of viruses known as the Old World arenaviruses. Using this structural information we elucidate the pH switch and binding mechanisms of Lassa virus to LAMP1, a recently identified host receptor that is critical for successful infection. Lastly, our structural analysis suggests two novel immune evasion mechanisms that Lassa virus may utilize to escape antibody-based immune response.

  • High-throughput analysis of human cytomegalovirus genome diversity highlights the widespread occurrence of gene-disrupting mutations and pervasive recombination [PublishAheadOfPrint]

  • Human cytomegalovirus is a widespread pathogen of major medical importance. It causes significant morbidity and mortality in the immunocompromised and congenital infections can result in severe disabilities or stillbirth. Development of a vaccine is prioritized, but no candidate is close to release. Although correlations of viral genetic variability with pathogenicity are suspected, knowledge about strain diversity of the 235kb genome is still limited. In this study, 96 full-length human cytomegalovirus genomes from clinical isolates were characterized, quadrupling the available information for full-genome analysis. These data provide the first high-resolution map of human cytomegalovirus interhost diversity and evolution. We show that cytomegalovirus is significantly more divergent than all other human herpesviruses and highlight hotspots of diversity in the genome. Importantly, 75% of strains are not genetically intact, but contain disruptive mutations in a diverse set of 26 genes, including immunomodulative genes UL40 and UL111A. These mutants are independent from culture passaging artifacts and circulate in natural populations. Pervasive recombination, which is linked to the widespread occurrence of multiple infections, was found throughout the genome. Recombination density was significantly higher than in other human herpesviruses and correlated with strain diversity. While the overall effects of strong purifying selection on virus evolution are apparent, evidence of diversifying selection was found in several genes encoding proteins that interact with the host immune system, including UL18, UL40, UL142 and UL147. These residues may present phylogenetic signatures of past and ongoing virus-host interactions.

    IMPORTANCE Human cytomegalovirus has the largest genome of all viruses that infect humans. Currently, there is a great interest in establishing associations between genetic variants and strain pathogenicity of this herpesvirus. Since the number of publicly available full-genome sequences is limited, knowledge about strain diversity is highly fragmented and biased towards a small set of loci. Combined with our previous work, we have now contributed 101 complete genome sequences. We have used these data to conduct the first high-resolution analysis of interhost genome diversity, providing an unbiased and comprehensive overview of cytomegalovirus variability. These data are of major value to the development of novel antivirals and a vaccine and to identify potential targets for genotype-phenotype experiments. Furthermore, they have enabled a thorough study of the evolutionary processes that have shaped cytomegalovirus diversity.

  • Cooperativity of HIV-specific cytolytic CD4+ T cells and CD8+ T cells in control of HIV viremia [PublishAheadOfPrint]

  • CD4+ T cells play a pivotal role in the control of chronic viral infections. Recently, non-traditional CD4+ T cell functions beyond helper effects have been described and a role of cytolytic CD4+ T cells in the control of HIV infection suggested. Here we define the transcriptional, phenotypic, and functional profiles of HIV-specific cytolytic CD4+ T cells. Fluidigm BioMark and multiparameter flow cytometric analysis of HIV-specific cytolytic CD4+ T cells revealed a distinct transcriptional signature compared to Th1 CD4+ cells, but shared similar features with HIV-specific cytolytic CD8+ T cells. Furthermore, HIV-specific cytolytic CD4+ T cells showed comparable killing activity relative to HIV-specific CD8+ T cells and worked cooperatively in the elimination of virally infected cells. Interestingly, we found that cytolytic CD4+ T cells emerge early during acute HIV infection and tightly follow acute viral load trajectory. This emergence was associated to the early viral set point, suggesting an involvement in early control, in spite of their susceptibility to HIV infection. Our data suggest cytolytic CD4+ T cells as an independent subset distinct from Th1 cells that show combined activity with CD8+ T cells in the long-term control of HIV infection.

    Importance The ability of the immune system to control chronic HIV infection is of critical interest to both vaccine design and therapeutic approaches. Much research has focused on the effect of the ability of CD8+ T cells to control the virus, while CD4+ T cells have been overlooked as effectors in HIV control due to the fact that they are preferentially infected. Here we show that a subset of HIV-specific CD4+ T cells cooperate in the cytolytic control of HIV viral replication. Moreover, these cells represent a distinct subset of CD4+ T cells showing significant transcriptional and phenotypical differences to HIV-specific Th1 cells, but have similarities to CD8+ T cells. These findings are important for our understanding of HIV immunopathology.

  • Comparable antigenicity and immunogenicity of oligomeric forms of a novel, acute HIV-1 subtype C gp145 envelope for use in preclinical and clinical vaccine research [PublishAheadOfPrint]

  • Eliciting broadly reactive functional antibodies remains a challenge in HIV-1 vaccine development, complicated by variations in envelope (Env) subtype and structure. The majority of new global HIV-1 infections are subtype C and novel antigenic properties have been described for C Envs. Thus, an HIV-1 subtype C Env protein (CO6980v0c22) from an acutely infected (Fiebig stage I/II) subject was developed as a research reagent and candidate immunogen. The gp145 envelope is a novel immunogen with a fully intact membrane proximal external region (MPER), extended by a poly-lysine tail. Soluble gp145 was enriched for trimers that yielded the expected "fan-blade" motifs when visualized by cryo-electron microscopy. The CO6980v0c22 gp145 reacts with the 4E10, PG9, PG16 and VRC01 HIV-1 neutralizing monoclonal antibodies (mAbs), as well as the V1/V2 specific PGT121, 697, 2158 and 2297 mAbs. Different gp145 oligomers were tested for immunogenicity in rabbits, and purified dimers, trimers and larger multimers elicited similar levels of cross-clade binding and neutralizing antibodies to tier 1, and some tier 2 viruses. Immunized rabbit sera did not neutralize the highly resistant CO6980v0c22 pseudovirus, but did inhibit the homologous infectious molecular clone (IMC) in a PBMC assay. This Env is currently in GMP production to be made available for use as a clinical research tool and further evaluation as a candidate vaccine.

    IMPORTANCE At present, the product pipeline for HIV vaccines is insufficient and is limited by inadequate capacity to produce large quantities of vaccine to standards required for human clinical trials. Such products are required to evaluate critical questions of vaccine formulation, route, dosing and schedule as well as to establish vaccine efficacy. The gp145 Env protein presented in this study forms physical trimers, binds to many of the well-characterized broad neutralizing monoclonal antibodies that target conserved Env epitopes, and induce cross-subtype neutralizing antibodies as measured in both cell line and primary cell assays. This subtype C Env gp145 protein is currently undergoing GMP production for use as a reagent for preclinical studies and for human clinical research. This product will serve as a reagent for comparative studies and may represent a next generation, candidate HIV immunogen.

  • A Two-year Follow-up of Macaques Developing Intermittent Control of the HIV Homolog SIVmac251 in the Chronic Phase of the Infection [PublishAheadOfPrint]

  • Off-therapy control of viremia by HIV-infected individuals has been associated with two likely players: a restricted viral reservoir and an efficient cell-mediated immune response. We previously showed that a combination of highly suppressive antiretroviral therapy, and two experimental drugs, i.e. auranofin and buthionine sulfoximine, was able to reduce the viral reservoir, elicit efficient cell-mediated antiviral responses, and induce intermittent post-therapy viral load control in chronically SIVmac251-infected macaques. We here show that the macaques that had received this drug combination and then stopped antiretroviral therapy were also able to maintain low numbers of activated CD4+ T-cells at viral rebound. Moreover, these macaques consistently displayed low-level SIV diversity, which was in line with the strong and broadly reactive cell-mediated immune responses against conserved Gag antigens. Extended follow-up showed that the two macaques that had received the complete drug combination remained healthy and did not develop AIDS in two years of follow-up after therapy suspension. This disease-free survival is longer than twice the average time of progression to AIDS in SIVmac251-infected rhesus macaques. These results suggest that limited numbers of activated T-cells at viral rebound and subsequent development of broadly reactive cell-mediated responses may be interrelated in reducing the viral reservoir.

    Importance The HIV reservoir in CD4+ T-cells represents one main obstacle to HIV eradication. Recent studies, however, show that a drastic reduction of this reservoir is insufficient for inducing a functional cure of AIDS. In the present report, we thoroughly studied and subjected to long-term follow-up two macaques showing intermittent control of the virus following suspension of antiretroviral therapy plus an experimental anti-reservoir treatment, i.e. the gold salt auranofin and the investigational chemotherapeutic agent buthionione sulfoximine (BSO). We found that these drugs were able to decrease the number of activated CD4+ T-cells, which are preferential targets for HIV infection. Then, efficient immune responses against the virus were developed in the macaques, which remained healthy during two years of follow-up. This result may furnish another building block to future attempts to cure HIV/AIDS.

  • Phage therapy: a step forward in the treatment of Pseudomonas aeruginosa infections [PublishAheadOfPrint]

  • Antimicrobial resistance constitutes one of the major worldwide public health concerns. Bacteria are becoming resistant to the vast majority of antibiotics and nowadays, a common infection can be fatal. To revert this situation, the use of phages for the treatment of bacterial infections has been extensively studied as an alternative therapeutic strategy. Since P. aeruginosa is one of the most common causes of healthcare-associated infections, many studies have reported the in vitro and in vivo antibacterial efficacy of phage therapy against this bacterium. This review collects data of all the P. aeruginosa phages sequenced to date, providing a better understanding about their biodiversity. This review will further address the in vitro and in vivo results obtained by using phages to treat or prevent P. aeruginosa infections as well as the major hurdles associated with this therapy.

  • A single point mutation creating a furin cleavage site in the spike protein renders porcine epidemic diarrhea coronavirus trypsin-independent for cell entry and fusion. [PublishAheadOfPrint]

  • The emerging porcine epidemic diarrhea virus (PEDV) requires trypsin supplementation to activate its S protein for membrane fusion and virus propagation in cell culture. By substitution of a single amino acid in the S protein we created a recombinant PEDV (PEDV-SFCS) with an artificial furin protease cleavage site N-terminal of the putative fusion peptide. PEDV-SFCS exhibited trypsin-independent cell-cell fusion and was able to replicate in culture cells independent of trypsin, though to low titer.

  • Respiratory syncytial virus infection upregulates NLRC5 and MHC I expression through RIG-I induction in airway epithelial cells [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) is the leading cause of acute respiratory tract viral infection in infants, causing bronchiolitis and pneumonia. The host antiviral response to RSV acts via retinoic acid inducible gene I (RIG-I). We show here that RSV infection upregulates MHC I expression through the induction of NLRC5, a NOD-like, CARD domain-containing intracellular protein that has recently been identified as a class I MHC transactivator (CITA). RSV infection of A549 cells promotes upregulation of NLRC5 via IFNbbeta; production since the NLRC5-inducing activity in a conditioned medium from RSV-infected A549 cells was removed by antibody to IFNbbeta;, but not to IFN. RSV infection resulted in RIG-I upregulation, induction of NLRC5, and MHC I. Suppression of RIG-I induction significantly blocked NLRC5 as well as MHC I upregulation and diminished IRF3 activation. Importantly, Vero cells deficient in interferon production still upregulated MHC I following the introduction of RSV genome by infection or transfection, further supporting a key role for RIG-I. A model is therefore formed in which the host upregulates MHC I expression during RSV infection directly via the induction of RIG-I and NLRC5 expression. Since elevated expression of MHC I molecules can sensitize host cells to T lymphocyte-mediated cytotoxicity or immunopathologic damage, the results have significant implications for the modification of immunity in RSV disease.

    Importance Human respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in infants and young children worldwide. Early life infection is linked to persistent wheezing and allergic asthma in later life, possibly related to upregulation of major histocompatibility class I on the cell surface which facilitates molecules cytotoxic T cell activation and antiviral immunity. Here we show that RSV infection of lung epithelial cells induces expression of RIG-I, resulting in induction of a class I MHC transactivator NLRC5 and subsequent upregulation of MHC I. Suppression of RIG-I induction blocked RSV-induced NLRC5 expression and MHC I upregulation. Increased MHC I expression may exacerbate the disease condition of RSV condition due to immunopathologic damage, linking innate immune response to RSV disease.

  • Post-translational Modifications of Baculovirus Protamine-like Protein P6.9 and the Significance of Its Hyperphosphorylation for Viral Very Late Gene Hyperexpression [PublishAheadOfPrint]

  • Many viruses utilize viral or cellular chromatin machinery for efficient infection. Baculoviruses encode a conserved protamine-like protein P6.9. This protein plays essential roles in various viral physiological processes during infection. However, the mechanism by which P6.9 regulates transcription remains unknown. In this study, 7 phosphorylated species of P6.9 were resolved in Sf9 cells infected with the baculovirus type species Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Mass spectrometry identified 22 phosphorylation and 10 methylation sites but no acetylation sites in P6.9. Immunofluorescence demonstrated that the P6.9 and virus-encoded serine/threonine kinase PK1 exhibited a similar distribution pattern in infected cells, and co-immunoprecipitation confirmed the interaction between them. Upon pk1 deletion, nucleocapsid assembly and polyhedron formation were interrupted and the transcription of viral very late genes were down-regulated. Interestingly, we found that the 3 most phosphorylated P6.9 species vanished in Sf9 cells transfected with the pk1-deletion mutant, suggesting that PK1 is involved in the hyperphosphorylation of P6.9. Mass spectrometry suggested that the phosphorylation of the 7 Ser/Thr and 5 Arg residues in P6.9 was PK1-dependent. Substitution of the 7 Ser/Thr residues with Ala resulted in a P6.9 phosphorylation pattern similar to that of the pk1-deletion mutant. Importantly, the decreases in the transcription level of viral very late genes and the viral infectivity were consistent. Our findings reveal that P6.9 hyperphosphorylation is a precondition for the maximal hyperexpression of baculovirus very late genes and provide the first experimental insight into the function of the baculovirus protamine-like protein and the related protein kinase in epigenetics.

    IMPORTANCE Diverse post-translational modifications (PTMs) of histones constitute a code that creates binding platforms that recruit transcription factors to regulate gene expression. Many viruses also utilize host- or virus-induced chromatin machinery to promote efficient infections. Baculoviruses encode a protamine-like protein P6.9, which is required for a variety of processes in the infection cycle. Currently, P6.9's PTM sites and its regulating factors remain unknown. Here, we found that P6.9 could be categorized as unphosphorylated, hypophosphorylated, and hyperphosphorylated species and that a virus-encoded serine/threonine kinase PK1 was essential for P6.9 hyperphosphorylation. Abundant PTM sites on P6.9 were identified, among which 7 Ser/Thr phosphorylated sites were PK1-dependent. Mutation of these Ser/Thr sites reduced very late viral genes transcription and viral infectivity, indicating that the PK1-mediated P6.9 hyperphosphorylation contributes to viral proliferation. These data suggest that a potential "code" might exist in the sophisticated PTM of viral protamine-like proteins and participate in viral gene transcription.

  • Transcriptome profiling of the virus-induced innate immune response in Pteropus vampyrus and its attenuation by Nipah virus interferon antagonist functions [PublishAheadOfPrint]

  • Bats are important reservoirs for several viruses, many of which cause lethal infections in humans but have reduced pathogenicity in bats. As the innate immune response is critical for controlling viruses, the nature of this response in bats, and how it may differ from other mammals, is of great interest. Using next generation mRNAseq, we profiled the transcriptional response of Pteropus vampyrus bat kidney (PVK) cells to Newcastle disease virus (NDV), an avian paramyxovirus known to elicit a strong innate immune response in mammalian cells. This bat species is a known reservoir of Nipah virus (NiV) and Hendra virus (HeV). Analysis of the 200-300 regulated genes showed that interferon (IFN) and antiviral pathways are highly upregulated in NDV infected PVK cells, including genes such as IFN bbeta;, RIGI, MDA5, ISG15, and IRF1. NDV infected cells also upregulated several genes not previously characterized as antiviral such as RND1, SERTAD1, CHAC1, and MORC3. In fact, we show that MORC3 is induced by both IFN and NDV infection in PVK cells, but by neither stimulus in human A549 cells. In contrast to NDV, HeV and NiV infection of PVK cells failed to induce these innate immune genes. Likewise, an attenuated response was observed in PVK cells infected with recombinant NDVs expressing the NiV IFN antagonist proteins V and W. This study provides the first global profile of a robust virus-induced innate immune response in bats and indicates that henipavirus IFN antagonist mechanisms are likely active in bat cells.

    Importance Bats are the reservoir host for many highly pathogenic human viruses, including henipaviruses, lyssaviruses, SARS coronavirus, and filoviruses, and many other viruses have also been isolated from bats. Viral infections are reportedly asymptomatic or heavily attenuated in bat populations. Despite their ecological importance to viral maintenance, research into their immune system and mechanisms for viral control has only recently begun. Nipah virus and Hendra virus are two paramyxoviruses associated with high mortality rates in humans and whose reservoir is the Pteropus genus of bats. Greater knowledge of the innate immune response of P. vampyrus to viral infection may elucidate how bats serve as a reservoir for so many viruses.

  • The minor capsid protein VP11 of thermophilic bacteriophage P23-77 facilitates virus assembly using lipid-protein interactions [PublishAheadOfPrint]

  • Thermus thermophilus bacteriophage P23-77 is the type member of a new virus family of icosahedral, tailless, inner membrane-containing dsDNA viruses infecting thermophilic bacteria and halophilic archaea. Those viruses have a unique capsid architecture consisting of two major capsid proteins assembled in various building blocks. We analyzed the function of minor capsid protein VP11, which is the third known capsid component in bacteriophage P23-77. Our findings show that VP11 is a dynamically elongated dimer with predominantly aalpha;-helical secondary structure and a high thermal stability. A high proportion of basic amino acids in the protein enable electrostatic interaction with negatively charged molecules including nucleic acid and large unilamellar lipid vesicles (LUVs). The plausible biological function for VP11 is elucidated by demonstrating the interactions of VP11 with Thermus derived LUVs and with the major capsid proteins by means of dynamic light scattering technique. In particular, the major capsid protein VP17 was able to link VP11-complexed LUVs into larger particles, whereas the other P23-77 major capsid protein, VP16, was unable to link VP11-comlexed LUVs. Our results rule out a previously suggested penton function for VP11. Instead, the electrostatic membrane association of VP11 triggers binding of major capsid protein VP17, thus facilitating a controlled incorporation of the two different protein species into the assembling capsid.

    Importance The study of thermophilic viruses with inner membranes provides valuable insights into the mechanisms used for stabilization and assembly of protein-lipid systems at high temperatures. Our results reveal a novel way by which an internal membrane and outer capsid shell are linked in a virus that uses two different major protein species for capsid assembly. We show that a positive protein-charge is important to form electrostatic interactions with the lipid surface, thereby facilitating the incorporation of other capsid proteins on the membrane surface. This implies an alternative function for basic proteins present in the virions of other lipid-containing thermophilic viruses, whose proposed role in genome packaging is based on their capability to bind DNA. The unique minor capsid protein of bacteriophage P23-77 resembles in its characteristics the scaffolding proteins of tailed phages, though it constitutes a substantial part of the mature virion.

  • The Nuclear DNA Sensor IFI16 Acts as a Restriction Factor for Human Papillomavirus Replication through Epigenetic Modifications of the Viral Promoters [PublishAheadOfPrint]

  • The human interferon-inducible IFI16 protein, an innate immune sensor of intracellular DNA, was recently demonstrated to act as a restriction factor for human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) infection by inhibiting both viral DNA replication and transcription. Through the use of two distinct cellular models, this study provides strong evidence in support of the notion that IFI16 can also restrict HPV18 replication. In the first model, an immortalized keratinocyte cell line (NIKS) was used in which the IFI16 protein was knocked down through the use of siRNA technology, and overexpressed following transduction with the AdVIFI16 vector. The second model consisted of U2OS cells transfected by electroporation with HPV18 minicircles. In differentiated IFI16-silenced NIKS-HPV18 cells, viral load values were significantly increased compared with differentiated control cells. Consistent with this, IFI16 overexpression severely impaired HPV18 replication in both NIKS and U2OS cells, thus confirming its antiviral restriction activity. In addition to the inhibition of viral replication, IFI16 was also able to reduce viral transcription, as demonstrated by viral gene expression analysis in U2OS cells carrying episomal HPV18 minicircles and HeLa cells. We also provide evidence that IFI16 promotes the addition of heterochromatin marks and the reduction of euchromatin marks on viral chromatin at both early and late promoters, thus reducing both viral replication and transcription. Altogether, these results argue that IFI16 restricts chromatinised HPV DNA through epigenetic modifications and executes a broad surveillance role against viral DNA in the nucleus that is not restricted to Herpesviruses.

    IMPORTANCE Intrinsic immunity is mediated by cellular restriction factors that are constitutively expressed and active even before a pathogen enters the cell. The host nuclear factor IFI16 acts as sensors of foreign DNA and antiviral restriction factors, as recently demonstrated by our group for human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1). Here, we provide the first evidence that IFI16 inhibits HPV18 replication by repressing viral gene expression and replication. This antiviral restriction activity was observed in immortalized keratinocytes transfected with the religated genomes and in U2OS transfected with HPV18 minicircles suggesting that it is not cell-type specific. We also show that IFI16 promotes the assembly of heterochromatin on HPV DNA. These changes in viral chromatin structure lead to the generation of a repressive state at both early and late HPV18 promoters, thus implicating a role of this protein in the epigenetic regulation of HPV gene expression and replication.

  • Human memory B cells producing potent cross-neutralizing antibodies against human parechovirus; implications for prevalence, treatment and diagnosis [PublishAheadOfPrint]

  • The family of Picornaviridae is a large and diverse group of positive sense RNA viruses, including the human enteroviruses (EVs) and human parechoviruses (HPeVs). The human immune response against EVs and HPeVs is thought to be mainly humoral and an insufficient neutralizing antibody (Ab) response during infection is a risk factor and can ultimately be life threatening. The accessibility of different antigenic sites and observed cross-reactivity makes HPeVs a good target for development of therapeutic human monoclonal antibodies (mAbs). In this study we generated two different human mAbs specific for HPeV by screening culture supernatants of Ab producing human B cell cultures for direct neutralization of HPeV1. Both mAbs showed HPeV1-specific neutralization, as well as neutralization of HPeV2. One antibody, AM18, cross-neutralized HPeV4, 5 and 6 and coxsackievirus A9 (CV-A9). VP1 capsid protein specific assays confirmed that AM18 bound VP1 of HPeV1, 2 and 4 with high affinity (11.5 pM). In contrast, the HPeV1specific mAb AM28, which neutralized HPeV1 even more efficiently compared to AM18, showed no cross-reactivity with HPeV3-6 or other EVs and did not bind any of the capsid proteins, suggesting AM28 is specific for a conformation dependent, non-linear epitope on the virus. The discovery of mAbs cross-reactive between HPeVs may help development of HPeV treatment options by antibodies and vaccine design based on epitopes recognized by these antibodies.

    Importance HPeV infections are widespread among young children and adults, causing a broad range of disease. Infections can be severe and life threatening, while no antiviral treatment is available. Given the absence of neutralizing Abs is a risk factor for severe disease in infants, treatment of picornavirus infections with mAbs would be a therapeutic option. To study antibody neutralization of HPeV in more detail, we generated two different HPeV1-specific human mAbs. Both mAbs show HPeV1-specific neutralization and cross-neutralized HPeV2. One mAb also cross-neutralized with other HPeVs. Surprisingly, this mAb also neutralized CV-A9. These mAbs provide a unique tool for further research and for diagnosis (antigen detection) and possible treatment of HPeV infections.

  • LMP1-induced Sumoylation Influences the Maintenance of EBV Latency Through KAP1. [PublishAheadOfPrint]

  • As a herpesvirus, EBV establishes a latent infection that can periodically undergo reactivation, resulting in lytic replication and the production of new infectious virus. Latent membrane protein-1 (LMP1), the principal viral oncoprotein, is a latency-associated protein implicated in regulating viral reactivation and the maintenance of latency. We recently found that LMP1 hijacks the SUMO-conjugating enzyme, Ubc9, via its C-terminal activating region-3 (CTAR3), and induces the sumoylation of cellular proteins. Because protein sumoylation can promote transcriptional repression, we hypothesized that LMP1-induced protein sumoylation induces the repression of EBV lytic promoters and helps maintain the viral genome in its latent state. We now show that with inhibition of LMP1-induced protein sumoylation, the latent state becomes less stable or "leakier" in EBV-transformed LCLs. The cells are also more sensitive to viral reactivation induced by irradiation, which results in increased production and release of infectious virus, as well as increased susceptibility to ganciclovir treatment. We have identified a target of LMP1-mediated sumoylation that contributes to the maintenance of latency in this context: KRAB-associated protein-1 (KAP1). LMP1 CTAR3-mediated sumoylation regulates the function of KAP1. KAP1 also binds to EBV oriLyt and immediate early promoters in a CTAR3-dependent manner, and inhibition of sumoylation processes abrogates the binding of KAP1 to these promoters. These data provide an additional line of evidence that supports our findings that CTAR3 is a distinct functioning regulatory region of LMP1 and confirm that LMP1-induced sumoylation may help stabilize the maintenance of EBV latency.

    Importance Epstein-Barr virus (EBV) Latent Membrane Protein-1 (LMP1) plays an important role in the maintenance of viral latency. Previously, we documented that LMP1 targets cellular proteins to be modified by an ubiquitin-like protein (SUMO). We have now identified a function for this LMP1-induced modification of cellular proteins in the maintenance of EBV latency. Because latently infected cells have to undergo viral reactivation in order to be vulnerable to anti-viral drugs, these findings identify a new way to increase the rate of EBV reactivation, which increases cell susceptibility to anti-viral therapies.

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