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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]

  • What DNA Viral Genomic Rearrangements Tell Us about Persistence [Gems]

  • Understanding the life cycle and pathogenesis of animal viruses requires that we have systems in which the viruses can replicate and cause disease. For the latter, we rely upon animal models or information that we can obtain from studying natural infections of humans and other animals. For the former, however, we are largely dependent on the availability of cell culture systems in which viruses can be propagated to investigate the molecular mechanisms of viral replication. For many years, it was assumed that replication in culture provided an accurate description of the life cycle of the organism. In this Gem, we will discuss two viruses, polyomavirus and cytomegalovirus, in which cell culture systems have accidentally provided unique potential insights into viral replication and persistence in their hosts.

  • Expanding the Role of the Virome: Commensalism in the Gut [Gems]

  • Viruses affect host physiology beyond causing acute disease, thereby giving rise to the concept that the virome is a component of the microbiome. However, the role of the enteric virome is understudied relative to the fast-paced research examining commensal bacteria in the intestine. In this article, I discuss our recent work on murine norovirus indicating that an animal virus in the intestine can provide many of the signals to the host that have been attributed to commensal bacteria. Our findings suggest that the surge in microbiome research should incorporate examination of the enteric virome.

  • Receptor Recognition Mechanisms of Coronaviruses: a Decade of Structural Studies [Minireviews]

  • Receptor recognition by viruses is the first and essential step of viral infections of host cells. It is an important determinant of viral host range and cross-species infection and a primary target for antiviral intervention. Coronaviruses recognize a variety of host receptors, infect many hosts, and are health threats to humans and animals. The receptor-binding S1 subunit of coronavirus spike proteins contains two distinctive domains, the N-terminal domain (S1-NTD) and the C-terminal domain (S1-CTD), both of which can function as receptor-binding domains (RBDs). S1-NTDs and S1-CTDs from three major coronavirus genera recognize at least four protein receptors and three sugar receptors and demonstrate a complex receptor recognition pattern. For example, highly similar coronavirus S1-CTDs within the same genus can recognize different receptors, whereas very different coronavirus S1-CTDs from different genera can recognize the same receptor. Moreover, coronavirus S1-NTDs can recognize either protein or sugar receptors. Structural studies in the past decade have elucidated many of the puzzles associated with coronavirus-receptor interactions. This article reviews the latest knowledge on the receptor recognition mechanisms of coronaviruses and discusses how coronaviruses have evolved their complex receptor recognition pattern. It also summarizes important principles that govern receptor recognition by viruses in general.

  • Generation and Evaluation of Clade C Simian-Human Immunodeficiency Virus Challenge Stocks [Vaccines and Antiviral Agents]

  • The development of a panel of mucosally transmissible simian-human immunodeficiency virus (SHIV) challenge stocks from multiple virus clades would facilitate preclinical evaluation of candidate HIV-1 vaccines and therapeutics. The majority of SHIV stocks that have been generated to date have been derived from clade B HIV-1 env sequences from viruses isolated during chronic infection and typically required serial animal-to-animal adaptation for establishing mucosal transmissibility and pathogenicity. To capture essential features of mucosal transmission of clade C viruses, we produced a series of SHIVs with early clade C HIV-1 env sequences from acutely HIV-1-infected individuals from South Africa. SHIV-327c and SHIV-327cRM expressed env sequences that were 99.7 to 100% identical to the original HIV-1 isolate and did not require in vivo passaging for mucosal infectivity. These challenge stocks infected rhesus monkeys efficiently by both intrarectal and intravaginal routes, replicated to high levels during acute infection, and established chronic setpoint viremia in 13 of 17 (76%) infected animals. The SHIV-327cRM challenge stock was also titrated for both single, high-dose intrarectal challenges and repetitive, low-dose intrarectal challenges in rhesus monkeys. These SHIV challenge stocks should facilitate the preclinical evaluation of vaccines and other interventions aimed at preventing clade C HIV-1 infection.

    IMPORTANCE We describe the development of two related clade C SHIV challenge stocks. These challenge stocks should prove useful for preclinical testing of vaccines and other interventions aimed at preventing clade C HIV-1 infection.

  • Influenza Virus M2 Protein Ion Channel Activity Helps To Maintain Pandemic 2009 H1N1 Virus Hemagglutinin Fusion Competence during Transport to the Cell Surface [Virus-Cell Interactions]

  • The influenza virus hemagglutinin (HA) envelope protein mediates virus entry by first binding to cell surface receptors and then fusing viral and endosomal membranes during endocytosis. Cleavage of the HA precursor (HA0) into a surface receptor-binding subunit (HA1) and a fusion-inducing transmembrane subunit (HA2) by host cell enzymes primes HA for fusion competence by repositioning the fusion peptide to the newly created N terminus of HA2. We previously reported that the influenza virus M2 protein enhances pandemic 2009 influenza A virus [(H1N1)pdm09] HA-pseudovirus infectivity, but the mechanism was unclear. In this study, using cell-cell fusion and HA-pseudovirus infectivity assays, we found that the ion channel function of M2 was required for enhancement of HA fusion and HA-pseudovirus infectivity. The M2 activity was needed only during HA biosynthesis, and proteolysis experiments indicated that M2 proton channel activity helped to protect (H1N1)pdm09 HA from premature conformational changes as it traversed low-pH compartments during transport to the cell surface. While M2 has previously been shown to protect avian influenza virus HA proteins of the H5 and H7 subtypes that have polybasic cleavage motifs, this study demonstrates that M2 can protect HA proteins from human H1N1 strains that lack a polybasic cleavage motif. This finding suggests that M2 proton channel activity may play a wider role in preserving HA fusion competence among a variety of HA subtypes, including HA proteins from emerging strains that may have reduced HA stability.

    IMPORTANCE Influenza virus infects cells when the hemagglutinin (HA) surface protein undergoes irreversible pH-induced conformational changes after the virus is taken into the cell by endocytosis. HA fusion competence is primed when host cell enzymes cleave the HA precursor. The proton channel function of influenza virus M2 protein has previously been shown to protect avian influenza virus HA proteins that contain a polybasic cleavage site from pH-induced conformational changes during biosynthesis, but this effect is less well understood for human influenza virus HA proteins that lack polybasic cleavage sites. Using assays that focus on HA entry and fusion, we found that the M2 protein also protects (H1N1)pdm09 influenza A virus HA from premature conformational changes as it transits low-pH compartments during biosynthesis. This work suggests that M2 may play a wider role in preserving HA function in a variety of influenza virus subtypes that infect humans and may be especially important for HA proteins that are less stable.

  • Lentiviral Nef Proteins Manipulate T Cells in a Subset-Specific Manner [Virus-Cell Interactions]

  • The role of the accessory viral Nef protein as a multifunctional manipulator of the host cell that is required for effective replication of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) in vivo is well established. It is unknown, however, whether Nef manipulates all or just specific subsets of CD4+ T cells, which are the main targets of virus infection and differ substantially in their state of activation and importance for a functional immune system. Here, we analyzed the effect of Nef proteins differing in their T cell receptor (TCR)-CD3 downmodulation function in HIV-infected human lymphoid aggregate cultures and peripheral blood mononuclear cells. We found that Nef efficiently downmodulates TCR-CD3 in naive and memory CD4+ T cells and protects the latter against apoptosis. In contrast, highly proliferative CD45RA+ CD45RO+ CD4+ T cells were main producers of infectious virus but largely refractory to TCR-CD3 downmodulation. Such T cell subset-specific differences were also observed for Nef-mediated modulation of CD4 but not for enhancement of virion infectivity. Our results indicate that Nef predominantly modulates surface receptors on CD4+ T cell subsets that are not already fully permissive for viral replication. As a consequence, Nef-mediated downmodulation of TCR-CD3, which distinguishes most primate lentiviruses from HIV type 1 (HIV-1) and its vpu-containing simian precursors, may promote a selective preservation of central memory CD4+ T cells, which are critical for the maintenance of a functional immune system.

    IMPORTANCE The Nef proteins of human and simian immunodeficiency viruses manipulate infected CD4+ T cells in multiple ways to promote viral replication and immune evasion in vivo. Here, we show that some effects of Nef are subset specific. Downmodulation of CD4 and TCR-CD3 is highly effective in central memory CD4+ T cells, and the latter Nef function protects this T cell subset against apoptosis. In contrast, highly activated/proliferating CD4+ T cells are largely refractory to receptor downmodulation but are main producers of infectious HIV-1. Nef-mediated enhancement of virion infectivity, however, was observed in all T cell subsets examined. Our results provide new insights into how primate lentiviruses manipulate their target cells and suggest that the TCR-CD3 downmodulation function of Nef may promote a selective preservation of memory CD4+ T cells, which are critical for immune function, but has little effect on activated/proliferating CD4+ T cells, which are the main targets for viral replication.

  • Functional Characterization of Nuclear Trafficking Signals in Pseudorabies Virus pUL31 [Structure and Assembly]

  • The herpesviral nuclear egress complex (NEC), consisting of pUL31 and pUL34 homologs, mediates efficient translocation of newly synthesized capsids from the nucleus to the cytosol. The tail-anchored membrane protein pUL34 is autonomously targeted to the nuclear envelope, while pUL31 is recruited to the inner nuclear membrane (INM) by interaction with pUL34. A nuclear localization signal (NLS) in several pUL31 homologs suggests importin-mediated translocation of the protein. Here we demonstrate that deletion or mutation of the NLS in pseudorabies virus (PrV) pUL31 resulted in exclusively cytosolic localization, indicating active nuclear export. Deletion or mutation of a predicted nuclear export signal (NES) in mutant constructs lacking a functional NLS resulted in diffuse nuclear and cytosolic localization, indicating that both signals are functional. pUL31 molecules lacking the complete NLS or NES were not recruited to the INM by pUL34, while site-specifically mutated proteins formed the NEC and partially complemented the defect of the UL31 deletion mutant. Our data demonstrate that the N terminus of pUL31, encompassing the NLS, is required for efficient nuclear targeting but not for pUL34 interaction, while the C terminus, containing the NES but not necessarily the NES itself, is required for complex formation and efficient budding of viral capsids at the INM. Moreover, pUL31-NLS displayed a dominant negative effect on wild-type PrV replication, probably by diverting pUL34 to cytoplasmic membranes.

    IMPORTANCE The molecular details of nuclear egress of herpesvirus capsids are still enigmatic. Although the key players, homologs of herpes simplex virus pUL34 and pUL31, which interact and form the heterodimeric nuclear egress complex, are well known, the molecular basis of this interaction and the successive budding, vesicle formation, and scission from the INM, as well as capsid release into the cytoplasm, remain largely obscure. Here we show that classical cellular targeting signals for nuclear import and export are important for proper localization and function of the NEC, thus regulating herpesvirus nuclear egress.

  • Lethal Coinfection of Influenza Virus and Streptococcus pneumoniae Lowers Antibody Response to Influenza Virus in Lung and Reduces Numbers of Germinal Center B Cells, T Follicular Helper Cells, and Plasma Cells in Mediastinal Lymph Node [Cellular Response to Infection]

  • Secondary Streptococcus pneumoniae infection after influenza is a significant clinical complication resulting in morbidity and sometimes mortality. Prior influenza virus infection has been demonstrated to impair the macrophage and neutrophil response to the subsequent pneumococcal infection. In contrast, how a secondary pneumococcal infection after influenza can affect the adaptive immune response to the initial influenza virus infection is less well understood. Therefore, this study focuses on how secondary pneumococcal infection after influenza may impact the humoral immune response to the initial influenza virus infection in a lethal coinfection mouse model. Compared to mice infected with influenza virus alone, mice coinfected with influenza virus followed by pneumococcus had significant body weight loss and 100% mortality. In the lung, lethal coinfection significantly increased virus titers and bacterial cell counts and decreased the level of virus-specific IgG, IgM, and IgA, as well as the number of B cells, CD4 T cells, and plasma cells. Lethal coinfection significantly reduced the size and weight of spleen, as well as the number of B cells along the follicular developmental lineage. In mediastinal lymph nodes, lethal coinfection significantly decreased germinal center B cells, T follicular helper cells, and plasma cells. Adoptive transfer of influenza virus-specific immune serum to coinfected mice improved survival, suggesting the protective functions of anti-influenza virus antibodies. In conclusion, coinfection reduced the B cell response to influenza virus. This study helps us to understand the modulation of the B cell response to influenza virus during a lethal coinfection.

    IMPORTANCE Secondary pneumococcal infection after influenza virus infection is an important clinical issue that often results in excess mortality. Since antibodies are key mediators of protection, this study aims to examine the antibody response to influenza virus and demonstrates that lethal coinfection reduced the B cell response to influenza virus. This study helps to highlight the complexity of the modulation of the B cell response in the context of coinfection.

  • Human Noroviruses' Fondness for Histo-Blood Group Antigens [Structure and Assembly]

  • Human noroviruses are the dominant cause of outbreaks of gastroenteritis around the world. Human noroviruses interact with the polymorphic human histo-blood group antigens (HBGAs), and this interaction is thought to be important for infection. Indeed, synthetic HBGAs or HBGA-expressing enteric bacteria were shown to enhance norovirus infection in B cells. A number of studies have found a possible relationship between HBGA type and norovirus susceptibility. The genogroup II, genotype 4 (GII.4) noroviruses are the dominant cluster, evolve every other year, and are thought to modify their binding interactions with different HBGA types. Here we show high-resolution X-ray crystal structures of the capsid protruding (P) domains from epidemic GII.4 variants from 2004, 2006, and 2012, cocrystallized with a panel of HBGA types (H type 2, Lewis Y, Lewis B, Lewis A, Lewis X, A type, and B type). Many of the HBGA binding interactions were found to be complex, involving capsid loop movements, alternative HBGA conformations, and HBGA rotations. We showed that a loop (residues 391 to 395) was elegantly repositioned to allow for Lewis Y binding. This loop was also slightly shifted to provide direct hydrogen- and water-mediated bonds with Lewis B. We considered that the flexible loop modulated Lewis HBGA binding. The GII.4 noroviruses have dominated outbreaks over the past decade, which may be explained by their exquisite HBGA binding mechanisms, their fondness for Lewis HBGAs, and their temporal amino acid modifications.

    IMPORTANCE Our data provide a comprehensive picture of GII.4 P domain and HBGA binding interactions. The exceptionally high resolutions of our X-ray crystal structures allowed us to accurately recognize novel GII.4 P domain interactions with numerous HBGA types. We showed that the GII.4 P domain-HBGA interactions involved complex binding mechanisms that were not previously observed in norovirus structural studies. Many of the GII.4 P domain-HBGA interactions we identified were negative in earlier enzyme-linked immunosorbent assay (ELISA)-based studies. Altogether, our data show that the GII.4 norovirus P domains can accommodate numerous HBGA types.

  • Hepatitis B Virus Core Protein Sensitizes Hepatocytes to Tumor Necrosis Factor-Induced Apoptosis by Suppression of the Phosphorylation of Mitogen-Activated Protein Kinase Kinase 7 [Virus-Cell Interactions]

  • Hepatitis B, which caused by hepatitis B virus (HBV) infection, remains a major health threat worldwide. Hepatic injury and regeneration from chronic inflammation are the main driving factors of liver fibrosis and cirrhosis in chronic hepatitis B. Proinflammatory tumor necrosis factor alpha (TNF-aalpha;) has been implicated as a major inducer of liver cell death during viral hepatitis. Here, we report that in hepatoma cell lines and in primary mouse and human hepatocytes, expression of hepatitis B virus core (HBc) protein made cells susceptible to TNF-aalpha;-induced apoptosis. We found by tandem affinity purification and mass spectrometry that receptor of activated protein kinase C 1 (RACK1) interacted with HBc. RACK1 was recently reported as a scaffold protein that facilitates the phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7) by its upstream activators. Our study showed that HBc abrogated the interaction between MKK7 and RACK1 by competitively binding to RACK1, thereby downregulating TNF-aalpha;-induced phosphorylation of MKK7 and the activation of c-Jun N-terminal kinase (JNK). In line with this finding, specific knockdown of MKK7 increased the sensitivity of hepatocytes to TNF-aalpha;-induced apoptosis, while overexpression of RACK1 counteracted the proapoptotic activity of HBc. Capsid particle formation was not obligatory for HBc proapoptotic activity, as analyzed using an assembly-defective HBc mutant. In conclusion, the expression of HBc sensitized hepatocytes to TNF-aalpha;-induced apoptosis by disrupting the interaction between MKK7 and RACK1. Our study is thus the first indication of the pathogenic effects of HBc in liver injury during hepatitis B.

    IMPORTANCE Our study revealed a previously unappreciated role of HBc in TNF-aalpha;-mediated apoptosis. The proapoptotic activity of HBc is important for understanding hepatitis B pathogenesis. In particular, HBV variants associated with severe hepatitis may upregulate apoptosis of hepatocytes through enhanced HBc expression. Our study also found that MKK7 is centrally involved in TNF-aalpha;-induced hepatocyte apoptosis and revealed a multifaceted role for JNK signaling in this process.

  • Hydrophobic and Charged Residues in the C-Terminal Arm of Hepatitis C Virus RNA-Dependent RNA Polymerase Regulate Initiation and Elongation [Genome Replication and Regulation of Viral Gene Expression]

  • The RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is essential for viral genome replication. Crystal structures of the HCV RdRp reveal two C-terminal features, a bbeta;-loop and a C-terminal arm, suitably located for involvement in positioning components of the initiation complex. Here we show that these two elements intimately regulate template and nucleotide binding, initiation, and elongation. We constructed a series of bbeta;-loop and C-terminal arm mutants, which were used for in vitro analysis of RdRp de novo initiation and primer extension activities. All mutants showed a substantial decrease in initiation activities but a marked increase in primer extension activities, indicating an ability to form more stable elongation complexes with long primer-template RNAs. Structural studies of the mutants indicated that these enzyme properties might be attributed to an increased flexibility in the C-terminal features resulting in a more open polymerase cleft, which likely favors the elongation process but hampers the initiation steps. A UTP cocrystal structure of one mutant shows, in contrast to the wild-type protein, several alternate conformations of the substrate, confirming that even subtle changes in the C-terminal arm result in a more loosely organized active site and flexible binding modes of the nucleotide. We used a subgenomic replicon system to assess the effects of the same mutations on viral replication in cells. Even the subtlest mutations either severely impaired or completely abolished the ability of the replicon to replicate, further supporting the concept that the correct positioning of both the bbeta;-loop and C-terminal arm plays an essential role during initiation and in HCV replication in general.

    IMPORTANCE HCV RNA polymerase is a key target for the development of directly acting agents to cure HCV infections, which necessitates a thorough understanding of the functional roles of the various structural features of the RdRp. Here we show that even highly conservative changes, e.g., Tyr-ggt;Phe or Asp-ggt;Glu, in these seemingly peripheral structural features have profound effects on the initiation and elongation properties of the HCV polymerase.

  • Novel Mechanism of Regulation of Tomato Bushy Stunt Virus Replication by Cellular WW-Domain Proteins [Virus-Cell Interactions]

  • Replication of (+)RNA viruses depends on several co-opted host proteins but is also under the control of cell-intrinsic restriction factors (CIRFs). By using tombusviruses, small model viruses of plants, we dissect the mechanism of inhibition of viral replication by cellular WW-domain-containing proteins, which act as CIRFs. By using fusion proteins between the WW domain and the p33 replication protein, we show that the WW domain inhibits the ability of p33 to bind to the viral RNA and to other p33 and p92 replication proteins leading to inhibition of viral replication in yeast and in a cell extract. Overexpression of WW-domain protein in yeast also leads to reduction of several co-opted host factors in the viral replicase complex (VRC). These host proteins, such as eEF1A, Cdc34 E2 ubiquitin-conjugating enzyme, and ESCRT proteins (Bro1p and Vps4p), are known to be involved in VRC assembly. Simultaneous coexpression of proviral cellular factors with WW-domain protein partly neutralizes the inhibitory effect of the WW-domain protein. We propose that cellular WW-domain proteins act as CIRFs and also as regulators of tombusvirus replication by inhibiting the assembly of new membrane-bound VRCs at the late stage of infection. We suggest that tombusviruses could sense the status of the infected cells via the availability of cellular susceptibility factors versus WW-domain proteins for binding to p33 replication protein that ultimately controls the formation of new VRCs. This regulatory mechanism might explain how tombusviruses could adjust the efficiency of RNA replication to the limiting resources of the host cells during infections.

    IMPORTANCE Replication of positive-stranded RNA viruses, which are major pathogens of plants, animals, and humans, is inhibited by several cell-intrinsic restriction factors (CIRFs) in infected cells. We define here the inhibitory roles of the cellular Rsp5 ubiquitin ligase and its WW domain in plant-infecting tombusvirus replication in yeast cells and in vitro using purified components. The WW domain of Rsp5 binds to the viral RNA-binding sites of p33 and p92 replication proteins and blocks the ability of these viral proteins to use the viral RNA for replication. The WW domain also interferes with the interaction (oligomerization) of p33 and p92 that is needed for the assembly of the viral replicase. Moreover, WW domain also inhibits the subversion of several cellular proteins into the viral replicase, which otherwise play proviral roles in replication. Altogether, Rsp5 is a CIRF against a tombusvirus, and it possibly has a regulatory function during viral replication in infected cells.

  • Mutations across Murine Hepatitis Virus nsp4 Alter Virus Fitness and Membrane Modifications [Virus-Cell Interactions]

  • A common feature of infection by positive-sense RNA virus is the modification of host cell cytoplasmic membranes that serve as sites of viral RNA synthesis. Coronaviruses induce double-membrane vesicles (DMVs), but the role of DMVs in replication and virus fitness remains unclear. Coronaviruses encode 16 nonstructural proteins (nsps), three of which, nsp3, nsp4, and nsp6, are necessary and sufficient for DMV formation. It has been shown previously that mutations in murine hepatitis virus (MHV) nsp4 loop 1 that alter nsp4 glycosylation are associated with disrupted DMV formation and result in changes in virus replication and RNA synthesis. However, it is not known whether DMV morphology or another function of nsp4 glycosylation is responsible for effects on virus replication. In this study, we tested whether mutations across nsp4, both alone and in combination with mutations that abolish nsp4 glycosylation, affected DMV formation, replication, and fitness. Residues in nsp4 distinct from glycosylation sites, particularly in the endoplasmic reticulum (ER) luminal loop 1, independently disrupted both the number and morphology of DMVs and exacerbated DMV changes associated with loss of glycosylation. Mutations that altered DMV morphology but not glycosylation did not affect virus fitness while viruses lacking nsp4 glycosylation exhibited a loss in fitness. The results support the hypothesis that DMV morphology and numbers are not key determinants of virus fitness. The results also suggest that nsp4 glycosylation serves roles in replication in addition to the organization and stability of MHV-induced double-membrane vesicles.

    IMPORTANCE All positive-sense RNA viruses modify host cytoplasmic membranes for viral replication complex formation. Thus, defining the mechanisms of virus-induced membrane modifications is essential for both understanding virus replication and development of novel approaches to virus inhibition. Coronavirus-induced membrane changes include double-membrane vesicles (DMVs) and convoluted membranes. Three viral nonstructural proteins (nsps), nsp3, nsp4, and nsp6, are known to be required for DMV formation. It is unknown how these proteins induce membrane modification or which regions of the proteins are involved in DMV formation and stability. In this study, we show that mutations across nsp4 delay virus replication and disrupt DMV formation and that loss of nsp4 glycosylation is associated with a substantial fitness cost. These results support a critical role for nsp4 in DMV formation and virus fitness.

  • The P25 Protein of Potato Virus X (PVX) Is the Main Pathogenicity Determinant Responsible for Systemic Necrosis in PVX-Associated Synergisms [Pathogenesis and Immunity]

  • Most plant viruses counter the RNA silencing-based antiviral defense by expressing viral suppressors of RNA silencing (VSRs). In this sense, VSRs may be regarded as virulence effectors that can be recognized by the host as avirulence (avr) factors to induce R-mediated resistance. We made use of Agrobacterium-mediated transient coexpression of VSRs in combination with Potato virus X (PVX) to recapitulate in local tissues the systemic necrosis (SN) caused by PVX-potyvirus synergistic infections in Nicotiana benthamiana. The hypersensitive response (HR)-like response was associated with an enhanced accumulation of PVX subgenomic RNAs. We further show that expression of P25, the VSR of PVX, in the presence of VSR from different viruses elicited an HR-like response in Nicotiana spp. Furthermore, the expression of P25 by a Plum pox virus (PPV) vector was sufficient to induce an increase of PPV pathogenicity that led to necrotic mottling. A frameshift mutation in the P25 open reading frame (ORF) of PVX did not lead to necrosis when coexpressed with VSRs. These findings indicate that P25 is the main PVX determinant involved in eliciting a systemic HR-like response in PVX-associated synergisms. Moreover, we show that silencing of SGT1 and RAR1 attenuated cell death in both PVX-potyvirus synergistic infection and the HR-like response elicited by P25. Our study underscores that P25 variants that have impaired ability to suppress RNA silencing cannot act as elicitors when synergized by the presence of other VSRs. These findings highlight the importance of RNA silencing suppression activity in the HR-like response elicited by VSRs in certain hosts.

    IMPORTANCE The work presented here describes how the activity of the PVX suppressor P25 elicits an HR-like response in Nicotiana spp. when overexpressed with other VSR proteins. This finding suggests that the SN response caused by PVX-associated synergisms is a delayed immune response triggered by P25, once it reaches a threshold level by the action of other VSRs. Moreover, this work supports the contention that the silencing suppressor activity of PVX P25 protein is a prerequisite for HR elicitation. We propose that unidentified avr determinants could be involved in other cases of viral synergisms in which heterologous "helper" viruses encoding strong VSRs exacerbate the accumulation of the avr-encoding virus.

  • Host Genetic and Viral Determinants of HIV-1 RNA Set Point among HIV-1 Seroconverters from Sub-Saharan Africa [Pathogenesis and Immunity]

  • We quantified the collective impact of source partner HIV-1 RNA levels, human leukocyte antigen (HLA) alleles, and innate responses through Toll-like receptor (TLR) alleles on the HIV-1 set point. Data came from HIV-1 seroconverters in African HIV-1 serodiscordant couple cohorts. Linear regression was used to determine associations with set point and R2 to estimate variation explained by covariates. The strongest predictors of set point were HLA alleles (B*53:01, B*14:01, and B*27:03) and plasma HIV-1 levels of the transmitting partner, which explained 13% and 10% of variation in set point, respectively. HLA-A concordance between partners and TLR polymorphisms (TLR2 rs3804100 and TLR7 rs179012) also were associated with set point, explaining 6% and 5% of the variation, respectively. Overall, these factors and genital factors of the transmitter (i.e., male circumcision, bacterial vaginosis, and use of acyclovir) explained 46% of variation in set point. We found that both innate and adaptive immune responses, together with plasma HIV-1 levels of the transmitting partner, explain almost half of the variation in viral load set point.

    IMPORTANCE After HIV-1 infection, uncontrolled virus replication leads to a rapid increase in HIV-1 concentrations. Once host immune responses develop, however, HIV-1 levels reach a peak and subsequently decline until they reach a stable level that may persist for years. This stable HIV-1 set point represents an equilibrium between the virus and host responses and is predictive of later disease progression and transmission potential. Understanding how host and virus factors interact to determine HIV-1 set point may elucidate novel mechanisms or biological pathways for treating HIV-1 infection. We identified host and virus factors that predict HIV-1 set point in people who recently acquired HIV-1, finding that both innate and adaptive immune responses, along with factors that likely influence HIV-1 virulence and inoculum, explain ~46% of the variation in HIV-1 set point.

  • Regulatory T Cells Resist Virus Infection-Induced Apoptosis [Cellular Response to Infection]

  • Regulatory T (Treg) cells are important in the maintenance of self-tolerance, and the depletion of Treg cells correlates with autoimmune development. It has been shown that type I interferon (IFN) responses induced early in the infection of mice can drive memory (CD44hi) CD8 and CD4 T cells into apoptosis, and we questioned here whether the apoptosis of CD44-expressing Treg cells might be involved in the infection-associated autoimmune development. Instead, we found that Treg cells were much more resistant to apoptosis than CD44hi CD8 and CD4 T cells at days 2 to 3 after lymphocytic choriomeningitis virus infection, when type I IFN levels are high. The infection caused a downregulation of the interleukin-7 (IL-7) receptor, needed for survival of conventional T cells, while increasing on Treg cells the expression of the high-affinity IL-2 receptor, needed for STAT5-dependent survival of Treg cells. The stably maintained Treg cells early during infection may explain the relatively low incidence of autoimmune manifestations among infected patients.

    IMPORTANCE Autoimmune diseases are controlled in part by regulatory T cells (Treg) and are thought to sometimes be initiated by viral infections. We tested the hypothesis that Treg may die off at early stages of infection, when virus-induced factors kill other lymphocyte types. Instead, we found that Treg resisted this cell death, perhaps reducing the tendency of viral infections to cause immune dysfunction and induce autoimmunity.

  • The Amphipathic Helix of Adenovirus Capsid Protein VI Contributes to Penton Release and Postentry Sorting [Structure and Assembly]

  • Nuclear delivery of the adenoviral genome requires that the capsid cross the limiting membrane of the endocytic compartment and traverse the cytosol to reach the nucleus. This endosomal escape is initiated upon internalization and involves a highly coordinated process of partial disassembly of the entering capsid to release the membrane lytic internal capsid protein VI. Using wild-type and protein VI-mutated human adenovirus serotype 5 (HAdV-C5), we show that capsid stability and membrane rupture are major determinants of entry-related sorting of incoming adenovirus virions. Furthermore, by using electron cryomicroscopy, as well as penton- and protein VI-specific antibodies, we show that the amphipathic helix of protein VI contributes to capsid stability by preventing premature disassembly and deployment of pentons and protein VI. Thus, the helix has a dual function in maintaining the metastable state of the capsid by preventing premature disassembly and mediating efficient membrane lysis to evade lysosomal targeting. Based on these findings and structural data from cryo-electron microscopy, we suggest a refined disassembly mechanism upon entry.

    IMPORTANCE In this study, we show the intricate connection of adenovirus particle stability and the entry-dependent release of the membrane-lytic capsid protein VI required for endosomal escape. We show that the amphipathic helix of the adenovirus internal protein VI is required to stabilize pentons in the particle while coinciding with penton release upon entry and that release of protein VI mediates membrane lysis, thereby preventing lysosomal sorting. We suggest that this dual functionality of protein VI ensures an optimal disassembly process by balancing the metastable state of the mature adenovirus particle.

  • Model of the TVA Receptor Determinants Required for Efficient Infection by Subgroup A Avian Sarcoma and Leukosis Viruses [Virus-Cell Interactions]

  • The study of the interactions of subgroup A avian sarcoma and leucosis viruses [ASLV(A)] with the TVA receptor required to infect cells offers a powerful experimental model of retroviral entry. Several regions and specific residues in the TVA receptor have previously been identified to be critical determinants of the binding affinity with ASLV(A) envelope glycoproteins and to mediate efficient infection. Two homologs of the TVA receptor have been cloned: the original quail TVA receptor, which has been the basis for most of the initial characterization of the ASLV(A) TVA, and the chicken TVA receptor, which is 65% identical to the quail receptor overall but identical in the region thought to be critical for infection. Our previous work characterized three mutant ASLV(A) isolates that could efficiently bind and infect cells using the chicken TVA receptor homolog but not using the quail TVA receptor homolog, with the infectivity of one mutant virus being ggt;500-fold less with the quail TVA receptor. The mutant viruses contained mutations in the hr1 region of the surface glycoprotein. Using chimeras of the quail and chicken TVA receptors, we have identified new residues of TVA critical for the binding affinity and entry of ASLV(A) using the mutant glycoproteins and viruses to probe the function of those residues. The quail TVA receptor required changes at residues 10, 14, and 31 of the corresponding chicken TVA residues to bind wild-type and mutant ASLV(A) glycoproteins with a high affinity and recover the ability to mediate efficient infection of cells. A model of the TVA determinants critical for interacting with ASLV(A) glycoproteins is proposed.

    IMPORTANCE A detailed understanding of how retroviruses enter cells, evolve to use new receptors, and maintain efficient entry is crucial for identifying new targets for combating retrovirus infection and pathogenesis, as well as for developing new approaches for targeted gene delivery. Since all retroviruses share an envelope glycoprotein organization, they likely share a mechanism of receptor triggering to begin the entry process. Multiple, noncontiguous interaction determinants located in the receptor and the surface (SU) glycoprotein hypervariable domains are required for binding affinity and to restrict or broaden receptor usage. In this study, further mechanistic details of the entry process were elucidated by characterizing the ASLV(A) glycoprotein interactions with the TVA receptor required for entry. The ASLV(A) envelope glycoproteins are organized into functional domains that allow changes in receptor choice to occur by mutation and/or recombination while maintaining a critical level of receptor binding affinity and an ability to trigger glycoprotein conformational changes.

  • Pseudorabies Virus Triggers Glycoprotein gE-Mediated ERK1/2 Activation and ERK1/2-Dependent Migratory Behavior in T Cells [Virus-Cell Interactions]

  • The interaction between viruses and immune cells of the host may lead to modulation of intracellular signaling pathways and to subsequent changes in cellular behavior that are of benefit for either virus or host. ERK1/2 (extracellular signal regulated kinase 1/2) signaling represents one of the key cellular signaling axes. Here, using wild-type and gE null virus, recombinant gE, and gE-transfected cells, we show that the gE glycoprotein of the porcine Varicellovirus pseudorabies virus (PRV) triggers ERK1/2 phosphorylation in Jurkat T cells and primary porcine T lymphocytes. PRV-induced ERK1/2 signaling resulted in homotypic T cell aggregation and increased motility of T lymphocytes. Our study reveals a new function of the gE glycoprotein of PRV and suggests that PRV, through activation of ERK1/2 signaling, has a substantial impact on T cell behavior.

    IMPORTANCE Herpesviruses are known to be highly successful in evading the immune system of their hosts, subverting signaling pathways of the host to their own advantage. The ERK1/2 signaling pathway, being involved in many cellular processes, represents a particularly attractive target for viral manipulation. Glycoprotein E (gE) is an important virulence factor of alphaherpesviruses, involved in viral spread. In this study, we show that gE has the previously uncharacterized ability to trigger ERK1/2 phosphorylation in T lymphocytes. We also show that virus-induced ERK1/2 signaling leads to increased migratory behavior of T cells and that migratory T cells can spread the infection to susceptible cells. In conclusion, our results point to a novel function for gE and suggest that virus-induced ERK1/2 activation may trigger PRV-carrying T lymphocytes to migrate and infect other cells susceptible to PRV replication.

  • The Inability of Wild-Type Rabies Virus To Activate Dendritic Cells Is Dependent on the Glycoprotein and Correlates with Its Low Level of the De Novo-Synthesized Leader RNA [Cellular Response to Infection]

  • Dendritic cells (DCs) are the most efficient antigen-presenting cells, playing a key role in the adaptive immune responses to viral infections. Our studies demonstrate that wild-type (wt) rabies virus (RABV) does not activate DCs. Adoptive transfer of DCs primed with wt RABV did not activate DCs, stimulate virus neutralizing antibodies (VNA), or protect recipients against challenge. However, adoptive transfer of DCs primed with laboratory-attenuated RABV resulted in DC activation, production of VNA, and protection against challenge. In vitro studies with recombinant RABV (laboratory-attenuated RABV expressing the glycoprotein or the phosphoprotein from wt RABV) demonstrate that DC activation is dependent on the glycoprotein and involves the IPS-1 pathway. Furthermore, binding to and entry into DCs by wt RABV is severely blocked, and the copy number of de novo-synthesized leader RNA was two logs lower in DCs infected with the wt than in DCs treated with laboratory-attenuated RABV. However, transient transfection of DCs with synthesized leader RNA from either wt or attenuated RABV is capable of activating DCs in a dose-dependent manner. Thus, the inability of wt RABV to activate DCs correlates with its low level of the de novo-synthesized leader RNA.

    IMPORTANCE Rabies remains a public health threat, with more than 55,000 fatalities each year around the world. Since DCs play a key role in the adaptive immune responses to viral infections, we investigated the ability of rabies virus (RABV) to activate DCs. It was found that the adoptive transfer of DCs primed with wt RABV did not activate DCs, stimulate VNA, or protect mice against lethal challenge. However, laboratory-attenuated RABV mediates the activation of DCs via the IPS-1 pathway and is glycoprotein dependent. We further show that wt RABV evades DC-mediated immune activation by inefficient binding/entry into DCs and as a result of a reduced level of de novo-synthesized leader RNA. These findings may have important implications in the development of efficient rabies vaccines.

  • Structural Flexibility of a Conserved Antigenic Region in Hepatitis C Virus Glycoprotein E2 Recognized by Broadly Neutralizing Antibodies [Structure and Assembly]

  • Neutralizing antibodies (NAbs) targeting glycoprotein E2 are important for the control of hepatitis C virus (HCV) infection. One conserved antigenic site (amino acids 412 to 423) is disordered in the reported E2 structure, but a synthetic peptide mimicking this site forms a bbeta;-hairpin in complex with three independent NAbs. Our structure of the same peptide in complex with NAb 3/11 demonstrates a strikingly different extended conformation. We also show that residues 412 to 423 are essential for virus entry but not for E2 folding. Together with the neutralizing capacity of the 3/11 Fab fragment, this indicates an unexpected structural flexibility within this epitope. NAbs 3/11 and AP33 (recognizing the extended and bbeta;-hairpin conformations, respectively) display similar neutralizing activities despite converse binding kinetics. Our results suggest that HCV utilizes conformational flexibility as an immune evasion strategy, contributing to the limited immunogenicity of this epitope in patients, similar to the conformational flexibility described for other enveloped and nonenveloped viruses.

    IMPORTANCE Approximately 180 million people worldwide are infected with hepatitis C virus (HCV), and neutralizing antibodies play an important role in controlling the replication of this major human pathogen. We show here that one of the most conserved antigenic sites within the major glycoprotein E2 (amino acids 412 to 423), which is disordered in the recently reported crystal structure of an E2 core fragment, can adopt different conformations in the context of the infectious virus particle. Recombinant Fab fragments recognizing different conformations of this antigenic site have similar neutralization activities in spite of converse kinetic binding parameters. Of note, an antibody response targeting this antigenic region is less frequent than those targeting other more immunogenic regions in E2. Our results suggest that the observed conformational flexibility in this conserved antigenic region contributes to the evasion of the humoral host immune response, facilitating chronicity and the viral spread of HCV within an infected individual.

  • Induction of an Embryonic Mouse Innate Immune Response following Inoculation In Utero with Minute Virus of Mice [Cellular Response to Infection]

  • We used an embryonic-infection model system to show that MVMp, the prototypic minute virus of mice (MVM) serotype and a member of the genus Protoparvovirus, triggers a comprehensive innate immune response in the developing mouse embryo. Direct inoculation of the midtrimester embryo in utero with MVMp results in a widespread, productive infection. During a 96-h infection course, embryonic beta interferon (IFN-bbeta;) and IFN- transcription were induced 90- and 60-fold, respectively. IFN-bbeta; levels correlated with the embryo viral burden, while IFN- levels first increased and then decreased. Production of proinflammatory cytokines, interleukin 1bbeta; (IL-1bbeta;) and tumor necrosis factor alpha (TNF-aalpha;), also increased, but by smaller amounts, approximately 7-fold each. We observed increased levels of downstream antiviral effector molecules, PKR and phosphorylated STAT2. Finally, we showed that there is an immune cell response to the virus infection. Infected tissues in the embryo exhibited an increased density of mature leukocytes compared to the same tissues in uninfected embryos. The responses we observed were almost completely restricted to the infected embryos. Uninfected littermates routinely exhibited small increases in innate immune components that rarely reached statistical significance compared to negative controls. Similarly, the placentae of infected embryos did not show any significant increase in transcription of innate immune cytokines. Since the placenta has both embryonic and maternal components, we suggest there is minimal involvement of the dam in the response to infection.

    IMPORTANCE Interaction between the small single-stranded vertebrate DNA viruses, the protoparvoviruses, and the host innate immune system has been unclear. The issue is important practically given the potential use of these viruses as oncotherapeutic agents. The data reported here stand in contrast to studies of innate immune response during protoparvovirus infection of adult hosts, which invariably reported no or minimal and sporadic induction of an interferon response during infection. We conclude that under conditions of robust and productive MVM infection, a normal murine host is able to mount a significant and broad innate immune response.

  • Live-Attenuated Measles Virus Vaccine Targets Dendritic Cells and Macrophages in Muscle of Nonhuman Primates [Vaccines and Antiviral Agents]

  • Although live-attenuated measles virus (MV) vaccines have been used successfully for over 50 years, the target cells that sustain virus replication in vivo are still unknown. We generated a reverse genetics system for the live-attenuated MV vaccine strain Edmonston-Zagreb (EZ), allowing recovery of recombinant (r)MVEZ. Three recombinant viruses were generated that contained the open reading frame encoding enhanced green fluorescent protein (EGFP) within an additional transcriptional unit (ATU) at various positions within the genome. rMVEZEGFP(1), rMVEZEGFP(3), and rMVEZEGFP(6) contained the ATU upstream of the N gene, following the P gene, and following the H gene, respectively. The viruses were compared in vitro by growth curves, which indicated that rMVEZEGFP(1) was overattenuated. Intratracheal infection of cynomolgus macaques with these recombinant viruses revealed differences in immunogenicity. rMVEZEGFP(1) and rMVEZEGFP(6) did not induce satisfactory serum antibody responses, whereas both in vitro and in vivo rMVEZEGFP(3) was functionally equivalent to the commercial MVEZ-containing vaccine. Intramuscular vaccination of macaques with rMVEZEGFP(3) resulted in the identification of EGFP+ cells in the muscle at days 3, 5, and 7 postvaccination. Phenotypic characterization of these cells demonstrated that muscle cells were not infected and that dendritic cells and macrophages were the predominant target cells of live-attenuated MV.

    IMPORTANCE Even though MV strain Edmonston-Zagreb has long been used as a live-attenuated vaccine (LAV) to protect against measles, nothing is known about the primary cells in which the virus replicates in vivo. This is vital information given the push to move toward needle-free routes of vaccination, since vaccine virus replication is essential for vaccination efficacy. We have generated a number of recombinant MV strains expressing enhanced green fluorescent protein. The virus that best mimicked the nonrecombinant vaccine virus was formulated according to protocols for production of commercial vaccine virus batches, and was subsequently used to assess viral tropism in nonhuman primates. The virus primarily replicated in professional antigen-presenting cells, which may explain why this LAV is so immunogenic and efficacious.

  • Thymic HIV-2 Infection Uncovers Posttranscriptional Control of Viral Replication in Human Thymocytes [Pathogenesis and Immunity]

  • A unique HIV-host equilibrium exists in untreated HIV-2-infected individuals. This equilibrium is characterized by low to undetectable levels of viremia throughout the disease course, despite the establishment of disseminated HIV-2 reservoirs at levels comparable to those observed in untreated HIV-1 infection. Although the clinical spectrum is similar in the two infections, HIV-2 infection is associated with a much lower rate of CD4 T-cell decline and has a limited impact on the mortality of infected adults. Here we investigated HIV-2 infection of the human thymus, the primary organ for T-cell production. Human thymic tissue and suspensions of total or purified CD4 single-positive thymocytes were infected with HIV-2 or HIV-1 primary isolates using either CCR5 or CXCR4 coreceptors. We found that HIV-2 infected both thymic organ cultures and thymocyte suspensions, as attested to by the total HIV DNA and cell-associated viral mRNA levels. Nevertheless, thymocytes featured reduced levels of intracellular Gag viral protein, irrespective of HIV-2 coreceptor tropism and cell differentiation stage, in agreement with the low viral load in culture supernatants. Our data show that HIV-2 is able to infect the human thymus, but the HIV-2 replication cycle in thymocytes is impaired, providing a new model to identify therapeutic targets for viral replication control.

    IMPORTANCE HIV-1 infects the thymus, leading to a decrease in CD4 T-cell production that contributes to the characteristic CD4 T-cell loss. HIV-2 infection is associated with a very low rate of progression to AIDS and is therefore considered a unique naturally occurring model of attenuated HIV disease. HIV-2-infected individuals feature low to undetectable plasma viral loads, in spite of the numbers of circulating infected T cells being similar to those found in patients infected with HIV-1. We assessed, for the first time, the direct impact of HIV-2 infection on the human thymus. We show that HIV-2 is able to infect the thymus but that the HIV-2 replication cycle in thymocytes is impaired. We propose that this system will be important to devise immunotherapies that target viral production, aiding the design of future therapeutic strategies for HIV control.

  • Structure-Function Analysis of Vaccinia Virus H7 Protein Reveals a Novel Phosphoinositide Binding Fold Essential for Poxvirus Replication [Structure and Assembly]

  • Phosphoinositides and phosphoinositide binding proteins play a critical role in membrane and protein trafficking in eukaryotes. Their critical role in replication of cytoplasmic viruses has just begun to be understood. Poxviruses, a family of large cytoplasmic DNA viruses, rely on the intracellular membranes to develop their envelope, and poxvirus morphogenesis requires enzymes from the cellular phosphoinositide metabolic pathway. However, the role of phosphoinositides in poxvirus replication remains unclear, and no poxvirus proteins show any homology to eukaryotic phosphoinositide binding domains. Recently, a group of poxvirus proteins, termed viral membrane assembly proteins (VMAPs), were identified as essential for poxvirus membrane biogenesis. A key component of VMAPs is the H7 protein. Here we report the crystal structure of the H7 protein from vaccinia virus. The H7 structure displays a novel fold comprised of seven aalpha;-helices and a highly curved three-stranded antiparallel bbeta;-sheet. We identified a phosphoinositide binding site in H7, comprised of basic residues on a surface patch and the flexible C-terminal tail. These residues were found to be essential for viral replication and for binding of H7 to phosphatidylinositol-3-phosphate (PI3P) and phosphatidylinositol-4-phosphate (PI4P). Our studies suggest that phosphoinositide binding by H7 plays an essential role in poxvirus membrane biogenesis.

    IMPORTANCE Poxvirus viral membrane assembly proteins (VMAPs) were recently shown to be essential for poxvirus membrane biogenesis. One of the key components of VMAPs is the H7 protein. However, no known structural motifs could be identified from its sequence, and there are no homologs of H7 outside the poxvirus family to suggest a biochemical function. We have determined the crystal structure of the vaccinia virus (VACV) H7 protein. The structure displays a novel fold with a distinct and positively charged surface. Our data demonstrate that H7 binds phosphatidylinositol-3-phosphate and phosphatidylinositol-4-phosphate and that the basic surface patch is indeed required for phosphoinositide binding. In addition, mutation of positively charged residues required for lipid binding disrupted VACV replication. Phosphoinositides and phosphoinositide binding proteins play critical roles in membrane and protein trafficking in eukaryotes. Our study demonstrates that VACV H7 displays a novel fold for phosphoinositide binding, which is essential for poxvirus replication.

  • Targeting Cellular Squalene Synthase, an Enzyme Essential for Cholesterol Biosynthesis, Is a Potential Antiviral Strategy against Hepatitis C Virus [Virus-Cell Interactions]

  • Hepatitis C virus (HCV) exploits host membrane cholesterol and its metabolism for progeny virus production. Here, we examined the impact of targeting cellular squalene synthase (SQS), the first committed enzyme for cholesterol biosynthesis, on HCV production. By using the HCV JFH-1 strain and human hepatoma Huh-7.5.1-derived cells, we found that the SQS inhibitors YM-53601 and zaragozic acid A decreased viral RNA, protein, and progeny production in HCV-infected cells without affecting cell viability. Similarly, small interfering RNA (siRNA)-mediated knockdown of SQS led to significantly reduced HCV production, confirming the enzyme as an antiviral target. A metabolic labeling study demonstrated that YM-53601 suppressed the biosynthesis of cholesterol and cholesteryl esters at antiviral concentrations. Unlike YM-53601, the cholesterol esterification inhibitor Sandoz 58-035 did not exhibit an antiviral effect, suggesting that biosynthesis of cholesterol is more important than that of cholesteryl esters for HCV production. YM-53601 inhibited transient replication of a JFH-1 subgenomic replicon and entry of JFH-1 pseudoparticles, suggesting that at least suppression of viral RNA replication and entry contributes to the antiviral effect of the drug. Collectively, our findings highlight the importance of the cholesterol biosynthetic pathway in HCV production and implicate SQS as a potential target for antiviral strategies against HCV.

    IMPORTANCE Hepatitis C virus (HCV) is known to be closely associated with host cholesterol and its metabolism throughout the viral life cycle. However, the impact of targeting cholesterol biosynthetic enzymes on HCV production is not fully understood. We found that squalene synthase, the first committed enzyme for cholesterol biosynthesis, is important for HCV production, and we propose this enzyme as a potential anti-HCV target. We provide evidence that synthesis of free cholesterol is more important than that of esterified cholesterol for HCV production, highlighting a marked free cholesterol dependency of HCV production. Our findings also offer a new insight into a role of the intracellular cholesterol pool that is coupled to its biosynthesis in the HCV life cycle.

  • TRIM5{alpha} Restriction Affects Clinical Outcome and Disease Progression in Simian Immunodeficiency Virus-Infected Rhesus Macaques [Pathogenesis and Immunity]

  • Tripartite motif-containing protein 5aalpha; (TRIM5aalpha;) is considered to be a potential target for cell-based gene modification therapy against human immunodeficiency virus type 1 (HIV-1) infection. In the present study, we used a relevant rhesus macaque model of infection with simian immunodeficiency virus from sooty mangabey (SIVsm) to evaluate the effect of TRIM5aalpha; restriction on clinical outcome. For macaques expressing a restrictive TRIM5 genotype, the disease outcomes of those infected with the wild-type TRIM-sensitive SIVsm strain and those infected with a virus with escape mutations in the capsid were compared. We found that TRIM5aalpha; restriction significantly delayed disease progression and improved the survival rate of SIV-infected macaques, supporting the feasibility of exploiting TRIM5aalpha; as a target for gene therapy against HIV-1. Furthermore, we also found that preservation of memory CD4 T cells was associated with protection by TRIM5aalpha; restriction, suggesting memory CD4 T cells or their progenitor cells as an ideal target for gene modification. Despite the significant effect of TRIM5aalpha; restriction on survival, SIV escape from TRIM5aalpha; restriction was also observed; therefore, this may not be an effective stand-alone strategy and may require combination with other targets.

    IMPORTANCE Recent studies suggest that it may be feasible not only to suppress viral replication with antiviral drugs but also potentially to eliminate or "cure" human immunodeficiency virus (HIV) infection. One approach being explored is the use of gene therapy to introduce genes that can restrict HIV replication, including a restrictive version of the host factor TRIM5aalpha;. TRIM5 was identified as a factor that restricts HIV replication in macaque cells. The rhesus gene is polymorphic, and some alleles are restrictive for primary SIVsm isolates, although escape mutations arise late in infection. Introduction of these escape mutations into the parental virus conferred resistance to TRIM5 on macaques. The present study evaluated these animals for long-term outcomes and found that TRIM5aalpha; restriction significantly delayed disease progression and improved the survival rate of SIV-infected macaques, suggesting that this could be a valid gene therapy approach that could be adapted for HIV.

  • The Nucleoprotein of Newly Emerged H7N9 Influenza A Virus Harbors a Unique Motif Conferring Resistance to Antiviral Human MxA [Pathogenesis and Immunity]

  • Interferon-induced Mx proteins show strong antiviral activity against influenza A viruses (IAVs). We recently demonstrated that the viral nucleoprotein (NP) determines resistance of seasonal and pandemic human influenza viruses to Mx, while avian isolates retain Mx sensitivity. We identified a surface-exposed cluster of amino acids in NP of pandemic A/BM/1/1918 (H1N1), comprising isoleucine-100, proline-283, and tyrosine-313, that is essential for reduced Mx sensitivity in cell culture and in vivo. This cluster has been maintained in all descendant seasonal strains, including A/PR/8/34 (PR/8). Accordingly, two substitutions in the NP of PR/8 [PR/8(mut)] to the Mx-sensitive amino acids (P283L and Y313F) led to attenuation in Mx1-positive mice. Serial lung passages of PR/8(mut) in Mx1 mice resulted in a single exchange of tyrosine to asparagine at position 52 in NP (in close proximity to the amino acid cluster at positions 100, 283, and 313), which partially compensates loss of Mx resistance in PR/8(mut). Intriguingly, the NP of the newly emerged avian-origin H7N9 virus also contains an asparagine at position 52 and shows reduced Mx sensitivity. N52Y substitution in NP results in increased sensitivity of the H7N9 virus to human Mx, indicating that this residue is a determinant of Mx resistance in mammals. Our data strengthen the hypothesis that the human Mx protein represents a potent barrier against zoonotic transmission of avian influenza viruses. However, the H7N9 viruses overcome this restriction by harboring an NP that is less sensitive to Mx-mediated host defense. This might contribute to zoonotic transmission of H7N9 and to the severe to fatal outcome of H7N9 infections in humans.

    IMPORTANCE The natural host of influenza A viruses (IAVs) are aquatic birds. Occasionally, these viruses cross the species barrier, as in early 2013 when an avian H7N9 virus infected humans in China. Since then, multiple transmissions of H7N9 viruses to humans have occurred, leaving experts puzzled about molecular causes for such efficient crossing of the species barrier compared to other avian influenza viruses. Mx proteins are known restriction factors preventing influenza virus replication. Unfortunately, some viruses (e.g., human IAV) have developed some resistance, which is associated with specific amino acids in their nucleoproteins, the target of Mx function. Here, we demonstrate that the novel H7N9 bird IAV already carries a nucleoprotein that overcomes the inhibition of viral replication by human MxA. This is the first example of an avian IAV that is naturally less sensitive to Mx-mediated inhibition and might explain why H7N9 viruses transmitted efficiently to humans.

  • Identification and Functional Comparison of Seven-Transmembrane G-Protein-Coupled BILF1 Receptors in Recently Discovered Nonhuman Primate Lymphocryptoviruses [Genetic Diversity and Evolution]

  • Coevolution of herpesviruses with their respective host has resulted in a delicate balance between virus-encoded immune evasion mechanisms and host antiviral immunity. BILF1 encoded by human Epstein-Barr virus (EBV) is a 7-transmembrane (7TM) G-protein-coupled receptor (GPCR) with multiple immunomodulatory functions, including attenuation of PKR phosphorylation, activation of G-protein signaling, and downregulation of major histocompatibility complex (MHC) class I surface expression. In this study, we explored the evolutionary and functional relationships between BILF1 receptor family members from EBV and 12 previously uncharacterized nonhuman primate (NHP) lymphocryptoviruses (LCVs). Phylogenetic analysis defined 3 BILF1 clades, corresponding to LCVs of New World monkeys (clade A) or Old World monkeys and great apes (clades B and C). Common functional properties were suggested by a high degree of sequence conservation in functionally important regions of the BILF1 molecules. A subset of BILF1 receptors from EBV and LCVs from NHPs (chimpanzee, orangutan, marmoset, and siamang) were selected for multifunctional analysis. All receptors exhibited constitutive signaling activity via G protein Gaalpha;i and induced activation of the NF-B transcription factor. In contrast, only 3 of 5 were able to activate NFAT (nuclear factor of activated T cells); chimpanzee and orangutan BILF1 molecules were unable to activate NFAT. Similarly, although all receptors were internalized, BILF1 from the chimpanzee and orangutan displayed an altered cellular localization pattern with predominant cell surface expression. This study shows how biochemical characterization of functionally important orthologous viral proteins can be used to complement phylogenetic analysis to provide further insight into diverse microbial evolutionary relationships and immune evasion function.

    IMPORTANCE Epstein-Barr virus (EBV), known as an oncovirus, is the only human herpesvirus in the genus Lymphocryptovirus (LCV). EBV uses multiple strategies to hijack infected host cells, establish persistent infection in B cells, and evade antiviral immune responses. As part of EBV's immune evasion strategy, the virus encodes a multifunctional 7-transmembrane (7TM) G-protein-coupled receptor (GPCR), EBV BILF1. In addition to multiple immune evasion-associated functions, EBV BILF1 has transforming properties, which are linked to its high constitutive activity. We identified BILF1 receptor orthologues in 12 previously uncharacterized LCVs from nonhuman primates (NHPs) of Old and New World origin. As 7TM receptors are excellent drug targets, our unique insight into the molecular mechanism of action of the BILF1 family and into the evolution of primate LCVs may enable validation of EBV BILF1 as a drug target for EBV-mediated diseases, as well as facilitating the design of drugs targeting EBV BILF1.

  • Kaposi's Sarcoma-Associated Herpesvirus Induces Nrf2 Activation in Latently Infected Endothelial Cells through SQSTM1 Phosphorylation and Interaction with Polyubiquitinated Keap1 [Virus-Cell Interactions]

  • Nuclear factor erythroid 2-related factor 2 (Nrf2), the cellular master regulator of the antioxidant response, dissociates from its inhibitor Keap1 when activated by stress signals and participates in the pathogenesis of viral infections and tumorigenesis. Early during de novo infection of endothelial cells, KSHV induces Nrf2 through an intricate mechanism involving reactive oxygen species (ROS) and prostaglandin E2 (PGE2). When we investigated the Nrf2 activity during latent KSHV infection, we observed increased nuclear serine-40-phosphorylated Nrf2 in human KS lesions compared to that in healthy tissues. Using KSHV long-term-infected endothelial cells (LTC) as a cellular model for KS, we demonstrated that KSHV infection induces Nrf2 constitutively by extending its half-life, increasing its phosphorylation by protein kinase C (PKC) via the infection-induced cyclooxygenase-2 (COX-2)/PGE2 axis and inducing its nuclear localization. Nrf2 knockdown in LTC decreased expression of antioxidant genes and genes involved in KS pathogenesis such as the NAD(P)H quinone oxidase 1 (NQO1), gamma glutamylcysteine synthase heavy unit (GCSH), the cysteine transporter (xCT), interleukin 6 (IL-6), and vascular endothelial growth factor A (VEGF-A) genes. Nrf2 activation was independent of oxidative stress but dependent on the autophagic protein sequestosome-1 (SQSTM1; p62). SQSTM1 levels were elevated in LTC, a consequence of protein accumulation due to decreased autophagy and Nrf2-mediated transcriptional activation. SQSTM1 was phosphorylated on serine-351 and -403, while Keap1 was polyubiquitinated with lysine-63nndash;ubiquitin chains, modifications known to increase their mutual affinity and interaction, leading to Keap1 degradation and Nrf2 activation. The latent KSHV protein Fas-associated death domain-like interleukin-1bbeta;-converting enzyme-inhibitory protein (vFLIP) increased SQSTM1 expression and activated Nrf2. Collectively, these results demonstrate that KSHV induces SQSTM1 to constitutively activate Nrf2, which is involved in the regulation of genes participating in KSHV oncogenesis.

    IMPORTANCE The transcription factor Nrf2 is activated by stress signals, including viral infection, and responds by activating the transcription of cytoprotective genes. Recently, Nrf2 has been implicated in oncogenesis and was shown to be activated during de novo KSHV infection of endothelial cells through ROS-dependent pathways. The present study was undertaken to determine the mechanism of Nrf2 activation during prolonged latent infection of endothelial cells, using an endothelial cell line latently infected with KSHV. We show that Nrf2 activation was elevated in KSHV latently infected endothelial cells independently of oxidative stress but dependent on the autophagic protein sequestosome-1 (SQSTM1), which was involved in the degradation of the Nrf2 inhibitor Keap1. Furthermore, our results indicated that the KSHV latent protein vFLIP participates in Nrf2 activation. This study suggests that KSHV hijacks the host's autophagic protein SQSTM1 to induce Nrf2 activation, thereby manipulating the infected host gene regulation to promote KS pathogenesis.

  • Hepatitis B Virus Polymerase Disrupts K63-Linked Ubiquitination of STING To Block Innate Cytosolic DNA-Sensing Pathways [Virus-Cell Interactions]

  • The cellular innate immune system recognizing pathogen infection is essential for host defense against viruses. In parallel, viruses have developed a variety of strategies to evade the innate immunity. The hepatitis B virus (HBV), a DNA virus that causes chronic hepatitis, has been shown to inhibit RNA helicase RIG-I-mediated interferon (IFN) induction. However, it is still unknown whether HBV could affect the host DNA-sensing pathways. Here we report that in transiently HBV-transfected Huh7 cells, the stably HBV-producing cell line HepAD38, and HBV-infected HepaRG cells and primary human hepatocytes, HBV markedly interfered with IFN-bbeta; induction and antiviral immunity mediated by the stimulator of interferon genes (STING), which has been identified as a central factor in foreign DNA recognition and antiviral innate immunity. Screening analysis demonstrated that the viral polymerase (Pol), but not other HBV-encoded proteins, was able to inhibit STING-stimulated interferon regulatory factor 3 (IRF3) activation and IFN-bbeta; induction. Moreover, the reverse transcriptase (RT) and the RNase H (RH) domains of Pol were identified to be responsible for the inhibitory effects. Furthermore, Pol was shown to physically associate with STING and dramatically decrease the K63-linked polyubiquitination of STING via its RT domain without altering the expression level of STING. Taken together, these observations suggest that besides its inherent catalytic function, Pol has a role in suppression of IFN-bbeta; production by direct interaction with STING and subsequent disruption of its K63-linked ubiquitination, providing a new mechanism for HBV to counteract the innate DNA-sensing pathways.

    IMPORTANCE Although whether and how HBV infection induces the innate immune responses are still controversial, it has become increasingly clear that HBV has developed strategies to counteract the pattern recognition receptor-mediated signaling pathways. Previous studies have shown that type I IFN induction activated by the host RNA sensors could be inhibited by HBV. However, it remains unknown whether HBV as a DNA virus utilizes evasion mechanisms against foreign DNA-elicited antiviral signaling. In recent years, the cytosolic DNA sensor and key adaptor STING has been demonstrated to be essential in multiple foreign DNA-elicited innate immune signalings. Here, for the first time, we report STING as a new target of HBV to antagonize IFN induction and identify the viral polymerase responsible for the inhibitory effect, thus providing an additional molecular mechanism by which HBV evades the innate immunity; this implies that in addition to its inherent catalytic function, HBV polymerase is a multifunctional immunomodulatory protein.

  • Epstein-Barr Virus Type 2 Latently Infects T Cells, Inducing an Atypical Activation Characterized by Expression of Lymphotactic Cytokines [Cellular Response to Infection]

  • Epstein-Barr virus (EBV) is a well-established B-cell-tropic virus associated with various lymphoproliferative diseases of both B-cell and non-B-cell origin. EBV is associated with a number of T-cell lymphomas; however, in vitro studies utilizing prototypical EBV type 1 (EBV-1) laboratory strains have generally failed to readily infect mature T cells in culture. The difficulties in performing in vitro T-cell experiments have left questions regarding the role of EBV in the pathogenesis of EBV-positive T-cell lymphoproliferative diseases largely unresolved. We report here that the EBV type 2 (EBV-2) strain displays a unique cell tropism for T cells. In remarkable contrast to EBV-1, EBV-2 readily infects primary T cells in vitro, demonstrating a propensity for CD8+ T cells. EBV-2 infection of purified T cells results in expression of latency genes and ultimately leads to T-cell activation, substantial proliferation, and profound alteration of cytokine expression. The pattern of cytokine production is strikingly skewed toward chemokines with roles in lymphocyte migration, demonstrating that EBV-2 has the ability to modulate normal T-cell processes. Collectively, these novel findings identify a previously unknown cell population potentially utilized by EBV-2 to establish latency and lay the foundation for further studies to elucidate the role of EBV in the pathogenesis of T-cell lymphoproliferative diseases.

    IMPORTANCE The ability of EBV to infect T cells is made apparent by its association with a variety of T-cell lymphoproliferative disorders. However, studies to elucidate the pathogenic role of EBV in these diseases have been limited by the inability to conduct in vitro T-cell infection experiments. Here, we report that EBV-2 isolates, compromised in the capacity to immortalize B cells, infect CD3+ T cells ex vivo and propose a working model of EBV-2 persistence where alteration of T-cell functions resulting from EBV-2 infection enhances the establishment of latency in B cells. If indeed EBV-2 utilizes T cells to establish a persistent infection, this could provide one mechanism for the association of EBV with T-cell lymphomas. The novel finding that EBV-2 infects T cells in culture will provide a model to understand the role EBV plays in the development of T-cell lymphomas.

  • Herpes Simplex Virus 1 gN Partners with gM To Modulate the Viral Fusion Machinery [Structure and Assembly]

  • Herpes simplex virus 1 (HSV-1) capsids are assembled in the nucleus, where they incorporate the viral genome. They then transit through the two nuclear membranes and are wrapped by a host-derived envelope. In the process, several HSV-1 proteins are targeted to the nuclear membranes, but their roles in viral nuclear egress are unclear. Among them, glycoprotein M (gM), a known modulator of virus-induced membrane fusion, is distributed on both the inner and outer nuclear membranes at the early stages of the infection, when no other viral glycoproteins are yet present there. Later on, it is found on perinuclear virions and ultimately redirected to the trans-Golgi network (TGN), where it cycles with the cell surface. In contrast, transfected gM is found only at the TGN and cell surface, hinting at an interaction with other viral proteins. Interestingly, many herpesvirus gM analogs interact with their gN counterparts, which typically alters their intracellular localization. To better understand how HSV-1 gM localization is regulated, we evaluated its ability to bind gN and discovered it does so in both transfected and infected cells, an interaction strongly weakened by the deletion of the gM amino terminus. Functionally, while gN had no impact on gM localization, gM redirected gN from the endoplasmic reticulum (ER) to the TGN. Most interestingly, gN overexpression stimulated the formation of syncytia in the context of an infection by a nonsyncytial strain, indicating that gM and gN not only physically but also functionally interact and that gN modulates gM's activity on membrane fusion.

    IMPORTANCE HSV-1 gM is an important modulator of virally induced cell-cell fusion and viral entry, a process that is likely finely modulated in time and space. Until now, little was known of the proteins that regulate gM's activity. In parallel, gM is found in various intracellular locations at different moments, ranging from nuclear membranes, perinuclear virions, the TGN, cell surface, and mature extracellular virions. In transfected cells, however, it is found only on the TGN and cell surface, hinting that its localization is modulated by other viral proteins. The present study identifies HSV-1 gN as a binding partner for gM, in agreement with their analogs in other herpesviruses, but most excitingly shows that gN modulates gM's impact on HSV-1-induced membrane fusion. These findings open up new research avenues on the viral fusion machinery.

  • The Progressive Adaptation of a Georgian Isolate of African Swine Fever Virus to Vero Cells Leads to a Gradual Attenuation of Virulence in Swine Corresponding to Major Modifications of the Viral Genome [Pathogenesis and Immunity]

  • African swine fever virus (ASFV) causes a contagious and often lethal disease of feral and domestic swine. Experimental vaccines derived from naturally occurring, genetically modified, or cell culture-adapted ASFV have been evaluated, but no commercial vaccine is available to control African swine fever (ASF). We report here the genotypic and phenotypic analysis of viruses obtained at different passages during the process of adaptation of a virulent ASFV field isolate from the Republic of Georgia (ASFV-G) to grow in cultured cell lines. ASFV-G was successively passaged 110 times in Vero cells. Viruses obtained at passages 30, 60, 80, and 110 were evaluated in vitro for the ability to replicate in Vero cells and primary swine macrophages cultures and in vivo for assessing virulence in swine. Replication of ASFV-G in Vero cells increased with successive passages, corresponding to a decreased replication in primary swine macrophages cultures. In vivo, progressive loss of virus virulence was observed with increased passages in Vero cells, and complete attenuation of ASFV-G was observed at passage 110. Infection of swine with the fully attenuated virus did not confer protection against challenge with virulent parental ASFV-G. Full-length sequence analysis of each of these viruses revealed significant deletions that gradually accumulated in specific areas at the right and left variable ends of the genome. Mutations that result in amino acid substitutions and frameshift mutations were also observed, though in a rather limited number of genes. The potential importance of these genetic changes in virus adaptation/attenuation is discussed.

    IMPORTANCE The main problem in controlling ASF is the lack of vaccines. Attempts to produce vaccines by adaptation of ASFV to cultured cell lines have been made. These attempts led to the production of attenuated viruses that conferred only homologous protection. Specifics regarding adaptation of these isolates to cell cultures have been insufficiently described. Details like the numbers of passages required to obtain attenuated viruses, genetic modifications introduced into the virus genomes along passages, and the extent of attenuation and induced protective efficacy are not readily available. In this study, we assessed the changes that lead to decreased growth in swine macrophages and to attenuation in swine. Loss of virulence, probably associated with limited replication in vivo, may lead to the lack of protective immunity in swine observed after challenge. This report provides valuable information that can be used to further the understanding of ASFV gene function, virus attenuation, and protection against infection.

  • MicroRNA-Mediated Transformation by the Kaposi's Sarcoma-Associated Herpesvirus Kaposin Locus [Transformation and Oncogenesis]

  • The human oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) expresses a set of ~20 viral microRNAs (miRNAs). miR-K10a stands out among these miRNAs because its entire stem-loop precursor overlaps the coding sequence for the Kaposin (Kap) A/C proteins. The ectopic expression of KapA has been reported to lead to transformation of rodent fibroblasts. However, these experiments inadvertently also introduced miR-K10a, which raises the question whether the transforming activity of the locus could in fact be due to miR-K10a expression. To answer this question, we have uncoupled miR-K10a and KapA expression. Our experiments revealed that miR-K10a alone transformed cells with an efficiency similar to that when it was coexpressed with KapA. Maintenance of the transformed phenotype was conditional upon continued miR-K10a but not KapA protein expression, consistent with its dependence on miRNA-mediated changes in gene expression. Importantly, miR-K10a taps into an evolutionarily conserved network of miR-142-3p targets, several of which are expressed in 3T3 cells and are also known inhibitors of cellular transformation. In summary, our studies of miR-K10a serve as an example of an unsuspected function of an mRNA whose precursor is embedded within a coding transcript. In addition, our identification of conserved miR-K10a targets that limit transformation will point the way to a better understanding of the role of this miRNA in KSHV-associated tumors.

    IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus. The viral Kaposin locus has known oncogenic potential, which has previously been attributed to the encoded KapA protein. Here we show that the virally encoded miR-K10a miRNA, whose precursor overlaps the KapA-coding region, may account for the oncogenic properties of this locus. Our data suggest that miR-K10a mimics the cellular miRNA miR-142-3p and thereby represses several known inhibitors of oncogenic transformation. Our work demonstrates that functional properties attributed to a coding region may in fact be carried out by an embedded noncoding element and sheds light on the functions of viral miR-K10a.

  • N-Linked Glycosylation Protects Gammaretroviruses against Deamination by APOBEC3 Proteins [Pathogenesis and Immunity]

  • Retroviruses are pathogens with rapid infection cycles that can be a source of disease, genome instability, and tumor development in their hosts. Host intrinsic restriction factors, such as APOBEC3 (A3) proteins, are constitutively expressed and dedicated to interfering with the replication cycle of retroviruses. To survive, propagate, and persist, retroviruses must counteract these restriction factors, often by way of virus genome-encoded accessory proteins. Glycosylated Gag, also called glycosylated Pr80 Gag (gPr80), is a gammaretrovirus genome-encoded protein that inhibits the antiretroviral activity of mouse A3 (mA3). Here we show that gPr80 exerts two distinct inhibitory effects on mA3: one that antagonizes deamination-independent restriction and another one that inhibits its deaminase activity. More specifically, we find that the number of N-glycosylated residues in gPr80 inversely correlates with the sensitivity of a gammaretrovirus to deamination by mouse A3 and also, surprisingly, by human A3G. Finally, our work highlights that retroviruses which have successfully integrated into the mouse germ line generally express a gPr80 with fewer glycosylated sites than exogenous retroviruses. This observation supports the suggestion that modulation of A3 deamination intensity could be a desirable attribute for retroviruses to increase genetic diversification and avoid immune detection. Overall, we present here the first description of how gammaretroviruses employ posttranslational modification to antagonize and modulate the activity of a host genome-encoded retroviral restriction factor.

    IMPORTANCE APOBEC3 proteins are host factors that have a major role in protecting humans and other mammals against retroviruses. These enzymes hinder their replication and intensely mutate their DNA, thereby inactivating viral progeny and the spread of infection. Here we describe a newly recognized way in which some retroviruses protect themselves against the mutator activity of APOBEC3 proteins. We show that gammaretroviruses expressing an accessory protein called glycosylated Gag, or gPr80, use the host's posttranslational machinery and, more specifically, N-linked glycosylation as a way to modulate their sensitivity to mutations by APOBEC3 proteins. By carefully controlling the amount of mutations caused by APOBEC3 proteins, gammaretroviruses can find a balance that helps them evolve and persist.

  • Dengue Virus Induces and Requires Glycolysis for Optimal Replication [Virus-Cell Interactions]

  • Viruses rely on host cellular metabolism to provide the energy and biosynthetic building blocks required for their replication. Dengue virus (DENV), a member of the Flaviviridae family, is one of the most important arthropod-borne human pathogens worldwide. We analyzed global intracellular metabolic changes associated with DENV infection of primary human cells. Our metabolic profiling data suggested that central carbon metabolism, particularly glycolysis, is strikingly altered during a time course of DENV infection. Glucose consumption is increased during DENV infection and depriving DENV-infected cells of exogenous glucose had a pronounced impact on viral replication. Furthermore, the expression of both glucose transporter 1 and hexokinase 2, the first enzyme of glycolysis, is upregulated in DENV-infected cells. Pharmacologically inhibiting the glycolytic pathway dramatically reduced DENV RNA synthesis and infectious virion production, revealing a requirement for glycolysis during DENV infection. Thus, these experiments suggest that DENV induces the glycolytic pathway to support efficient viral replication. This study raises the possibility that metabolic inhibitors, such as those that target glycolysis, could be used to treat DENV infection in the future.

    IMPORTANCE Approximately 400 million people are infected with dengue virus (DENV) annually, and more than one-third of the global population is at risk of infection. As there are currently no effective vaccines or specific antiviral therapies for DENV, we investigated the impact DENV has on the host cellular metabolome to identify metabolic pathways that are critical for the virus life cycle. We report an essential role for glycolysis during DENV infection. DENV activates the glycolytic pathway, and inhibition of glycolysis significantly blocks infectious DENV production. This study provides further evidence that viral metabolomic analyses can lead to the discovery of novel therapeutic targets to block the replication of medically important human pathogens.

  • Phosphatidylserine-Specific Phospholipase A1 Involved in Hepatitis C Virus Assembly through NS2 Complex Formation [Virus-Cell Interactions]

  • Several members of the phospholipase family have been reported to be involved in hepatitis C virus (HCV) replication. Here, we identified another phospholipase, phosphatidylserine-specific phospholipase A1 (PLA1A), as a host factor involved in HCV assembly. PLA1A was upregulated by HCV infection, and PLA1A knockdown significantly reduced J399EM (genotype 2a) HCV propagation at the assembly step but not the entry, RNA replication, and protein translation steps of the life cycle. Protein localization and interaction analysis further revealed a role of PLA1A in the interaction of NS2-E2 and NS2-NS5A, as the formation of the NS2-E2 and NS2-NS5A complexes was weakened in the absence of PLA1A. In addition, PLA1A stabilized the NS2/NS5A dotted structure during infection. These data suggest that PLA1A plays an important role in bridging the membrane-associated NS2-E2 complex and the NS5A-associated replication complex via its interaction with E2, NS2, and NS5A, which leads to a coordinating interaction between the structural and nonstructural proteins and facilitates viral assembly.

    IMPORTANCE Hepatitis C virus (HCV) genomic replication is driven by the replication complex and occurs at the membranous web, while the lipid droplet is the organelle in which virion assembly is initiated. In this study, we identified phosphatidylserine-specific phospholipase A1 (PLA1A), a member of phospholipase A 1 family, as a novel host factor involved in the assembly process of HCV. PLA1A, which is induced by HCV infection at a late infection stage, interacts with HCV E2, NS2, and NS5A proteins and enhances and stabilizes the NS2-E2 and NS2-NS5A complex formation, which is essential for viral assembly. Thus, PLA1A is an important host factor which is involved in the initiation of the viral assembly in close proximity to Core-decorated lipid droplets through bringing together the HCV replication complex and envelope complex.

  • Structural Analysis of a Rabbit Hemorrhagic Disease Virus Binding to Histo-Blood Group Antigens [Structure and Assembly]

  • Rabbit hemorrhagic disease virus (RHDV) is a member of the Caliciviridae family (Lagovirus genus). RHDV is highly contagious and attaches to epithelial cells in the digestive or respiratory tract, leading to massive lesions with high mortality rates. A new variant of RHDV (termed RHDVb) recently has emerged, and previously vaccinated rabbits appear to have little protection against this new strain. Similar to human norovirus (Caliciviridae, Norovirus genus), RHDV binds histo-blood group antigens (HBGAs), and this is thought to be important for infection. Here, we report the HBGA binding site on the RHDVb capsid-protruding domain (P domain) using X-ray crystallography. The HBGA binding pocket was located in a negatively charged patch on the side of the P domain and at a dimeric interface. Residues from both monomers contributed to the HBGA binding and involved a network of direct hydrogen bonds and water-mediated interactions. An amino acid sequence alignment of different RHDV strains indicated that the residues directly interacting with the ABH-fucose of the HBGAs (Asp472, Asn474, and Ser479) were highly conserved. This result suggested that different RHDV strains also could bind HBGAs at the equivalent pocket. Moreover, several HBGA binding characteristics between RHDVb and human genogroup II norovirus were similar, which indicated a possible convergent evolution of HBGA binding interactions. Further structural studies with other RHDV strains are needed in order to better understand the HBGA binding mechanisms among the diverse RHDV strains.

    IMPORTANCE We identified, for the first time, the HBGA binding site on an RHDVb P domain using X-ray crystallography. Our results showed that RHDVb and human genogroup II noroviruses had similar HBGA binding interactions. Recently, it was discovered that synthetic HBGAs or HBGA-expressing enteric bacteria could enhance human genogroup II norovirus infection in B cells. Considering that RHDVb and genogroup II norovirus similarly interacted with HBGAs, it may be possible that a comparable cell culture system also could work with RHDVb. Taken together, these new findings will extend our understanding of calicivirus HBGA interactions and may help to elucidate the specific roles of HBGAs in calicivirus infections.

  • Prion Infection of Mouse Brain Reveals Multiple New Upregulated Genes Involved in Neuroinflammation or Signal Transduction [Prions]

  • Gliosis is often a preclinical pathological finding in neurodegenerative diseases, including prion diseases, but the mechanisms facilitating gliosis and neuronal damage in these diseases are not understood. To expand our knowledge of the neuroinflammatory response in prion diseases, we assessed the expression of key genes and proteins involved in the inflammatory response and signal transduction in mouse brain at various times after scrapie infection. In brains of scrapie-infected mice at pre- and postclinical stages, we identified 15 previously unreported differentially expressed genes related to inflammation or activation of the STAT signal transduction pathway. Levels for the majority of differentially expressed genes increased with time postinfection. In quantitative immunoblotting experiments of STAT proteins, STAT1aalpha;, phosphorylated-STAT1aalpha; (pSTAT1aalpha;), and pSTAT3 were increased between 94 and 131 days postinfection (p.i.) in brains of mice infected with strain 22L. Furthermore, a select group of STAT-associated genes was increased preclinically during scrapie infection, suggesting early activation of the STAT signal transduction pathway. Comparison of inflammatory markers between mice infected with scrapie strains 22L and RML indicated that the inflammatory responses and gene expression profiles in the brains were strikingly similar, even though these scrapie strains infect different brain regions. The endogenous interleukin-1 receptor antagonist (IL-1Ra), an inflammatory marker, was newly identified as increasing preclinically in our model and therefore might influence scrapie pathogenesis in vivo. However, in IL-1Ra-deficient or overexpressor transgenic mice inoculated with scrapie, neither loss nor overexpression of IL-1Ra demonstrated any observable effect on gliosis, protease-resistant prion protein (PrPres) formation, disease tempo, pathology, or expression of the inflammatory genes analyzed.

    IMPORTANCE Prion infection leads to PrPres deposition, gliosis, and neuroinflammation in the central nervous system before signs of clinical illness. Using a scrapie mouse model of prion disease to assess various time points postinoculation, we identified 15 unreported genes that were increased in the brains of scrapie-infected mice and were associated with inflammation and/or JAK-STAT activation. Comparison of mice infected with two scrapie strains (22L and RML), which have dissimilar neuropathologies, indicated that the inflammatory responses and gene expression profiles in the brains were similar. Genes that increased prior to clinical signs might be involved in controlling scrapie infection or in facilitating damage to host tissues. We tested the possible role of the endogenous IL-1Ra, which was increased at 70 days p.i. In scrapie-infected mice deficient in or overexpressing IL-1Ra, there was no observable effect on gliosis, PrPres formation, disease tempo, pathology, or expression of inflammatory genes analyzed.

  • MCL1 Enhances the Survival of CD8+ Memory T Cells after Viral Infection [Pathogenesis and Immunity]

  • Viral infection results in the generation of massive numbers of activated effector CD8+ T cells that recognize viral components. Most of these are short-lived effector T cells (SLECs) that die after clearance of the virus. However, a small proportion of this population survives and forms antigen-specific memory precursor effector cells (MPECs), which ultimately develop into memory cells. These can participate in a recall response upon reexposure to antigen even at protracted times postinfection. Here, antiapoptotic myeloid cell leukemia 1 (MCL1) was found to prolong survival upon T cell stimulation, and mice expressing human MCL1 as a transgene exhibited a skewing in the proportion of CD8+ T cells, away from SLECs toward MPECs, during the acute phase of vaccinia virus infection. A higher frequency and total number of antigen-specific CD8+ T cells were observed in MCL1 transgenic mice. These findings show that MCL1 can shape the makeup of the CD8+ T cell response, promoting the formation of long-term memory.

    IMPORTANCE During an immune response to a virus, CD8+ T cells kill cells infected by the virus, and most die when the infection resolves. However, a small proportion of cells survives and differentiates into long-lived memory cells that confer protection from reinfection by the same virus. This report shows that transgenic expression of an MCL1 protein enhances survival of memory CD8+ T cells following infection with vaccinia virus. This is important because it shows that MCL1 expression may be an important determinant of the formation of long-term CD8+ T cell memory.

  • Genetic Analysis of the Localization of APOBEC3F to Human Immunodeficiency Virus Type 1 Virion Cores [Pathogenesis and Immunity]

  • Members of the APOBEC3 family of cytidine deaminases vary in their proportions of a virion-incorporated enzyme that is localized to mature retrovirus cores. We reported previously that APOBEC3F (A3F) was highly localized into mature human immunodeficiency virus type 1 (HIV-1) cores and identified that L306 in the C-terminal cytidine deaminase (CD) domain contributed to its core localization (C. Song, L. Sutton, M. Johnson, R. D'Aquila, J. Donahue, J Biol Chem 287:16965nndash;16974, 2012, http://dx.doi.org/10.1074/jbc.M111.310839). We have now determined an additional genetic determinant(s) for A3F localization to HIV-1 cores. We found that one pair of leucines in each of A3F's C-terminal and N-terminal CD domains jointly determined the degree of localization of A3F into HIV-1 virion cores. These are A3F L306/L368 (C-terminal domain) and A3F L122/L184 (N-terminal domain). Alterations to one of these specific leucine residues in either of the two A3F CD domains (A3F L368A, L122A, and L184A) decreased core localization and diminished HIV restriction without changing virion packaging. Furthermore, double mutants in these leucine residues in each of A3F's two CD domains (A3F L368A plus L184A or A3F L368A plus L122A) still were packaged into virions but completely lost core localization and anti-HIV activity. HIV virion core localization of A3F is genetically separable from its virion packaging, and anti-HIV activity requires some core localization.

    IMPORTANCE Specific leucine-leucine interactions are identified as necessary for A3F's core localization and anti-HIV activity but not for its packaging into virions. Understanding these signals may lead to novel strategies to enhance core localization that may augment effects of A3F against HIV and perhaps of other A3s against retroviruses, parvoviruses, and hepatitis B virus.

  • GB Virus C Coinfections in West African Ebola Patients [Pathogenesis and Immunity]

  • In 49 patients with known Ebola virus disease outcomes during the ongoing outbreak in Sierra Leone, 13 were coinfected with the immunomodulatory pegivirus GB virus C (GBV-C). Fifty-three percent of these GBV-C+ patients survived; in contrast, only 22% of GBV-Cnndash; patients survived. Both survival and GBV-C status were associated with age, with older patients having lower survival rates and intermediate-age patients (21 to 45 years) having the highest rate of GBV-C infection. Understanding the separate and combined effects of GBV-C and age on Ebola virus survival may lead to new treatment and prevention strategies, perhaps through age-related pathways of immune activation.

  • Analysis of Human T-Cell Leukemia Virus Type 1 Particles by Using Cryo-Electron Tomography [Structure and Assembly]

  • The particle structure of human T-cell leukemia virus type 1 (HTLV-1) is poorly characterized. Here, we have used cryo-electron tomography to analyze HTLV-1 particle morphology. Particles produced from MT-2 cells were polymorphic, roughly spherical, and varied in size. Capsid cores, when present, were typically poorly defined polyhedral structures with at least one curved region contacting the inner face of the viral membrane. Most of the particles observed lacked a defined capsid core, which likely impacts HTLV-1 particle infectivity.

  • Deletion of the ORF9p Acidic Cluster Impairs the Nuclear Egress of Varicella-Zoster Virus Capsids [Structure and Assembly]

  • The protein encoded by ORF9 is essential for varicella-zoster virus (VZV) replication. Previous studies documented its presence in the trans-Golgi network and its involvement in secondary envelopment. In this work, we deleted the ORF9p acidic cluster, destroying its interaction with ORF47p, and this resulted in a nuclear accumulation of both proteins. This phenotype results in an accumulation of primary enveloped capsids in the perinuclear space, reflecting a capsid de-envelopment defect.

  • Adaptation of Pandemic H2N2 Influenza A Viruses in Humans [Genetic Diversity and Evolution]

  • The 1957 A/H2N2 influenza virus caused an estimated 2 million fatalities during the pandemic. Since viruses of the H2 subtype continue to infect avian species and pigs, the threat of reintroduction into humans remains. To determine factors involved in the zoonotic origin of the 1957 pandemic, we performed analyses on genetic sequences of 175 newly sequenced human and avian H2N2 virus isolates and all publicly available influenza virus genomes.

  • Characterization of Novel Transcripts of Human Papillomavirus Type 16 Using Cap Analysis Gene Expression Technology [Genome Replication and Regulation of Viral Gene Expression]

  • We have performed cap-analysis gene expression (CAGE) sequencing to identify the regulatory networks that orchestrate genome-wide transcription in human papillomavirus type 16 (HPV16)-positive cervical cell lines of different grades: W12E, SiHa, and CaSki. Additionally, a cervical intraepithelial neoplasia grade 1 (CIN1) lesion was assessed for identifying the transcriptome expression profile. Here we have precisely identified a novel antisense noncoding viral transcript in HPV16. In conclusion, CAGE sequencing should pave the way for understanding a diversity of viral transcript expression.

  • Erratum for Tesfay et al., PEGylation of Vesicular Stomatitis Virus Extends Virus Persistence in Blood Circulation of Passively Immunized Mice [Errata]

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    JVI Accepts: Articles Published Ahead of Print

  • KSHV microRNAs repress breakpoint cluster region protein expression, enhance Rac1 activity, and increase in vitro angiogenesis [PublishAheadOfPrint]

  • MicroRNAs (miRNAs) are small ~22-nt long RNAs that regulate gene expression post-transcriptionally. KSHV encodes 12 pre-miRNAs during latency and the functional significance of these microRNAs during KSHV infection and their cellular targets are emerging only recently. Using a previously reported microarray profiling analysis, we identified breakpoint cluster region mRNA (Bcr) as a cellular target of the KSHV-encoded miRNA, miR-K12-6-5p (miR-K6-5). Bcr protein levels were repressed in human umbilical vein endothelial cells (HUVECs) upon transfection with miR-K6-5 and during KSHV infection. Luciferase assays, wherein, the Bcr 3rrsquo; -UTR was cloned downstream of a luciferase reporter showed repression in the presence of miR-K6-5 and mutation of one of the two predicted miR-K6-5 binding sites relieved this repression. Further, inhibition or deletion of miR-K6-5 in KSHV-infected cells showed increased Bcr protein levels. Together, these results show that Bcr is a direct target of the KSHV miRNA, miR-K6-5. To understand the functional significance of Bcr knockdown in the context of KSHV-associated disease, we hypothesized that the knockdown of Bcr, a negative regulator of Rac1, might enhance Rac1-mediated angiogenesis. We found that HUVECs transfected with miR-K6-5 had increases in Rac1-GTP levels and tube formation, compared to control miRNAs. Knockdown of Bcr in KSHV-latently infected BCBL-1 cells increased the levels of viral RTA, suggesting that Bcr repression by KSHV might aid lytic reactivation. Together, our results reveal a new function for both KSHV miRNAs and Bcr in KSHV infection and suggest that KSHV miRNAs, in part, promote angiogenesis and lytic reactivation.

    Importance: Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) infection is linked to multiple human cancers and lymphomas. KSHV encodes small nucleic acids (microRNAs) that can repress expression of specific human genes, the biological functions for which are still emerging. This report uses a variety of approaches to show that a KSHV microRNA represses the expression of the human gene called breakpoint cluster region (Bcr). Repression of Bcr correlated with activation of a protein previously shown to cause KS-like lesions in mice (Rac1), an increase in KS-associated phenotypes (tube formation in endothelial cells, VEGF synthesis) and modifying the life cycle of the virus (lytic replication). Our results suggest that KSHV-encoded microRNAs suppress host proteins and contribute to KS-associated pathogenesis.

  • Identification of Three Redundant Segments Responsible for Herpes Simplex Virus 1 Infected Cell Protein 0 to Fuse with ND10 Nuclear Bodies [PublishAheadOfPrint]

  • Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is a key regulator in both lytic and latent infection. In lytic infection an important early event is the colocalization of ICP0 to nuclear domains 10 (ND10s) that impose restrictions on viral expression. ICP0 contains an E3 ubiquitin ligase that degrades PML and Sp100, two major components of ND10s, and disperses ND10s to alleviate the repression. We have previously reported that association between ICP0 and ND10 is a dynamic process that includes three steps, adhesion, fusion and retention. ICP0 residues 245-474, defined as ND10-entry signal (ND10-ES), is a region required for the fusion step. Without ND10-ES, ICP0 adheres at ND10 surface but fails to enter. In the present study we focus on characterizing ND10-ES. Here we report that (i) fusion of ICP0 with ND10 relies on specific sequences located within ND10-ES. Replacement of ND10-ES by corresponding region from ORF61 of varicella-zoster virus did not rescue ND10 fusion; (ii) three tandem ND10-fusion segments (ND10-FS1, ND10-FS2 and ND10-FS3) encompassing 200 amino acids within ND10-ES redundantly facilitate the fusion. Each of the three segments is sufficient to independently drive the fusion process, but none of the segments by itself is necessary for ND10 fusion. Only when all three segments are deleted will fusion be blocked; (iii) SUMO interaction motif located within ND10-FS2 is not required for ND10 fusion but is required for the complete degradation of PML, suggesting that PML degradation and ND10 fusion are regulated by different molecular mechanisms.

    Importance Nuclear bodies ND10s are part of the cell intrinsic anti-viral defenses that restrict viral gene expression upon virus infection. As a countermeasure, infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) localizes to ND10s, degrades ND10 organizer and disperses ND10 components in order to alleviate the repression. We study ICP0-ND10 association to delineate elements important for this dynamic interaction and to understand its role in viral replication and host defense. In this work, we show that ICP0 contains three redundant segments to ensure an effective mergence of ICP0 with the ND10 nuclear bodies. This is the first study to systematically investigate ICP0 elements that are important for ICP0-ND10 fusion.

  • Clathrin mediates infectious hepatitis C virus particle egress [PublishAheadOfPrint]

  • Although it is well established that hepatitis C virus (HCV) entry into hepatocytes depends on clathrin-mediated endocytosis, the possible roles of clathrin in other steps of the viral cycle remain unexplored. Thus, we studied whether cell culture-derived HCV (HCVcc) exocytosis was altered after clathrin interference. Knockdown of clathrin or the clathrin adaptor AP-1 in HCVcc-infected human hepatoma cell cultures impaired viral secretion without altering intracellular HCVcc levels or apolipoprotein B (apoB) and apoE exocytosis. Similar reduction in HCVcc secretion was observed after treatment with specific clathrin and dynamin inhibitors. Furthermore, detergent-free immunoprecipitation assays, neutralization experiments and immunofluorescence analyses suggested that whereas apoE associated with infectious intracellular HCV precursors in endoplasmic reticulum (ER)-related structures, AP-1 participated in HCVcc egress in a post-ER compartment. Finally, we observed that clathrin and AP-1 knockdown altered the endosomal distribution of HCV core, reducing and increasing its co-localization with early endosome and lysosome markers, respectively. Our data support a model in which nascent HCV particles associate with apoE in the ER and exit cells following a clathrin-dependent transendosomal secretory route.

    IMPORTANCE. HCV entry into hepatocytes depends on clathrin-mediated endocytosis. Herein, we demonstrate for the first time that clathrin also participates in HCV exit from infected cells. Our data uncover important features of HCV egress, which could lead to the development of new therapeutic interventions. Interestingly, we show that secretion of the VLDL components apoB and apoE is not impaired after clathrin interference. This is a significant finding, since to date it has been proposed that HCV and VLDL follow similar exocytic routes. Given that lipid metabolism has recently emerged as a potential target against HCV infection, our data could help to design new strategies to interfere specifically with HCV exocytosis without perturbing cellular lipid homeostasis, with the aim of achieving more efficient, selective and safe antivirals.

  • HIV-1 fitness cost associated with escape from the VRC01 class of CD4 binding site neutralizing antibodies [PublishAheadOfPrint]

  • Broadly neutralizing antibodies (bNAbs) have been isolated from selected HIV-1 infected individuals and shown to bind to conserved sites on the envelope glycoprotein (Env). However, circulating plasma virus in these donors is usually resistant to the autologous isolated bNAbs, indicating that during chronic infection HIV-1 can escape from even broadly cross-reactive antibodies. Here, we evaluate if such viral escape is associated with an impairment of viral replication. The VRC01 class of antibodies target the functionally conserved CD4 binding site and share a structural mode of gp120 recognition that includes mimicry of the CD4 receptor. We examined naturally occurring VRC01-sensitive and resistant viral strains, as well as their mutated sensitive or resistant variants, and tested point mutations in the backbone of the VRC01-sensitive isolate YU2. In several cases, VRC01-resistance was associated with a reduced efficiency of CD4-mediated viral entry and diminished viral replication. Several mutations, alone or in combination, in the loop D or bbeta;23-V5 region of Env conferred high-level of resistance to VRC01 class antibodies, suggesting a preferred escape pathway. We further mapped the VRC01-induced escape pathway in vivo using Envs from donor 45, from whom antibody VRC01 was isolated. Initial escape mutations, including the addition of a key glycan, occurred in loop D and were associated with impaired viral replication; however, compensatory mutations restored full replicative fitness. These data demonstrate that escape from VRC01 class antibodies can diminish viral replicative fitness, but compensatory changes may explain the limited impact of neutralizing antibodies during the course of HIV-1 natural infection.

    Importance: Some antibodies that arise during natural HIV-1 infection bind to conserved regions on the virus envelope glycoprotein and potently neutralize the majority of diverse HIV-1 strains. The VRC01 class of antibodies blocks the conserved CD4 receptor binding site interaction that is necessary for viral entry, raising the possibility that viral escape from antibody neutralization might exert detrimental effects on viral function. Here we show that escape from VRC01 class antibodies can be associated with impaired viral entry and replication; however, during the course of natural infection, compensatory mutations restore the ability of the virus to replicate normally.

  • ANALYSIS OF HIV-1 GAG PROTEIN INTERACTIONS VIA BIOTIN LIGASE TAGGING [PublishAheadOfPrint]

  • We have examined the interactions of wild type (WT) and matrix protein-deleted (MA) HIV-1 precursor Gag (PrGag) proteins in virus-producing cells using a biotin ligase tagging approach. To do so, WT and MA PrGag proteins were tagged with the E. coli promiscuous biotin ligase (BirA*), expressed in cells, and examined. Localization patterns of PrGag proteins and biotinylated proteins overlapped, consistent with observations that BirA*-tagged proteins biotinylate neighbor proteins that are in close proximity. Results indicate that BirA*-tagged PrGag proteins biotinylated themselves, as well as WT PrGag proteins in trans. Previous data have shown that the HIV-1 Envelope (Env) protein requires an interaction with MA for assembly into virions. Unexpectedly, MA proteins biotinylated Env, whereas WT BirA*-tagged proteins did not, suggesting the presence of MA made Env inaccessible to biotinylation. We also identified over fifty cellular proteins that were biotinylated by BirA*-tagged PrGag proteins. These included membrane proteins, cytoskeleton-associated proteins, nuclear transport factors, lipid metabolism regulators, translation factors, and RNA processing proteins. The identification of these biotinylated proteins offers new insights as to HIV-1 Gag protein trafficking and activities, and provide new potential targets for antiviral interference.

    IMPORTANCE We have employed a novel strategy to analyze the interactions of the HIV-1 structural Gag proteins, which involved tagging wild type and mutant Gag proteins with a biotin ligase. Expression of the tagged proteins in cells allowed us to analyze proteins that came in close proximity to the Gag proteins as they were synthesized, transported, assembled and released from cells. The tagged proteins biotinylated proteins encoded by the HIV-1 pol gene and neighbor Gag proteins, but surprisingly, only the mutant Gag protein biotinylated the HIV-1 Envelope protein. We also identified over fifty cellular proteins that were biotinylated, including membrane and cytoskeletal proteins, and proteins involved in lipid metabolism, nuclear import, translation and RNA processing. Our results offer new insights as to HIV-1 Gag protein trafficking and activities, and provide new potential targets for antiviral interference.

  • The ER membrane J protein C18 executes a distinct role in promoting SV40 membrane penetration [PublishAheadOfPrint]

  • The non-enveloped SV40 hijacks the three endoplasmic reticulum (ER) membrane-bound J proteins B12, B14, and C18 to escape from the ER into the cytosol en route for successful infection. How C18 controls SV40 ER-to-cytosol membrane penetration is the least understood. We previously found that SV40 triggers B12 and B14 to reorganize into discrete puncta in the ER membrane called foci, a structure postulated to represent the cytosol entry site. We now find that SV40 also recruits C18 to the virus-induced B12/B14 foci. Importantly, the C18 foci harbor membrane penetration-competent SV40, further implicating this structure as the membrane penetration site. Consistent with this, a mutant SV40 that cannot penetrate the ER membrane and promote infection fails to induce C18 foci. C18 also regulates the recruitment of B12/B14 into the foci. By contrast to B14, C18's cytosolic Hsc70-binding J- but not lumenal domain is essential for its targeting to the foci; this J-domain is likewise necessary to support SV40 infection. Knockdown-rescue experiments reveal that C18 executes a nonredundant role from B12/B14 during SV40 infection. Collectively, our data illuminate C18's contribution to SV40 ER membrane penetration, strengthening the idea that SV40-triggered foci is critical for cytosol entry.

    Importance Polyomaviruses (PyVs) cause devastating human diseases, particularly in immunocompromised patients. As this virus family continues to be a significant human pathogen, clarifying the molecular basis of their cellular entry pathway remains a high priority. To infect cells, PyV traffics from the cell surface to the ER where it penetrates the ER membrane to reach the cytosol. In the cytosol, the virus moves to the nucleus to cause infection. ER-to-cytosol membrane penetration is a critical yet mysterious infection step. In this study, we clarify the role of an ER membrane protein called C18 in mobilizing the simian PyV SV40, a PyV archetype, from the ER into the cytosol. Our findings also support the hypothesis that SV40 induces the formation of a punctate structure in the ER membrane called foci that serve as the portal for cytosol entry of the virus.

  • Mammalian Adaptive Mutations of the PA protein of Highly Pathogenic Avian H5N1 Influenza Virus [PublishAheadOfPrint]

  • Highly pathogenic H5N1 influenza A viruses continue to circulate among avian species and cause sporadic cases of human infection. Therefore, the threat of a pandemic persists. Yet the human cases of H5N1 infection have been limited mainly to individuals in close contact with infected poultry. These findings suggest that the H5N1 viruses need to acquire adaptive mutations to gain a replicative advantage in mammalian cells to break through the species barrier. Many amino acid mutations of the polymerase complex have been reported to enhance H5N1 virus growth in mammalian cells; however, the mechanism for H5N1 virus of adaptation to humans remains unclear. Here, we propose that the PA of an H5N1 influenza virus isolated from a human in Vietnam (A/Vietnam/UT36285/2010 (36285)) increased the ability of an avian H5N1 virus (A/chicken/Vietnam/TY31/2005, (Ck/TY31)) to grow in human lung epithelial A549 cells. The five PA amino acid substitutions V44I, V127A, C241Y, A343T, and I573V, which are rare in H5N1 viruses from human and avian sources, enhanced the growth capability of this virus in A549 cells. Moreover, these mutations increased the pathogenicity of the virus in mice, suggesting that they contribute to adaptation to mammalian hosts. Intriguingly, PA-241Y, which 36285 encodes, is conserved in more than 90% of human seasonal H1N1 viruses, suggesting that PA-241Y contributes to virus adaptation to human lung cells and mammalian hosts.

    Importance Many amino acid substitutions in highly pathogenic H5N1 avian influenza viruses have been shown to contribute to adaptation to mammalian hosts. However, no naturally isolated H5N1 virus has caused extensive human-to-human transmission, suggesting that additional, as yet unidentified, amino acid mutations are needed for adaptation to humans. Here, we report that five amino acid substitutions in PA (V44I, V127A, C241Y, A343T, and I573V) contribute to the replicative efficiency of H5N1 viruses in human lung cells and to high virulence in mice. These results are helpful for assessing the pandemic risk of isolates and further our understanding of the mechanism of H5N1 virus adaptation to mammalian hosts.

  • HeLa nucleic acid contamination in The Cancer Genome Atlas leads to the misidentification of HPV18 [PublishAheadOfPrint]

  • We searched the TCGA database for viruses by comparing non-human reads present in RNA-seq and DNA-seq data to viral sequence databases. Human papillomavirus type 18 (HPV18) is an etiologic agent of cervical cancer, and as expected we found robust expression of HPV18 genes in cervical cancer samples. In agreement with previous studies we also found HPV18 transcripts in non-cervical cancer samples including colon, rectum, and normal kidney. However, in each of these cases, HPV18 gene expression was low and single nucleotide variants and position of genomic alignments matched the integrated portion of HPV18 present in HeLa cells. Chimeric reads that match a known virus-cell junction of HPV18 integrated in HeLa were also present in some samples. We hypothesize that HPV18 sequences in these non-cervical samples are due to nucleic acid contamination from HeLa cells. This finding highlights the problems that contamination presents in computational virus detection pipelines.

    Importance Viruses associated with cancer can be detected by searching tumor sequence databases. Several studies searching The Cancer Genome Atlas (TCGA) databases have reported the presence of Human papillomavirus type 18 (HPV18), a known cause of cervical cancer, in a small number of additional cancers including rectum, kidney, and colon. We have determined that the sequences related to HPV18 in non-cervical samples are due to nucleic acid contamination from HeLa cells. To our knowledge this is the first report of the misidentification of viruses in next generation sequencing data of tumors due to contamination with a cancer cell line. These results raise awareness of the difficulty to accurately identify viruses in human sequence databases.

  • Species-Specific Transmission of Novel Picornaviruses in Lemurs [PublishAheadOfPrint]

  • The roles of host genetics versus exposure and contact frequency in driving cross-species transmission remains debated. Here, we used a multi-taxa lemur collection at the Saint Louis Zoo in the USA as a model to gain insight into viral transmission in a high inter-species contact setting. Lemurs are a diverse and understudied group of primates that are highly endangered. Endemic to the island of Madagascar, the speciation of lemurs occurred in geographic isolation apart from continental African primates. Although evidence of endogenized viruses in lemur genomes exist, no exogenous viruses of lemurs have been described to date. Here we identified two novel picornaviruses in fecal specimens of ring-tailed lemurs (Lemur catta) and black-and-white ruffed lemurs (Varecia variegata). We found that the viruses were transmitted in a species-specific manner (lesavirus 1 was detected only in ring-tailed lemurs while lesavirus 2 was detected only in black-and-white ruffed lemurs). Longitudinal sampling over a one year interval demonstrated ongoing infection in the collection. This was supported by evidence of viral clearance in some animals and new infections in previously uninfected animals, including a set of newly-born triplets that acquired the infection. While both viruses were found to be co-circulating in a mixed species exhibit of ring-tailed lemurs, black-and-white ruffed lemurs and black lemurs, there was no evidence of cross-species transmission. This suggests that despite high intensity contact, host species barriers can prevent cross-species transmissions of these viruses.

    Importance Up to seventy-five percent of emerging infectious diseases in humans today are the result of zoonotic transmission. However, a challenge in understanding transmission dynamics has been the limited models of cross-species transmission. Zoos provide a unique opportunity to explore parameters defining viral transmission. We demonstrated that ongoing virus transmission in a mixed lemur species exhibit was species-specific. This suggests that despite high contact intensity, host species barriers contribute to protection from cross-species transmission of these viruses. While the combination of species might differ, most zoological parks worldwide commonly feature mixed species exhibits. Collectively, this study demonstrates a widely applicable approach towards understanding infectious disease transmission.

  • The Avian-Origin PB1 Gene Segment Facilitated Replication and Transmissibility of the H3N2/1968 Pandemic Influenza Virus [PublishAheadOfPrint]

  • The H2N2/1957 and H3N2/1968 pandemic influenza viruses emerged via the exchange of genomic RNA segments between human and avian viruses. The avian hemagglutinin (HA) allowed the hybrid viruses to escape pre-existing immunity in the human population. Both pandemic viruses further received the PB1 gene segment from the avian parent (Y.Kawaoka, S.Krauss and R.G.Webster, J Virol 63:4603-4608, 1989), but the biological significance of this observation was not understood. To assess whether the avian-origin PB1 segment provided pandemic viruses with some selective advantage, either on its own or via cooperation with the homologous HA segment, we modeled by reverse genetics the reassortment event that led to the emergence of the H3N2/1968 pandemic virus. Using seasonal H2N2 virus A/California/1/66 (Cal) as a surrogate precursor human virus and pandemic virus A/Hong Kong/1/68 (H3N2) (HK) as a source of avian-derived PB1 and HA gene segments, we generated four reassortant recombinant viruses and compared pairs of viruses which differed solely by the origin of PB1. Substitution of the PB1 segment of Cal by PB1 of HK facilitated viral polymerase activity, replication efficiency in human cells and contact transmission in guinea pigs. A combination of PB1 and HA segments of HK did not enhance replicative fitness of the reassortant virus in comparison with the single-gene PB1 reassortant. Our data suggest that the avian PB1 segment of the 1968 pandemic virus served to enhance viral growth and transmissibility, likely, by enhancing activity of the viral polymerase complex.

    Importance Despite the high impact of influenza pandemics on human health, some mechanisms underlying the emergence of pandemic influenza viruses are still poorly understood. Thus, it was unclear why both H2N2/1957 and H3N2/1968 reassortant pandemic viruses contained, in addition to the avian HA, the PB1 gene segment of the avian parent. Here we addressed this long-standing question by modeling the emergence of the H3N2/1968 virus from its putative human and avian precursors. We show that the avian PB1 segment increased activity of the viral polymerase and facilitated viral replication. Our results suggest that in addition to acquisition of antigenically novel HA ("antigenic shift") enhanced viral polymerase activity may be required for the emergence of pandemic influenza viruses from their seasonal human precursors.

  • The Mechanism of Angiopoietin-1 Up-regulation in KSHV-infected PEL Cell Lines [PublishAheadOfPrint]

  • Angiopoietin-1 (ANGPT-1) is a secreted glycoprotein that was first characterized as a ligand of the Tie-2 receptor. In a previous study using microarray analysis, we reported that the expression of ANGPT-1 was up-regulated in Kaposi's sarcoma-associated herpesvirus (KSHV)-infected primary effusion lymphoma (PEL) cell lines compared with uninfected Burkitt and other leukemic cell lines. Other authors have also reported focal expression of ANGPT-1 mRNA in biopsies of Kaposi's sarcoma (KS) from patients with acquired immune deficiency syndrome (AIDS). Here, to confirm these findings, we examined the expression and secretion levels of ANGPT-1 in KSHV-infected PEL cell lines and address the transcriptional regulation mechanisms of ANGPT-1. We also showed that ANGPT-1 was expressed and localized in the cytoplasm and was secreted into the supernatant in KSHV-infected PEL cells. Deletion studies of the regulatory region revealed that a -143 to -125 nt region of the ANGPT-1-regulating sequence was responsible for the up-regulation. Moreover, an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) followed by qPCR suggested that some KSHV-infected PEL cell line-specific DNA-binding factors, such as OCT-1, should be involved in the up-regulation of ANGPT-1 in a sequence-dependent manner.

    Importance We confirmed that ANGPT-1 was expressed and secreted in and from KSHV-infected PEL cells, respectively, and the transcriptional activity of ANGPT-1 was up-regulated. A 19 bp fragment was identified as a responsible region for ANGPT-1 up-regulation, through binding with OCT-1 as a core factor in the PEL cells. This study suggests that ANGPT-1 is overproduced in KSHV-infected PEL cells, which could affect the pathophysiology of patients harboring PEL under AIDS setting.

  • NECTIN-LIKE INTERACTIONS BETWEEN POLIOVIRUS AND ITS RECEPTOR TRIGGER CONFORMATIONAL CHANGES ASSOCIATED WITH CELL ENTRY [PublishAheadOfPrint]

  • Poliovirus infection is initiated by attachment to a receptor on the cell surface called Pvr or CD155. At physiological temperature the receptor catalyzes an irreversible expansion of the virus to form an expanded form of the capsid called the 135S particle. This expansion results in the externalization of the myristoylated capsid protein VP4 and the N-terminal extension of the capsid protein VP1, both of which become inserted into the cell membrane. Structures of the expanded forms of poliovirus and of several related viruses have recently been reported. However, until now, it has been unclear how receptor binding triggers viral expansion at physiological temperature. Here, we report poliovirus in complex with an enzymatically partially deglycosylated form of the 3-domain ectodomain of Pvr at 4AAring; resolution, as determined by electron cryomicroscopy. The interaction of the receptor with the virus in this structure is reminiscent of the interactions of Pvr with its natural ligands. At low temperature, the receptor induces very few changes in the structure of the virus, with the largest changes occurring within the footprint of the receptor, and in a loop of the internal protein VP4. Changes in the vicinity of the receptor include the displacement of a natural lipid ligand (called "pocket factor"), demonstrating that the loss of this ligand, alone, is not sufficient to induce particle expansion. Finally, analogies with naturally occurring ligand binding in the nectin family suggest which specific structural rearrangements in the virus-receptor complex could help to trigger the irreversible expansion of the capsid.

    IMPORTANCE The cell-surface receptor (Pvr) catalyzes a large structural change in the virus that exposes membrane-binding protein chains. We fitted known atomic models of the virus and Pvr into three-dimensional experimental maps of the receptor-virus complex. The molecular interactions we see between poliovirus and its receptor are reminiscent of the nectin family, by involving the burying of otherwise-exposed hydrophobic groups. Importantly, poliovirus expansion is regulated by the binding of a lipid molecule within the viral capsid. We show that receptor binding either causes this molecule to be expelled or requires it, but that its loss is not sufficient to trigger irreversible expansion. Based on our model, we propose testable hypotheses to explain how the viral shell becomes destabilized, leading to RNA uncoating. These findings give us a better understanding of how poliovirus has evolved to exploit a natural process of its host to penetrate the membrane barrier.

  • Unique glycan signatures regulate adeno-associated viral tropism in the developing brain [PublishAheadOfPrint]

  • Adeno-associated viruses (AAV) are thought to spread through the central nervous system (CNS) by exploiting cerebrospinal fluid (CSF) flux and hijacking axonal transport pathways. The role of host receptors that mediate these processes is not well understood. In the current study, we utilized AAV serotype 4 as a model to evaluate whether ubiquitously expressed 2,3-linked sialic acid and the developmentally regulated marker, 2,8-linked polysialic acid (PSA) regulate viral transport and tropism in the neonatal brain. Modulation of the levels of SA and PSA in cell culture studies using specific neuraminidases revealed possibly opposing roles of the two glycans on AAV4 transduction. Interestingly, upon intracranial injection into lateral ventricles of the neonatal mouse brain, a low affinity AAV4 mutant (AAV4.18) displayed a striking shift in cellular tropism from 2,3-linked SA+ ependymal lining to 2,8-linked PSA+ migrating progenitors in the rostral migratory stream and olfactory bulb. In addition, this gain-of-function phenotype correlated with robust CNS spread of AAV4.18 through paravascular transport pathways. Consistent with these observations, altering glycan dynamics within the brain by co-administering SA and PSA specific neuraminidases resulted in striking changes to the cellular tropisms and transduction efficiencies of both parental and mutant vectors. We postulate that glycan signatures associated with host development can be exploited to redirect novel AAV vectors to specific cell types in the brain.

    IMPORTANCE Viruses invade the CNS through various mechanisms. In the current study, we utilize AAV as a model to study the dynamics of virus-carbohydrate interactions in the developing brain and its impact on viral tropism. Our findings suggest that carbohydrate content can be exploited to regulate viral transport and tropism in the brain.

  • Co-translational CP-mediated inhibition of potyviral RNA translation [PublishAheadOfPrint]

  • Potato Virus A (PVA) is a single-stranded positive-sense RNA virus and a member of the family Potyviridae. The PVA coat protein (CP) has an intrinsic capacity to self-assemble into filamentous virus-like particles, but the mechanism responsible for the initiation of viral RNA encapsidation in vivo remains unclear. Apart from virion assembly, PVA CP is also involved in the inhibition of viral RNA translation. In this study, we show that CP inhibits PVA RNA translation in a dose-dependent manner through a mechanism involving the CP-encoding region. Analysis of this region, however, failed to identify any RNA secondary structure(s) preferentially recognized by CP, suggesting that the inhibition depends on CP-CP, rather than CP-RNA, interactions. In agreement with this possibility, insertion of an in-frame stop codon upstream of the CP sequence led to a marked decrease in the inhibition of viral RNA translation. Based on these results, we propose a model in which the co-translational interactions between excess CP provided in trans and CP translated from viral RNA in cis are required to initiate the translational repression. This model suggests a mechanism for how viral RNA can be sequestered from translation and specifically selected for encapsidation at the late stages of viral infection.

    Importance The main function of the coat protein during potyvirus infection is to protect viral RNA from degradation and transport it locally, systemically, and from host to host. Although virion assembly is a key step in the potyviral infectious cycle, little is known about how it is initiated and how viral RNA is selected for encapsidation. The results presented here suggest that CP-CP, rather than CP-RNA, interactions are predominantly involved in the sequestration of viral RNA away from translation. We propose that the co-translational nature of these interactions may represent a mechanism for the selection of viral RNA for encapsidation. A better understanding of the mechanism of virion assembly may lead to development of crops resistant to potyviruses on the level of viral RNA encapsidation, thereby reducing the detrimental effects of potyvirus infections on food production.

  • DNA vaccine molecular adjuvants SP-D-BAFF and SP-D-APRIL enhance anti-gp120 immune response and increase HIV-1 neutralizing antibody titers [PublishAheadOfPrint]

  • Broadly neutralizing antibodies (bNAbs) specific for conserved epitopes on HIV-1 Envelope (Env) are believed to be essential for protection against multiple HIV-1 clades. However, vaccines capable of stimulating production of bNAbs remain a major challenge. Given that polyreactivity and autoreactivity are considered important characteristics of anti-HIV bNAbs, we designed an HIV vaccine incorporating B cell activating factor (BAFF) and A Proliferation-Inducing Ligand (APRIL) molecular adjuvants with the potential to facilitate maturation of polyreactive and autoreactive B cells as well as enhance the affinity and/or avidity of Env-specific antibodies. We designed recombinant DNA plasmids encoding soluble multi-trimers of BAFF and APRIL using Surfactant protein D as a scaffold, and vaccinated mice with these molecular adjuvants in DNA and DNA/protein vaccine strategies. We found that immunization of mice with a DNA vaccine encoding BAFF or APRIL multi-trimers, together with IL-12 and membrane-bound HIV-1 Env gp140, induced tier 1 and vaccine strain tier 2 neutralizing antibodies. The APRIL-containing vaccine was particularly effective in generating tier 2 neutralizing antibodies following protein boost. These BAFF and APRIL effects coincided with enhanced GC reaction, increased anti-gp120-specific antibody secreting cells, and increased anti-gp120 functional avidity. Notably, BAFF and APRIL did not cause indiscriminate B cell expansion or an increase in total IgG. We propose that BAFF and APRIL multi-trimers are promising molecular adjuvants for vaccines designed to induce bNAbs against HIV-1.

    IMPORTANCE: Recent identification of antibodies that neutralize most HIV-1 strains has revived hopes and efforts to create novel vaccines that can effectively stimulate HIV-1 neutralizing antibodies. However, multiple immune evasion properties of HIV have hampered these efforts. These include instability of gp120 trimer, inaccessibility of the conserved sequences, highly variable protein sequences and the loss of HIV-1-specific antibody-producing cells during development. We have previously shown that TNF superfamily ligands, including BAFF and APRIL, can be multi-trimerized using the lung protein Surfactant Protein D (SP-D), enhancing immune responses. Here we show that DNA or DNA/protein vaccines encoding BAFF or APRIL multi-trimers, IL-12p70, and membrane-bound HIV-1 Env gp140 induced tier 1 and tier 2 neutralizing antibodies in a mouse model. BAFF and APRIL enhanced the immune reaction, improved antibody binding, and increased numbers of anti-HIV-1 antibody-secreting cells. Adaptation of this vaccine design may prove useful in designing preventive HIV-1 vaccines in humans.

  • Modulation of Flavivirus Population Diversity by RNA Interference [PublishAheadOfPrint]

  • To test the hypothesis that RNAi imposes diversifying selection on RNA virus genomes, we quantified West Nile virus (WNV) quasispecies diversity after passage in Drosophila cells in which RNAi was left intact, depleted, or stimulated against WNV. As predicted, WNV diversity was significantly lower in RNAi-depleted cells and significantly greater in RNAi-stimulated cells relative to controls. These findings reveal that an innate immune defense can shape viral population structure.

  • PA-X Decreases the Pathogenicity of Highly Pathogenic H5N1 Influenza A Virus in Avian Species by Inhibiting Virus replication and Host Response [PublishAheadOfPrint]

  • PA-X is a newly discovered protein that decreases the virulence of 1918 H1N1 virus in a mouse model. However, the role of PA-X in the pathogenesis of highly pathogenic avian influenza virus (HPAIV) H5N1 subtype in avian species is totally unknown. By generating two PA-X-deficient viruses and evaluating their virulence in different animal models, we here showed that PA-X diminishes the virulence of a HPAIV H5N1 strain A/Chicken/Jiangsu/k0402/2010 (CK10) in mice, chickens and ducks. Expression of PA-X dampens the polymerase activity and virus replication both in vitro and in vivo. Using microarray analysis, we found that PA-X blunts the global host response in chicken lungs, which includes markedly down-regulates genes associated with the inflammatory and cell death response. Correspondingly, decreased cytokine response was recapitulated in multiple organs of chickens and ducks infected with the wild type virus when compared to infect with the PA-X-deficient virus. In addition, the PA-X protein exhibits anti-apoptotic activity in CEF and DEF cells. Thus, our results demonstrated that PA-X acts as a negative virulence regulator and decreases the virulence through inhibiting viral replication and the host innate immune response. Therefore, we here define the role of PA-X in the pathogenicity of H5N1 HPAIV, furthering our understanding of the intricate pathogenesis of influenza A virus.

    IMPORTANCE Influenza A virus (IAV) continues to pose a huge threat to global public health. Eight gene segments of the IAV genome encode up to 17 proteins, including 8 main viral proteins and 9 accessory proteins. The presence of these accessory proteins may further complicate the pathogenesis of IAV. PA-X is a newly-identified protein in segment 3 and acts to decrease the virulence of 1918 H1N1 virus in mice through modulating host gene expression. Our study extends these functions of PA-X to HPAIV H5N1 virus. We demonstrated that loss of PA-X expression increases the virulence and virus replication of H5N1 virus in mice and avian species, and alters the host innate immune and cell death response. Our study is the first report delineating the role of the novel PA-X protein in the pathogenesis of H5N1 in avian species and promotes our understanding of HPAIV H5N1 virus.

  • Zombies in TCGA [PublishAheadOfPrint]

  • Next-generation sequencing results obtained to detect somatic mutations in human cancers can also be searched for viruses that contribute to cancer. Recently, human papillomavirus 18 RNA was detected in tumor types not typically associated with HPV infection. Analyses reported in this issue of Journal of Virology demonstrate that the apparent presence of HPV18 RNA in these atypical tumors is due in at least some cases to contamination of samples with HeLa cells, which harbor HPV18.

  • Restricted Protein Phosphatase 2A Targeting by Merkel Cell Polyomavirus Small T Antigen [PublishAheadOfPrint]

  • Merkel cell polyomavirus (MCV) is a newly discovered human cancer virus encoding a small T (sT) oncoprotein. We performed MCV sT FLAG-affinity purification followed by mass spectroscopy (MS) analysis, which identified several protein phosphatases (PP) including PP2A A and C subunits and PP4C as potential cellular interacting proteins. PP2A targeting is critical for the transforming properties of non-human polyomaviruses, such as simian virus 40 (SV40), but is not required for MCV sT-induced rodent cell transformation. We compared similarities and differences in PP2A binding between MCV and SV40 sT. While SV40 sT co-immunopurified with subunits PP2A Aaalpha; and PP2A C, MCV sT co-immunopurified with PP2A Aaalpha;, PP2A Abbeta; and PP2A C. Scanning alanine mutagenesis at 29 sites across the MCV protein revealed that PP2A-binding domains lie on the opposite molecular surface from a previously-described large T stabilization domain (LSD) loop that binds E3 ligases, such as Fbw7. MCV sT-PP2A interactions can be functionally distinguished by mutagenesis from MCV sT LSD-dependent 4E-BP1 hyperphosphorylation and viral DNA replication enhancement. MCV sT has a restricted range for PP2A B subunit substitution, inhibiting only the assembly of B56aalpha; into the phosphatase holoenzyme. In contrast, SV40 sT inhibits the assembly of B55aalpha;, B56aalpha; and B56 into PP2A. We conclude that MCV sT is required for Merkel cell carcinoma growth but its in vitro transforming activity depends on LSD interactions rather than PP2A targeting.

    IMPORTANCE Merkel cell polyomavirus is a newly-discovered human cancer virus that promotes cancer, in part, through expression of its small T (sT) oncoprotein. Animal polyomavirus sT oncoproteins have been found to cause experimental tumors by blocking the activities of a group of phosphatases called PP2A. Our structural analysis reveals that MCV sT also displaces the B subunit of PP2A to inhibit PP2A activity. MCV sT, however, only displaces a restricted subset of PP2A B subunits, which is insufficient to cause tumor cell formation in vitro. MCV sT in stead transforms tumor cells through another region called the large T stabilization domain. The PP2A targeting and transforming activities lie on opposite faces of the MCV sT molecule and can be genetically separated from each other.

  • Disruption of Type I Interferon Signaling by NSs Protein of Severe Fever with Thrombocytopenia Syndrome Virus via Hijacking STAT2 and STAT1 into Inclusion Bodies [PublishAheadOfPrint]

  • Type I interferon (IFN) system including IFN induction and signaling is the critical component of the host defense line against viral infection, which in turn, is also a vulnerable target for viral immune evasion. Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging bunyavirus. Previous data have shown that SFTSV can interfere with the early induction of type I IFNs through targeting host kinases TBK1/IKK. In this study, we demonstrated that SFTSV can also suppress type I IFN-triggered signaling and hence interferon-stimulated gene (ISG) expression. Interestingly, we observed the significant inhibition of IFN signaling in cells transfected with the plasmids encoding the nonstructural protein (NSs) but not the nucleocapsid protein (NP), indicating the role of NSs as an antagonist of IFN signaling. Furthermore, co-immunoprecipitation (Co-IP) and pull-down assays indicated that NSs interacts with the cellular signal transducer and activator of transcription 2 (STAT2) and the DNA-binding domain of STAT2 may contribute to the NSs-STAT2 interaction. Combined with confocal microscopy analyses we demonstrated that NSs sequesters STAT2 and STAT1 into viral inclusion bodies (IBs) and impairs IFN-induced STAT2 phosphorylation and nuclear translocation of both STATs, resulting in the inhibition of IFN signaling and ISG expression. SFTSV NSs-mediated hijacking of STATs in IBs represents a novel mechanism of viral suppression of IFN signaling, highlighting the role of viral IBs as the virus-built "jail" sequestering some crucial host factors and thus interfering with the corresponding cellular processes.

    Importance SFTSV is an emerging bunyavirus which can cause a severe haemorrhagic fever-like disease with high case fatality rates in humans, posing a serious health threat. However, there are no specific antivirals available and the pathogenesis and virus-host interactions are largely unclear. Here, we demonstrated that SFTSV can inhibit type I interferon (IFN) antiviral signaling by the nonstructral protein (NSs)-mediated hijacking of STAT2 and STAT1 into viral inclusion bodies (IBs), highlighting the interesting role of viral IBs in virus-host interactions as the virus-built "jail". Sequestering signaling molecules into IBs represents a novel and perhaps also general mechanism of viral suppression of IFN signaling, understanding of which may benefit the study on the viral pathogenesis and the development of antiviral therapies.

  • Characterization of hepatitis C virus interaction with heparan sulfate proteoglycans [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) entry involves binding to cell surface heparan sulfate (HS) structures. However, due to the lipoprotein-like structure of HCV, the exact contribution of virion components to this interaction remains controversial. Here, we investigated the relative contribution of HCV envelope proteins and apolipoprotein E in the HS-binding step. Deletion of hypervariable region 1, a region previously proposed to be involved in HS-binding, did not alter HCV virion binding to HS, indicating that this region is not involved in this interaction in the context of a viral infection. Patient sera and monoclonal antibodies recognizing different regions of HCV envelope glycoproteins were also used in a pull-down assay with beads coated with heparin, a close HS structural homologue. Although isolated HCV envelope glycoproteins could interact with heparin, none of these antibodies was able to interfere with virion-heparin interaction, strongly suggesting that, at the virion surface HCV envelope glycoproteins are not accessible for HS binding. In contrast, results from kinetic studies, heparin pull-down and inhibition experiments with anti-apolipoprotein E antibodies indicate that this apolipoprotein plays a major role in HCV-HS interaction. Finally, characterization of HS structural determinants required for HCV infection by silencing enzymes involved in the HS biosynthesis pathway and by competition with modified heparin indicated that N- and 6-O-sulfation but not 2-O-sulfation are required for HCV infection, and that the minimum HS oligosaccharide length required for HCV infection is a decasaccharide. Together, these data indicate that HCV hijacks apolipoprotein E to initiate its interaction with specific HS structures.

    IMPORTANCE Hepatitis C is a global health problem. Hepatitis C virus (HCV) infects approximately 130 millions individuals worldwide with the majority remaining undiagnosed and untreated. In most infected individuals, the virus evades the immune system and establishes a chronic infection. As a consequence, hepatitis C is the leading cause for cirrhosis, end-stage liver disease, hepatocellular carcinoma and liver transplantation. Virus infection is initiated by entry of the virus in the host cell. In this study, we provide new insights on the viral and cellular determinants involved in the first step of HCV entry, the binding of the virus to host cells. We show that the apolipoprotein E is likely responsible for virus binding to heparan sulfate and that N- and 6-O-sulfation of the heparan sulfate proteoglycans are required for HCV infection. In addition, a decasaccharide is the minimal HS chain length necessary.

  • Simian Retrovirus 4 Induces Lethal Acute Thrombocytopenia in Japanese Macaques [PublishAheadOfPrint]

  • In 2001-2002, six out of seven Japanese macaques (Macaca fuscata) died after developing hemorrhagic syndrome at the Kyoto University Primate Research Institute (KUPRI). While the cause of death was unknown at the time, we detected simian retrovirus 4 (SRV-4) in samples obtained from a similar outbreak in 2008-2011, during which 42 out of 43 Japanese macaques died after exhibiting hemorrhagic syndrome. In this study, we isolated SRV-4 strain PRI-172 from a Japanese macaque showing severe thrombocytopenia. When inoculated into four Japanese macaques, the isolate induced severe thrombocytopenia in all within 37 days. We then constructed an infectious molecular clone of strain PRI-172, termed pSR415, and inoculated the clone-derived virus into two Japanese macaques. These animals also developed severe thrombocytopenia in just 31 days after inoculation, and the virus was re-isolated from blood, bone marrow and stool. At necropsy, we observed bleeding from gingiva, and subcutaneous bleeding in all animals. SRV-4 infected a variety of tissues especially in digestive organs including colon and stomach, determined by real-time PCR, RT-PCR and immunohistochemical staining. Furthermore, we identified the SRV-4 receptor as ASCT2, a neutral amino acid transporter. ASCT2 mRNA was expressed in a variety of tissues and the distribution of SRV-4 proviruses in infected Japanese macaques correlated well with the expression levels of ASCT2 mRNA. From these results, we conclude that the causative agent of hemorrhagic syndrome in KUPRI Japanese macaques was SRV-4, and its receptor is ASCT2.

    IMPORTANCE During two separate outbreaks at the KUPRI, in 2001-2002 and 2008-2011, 96% of Japanese macaques (JM) that developed an unknown hemorrhagic syndrome died. Here, we isolated SRV-4 from a JM developing thrombocytopenia. The SRV-4 isolate and a molecularly cloned SRV-4 induced severe thrombocytopenia in virus-inoculated JMs within 37 days. At necropsy, we observed bleeding from gingiva and subcutaneous bleeding in all affected JMs, and re-isolated SRV-4 from blood, bone marrow and stool. The distribution of SRV-4 proviruses in tissues correlated with the mRNA expression levels of ASCT2, which we identified as the SRV-4 receptor. From these results, we conclude that SRV-4 was the causative agent of hemorrhagic syndrome in JMs in KUPRI.

  • High secretion of interferons by human plasmacytoid dendritic cells upon recognition of MERS-CoV [PublishAheadOfPrint]

  • The Middle East Respiratory Syndrome coronavirus (MERS-CoV) emerged in 2012 as causative agent of a severe respiratory disease with a fatality rate of approx. 30%. The high virulence and mortality rate prompted us to analyze aspects of MERS-CoV pathogenesis, especially its interaction with innate immune cells such as antigen-presenting cells (APCs). Particularly, we analyzed secretion of type I and type III interferons (IFNs) by APCs, i.e. B cells, macrophages, myeloid dendritic cells (MDDCs/mDCs), and by plasmacytoid dendritic cells (pDCs) of human and murine origin after inoculation with MERS-CoV. Production of high amounts of type I and III IFNs was induced exclusively in human pDCs, which was significantly higher than IFN induction by SARS-CoV. Of note, IFNs were secreted in absence of productive replication. However, receptor binding, endosomal uptake, and probably signaling via TLR7 were critical for sensing of MERS-CoV by pDCs. Furthermore, active transcription of MERS-CoV N RNA and subsequent N protein expression was evident in infected pDCs, indicating abortive infection. Taken together, our results point toward DPP4-dependent endosomal uptake and subsequent infection of human pDCs by MERS-CoV. However, the replication cycle is stopped after early gene expression. In parallel, human pDCs are potent IFN-producing cells upon MERS-CoV infection. Realization of such IFN responses supports understanding of MERS-CoV pathogenesis and is critical for the choice of treatment options.

    Importance MERS-CoV is causing a severe respiratory disease with high fatality rates in human patients. Recently, confirmed human cases have increased dramatically, both in number and geographic distribution. Understanding the pathogenesis of this highly pathogenic CoV is crucial for developing successful treatment strategies. This study elucidates the interaction of MERS-CoV with APCs and pDCs particularly the induction of type I and III IFN secretion. Human pDCs are the immune cell population sensing MERS-CoV, but compared to SARS-CoV, secrete significantly higher amounts of IFNs, especially IFN-aalpha;. A model for molecular virus-host interactions is presented outlining IFN induction in pDCs. The massive IFN secretion upon contact suggests a critical role of this mechanism for the high immune activation observed during MERS-CoV infection.

  • Complete genome sequence of MneRV2, the pig-tailed macaque RV2 rhadinovirus, and evolutionary relationship with rhesus macaque RRV and human herpesvirus 8/KSHV [PublishAheadOfPrint]

  • Two Rhadinovirus lineages have been identified in Old World primates. The Rhadinovirus 1 (RV1) lineage consists of human herpesvirus 8, the Kaposi's sarcoma-associated herpesvirus (KSHV), and closely related rhadinoviruses of chimpanzees, gorillas, macaques and other Old World primates. The RV2 rhadinovirus lineage is distinct and consists of closely related viruses from the same Old World primate species. Rhesus macaque rhadinovirus (RRV) is the RV2 prototype, and two RRV isolates, 26-95 and 17577, were sequenced. We determined that the pig-tailed macaque RV2 rhadinovirus, MneRV2, is highly associated with lymphomas in macaques with simian AIDS. To further study the role of rhadinoviruses in the development of lymphoma, we sequenced the complete genome of MneRV2, and identified 87 protein coding genes and 17 candidate miRNAs. A strong genome colinearity and sequence homology was observed between MneRV2 and RRV26-95, although the open reading frame encoding the KSHV ORFK15 homolog was disrupted in RRV26-95. Comparison with MneRV2 revealed several genomic anomalies in RRV17577 that were not present in other rhadinovirus genomes, including a N-terminal duplication in ORF4 and a recombinative exchange of more distantly related homologs of the ORF22/ORF47 interacting glycoprotein genes. The comparison with MneRV2 has revealed novel genes and important conservation of protein coding domains, transcription initiation, termination and splicing signals, which have added to our knowledge of RV2 rhadinovirus genetics. Further comparisons with KSHV and other RV1 rhadinoviruses will provide important avenues for dissecting the biology, evolution and pathology of these closely related tumor-inducing viruses in humans and other Old World primates.

    IMPORTANCE This manuscript provides the sequence characterization of MneRV2, the pig-tailed macaque homolog of rhesus rhadinovirus RRV. MneRV2 and RRV belong to the RV2 rhadinovirus lineage of Old World primates, and are distinct but related to Kaposi's sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi's sarcoma. Pig-tailed macaques provide important models of human disease, and our previous studies have indicated that MneRV2 plays a causal role in AIDS related lymphomas in macaques. Delineation of the MneRV2 sequence has allowed a detailed characterization of the genome structure, and evolutionary comparisons with RRV and KSHV have identified conserved promoters, splice junctions and novel genes. This comparison provides insight into RV2 rhadinovirus biology and sets the groundwork for more intensive Next-Gen transcript and genetic analysis of this class of tumor-inducing herpesvirus. This study supports the use of MneRV2 in pig-tailed macaques as an important model for studying rhadinovirus biology, transmission and pathology.

  • The Lysine Residues within the Human Ribosomal Protein S17 (RPS17) Sequence Naturally Inserted into the Viral Nonstructural Protein of a Unique Strain of Hepatitis E Virus are Important for Enhanced Virus Replication [PublishAheadOfPrint]

  • Hepatitis E virus (HEV) is an important but extremely understudied human pathogen. Due largely to the lack of an efficient cell culture system for HEV, the molecular mechanisms of HEV replication and pathogenesis are poorly understood. Recently, a unique genotype 3 strain of HEV recovered from a chronically-infected patient was adapted for growth in human hepatoma cells HepG2/C3A. The adaptation of the Kernow C-1 P6 HEV to propagate in HepG2/C3A cells selected for a rare virus recombinant that contains an insertion of 171-nucleotide sequence encoding amino acids 21-76 of the human ribosomal protein S17 (RPS17) within the hypervariable region (HVR) of HEV ORF1. When the RPS17 insertion was placed into a strain of genotype 1 HEV which infects only humans, it expanded the host range allowing it to infect cell lines from multiple animal species including cow, dog, cat, chicken, and hamster. In this study, we utilized forward and reverse genetics attempting to define what aspects of the RPS17 insertion allow for the ability of the Kernow C-1 P6 HEV to adapt in cell culture and allow for expanded host tropism. We demonstrate that the RPS17 sequence insertion in HEV bestows novel nuclear/nucleolar trafficking capabilities to the ORF1 protein of Kernow P6 HEV, and that lysine residues within the RPS17 insertion, but not nuclear localization of ORF1, correlate to the enhanced replication of the HEV Kernow C-1 P6 strain. The results from this study have important implications for understanding the mechanism of cross-species infection and replication of HEV.

    IMPORTANCE HEV is an important pathogen worldwide. The virus causes high mortality (up to 30%) in pregnant women and has been recognized to cause chronic hepatitis in the immunocompromised population. The life cycle of HEV has been understudied due to a lack of sufficient cell culture systems to propagate the virus. Recently insertions and rearrangements of the hypervariable region (HVR) within the HEV genome allowing for cell culture adaptation and expansion in host range have been reported. We utilize these cell culture-adapted HEV strains to assess how the HVR may be involved in virus replication and host range. We provide evidence that insertion of the RPS17 sequence in HEV likely confers nuclear trafficking capabilities to the nonstructural protein of the virus, and that lysine residues within the RPS17 insertion are important for enhanced replication of the virus. These data will help elucidate the mechanism of cross-species infection of HEV in the future.

  • SARS coronaviruses with mutations in E protein are attenuated and promising vaccine candidates [PublishAheadOfPrint]

  • Severe acute respiratory syndrome coronavirus (SARS-CoV) causes a respiratory disease with a mortality rate of 10%. A mouse-adapted SARS-CoV (SARS-CoV-MA15) lacking the envelope (E) protein (rSARS-CoV-MA15-E) is attenuated in vivo. To identify E protein regions and host responses that contribute to rSARS-CoV-MA15-E attenuation, several mutants (rSARS-CoV-MA15-E*) containing point mutations or deletions in the amino-terminal or the carboxy-terminal regions of the E protein were generated. Amino acid substitutions in the amino terminus, or deletion of regions in the internal carboxy terminal region of E protein led to virus attenuation. Attenuated viruses induced minimal lung injury, diminished limited neutrophil influx and increased CD4+ and CD8+ T cell counts in the lungs of BALB/c mice, when compared to mice infected with wild type virus. To analyze the host responses leading to rSARS-CoV-MA15-E* attenuation, differences in gene expression elicited by the native and mutant viruses in the lungs of infected mice were determined. Expression levels of a large number of proinflammatory cytokines associated with lung injury were reduced in the lungs of rSARS-CoV-MA15-E* infected mice, whereas the levels of anti-inflammatory cytokines were increased, both at the mRNA and protein levels. These results suggested that the reduction in lung inflammation together with a more robust antiviral T cell response contributed to rSARS-CoV-MA15-E* attenuation. The attenuated viruses completely protected mice against challenge with the lethal parental virus, indicating that these viruses are promising vaccine candidates.

    Importance Human coronaviruses are important zoonotic pathogens. SARS-CoV caused a worldwide epidemic infecting more than 8000 people with a mortality of around 10%. Therefore, understanding the virulence mechanisms of this pathogen and developing efficacious vaccines is of high importance to prevent epidemics from this, and other human coronaviruses. Previously, we demonstrated that a SARS-CoV lacking the E protein was attenuated in vivo. Here, we show that small deletions and modifications within E protein led to virus attenuation, manifested by minimal lung injury, limited neutrophil influx to the lungs, reduced expression of proinflammatory cytokines, increased anti-inflammatory cytokine levels and enhanced CD4+ and CD8+ T cell counts in vivo, suggesting that these phenomena contribute to virus attenuation. The attenuated mutants fully protected mice from challenge with virulent virus. These studies show that mutations in the E protein are not well tolerated and indicate that this protein is an excellent target for vaccine development.

  • Evolution of the Bunyamwera virus polymerase to accommodate deletions within genomic untranslated region sequences. [PublishAheadOfPrint]

  • The untranslated regions (UTR) present at the ends of bunyavirus genome segments are required for essential steps in the virus life cycle, and provide signals for encapsidation by nucleocapsid protein, the promoters for RNA transcription and replication, as well as mRNA transcription termination. For the prototype bunyavirus, Bunyamwera virus (BUNV), only the terminal 11 nucleotide (nt) are identical between each segment. Thereafter, the UTRs are highly variable both in length and in sequence. Furthermore, apart from the conserved termini, the UTRs are highly variable between different viruses. We previously generated recombinant BUNV carrying the minimal UTRs on all three segments that were attenuated for growth in cell culture. Following serial passage of these viruses, the viruses acquired increased fitness, and amino acid changes were observed to accumulate in the viral polymerase (L protein) of most mutant viruses, the vast majority of the amino acid changes occurring in the C-terminal region. The function of this domain within L remains unknown, but by using a minigenome assay we showed that it might be involved in UTR recognition. Moreover, we identified an amino acid mutation within the polymerase that, when introduced into an otherwise wild-type BUNV, resulted in a virus with a temperature-sensitive phenotype. Viruses carrying temperature-sensitive mutations are good candidates for the design of live-attenuated vaccines. We suggest that a combination of stable deletions of the UTRs together with the introduction of temperature-sensitive mutations in both the nucleocapsid and the polymerase could be used to design live-attenuated vaccines against serious pathogens within the family Bunyaviridae.

    Importance. Virus growth in tissue culture can be attenuated by introduction of mutations in both coding and noncoding sequences. We generated attenuated Bunyamwera viruses by deleting sequences within both the 3rrsquo; and 5rrsquo; untranslated regions (UTR) on each genome segment, and showed that the viruses regained fitness following serial passage in cell culture. The fitter viruses had acquired amino acid changes predominantly in the C-terminal domain of the viral polymerase (L protein), and by using minigenome assays we showed that the mutant polymerases were better adapted to recognising the mutant UTRs. We suggest that making deletions within the UTRs should be incorporated along with other specific mutations, including deletion of the major virulence gene NSs and introducing temperature-sensitive mutations, in the design of attenuated bunyaviruses that could have potential as vaccines.

  • Phenotypic and Functional Characterization of Herpes Simplex Virus Glycoprotein B Epitope-specific Effector and Memory CD8+ T Cells from Ocular Herpes Symptomatic and Asymptomatic Individuals [PublishAheadOfPrint]

  • Herpes Simplex Virus type 1 (HSV-1) glycoprotein B- (gB-) specific CD8+ T cells protect mice from herpes infection and disease. However, whether and which HSV-1 gB-specific CD8+ T cells play a key role in the "natural" protection seen in HSV-1 seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we have dissected the phenotype and the function of HSV-1 gB-specific CD8+ T cells from HLA-A*02:01 positive, HSV-1 seropositive ASYMP and symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent ocular herpes disease). We found that: (i) healthy ASYMP individuals maintained a significantly higher proportion of differentiated HSV-1 gB-specific effector memory CD8+ T cells (TEM, CD45RAlowCCR7lowCD44highCD62Llow). In contrast, SYMP patients had frequent less-differentiated central memory CD8+ T cells (TCM, CD45RAlowCCR7highCD44lowCD62Lhigh); (ii) ASYMP individuals had significantly higher proportions of multi-functional effector CD8+ T cells, which responded mainly to gB342-350 and gB561-569 "ASYMP" epitopes, and simultaneously produced IFN-, CD107a/b, granzyme B and perforin. In contrast, effector CD8+ T cells from SYMP individuals were mostly mono-functional and were directed mainly against non-overlapping gB17-25 and gB183-191 "SYMP" epitopes; (iii) immunization of HLA-A*02:01 transgenic mouse model of ocular herpes with "ASYMP" CD8+ TEM cell epitopes, but not with "SYMP" CD8+ TCM cell epitopes, induced a strong CD8+ T cell-dependent protective immunity against ocular herpes infection and disease. Our findings provide insights into the role of HSV-specific CD8+ TEM cells in protection against herpes and should be considered in the development of an effective vaccine.

    IMPORTANCE A significantly higher proportion of differentiated and multi-functional HSV-1 gB-specific effector memory CD8+ T cells (TEM, CD45RAlowCCR7lowCD44highCD62Llow) were found in healthy ASYMP individuals who are seropositive for HSV-1 but never had any recurrent herpetic disease, compared to frequent less-differentiated and mono-functional central memory CD8+ T cells (TCM, CD45RAlowCCR7highCD44lowCD62Lhigh) in SYMP patients. Immunization with "ASYMP" CD8+ TEM cell epitopes, but not with "SYMP" CD8+ TCM cell epitopes, induced a strong protective HSV-specific CD8+ T cells in HLA-A*02:01 transgenic mice. These findings are important for the development of a safe and effective T cell-based herpes vaccine.

  • Local evolutionary patterns of Human Respiratory Syncytial Virus derived from whole genome sequencing [PublishAheadOfPrint]

  • Human Respiratory Syncytial Virus (RSV) is associated with severe childhood respiratory infections. A clear description of local RSV molecular epidemiology, evolution and transmission requires detailed sequence data and can inform new strategies for virus control and vaccine development. We have generated 27 complete or nearly complete genomes of RSV from hospitalized children attending a rural coastal district hospital in Kilifi, Kenya over a 10-year period using a novel full genome deep sequencing process. Phylogenetic analysis of the new genomes demonstrated the existence and co-circulation of multiple genotypes in both RSV A and B groups in Kilifi. Comparison of local versus global strains demonstrated that most RSV A variants observed locally in Kilifi were also seen in other parts of the world, while the Kilifi RSV B genomes encoded a high degree of variation that was not observed in other parts of the world. The nucleotide substitution rates for the individual open reading frames (ORFs) were highest in the regions encoding the attachment (G) glycoprotein and the NS2 protein. The analysis of RSV full genomes, compared to subgenomic regions, provided more precise estimates of the RSV sequence changes and revealed important patterns of RSV genomic variation and global movement. The novel sequencing method and the new RSV genomic sequences reported in this study expand our knowledge base for large-scale RSV epidemiological and transmission studies.

    Importance The new RSV genomic sequences and the novel sequencing method reported in this study provide important data for understanding RSV transmission and vaccine development. Given the complex interplay between RSV A and RSV B infections, the existence of local RSV B evolution is an important factor in vaccine deployment.

  • Identification of PB2 mutations responsible for the efficient replication of H5N1 influenza viruses in human lung epithelial cells [PublishAheadOfPrint]

  • Highly pathogenic H5N1 avian influenza viruses have caused outbreaks among poultry worldwide, resulting in sporadic infections in humans, approximately 60% mortality of which have been fatal. However, efficient transmission of H5N1 viruses among humans has yet to occur, suggesting that further adaptation of H5N1 viruses to humans is required for their efficient transmission among humans. Viral determinants for efficient replication in humans are currently poorly understood. Here, we report that the polymerase PB2 protein of an H5N1 influenza virus isolated from a human in Vietnam (A/Vietnam/UT36285/2010, 36285) increased the growth ability of an avian H5N1 virus (A/wild bird/Anhui/82/2005, Wb/AH82) in human lung epithelial A549 cells (although, the reassortant virus did not replicate more efficiently than human 36285 virus). Furthermore, we demonstrate that amino acid residues at positions 249, 309, and 339 of the PB2 protein from this human isolate were responsible for its efficient replication in A549 cells. PB2-249G and 339M, which are found in the human H5N1 virus, are rare in H5N1 viruses from both human and avian sources. Interestingly, PB2-249G is found in over 30% of human seasonal H3N2 viruses, which suggests that H5N1 viruses may replicate well in human cells when they acquire this mutation. Our data are of value to H5N1 virus surveillance.

    Importance Highly pathogenic H5N1 avian influenza viruses must acquire mutations to overcome the species barrier between avian species and humans. When H5N1 viruses replicate in human respiratory cells, they can acquire amino acid mutations that allow them to adapt to humans through continuous selective pressure. Several amino acid mutations have been shown to be advantageous for virus adaptation to mammalian hosts. Here, we found that amino acid changes at positions 249, 309, and 339 of PB2 contribute to efficient replication of avian H5N1 viruses in human lung cells. These findings are beneficial for evaluating the pandemic risk of circulating avian viruses and for further functional analysis of PB2.

  • The Susceptibility of Primate Lentiviruses to Nucleosides and Vpx during the Infection of DCs is Regulated by CA [PublishAheadOfPrint]

  • The block towards HIV-1 infection of DCs can be relieved by Vpx that degrades SAMHD1 or by exogenously added dNs, lending support to the hypothesis that SAMHD1 acts by limiting dNTPs. This notion has however been questioned. We show that while dNs and Vpx increase the infectivity of HIV-1, only the latter restores the infectivity of a VpxSIVMAC virus. This distinct behavior seems to map to CA, suggesting that species-specific CA-interactors modulate infection of DCs.

  • Transmission properties of atypical Creutzfeldt-Jakob disease: a clue to disease etiology? [PublishAheadOfPrint]

  • The genotype at polymorphic codon 129 of the PRNP gene has a profound influence on both phenotypic expression and prion strain susceptibility in humans. For example, while the most common sporadic Creutzfeldt-Jakob disease (CJD) subtype, sporadic CJD-MM1 (M1 strain), induces a single phenotype after experimental transmission regardless of the codon 129 genotype of the recipient animal, the phenotype elicited by sporadic CJD-VV2 (V2 strain), the second most common subtype, varies according to the host codon 129 genotype. In particular, the propagation of the V2 strain in codon 129 methionine homozygotes has only been linked to acquired forms of CJD such as plaque-type dura mater graft-associated CJD (dCJD), a subgroup of iatrogenic CJD with distinctive phenotypic features, but never observed in sporadic CJD. In the present study, we describe atypical CJD cases carrying the codon 129 methionine homozygosity, in a neurosurgeon and in a patient with a medical history of neurosurgery without dural grafting, showing the distinctive phenotypic features and transmission properties of plaque-type dCJD. These findings raise the possibility that the two cases, previously considered as sporadic CJD, might actually be acquired CJD caused by infection with the V2 strain. Thus, careful analyses of phenotypic features and transmission properties in atypical cases may be useful to distinguish acquired from sporadic cases of CJD.

    IMPORTANCE Susceptibility and phenotypic expression of Creutzfeldt-Jakob disease (CJD) depend on both the prion strain and genotype at polymorphic codon 129 of the PRNP gene. For example, propagation of the second most common sporadic CJD strain (V2 strain) into the codon 129 methionine homozygotes has been linked to plaque-type dura mater graft-associated CJD (dCJD), a subgroup of iatrogenic CJD with distinctive phenotypic features, but never observed in sporadic CJD. In the present study, we describe atypical CJD cases in a neurosurgeon and in a patient with a medical history of neurosurgery without dural grafting, showing the distinctive phenotypic features and transmission properties of plaque-type dCJD. These findings raise the possibility that the two cases, previously considered as sporadic CJD, might actually be acquired CJD caused by infection with the V2 strain.

  • Control of HCV replication in mouse liver derived cells by MAVS-dependent production of type-I and -III interferons [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) efficiently infects only humans and chimpanzees. Although the detailed mechanisms responsible for this narrow species tropism remain elusive, recent evidence has shown that murine innate immune responses efficiently suppress HCV replication. Therefore, poor adaptation of HCV to evade and/or counteract innate immune responses may prevent HCV replication in mice. The HCV NS3-4A protease cleaves human MAVS, a key cellular adaptor protein required for RIG-I like receptor (RLRs) dependent innate immune signalling. However, it is unclear if HCV interferes with mouse MAVS function equally well. Moreover, MAVS-dependent signalling events that restrict HCV replication in mouse cells were incompletely defined. Thus, we quantified the ability of HCV NS3-4A to counteract mouse and human MAVS. HCV NS3-4A similarly diminished both human and mouse MAVS-dependent signalling in human and mouse cells. Moreover, replicon-encoded protease cleaved a similar fraction of both MAVS variants. Finally, FLAG-tagged MAVS proteins repressed HCV replication to similar degree. Depending on MAVS expression, HCV replication in mouse liver cells triggered not only type-I but also type-III IFNs, which cooperatively repressed HCV replication. Mouse liver cells lacking both type-I and -III IFN receptors were refractory to MAVS-dependent antiviral effects indicating that the HCV-induced MAVS-dependent antiviral state depends on both type-I and -III IFN-receptor signalling.

    IMPORTANCE: Here we found that HCV NS3-4A similarly diminished both human and mouse MAVS-dependent signalling in human and mouse cells. Therefore, it is unlikely that ineffective cleavage of mouse MAVS per se precludes HCV propagation in immune competent mouse liver cells. Hence, approaches to reinforce HCV replication in mouse liver cells (e.g. by expression of essential human replication co-factors) should not be thwarted by the poor ability of HCV to counteract MAVS-dependent antiviral signalling. In addition we show that that mouse MAVS induces both type-I and type-III IFNs, which together control HCV replication. Characterization of type-I or type-III-dependent interferon stimulated genes in these cells should help to identify key murine restriction factors that preclude HCV propagation in immune competent mouse liver cells.

  • Interleukin 1- and type I interferon-dependent enhancement of the innate immune profile of a NYVAC-HIV-1 Env-Gag-Pol-Nef vaccine vector with dual deletion of type I and type II interferon-binding proteins [PublishAheadOfPrint]

  • NYVAC, a highly attenuated, replication-restricted poxvirus, is a safe and immunogenic vaccine vector. Deletion of immune evasion genes encoded by poxviruses is an attractive strategy for improving their immunogenic properties. Using system biology approaches, we herein describe the enhanced immunological profile of NYVAC vectors expressing the HIV-1 clade C env-gag-pol-nef genes (NYVAC-C) with single or double deletion of genes encoding type I (B19R) or type II (B8R) interferon (IFN)-binding proteins. Transcriptomic analyses of human monocytes infected with NYVAC-C, NYVAC-C-B19R or NYVAC-C-B8RB19R revealed a concerted up-regulation of innate immune pathways (IFN-stimulated genes [ISGs]) of increasing magnitude with NYVAC-C-B19R and NYVAC-C-B8RB19R relative to NYVAC-C. Deletion of B8R and B19R resulted in an enhanced activation of IRF3, IRF7 and STAT1, robust production of type I IFN and of ISGs whose expressions were inhibited by anti-type I IFN antibodies. Interestingly, NYVAC-C -B8RB19R induced the production of much higher levels of pro-inflammatory cytokines (TNF, IL-6 and IL-8) than NYVAC-C or NYVAC-C-B19R as well as a strong inflammasome response (caspase-1 and IL-1bbeta;) in infected monocytes. Top network analyses showed that this broad B8RB19R-mediated response was organized around two up-regulated gene expression nodes (TNF and IRF7). Consistent with these findings, monocytes infected with NYVAC-C-B8RB19R induced a stronger type I IFN-dependent and IL-1-dependent allogenic CD4+-T-cell response than NYVAC-C or NYVAC-C-B19R. Dual deletion of type I and type II IFN immune evasion genes in NYVAC markedly enhanced its immunogenic properties via an increased expression of type I IFNs and IL-1bbeta; and make it an attractive candidate HIV vaccine vector.

    IMPORTANCE NYVAC is a replication-deficient poxvirus developed as a vaccine vector against HIV. NYVAC expresses several genes known to impair the host immune defenses by interfering with innate immune receptors, cytokines or interferons. Given the crucial role played by interferons against viruses, we postulated that targeting type I and type II decoy receptors used by poxvirus to subvert the host innate immune response would be an attractive approach to improve the immunogenicity of NYVAC vectors. Using system biology approaches, we report that deletion of type I and type II IFN immune evasion genes in NYVAC poxvirus resulted in a robust expression of type I IFNs and interferon-stimulated genes (ISG), a strong activation of the inflammasome and up-regulated expression of IL-1bbeta; and pro-inflammatory cytokines. Dual deletion of type I and type II IFN immune evasion genes in NYVAC poxvirus improved its immunogenic profile and makes it an attractive candidate HIV vaccine vector.

  • A trans-dominant form of Gag restricts Ty1 retrotransposition and mediates copy number control [PublishAheadOfPrint]

  • Saccharomyces cerevisiae and S. paradoxus lack the conserved RNA interference pathway and utilize a novel form of copy number control (CNC) to inhibit Ty1 retrotransposition. Although noncoding transcripts have been implicated in CNC, here we present evidence that a truncated form of the Gag capsid protein (p22) or its processed form (p18) is necessary and sufficient for CNC and likely encoded by Ty1 internal transcripts. Coexpression of p22/p18 and Ty1 decreases mobility more than 30,000-fold. p22/p18 cofractionate with Ty1 virus-like particles (VLPs) and affect VLP yield, protein composition and morphology. Although p22/p18 and Gag colocalize in the cytoplasm, p22/p18 disrupt sites used for VLP assembly. GST affinity pull-downs also suggest that p18 and Gag interact. Therefore, this intrinsic Gag-like restriction factor confers CNC by interfering with VLP assembly and function, and expands the strategies used to limit retroelement propagation.

    IMPORTANCE Retrotransposons dominate the chromosomal landscape in many eukaryotes, can cause mutations by insertion or genome rearrangement and are evolutionarily related to retroviruses such as HIV. Thus, understanding factors that limit transposition and retroviral replication are fundamentally important. The present manuscript describes a retrotransposon-encoded restriction protein derived from the capsid gene of the yeast Ty1 element that disrupts virus-like particle assembly in a dose dependent manner. This form of copy number control acts as a molecular rheostat, allowing high levels of retrotransposition when few Ty1 elements are present and inhibiting transposition as copy number increases. Thus, yeast and Ty1 have co-evolved a form of copy number control that is beneficial to both "host and parasite". To our knowledge, this is the first Gag-like retrotransposon restriction factor described in the literature and expands the ways restriction proteins modulate retroelement replication.

  • Narrowing of human influenza A virus specific T cell receptor {alpha} and {beta} repertoire with increasing age [PublishAheadOfPrint]

  • Alterations in memory CD8 T cell responses may contribute to the high morbidity and mortality caused by seasonal influenza A virus (IAV) infections in older individuals. We questioned whether memory CD8 responses changed over time with increasing age to this non-persistent virus, to which recurrent exposure with new strains is common. Here, we show a direct correlation between increasing age and narrowing of the HLA-A2-restricted IAV M158-66-specific Vaalpha; and Vbbeta; T cell repertoires simultaneously leading to oligoclonal expansions including the usage of a single identical VA12-JA29 clonotype in all 8 older donors. The VA repertoire of older individuals also had longer CDR3 regions with increased usage of G/A runs, whose molecular flexibility may enhance TCR promiscuity. Collectively, these results suggest that CD8 memory T cell responses in humans to non-persistent viruses like IAV are dynamic, and with aging there is reduced diversity but preferential retention of T cell repertoires with features of enhanced cross-reactivity.

    Importance With increasing age the immune system undergoes drastic changes and older individuals have declined resistance to infections. Vaccinations become less effective and infection with influenza A virus in older individuals is associated with higher morbidity and mortality. Here, we questioned whether T cell responses directed against the highly conserved HLA-A2-restricted M158-66 peptide of IAV evolves with increasing age. Specifically, we postulated that CD8 T cell repertoires will narrow with recurrent exposure and thus may be less efficient in response to new infections with new strains of IAV. Detailed analyses of VA and VB TCR repertoire simultaneously showed a direct correlation between increasing age and narrowing of TCR repertoire. Features of the TCRs indicated potentially enhanced cross-reactivity in all older donors. In summary, T cell repertoire analysis in older individuals maybe useful as one of the predictors of protection after vaccination.

  • A novel humanized antibody neutralizes H5N1 via two different mechanisms [PublishAheadOfPrint]

  • H5N1 HPAI virus continues to be a severe threat to public health, as well as the poultry industry, due to its high mortality and antigenic drift rate. Neutralizing monoclonal antibodies can serve as a useful tool in preventing, treating and detecting H5N1. In the present study, a humanized H5 antibody 8A8 was developed from a murine H5 monoclonal antibody (Mab). Both the humanized and mouse Mabs presented positive activity in HI, virus neutralization and IFA against a wide range of H5N1s. Interestingly, both human and murine 8A8 were able to detect H5 in western blotting under reduction conditions. Further, by sequencing escape mutants, the conformational epitope of 8A8 was found to be located within the receptor binding domain (RBD) of H5. The linear epitope of 8A8 was identified by western blotting overlapping fragments and substitution mutants of HA1. RG H5N1s with individual mutations in either the conformational or linear epitope were generated and characterized in a series of assays including HI, post-attachment and cell-cell fusion inhibition assay. The results indicate that for 8A8, virus neutralization mediated by RBD-blocking relies on the conformational epitope while binding to the linear epitope contributes to the neutralization by inhibiting membrane fusion. Taken together, we report in this study that a novel humanized H5 Mab binds to two types of epitopes on HA, leading to virus neutralization via two mechanisms.

    Importance Recurrence of the highly pathogenic avian influenza (HPAI) virus subtype H5N1 in humans and poultry continues to be a serious concern to public health. Preventive and therapeutic measures against influenza A viruses have received much interest in the context of global efforts to combat the current and future pandemic. Passive immune therapy is considered to be the most effective and economically prudent preventive strategy against influenza besides vaccination. It is important to develop a humanized neutralizing Mab against all clades of H5N1. For the first time, we report in this study that a novel humanized H5 Mab binds to two types of epitopes on HA, leading to virus neutralization via two mechanisms. These findings further deepen our understanding of influenza neutralization.

  • Identification of amino acid substitutions supporting antigenic change of A(H1N1)pdm09 viruses. [PublishAheadOfPrint]

  • The majority of currently circulating influenza A(H1N1) viruses are antigenically similar to the virus that caused the 2009 influenza pandemic. However, antigenic variants are expected to emerge as population immunity increases. Amino acid substitutions in the hemagglutinin protein can result in escape from neutralizing antibodies, affect viral fitness, and change receptor preference. Here we constructed mutants with substitutions in the hemagglutinin of A/Netherlands/602/09 in an attenuated backbone to explore amino acid changes that may contribute to emergence of antigenic variants in the human population. Our analysis revealed that single substitutions affecting the 151 nndash; 159 loop located adjacent to the receptor binding site caused escape from ferret and human antibodies elicited after primary A(H1N1)pdm09 virus infection. The majority of these substitutions resulted in similar or increased replication efficiency in vitro compared to the virus carrying the wildtype hemagglutinin, and did not result in a change of receptor preference. However, none of the substitutions was sufficient to escape from the antibodies in sera from individuals that experienced both seasonal and pandemic A(H1N1) virus infections. These results suggest that antibodies directed against epitopes on seasonal A(H1N1) viruses contribute to neutralization of A(H1N1)pdm09 antigenic variants, thereby limiting the number of possible substitutions that could lead to escape from population immunity.

    Importance Influenza A viruses can cause significant morbidity and mortality in humans. Amino acid substitutions in the hemagglutinin protein can result in escape from antibody-mediated neutralization. This allows the virus to re-infect individuals that have acquired immunity to previously circulating strains through infection or vaccination. To date, the vast majority of A(H1N1)pdm09 strains remain antigenically similar to the virus that caused the 2009 influenza pandemic. However, antigenic variants are expected to emerge as a result of increasing population immunity. We show that single amino acid substitutions near the receptor binding site were sufficient to escape from antibodies specific for A(H1N1)pdm09 viruses, but not from antibodies elicited in response to infections with seasonal A(H1N1) and A(H1N1)pdm09 viruses. This study identifies substitutions in A(H1N1)pdm09 viruses that support escape from population immunity, but also suggests that the number of potential escape variants is limited by previous exposure to seasonal A(H1N1) viruses.

  • Tom70 mediates Sendai virus-induced apoptosis on mitochondria [PublishAheadOfPrint]

  • Virus infection triggers immediate innate immune responses. Apoptosis represents another effective means to restrict the virus invasion, besides the robust expression of the host cytokines and chemokines. IRF3 is recently demonstrated to be indispensable for SeV- (Sendai virus) induced apoptosis, but the underlying mechanism is not fully understood. Here, we report that a dynamic protein complex, Tom70/Hsp90/IRF3/Bax, mediate the SeV-induced apoptosis. The cytosolic pro-apoptotic protein Bax interacts specifically with IRF3 upon virus infection. The mitochondrial outer membrane protein Tom70 recruits IRF3 onto mitochondrion, via Hsp90. Consequently, the relocation of Bax onto mitochondrion induces the leakage of cytochrome C into cytosol and initiates the corresponding apoptosis. Interestingly, IKK-i is essential for the apoptosis, whereas TBK1 is dispensable. Collectively, our study characterizes a novel protein complex important for the SeV-induced apoptosis.

    Importance Apoptosis is an effective means to sacrifice virus-infected cells and restrain the spread of virus. In this study, we demonstrate that IRF3 associates with Bax upon virus infection. Tom70 recruits this protein complex onto the mitochondrial outer membrane through Hsp90, which thus induces the release of cytochrome C into cytosol, initiating the virus-induced apoptosis. Interestingly, IKKi plays an essential role in this activation. This study uncovers a novel mechanism of the SeV-induced apoptosis.

  • Requirement for Chloride Channel Function during the Hepatitis C Virus Lifecycle [PublishAheadOfPrint]

  • Hepatocytes express an array of plasma membrane and intracellular ion channels yet their role during the Hepatitis C virus (HCV) lifecycle remains largely undefined. Here, we show that HCV increases intracellular hepatic chloride (Cl-) influx that can be inhibited by selective Cl- channel blockers. Through pharmacological and siRNA mediated silencing we demonstrate that Cl- channel inhibition is detrimental to HCV replication. This represents the first observation of the involvement of Cl- channels during the HCV lifecycle.

  • Structure of the Extracellular Domain of Matrix Protein 2 of Influenza A Virus in Complex with a Protective Monoclonal Antibody [PublishAheadOfPrint]

  • The extracellular domain of influenza A virus matrix protein 2 (M2, M2e) is conserved and is being evaluated as a quasi-universal influenza A vaccine candidate. We describe the crystal structure at 1.6 AAring; resolution of M2e in complex with the Fab fragment of an M2e-specific monoclonal antibody that protects against influenza A virus challenge. This antibody binds M2 expressed on the surface of cells infected with influenza A virus. Five out of six complementary determining regions interact with M2e, and three highly conserved M2e residues are critical for this interaction. In this complex, M2e adopts a compact U-shaped conformation stabilized in the centre by the highly conserved tryptophan residue in M2e. This is the first description of the three dimensional structure of M2e.

    IMPORTANCE M2e of influenza A is under investigation as a universal influenza A vaccine, but its three dimensional structure is unknown. We describe the structure of M2e stabilized with an M2e-specific monoclonal antibody that recognizes natural M2. We found that the conserved tryptophan is positioned in the centre of the U-shaped structure of M2e and stabilizes its conformation. The structure also explains why previously reported in vivo escape viruses, selected with a similar monoclonal antibody, carried proline residue substitutions at position 10 in M2.

  • Modulation of a pore in the capsid of JC polyomavirus reduces infectivity and prevents exposure of the minor capsid proteins [PublishAheadOfPrint]

  • JC polyomavirus (JCPyV) infection of immunocompromised individuals results in the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). The viral capsid of JCPyV is composed primarily of the major capsid protein, virus protein 1 (VP1), and pentameric arrangement of VP1 monomers results in the formation of a pore at the five-fold axis of symmetry. While the presence of this pore is conserved among polyomaviruses, its functional role in infection or assembly is unknown. Here, we investigate the role of the five-fold pore in assembly and infection of JCPyV by generating a panel of mutant viruses containing amino acid substitutions to the residues lining this pore. Multicycle growth assays demonstrated that the relative fitness of all mutants was reduced, as compared to wild type virus. Bacterial expression of VP1 pentamers containing substitutions to residues lining the five-fold pore did not affect pentamer assembly or prevent association with the minor capsid protein VP2. The X-ray crystal structures of selected pore mutants contained subtle changes to the five-fold pore, and no other changes to VP1 were observed. Pore-mutant pseudoviruses were not deficient in assembly, packaging of the minor capsid proteins, binding to cells, or in transport to the host cell endoplasmic reticulum. Instead, these mutant viruses were unable to expose VP2 upon arrival to the endoplasmic reticulum, a step that is critical for infection. This study demonstrates that the five-fold pore is an important structural feature of JCPyV and minor modifications to this structure has significant impacts on infectious entry.

    IMPORTANCE JCPyV is an important human pathogen that causes a severe neurological disease in immunocompromised individuals. While the high resolution X-ray structure of the major capsid protein of JCPyV has been solved, the importance of a major structural feature of the capsid, the five-fold pore, remains poorly understood. This pore is conserved across polyomaviruses and suggests that these viruses have either limited structural plasticity in this region or that this pore is important in infection or assembly. Using a structure-guided mutational approach, we showed that modulation of this pore severely inhibits JCPyV infection. These mutants do not appear deficient in assembly or early steps in infectious entry and are instead reduced in their ability to expose a minor capsid protein in the host cell endoplasmic reticulum. Our work demonstrates that the five-fold pore is an important structural feature for JCPyV.

  • Antibody Titer Has Positive Predictive Value for Vaccine Protection against Challenge with Natural Antigenic Drift Variants of H5N1 High Pathogenicity Avian Influenza Viruses from Indonesia [PublishAheadOfPrint]

  • Vaccines are used in integrated control strategies to protect poultry against H5N1 high pathogenicity avian influenza (HPAI). H5N1 HPAI was first reported in Indonesia in 2003 and vaccination was initiated in 2004, but reports of vaccine failures began to emerge in mid-2005. This study investigated the role of Indonesian licensed vaccines, specific vaccine seed strains and emerging variant field viruses as causes of vaccine failures. Eleven of 14 licensed vaccines contained the manufacturer's listed vaccine seed strains, but three vaccines contained a different seed strain than listed on the label. Vaccines containing A/turkey/Wisconsin/1968 (WI/68), A/chicken/Mexico/28159-232/1994 (Mex/94) and Eng/73 seed strains had high serological potency in chickens (geometric mean HI titers gge; 1:169), but vaccines containing reverse genetic (rg) A/chicken/Guangdong/1/1996 (rgGD/96), A/chicken/Legok/2003 (Legok/03), rgA/chicken/Vietnam/C57/2004 (rgVN/04) or rgA/chicken/Legok/2003 (rgLegok/03) had lower serological potency (geometric mean HI titers lle; 1:95). In challenge studies, chickens immunized with any of the H5 AI vaccines were protected against A/chicken/West Java/SMI-HAMD/2006 (SMI-HAMD/06), partially protected against A/chicken/Papua/TA5/2006 (Papua/06), but were not protected against A/chicken/West Java/PWT-WIJ/2006 (PWT/06). Experimental inactivated vaccines made with PWT/06 HPAI or rgPWT/06 LPAI seed strains protected chickens from lethal challenge as did a combination of a commercially available live fowl poxvirus vaccine expressing the H5 influenza gene and inactivated Legok/03 vaccine. These studies indicate that antigenic variants did emerge in Indonesia following widespread H5 avian influenza vaccine usage, and efficacious inactivated vaccines can be developed using antigenic variant wild type viruses or rgLPAI seed strains containing hemagglutinin and neuraminidase genes of wild type viruses.

    IMPORTANCE H5N1 high pathogenicity avian influenza (HPAI) has become endemic in Indonesian poultry and such poultry are the source of virus for birds and mammals including humans. Vaccination has become a part of the poultry control strategy but vaccine failures have occurred in the field. This study identified possible causes of vaccine failure which included use of an unlicensed virus seed strain and induction of low levels of protective antibody because of insufficient quantity of vaccine antigen. However, the most important cause of vaccine failure was the appearance of drift variant field viruses that partially or completely overcome commercial vaccine induced immunity. Furthermore, experimental vaccines using inactivated wild type or reverse genetic generated vaccines containing hemagglutinin and neuraminidase genes of wild type drift variant field viruses were protective. These studies indicate the need for surveillance to identify drift variant viruses in the field and update licensed vaccines when such variants appear.

  • Influenza viruses with receptor-binding N1 neuraminidases occur sporadically in several lineages and show no attenuation in cell culture or mice [PublishAheadOfPrint]

  • In nearly all characterized influenza viruses, hemagglutinin (HA) is the receptor-binding protein while neuraminidase (NA) is a receptor-cleaving protein that aids in viral release. However in recent years, several groups have described point mutations that confer receptor-binding activity on NA, albeit in laboratory rather than natural settings. One of these mutations, D151G, appears to arise in the NA of recent human H3N2 viruses upon passage in tissue culture. We inadvertently isolated the second of these mutations, G147R, in the NA of the lab-adapted A/WSN/33 (H1N1) strain, while we were passaging a heavily engineered virus in the lab. G147R also occurs at low frequency in the reported sequences of viruses from three different lineages: human 2009 pandemic H1N1 (pdmH1N1), human seasonal H1N1, and chicken H5N1. Here we reconstruct a representative G147R NA from each of these lineages, and find that all the proteins have acquired the ability to bind an unknown cellular receptor while retaining substantial sialidase activity. We then reconstruct a virus with the HA and NA of a reported G147R pdmH1N1 variant, and find no attenuation of viral replication in cell culture or change in pathogenesis in mice. Furthermore, the G147R virus has modestly enhanced resistance to neutralization by the Fab of an antibody against the receptor-binding pocket of HA, although it remains completely sensitive to the full-length IgG. Overall, our results suggest that circulating N1 viruses occasionally may acquire the G147R NA receptor-binding mutation without impairment of replicative capacity.

    IMPORTANCE Influenza viruses have two main proteins on their surface: one (hemagglutinin) binds incoming viruses to cells, while the other (neuraminidase) helps release newly formed viruses from these same cells. Here we characterize unusual mutant neuraminidases that have acquired the ability to bind to cells. We show that the mutation that allows neuraminidase to bind cells has no apparent adverse effect on viral replication, but does make the virus modestly more resistant to a fragment of an antibody that blocks the normal hemagglutinin-mediated mode of viral attachment. Our results suggest that viruses with receptor-binding neuraminidases may occur at low levels in circulating influenza lineages.

  • Soluble Tim-3 is shed from CD8+ T cells by the sheddase ADAM10, is increased in plasma during untreated HIV infection, and correlates with HIV disease progression. [PublishAheadOfPrint]

  • Chronic HIV infection results in a loss of HIV-specific CD8+ T cell effector function, termed "exhaustion", which is mediated, in part, by the membrane co-inhibitory receptor T cell immunoglobulin mucin domain-3 (Tim-3). Like many other receptors, a soluble form of this protein has been described in human blood plasma. However, soluble Tim-3 (sTim-3) is poorly characterized and its role in HIV disease is unknown. Here we show that Tim-3 is shed from the surface of responding CD8+ T cells by the matrix metalloproteinase, ADAM10, producing a soluble form of the co-inhibitory receptor. Despite previous reports in the mouse model, no alternatively spliced, soluble form of Tim-3 was observed in humans. Shed sTim-3 was found in human plasma, and was significantly elevated during early and chronic untreated HIV infection, but was not found differentially modulated in HAART treated HIV-infected subjects or in elite controllers, when compared to HIV-uninfected subjects. Plasma sTim-3 levels positively correlated with HIV viral load and negatively correlated with CD4 counts. Thus, plasma sTim-3 shedding correlated with HIV disease progression. Despite these correlations, we found that shedding Tim-3 did not improve the function of CD8+ T cells in terms of IFN- production or prevent their apoptosis through galectin-9. Further characterization studies of sTim-3 function are needed to understand the contribution of sTim-3 in HIV disease pathogenesis with implications for novel therapeutic interventions. (224/250)

    Importance Despite the overall success of HAART slowing the progression to AIDS in HIV-infected subjects, chronic immune activation and T cell exhaustion contribute to the eventual deterioration of the immune system. Understanding these processes will aid in the development of interventions and therapeutics to be used in combination with HAART to slow or reverse this deterioration. Here, we show that a soluble form of T cell exhaustion associated co-inhibitory molecule, Tim-3, is shed from the surface of T cells. Furthermore, sTim-3 is elevated in the plasma of treatment naiiuml;ve subjects with acute and chronic HIV infection and is associated with markers of disease progression. This is the first study to characterize sTim-3 in human plasma, its source and mechanism of production. While it is still unclear whether sTim-3 contributes to HIV pathogenesis, sTim-3 may represent a new correlate of HIV disease progression. (141/150)

  • Long-term SIV control in cynomolgus macaques not associated with efficient SIV-specific CD8 T-cell responses [PublishAheadOfPrint]

  • The spontaneous control of human and simian immunodeficiency viruses (HIV/SIV) is typically associated with specific MHC-class I alleles and efficient CD8 T-cell responses, but many controllers maintain viral control despite a non-protective MHC background and weak CD8 T-cell responses. Therefore, the contribution of this response to maintaining long-term viral control remains unclear. To address this question, we transiently depleted CD8 T cells from five SIV-infected cynomolgus macaques with long-term viral control and weak CD8 T-cell responses. Among them, only one carried the protective MHC allele H6. After depletion, four of five controllers experienced a transient rebound of viremia. The return to undetectable viremia was accompanied by only modest expansion of SIV-specific CD8+ T cells that lacked efficient SIV-suppression capacity ex vivo. In contrast, the depletion was associated with homeostatic activation/expansion of CD4+ T cells that correlated with viral rebound. In one macaque, viremia remained undetectable despite efficient CD8+ cell depletion and inducible SIV replication from its CD4+ T cells in vitro. Altogether, our results suggest that CD8+ T cells are not unique contributors to the long-term maintenance of low viremia in this SIV controller model and that other mechanisms such as weak viral reservoirs or control of activation may be important players in control.

    IMPORTANCE Spontaneous control of HIV-1 to undetectable levels is associated to efficient anti-HIV CD8 T-cell responses. However, in some cases this response fades overtime although viral control is maintained, and many HIV controllers (llaquo; weak responders rraquo;) have very low frequencies of HIV-specific CD8 T-cells. In these cases, the importance of CD8 T-cells in the maintenance of HIV-1 control is questionable. We developed a non-human primate model of durable SIV control with an immune profile resembling that of weak responders. Transient depletion of CD8+ cells induced a raise in viral load. However, viremia was correlated to CD4 T-cell activation consecutive to CD8 cell depletion. Regain of viral control to pre-depletion levels was not associated to a recall of anti-SIV capacities of CD8 T-cells. Our results suggest that CD8 T-cells may not be involve in maintenance of viral control in weak responders and highlight that additional mechanisms should not be underestimated.

  • Viral semaphorin inhibits dendritic cell phagocytosis and migration but is not essential for {gamma}-herpesvirus-induced lymphoproliferation in malignant catarrhal fever [PublishAheadOfPrint]

  • Viral semaphorins are semaphorin 7A (sema7A) mimics found in pox- and herpesviruses. Among herpesviruses, semaphorins are encoded by -herpesviruses of the Macavirus genus only. Alcelaphine herpesvirus 1 (AlHV-1) is a Macavirus that persistently infects wildebeest asymptomatically but induces malignant catarrhal fever (MCF) when transmitted to several species of susceptible ruminants and the rabbit model. MCF is caused by the activation/proliferation of latently infected T lymphocytes. Viral semaphorins have been suggested to mediate immune evasion mechanisms and/or directly alter host T cell function. Here we studied AlHV-sema, the viral semaphorin encoded by the A3 gene of AlHV-1. Phylogenetic analyses revealed independent acquisition of pox- and herpesvirus semaphorins, suggesting that these proteins might have distinct functions. AlHV-sema showed a predicted 3-D structure very similar to sema7A and conserved key residues in sema7A-plexinC1 interaction. Expression analyses revealed that AlHV-sema is a secreted 93 kDa glycoprotein expressed during the early phase of virus replication. Purified AlHV-sema was able to bind to fibroblasts and dendritic cells and induce F-actin condensation and cell retraction through a plexinC1 and Rho/cofilin dependent mechanism. Cytoskeleton rearrangement was further associated with inhibition of phagocytosis by dendritic cells and migration to the draining lymph node. Next, we generated recombinant viruses and demonstrated that the lack of A3 did not significantly affect virus growth in vitro and did not impair MCF induction and associated lymphoproliferative lesions. In conclusion, AlHV-sema has immune evasion functions through mechanisms similar to poxvirus semaphorin but is not directly involved in host T cell activation during MCF.

    Importance Whereas most poxviruses encode viral semaphorins, semaphorin-like genes have only been identified in few -herpesviruses belonging to the Macavirus genus. Alcelaphine herpesvirus 1 (AlHV-1) is a Macavirus carried asymptomatically by wildebeest but induces a latency-associated lymphoproliferative disease of T lymphocytes in various ruminant species, namely malignant catarrhal fever (MCF). Viral semaphorins have been hypothesized to have immune evasion functions and/or be involved in activating latently infected T cells. Here we showed evidence that the viral semaphorin AlHV-sema inhibits dendritic cell phagocytosis and migration to the draining lymph node, both being indispensable mechanisms for protective anti-viral responses. Next, we engineered recombinant viruses unable to express AlHV-sema and demonstrated that this protein is dispensable for the induction of MCF. In conclusion, this study suggests that herpesvirus and poxvirus semaphorins have independently evolved similar functions to thwart the immune system of the host while AlHV-sema is not directly involved in MCF-associated T-cell activation.

  • Sp1 sites in the non-coding control region of BK polyomavirus are key regulators of bidirectional viral early and late gene expression [PublishAheadOfPrint]

  • In kidney transplant patients with BK polyomavirus (BKPyV) nephropathy, viral variants arise bearing rearranged (rr) non-coding control regions (NCCR) that increase viral early gene expression, replicative fitness and cytopathology. rr-NCCRs result from various deletions and duplications of archetype (ww-)NCCR sequences, that alter transcription factor binding sites (TFBS). However, the role of specific TFBS is unclear. We inactivated 24 TFBS in the archetype NCCR by selective point mutations and examined viral gene expression in bi-directional reporter constructs. Compared to archetype, Group-1 mutations increased viral early gene expression similar to rr-NCCR and resulted from inactivating one Sp1 or one Ets1 TFBS near the late transcription start site (TSS). Group-2 mutations conferred intermediate early gene activation and affected NF1, YY1, and p53 sites between early and late TSS. Group-3 mutations decreased early and late gene expression and included two other Sp1 sites near the early TSS. Recombinant viruses bearing Group-1 NCCRs showed increased replication in human renal epithelial cells similar to clinical rr-NCCR variants. Group-2 and -3 viruses showed intermediate or no replication, respectively. Literature search revealed unnoticed Group-1 mutations in BKPyV nephropathy, hemorrhagic cystitis, and disseminated disease.

    IMPORTANCE The NCCRs of polyomaviruses mediate silent persistence of the viral genome as well as the appropriately timed (re-)activation of the viral life cycle. This study indicates that the basal BKPyV NCCR is critically controlled by a hierarchy of single TFBS in the archetype NCCR that direct, modulate and execute the bidirectional EVGR and LVGR expression. The results provide new insights into how BKPyV NCCR functions as viral sensor of host cell signals and shed new light on how transcription factors like Sp1 control bidirectional viral gene expression, and contribute to replication and pathology.

  • A host susceptibility gene, DR1, Facilitates Influenza A Virus Replication via Suppressing Host Innate Immunity and Enhancing Viral RNA Replication. [PublishAheadOfPrint]

  • Influenza A virus (IAV) depends on cellular factors to complete its replication cycle; thus, investigation of the factors utilized by IAV could facilitate antiviral drug development. To this end, a cellular transcriptional repressor, DR1, was identified from a genome-wide RNAi screen. Knockdown (KD) of DR1 resulted in reduction of the viral RNA and protein production, demonstrating that DR1 acts as a positive host factor in IAV replication. Genome-wide transcriptomic analysis showed that there was strong induction of interferon-stimulated genes (ISGs) expression after prolonged DR1 KD. We found that IFNbbeta; was induced by DR1 KD, thereby activating the JAK-STAT pathway to turn on ISGs expression, which led to strong inhibition of IAV replication. This result suggests that DR1 in normal cells suppresses IFN induction probably to prevent undesired cytokine production, but this suppression could create a milieu that favors IAV replication once cells are infected. Furthermore, biochemical assays of the viral RNA replication showed that DR1 KD suppressed the viral RNA replication. We also showed that DR1 associated with all three subunits of the viral RNA-dependent RNA polymerase (RdRp) complex, indicating that DR1 may be through interacting with individual components of the viral RdRp complex to enhance the viral RNA replication. Thus, DR1 could be considered a novel host susceptibility gene for IAV replication via dual mechanisms, which not only suppresses host defense to indirectly favor IAV replication but also directly facilitates the viral RNA replication.

    Importance Investigation of virus-host interactions involved in influenza A virus replication is important for understanding the viral pathogenesis and the host defenses, which could manipulate influenza virus infection or prevent the emergence of drug resistance caused by high error rate during the viral RNA replication. For this purpose, a cellular transcriptional repressor, DR1, was identified from a genome-wide RNAi screen as a positive regulator in IAV replication. In the current studies, we showed that DR1 suppresses the gene expression of a large set of host innate immunity, which indirectly facilitates IAV replication in the event of IAV infection. Besides this scenario, DR1 also directly enhanced the viral RdRp activity likely through associating with individual components of the viral RdRp complex. Thus, DR1 represents a novel host susceptibility gene for IAV replication via multiple functions, which not only suppresses host defense but also enhances the viral RNA replication. DR1 may be a potential target for drug development against influenza infection.

  • Relationships between dissemination of primary parainfluenza virus infection in the respiratory tract, mucosal and peripheral immune responses, and protection from reinfection: a non-invasive bioluminescence imaging study [PublishAheadOfPrint]

  • Respiratory paramyxoviruses such as respiratory syncytial virus (RSV) and the human parainfluenza viruses (HPIV1-4) infect virtually all children by ages 2-5, leading to partial but incomplete protection from reinfection. Here, we used luciferase-expressing reporter Sendai viruses (the murine counterpart of HPIV1) to non-invasively measure in living mice primary infection, immune responses, and protection from reinfection by either lethal challenge or natural transmission. Both a non-attenuated and an attenuated reporter Sendai virus were used and three inoculation strategies were employed: intramuscular (IM), intranasal (IN) at a low dose and volume (low d/v), and IN at a high d/v. High d/v IN inoculation resulted in the highest levels of antibody responses and protection from reinfection. Low d/v IN inoculation afforded complete protection from contact transmission and protected from morbidity, mortality, and viral growth during lethal challenge. IM inoculation was inferior to IN at inducing antibody responses and protecting from challenge. For individual mice and across groups, the levels of serum binding and neutralizing antibody responses correlated with primary infection and protection from reinfection in the lungs. Contact transmission, the predominant mode of parainfluenza virus transmission, was modeled accurately by direct IN inoculation of Sendai virus at a low d/v, and was completely preventable by IN vaccination of an attenuated virus at a low d/v. The data highlight differences in infection and protection from challenge in the upper versus lower respiratory tract and bear upon live attenuated vaccine development.

    IMPORTANCE There are currently no licensed vaccines against the HPIVs and HRSV, important respiratory pathogens of infants and children. Natural infection leads to partial but incomplete protective immunity, resulting in subsequent reinfections even in the absence of antigenic drift. Here, we used non-invasive bioluminescence imaging in a mouse model to dissect relationships between (i) the mode of inoculation, (ii) the dynamics of primary infection, (iii) consequent immune responses, and (iv) protection from high d/v lethal challenge and contact transmission, which we find here to be similar to a mild low d/v URT-biased infection. Our studies demonstrate the superiority of IN versus IM vaccination in protecting from both lethal challenge and contact transmission. In addition to providing correlates of protection that will assist respiratory virus vaccine development, these studies extend the development of a growing technique used to study viral infection and immunity: non-invasive bioluminescence imaging.

  • The nsp1, nsp13, and M proteins contribute to the hepatotropism of murine coronavirus JHM.WU [PublishAheadOfPrint]

  • Mouse hepatitis virus (MHV) isolates JHM.WU and JHM.SD promote severe central nervous system disease. However, while JHM.WU replicates robustly and induces hepatitis, JHM.SD fails to replicate or induce pathology in the liver. These two JHM variants encode homologous proteins with few polymorphisms, and little is known about which viral proteins(s) are responsible for the liver tropism of JHM.WU. We constructed reverse genetic systems for JHM.SD and JHM.WU and, utilizing these full-length cDNA clones, constructed chimeric viruses and mapped the virulence factors involved in liver tropism. Exchanging the spike proteins of the two viruses neither increased replication of JHM.SD in the liver nor attenuated JHM.WU. By further mapping, we found that polymorphisms in JHM.WU structural protein M and nonstructural replicase proteins nsp1 and nsp13 are essential for liver pathogenesis. M protein and nsp13, the helicase, of JHM.WU are required for efficient replication in vitro and in the liver in vivo. The JHM.SD nsp1 protein contains a K194R substitution of Lys194, a residue conserved among all other MHV strains. The K194R polymorphism has no effect on in vitro replication but influences hepatotropism and introduction of R194K into JHM.SD promotes replication in the liver. Conversely, a K194R substitution in nsp1 of JHM.WU or A59, another hepatotropic strain, significantly attenuates replication of each strain in the liver and increases IFN-bbeta; expression in macrophages in culture. Our date indicate that both structural and nonstructural proteins contribute to MHV liver pathogenesis and support previous reports that nsp1 is a Betacoronavirus virulence factor.

    IMPORTANCE The Betacoronaviruses genus includes human pathogens, some of which cause severe respiratory disease. The spread of SARS-CoV and MERS-CoV into human populations demonstrates the zoonotic potential of emerging coronaviruses, and there are currently no vaccines or effective antivirals for human coronaviruses. Thus it is important to understand the virus-host interaction that regulates coronavirus pathogenesis. Murine coronavirus infection of mice provides a useful model for the study of coronavirus-host interactions, including the determinants of tropism and virulence. We found that very small changes in coronavirus proteins can profoundly affect tropism and virulence. Furthermore, the hepatotropism of MHV-JHM depends not on the spike protein and viral entry but rather on a combination of the structural protein M and nonstructural replicase-associated proteins nsp1 and nsp13, which are conserved among Betacoronaviruses. Understanding virulence determinants will aid in the design of vaccines and antiviral strategies.

  • Early function of the Abutilon mosaic virus AC2 gene as a replication brake [PublishAheadOfPrint]

  • The C2/AC2 genes of monopartite/bipartite geminiviruses of the genera Begomovirus and Curtovirus encode important pathogenicity factors with multiple functions described so far. A novel function as a replication brake of Abutilon mosaic virus (AbMV) AC2 is described utilizing transgenic plants with dimeric inserts of DNA B or with a reporter construct to express green fluorescent protein (GFP). Their replicational release upon AbMV superinfection or individual and combined expression of epitope-tagged AbMV AC1, AC2 and AC3 were studied. In addition, the effects were compared in the presence or absence of an unrelated tombusvirus suppressor of silencing (P19). The results show that AC2 suppresses replication reproducibly in all test assays and AC3 counteracts this effect. Examination of the topoisomer distribution of supercoiled DNA, which indicates changes in the viral minichromosome structure, did not support any influence of AC2 on transcriptional gene silencing and DNA methylation. Geminiviral AC2 protein has been detected here for the first time in plants. The experiments revealed an extremely low level which was slightly increased, if constructs with an intron and a HA tag in addition to P19 expression were used. AbMV AC2 properties are discussed with reference to those of other geminiviruses with respect to charge, modification and size in order to delimit possible reasons of different behaviors.

    Importance The (A)C2 genes encode a key pathogenicity factor of begomoviruses and curtoviruses in the plant virus family Geminiviridae. It has been implicated in the resistance breaking observed in agricultural cotton production. AC2 is a multifunctional protein involved in transcriptional control, gene silencing and regulation of basal biosynthesis. Here, a new function of Abutilon mosaic virus AC2 in replication control is added as a feature of this protein in viral multiplication providing a novel finding on geminiviral molecular biology.

  • Effective lethal mutagenesis of influenza virus by three nucleoside analogs [PublishAheadOfPrint]

  • Lethal mutagenesis is a broad-spectrum antiviral strategy that exploits the high mutation rate and low mutational tolerance of many RNA viruses. This approach uses mutagenic drugs to increase viral mutation rates and burden viral populations with mutations that reduce the number of infectious progeny. We investigated the effectiveness of lethal mutagenesis as a strategy against influenza virus using three nucleoside analogs, ribavirin, 5-azacytidine, and 5-fluorouracil. All three drugs were active against a panel of seasonal H3N2 and laboratory-adapted H1N1 strains. We found that each drug increased the frequency of mutations in influenza virus populations and decreased the virus' specific infectivity, indicating a mutagenic mode of action. We were able to drive viral populations to extinction by passaging influenza virus in the presence of each drug, indicating that complete lethal mutagenesis of influenza populations can be achieved when a sufficient mutational burden is applied. Population-wide resistance to these mutagenic agents did not arise after serial passage of influenza populations in sub-lethal concentrations of drug. Sequencing of these drug-passaged viral populations revealed genome-wide accumulation of mutations at low frequency. The replicative capacity of drug-passaged populations was reduced at higher multiplicities of infection, suggesting the presence of defective interfering particles and a possible barrier to the evolution of resistance. Together, our data suggest that lethal mutagenesis may be a particularly effective therapeutic approach with a high genetic barrier to resistance for influenza virus.

    Importance Influenza virus is an RNA virus that causes significant morbidity and mortality during annual epidemics. Novel therapies for RNA viruses are needed due to the ease with which these viruses evolve resistance to existing therapeutics. Lethal mutagenesis is a broad-spectrum strategy that exploits the high mutation rate and the low mutational tolerance of most RNA viruses. It is thought to possess a higher barrier to resistance than conventional antiviral strategies. We investigated the effectiveness of lethal mutagenesis against influenza virus using three different drugs. We showed that influenza virus was sensitive to lethal mutagenesis by demonstrating that all three drugs induced mutations and led to an increase in the generation of defective viral particles. We also found that it may be difficult for resistance to these drugs to arise at a population-wide level. Our data suggest that lethal mutagenesis may be an attractive anti-influenza strategy that warrants further investigation.

  • A native-like SOSIP.664 trimer based on a HIV-1 subtype B env gene [PublishAheadOfPrint]

  • Recombinant trimeric mimics of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) spike should expose as many epitopes as possible for broadly neutralizing antibodies (bNAbs), but few if any for non-neutralizing antibodies (non-NAbs). Soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A strain, BG505, approach this ideal and are therefore plausible vaccine candidates. Here, we report on the production and in vitro properties of a new SOSIP.664 trimer derived from a subtype B env gene, B41, including how to make this protein in low-serum media without proteolytic damage (clipping) to the V3 region. We also show that non-clipped trimers can be purified successfully via a positive selection affinity column using the bNAb PGT145, which recognizes a quaternary epitope at the trimer apex. Negative-stain electron microscopy imaging shows that the purified, non-clipped, native-like B41 SOSIP.664 trimers contain two sub-populations, which we propose represent an equilibrium between the fully closed and a more open conformation. The latter is different from the fully open, CD4 receptor-bound conformation and may represent an intermediate state of the trimer. This new subtype B trimer adds to the repertoire of native-like Env proteins that are suitable for immunogenicity and structural studies.

    Importance The cleaved, trimeric envelope protein complex is the only neutralizing antibody target on the HIV-1 surface. Many vaccine strategies are based on inducing neutralizing antibodies. For HIV-1, one approach involves using recombinant, soluble protein mimics of the native trimer. At present, the only reliable way to make native-like, soluble trimers in practical amounts is via the introduction of specific sequence changes that confer stability to the cleaved form of Env. The resulting trimers are known as SOSIP.664 gp140s, and the current paradigm is based on the BG505 subtype A env gene. Here, we describe the production and characterization of a SOSIP.664 trimer derived from a subtype B gene (B41), together with a simple, one-step method to purify native-like trimers by affinity chromatography with a quaternary epitope-specific bNAb, PGT145. The resulting trimers will be useful for structural and immunogenicity experiments aimed at devising ways to make an effective HIV-1 vaccine.

  • Mapping the interactions between the NS4B and NS3 proteins of dengue virus [PublishAheadOfPrint]

  • Flavivirus RNA synthesis is mediated by a multi-protein complex associated with the endoplasmic reticulum membrane, named the replication complex (RC). Within the flavivirus RC, NS4B, an integral membrane protein with a role in virulence and regulation of the innate immune response, binds to the NS3 protease-helicase. NS4B modulates the RNA helicase activity of NS3, but the molecular details of their interaction remain elusive. Here we used dengue virus (DENV) to map the determinants for the NS3/NS4B interaction. Co-immunoprecipitation and in situ proximity ligation assay confirmed that NS3 colocalizes with NS4B both in DENV-infected cells and in cells co-expressing both proteins. Surface plasmon resonance demonstrated that subdomains 2 and 3 of the NS3 helicase region and the cytoplasmic loop of NS4B are required for binding. Using NMR, we found that the isolated cytoplasmic loop of NS4B is flexible with a tendency to form a three-turn aalpha;-helix and two short bbeta;-strands. Upon binding to the NS3 helicase, twelve amino acids within the cytoplasmic loop of NS4B exhibited line broadening, suggesting a participation in the interaction. Sequence alignment showed that four of these twelve residues are strictly conserved across different flaviviruses. Mutagenesis analysis showed that three (Q134, G140, and N144) of the four evolutionary conserved NS4B residues are essential for DENV replication. The mapping of the NS3/NS4B interacting regions described here can assist the design of inhibitors that disrupt their interface for antiviral therapy.

    SIGNIFICANCE NS3 and NS4B are essential components of the flavivirus RC. Using DENV as a model, we mapped the interaction between the viral NS3 and NS4B proteins. The subdomains 2 and 3 of NS3 helicase as well as the cytoplasmic loop of NS4B are critical for the interaction. Functional analysis delineated residues within the NS4B cytoplasmic loop that are crucial for DENV replication. Our findings reveal molecular details on how flavivirus NS3 protein cooperates with NS4B within the RC. In addition, this study has established the rationale and assays to search for inhibitors disrupting the NS3/NS4B interaction for antiviral drug discovery.

  • Immune screening identifies novel T cell targets encoded by anti-sense reading frames of HIV-1 [PublishAheadOfPrint]

  • Cytotoxic T-lymphocyte (CTL) responses to epitopes in alternative HIV reading frames have been reported. However, the extent of CTL responses to putative proteins encoded in anti-sense reading frames is unknown. Using sequence alignments and computational approaches, we here predict five potential anti-sense HIV proteins and characterize common CTL responses against them. Results suggest that anti-sense-derived sequences are commonly transcribed and translated and could encode functional proteins that contain important targets of the anti-HIV cellular immunity.

  • Generation of Transgenic Mouse Model of Middle East Respiratory Syndrome-Coronavirus Infection and Disease [PublishAheadOfPrint]

  • The emergence of Middle East respiratory syndrome-coronavirus (MERS-CoV) in the Middle East since 2012 has caused more than 900 human infections with ~40% mortality to date. Animal models are needed for studying pathogenesis and for development of preventive and therapeutic agents against MERS-CoV infection. Nonhuman primates (rhesus macaques and marmosets) are expensive models of limited availability. Although a mouse lung infection model has been described using adenovirus vectors expressing human CD26/dipeptidyl peptidase 4 (DPP4), it is believed that a transgenic mouse model is needed for MERS-CoV research. We have developed this transgenic mouse model as indicated in this study. We show that transgenic mice globally expressing hCD26/DPP4 were fully permissive to MERS-CoV infection, resulting in relentless weight loss and death within days post infection (dpi). High infectious viral titers were recovered primarily from the lungs and brains of mice at 2 and 4 dpi, respectively, whereas viral RNAs were also detected in the heart, spleen, and intestine, indicating a disseminating viral infection. Infected Tg+ mice developed a progressive pneumonia, characterized by extensive inflammatory infiltration. In contrast, an inconsistent mild perivascular cuffing was the only pathological change associated with the infected brains. Moreover, infected Tg+ mice were able to activate genes encoding for many antiviral and inflammatory mediators within the lungs and brains, coinciding with the high levels of viral replication. This new and unique transgenic mouse model will be useful for furthering knowledge of MERS pathogenesis and for the development of vaccine and treatments against MERS-CoV infection.

    Importance Small and economical animal models are required for the controlled and extensive studies needed for elucidating pathogenesis and development of vaccines and antivirals against MERS. Mice are the most desirable small animal species for this purpose because of availability and existence of a thorough knowledge base, particularly of genetics and immunology. The standard small animals, mice, hamsters and ferrets, all lack the functional MERS-CoV receptor and are not susceptible to infection. So, initial studies were done with nonhuman primates, expensive models of limited availability. A mouse lung infection model was described where a mouse adenovirus was used to transfect lung cells for receptor expression. Nevertheless, all generally agree that a transgenic mouse model expressing the DPP4 receptor is needed for MERS-CoV research. We have developed this transgenic mouse model as indicated in this study. This new and unique transgenic mouse model will be useful for furthering MERS research.

  • Development of live-attenuated arenavirus vaccines based on codon deoptimization [PublishAheadOfPrint]

  • Arenaviruses have a significant impact in public health and pose a credible biodefense threat, but the development of safe and effective arenavirus vaccines has remained elusive and currently no Food and Drug Administration (FDA)-licensed arenavirus vaccines are available. Here, we have explored the use of a codon deoptimization (CD)-based approach as a novel strategy to develop live-attenuated arenavirus vaccines. We recoded the nucleoprotein (NP) of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) with the least frequently used codons in mammalian cells, which caused lower LCMV NP expression levels in transfected cells that correlated with decreased NP activity in cell-based functional assays. We used reverse genetic approaches to rescue a battery of recombinant (r)LCM viruses encoding CD NPs (rLCMV/NPCD) that showed attenuated growth kinetics in vitro. Moreover, experiments using the well-characterized mouse model of LCMV infection revealed that rLCMV/NPCD1 and rLCMV/NPCD2 were highly attenuated in vivo but, upon a single immunization, conferred complete protection against a subsequent lethal challenge with wild-type (WT) recombinant LCMV (rLCMV/WT). Both rLCMV/NPCD1 and rLCMV/NPCD2 were genetically and phenotypically stable during serial passages in FDA vaccine-approved Vero cells. These results provide proof of concept of the safety, efficacy and stability of a CD-based approach for developing live-attenuated vaccine candidates against human pathogenic arenaviruses.

    IMPORTANCE Several arenaviruses cause severe hemorrhagic fever in humans and pose a credible bioterrorism threat. Currently, no FDA-licensed vaccines are available to combat arenavirus infections, whereas anti-arenaviral therapy is limited to the off-label use of ribavirin that is only partially effective and associated with side effects. Here we describe the generation of recombinant versions of the prototypic arenavirus LCMV encoding codon deoptimized viral nucleoproteins (rLCMV/NPCD). We identified rLCMV/NPCD1 and rLCMV/NPCD2 as being highly attenuated in vivo but able to confer protection against a subsequent lethal challenge with wild-type LCMV. These viruses displayed an attenuated phenotype during serial amplification passages in cultured cells. Our findings support the use of this approach for the development of safe, stable, and protective live-attenuated arenavirus vaccines.

  • Requirement for Fc effector mechanisms in the APOBEC3/Rfv3 dependent neutralizing antibody response [PublishAheadOfPrint]

  • Antiretroviral neutralizing antibody (NAb) responses are often evaluated in the absence of Fc-dependent immune effectors. In murine Friend retrovirus infection, Apobec3/Rfv3 promotes a potent polyclonal NAb response. Here, we show that the Apobec3/Rfv3-dependent NAb response correlated with virus-specific IgG2 titers, and the in vivo neutralization potency of Apobec3/Rfv3 resistant antisera was dependent on activating Fc receptors but not complement. The data strengthen retroviral vaccine strategies aimed at eliciting NAbs that activate specific Fc receptors.

  • Post-challenge administration of Brincidofovir protects normal and immune-deficient mice reconstituted with limited numbers of T cells from lethal challenge with IHD-J-Luc vaccinia virus [PublishAheadOfPrint]

  • Protection from lethality by post-challenge administration of brincidofovir (BCV, CMX001) was studied in normal and immune-deficient (nu-/nu-) BALB/c mice infected with vaccinia virus (VACV). Whole body bioluminescence imaging was used to record total fluxes in the nasal cavity, lungs, spleen, and liver and to enumerate pox lesions on tails of mice infected via the intranasal route with 105 pfu of recombinant IHD-J-Luc VACV expressing luciferase. Areas Under the flux Curve (AUC) were calculated for individual mice to assess viral loads. A 3 dose regimen of 20 mg/kg BCV administered every 48 hours starting either on Day 1 or Day 2 post-challenge protected 100% of mice. Initiating BCV treatment earlier was more efficient in reducing viral loads and in protecting from pox lesion development. All BCV-treated mice that survived challenge were also protected from re-challenge with IHD-J-Luc or WRvFire VACV without additional treatment. In immune-deficient mice, BCV protected animals from lethality and reduced viral loads while on drug. Viral recrudescence occurred within 4-9 days and mice succumbed ~10-20 days after treatment termination. Nude mice reconstituted with 105 T cells prior to challenge with 104 pfu of IHD-J-Luc and treated with BCV post-challenge survived the infection, cleared the virus from all organs, and survived re-challenge with 105 pfu of IHD-J-Luc VACV without additional BCV treatment. Together, these data suggest that BCV protects immune competent and partially T-cell reconstituted immune-deficient mice from lethality, reduces viral dissemination in organs, prevents pox lesion development, and permits generation of VACV-specific memory.

    Importance Mass vaccination is the primary element of the public health response to a smallpox outbreak. In addition to vaccination, however, antiviral drugs are required for individuals with uncertain exposure status to smallpox, or for whom vaccination is contraindicated. Whole body bioluminescence imaging was used to study the effect of brincidofovir (BCV) in normal and immune-deficient (nu-/nu-) mice infected with vaccinia virus, a model of smallpox. Post-challenge administration of 20 mg/kg BCV rescued normal and immune-deficient mice partially reconstituted with T cells from lethality and significantly reduced viral loads in organs. All BCV-treated mice that survived infection were protected from re-challenge without additional treatment. In immune-deficient mice, BCV extended survival. The data show that BCV controls viral replication at the site of challenge and reduces viral dissemination to internal organs thus providing a shield for the developing adaptive immunity that clears the host of virus and builds virus-specific immunological memory.

  • Demographics of natural oral infection of mosquitos by Venezuelan equine encephalitis virus [PublishAheadOfPrint]

  • The within-host diversity of virus populations can be drastically limited during between-host transmission, with primary infection of hosts representing a major constraint to diversity maintenance. However, there is an extreme paucity of quantitative data on the demographic changes experienced by virus populations during primary infection. Here, the multiplicity of cellular infection (MOI) and population bottlenecks were quantified during mosquito primary infection by Venezuelan equine encephalitis virus, an arbovirus causing neurological disease in humans and equids.

  • Conserved molecular signatures in gp120 are associated with the genetic bottleneck during SIV, SHIV, and HIV-1 transmission [PublishAheadOfPrint]

  • HIV Transmission typically results from infection by a single transmitted/founder (T/F) variant. Are T/F variants chosen uniformly at random from the donor pool or are they selected based on advantageous traits facilitating transmission? Finding evidence for selection during transmission is of particular interest because it would indicate that phenotypic and/or genetic properties of the viruses might be harnessed as potential vaccine targets or immunotherapies. Here, we systematically evaluate the differences between the Env proteins of SIV/SHIV stock and T/F variants in search of "signature" sites of transmission. We also survey residue preferences in HIV at the SIV/SHIV signature sites. Four sites of gp120 showed significant selection, and an additional two sites showed a similar trend. The six sites therefore clearly differentiate T/F viruses from the majority of circulating variants in the stocks. Selection of SIV/SHIV viruses could be inferred reasonably across both vaccinated and unvaccinated subjects with infections resulting from vaginal, rectal, and intravenous routes of transmission, and regardless of viral dosage. The evidence for selection in SIV and SHIV T/F variants is strong and plentiful; and in HIV, suggestive, though commensurate with the availability of suitable data for analysis. Two of the signature residues are completely conserved across the SIV, SHIV, and HIV variants we examined. Five of the signature residues map to the C1 region of gp120 and one to the signal peptide. Our data raise the possibility that C1, while governing the association between gp120 and gp41, might modulate transmission efficiency, replicative fitness and/or host cell tropism at the level of virus-cell attachment and entry.

    Importance The present study finds significant evidence of selection on gp120 molecules of SIV/SHIV T/F viruses. The data provide ancillary evidence suggesting the same sites are under selection in HIV. Our findings suggest that the signature residues are involved in increasing the transmissibility of infecting viruses and are therefore potential targets for developing a vaccine or other protective measures. A recent study identified the same T/F signature motif but interpreted it as an effect of neutralization resistance. Here, we show that the T/F motif has broader functional significance beyond neutralization sensitivity, because it is present in non-immune subjects. Also, a vaccine regimen popular in animal trials might have possibly increased the transmission of variants with otherwise low transmission fitness. Our observations might explain why many animal vaccine trials have not faithfully predicted outcomes in human vaccine trials and suggest that current practices in vaccine design might need to be re-examined accordingly.

  • Mining the human complexome database identifies RBM14 as an XPO1-Associated Protein involved in HIV-1 Rev function [PublishAheadOfPrint]

  • By recruiting the host protein XPO1 (CRM1), the HIV-1 Rev protein mediates the nuclear export of incompletely spliced viral transcripts. We mined data from the recently described human nuclear complexome to identify a host protein, RBM14, which associates with XPO1 and Rev and is involved in Rev function. Using a Rev-dependent p24 reporter plasmid, we found that RBM14 depletion decreased Rev activity and Rev-mediated enhancement of the cytoplasmic levels of unspliced viral transcripts. RBM14 depletion also reduced p24 expression during viral infection, indicating that RBM14 is limiting for Rev function. RBM14 has previously been shown to localize to nuclear paraspeckles, a structure implicated in retaining unspliced HIV-1 transcripts for either Rev-mediated nuclear export or degradation. We found that depletion of NEAT1 RNA, a long non-coding RNA required for paraspeckle integrity, abolished the ability of over-expressed RBM14 to enhance Rev function, indicating the dependence of RBM14 function on paraspeckle integrity. Our study extends the known host cell interactome of Rev and XPO1 and further substantiates a critical role for paraspeckles in the mechanism of action of Rev. Our study also validates the nuclear complexome as a database from which viral cofactors can be mined.

    Importance This study mined a database of nuclear protein complexes to identify a cellular protein named RBM14 that is associated with XPO1(CRM1), a nuclear protein that binds to the HIV-1 Rev protein and mediates nuclear export of incompletely splice viral RNAs. Functional assays demonstrated that RBM14, a protein found in paraspeckle structures in the nucleus, is involved in HIV-1 Rev function. This study validates the nuclear complexome database as a reference that can be mined to identify viral cofactors.

  • On the stability of parainfluenza virus 5 F proteins [PublishAheadOfPrint]

  • The crystal structure of the F protein (prefusion form) of paramyxovirus parainfluenza virus 5 (PIV5), WR isolate, was determined. We investigated the basis by which point mutations affect fusion in PIV5 isolates W3A and WR, which differ by two residues in the F ectodomain. The P22 stabilizing site acts through a local conformational change and a hydrophobic pocket interaction, whereas the S443 destabilizing site appears sensitive to both conformational effects and amino acid charge/polarity changes.

  • Antigenic Relationships among Porcine Epidemic Diarrhea Virus and Transmissible Gastroenteritis Virus Strains [PublishAheadOfPrint]

  • Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are economically important swine enteropathogenic coronaviruses. These two viruses belong to two distinct species of the Alphacoronavirus genus within Coronaviridae, and induce similar clinical signs and pathological lesions in newborn piglets, but are presumed to be antigenically distinct. In the present study, two-way antigenic cross reactivity examinations between the prototype PEDV CV777 strain, three distinct US PEDV strains (the original highly virulent PC22A, S INDEL Iowa106 and S 197DEL PC177) and two representative US TGEV strains (Miller and Purdue) were conducted by cell culture immunfluorescent (CCIF) and viral neutralization assays. None of the pig TGEV antisera neutralized PEDV and vice versa. One-way cross reactions were observed by CCIF between TGEV Miller hyperimmune pig antisera and all PEDV strains. Enzyme-linked immunosorbent assays, immunoblot using monoclonal antibodies and Escherichia coli-expressed recombinant PEDV and TGEV nucleocapsid (N) proteins and sequence analysis suggested at least one epitope on the N-terminal region of PEDV/TGEV N protein that contributed to this cross-reactivity. Biologically, PEDV CV777 strain induced greater cell fusion in Vero cells than US PEDV strains. Consistent with the reported genetic differences, the results of CCIF and VN assays also revealed higher antigenic variation between PEDV CV777 and other US strains.

    Importance: Evidence of antigenic cross-reactivity between porcine enteric coronaviruses, PEDV and TGEV, in CCIF assays supports that these two species are evolutionarily related, but distinct species defined by VN assays. Identification of PEDV- or TGEV-specific antigenic regions allows the development of more specific immunoassays for each virus. Antigenic and biologic variations between the prototype and current PEDV strains could explain, at least partially, the recurrence of PEDV epidemics. Information on the conserved antigenicity among PEDV strains is important for the development of PEDV vaccines to protect swine from current highly virulent PEDV infections.

  • Emergence and evolution of H10 subtype influenza viruses in poultry in China [PublishAheadOfPrint]

  • The H10N8 human infection cases identified in late 2013 and early 2014 in Jiangxi, China have raised concerns over its origin, prevalence and development in this region. Our long term influenza surveillance in the past 12 years on poultry and migratory birds in southern China showed that H10 influenza viruses have been introduced from migratory to domestic ducks over several winter seasons at sentinel duck farms at Poyang Lake where domestic ducks share their water body with over-wintering migratory birds. H10 viruses were never detected in terrestrial poultry in our survey areas until August 2013 when they were identified at live poultry markets in Jiangxi. Since then, we have isolated 124 H10N8 or H10N6 viruses from chickens at the local markets, revealing an ongoing outbreak. Phylogenetic analysis of H10 and related viruses showed that the chicken H10N8 viruses were generated through multiple reassortments between H10 and N8 viruses from domestic ducks and the enzootic chicken H9N2 viruses. These chicken reassortant viruses were highly similar to the human isolate, indicating the market chickens were the source of the human infection. Recently, the H10 viruses further reassorted, apparently with H5N6 viruses, and generated an H10N6 variant. The emergence and prevalence of H10 viruses in chickens and the occurrence of human infections provide direct evidence of the threat from the current influenza ecosystem in China.

    Importance After the outbreak of avian-origin H7N9 influenza viruses in China, fatal human infections with a novel H10N8 virus were reported. Utilizing data from twelve years of influenza surveillance in southern China, we showed that H10 viruses were regularly introduced by migratory ducks to domestic ducks on Poyang Lake, a major aggregative site of migratory birds in Asia. The H10 viruses were maintained and amplified in domestic ducks, then transmitted to chickens and reassorted with enzootic H9N2 viruses, leading to an outbreak and human infections at live poultry markets. The emergence of the H10N8 virus, following a similar pathway to the recent H7N9 virus, highlights the role of domestic ducks and the current influenza ecosystem in China that facilitates influenza viruses moving from their reservoir hosts through the live poultry system to cause severe consequences for public health.

  • The Aberrant Gene-End Transcription Signal of the Matrix M Gene of Human Parainfluenza Virus Type 3 Down-regulates Fusion F Protein Expression and the F-specific Antibody Response In Vivo [PublishAheadOfPrint]

  • Human parainfluenza virus type 3 (HPIV3), a paramyxovirus, is a major viral cause of severe lower respiratory tract disease in infants and children. The gene end (GE) transcription signal of the HPIV3 matrix (M) protein gene is identical to those of the nucleoprotein and phosphoprotein genes except that it contains an apparent 8-nucleotide insert. This was associated with an increased synthesis of a read-through transcript of the M gene and the downstream fusion (F) protein gene. We hypothesized that this insert may function to down-regulate expression of F protein by interfering with termination/re-initiation at the M-F gene junction, thus promoting the production of M-F read-through mRNA at the expense of monocistronic F mRNA. To test this hypothesis, two similar recombinant HPIV3 viruses were generated from which this insert in the M-GE signal was removed. The M-GE mutants exhibited a reduction in M-F read-through mRNA and an increase in monocistronic F mRNA. This resulted in a substantial increase in F protein synthesis in the infected cells as well as enhanced incorporation of F protein into virions. The efficiency of mutant virus replication was similar to that of wt HPIV3 both in vitro and in vivo. However, the F protein-specific serum antibody response in hamsters was increased for the mutants as compared to wt HPIV3. This study identifies a previously undescribed viral mechanism for attenuating the host adaptive immune response. Repairing the M-GE signal should provide a means to increase the antibody response to a live attenuated HPIV3 vaccine without affecting viral replication and attenuation.

    Importance The HPIV3 M-GE signal was previously shown to contain an apparent 8 nucleotide insert that was associated with increased synthesis of a read-through mRNA of the M gene and the downstream F gene. However, whether this had any significant effect on the synthesis of monocistronic F mRNA or F protein, virus replication, virion morphogenesis, and immunogenicity was unknown. Here, we show that removing this insert shifts F gene transcription from read-through M-F mRNA to monocistronic F mRNA. This resulted in a substantial increase in F protein expressed in the cell and packaged in the virus particle. This did not affect virus replication but increased the F-specific antibody response in hamsters. Thus, in wild-type HPIV3 the aberrant M-GE signal operates a previously undescribed mechanism that reduces the expression of a major neutralization and protective antigen, resulting in reduced immunogenicity. This has implications in the design of live attenuated HPIV3 vaccines; specifically, the antibody response against F can be elevated by "repairing" the M-GE signal to achieve higher F antigen expression, with no effect on attenuation.

  • New insights into the evolutionary rate of hepatitis B virus at different biological scales [PublishAheadOfPrint]

  • The evolution rates of the hepatitis B virus (HBV) estimated using contemporary sequences are 102-104 times faster than those derived from archaeological and genetic evidence. This discrepancy makes the origin of HBV and time scale of its spread, both of which are critical for studying the burden of HBV pathogenicity, remains largely unresolved. To evaluate whether the dual demands (i.e., adaptation within hosts and colonization between hosts) of the viral life cycle affect this conundrum, the HBV quasispecies dynamics within and among hosts from a family consisting of a grandmother, her 5 children, and her 2 granddaughters, all of whom presumably acquired chronic HBV through mother-to-infant transmission, were examined by PCR cloning and next generation sequencing methods. We found that evolutionary rate of HBV between hosts was considerably lower than that of within hosts. Moreover, the between-host substitution rates of the HBV decreased as transmission numbers between individual increased. Both observations were primarily due to changes at nonsynonymous rather than synonymous sites. There were significantly more multiple substitutions than expected under random mutation process and 97% of substitutions were changed from common to rare amino acid residues in the database. Continual switching between colonization and adaptation resulted in the rapid accumulation of mutations at a limited number of positions, which quickly became saturated, whereas substitutions at the remaining regions occurred at a much slower rate. Our study may help to explain the time-dependent HBV substitution rates reported in the literature and provide new insights into the origin of the virus.

    IMPORTANCE It is known that the estimated hepatitis B virus (HBV) substitution rate is time-dependent, but the reason behind is still elusive. We hypothesize that, owing to their small genome size, the transmission between hosts and adaptation within hosts must exhibit high levels of fitness tradeoffs for the viruses. By studying the HBV quasispecies dynamics from a chain of sequentially infected transmissions within a family, we found HBV substitution rate between patients to be negatively correlated with the number of transmissions. Continual switching between hosts resulted in the rapid accumulation of mutations at a limited number of genomic sites, which quickly became saturated in the short-term. Nevertheless, substitutions at the remaining regions occurred at a much slower rate. Therefore, the HBV substitution rate decreased as the divergence time increased.

  • Absence of the Uracil DNA Glycosylase of Murine Gammaherpesvirus 68 Impairs Replication and Delays the Establishment of Latency In Vivo. [PublishAheadOfPrint]

  • Uracil DNA glycosylases (UNG) are highly conserved proteins that preserve DNA fidelity by catalyzing the removal of mutagenic uracils. All herpesviruses encode a viral UNG (vUNG), yet the role of the vUNG in a pathogenic course of gammaherpesvirus infection is not known. First, we demonstrated that the vUNG of murine gammaherpesvirus 68 (MHV68) retains the enzymatic function of host UNG in an in vitro class switch recombination assay. Next, we generated a recombinant MHV68 with a stop codon in ORF46/UNG (UNG) that led to loss of UNG activity in infected cells and a replication defect in primary fibroblasts. Acute replication of MHV68UNG in the lungs of infected mice was reduced a hundred-fold and was accompanied by a substantial delay in the establishment of splenic latency. Latency was largely, yet not fully, restored by an increase in virus inoculum or by altering the route of infection. MHV68 reactivation from latent splenocytes was not altered in the absence of the vUNG. A survey of host UNG activity in cells and tissues targeted by MHV68 indicated that the lung tissue has a lower level of UNG enzymatic activity than the spleen. Taken together, these results indicate that the vUNG plays a critical role in the replication of MHV68 in tissues with limited host UNG activity and this vUNG-dependent expansion, in turn, influences the kinetics of latency establishment in distal reservoirs.

    IMPORTANCE Herpesviruses establish chronic life-long infections using a strategy of replicative expansion, dissemination to latent reservoirs, and subsequent reactivation for transmission and spread. We examined the role of the viral uracil DNA glycosylase, a protein conserved among all herpesviruses, in replication and latency of murine gammaherpesvirus 68. We report that the viral UNG of this murine pathogen retains catalytic activity and influences replication in culture. The viral UNG was impaired for productive replication in the lung. This defect in expansion at the initial site of acute replication was associated with a substantial delay of latency establishment in the spleen. Levels of host UNG were substantially lower in the lung compared to the spleen, suggesting that herpesviruses encode a viral UNG to compensate for reduced host enzyme levels in some cell types and tissues. These data suggest that intervention at the site of initial replicative expansion can delay the establishment of latency, a hallmark of chronic herpesvirus infection.

  • Broadly-Neutralizing Anti-Influenza Virus Antibodies: Enhancement of Neutralizing Potency in Polyclonal Mixtures and IgA Backbones [PublishAheadOfPrint]

  • Current-generation influenza virus vaccines rely upon the accurate prediction of circulating virus strains months in advance of the actual influenza season in order to allow time for vaccine manufacture. Unfortunately, mismatches occur frequently and even when perfect matches are achieved, suboptimal vaccine efficacy leaves several high-risk populations vulnerable to infection. However, the recent discovery of broadly-neutralizing antibodies that target the hemagglutinin stalk domain has renewed hope that the development of "universal" influenza virus vaccines may be within reach. Here, we examine the functions of influenza A virus hemagglutinin stalk-binding antibodies in an endogenous setting - as polyclonal preparations isolated from human sera. Relative to monoclonal antibodies that bind to the HA head domain, the neutralization potency of monoclonal stalk-binding antibodies was vastly inferior in vitro, but was enhanced by several orders of magnitude in the polyclonal context. Furthermore, we demonstrated a surprising enhancement in IgA-mediated HA stalk neutralization relative to that achieved by antibodies of IgG isotypes. Mechanistically, this could be explained in two ways. Identical variable regions consistently neutralized virus more potently when in an IgA backbone compared to an IgG backbone. Additionally, HA-specific memory B cells isolated from human peripheral blood were more likely to be stalk-specific when secreting antibodies of IgA isotypes compared to those secreting IgG. Taken together, our data provide strong evidence that HA stalk-binding antibodies perform optimally when in a polyclonal context, and that the targeted elicitation of HA stalk-specific IgA should be an important consideration during "universal" influenza virus vaccine design.

    IMPORTANCE Influenza viruses remain one of the most worrisome global public health threats due to their capacity to cause pandemics. While seasonal vaccines fail to protect against the emergence of pandemic strains, a new class of broadly-neutralizing antibodies has been recently discovered and may be the key to developing a "universal" influenza virus vaccine. While much has been learned about the biology of these antibodies, most studies have focused only on monoclonal antibodies of IgG subtypes. However, the study of monoclonal antibodies often fails to capture the complexity of antibody functions that occur during natural polyclonal responses. Here, we provide the first detailed analyses of the biological activity of these antibodies in polyclonal contexts, comparing both IgG and IgA isotypes isolated from human donors. The striking differences observed in the functional properties of broadly-neutralizing antibodies in polyclonal contexts will be essential for guiding design of "universal" influenza virus vaccines and therapeutics.

  • Hepatitis C Virus Mediated Enhancement of miR-373 Impairs JAK/STAT Signaling Pathway [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) is a serious global health problem which establishes chronic infection in a significant number of infected humans worldwide. IFN and IFN-stimulated genes (ISGs) are amplified during HCV infection but fail to eliminate virus from the liver in a large number of infected patients and the mechanism is not fully understood. MicroRNAs (miRNAs) have been implicated in the control of many biological processes including IFN signaling. To gain more insights on the role of cellular miRNAs in possible counter measures of HCV for suppression of host antiviral response, a miRNA array was performed using primary human hepatocytes (PHH) infected with in vitro cell culture grown HCV. A group of miRNAs were modulated in HCV infected PHH. We focused on miR-373 as this miRNA was significantly upregulated in HCV infected PHH. Here, we analyzed the function of miR-373 in context to HCV infection. HCV infection upregulates miR-373 expression in hepatocytes and HCV infected liver biopsy specimens. Further, we discovered that miR-373 directly targets Janus kinase1 (JAK1) and IFN regulating factor 9 (IRF9), important factors in the IFN signaling pathway. Upregulation of miR-373 by HCV also inhibited STAT1 phosphorylation, which is involved in ISGF3 complex formation and ISGs expression. Knockdown of miR-373 in hepatocytes enhanced JAK1 and IRF9 expression, and reduced HCV RNA replication. Taken together, our results demonstrated that miR-373 is upregulated during HCV infection and negatively regulated type I IFN signaling pathway by suppressing JAK1 and IRF9. Our results offer a potential therapeutic approach for antiviral intervention.

    Importance Chronic HCV infection is one of the major causes of end stage liver disease worldwide. Although recent introduction of DAA therapy is extremely encouraging, some infected individuals do not respond to this therapy. Further, these drugs target HCV nonstructural proteins and with selective pressure, virus may develop resistant strain. Therefore, understanding the impairment of IFN signals will help in designing additional therapeutic modalities. In this study, we provide evidence of HCV mediated upregulation of miR-373 and show that miR-373 impairs IFN signaling by targeting JAK1/IRF9 molecules. Knockdown of miR-373 inhibited HCV replication by upregulating interferon stimulating genes expression. Together, these results provided new mechanistic insights in understanding the role of miR-373 in HCV infection, and suggest a new potential target against HCV infection.

  • Constitutive But Not Inducible Attenuation Of Transforming Growth Factor-{beta} Signaling Increases Natural Killer Cell Responses Without Directly Affecting Dendritic Cells Early After Chronic Viral Infection [PublishAheadOfPrint]

  • Rapid innate responses to viral encounters are crucial to shaping the outcome of infection, from viral clearance to persistence. Transforming Growth Factor-Beta (TGFbbeta;) is a potent immune suppressor that is up-regulated early upon viral infection and maintained during chronic infections in both mice and humans. However, the role of TGFbbeta; signaling in regulating individual cell types in vivo is still unclear. Using two different infections with persistent viruses, Murine Cytomegalovirus (MCMV) and Lymphocytic Choriomeningitis Virus (LCMV Cl 13), in their natural rodent host, we observed that TGFbbeta; signaling on Dendritic cells (DCs) did not dampen DC maturation or cytokine production in the early stages of either chronic viral infection in vivo. In contrast, TGFbbeta; signaling prior to (but not during) chronic viral infection directly restricted Natural Killer (NK) cell number and effector function. This restriction likely compromised both early control and host survival upon MCMV infection but not long-term control of LCMV infection. These data highlight the context and timing of TGFbbeta; signaling on different innate cells that contribute to the early host response, which ultimately influences the outcome of chronic viral infection in vivo.

    Significance In vivo host responses to pathogens are complex processes involving the cooperation of many different immune cells migrating to specific tissues over time, events that cannot be replicated in vitro. Chronic viruses are able to subvert this immune response and represent a human health burden. Here we use two well-characterized viruses that are able to persist in their natural mouse host to dissect the role of the suppressive molecule TGFbbeta; in dampening host responses to infection in vivo. This manuscript increases the understanding of long-studied TGFbbeta; signaling by examining its direct effect on different immune cells that are activated very early after in vivo viral infection and may aid development of new anti-viral therapeutic strategies.

  • Structural Basis for Host Membrane Remodeling Induced by Protein 2B of Hepatitis A Virus [PublishAheadOfPrint]

  • The complexity of viral RNA synthesis and the numerous participating factors require a mechanism to topologically coordinate and concentrate these multiple viral and cellular components, ensuring a concerted function. Similar to all other positive-strand RNA viruses, picornaviruses induce rearrangements of host intracellular membranes to create structures, which act as functional scaffolds for genome replication. The membrane-targeting proteins 2B, 2C, their precursor 2BC, and protein 3A appear primarily involved in membrane remodeling. Little is known about the structure of these proteins and the mechanisms by which they induce massive membrane remodeling. Here we report the crystal structure of the soluble region of hepatitis A virus (HAV) protein 2B, consisting of two domains: a C-terminal helical bundle, preceded by a N-terminal curved five-stranded anti-parallel bbeta;-sheet that displays striking structural similarity to the bbeta;-barrel domain of enteroviral 2A proteins. Moreover, the helicoidal arrangement of the protein molecules in the crystal provides a model for 2B-induced host membrane remodeling during HAV infection.

    IMPORTANCE No structural information is currently available for the 2B protein of any picornavirus, despite being involved in a critical process in viral factories formation: the rearrangement of host intracellular membranes. Here we present the structure of the soluble domain of the 2B protein of HepatititsA virus (HAV). Its arrangement, both in crystals and in solution under physiological conditions can help to understand its function and sheds some light on the membrane rearrangement process, a putative target of future antiviral drugs.

    Moreover, this first structure of a picornaviral2B protein also unveils a closer evolutionary relationship between the hepatovirus and enterovirus genres within the Picornaviridae family.

  • High preexisting serological antibody levels correlate with diversification of the influenza vaccine response [PublishAheadOfPrint]

  • Reactivation of memory B cells allows for a rapid and robust immune response upon challenge with the same antigen. Variant influenza strains generated through antigenic shift or drift are encountered multiple times over the lifetime of an individual. One might predict, then, that upon vaccination with the trivalent influenza vaccine across multiple years, the antibody response would become more and more dominant towards strains consistently present in the vaccine at the expense of more divergent strains. However, when we analyzed the vaccine-induced plasmablast, memory and serological response to the trivalent influenza vaccine between 2006 and 2013, we found that the B cell response was most robust against more divergent strains. Overall the antibody response was highest when one or more strains contained in the vaccine varied from year to year. This suggests that in the broader immunological context of viral antigen exposure, the B cell response to variant influenza strains is not dictated by the composition of the memory B cell precursor pool. The outcome is instead a diversified B cell response.

    Importance Vaccine strategies are being designed to boost broadly-reactive B cells present in the memory repertoire to provide universal protection to influenza. It is important to understand how past exposure to influenza strains affects the response to subsequent immunizations. The viral epitopes targeted by B cells responding to the vaccine may be a direct reflection of the B cell memory specificities abundant in the preexisting immune repertoire or other factors may influence the vaccine response. Herein we demonstrate that high preexisting serological antibody levels to a given influenza strain correlate with low production of antibody-secreting cells and memory B cells recognizing that strain upon revaccination. In contrast, introduction of antigenically novel strains generates a robust B cell response. Thus, both the preexisting memory B cell repertoire and serological antibody levels must be taken into consideration when predicting the quality of the B cell response to new prime-boost vaccine strategies.

  • Overlapping Local and Long Range RNA-RNA Interactions Modulate Dengue Virus Genome Cyclization and Replication [PublishAheadOfPrint]

  • The dengue virus genome is a dynamic molecule that adopts different conformations in the infected cell. Here, using RNA folding predictions, chemical probing analysis, RNA binding assays and functional studies, we identified new cis-acting elements present in the capsid coding sequence that facilitate cyclization of the viral RNA by hybridization with a sequence involved in a local dumbbell structure at the viral 3rrsquo; UTR. The identified interaction differentially enhances viral replication in mosquito and mammalian cells.

  • Contribution of MxB Oligomerization to HIV-1 Capsid Binding and Restriction [PublishAheadOfPrint]

  • The IFN-aalpha;-inducible restriction factor MxB blocks HIV-1 infection after reverse transcription but prior to integration. MxB binds to the HIV-1 core, which is composed of capsid protein, and this interaction leads to inhibition of the uncoating process of HIV-1. Previous studies suggested that HIV-1 restriction by MxB requires binding to capsid. This work tests the hypothesis that MxB oligomerization is important for the ability of MxB to bind to the HIV-1 core. For this purpose, we modeled the structure of MxB using the published tertiary structure of MxA. The modeled structure of MxB guided our mutagenic studies, and led to the discovery of several MxB variants that lose the capacity to oligomerize. In agreement with our hypothesis, MxB variants that lost oligomerization also lost the ability to bind to the HIV-1 core. MxB variants deficient for oligomerization were not able to block HIV-1 infection. Overall, our work showed that oligomerization is required for the ability of MxB to bind to the HIV-1 core and block HIV-1 infection.

    IMPORTANCE MxB is a novel restriction factor that blocks infection of HIV-1. MxB is inducible by IFN-aalpha;, particularly in T cells. The current work studies the oligomerization determinants of MxB, and carefully explore the contribution of oligomerization to capsid binding and restriction. This work takes advantage of the current structure of MxA, and models the structure of MxB, which is used to guide structure-function studies. This work leads to the conclusion that MxB oligomerization is important for HIV-1 capsid binding and restriction.

  • Stability of structured Kaposi sarcoma-associated herpesvirus ORF57 protein is regulated by protein phosphorylation and homodimerization [PublishAheadOfPrint]

  • Kaposi sarcoma-associated herpesvirus (KSHV) ORF57 plays an essential role in KSHV lytic infection by promoting viral gene expression at the posttranscriptional level. Using bioinformatic and biochemical approaches we identified that ORF57 contains two structurally and functionally distinct domains, a disordered non-structural N-terminal (aa 1-152) domain and a structured aalpha;-helix-rich C-terminal (aa 153-455) domain. The N-terminal domain mediates ORF57 interaction with several RNA-protein complexes essential for ORF57 to function. The N-terminal phosphorylation by cellular casein kinase II at S21, T32, S43, S95 and S97 residues in proximity of the caspase 7 cleavage site, 30-DETD-33, inhibits caspase 7 digestion of ORF57. The structured C-terminal domain mediates homodimerization of ORF57 and the critical region for this function was mapped carefully to aalpha;-helixes 7 to 9. Introduction of point mutations into the aalpha;-helix 7 at ORF57 aa 280-299, a region being highly conserved among ORF57 homologues from other herpesviruses, inhibited ORF57 homodimerization and led to proteasome-mediated degradation of ORF57 protein. Thus, homodimerization of ORF57 via its C-terminus is to prevent ORF57 from degrading and to allow two structure-free N-termini of the dimerized ORF57 to work coordinately together for host factor interactions, consequently leading to productive KSHV lytic infection and pathogenesis.

    IMPORTANCE KSHV is a human oncogenic virus linked to the development of several malignancies. KSHV-mediated oncogenesis requires both latent and lytic infection. KSHV ORF57 protein is essential for KSHV lytic replication by regulating the expression of viral lytic genes at the posttranscriptional level. This report provides the evidence that structural conformation of ORF57 protein plays a critical role in regulation of ORF57 stability. Phosphorylation by CKII on the identified serine/threonine residues at the N-terminal unstructured domain of ORF57 prevents its digestion by caspase 7. The C-terminal domain of ORF57, which is rich in aalpha;-helixes, contributes to homodimerization of ORF57 to prevent proteasome-mediated protein degradation. Elucidation of ORF57 structure not only enables us to better understand ORF57 stability and functions, but also provides an important tool for us to modulate ORF57's activity in aims to inhibit KSHV lytic replication.

  • Vpu is the main determinant for tetraspanin downregulation in HIV-1 infected cells [PublishAheadOfPrint]

  • Tetraspanins constitute a family of cellular proteins that organize various membrane-based processes. Several members of this family, including CD81, are actively recruited by HIV-1 Gag to viral assembly and release sites. Despite their enrichment at viral exit sites, the overall levels of tetraspanins are decreased in HIV-1-infected cells. Here, we identify Vpu as the main viral determinant for tetraspanin downregulation. We also show that reduction of CD81 levels by Vpu is not a by-product of CD4 or BST-2/Tetherin elimination from the surface of infected cells and likely occurs through an interaction between Vpu and CD81. Finally, we document that Vpu-mediated downregulation of CD81 from the surface of infected T cells can contribute to preserving the infectiousness of viral particles, thus revealing a novel Vpu function that promotes virus propagation by modulating the host cell environment.

    Importance The HIV-1 accessory protein Vpu has previously been shown to downregulate various host-cell factors, thus helping the virus to overcome restriction barriers, evade immune attack, and maintain the infectivity of viral particles. Our study identifies tetraspanins as an additional group of host factors whose expression at the surface of infected cells is lowered by Vpu. While the downregulation of these integral membrane proteins, including CD81 and CD82, likely affects more than one function of HIV-1-infected cells, we document that Vpu-mediated lowering of CD81 levels in viral particles can be critical to maintaining their infectiousness.

  • Identification and characterization of the binding site of the respiratory syncytial virus phosphoprotein to RNA-free nucleoprotein [PublishAheadOfPrint]

  • The RNA genome of respiratory syncytial virus (RSV) is constitutively encapsidated by the viral nucleoprotein N thus forming a helical nucleocapsid. Polymerization of N along the genomic and anti-genomic RNAs is concomitant to replication and requires the preservation of an unassembled monomeric nucleoprotein pool. To this end, and by analogy with Paramyxoviridae and Rhabdoviridae, it is expected that the viral phosphoprotein P acts as a chaperone protein, forming a soluble complex with the RNA-free form of N (N0-P complex). Here, we have engineered a mutant form of N which is monomeric, unable to bind RNA, still interacts with P, and could thus mimic the N0 monomer. We used this N mutant designated Nmono as a substitute for N0 in order to characterize P regions involved in the N0-P complex formation. Using a series of P fragments, we determined by GST pulldown assays that the N- and C-termini of P are able to interact with Nmono. We analyzed the functional role of amino-terminal residues of P by site-directed mutagenesis, using an RSV polymerase activity assay based on a human RSV minireplicon, and found that several residues were critical for viral RNA synthesis. Using GST-pulldown and surface plasmon resonance assays, we showed that these critical residues are involved in the interaction between P[1-40] peptide and Nmono in vitro. Finally, we showed that overexpression of the peptide P[1-29] can inhibit the polymerase activity in the context of the RSV minireplicon, thus demonstrating that targeting the N0-P interaction could constitute a potential antiviral strategy.

    Importance. Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine or efficient antiviral treatment are available against RSV, it is essential to better understand how the viral machinery functions in order to develop new antiviral strategies. RSV phosphoprotein P, the main RNA polymerase cofactor, is believed to function as a chaperon protein, maintaining N as a non-assembled, RNA free protein (N0) competent for RNA encapsidation. In this paper, we provide the first evidence, to our knowledge, that the N-terminus of P contains a domain that binds specifically to this RNA-free form of N. We further show that overexpression of a small peptide spanning this region of P can inhibit viral RNA synthesis. These findings extend our understanding of the function of RSV RNA polymerase and point to a new target for the development of drugs against this virus.

  • The interface between HBV capsid proteins affects self-assembly, pgRNA packaging, and reverse transcription [PublishAheadOfPrint]

  • Hepatitis B virus (HBV) capsid proteins (Cp) assemble around the pregenomic RNA (pgRNA) and viral reverse transcriptase (P). pgRNA is then reverse transcribed to double stranded DNA (dsDNA) within the capsid. The Cp assembly domain, which forms the shell of capsid, regulates assembly kinetics and capsid stability. The Cp, via its nucleic acid binding C-terminal domain, also affects nucleic acid organization. We hypothesize that the structure of the capsid may also have a direct affect on nucleic acid processing. Using structure-guided design, we made a series of mutations at the interface between Cp subunits that change capsid assembly kinetics and thermodynamics in a predictable manner. Assembly in cell culture mirrored in vitro activity. However, all of these mutations led to defects in pgRNA packaging. Synthesis of first-strand DNA was roughly proportional to the amount of RNA packaged. However, synthesis of second-strand DNA, which requires two template switches, was not supported by any of the substitutions. These data demonstrate that the HBV capsid is far more than an inert container as mutations in the assembly domain, distant from packaged nucleic acid, affect reverse transcription. We suggest that capsid molecular motion plays a role in regulating genome replication.

    Importance The Hepatitis B virus (HBV) capsid plays a central role in the virus life cycle and has been studied as a potential antiviral target. The capsid protein (Cp) packages the viral pregenomic RNA (pgRNA) and polymerase to form the HBV core. The role of the capsid in subsequent nucleic acid metabolism is unknown. Here, guided by the structure of capsid with bound antiviral molecules, we designed Cp mutants that enhanced or attenuated assembly of purified Cp in vitro. In cell culture, assembly of mutants was consistent with their in vitro biophysical properties. However, all of these mutations inhibited HBV replication. Specifically, changing the biophysical chemistry of Cp caused defects in pgRNA packaging and synthesis of the second strand of DNA. These results suggest that the HBV Cp assembly domain potentially regulates reverse transcription, extending the activities of the capsid protein beyond its presumed role as an inert compartment.

  • Characterization of dengue virus NS4A and NS4B protein interaction [PublishAheadOfPrint]

  • Flavivirus replication is mediated by a membrane-associated replication complex where viral membrane proteins NS2A, NS2B, NS4A, and NS4B serve as the scaffold for the replication complex formation. Here we used dengue virus serotype-2 (DENV-2) as a model to characterize viral NS4A/NS4B interaction. NS4A interacts with NS4B in virus-infected cells and in cells transiently expressing NS4A and NS4B in the absence of other viral proteins. Recombinant NS4A and NS4B proteins directly bind to each other with an estimated Kd of 50 nM. Amino acids 40-76 (spanning the first transmembrane domain [amino acids 50-73]) of NS4A and amino acids 84-146 (also spanning the first transmembrane domain [amino acids 101-129]) of NS4B are the determinants for NS4A/NS4B interaction. NMR analysis suggests that NS4A residues17-80 form two amphipathic helices (helix aalpha;1 [residues 17-32] and helix aalpha;2 [residues 40-47]) that associate with the cytosolic side of endoplasmic reticulum (ER) membrane and helix aalpha;3 (residues 52-75) that transverses ER membrane. In addition, NMR analysis identified NS4A residues that may participate in NS4A/NS4B interaction. Amino acid-substitution of these NS4A residues exhibited distinct effects on viral replication. Three of the four NS4A mutations (L48A, T54A, and L60A) that affected NS4A/NS4B interaction abolished or severely reduced viral replication; in contrast, two NS4A mutations (F71A and G75A) that did not affect NS4A/NS4B interaction had marginal effects on viral replication, demonstrating the biological relevance of NS4A/NS4B interaction to DENV-2 replication. Taken together, the study has provided experimental evidence to argue that blocking the NS4A/NS4B interaction could be a potential antiviral approach.

    SIGNIFICANCE Flavivirus NS4A and NS4B proteins are essential components of the ER membrane-associated replication complex. The current study systematically characterizes the interaction between flavivirus NS4A and NS4B. Using DENV-2 as a model, we show that NS4A interacts with NS4B in virus-infected cell, in cells that transiently expressing NS4A and NS4B proteins, or in vitro with recombinant NS4A and NS4B proteins. We mapped the minimal regions required for NS4A/NS4B interaction to be amino acids 40-76 of NS4A and amino acids 84-146 of NS4B. NMR analysis revealed the secondary structure of amino acids 17-80 of NS4A and the NS4A amino acids that may participate in the NS4A/NS4B interaction. Functional analysis showed a correlation between viral replication and NS4A/NS4B interaction, demonstrating the biological importance of NS4A/NS4B interaction. The study has advanced our knowledge of the molecular function of flavivirus NS4A and NS4B proteins. The results also suggest that inhibitors of NS4A/NS4B interaction could be pursued for flavivirus antiviral development.

  • Conserved Motifs in a Tombusvirus Polymerase Modulate Genome Replication, Subgenomic Transcription and Amplification of DI RNAs [PublishAheadOfPrint]

  • The replication of plus-strand RNA virus genomes is mediated by virally-encoded RNA-dependent RNA polymerases (RdRps). We have investigated the role of the C-proximal region in the RdRp of Tomato bushy stunt virus (TBSV) in mediating viral RNA synthesis. TBSV is the prototype species in the genus Tombusvirus, family Tombusviridae, and its RdRp is responsible for replicating the viral genome, transcribing two subgenomic mRNAs, and supporting replication of defective interfering RNAs. Comparative sequence analysis of the RdRps of tombusvirids identified three highly conserved motifs in their C-proximal regions and these sequences were subsequently targeted for mutational analysis in TBSV. The results revealed that these motifs are important for (i) synthesizing viral genomic RNA and subgenomic mRNAs, (ii) facilitating plus- and/or minus-strand synthesis, and (iii) modulating trans-replication of a defective interfering RNA. These motifs were also found to be conserved in other plant viruses, as well as in a fungal and insect virus. The collective findings are discussed in relation to viral RNA synthesis and taxonomy.

    IMPORTANCE Little is currently known about the structure and function of the viral polymerases that replicate the genomes of RNA plant viruses. Tombusviruses, the prototype of the tombusvirids, have been model plus-stand RNA plant viruses for understanding many of the steps in the infectious process; however, their polymerases remain poorly characterized. To help address this issue, the function of the C-terminal region of the polymerase of a tombusvirus was investigated. Three conserved motifs were identified and targeted for mutational analysis. The results revealed that these polymerase motifs are important for determining what type of viral RNA is produced, facilitating different steps in viral RNA production, and amplifying sub-genomic RNA replicons. Accordingly, the C-terminal region of the tombusvirus polymerase is needed for a variety of fundamental activities. Furthermore, as these motifs are also present in distantly related viruses, the significance of these results extend beyond tombusvirids.

  • Human Immunodeficiency Virus Type 1 Employs the Cellular Dynein Light Chain 1 Protein for Reverse Transcription Through Interaction with its Integrase Protein [PublishAheadOfPrint]

  • In this study, we examined the requirement for host dynein adapter proteins such as dynein light chain 1 (DYNLL1), dynein light chain Tctex-type 1 (DYNLT1), and p150Glued, in early steps of human immunodeficiency virus type 1 (HIV-1) replication. We found that the knockdown (KD) for DYNLL1, but not DYNLT1 or p150Glued, resulted in significantly lower levels of HIV-1 reverse transcription in cells. Following an attempt to determine how DYNLL1 could impact HIV-1 reverse transcription, we detected the DYNLL1 interaction with HIV-1 integrase (IN), but not with capsid (CA), matrix (MA), or reverse transcriptase (RT) protein. Furthermore, by mutational analysis of putative DYNLL1 interaction motifs in IN, we identified the motifs "52GQVD" and "250VIQD" in IN as essential for DYNLL1 interaction. The DYNLL1 interaction-defective IN mutant HIV-1 (HIV-1INQ53A/Q252A) exhibited impaired reverse transcription. Through further investigations, we have also detected the relatively lower amounts of particulate CA in DYNLL1-KD cells or in infections with HIV-1INQ53A/Q252A mutant virus. Over all, our study demonstrates the novel interaction between HIV-1 IN and cellular DYNLL1 proteins and suggests the requirement of this viral-cellular interaction for proper uncoating and efficient reverse transcription of HIV-1.

    Importance: Host cellular DYNLL1, DYNLT1, and p150Glued proteins have been implicated in the replication of several viruses. However, their roles in HIV-1 replication have not been investigated. For the first time, we demonstrated that during viral infection, HIV-1 IN interacts with DYNLL1 and their interaction is found to have a role in proper uncoating and efficient reverse transcription of HIV-1. Thus, IN and DYNLL1 interaction may be a potential target for future anti-HIV therapy. Moreover, while our study has evaluated the involvement of IN in HIV-1 uncoating and reverse transcription, it also predicts a possible mechanism by which IN contributes to these early viral replication steps.

  • Synthetic TLR4 and TLR7 ligands as influenza virus vaccine adjuvants induce rapid, sustained and broadly protective responses [PublishAheadOfPrint]

  • Current vaccines against influenza virus infection rely on the induction of neutralizing antibodies targeting the globular head of the viral hemagglutinin (HA). Protection against seasonal antigenic drift or sporadic pandemic outbreaks requires further vaccine development to induce cross protective humoral responses, potentially to the more conserved HA stalk region. Here, we present a novel viral vaccine adjuvant comprised of two synthetic ligands for Toll-like receptor (TLR) 4 and TLR7. 1Z105 is a substituted pyrimido[5,4-b]indole specific for the TLR4/MD2 complex, and 1V270 is a phospholipid conjugated TLR7 agonist. Separately, 1Z105 induces rapid Th2 associated IgG1 responses, and 1V270 potently generates Th1 cellular immunity. 1Z105 and 1V270 in combination with recombinant HA from the A/Puerto Rico/8/1934 strain (rPR/8 HA) effectively induces rapid and sustained humoral immunity that is protective against lethal challenge with a homologous virus. More importantly, immunization with the combined adjuvant and rPR/8 HA, a commercially available split vaccine, or chimeric rHA antigens significantly improves protection against both heterologous or heterosubtypic challenge viruses. Heterosubtypic protection is associated with broadly reactive antibodies to HA stalk epitopes. Histological examination and cytokine profiling reveal that IM administration of 1Z105 and 1V270 is less reactogenic than a squalene-based adjuvant, AddaVax. In summary, the combination of 1Z105 and 1V270 with a recombinant HA induces rapid, long lasting and balanced Th1 and Th2-type immunity, demonstrates efficacy in a variety of murine influenza vaccine models assaying homologous, heterologous and heterosubtypic challenge viruses, and has an excellent safety profile.

    Importance Novel adjuvants are needed to enhance immunogenicity and increase protective breadth of influenza virus vaccines to reduce seasonal disease burden and ensure pandemic preparedness. We show here that the combination of synthetic Toll-like receptor (TLR) 4 and TLR7 ligands is a potent adjuvant for recombinant influenza virus hemagglutinin, inducing rapid and sustained immunity that is protective against influenza viruses in homologous, heterologous and heterosubtypic challenge models. Combining TLR4 and TLR7 ligands balances Th1- and Th2-type immune responses for long-lived cellular and neutralizing humoral immunity against the viral hemagglutinin. The combined adjuvant has an attractive safety profile and the potential to augment seasonal vaccine breadth, contribute to a broadly neutralizing universal vaccine formulation, and improve response time in an emerging pandemic.

  • Impact of the adenoviral E4 Orf3 protein on the activity and post-translational modification of p53 [PublishAheadOfPrint]

  • Our previous studies have established that the p53 populations that accumulate in normal human cells exposed to etoposide or infected by an E1B 55 kDa protein-null mutant of human adenovirus type 5 carry a large number of post-translational modifications at numerous residues (DeHart et al. (2014) Mol. Cell. Proteomics 13:1-17). In the absence of this E1B protein, the p53 transcriptional program is not induced, and it has been reported that the viral E4 Orf3 protein inactivates p53 (Soria et al. (2010) Nature 466:1076-1081). As the latter protein disrupts nuclear Pml bodies, sites at which p53 is modified, we used mass spectrometry to catalogue the post-transcriptional modifications of the p53 population that accumulates when neither the E1B 55 kDa nor the E4 Orf3 protein is made in infected cells. Eighty-five residues carrying 163 modifications were identified. The overall patterns of post-translational modification of this population and p53 present in cells infected by an E1B 55 kDa-null mutant were similar. The efficiencies with which these two forms of p53 bound to a consensus DNA recognition sequence could not be distinguished, and were lower than that of transcriptionally active p53. The absence of the E4 Orf3 protein increased expression of several p53-responsive genes when the E1B protein was also absent from infected cells. However, expression of these genes did not attain the levels observed when p53 was activated in response to etoposide treatment, and remained lower than those measured in mock-infected cells.

    Importance. The tumor suppressor p53, a master regulator of cellular responses to stress, is inactivated and destroyed in cells infected by species C human adenoviruses, such as type 5. It is targeted for proteasomal degradation by the action of a virus-specific E3 ubiquitin ligase that contains the viral E1B 55 kDa and E4 Orf6 proteins, while the E4 Orf3 protein has been reported to block its ability to stimulate expression of p53-dependent genes. The comparisons reported here of the post-translational modifications and activities of p53 populations that accumulate in infected normal human cells in the absence of both mechanisms of inactivation or of only the E3 ligase revealed little impact of the E4 Orf3 protein. These observations indicate that E4 Orf3-dependent disruption of Pml bodies does not have a major effect on the pattern of p53 posttranslational modifications in adenovirus-infected cells. Furthermore, they suggest that suggest that one or more additional viral proteins contribute to blocking p53 activation and the consequences deleterious for viral reproduction, such as apoptosis or cell cycle arrest.

  • Parainfluenza virus 5 expressing the G protein of rabies virus protected mice after rabies virus infection [PublishAheadOfPrint]

  • Rabies remains a major public health threat around the world. Once symptoms appear, there is no effective treatment to prevent death. In this work, we tested a recombinant parainfluenza virus 5 (PIV5) expressing the glycoprotein (G) of rabies (PIV5-G) as a therapy for rabies virus infection: we have found that PIV5-G protected mice as late as 6 days after rabies virus infection. PIV5-G is a promising vaccine for prevention and treatment of rabies virus infection.

  • Analysis of the functional interchange between the IE1 and pp71 proteins of HCMV and ICP0 of HSV-1 [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) immediate-early protein IE1 and the tegument protein pp71 are required for efficient infection. These proteins have some functional similarities with herpes simplex virus type 1 (HSV-1) immediate-early protein ICP0, which stimulates lytic HSV-1 infection and de-represses quiescent HSV-1genomes. All three proteins counteract antiviral restriction mediated by one or more components PML nuclear bodies, and IE1 and pp71, acting together, almost completely complement ICP0-null mutant HSV-1. Here we investigated whether ICP0 might substitute for IE1 or pp71 during HCMV infection. Using human fibroblasts that express ICP0, IE1 or pp71 in an inducible manner, we found that ICP0 stimulated replication of both wt and pp71 mutant HCMV, while IE1 increased wt HCMV plaque formation and completely complemented the IE1 mutant. Although ICP0 stimulated IE2 expression from IE1 mutant HCMV and increased the number of IE2 positive cells, it could not compensate for IE1 in full lytic replication. These results are consistent with previous evidence that both IE1 and IE2 are required for efficient HCMV gene expression, but they also imply that IE2 functionality is influenced specifically by IE1, either directly or indirectly, and that IE1 may include sequences that have HCMV-specific functions. We discovered a mutant form of IE1 (YL2) that fails to stimulate HCMV infection while retaining 30-80% of the activity of the wt protein in complementing ICP0 null mutant HSV-1. It is intriguing that the YL2 mutation is situated in the region of IE1 that is shared with IE2, and which is highly conserved amongst primate cytomegaloviruses.

    IMPORTANCE Herpesvirus gene expression can be repressed by cellular restriction factors, of which one group is associated with structures known as ND10 or PML Nuclear Bodies (PML NBs). Regulatory proteins of several herpesviruses interfere with PML NB mediated repression, and in some cases their activities are transferrable between different viruses. For example, the requirement for ICP0 during herpes simplex virus type 1 (HSV-1) infection can be largely replaced by ICP0-related proteins expressed by other alphaherpes viruses, and even by a combination of the unrelated IE1 and pp71 proteins of human cytomegalovirus (HCMV). Here we report that ICP0 stimulates gene expression and replication of wt HCMV, but cannot replace the need for IE1 during infection by IE1 defective HCMV mutants. Therefore IE1 includes HCMV specific functions that cannot be replaced by ICP0.

  • A reverse genetic system for severe fever with thrombocytopenia syndrome virus. [PublishAheadOfPrint]

  • Severe fever with thrombocytopenia syndrome virus (SFTS virus) is an emerging tick-borne pathogen first reported in China in 2009. Phylogenetic analysis of the viral genome showed it represents a new lineage within the Phlebovirus genus, distinct from the existing sandfly fever and Uukuniemi virus groups, in the family Bunyaviridae. SFTS disease is characterized by gastrointestinal symptoms, chills, joint pain, myalgia, thrombocytopenia, leukocytopenia and some haemorrhagic manifestations with a case fatality rate of about 2-15%. Here we report the development of reverse genetics systems to study STFSV replication and pathogenesis. We developed and optimized functional T7 polymerase-based M- and S-segment minigenome assays, which revealed errors in the published terminal sequences of the S segment, of the Hubei 29 strain of SFTSV. We then generated recombinant viruses from cloned cDNAs prepared to the antigenomic RNAs of both the minimally passaged virus (HB29) and of a cell-culture adapted strain designated HB29pp. The growth properties, pattern of viral protein synthesis and subcellular localization of viral N and NSs proteins of wtHB29pp and recombinant HB29pp viruses were indistinguishable. We also show the viruses fail to shut-off host cell polypeptide production. The robust reverse genetics system described will be a valuable tool for the design of therapeutics and the development of killed and attenuated vaccines against this important emerging pathogen.

    Importance SFTSV and related tick-borne phleboviruses such as Heartland virus are emerging viruses shown to cause severe disease in man in the Far East and the USA respectively. Study of these novel pathogens would be facilitated by technology to manipulate these viruses in a laboratory setting using reverse genetics. Here, we report the generation of infectious SFTSV from cDNA clones and demonstrate that recombinant viruses behave similarly to wild-type. This advance will allow for further dissection of the roles of each of the viral proteins in the context of virus infection, as well as help in the development of antiviral drugs and protective vaccines.

  • Human Cytomegalovirus Exploits Interferon-Induced Transmembrane Proteins to Facilitate Morphogenesis of the Virion Assembly Compartment [PublishAheadOfPrint]

  • Recently, interferon-induced transmembrane proteins (IFITMs) have been identified as key effector molecules in the host type I interferon defense system. The invasion of host cells by a large range of RNA viruses is inhibited by IFITMs during the entry step. However, the roles of IFITMs in DNA virus infections have not been studied in detail. In this study, we report that human cytomegalovirus (HCMV), a large human DNA virus, exploits IFITMs to facilitate the formation of the virion assembly compartment (vAC) during the infection of human fibroblasts. We found that IFITMs were expressed constitutively in human embryonic lung fibroblasts (MRC5). HCMV infection inhibited IFITM protein accumulation in the later stages of infection. Overexpression of an IFITM protein in MRC5 cells slightly enhanced HCMV production and RNAi knockdown of IFITMs reduced the virus titer by about 100-fold on day 8 post-infection according to a virus yield assay at a low MOI infection. Virus gene expression and DNA synthesis were not affected, but the typical round structure of vAC was not formed after the suppression of IFITMs, thereby resulting in defective virion assembly and the production of less infectious virion particles. Interestingly, the replication of herpes simplex virus, a human herpesvirus that is related closely to HCMV, was not affected by the suppression of IFITMs in MRC5 cells. These results indicate that IFITMs are involved in a specific pathway required for HCMV replication.

    Importance HCMV is known to repurpose interferon-stimulated genes (ISGs) viperin and tetherin to facilitate its replication. Our results expand the range of ISGs that can be exploited by HCMV for its replication. This is also the first report of a pro-viral function of IFITMs in DNA virus replication. In addition, whereas previous studies showed that IFITMs modulate virus entry, which is a very early stage in the virus life cycle, we identified a new function of IFITMs during the very late stage of the virus replication, i.e., virion assembly. Virus entry and assembly both involve vesicle transport and membrane fusion, thus a common biochemical activity of IFITMs is likely to be involved. Therefore, our findings may provide a new platform for dissecting the molecular mechanism of IFITMs during the blocking or enhancement of virus infection, which are under intense investigation in this field.

  • Ebola and SARS Display Late Cell Entry Kinetics: Evidence that Transport to NPC1+ Endolysosomes is a Rate-Defining Step [PublishAheadOfPrint]

  • Ebolavirus (EBOV) causes hemorrhagic fevers with high mortality rates. During cellular entry, the virus is internalized by macropinocytosis and trafficked through endosomes until fusion between the viral and an endosomal membrane is triggered, releasing the RNA genome into the cytoplasm. We found that while macropinocytotic uptake of filamentous EBOV virus-like particles (VLPs) expressing the EBOV glycoprotein (GP) occurs relatively quickly, VLPs only begin to enter the cytoplasm after a 30 min lag, considerably later than particles bearing the influenza hemagglutinin or GP from lymphocytic choriomeningitis virus, which enter through late endosomes (LE). For EBOV, the long lag is not due to the large size or unusual shape of EBOV filaments, the need to prime EBOV GP to the 19-kDa receptor-binding species, or a need for unusually low endosomal pH. In contrast, since we observed that EBOV entry occurs upon arrival in NPC1+ endolysosomes (LE/Lys), we propose that trafficking to LE/Lys is a key rate-defining step. Additional experiments revealed, unexpectedly, that SARS S-mediated entry also only begins after a 30 min lag. Furthermore, although SARS does not require NPC1 for entry, SARS entry also begins after colocalization with NPC1. Since the only endosomal requirement for SARS entry is cathepsin L activity, we tested, and provide evidence that NPC1+ LE/Lys have higher cathepsin L activity than LE, with no detectable activity in earlier endosomes. Our findings suggest that both EBOV and SARS traffic deep into the endocytic pathway for entry and that they do so to access higher cathepsin activity.

    IMPORTANCE Ebolavirus is a hemorrhagic fever virus that causes high fatality rates when it spreads from zoonotic vectors into the human population. Infection by SARS CoV causes severe respiratory distress in infected patients. A devastating outbreak of EBOV occurred in West Africa in 2014, and there was a significant outbreak of SARS in 2003. No effective vaccine or treatment has yet been approved for either virus. We present evidence that both viruses traffic late into the endocytic pathway, to NPC1+ LE/Lys, in order to enter host cells, and that they do so to access high levels of cathepsin activity, which both viruses use in their fusion triggering mechanisms. This unexpected similarity suggests an unexplored vulnerabilitymmdash;trafficking to NPC1+ LE/Lysmmdash;as a therapeutic target for SARS and EBOV.

  • Differential effects of the G118R, H51Y and E138K resistance substitutions in HIV integrase of different subtypes [PublishAheadOfPrint]

  • Dolutegravir (DTG) is the latest antiviral (ARV) approved for treatment of human immunodeficiency virus (HIV) infection. The G118R substitution, previously identified with MK-2048 and raltegravir may represent the initial substitution in a dolutegravir resistance pathway. We have found that subtype C integrase proteins have a lower enzymatic cost associated with the G118R substitution, mostly at the strand transfer step of integration, when compared to either subtype B or recombinant CRF02_AG proteins. Subtype B and circulating recombinant form AG (CRF02_AG) clonal viruses encoding G118R-bearing integrase were severely restricted in viral replication capacity, and G118R/E138K-bearing viruses had varying levels of resistance to dolutegravir, raltegravir and elvitegravir. In cell-free, the impacts of the H51Y and E138K substitutions on resistance and enzyme efficiency, when present with G118R, were highly dependent on viral subtype. Sequence alignment and homology modeling showed that the subtype-specific effects of these mutations were likely due to differential amino acid residue networks in the different integrase proteins, caused by polymorphic residues, which do significantly affect native protein activity, structure or function and are important for drug-mediated inhibition of enzyme activity. This preemptive study will aid in interpretation of resistance patterns in dolutegravir-treated patients.

    IMPORTANCE Recognized drug resistance mutations have never been reported in naiiuml;ve patients treated with dolutegravir. Additionally, in integrase inhibitor experienced patients, only R263K and other previously known integrase resistance substitutions have been reported. Here we suggest that alternate resistance pathways may develop in non-B HIV-1 subtypes and explain how "minor" polymorphisms and substitutions in HIV integrase that are associated with these subtypes can influence resistance against dolutegravir. This manuscript also highlights the importance of phenotyping versus genotyping -when a strong inhibitor is such as dolutegravir is being used. By characterizing G118R, this manuscript also preemptively defines parameters for a potentially important pathway in some HIV non-B subtype viruses treated with dolutegravir, and will aid in the inhibition of such virus, if detected. The general inability of strand transfer -related substitutions to diminish 3rrsquo; processing indicates the importance of the 3rrsquo; processing step and highlights a therapeutic angle that needs to be better exploited.

  • The Z proteins of pathogenic but not non-pathogenic arenaviruses inhibit the RIG-i-like receptor (RLR)-dependent interferon production [PublishAheadOfPrint]

  • Arenavirus pathogens cause a wide spectrum of diseases in humans ranging from central nervous disease to lethal hemorrhagic fevers with little treatment options. The reason why some arenaviruses can cause severe human diseases while others cannot is unknown. We find that the Z proteins of all known pathogenic arenaviruses, Lymphocytic choriomeningitis virus (LCMV), Lassa, Junin, Machupo, Sabia, Guanarito, Chapare, Dandenong, and Lujo viruses, can inhibit RIG-i and MDA5, in sharp contrast to those of 14 other non-pathogenic arenaviruses. Inhibition of the RIG-i-like receptors (RLRs) by pathogenic Z proteins is mediated by the protein-protein interactions of Z and RLRs, which leads to the disruption of the interactions between RLRs and mitochondrial antiviral signaling (MAVS). The Z-RLR interactive interface are located within the N-terminal domain (NTD) of the Z protein and the N-terminal CARD domains of RLRs. Swapping of the LCMV Z NTD into the non-pathogenic Pichinde virus (PICV) genome does not affect virus growth in Vero cells, but significantly inhibits the type I IFN responses and increases viral replication in human primary macrophages. In summary, our results show for the first time an innate immune suppressive mechanism shared by the diverse pathogenic arenaviruses and thus shed important light on the pathogenic mechanism of human arenavirus pathogens.

    Authors' summary We show that all known human pathogenic arenaviruses share a common innate immune suppression mechanism that is based on the viral Z protein-mediated RLR inhibition. Our study sheds important insights into the potential mechanism of arenavirus pathogenesis, provides a convenient way to evaluate the pathogenic potential of known and emerging arenaviruses, and reveals a novel target for the development of broad-spectrum therapies to treat this group of diverse pathogens. More broadly, our study provides a better understanding of the mechanisms of viral immune suppression and host-pathogen interactions.

  • Heat shock protein 70 regulates degradation of the mumps virus phosphoprotein via the ubiquitin-proteasome pathway [PublishAheadOfPrint]

  • Mumps virus (MuV) infection induces formation of cytoplasmic inclusion bodies (IBs). Growing evidence indicates that IBs are the site where RNA viruses synthesize their viral RNA. However, in the case of MuV infection, little is known about the viral and cellular compositions and biological functions of the IBs. In this study, pulldown purification and N-terminal amino acid sequencing revealed that stress inducible heat shock protein 70 (Hsp72) was a binding partner of MuV phosphoprotein (P protein), which was an essential component of the IBs formation. Immunofluorescence and immunoblotting analyses revealed that Hsp72 was colocalized with the P protein in the IBs, and its expression was increased during MuV infection. Knockdown of Hsp72 using siRNAs had little, if any, effect on the viral propagation in cultured cells. Knockdown of Hsp72 caused accumulation of ubiquitinated P protein and delayed the P protein degradation. These results show that Hsp72 is recruited to IBs and regulates degradation of MuV P protein through the ubiquitin-proteasome pathway.

    Importance Formation of cytoplasmic inclusion bodies (IBs) is a common characteristic feature in mononegavirus infections. IBs are considered to be the site of viral RNA replication and transcription. However, there have been few studies focused on host factors recruited to the IBs and their biological functions. Here, we identified stress inducible heat shock protein 70 (Hsp72) as the first cellular partner of mumps virus (MuV) phosphoprotein (P), which is an essential component of the IBs and involved in viral RNA replication/transcription. We found that the Hsp72 mobilized to the IBs promoted degradation of the MuV P protein thorough the ubiquitin-proteasome pathway. Our data provide new insight into the role played by IBs in mononegavirus infection.

  • Efficient virus assembly, but not infectivity, determines the magnitude of hepatitis C virus induced interferon-{alpha} responses of plasmacytoid dendritic cells [PublishAheadOfPrint]

  • Worldwide approximately 160 million people are chronically infected with hepatitis C virus (HCV) of which seven distinct genotypes are discriminated. Hallmark of HCV is its genetic variability and the diverge courses of hepatitis C progression in patients. We assessed whether intra-genotypic HCV variations would differentially trigger host innate immunity. To this end, we stimulated human primary plasmacytoid dendritic cells (pDC) with crude preparations of different cell culture-derived genotype 2a HCV variants. Parental JFH1 did not induce IFN-aalpha;, whereas the intra-genotypic chimera Jc1 triggered massive IFN-aalpha; responses. Purified Jc1 retained full infectivity but no longer induced IFN-aalpha;. Co-culture of pDC with HCV infected hepatoma cells retrieved the capacity to induce IFN-aalpha;, whereas Jc1 infected cells triggered stronger responses than JFH1 infected cells. Since infectivity of virus particles did not seem to affect pDC activation, we next tested Jc1 mutants that were arrested at different steps of particle assembly. These experiments revealed that efficient assembly and core protein envelopment was critically needed to trigger IFN-aalpha;. Of note, sequences within domain 2 of the core that vitally affect virus assembly also crucially influenced IFN-aalpha; responses of pDC. These data showed that viral determinants shaped host innate IFN-aalpha; responses to HCV.

    Importance Although pegylated interferon-aalpha; plus Ribavirin currently is the standard of care for the treatment of chronic hepatitis C virus infection, not much is known about the relevance of early interferon responses in the pathogenesis of hepatitis C virus infection. Here we addressed whether intragenotypic variations of hepatitis C virus would account for differential induction of type I interferon responses mounted by primary blood derived plasmacytoid dendritic cells. Surprisingly, a chimeric genotype 2a virus carrying the non-structural genes of Japanese fulminant hepatitis C virus (JFH1) induced massive type I interferon responses, whereas the original genotype 2a JFH1 strain did not. Our detailed analyses revealed that not the virus infectivity, but efficiency of virus assembly and core protein envelopment, critically determined the magnitude of interferon responses. To our knowledge this is the first example of hepatitis C virus associated genetic variations that determine the magnitude of innate host responses.

  • Impact of natural IgM concentration on gene therapy with adenovirus type 5 vectors [PublishAheadOfPrint]

  • Natural IgM inhibits gene transfer by type 5 adenovirus (Ad5) vectors. We show that polyreactive natural IgM antibodies bind to Ad5 and that inhibition of liver transduction by IgM depends on Kupffer cells. By manipulating IgM concentration in vivo, we demonstrate that IgM inhibits liver transduction in a concentration-dependent manner. We further show that differences in natural IgM between BALB/c and C57BL/6 mice contribute to lower efficiency of Ad5 gene transfer in BALB/c mice.

  • Mutations conferring a non-cytotoxic phenotype on Chikungunya virus replicons compromise enzymatic properties of non-structural protein 2 [PublishAheadOfPrint]

  • Chikungunya virus (CHIKV, genus Alphavirus) has a positive-sense RNA genome. CHIKV non-structural protein 2 (nsP2) proteolytically processes the viral non-structural polyprotein; possesses NTPase, RNA triphosphatase, and RNA helicase activities; and induces cytopathic effects in vertebrate cells. Although alphaviral nsP2 mutations can result in a non-cytotoxic phenotype, the effects of such mutations on nsP2 enzymatic activities are not well understood. In this study, we introduced a P718G (PG) mutation and selected for additional mutations in CHIKV nsP2 that resulted in a CHIKV replicon with a non-cytotoxic phenotype in BHK-21 cells. Combinations of PG and either E116K (EK) substitution or a GEEGS sequence insertion after T648 residue (5A) markedly reduced RNA synthesis; however, neither PG nor 5A prevented nsP2 nuclear translocation. Introducing PG into recombinant nsP2 inhibited proteolytic cleavage of nsP1/nsP2 and nsP3/nsP4 sites, reduced GTPase and RNA helicase activities and abolished RNA-stimulation of GTPase activity. 5A and EK modulated the effects of PG. However, only the RNA helicase activity of nsP2 was reduced by both of these mutations, suggesting that defects in this activity may be linked to a non-cytotoxic phenotype. These results increase our understanding of the molecular basis for the cytotoxicity that accompanies alphaviral replication. Furthermore, adaptation of the CHIKV replicon containing both 5A and PG allowed the selection of a CHIKV replicon with adaptive mutations in nsP1 and nsP3 that enable persistence in human cell line. Such cell lines represent valuable experimental systems for discovering host factors and for screening inhibitors of CHIKV replication at lower biosafety levels.

    IMPORTANCE CHIKV is a medically important pathogen that causes febrile illness and can cause chronic arthritis. No approved vaccines or antivirals are available for CHIKV. The attenuation of CHIKV is critical to the establishment of experimental systems that can be used to conduct virus replication studies at a lower biosafety level. We applied a functional selection approach to develop, for the first time, a non-cytotoxic CHIKV replicon capable of persisting in human cell lines. We anticipate that this safe and efficient research tool will be valuable for screening CHIKV replication inhibitors and for identifying and analyzing host factors involved in viral replication. We also analyzed the functional defects caused by mutations conferring non-cytotoxic phenotypes, from virological and protein biochemistry perspectives; we found that all known enzymatic activities of CHIKV nsP2, as well as its RNA-binding capability, were compromised by these mutations, which led to a reduced capacity for replication.

  • Identification and characterization of nuclear and nucleolar localization signals in the adeno-associated virus serotype 2 assembly-activating protein [PublishAheadOfPrint]

  • Assembly-activating protein (AAP) of adeno-associated virus serotype 2 (AAV2) is a nucleolar localizing protein that plays a critical role in transporting viral capsid VP3 protein to the nucleolus for assembly. Here we identify and characterize AAV2 AAP (AAP2) nuclear and nucleolar localization signals (NLS and NoLS, respectively) near the carboxy-terminal region of AAP2 (amino acid positions 144-184 or AAP2144-184). This region contains five basic amino acid-rich (BR) clusters, KSKRSRR (AAP2BR1), RRR (AAP2BR2), RFR (AAP2BR3), RSTSSR (AAP2BR4) and RRIK (AAP2BR5) from the amino terminus to the carboxy terminus. We created 30 AAP2BR mutants by arginine/lysine-to-alanine mutagenesis or deletion of AAP2BRs and 8 and 1 GFP-AAP2BR and bbeta;-galactosidase-AAP2BR fusion proteins, respectively, and analyzed their intracellular localization in HeLa cells by immunofluorescence microscopy. The results showed that: AAP2144-184 has redundant multipartite NLSs and any combinations of 4 AAP2BRs, but not 3 or less, can constitute a functional NLS/NoLS; AAP2BR1 and AAP2BR2 play the most influential role for nuclear localization but either one of these two AAP2BRs is dispensable if all the other 4 AAP2BRs are present, resulting in 3 different, overlapping NLS motifs; and the NoLS is shared redundantly among the five AAP2BRs and functions in a context dependent manner. AAP2BR mutations not only resulted in aberrant intracellular localization but also attenuated AAP2 protein expression to various degrees, and both of these abnormalities have a significant negative impact on capsid production. Thus, this study reveals the organization of the intermingling NLSs and NoLSs in AAP2 and provides insights into their functional role in capsid assembly.

    Importance Adeno-associated virus (AAV) has become a popular and successful vector for in vivo gene therapy; however, its biology has yet to be fully understood. In this regard, the recent discovery of the assembly-activating protein (AAP), a non-structural, nucleolar localizing AAV protein essential for viral capsid assembly, has provided us a new opportunity to better understand the fundamental processes required for the virion formation. Here we identify clusters of basic amino acids in the carboxy-terminus of AAP from AAV serotype 2 (AAV2) that act as nuclear and nucleolar localization signals. We also demonstrate their importance in maintaining AAP expression levels and efficient production of viral capsids. Insights into the functions of AAP can elucidate the requirements and process for AAV capsid assembly, which may lead to improved vector production for use in gene therapy. This study also contributes to the growing body of work on nuclear and nucleolar localization signals.

  • Heterosubtypic antibodies to influenza A virus have limited activity against cell-bound virus but are not impaired by strain-specific serum antibodies. [PublishAheadOfPrint]

  • The majority of influenza virus-specific antibodies elicited by vaccination or natural infection is only effective against the eliciting or closely related viruses. Rare stem-specific heterosubtypic monoclonal antibodies (hmAbs) can neutralize multiple strains and subtypes by preventing hemagglutinin (HA)-mediated fusion of the viral with the endosomal membrane. The epitopes recognized by these hmAbs are therefore considered promising targets for the development of pan-influenza vaccines.

    Here we report the isolation of a novel human HA-stem-reactive monoclonal antibody, mAb 1.12, with exceptionally broad neutralizing activity encompassing viruses from 15 distinct HA subtypes. Using mAb 1.12 and two other monoclonal antibodies, we could demonstrate that neutralization by hmAbs is virtually irreversible but becomes severely impaired following virus attachment to cells. In contrast, no interference by human anti-influenza serum antibodies was found, indicating that apically binding antibodies do not impair access to the membrane-proximal heterosubtypic epitopes. Our findings therefore encourage development of new vaccine concepts aiming at the induction of stem-specific heterosubtypic antibodies, as we provide support for their effectiveness in individuals previously exposed to influenza virus.

    Importance The influenza A virus hemagglutinin (HA) can easily accommodate changes in its antigenic structures to escape pre-existing immunity. This variability is restricting breadth and long-term efficacy of influenza vaccines. Only a handful of heterosubtypic antibodies (hmAb), i.e. antibodies that can neutralize more than one subtype of influenza A virus, have been identified. The molecular interactions between these heterosubtypic antibodies and hemagglutinin are well characterized, yet little is known about the functional properties of these antibodies. Using a new extraordinarily broad hmAb, we show that virus neutralization by hmAbs is virtually irreversible, and that efficient neutralization is only possible if stem-specific hmAbs bind to HA before the virus attaches to the cell surface. No interference between strain-specific human serum immunoglobulin and hmAbs was found, indicating that pre-existing humoral immunity to influenza virus does not limit the efficacy of stem-reactive heterosubtypic antibodies. This knowledge supports the development of a pan-influenza vaccine.

  • Inhibition of H3K27me3-specific Histone Demethylases, JMJD3 and UTX, Blocks Reactivation of Herpes Simplex Virus 1 in Trigeminal Ganglion Neurons [PublishAheadOfPrint]

  • HSV-1 genomes are associated with the repressive heterochromatic marks H3K9me2/me3 and H3K27me3 during latency. Previous studies have demonstrated that inhibitors of H3K9me2/me3 histone demethylases reduce the ability of HSV-1 to reactivate from latency. Here we demonstrate that GSK-J4, a specific inhibitor of the H3K27me3 histone demethylases UTX and JMJD3, inhibits HSV-1 reactivation from sensory neurons in vitro. These results indicate that removal of the H3K27me3 mark plays a key role in HSV-1 reactivation.

  • Serine phosphorylation of the hepatitis C virus NS5A protein controls the establishment of replication complexes. [PublishAheadOfPrint]

  • The hepatitis C virus (HCV) non-structural 5A protein (NS5A) is highly phosphorylated and involved in both virus genome replication and virion assembly. We, and others, have identified serine 225 in NS5A as a phosphorylation site but the function of this post-translational modification in the virus lifecycle remains obscure. Here we describe the phenotype of mutations at serine 225, this residue was mutated to either alanine (S225A: phosphoablatant) or aspartic acid (S225D: phosphomimetic) in the context of both the JFH-1 cell culture infectious virus and a corresponding subgenomic replicon. S225A exhibited a 10-fold reduction in genome replication whereas S225D replicated as wildtype. By confocal microscopy we show that, in the case of S225A, the replication phenotype correlated with an altered subcellular distribution of NS5A. This phenotype was shared by other mutations in the low complexity sequence I (LCS I) mmdash; namely S229D, S232A and S235D, but not by mutations with a comparable replication defect that mapped to domain II of NS5A (P315A, L321A). Together with other components of the genome replication complex (NS3, double-stranded RNA and cellular lipids including phosphatidylinositol-4 phosphate), mutant NS5A was restricted to a perinuclear region. This phenotype was not due to cell confluency or another environmental factor, and could be partially transcomplemented by wildtype NS5A. We propose that serine phosphorylation within LCS I may regulate the assembly of an active genome replication complex.

    IMPORTANCE: The mechanisms by which hepatitis C virus replicates its RNA genome remain poorly characterised. We show here that phosphorylation of the viral non-structural protein NS5A at serine residues is important for the efficient assembly of a complex that is able to replicate the viral genome. This research implicates cellular protein kinases in control of virus replication and highlights the need to further understand the interplay between the virus and the host cell in order to develop potential avenues for future antiviral therapy.

  • The HSV-1 UL51 Protein Interacts with the UL7 Protein and Plays a Role in Its Recruitment into the Virion [PublishAheadOfPrint]

  • The alphaherpesvirus UL51 protein is a tegument component that interacts with the viral glycoprotein E and functions at multiple steps in virus assembly and spread in epithelial cells. We show here that pUL51 forms a complex in infected cells with another conserved tegument protein, pUL7. This complex can form in the absence of other viral proteins, and is largely responsible for recruitment of pUL7 to cytoplasmic membranes and into the virion tegument. Incomplete co-localization of pUL51 and pUL7 in infected cells, however, suggests that a significant fraction of the population of each protein is not complexed with the other, and that they may accomplish independent functions.

    IMPORTANCE The ability of herpesviruses to spread from cell to cell in the face of an immune response is critical for disease and shedding following reactivation from latency. Cell-to cell spread is a conserved ability of herpesviruses, and the identification of conserved viral genes that mediate this process will aid in the design of attenuated vaccines and of novel therapeutics. The conserved UL51 gene of HSV-1 plays important roles in cell-to-cell spread and in virus assembly in the cytoplasm, both of which likely depend on specific interactions with other viral and cellular proteins. Here we identify one of those interactions with the product of another conserved herpesvirus gene, UL7, and show that formation of this complex mediates recruitment of UL7 to membranes and to the virion.

  • Development of a mouse-adapted, live-attenuated influenza virus that permits in vivo analysis of enhancements to the safety of LAIV. [PublishAheadOfPrint]

  • The live attenuated influenza vaccine is preferentially recommended for use in persons 2 through 49 years of age, but not approved for children under 2 or asthmatics due to safety concerns. Therefore, increasing safety is desirable. Here we describe a murine LAIV with reduced pathogenicity that retains lethality at high doses and further demonstrate that we can enhance safety in vivo through mutations within NS1. This model may permit preliminary safety analysis of improved LAIVs.

  • Analysis of Geminivirus AL2 and L2 Proteins Reveals a Novel AL2 Silencing Suppressor Activity [PublishAheadOfPrint]

  • Both post-transcriptional and transcriptional gene silencing (PTGS and TGS) participate in defense against the DNA-containing geminiviruses. As a countermeasure, members of the genus Begomovirus (e.g. Cabbage leaf curl virus) encode an AL2 protein that is both a transcriptional activator and a silencing suppressor. The related L2 protein of Beet curly top virus (genus Curtovirus) lacks transcription activation activity. Previous studies showed that both AL2 and L2 suppress silencing by a mechanism that correlates with adenosine kinase (ADK) inhibition, while AL2 in addition activates transcription of cellular genes that negatively regulate silencing pathways. The goal of this study was to clarify the general means by which these viral proteins inhibit various aspects of silencing. We confirmed that AL2 inhibits systemic silencing spread by a mechanism that requires transcription activation activity. Surprisingly, we also found that reversal of PTGS and TGS by ADK inactivation depended on whether experiments were conducted in vegetative or reproductive Nicotiana benthamiana plants (i.e. before or after the vegetative to reproductive transition). While AL2 was able to reverse silencing in both vegetative and reproductive plants, L2 and ADK inhibition were effective only in vegetative plants. This suggests that silencing maintenance mechanisms can change during development, or in response to stress. Remarkably, we also observed that AL2 lacking its transcription activation domain could reverse TGS in reproductive plants, revealing a third, previously unsuspected AL2 suppression mechanism that depends neither on ADK inactivation nor transcription activation.

    IMPORTANCE RNA silencing in plants is a multivalent antiviral defense, and viruses respond by elaborating multiple and sometimes multifunctional proteins that inhibit various aspects of silencing. The studies described here add an additional layer of complexity to this interplay. By examining geminivirus AL2 and L2 suppressor activities, we show that L2 is unable to suppress silencing in Nicotiana benthamiana plants that have undergone the vegetative-to-reproductive transition. As L2 was previously shown to be effective in mature Arabidopsis plants, these results illustrate that silencing mechanisms can change during development, or in response to stress, in ways that may be species specific. The AL2 and L2 proteins are known to share a suppression mechanism that correlates with the ability of both proteins to inhibit ADK, while AL2 in addition can inhibit silencing by transcriptionally activating cellular genes. Here we also provide evidence for a third AL2 suppression mechanism that depends neither on transcription activation nor ADK inactivation. Besides revealing the remarkable versatility of AL2, this work highlights the utility of viral suppressors as probes for the analysis of silencing pathways.

  • Transcriptome Analysis of KSHV During De Novo Primary Infection of Human B and Endothelial Cells [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) infects many target cells (e.g., endothelial, epithelial, and B cells, keratinocytes, and monocytes) to establish lifelong latent infections. Viral latent-protein expression is critical in inducing and maintaining KSHV latency. Infected cells are programmed to retain the incoming viral genomes during primary infection. KSHV transcribes many lytic genes immediately after infections that modulate various cellular pathways to establish successful infection. Analysis of the virion particle showed that the virions contain viral mRNAs, micro-RNAs, and other noncoding RNAs that are transduced into the target cells during infection, but their biological functions are largely unknown. We performed a comprehensive analysis of the KSHV virions packaged transcripts and the profiles of viral genes transcribed after de novo infections of various cell types (human PBMCs, CD14+ monocytes, and TIVE cells), from viral entry until latency establishment. A next-generation sequence analysis of the total transcriptome showed that several viral RNAs (polyadenylated nuclear RNA, ORF58, ORF59, T0.7, and ORF17) were abundantly present in the KSHV virions and effectively transduced into the target cells. Analysis of the transcription profiles of each viral gene showed specific expression patterns in different cell lines with majority of the genes, other than latent genes, silencing after 24h post-infection. We differentiated the actively transcribing genes from the virions transduced transcripts using a nascent RNA capture approach (Click-It chemistry), which identified transcription of a number of viral genes during primary infection. Treating the infected cells with phosphonoacetic acid (PAA), to block the activity of viral DNA polymerase, confirmed the involvement of lytic DNA replication during primary infection. To further understand the role of DNA replication during primary infection, we performed de novo PBMC infections with a recombinant ORF59 deleted KSHV virus, which showed significantly reduced viral copies in the latently infected cells. In summary, the transduced KSHV RNAs as well as the actively transcribed genes control critical processes of early infection to establish KSHV latency.

    IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of multiple human malignancies in immunocompromised individuals. KSHV establishes a lifelong latency in the infected host, during which only a limited number of viral genes are expressed. However, a fraction of latently infected cells undergo spontaneous reactivation to produce virions that infect the surrounding cells. These newly infected cells are primed early on to retain the incoming viral genome and induce cell growth. KSHV transcribes a variety of lytic proteins during de novo infections that modulate various cellular pathways to establish the latent infection. Interestingly, a large number of viral proteins and RNA are encapsidated in the infectious virions and transduced into the infected cells during a de novo infection. This study determined the kinetics of the viral gene expression during de novo-KSHV infections and the functional role of the incoming viral transcripts in establishing latency.

  • Discovery of a novel coronavirus, China Rattus coronavirus HKU24, from Norway rats supports murine origin of Betacoronavirus 1 with implications on the ancestor of Betacoronavirus lineage A [PublishAheadOfPrint]

  • We discovered a novel Betacoronavirus lineage A coronavirus, China Rattus coronavirus HKU24 (ChRCoV HKU24), from Norway rats in China. ChRCoV HKU24 occupied a deep branch at the root of members of Betacoronavirus 1, being distinct from murine coronavirus and HCoV HKU1. Its unique putative cleavage sites at nsp1/2 and S, and low sequence identities to other lineage A bbeta;CoVs in conserved replicase domains, support ChRCoV HKU24 as a separate species. ChRCoV HKU24 possessed genome features that resemble both Betacoronavirus 1 and murine coronavirus, being closer to Betacoronavirus 1 in most predicted proteins, but closer to murine coronavirus by G+C content, a single NS4 and absent TRS for E. Its N-terminal domain (NTD) demonstrated higher sequence identity to BCoV than to MHV NTDs, with three of four critical sugar-binding residues in BCoV and two of 14 contact residues at MHV NTD/mCEACAM1a interface being conserved. Molecular clock analysis dated the tMRCA of ChRCoV HKU24, Betacoronavirus 1 and RbCoV HKU14 to ~1400. Cross reactivities were demonstrated between other lineage A and B bbeta;CoVs and ChRCoV HKU24 nucleocapsid but not spike polypeptide. Using the spike polypeptide-based western blot, we showed that only Norway rats and two Oriental house rats from Guangzhou were infected by ChRCoV HKU24. Other rats, including Norway rats from Hong Kong, only possessed antibodies against N protein but not spike, suggesting infection by bbeta;CoVs different from ChRCoV HKU24. ChRCoV HKU24 may represent the murine origin of Betacoronavirus 1 and rodents are likely an important reservoir for ancestors of lineage A bbeta;CoVs.

    IMPORTANCE While bats and birds are hosts for ancestors of most coronaviruses (CoVs), lineage A bbeta;CoVs have never been found in these animals and the origin of Betacoronavirus lineage A remains obscure. We discovered a novel lineage A bbeta;CoV, China Rattus coronavirus HKU24 (ChRCoV HKU24), from Norway rats in China, with a high seroprevalence. The unique genome features and phylogenetic analysis supported that ChRCoV HKU24 represents a novel CoV species, occupying a deep branch at the root of members of Betacoronavirus 1 and distinct from murine coronavirus. Nevertheless, ChRCoV HKU24 possessed genome characteristics that resemble both Betacoronavirus 1 and murine coronavirus. Our data suggest that ChRCoV HKU24 represents the murine origin of Betacoronavirus 1, with interspecies transmission from rodents to other mammals having occurred centuries ago before the emergence of HCoV OC43 in late 1800s. Rodents may be an important reservoir for ancestors of lineage A bbeta;CoVs.

  • Recombinant subgroup B human respiratory syncytial virus expressing EGFP efficiently replicates in primary human cells and is virulent in cotton rats [PublishAheadOfPrint]

  • Human respiratory syncytial virus (HRSV) is the most important viral cause of severe respiratory tract disease in infants. Two subgroups (A and B) have been identified, which co-circulate during, or alternate between yearly epidemics and cause indistinguishable disease. Existing in vitro and in vivo models of HRSV focus almost exclusively on subgroup A viruses. Here, a recombinant (r) subgroup B virus (rHRSVB05) was generated based on a consensus genome sequence obtained directly from an unpassaged clinical specimen from a hospitalized infant. An additional transcription unit containing the gene encoding enhanced green fluorescent protein (EGFP) was introduced between the phosphoprotein and matrix genes (position 5) of the genome to generate rHRSVB05EGFP (5). The recombinant viruses replicated efficiently in both HEp-2 cells and in well differentiated normal human bronchial cells grown at air-liquid interface. Intranasal infection of cotton rats (Sigmodon hispidus) resulted in high numbers of EGFP+ cells in epithelia of the nasal septum and conchae. When administered in a relatively large inoculum volume, the virus also replicated efficiently in bronchiolar epithelial cells and spread extensively in both the upper and lower respiratory tract. Virus replication was not observed in ciliated epithelial cells of the trachea. This is the first virulent rHRSV strain with the genetic composition of a currently circulating wild-type virus. In vivo tracking of infected cells by means of EGFP fluorescence in the absence of cytopathic changes increases the sensitivity of virus detection in HRSV pathogenesis studies.

    Importance Virology as a discipline has depended on monitoring cytopathic effects following virus culture in vitro. However, wild-type viruses isolated from patients often do not cause significant changes to infected cells, necessitating blind passage. This can lead to genetic and phenotypic changes and the generation of high-titer, laboratory-adapted viruses with diminished virulence in animal models of disease. To address this we determined the genome sequence of an unpassaged human respiratory syncytial virus from a sample obtained directly from an infected infant, assembled a molecular clone and recovered a wild-type recombinant virus. Addition of a gene encoding enhanced green fluorescent protein allowed this wild-type virus to be tracked in primary human cells and living animals in the absence of significant cytopathic effects. Imaging of fluorescent cells proved to be a highly valuable tool for monitoring the spread of virus, and may help improve assays for evaluating novel intervention strategies.

  • Human coagulation factor X-adenovirus type 5 complexes poorly stimulate an innate immune response in human mononuclear phagocytes [PublishAheadOfPrint]

  • One of the first lines of host defense against many viruses in vertebrates is the innate immune system, which detects pathogen associated molecular patterns (PAMPs) using pathogen recognition receptors (PRR). The dynamic interactions between pathogens and hosts create, in some cases, species-specific relationships. Recently, it was shown that murine factor X (mFX)-armored human adenovirus (HAd) stimulated a mFX-Toll-like receptor 4 (TLR4)-associated response in mouse macrophages in vitro and in vivo. Given the importance of studies using animal to better understand host-pathogen interactions, we asked if a human FX (hFX)-armored HAd type 5 (HAd5) was capable of activating innate immune sensors in primary human mononuclear phagocytes. To this end we assayed human mononuclear phagocytes for their ability to be stimulated by hFX-armored HAd5 via a TLR/NF-, and in particular a TLR4, pathway. In our hands, we found no significant interaction, activation, or maturation of human mononuclear phagocytes by hFX-armored HAd5.

    Importance Animals, and in particular mice, are often informative and powerful surrogates for how pathogens interact with natural host systems. When possible extended and targeted studies in the natural host can then be performed. Our data will help us understand the differences in preclinical testing in mice and clinical use in human in order to improve treatment for HAd diseases and Ad vector effectiveness.

  • Virus evolution towards limited dependence on the non-essential functions of the host: the bacteriophage SPP1 case [PublishAheadOfPrint]

  • All viruses are obligate intracellular parasites and depend on certain host cell functions for multiplication. However, the extent of such dependence and the exact nature of the functions provided by the host cell remain poorly understood. Here, we investigated if non-essential Bacillus subtilis genes are necessary for multiplication of bacteriophage SPP1. Screening of a collection of 2514 single-gene knockouts of non-essential B. subtilis genes yielded only a handful of genes necessary for efficient SPP1 propagation. Among these were genes belonging to the yuk operon, which codes for the Esat-6-like secretion system including the SPP1 receptor protein YueB. In addition, we found that SPP1 multiplication is negatively affected by the absence of two other genes putB and efp. Gene efp encodes elongation factor P, which enhances ribosome activity by alleviating translational stalling during the synthesis of polyproline containing proteins. PutB is an enzyme involved in the proline degradation pathway that is required for infection at the post-exponential growth phase of B. subtilis when the bacterium undergoes a complex genetic re-programming. The putB knock-out shortens significantly the window of opportunity for SPP1 infection during the host cell life cycle. This window is a critical parameter for competitive phage multiplication in the soil environment where B. subtilis rarely meets conditions for exponential growth. Our results in combination with those reported for other virus-host systems suggest that bacterial viruses have evolved towards limited dependence on the non-essential host functions.

    Importance A successful viral infection largely depends on the ability of the virus to highjack cellular machineries and to redirect the flow of building blocks and energy resources towards viral progeny production. However, the specific virus-host interactions underlying this fundamental transformation are poorly understood. Here, we report on the first systematic analysis of virus-host crosstalk during bacteriophage infection in Gram-positive bacteria. We show that lytic bacteriophage SPP1 is remarkably independent of non-essential genes of its host Bacillus subtilis, with only a handful of cellular genes being necessary for efficient phage propagation. We hypothesize that such limited dependence of the virus on its host results from a constant evolutionary arms-race and might be much more widespread than currently thought.

  • The unusual fold of HSV-1 UL21, a multifunctional tegument protein [PublishAheadOfPrint]

  • UL21 is a conserved protein in the tegument in alphaherpesviruses and has multiple important albeit poorly understood functions in viral replication and pathogenesis. To provide a roadmap for exploration of the multiple roles of UL21, we determined the crystal structure of its conserved N-terminal domain from Herpes Simplex virus Type 1 to 2.0-AAring; resolution, which revealed a novel sail-like protein fold. Evolutionarily conserved surface patches highlight residues of potential importance for future targeting by mutagenesis.

  • Natural Killer Cells can Inhibit the Transmission of Human Cytomegalovirus in Cell Culture Using Mechanisms from Innate and Adaptive Immune Response [PublishAheadOfPrint]

  • Human Cytomegalovirus (HCMV) transmission within the host is important for the pathogenesis of HCMV diseases. Natural Killer (NK) cells are well known to provide a first line of host defense against virus infections. However, the role of NK cells in the control of HCMV transmission is still unknown. Here, we provide the first experimental evidence that NK cells can efficiently control HCMV transmission in different cell types. NK cells engage different mechanisms to control the HCMV transmission both via soluble factors and by cell contact. NK cell produced interferon gamma (IFN-) suppresses HCMV production and induces resistance of bystander cells to HCMV infection. The viral gene UL16 contributes to an immune evasion from the NK cell mediated control of HCMV transmission. Furthermore, the efficacy of the antibody dependent NK cell mediated control of HCMV transmission is dependent on CD16-158V/F polymorphisms. Our findings indicate that NK cells may have a clinical relevance in HCMV infection and highlight the need to consider potential therapeutic strategies based on the manipulation of NK cells.

    Importance Human Cytomegalovirus (HCMV) infects 40% to 100% of the human population worldwide. After primary infection, mainly in childhood, the virus establishes a lifelong persistence with possible reactivations. Most infections remain asymptomatic; however, HCMV represents a major health problem since it is the most frequent cause of infection-induced birth defects and is responsible for a high morbidity and mortality in immunocompromised patients. The immune system normally controls the infection by antibodies and immune effector cells. One type of effector cells are the Natural Killer (NK) cells which provide a rapid response to virus-infected cells. NK cells participate in the viral clearance by inducing the death of infected cells. NK cells also secrete anti-viral cytokines as a consequence of the interaction with an infected cell. In this study, we investigated the mechanisms by which NK cells control HCMV transmission, from the perspectives of immune surveillance and immune evasion.

  • Chicken and duck myotubes are highly susceptible and permissive to influenza virus infection [PublishAheadOfPrint]

  • Skeletal muscle at 30 to 40% of body mass is the most abundant soft tissue in the body. Besides its primary function in movement and posture, skeletal muscle is a significant innate immune organ with the capacity to produce cytokines and chemokines, and respond to pro-inflammatory cytokines. Little is known about the role of skeletal muscle during systemic influenza A virus infection in any host and in particular avian species. Here we used primary chicken and duck multinucleated myotubes to examine their susceptibility and innate immune response to influenza virus infections. Both chicken and duck myotubes expressed avian and human sialic acid receptors, and were readily susceptible to low (H2N3 A/mallard duck/England/7277/06) and high (H5N1 A/turkey/England/50-92/91 and H5N1 A/turkey/Turkey/1/05) pathogenicity avian, and human H1N1 (A/USSR/77) influenza viruses. Both avian host species produced comparable levels of progeny H5N1 A/turkey/Turkey/1/05 virus. Notably, the rapid accumulation of viral nucleoprotein and matrix (M) gene RNA in chicken and duck myotubes, was accompanied by extensive cytopathic damage with marked myotube apoptosis (widespread microscopic blebs, caspase 3/7 activation and annexin-V binding at plasma membrane). Infected chicken myotubes produced significantly higher pro-inflammatory cytokines than corresponding duck cells. Additionally, in chicken myotubes infected with H5N1 viruses, induction of interferon (IFN)-bbeta; and IFN-inducible genes, including melanoma differentiation-associated protein 5 (MDA-5), was relatively weak in contrast to corresponding H2N3 virus infection. Our findings highlight avian skeletal muscle fibres as capable of productive influenza virus replication and a potential tissue source of infection.

    IMPORTANCE Infection with high pathogenicity H5N1 viruses in ducks is often asymptomatic and skeletal muscle from such birds could be a source of infection to humans and animals. Little is known about the ability of influenza A viruses to replicate in avian skeletal muscle fibres. We show here that cultured chicken and duck myotubes were highly susceptible to infection with both low and high pathogenicity avian influenza viruses. Infected myotubes of both avian species displayed rapid virus accumulation, apoptosis and extensive cellular damage. Our results indicate that avian skeletal muscle fibres of chicken and duck could be significant contributors to progeny production of highly pathogenic H5N1 viruses.

  • Regulation of Herpesvirus Reactivation by Host miRNAs [PublishAheadOfPrint]

  • The interplay between latent and lytic modes of infection is central to successful infection of all herpesviruses, yet knowledge is limited on the determinants that govern reactivation of these viruses from latent to lytic infection. Recently several studies have identified roles for specific cellular microRNAs in inhibiting reactivation of various herpesviruses, thereby promoting latent infections. These studies are discussed in the context of current knowledge on mechanisms of regulation of reactivation of specific herpesviruses.

  • Lymph node but not intradermal injection site macrophages are critical for germinal center formation and antibody responses to rabies vaccination [PublishAheadOfPrint]

  • Replication-deficient rabies virus (RABV)-based vaccines induce rapid and potent antibody responses via T cell-independent and T cell-dependent mechanisms. To further investigate early events in vaccine-induced antibody responses against RABV infections, we studied the role for macrophages as mediators of RABV-based vaccine immunogenicity. In this report, we show that a recombinant matrix gene-deleted RABV-based vaccine (rRABV-M) infects and activates primary murine macrophages in-vitro. Immunization of mice with live RABV-based vaccines results in accumulation of macrophages at the site of immunization, suggesting macrophages in tissues support the development of effective anti-RABV B cell responses. However, we show that draining lymph node macrophages, but not macrophages at the site of immunization, are essential for the generation of germinal center B cells, follicular T helper cells and RABV-specific antibodies. Our findings have implications for the design of new RABV-based vaccines where early immunological events are important for the protection against RABV in post-exposure settings.

    IMPORTANCE: Over two-thirds of the world's population lives in regions where rabies is endemic. Post-exposure prophylaxis is the primary means of treating humans. Identifying immunological principles that guide the development of rapid and potent antibody responses against rabies infections will greatly increase our ability to produce more effective rabies vaccines. Here we describe that macrophages in the draining lymph node, but not in the tissue at the site of immunization are important for vaccine-induced antibody responses to rabies. Information gleaned from this study, may help guide the development of a single-dose vaccine against rabies infections.

  • Pathogenicity and transmissibility of novel reassortant H3N2 influenza viruses with 2009 pandemic H1N1 genes in pigs [PublishAheadOfPrint]

  • At least 10 different genotypes of novel reassortant H3N2 influenza viruses with 2009 pandemic H1N1 [A(H1N1)pdm09] gene(s) have been identified in U.S. pigs including the H3N2 variant with single A(H1N1)pdm09 M gene which has infected more than 300 people. To date, only three genotypes of these viruses were evaluated in animal models, and the pathogenicity and transmissibility of the other seven genotype viruses remains unknown. Herein, we show that three H3N2 reassortant viruses that contain 3 (NP, M and NS) or 5 (PA, PB2, NP, M and NS) genes from A(H1N1)pdm09, were pathogenic in pigs, similar to the endemic H3N2 swine virus. However, the reassortant H3N2 virus with 3 A(H1N1)pdm09 genes and a recent human influenza N2 gene transmitted most efficiently among pigs whereas the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes transmitted less efficiently when compared to the endemic H3N2 virus. Interestingly, the polymerase complex of reassortant H3N2 virus with 5 A(H1N1)pdm09 genes showed significantly higher polymerase activity than those of endemic and reassortant H3N2 viruses with 3 A(H1N1)pdm09 genes. Further studies showed that an avian-like glycine at position 228 at the HA receptor binding site is responsible for inefficient transmission of the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes. Taken together, our results provide insights into the pathogenicity and transmissibility of novel reassortant H3N2 viruses in pigs and suggest that a mammalian-like serine at position 228 in the HA is critical for viral transmissibility of these reassortant H3N2 viruses.

    Importance Swine influenza is a highly contagious, zoonotic disease that threatens animal and public health. Introduction of 2009 pandemic H1N1 virus [A(H1N1)pdm09] into swine herds has resulted in novel reassortant influenza viruses in swine including H3N2 and H1N2 variants that have caused human infections in the USA. We showed that reassortant H3N2 influenza viruses with 3 or 5 genes from A(H1N1)pdm09 isolated from diseased pigs are pathogenic and transmissible in pigs, but the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes displayed less efficient transmissibility than the endemic and reassortant H3N2 viruses with 3 A(H1N1)pdm09 genes. Further studies revealed that an avian-like Glycine at the HA 228 receptor binding site of the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes is responsible for less efficient transmissibility in pigs. Our results provide insights into viral pathogenesis and transmission of novel reassortant H3N2 viruses that are circulating in U.S. swine herds and warrants future surveillance.

  • A Multivalent Clade C HIV-1 Env Trimer Cocktail Elicits a Higher Magnitude of Neutralizing Antibodies than Any Individual Component [PublishAheadOfPrint]

  • The sequence diversity of human immunodeficiency virus type 1 (HIV-1) presents a formidable challenge to the generation of an HIV-1 vaccine. One strategy to address such sequence diversity and to improve the magnitude of neutralizing antibodies (NAbs) is to utilize multivalent mixtures of HIV-1 envelope (Env) immunogens. Here we report the generation and characterization of three novel, acute clade C HIV-1 Env gp140 trimers (459C, 405C and 939C), each with unique antigenic properties. Among the single trimers tested, 459C elicited the most potent NAb responses in vaccinated guinea pigs. We evaluated the immunogenicity of various mixtures of clade C Env trimers and found that a quadrivalent cocktail of clade C trimers elicited a greater magnitude of NAbs against a panel of Tier 1A and 1B viruses than any single clade C trimer alone, demonstrating that the mixture had an advantage over all individual components of the cocktail. These data suggest that vaccination with a mixture of clade C Env trimers represents a promising strategy to augment vaccine-elicited NAb responses.

    Importance It is currently not known how to potent generate neutralizing antibodies (NAbs) to the diversity of circulating HIV-1 envelopes (Env) by vaccination. One strategy to address this diversity is to utilize mixtures of different soluble HIV-1 envelope proteins. In this study, we generated and characterized three distinct, novel acute clade C soluble trimers. We vaccinated guinea pigs with single trimers as well as mixtures of trimers, and we found that a mixture of four trimers elicited a greater magnitude of NAbs than any single trimer within the mixture. The results of this study suggest that further development of Env trimer cocktails is warranted.

  • The Epstein-Barr Virus BamHI C Promoter Is Not Essential For B Cell Immortalization In Vitro But Greatly Enhances B Cell Growth Transformation [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) infection of B cells leads to the sequential activation of two viral promoters, Wp and Cp, resulting in the expression of six EBV nuclear antigens (EBNAs) and the viral Bcl2 homologue BHRF1. The viral transactivator EBNA2 is required for this switch from Wp to Cp usage during the initial stages of infection. EBNA2-dependent Cp transcription is mediated by the EBNA2-response element (E2RE), a region that contains at least two binding sites for cellular factors; one of these sites, CBF1, interacts with RBP-JK which then recruits EBNA2 to the transcription initiation complex. Here we demonstrate that the B cell specific transcription factor BSAP/Pax5 binds to a second site, CBF2, in the E2RE. Deletion of the E2RE in the context of a recombinant virus greatly diminished levels of Cp-initiated transcripts during the initial stages of infection, but did not affect the levels of Wp-initiated transcripts or EBNA mRNAs. Consistent with this finding, viruses deleted for the E2RE were not markedly impaired in their ability to induce B cell transformation in vitro. By contrast, a larger deletion of the entire Cp region did reduce EBNA mRNA levels early after infection and subsequently almost completely ablated LCL outgrowth. Notably however, rare LCLs could be established following infection with Cp-deleted viruses and these were indistinguishable from wild type-derived LCLs in terms of steady state EBV gene transcription. These data indicate that, unlike Wp, Cp is dispensable for the virus' growth transforming activity.

    IMPORTANCE Epstein-Barr virus (EBV), a B lymphotropic herpesvirus aetiologically linked to several B cell malignancies, efficiently induces B cell proliferation leading to the outgrowth of lymphoblastoid cell lines (LCL). The initial stages of this growth transforming infection are characterized by the sequential activation of two viral promoters, Wp and Cp, both of which appear to be preferentially active in target B cells. In this work we have investigated the importance of Cp activity in initiating B cell proliferation and maintaining LCL growth. Using recombinant viruses we demonstrate that, while Cp is not essential for LCL outgrowth in vitro, it enhances transformation efficiency by more than 100-fold. We also show that Cp, like Wp, interacts with the B cell specific activator protein BSAP/Pax5. We suggest that EBV has evolved this two promoter system to ensure efficient colonization of the host B cell system in vivo.

  • Viromes, not gene markers for studying dsDNA viral communities [PublishAheadOfPrint]

  • Microbes are recently recognized as dominant forces in nature with studies benefiting from gene markers that can be quickly, informatively and universally surveyed. Viruses, where explored, have proven powerful modulators of locally and globally important microbes through mortality, horizontal gene transfer, and metabolic reprogramming. However, community-wide virus studies have been challenged by the lack of a universal marker. Here I propose that viral metagenomics has advanced to largely take over study of double-stranded DNA viruses.

  • Papillomavirus E7 Oncoproteins Share Functions with Polyomavirus Small T Antigens [PublishAheadOfPrint]

  • Many of the small DNA tumor viruses encode transforming proteins that function by targeting critical cellular pathways involved in cell proliferation and survival. In this study we have examined whether some of the functions of the polyomavirus small T antigens (ST) are shared by the E6 and E7 oncoproteins of two oncogenic papillomaviruses. Using three different assays we have found that E7 can provide some SV40 or murine polyomavirus (PyV) ST functions. Both HPV16 and BPV1 E7 proteins are capable of partially substituting for SV40 ST in a transformation assay that also includes SV40 large T antigen, the catalytic subunit of cellular telomerase, and oncogenic Ras. Like SV40 ST, HPV16 E7 has the ability to override a quiescence block induced by mitogen deprivation. Like PyV ST, it also has the ability to inhibit myoblast differentiation. At least two of these activities are dependent upon the interaction of HPV16 E7 with retinoblastoma protein family members. For small T antigens, interaction with PP2A is needed for each of these functions. Even though there is no strong evidence that E6 or E7 share the ability of small T to interact with PP2A, E7 provides these functions related to cellular transformation.

    IMPORTANCE DNA tumor viruses have provided major insights into how cancers develop. Some viruses, like the human papillomaviruses, can cause cancer directly. Both the papillomaviruses and the polyomaviruses have served as tools for understanding pathways that are often perturbed in cancer. Here we have compared the functions of transforming proteins from several DNA tumor viruses including two papillomaviruses and two polyomaviruses. We tested the papillomavirus E6 and E7 oncoproteins in three functional assays and found that E7 can provide some or all of the functions of the SV40 small T antigen (ST), another well-characterized oncoprotein, in two of these assays. In a third assay, the papillomavirus E7 has the same effect as the murine polyomavirus small T protein. In summary, we report several new functions for the papillomavirus E7 proteins, which will contribute new insights into the roles of viruses in cancer and the cellular pathways they perturb in carcinogenesis.

  • Suppression of TGF{beta}-R2 and Smad5 is Associated with High Levels of miR-155 in the Oral Mucosa during Chronic Simian Immunodeficiency Virus Infection [PublishAheadOfPrint]

  • Chronic human and simian immunodeficiency virus (HIV and SIV) infections are characterized by mucosal inflammation in the presence of anti-inflammatory cytokines such as TGFbbeta;. The mechanisms for refractiveness to TGFbbeta; is not clear. Here we show that the expression of miR-155 was significantly upregulated in the oropharyngeal mucosa during chronic SIV infection that was coincident with a down regulation of TGFbbeta;-R2 and SMAD5, key TGFbbeta; signaling genes that harbor putative target sites for miR-155. Ectopic expression of miR-155 in vitro was found to significantly down regulate TGFbbeta;-R2 and Smad5 expression suggesting a role for miR-155 in the suppression of TGFbbeta;-R2 and SMAD5 genes in vivo. The down regulation of TGFbbeta; signaling genes by miR-155 likely contributes to the non-responsiveness to TGFbbeta; during SIV infection and may inadvertently aid in increased immune activation during HIV and SIV infections.

  • A Viable Recombinant Rhabdovirus Lacking Its Glycoprotein Gene and Expressing Influenza Hemagglutinin and Neuraminidase Is a Potent Influenza Vaccine [PublishAheadOfPrint]

  • The emergence of novel influenza viruses that cause devastating human disease is an ongoing threat, and serves as an impetus for continued development of novel approaches to influenza vaccines. Influenza vaccine development has traditionally focused on producing humoral and/or cell-mediated immunity, often against the viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Here, we describe a new vaccine candidate that utilizes a replication-defective vesicular stomatitis virus (VSV) vector backbone that lacks the native G surface glycoprotein gene (VSVG). The expression of the H5 HA of an H5N1 highly pathogenic avian influenza virus (HPAIV), A/Vietnam/1203/04 (VN1203), and the NA of the mouse-adapted H1N1 influenza virus, A/Puerto Rico/8/34 (PR8), in the VSVG vector restored the ability of the recombinant virus to replicate in cell culture, without the requirement for addition of trypsin. We show here that this recombinant virus vaccine candidate was non-pathogenic in mice when given by either intramuscular or intranasal routes of immunization, and that in vivo replication of the VSVG-H5N1 virus is profoundly attenuated. It also provided protection against lethal H5N1 infection after a single dose. This novel approach to vaccination against HPAIVs may be widely applicable to other emerging strains of influenza.

    IMPORTANCE Preparation for a potentially catastrophic influenza pandemic requires novel influenza vaccines that are safe, can be produced and administered quickly, and are effective, both soon after administration, and for a long duration. We have created a new influenza vaccine that utilizes an attenuated vesicular stomatitis virus (VSV) vector, to deliver and express influenza proteins against which vaccinated animals develop potent antibody responses. The influenza hemagglutinin and neuraminidase proteins, expressed on the surface of VSV viral particles, allowed this vaccine to grow in cell culture, and induced a potent antibody response in mice that was effective against infection with a lethal influenza virus. The mice showed no adverse reactions to the vaccine, and they were protected against an otherwise lethal influenza infection after only 14 days post-vaccination, and after as many as 140 days post-vaccination. The ability to rapidly produce this safe and effective vaccine in cell culture is additionally advantageous.

  • Influenza A virus reservoirs and intermediate hosts: dogs, horses, and new possibilities for human infections [PublishAheadOfPrint]

  • Influenza A virus (IAV) infections in hosts outside the main aquatic bird reservoirs occur periodically. Although most such cross-species transmission events experience limited onward transmission in the new host, sustained influenza outbreaks have occurred in poultry and in a number of mammalian species including humans, pigs, horses, seals and mink. Recently, two distinct strains of IAV have emerged in domestic dogs, with each circulating widely for several years. Here, we briefly outline what is known about the role of intermediate hosts in influenza emergence, summarize our knowledge of the new canine influenza viruses (CIVs) and how they provide key new information on the process of host adaptation, and assess the risk these viruses pose to human populations.

  • Circovirus Transport Proceeds via Direct Interaction of the Cytoplasmic Dynein IC1 Subunit with the Viral Capsid Protein [PublishAheadOfPrint]

  • Microtubule transport of circovirus from the periphery of the cell to the nucleus is essential for viral replication in early infection. How the microtubule is recruited to the viral cargo remains unclear. In this study, we observed that circovirus trafficking is dependent on microtubule polymerization, and incoming circovirus particles colocalize with cytoplasmic dynein and endosomes. However, circovirus binding to dynein was independent of microtubular aalpha;-tubulin and translocated cytoplasmic dynein into the nucleus. The circovirus capsid (Cap) subunit enhanced microtubular acetylation and directly interacted with the intermediate chain 1 (IC1) of dynein. N-terminal residues 42-100 of the viral protein Cap were required for efficient binding to the dynein IC1 subunit and for retrograde transport. Knockdown of IC1 decreased virus transport and replication. These results demonstrate that Cap is a direct ligand of the cytoplasmic dynein IC1 subunit and an inducer of microtubule aalpha;-tubulin acetylation. Furthermore, Cap recruits the host dynein/microtubule machinery to facilitate transport towards the nucleus by an endosomal mechanism distinct from that used for physiological dynein cargo.

    IMPORTANCE Incoming viral particles hijack the intracellular trafficking machinery of the host in order to migrate from the cell surface to the replication sites. Better knowledge of the interaction between viruses and virus proteins and the intracellular trafficking machinery may provide new targets for antiviral therapies. Currently, little is known about the molecular mechanisms of circovirus transport. Here, we report that circovirus particles enter early endosomes and utilize the microtubule-associated molecular motor dynein to travel along microtubules. The circovirus capsid subunit enhances microtubular acetylation, and the N-terminal residues 42-100 directly interact with the dynein IC1 subunit during retrograde transport. These findings highlight a mechanism whereby circoviruses recruit dynein for transport to the nucleus via the dynein/microtubule machinery.

  • Regulation of Multiple Stages of Hepadnavirus Replication by Carboxyl-Terminal Domain of Viral Core Protein in Trans [PublishAheadOfPrint]

  • Mutational analyses have indicated that the carboxyl-terminal domain (CTD) of hepadnavirus core protein, and its state of phosphorylation, are critical for multiple steps in viral replication. Also, CTD interacts with host proteins in a phosphorylation state-dependent manner. To ascertain the role of CTD in viral replication without perturbing its sequence and the role of CTD-host interactions, CTD of the human hepatitis B virus (HBV) or duck hepatitis B virus (DHBV) core protein, either wild type (WT) or with alanine or glutamic acid/aspartic acid substitutions at the phosphorylation sites, were expressed in cells replicating DHBV with WT core protein. A dramatic decrease in phosphorylation of the DHBV core protein (DHBc) was observed when the WT and most HBV core protein CTD (HCTD) variants were co-expressed in trans, which was accompanied by a profound reduction of viral core DNA and in particular, the double-stranded DNA. One HCTD variant that failed to change DHBc phosphorylation also had no effect on DHBV core DNA. All WT and variant HCTDs and DHBc CTDs (DCTDs) decreased the DHBV covalently closed circular (CCC) DNA. Identification of CTD-host interactions indicated that CDK2 binding by CTD may mediate its inhibitory effect on DHBc phosphorylation and reverse transcription via competition with DHBc for the host kinase, whereas importin aalpha; binding by CTD may contribute to inhibition of CCC DNA production by competitively blocking the nuclear import of viral nucleocapsids. These results suggest the possibility of blocking multiple steps of viral replication, especially CCC DNA formation, via inhibition of CTD functions.

    Importance Mutational analyses have suggested that the carboxyl-terminal domain (CTD) of hepadnavirus core protein is critical for viral replication. However, results from mutational analyses are subject to alternative interpretations. Also, how CTD affects virus replication remains unclear. In this study, we took an alternative approach to mutagenesis by overexpressing CTD alone in cells replicating the virus with the wild type core protein to determine the roles of CTD in viral replication. Our results revealed that CTD can inhibit multiple stages of viral replication, and its effects may be mediated at least in part through specific host interactions. They suggest that CTD, or its mimics, may have therapeutic potential. Furthermore, our experimental approach should be broadly applicable as a complement to mutagenesis for studying protein functions and interactions while at the same time providing a means to identify the relevant interacting factors.

  • Post-entry restriction of Mason-Pfizer monkey virus in mouse cells [PublishAheadOfPrint]

  • Mason-Pfizer monkey virus (M-PMV) is a prototypical betaretrovirus responsible for simian acquired immunodeficiency syndrome (SAIDS) in rhesus macaques. It has been shown previously that mouse cells are resistant to infection by HIV-1 and other primate lentiviruses. However, the susceptibility of mouse cells to primate retroviruses such as M-PMV remains unexplored. In the present study, using single-round GFP reporter viruses, we show that various mouse cell lines are unable to support the early stages of M-PMV replication. The block to infection occurs post-entry and is independent of the viral envelope. Using quantitative real-time PCR, we show that the block to infection occurs after reverse transcription but before formation of circular DNA or proviral DNA. Finally, we show that the M-PMV block in mouse cells is not attributable to the previously characterized mouse restriction factor Fv1. Overall, these findings suggest that mouse cells exhibit a previously uncharacterized block to M-PMV infection.

    IMPORTANCE Here we document a novel post-entry restriction to M-PMV infection in mouse cells. The block occurs after reverse transcription but before the formation of circular or proviral DNA, and is independent of the previous characterized mouse restriction factor Fv1.

  • Functional comparison of HSV-1 and HSV-2 ICP27 homologs reveals a role for ICP27 in virion release [PublishAheadOfPrint]

  • Numerous studies have focused on the regulatory functions of ICP27, an immediate-early (IE) protein of herpes simplex virus type 1 (HSV-1). However, its homolog in HSV-2, termed ICP27t2, has been little studied. Here, we used two different approaches to functionally compare ICP27t2 and ICP27. In transfection-based assays, ICP27t2 closely resembled ICP27 in its capacity to enhance HSV-1 late gene expression, suppress the splicing of a viral intron, and complement the growth of an HSV-1 ICP27 null mutant. To study ICP27t2 in the context of viral infection, we engineered K2F1, an HSV-1 mutant that encodes ICP27t2 in place of ICP27. In Vero cells, K2F1 replicated with wild-type (WT) kinetics and yields, expressed delayed-early and late proteins normally, and was fully capable of activating several cellular signal transduction pathways that are ICP27-dependent. Thus, we conclude that ICP27t2 and ICP27 are functionally very similar, and that ICP27t2 can mediate all ICP27 activities that are required for HSV-1 replication in cell culture. Surprisingly, however, we found that K2F1 forms plaques that are morphologically different from those of WT HSV-1. Investigation of this trait demonstrated that it results from the decreased release of progeny virions into the culture media. This appears to be due to a reduction in the detachment of K2F1 progeny from the extracellular surface of the infected cell. We identified two HSV-1 ICP27 amino-terminal deletion mutants with a similar release defect. Together, these results demonstrate that ICP27 plays a heretofore unappreciated role in modulating the efficiency of progeny virion release.

    IMPORTANCE ICP27 is an essential, multifunctional regulatory protein that has a number of critical roles in the HSV-1 life-cycle. Although ICP27 homologs are encoded by all known members of the Herpesviridae, previous work with several of these homologs has shown that they cannot substitute for ICP27 in the context of HSV-1-infected cells. Here, we identify ICP27t2 as the first homolog that can efficiently replace ICP27 in HSV-1 infection. Unexpectedly, our results also reveal that the sequence of the ICP27 gene can affect the release of HSV-1 progeny virions from the infected cell. Thus, our comparative study has revealed a novel function for ICP27 in the regulation of virus release.

  • Ribonuclease L Targets Distinct Sites in Influenza A Virus RNAs [PublishAheadOfPrint]

  • Influenza A virus (IAV) infections are influenced by type 1 interferon-mediated antiviral defenses and by viral countermeasures to these defenses. When IAV NS1 protein is disabled, ribonuclease L (RNase L) restricts virus replication; however, the RNAs targeted for cleavage by RNase L under these conditions have not been defined. In this study, we used deep sequencing methods to identify RNase L cleavage sites within host and viral RNAs from IAV PR8NS1-infected A549 cells. ShRNA knockdown of RNase L allowed us to distinguish between RNase L-dependent and RNase L-independent cleavage sites. RNase L-dependent cleavage sites were evident at discrete locations in IAV RNA segments [both (+) and (-) strands]. Cleavage in PB2, PB1 and PA genomic RNAs suggests that viral RNPs are susceptible to cleavage by RNase L. Prominent amounts of cleavage mapped to specific regions within IAV RNAs, including some areas of increased synonymous site conservation. Among cellular RNAs, RNase L-dependent cleavage was most frequent at precise locations in ribosomal RNAs. Our data show that RNase L targets specific sites in both host and viral RNAs to restrict influenza virus replication when NS1 protein is disabled.

    IMPORTANCE Ribonuclease L (RNase L) is a critical component of interferon-regulated and dsRNA-activated antiviral host responses. We sought to determine how RNase L exerts its antiviral activity during influenza virus infection. We enhanced the antiviral activity of RNase L by disabling a viral protein, NS1, that inhibits the activation of RNase L. Then, using deep sequencing methods, we identified the host and viral RNAs targeted by RNase L. We found that RNase L cleaved viral RNAs and ribosomal RNAs at very precise locations. The direct cleavage of IAV RNAs by RNase L highlights an intimate battle between viral RNAs and an antiviral endonuclease.

  • Alpha-defensin HD5 Inhibits Furin Cleavage of HPV16 L2 to Block Infection [PublishAheadOfPrint]

  • Human papillomavirus (HPV) is a significant oncogenic virus, but the innate immune response to HPV is poorly understood. Human aalpha;-defensin 5 (HD5) is an innate immune effector peptide secreted by epithelial cells in the genitourinary tract. HD5 is broadly antimicrobial, exhibiting potent antiviral activity against HPV at physiologic concentrations; however, the specific mechanism of HD5-mediated inhibition against HPV is unknown. During infection, the HPV capsid undergoes several critical cell-mediated viral protein processing steps, including unfolding and cleavage of the minor capsid protein L2 by host cyclophilin B and furin. Using HPV16 pseudovirus, we show that HD5 interacts directly with the virus and inhibits the furin-mediated cleavage of L2 at the cell surface during infection at a step downstream of the cyclophilin B-mediated unfolding of L2. Importantly, HD5 does not affect the enzymatic activity of furin directly. Thus, our data supports a model in which HD5 prevents furin from accessing L2 by occluding the furin cleavage site via direct binding to the viral capsid.

    Importance Our study elucidates a new antiviral action for aalpha;-defensins against non-enveloped viruses in which HD5 directly interferes with a critical host-mediated viral processing step, furin cleavage of L2, at the cell surface. Blocking this key event has deleterious effects on the intracellular steps of virus infection. Thus, in addition to informing the antiviral mechanisms of aalpha;-defensins, our studies highlight the critical role of furin cleavage in HPV entry. Innate immune control, mediated in part by aalpha;-defensins expressed in the genital mucosa, may influence susceptibility to HPV infections that lead to cervical cancer. Moreover, understanding the mechanism of these natural antivirals may inform the design of therapeutics to limit HPV infection.

  • The genome of the nucleopolyhedrosis causing virus from Tipula oleracea (ToNV) sheds new light into the Nudiviridae family. [PublishAheadOfPrint]

  • A large dsDNA virus that produces occlusion bodies, typical of baculoviruses, has been described to infect crane fly larvae of the genus Tipula (Diptera, Tipulidae). Because of a lack of genomic data this virus has remained unclassified. Electron microscopy of an archival virus isolated from T. oleracea (ToNV) showed irregularly shaped occlusion bodies measuring from 2 to 5 mmu;m in length and 2 mmu;m in mid-diameter, filled with rode-shape virions containing single nucleocapsids within a bilayer envelope. Whole genome amplification and Roche-454 sequencing revealed a complete circular genome sequence of 145.7 kb, containing five direct repeat regions. We predicted 131 open reading frames, including a homolog of the polyhedrin gene encoding the major occlusion body protein of T. paludosa NPV. BLAST searches demonstrated that ToNV had 21 out of the 37 baculovirus core genes but shared 52 genes with nudiviruses. Phylogenomic analyses indicated that ToNV clearly belongs to the Nudiviridae family, but should probably be assigned to a new genus. Among nudiviruses, ToNV was most closely related to the Penaeus monodon NV and Heliothis zea NV clade but distantly related to Drosophila innubia NV, the other nudivirus infecting a Diptera. Lastly, ToNV was found most closely related to the nuvidirus ancestor of bracoviruses. This was also reflected in terms of gene content, as ToNV was the only known exogenous virus harboring homologs of the Cc50C22.6 and 27b (Cc50C22.7) genes found in the nudiviral genomic cluster involved in bracovirus particle production.

    IMPORTANCE The Nudiviridae is a family of arthropod dsDNA viruses from which striking cases of endogenization have been reported (i.e. symbiotic bracoviruses deriving from a nudivirus, and the endogenous nudivirus of the brown planthopper). Although related to baculoviruses, relatively little is known about the genomic diversity of exogenous nudiviruses. Here, we characterized, morphologically and genetically, an archival sample of the Tipula oleracea nudivirus (ToNV), which has the particularity of forming occlusion bodies. Comparative genomic and phylogenomic analyses showed ToNV to be to date the closest known relative of the exogenous ancestor of bracoviruses, and that ToNV should be assigned to a new genus. Moreover, we revised the homology relationships of nudiviral genes and identified a new set of 32 core genes for the Nudiviridae, of which 21 were also baculovirus core genes. These findings provide important insights into the evolutionary history of large arthropod dsDNA viruses.

  • HBV regulates apoptosis and tumorigenesis through miR15a-Smad7-TGF-{beta} pathway [PublishAheadOfPrint]

  • Hepatitis B virus (HBV) infection causes chronic hepatitis in hundreds of millions of people worldwide, which can eventually lead to hepatocellular carcinoma (HCC). Previously we found that HBV mRNAs can absorb miR-15a to affect apoptosis through Bcl-2 pathway. We asked whether HBV could inhibit apoptosis and promote tumorigenesis through different pathways. In this study, we identified that TGF-bbeta; pathway inhibitory factor Smad7 is a novel target of miR-15a. We demonstrated that HBV can up-regulate the level of Smad7 by down-regulating miR-15a. Furthermore we examined the level of Smad7 in liver samples from HBV-infected HCC patients, and found that HBV mRNAs are positively co-related with the level of Smad7. By taking the approach of immunoblotting and luciferase reporter assay, we revealed that HBV can abrogate TGF-bbeta; signaling via up-regulating Smad7. By using Annexin V staining and caspase 3/7 activity assay, we found that HBV can inhibit TGF-bbeta; induced apoptosis of HepG2 cells. We also showed that HBV can promote tumor growth in BALB/c nude mice through up-regulating the expression of Smad7. In conclusion, we demonstrated that HBV can up-regulate Smad7 expression and inhibit TGF-bbeta; signaling which makes the cells resistant to TGF-bbeta; induced apoptosis and promotes tumorigenesis.

    IMPORTANCE: Hepatitis B virus (HBV) infection causes chronic hepatitis, which can eventually lead to hepatocellular carcinoma (HCC). TGF-bbeta; signaling is closely linked to liver fibrosis, cirrhosis and subsequent HCC progression and plays a unique role in the pathogenesis of HCC. At the early stage of tumor formation, TGF-bbeta; functions as a tumor suppressor which inhibits the cell proliferation and induces apoptosis. Previously we found that HBV mRNAs can sponge off miR-15a to affect apoptosis through Bcl-2 pathway. In this study, we identified that TGF-bbeta; inhibitory factor Smad7 is a novel target of miR-15a. We reveal that HBV can abrogate TGF-bbeta; signaling via up-regulating Smad7 and inhibit TGF-bbeta; induced apoptosis as well as promote the tumor development. Our study provides the evidence to support the idea that viral RNAs can exert their functions as ceRNA towards miRNA and participate in important cellular process.

  • Structural and functional analysis of surface proteins from an A(H3N8) influenza virus isolated from New England harbor seals [PublishAheadOfPrint]

  • In late 2011, an A(H3N8) influenza virus infection resulted in the deaths of 162 New England harbor seals. Virus sequence analysis and virus receptor binding studies highlighted potential markers responsible for mammalian adaptation and a mixed receptor binding preference (Anthony et al. ; MBio 3:e00166-00112). Here we present a detailed structural and biochemical analysis of the surface antigens of this virus. Results obtained with recombinant proteins for both the hemagglutinin and neuraminidase, indicate a true avian receptor binding preference. Although the detection of this virus in new species highlights an increased potential for cross-species transmission with these viruses, our results indicate that the A(H3N8) virus currently poses a low risk to humans.

    Importance Cross-species transmission of zoonotic influenza viruses increases public health concerns. Here we report a molecular and structural study of the major surface proteins from an A(H3N8) influenza virus isolated from New England harbor seals. Results improve our understanding of these viruses as they evolve and provide important information to aid ongoing risk assessment analyses, as these zoonotic influenza viruses continue to circulate and adapt to new hosts.

  • Intracellular expression of camelid single domain antibodies specific for influenza virus nucleoprotein (NP) uncovers distinct features of its nuclear localization. [PublishAheadOfPrint]

  • Perturbation of protein-protein interactions relies mostly on genetic approaches or on chemical inhibition. Small RNA viruses, such as influenza A virus, do not easily lend themselves to the former approach, while chemical inhibition requires that the target protein be druggable. A lack of tools thus constrains the functional analysis of flu-encoded proteins. We generated a panel of camelid-derived single domain antibody fragments (VHHs) against influenza nucleoprotein (NP), a viral protein essential for nuclear trafficking and packaging of the influenza genome. We show that these VHHs can target NP in living cells and perturb NP's function during infection. Cytosolic expression of NP-specific VHHs (aalpha;NP-VHHs) disrupts virus replication at an early stage of the life cycle. Based on their specificity, these VHHs fall into two distinct groups. Both prevent nuclear import of the vRNP complex without disrupting nuclear import of NP alone. Different stages of the virus life cycle thus rely on distinct nuclear localization motifs of NP. Their molecular characterization may afford new means of intervention in the virus life cycle.

    Importance Many proteins encoded by RNA viruses are refractory to manipulation due to their essential role in replication. Thus, studying their function, and how to disrupt said function through pharmaceutical intervention, is difficult. We present a novel method based on single domain antibody technology that permits specific targeting and disruption of an essential flu protein in the absence of genetic manipulation of the flu virus itself. Characterization of such interactions may help identify new targets for pharmaceutical intervention. This approach can be extended to study proteins encoded by other viral pathogens.

  • Reduction of HIV-1 infectivity through ERAD-mediated Env depletion [PublishAheadOfPrint]

  • During HIV-1 replicative cycle the gp160 envelope is processed in the secretory pathway to mature into the gp41 and gp120 subunits. Misfolded proteins located within the endoplasmic reticulum (ER) are proteasomally degraded through the ER associated degradation (ERAD) pathway, a quality control system operating in this compartment. Here we exploited the ERAD pathway to induce the degradation of gp160 during viral production, thus leading to the release of gp120 depleted viral particles.

  • The proteasomal Rpn11 metalloprotease suppresses tombusvirus RNA recombination and promotes viral replication via facilitating the assembly of the viral replicase complex [PublishAheadOfPrint]

  • RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in evolutionary arms race with host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this paper, the authors have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in yeast and plants. Depletion or mutations of Rpn11p leads to the rapid formation of viral RNA recombinants in combination with reduced level of viral RNA replication in yeast or in vitro based on cell-free extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a "matchmaker" that brings the viral p92pol replication protein and the DDX3-like Ded1p/RH20 DEAD-box helicases into VRCs. Over-expression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs.

    Importance: RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in evolutionary arms race with host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, albeit the role of the host in virus evolution is still understudied. In this paper, the authors used a plant RNA virus, tombusvirus, to study the role of a cellular proteasomal protein, called Rpn11, in tombusvirus recombination in yeast model host, in plants and in vitro. They found that the cellular Rpn11 is subverted for tombusvirus replication and Rpn11 plays a proteasome-independent function in facilitating viral replication. When Rpn11 level is knocked down or a mutated Rpn11 is expressed, then tombusvirus RNA goes through rapid viral recombination and evolution. Taken together, the co-opted cellular Rpn11 is a critical host factor for tombusviruses by regulating viral replication and genetic recombination.

  • Upon intranasal VSV infection astrocytes in the olfactory bulb are important IFN-{beta} producers that protect from lethal encephalitis [PublishAheadOfPrint]

  • Previously we found that following intranasal (i.n.) infection with neurotropic vesicular stomatitis virus (VSV) type I interferon receptor (IFNAR) triggering of neuroectodermal cells was critically required to constrain intracerebral virus spread. To address whether locally active IFN-bbeta; was also induced proximally, we studied spatiotemporal conditions of VSV mediated IFN-bbeta; induction. To this end, we performed infection studies with IFN-bbeta; reporter mice. One day after intravenous (i.v.) VSV infection, luciferase induction was detected in lymph nodes. Upon i.n. infection luciferase induction was discovered at similar sites with delayed kinetics, whereas on day 3 and 4 post infection enhanced luciferase expression additionally was detected in the forehead of reporter mice. A detailed analysis of cell type-specific IFN-bbeta; reporter mice revealed that within the olfactory bulb IFN-bbeta; was expressed by neuroectodermal cells, primarily by astrocytes and to a lesser extent by neurons. Importantly, locally induced type I IFN triggered distal parts of the brain as indicated by the analysis of ISREeGFP mice which after i.n. VSV infection showed enhanced eGFP expression throughout the brain. Compared with WT mice, IFN-bbeta;-/- mice showed increased mortality to i.n. VSV infection, whereas upon i.v. infection no such differences were detected highlighting the biological significance of intra-cerebrally expressed IFN-bbeta;. In conclusion, upon i.n. VSV instillation, IFN-bbeta; responses mounted by astrocytes within the olfactory bulb critically contribute to the anti-viral defense by stimulating distal IFN-bbeta;-negative brain areas and thus arresting virus spread.

    Importance The central nervous system has long been considered as an immune privileged site. More recently it became evident that specialized immune mechanisms are active within the brain to control pathogens. Previously we showed that virus, which entered the brain via the olfactory route, was arrested within the olfactory bulb by a type I IFN dependent mechanism. Since peripheral type I IFN would not readily cross the blood brain barrier and within the brain so far no abundant type I IFN responses have been detected, here we addressed from where locally active IFN originated from. We found that upon intranasal VSV instillation primarily astrocytes, and to a lesser extent neurons, were stimulated within the olfactory bulb to mount IFN-bbeta; responses that also activated and protected distal brain areas. Our results are surprising because in other infection models astrocytes have not yet been identified as major type I IFN producers.

  • Inhibition of reactive oxygen species production ameliorates inflammation induced by influenza A viruses via up-regulation of SOCS1 and SOCS3 [PublishAheadOfPrint]

  • Highly pathogenic avian influenza infection is associated with severe mortality in both humans and poultry. The mechanisms of disease pathogenesis and immunity are poorly understood although recent evidence suggests that cytokine/chemokine dysregulation contributes to disease severity following H5N1 infection. Influenza A virus infection causes a rapid influx of inflammatory cells, resulting in increased reactive oxygen species production, cytokine expression and acute lung injury. Pro-inflammatory stimuli are known to induce intracellular reactive oxygen species by activating NADPH oxidase activity. We therefore hypothesised that inhibition of this activity would restore host cytokine homeostasis following avian influenza virus infection. A panel of airway epithelial and immune cells from mammalian and avian species were infected with A/Puerto Rico/8/1934 H1N1 virus, low pathogenic avian influenza H5N3 virus (A/duck/Victoria/0305-2/2012), highly pathogenic avian influenza H5N1 virus (A/chicken/Vietnam/0008/2004), or low pathogenic avian influenza H7N9 virus (A/Anhui/1/2013). Quantitative real-time reverse transcriptase PCR showed that H5N1 and H7N9 viruses significantly stimulated cytokine (IL-6, IFN-bbeta;, CXCL10 and CCL5) production. Among the influenza-induced cytokines, CCL5 was identified as a potential marker for overactive immunity. Apocynin, a Nox2 inhibitor, inhibited influenza-induced cytokines and reactive oxygen species production, although viral replication was not significantly altered in vitro. Interestingly, apocynin treatment significantly increased influenza virus-induced mRNA and protein expression of SOCS1 and SOCS3, enhancing negative regulation of cytokine signaling. These findings suggest that apocynin or its derivatives (targeting host responses) could be used in combination with anti-viral strategies (targeting viruses), as therapeutic agents to ameliorate disease severity in susceptible species.

    IMPORTANCE Highly pathogenic avian influenza virus infection causes severe morbidity and mortality in both humans and poultry. Wide-spread antiviral resistance necessitates the need for the development of additional novel therapeutic measures to modulate overactive host immune responses following infection. Disease severity following avian influenza virus infection can be attributed in part to hyper-induction of inflammatory mediators such as cytokines, chemokines and reactive oxygen species. Our study shows that highly pathogenic avian influenza H5N1 virus and low pathogenic avian influenza H7N9 virus (both associated with human fatalities) promote inactivation of FoxO3 and down-regulation of the TAM receptor tyrosine kinase, Tyro3, leading to augmentation of the inflammatory cytokine response. Inhibition of influenza-induced reactive oxygen species with apocynin activated FoxO3 and stimulated SOCS1 and SOCS3 proteins, restoring cytokine homeostasis. We conclude that modulation of host immune responses with antioxidant and/or anti-inflammatory agents in combination with antiviral therapy may have important therapeutic benefits.

  • Measles virus mutants possessing the fusion protein with enhanced fusion activity spread effectively in neuronal cells without causing strong cytopathology, but not in other cells [PublishAheadOfPrint]

  • Subacute sclerosing panencephalitis (SSPE) is caused by persistent measles virus (MV) infection in the central nervous system (CNS). Since human neurons, its main target cells, do not express known MV receptors (signaling lymphocyte activation molecule (SLAM) and nectin 4), it remains to be understood how MV infects and spreads in them. We have recently reported that fusion-enhancing substitutions in the extracellular domain of the MV fusion (F) protein (T461I and S103I/N462S/N465S), which are found in multiple SSPE virus isolates, promote MV spread in human neuroblastoma cell lines and brains of suckling hamsters. In this study, we show that hyperfusogenic viruses with these substitutions also spread efficiently in human primary neuron cultures without inducing syncytia. These substitutions were found to destabilize the pre-fusion conformation of the F protein trimer, thereby enhancing fusion activity. However, these hyperfusogenic viruses exhibited stronger cytopathology and produced lower titers at later time points in SLAM- or nectin 4-expressing cells, compared with the wild-type MV. Although these viruses spread efficiently in the brains of SLAM knock-in mice, they did not in the spleens. Taken together, the results suggest that enhanced fusion activity is beneficial for MV to spread in neuronal cells where no cytopathology occurs, but detrimental to other types of cells due to strong cytopathology. Acquisition of enhanced fusion activity through substitutions in the extracellular domain of the F protein may be crucial for MV's extensive spread in the CNS and development of SSPE.

    IMPORTANCE Subacute sclerosing panencephalitis (SSPE) is a fatal disease caused by persistent measles virus (MV) infection in the central nervous system (CNS). Its cause is not well understood, and no effective therapy is currently available. Recently, we have reported that enhanced fusion activity of MV through the mutations in its fusion protein is a major determinant of efficient virus spread in human neuronal cells and brains of suckling hamsters. In this study, we show that those mutations render the conformation of the fusion protein less stable, thereby making it hyperfusogenic. Our results also show that enhanced fusion activity is beneficial for MV to spread in the CNS, but rather detrimental to other types of cells in peripheral tissues, which are strongly damaged by the virus. Our findings provide important insight into the mechanism for the development of SSPE after MV infection.

  • MVA-infected Dendritic Cells Present CD4+ T-Cell Epitopes by Endogenous MHC Class II Presentation Pathways [PublishAheadOfPrint]

  • CD4+ T-lymphocytes play a central role in the immune system and mediate their function after recognition of their respective antigens presented on MHCII molecules on antigen presenting cells (APCs). Conventionally, phagocytosed antigens are loaded on MHCII for stimulation of CD4+ T-cells. Certain epitopes, however, can be directly processed from intracellular antigens and are presented on MHCII (endogenous MHCII presentation). Here we characterized the MHCII antigen presentation pathways being possibly involved in the immune response upon vaccination with MVA (modified vaccinia virus Ankara), a promising live viral vaccine vector. We established CD4+ T-cell lines specific for MVA-derived epitopes as tools for in vitro analysis of MHCII antigen processing and presentation in MVA-infected APCs. We provide evidence that infected APCs are able to directly transfer endogenous viral proteins into the MHCII pathway to efficiently activate CD4+ T-cells. By using knockout mice and chemical inhibitory compounds we further elucidate the molecular basis showing that among the various subcellular pathways investigated proteasomes and autophagy are key players in the endogenous MHCII presentation during MVA infection. Interestingly, although proteasomal processing plays an important role neither TAP nor LAMP-2 were found to be involved in the peptide transport.

    Defining the molecular mechanism of MHCII presentation during MVA infection provides a basis to improve MVA-based vaccination strategies aiming for enhanced CD4+ T-cell activation by targeting antigens into the responsible pathways.

    Importance Statement This work contributes significantly to our understanding of the immunogenic properties of pathogens by deciphering antigen processing pathways contributing to efficient activation of antigen-specific CD4+ T-cells. We identified autophagosome formation, proteasomal activity and lysosomal integrity to be crucial for endogenous CD4+ T-cell activation. Since poxvirus vectors such as MVA are already used in clinical trials as recombinant vaccines, the data provide important information for the future design of optimized poxviral vaccines for the study of advanced immunotherapy options.

  • Clustered microRNAs of the Epstein-Barr Virus Cooperatively Downregulate an Epithelial Cell-Specific Metastasis Suppressor [PublishAheadOfPrint]

  • The Epstein-Barr virus (EBV) encodes its own microRNAs (miRNAs); however, their biological roles remain elusive. The commonly used EBV B95-8 strain lacks a 12 kb genomic region, known as BamHI A Rightward Transcripts (BART) locus, where a number of BART miRNAs are encoded. Here, bacterial artificial chromosome (BAC) technology was used to generate an EBV B95-8 strain in which the 12 kb region was fully restored at its native locus [BART(+) virus)]. Epithelial cells were stably infected with either the parental B95-8 virus or the BART(+) virus, and BART miRNA expression was successfully reconstituted in the BART(+) virus-infected cells. Microarray analyses of cellular gene expression identified N-myc downstream regulated gene 1 (NDRG1) as a putative target of BART miRNAs. The NDRG1 protein was barely expressed in B-cells, highly expressed in epithelial cells, including primary epithelial cells, and strongly donwregulated in the BART(+) virus-infected epithelial cells of various origins. Although in vitro reporter assays identified BART22 as being responsible for the NDRG1 downregulation, EBV genetic analyses revealed that BART22 was not solely responsible; rather, the entire BART miRNA cluster 2 was responsible for the downregulation. Immunohistochemical analyses revealed that the expression level of the NDRG1 protein was downregulated significantly in EBV-positive nasopharyngeal carcinoma specimens. Considering that NDRG1 encodes an epithelial differentiation marker and a suppressor of metastasis, these data implicate a causative relationship between BART miRNA expression and epithelial carcinogenesis in vivo.

    Importance EBV-related epithelial cancers, such as nasopharyngeal carcinomas and EBV-positive gastric cancers, encompass more than 80% of EBV-related malignancies. Although it is known that they express high levels of virally-encoded BART miRNAs, how these miRNAs contribute to EBV-mediated epithelial carcinogenesis remains unknown. Although a number of screenings have been performed to identify targets of viral miRNAs, many targets likely have not been identified, especially in case of epithelial cell infection. This is the first study to use EBV genetics to perform unbiased screens of cellular genes that are differentially expressed in viral miRNA-positive and -negative epithelial cells. The result indicates that multiple EBV-encoded miRNAs cooperatively downregulate NDRG1, an epithelial differentiation marker and suppressor of metastasis. The experimental system described in this study should be useful for further clarifying the mechanism of EBV-mediated epithelial carcinogenesis.

  • Acanthamoeba polyphaga mimivirus prevents amoebal encystment-mediating serine proteinase expression and circumvents cell encystment [PublishAheadOfPrint]

  • Acanthamoeba is a genus of free-living amoebas distributed worldwide. Few studies have explored the interactions between these protozoa and their infecting giant virus, Acanthamoeba polyphaga mimivirus (APMV). Here we show that once the amoebal encystment is triggered, trophozoites become significantly resistant to APMV. Otherwise, upon infection, APMV is able to interfere with the expression of a serine proteinase related to amoebal encystment and the encystment can no longer be triggered.

  • Nanobody binding to a conserved epitope promoted human norovirus particle disassembly [PublishAheadOfPrint]

  • Human noroviruses are icosahedral single-stranded RNA viruses. The capsid protein is divided into shell (S) and protruding (P) domains, which are connected by a flexible hinge region. There are numerous genetically and antigenically distinct noroviruses and the dominant strains evolve every other year. Vaccine and antiviral development is hampered by the difficulties in growing human norovirus in cell culture and the continually evolving strains. Here, we show the X-ray crystal structures of human norovirus P domains in complex with two different Nanobodies. One Nanobody, Nano-85, was broadly reactive, while the other, Nano-25, was strain specific. We showed that both Nanobodies bound to the lower region on the P domain and had nanomolar affinities. The Nano-85 binding site mainly compromised of highly conserved amino acids among the genetically distinct genogroup II noroviruses. Several of the conserved residues were also recognized by a broadly reactive monoclonal antibody, which suggested this region contained a dominant epitope. Superposition of the P domain Nanobody complex structures into a cryo-EM particle structure revealed that both Nanobodies bound at occluded sites on the particles. The flexible hinge region, which contained ~10-12 amino acids, likely permitted a certain degree of P domain movement on the particles in order to accommodate the Nanobodies. Interestingly, the Nano-85 binding interaction with intact particles caused the particles to disassemble in vitro. Altogether, these results suggested that the highly conserved Nano-85 binding epitope contained a trigger mechanism for particle disassembly. Principally, this epitope represents a potential site of norovirus vulnerability.

    IMPORTANCE We characterized two different Nanobodies (Nano-85 and Nano-25) that bind to human noroviruses. Both Nanobodies bound with high affinities to the lower region of the P domain, which was occluded on intact particles. Nano-25 was specific for GII.10, whereas Nano-85 bound several different GII genotypes, including GII.4, GII.10, and GII.12. We showed that Nano-85 was able to detect norovirus virions in clinical stool specimens using a sandwich ELISA. Importantly, we found that Nano-85 binding to intact particles caused the particles to disassemble. We believe that with further testing, Nano-85 will not only work as a diagnostic reagent in norovirus detection systems, but could also function as a broadly reactive GII norovirus antiviral.

  • Structure of an Enteric Pathogen Bovine Parvovirus [PublishAheadOfPrint]

  • Bovine Parvovirus (BPV), the causative agent of respiratory and gastrointestinal disease in cows, is the type member of the Bocaparvovirus genus of the Parvoviridae. Towards efforts to obtain a template for the development of vaccines and small molecule inhibitors for this pathogen, the structure of the BPV capsid, assembled from the major capsid viral protein 2 (VP2), was determined using X-ray crystallography and cryo-electron microscopy and three-dimensional image reconstruction to 3.2 and 8.8 AAring; resolution, respectively. The VP2 region ordered in the crystal structure, residues 39-536, conserves the parvoviral eight-stranded jellyroll motif and an aalpha;A helix. The BPV capsid displays common parvovirus features: a channel at and depressions surrounding the 5-fold axes, and protrusions surrounding the 3-fold axes. However, rather than a depression centered at the 2-fold axes, a raised surface loop divides this feature in BPV. Additional observed density in the capsid interior in the cryo-reconstructed map, compared to the crystal structure, is interpreted as ten additional N-terminal residues, 29-38 that radially extends the channel under the 5-fold axis, as observed for Human Bocavirus 1. Surface loops of varying lengths and conformations extend from the core jellyroll motif of VP2. These confer the unique surface topology of the BPV capsid, making it strikingly different from HBoV1 as well as the type members of other Parvovirinae genera for which structures have been determined. For the type members, structurally analogous regions to those decorating the BPV capsid surface serve as determinants of receptor recognition, tissue and host tropism, pathogenicity, and antigenicity.

    Importance Bovine parvovirus (BPV), identified in the 1960s in diarrheic calves, is a type member of the Bocaparvovirus genus of the non-enveloped, ssDNA Parvoviridae family. Recent isolation of human bocaparvoviruses from children with severe respiratory and gastrointestinal infections has generated interest in understanding the lifecycle and pathogenesis of these emerging viruses. We have determined high-resolution structure of the BPV capsid assembled from its predominant capsid protein VP2, known to be involved in a myriad of functions during host cell entry, pathogenesis, and antigenicity for other Parvovirinae members. Our results indicate the conservation of the core secondary structural elements and the location of the N-terminal residues for the known bocaparvovirus capsid structures. However, surface loops with high variability in sequence and conformation give BPV a unique capsid surface topology. Similar analogous regions in other Parvovirinae type members are important as determinants of receptor recognition, tissue and host tropism, pathogenicity, and antigenicity.

  • Improving Neutralization Potency and Breadth by Combining Broadly Reactive HIV-1 Antibodies Targeting Major Neutralization Epitopes [PublishAheadOfPrint]

  • The isolation of broadly neutralizing HIV-1 monoclonal antibodies (mAbs) to distinct epitopes on the viral envelope glycoprotein (Env) provides the potential to use combinations of mAbs for prevention and treatment of HIV-1 infection. Since many of these mAbs have been isolated in the last few years, the potency and breadth of mAb combinations has not been well characterized. In two parallel experiments, we examined the in vitro neutralizing activities of double, triple and quadruple mAb combinations targeting four distinct epitopes, including the CD4-binding site, the V1V2-glycan region, the V3-glycan supersite and the gp41 membrane-proximal external region (MPER), using a panel of 125 Env-pseudotyped viruses. All mAb combinations showed substantially improved neutralization breadth compared to the corresponding single mAbs, while the neutralization potency of individual mAbs was maintained. At an IC50 cutoff of 1 mmu;g/ml per antibody, double mAb combinations neutralized 89-98% of viruses, and triple combinations neutralized 98-100%. Overall the improvement of neutralization breadth was closely predicted by an additive effect model and explained by complementary neutralization profiles of antibodies recognizing distinct epitopes. Subtle but consistent favorable interactions were observed in some mAb combinations whereas less favorable interactions were observed on a small subset of viruses that are highly sensitive to V3-glycan mAbs. These data demonstrate favorable in vitro combinations of broadly neutralizing HIV-1 mAbs and suggest that such combinations could have utility for HIV-1 prevention and treatment.

    Importance Over the last five years, numerous broadly reactive HIV-1 neutralizing mAbs have been isolated from B-cells of HIV-1 infected donors. Each of these mAbs binds to one of the major vulnerable sites (epitopes) on the surface of the viral envelope glycoprotein. Since antibodies to distinct viral epitopes could theoretically act together to provide greater potency and breadth of virus neutralization, we tested physical mixtures of double, triple and quadruple combinations of neutralizing mAbs targeting four major epitopes on the HIV-1 Env. When tested together, antibody combinations showed substantially improved neutralization breadth compared to single mAbs. This improvement could be explained by the complementary neutralization profiles of individual mAbs. We further demonstrated that each antibody maintained its full neutralization potency when used in combination with other mAbs. These data provided a rationale for clinical use of antibody based combinations for HIV-1 prevention and therapy.

  • COMMD1/Murr1 reinforces HIV-1 latent infection through I{kappa}B-{alpha} stabilization. [PublishAheadOfPrint]

  • The transcription factor NF-B is important for HIV-1 transcription initiation in HIV-1 primary infection and reactivation in HIV-1 latent infected cells. However, comparative analysis of the regulation and function of NF-B in HIV-1 latent infected cells has not been explored. Here we show that expression of IB-aalpha;, an endogenous inhibitor of NF-B, is enhanced by HIV-1 latent infection via induction of host-derived factor COMMD1/Murr1 in myeloid cells but not in lymphoid cells using 4 sets of HIV-1 latent infected cells and their respective parental cells. IB-aalpha; protein was stabilized by COMMD1, which attenuated NF-B signaling during Toll-like receptor ligands and TNFaalpha; treatment, and enhanced HIV-1 latency in HIV-1 latent infected cells. Activation of PI3K and JAK pathway is involved in COMMD1 induction in HIV-1 latent infected cells. Our findings indicate that COMMD1 induction is the inhibition mechanism of NF-B in HIV-1 latent infected cells, which contributes to innate immune deficiency and reinforcing the HIV-1 latency. Thus, COMMD1 might be a double-edged sword, beneficial in primary infection but not beneficial in latent infection when considering HIV-1 eradication.

    IMPORTANCE HIV-1 latency poses a major barrier to viral eradication at the era of combination of anti-retroviral therapy (cART). In this study, we found that COMMD1/Murr1, previously identified as an HIV-1 restriction factor, inhibits the proteasomal degradation of IB-aalpha; via increasing the interaction with IB-aalpha; in myeloid HIV-1 latent infected cells. IB-aalpha; protein was stabilized by COMMD1, which attenuated NF-B signaling during innate immune response and enhanced HIV-1 latency in HIV-1 latent infected cells. Activation of PI3K and JAK pathway is involved in COMMD1 induction in HIV-1 latent infected cells. Thus, host-derived factor COMMD1 is beneficial in suppressing primary infection, but it enhances latent infection, indicating a double-edged sword for HIV-1 eradication.

  • Structural basis for development of avian virus capsids to display influenza virus proteins for induction of protective immunity [PublishAheadOfPrint]

  • Bioengineering of viruses and virus-like particles (VLP) is a well-established approach in the development of new and improved vaccines to viral and bacterial pathogens. We report here that the capsid of a major avian pathogen, infectious bursal disease virus (IBDV), can accommodate heterologous proteins to induce protective immunity. The structural units of the ~70-nm-diameter T=13 IBDV capsid are trimers of VP2, which is made as a precursor (pVP2). The pVP2 C-terminal domain has an amphipathic aalpha;-helix that controls VP2 polymorphism. In the absence of VP3 scaffolding protein, pVP2 intermediates bearing this aalpha;-helix assemble into genuine VLP only when expressed with an N-terminal His tag (protein HT-VP2-466). HT-VP2-466 capsids are optimal for protein insertion, as they are large enough (~78,000 nm3 cargo space) and are assembled from a single protein. We explored HT-VP2-466-based chimeric capsids, initially using enhanced green fluorescent protein (EGFP). VLP assembly yield was efficient when we coexpressed EGFP-HT-VP2-466 and HT-VP2-466 from two recombinant baculoviruses. The native EGFP structure (~240 copies/virion) was successfully inserted in a functional form, as VLP were fluorescent, and three-dimensional cryo-electron microscopy showed EGFP molecules incorporated at the inner capsid surface. Immunization of mice with purified EGFP-VLP particles elicited anti-EGFP antibodies. We also inserted hemagglutinin (HA) and matrix (M2) protein epitopes derived from the mouse-adapted A/PR/8/34 influenza virus and engineered several HA- and M2-derived chimeric capsids. Mice immunized with VLP containing the HA stalk, an M2 fragment, or both antigens developed full protection against viral challenge.

    Importance Virus-like particles (VLP) are multimeric protein cages that mimic the infectious virus capsid and are potential candidates for non-living vaccines that induce long-lasting protection. Chimeric VLP can display or include foreign antigens, which could be a conserved epitope to elicit broadly neutralizing antibodies, or several variable epitopes effective against a large number of viral strains. We report the biochemical, structural and immunological characterization of chimeric VLP derived from infectious bursal disease virus (IBDV), an important poultry pathogen. To test the potential of IBDV VLP as a vaccine vehicle, we used the enhanced green fluorescent protein and two fragments derived from hemagglutinin and the M2 matrix protein of the human murine-adapted influenza virus. The IBDV capsid protein fused to influenza virus peptides formed assemblies able to protect mice against viral challenge. Our studies establish the basis for a new generation of multivalent IBDV-based vaccines.

  • SARS-CoV vaccines formulated with delta inulin adjuvants provide enhanced virus protection while ameliorating lung eosinophilic immunopathology [PublishAheadOfPrint]

  • Although the SARS coronavirus (SARS-CoV) epidemic was controlled by non-vaccine measures, coronaviruses remain a major threat to human health. Design of optimal coronavirus vaccines therefore remains a priority. Such vaccines present major challenges; coronavirus immunity often wanes rapidly, individuals needing to be protected include the elderly, and vaccines may exacerbate rather than prevent coronavirus lung immunopathology. To address these issues, we compared in a murine model a range of recombinant spike protein or inactivated whole virus vaccine candidates alone or adjuvanted with either alum, CpG or Advaxttrade;, a new delta inulin-based polysaccharide adjuvant. While all vaccines protected against lethal infection, addition of adjuvant significantly increased serum neutralizing antibody titers and reduced lung virus titers on day 3 post-challenge. Whereas unadjuvanted or alum-formulated vaccines were associated with significantly increased lung eosinophilic immunopathology on day 6 post-challenge, this was not seen in mice immunized with vaccines formulated with delta inulin adjuvant. Protection against eosinophilic immunopathology by vaccines containing delta inulin adjuvants correlated better with enhanced T-cell IFN- recall responses rather than reduced IL4 responses, suggesting immunopathology predominantly reflects an inadequate vaccine-induced Th1 response. This study highlights the critical importance for development of effective and safe coronavirus vaccines of selection of adjuvants based on ability to induce durable IFN- responses.

    Importance Coronaviruses such as SARS-CoV and MERS-CoV cause high case fatality rates and remain major human public health threats, creating a need for effective vaccines. Whilst coronavirus antigens that induce protective neutralizing antibodies have been identified, coronavirus vaccines present a unique problem in that immunized individuals when infected by virus can develop lung eosinophilic pathology, a problem that is further exacerbated by formulation of SARS-CoV vaccines with alum adjuvants. This study shows that formulation of SARS-CoV spike protein or inactivated whole virus vaccines with novel delta inulin-based polysaccharide adjuvants enhances neutralizing antibody titers and protection against clinical disease but at the same time also protects against development of lung eosinophilic immunopathology. It also shows that immunity achieved with delta inulin adjuvants is long-lived thereby overcoming the natural tendency for rapidly waning coronavirus immunity. Thus delta inulin adjuvants may offer a unique ability to develop safer and more effective coronavirus vaccines.

  • LMP1 promotes expression of IGF1 to selectively activate IGF1R and drive cell proliferation [PublishAheadOfPrint]

  • Epstein-Barr Virus (EBV) is a gammaherpes virus that infects the majority of the human population and it is linked to the development of multiple cancers including nasopharyngeal carcinoma. Latent membrane protein 1 (LMP1) is considered the primary oncoprotein of EBV and in epithelial cells it induces the expression and activation, or phosphorylation, of the epidermal growth factor receptor kinase. To identify effects on additional kinases, an unbiased screen of receptor tyrosine kinases potentially activated by LMP1 was performed. Using a protein array, it was determined that LMP1 selectively activates Insulin-like Growth Factor 1 Receptor (IGF1R). This activation takes place in fibroblast, epithelial, and nasopharyngeal cell lines that expressed LMP1 stably and transiently. Of note, LMP1 altered the phosphorylation, but not the expression, of IGF1R. LMP1 mutants with defective signaling domains revealed that the C-Terminal Activating Region 2 domain of LMP1 increased the mRNA expression and the secretion of the ligand IGF1, which promoted phosphorylation of IGF1R. IGF1R phosphorylation was dependent upon activation of canonical NF-B signaling and was suppressed by IBaalpha; and a dominant negative form of TRAF6. Inhibition of IGF1R activation with two small molecule inhibitors, AG1024 and PPP, or with shRNA directed against IGF1R selectively reduced proliferation, foci formation, and Akt activation in LMP1-positive cells but did not impair LMP1-induced cell migration. Expression of constitutively active Akt rescued cell proliferation in the presence of IGF1R inhibitors. These findings suggest that LMP1-mediated activation of IGF1R contributes to the ability of LMP1 to transform epithelial cells.

    Importance: EBV is linked to the development of multiple cancers in both lymphoid and epithelial cells, including nasopharyngeal carcinoma. Nasopharyngeal carcinoma is a major cancer that develops in specific populations with nearly 80,000 new cases reported annually. LMP1 is consistently expressed in early lesions and continues to be detected within 50-80% of these cancers at later stages. It is therefore of paramount importance to understand the mechanisms through which LMP1 alters cell growth and contributes to tumorigenesis. This study is the first to determine that LMP1 activates the IGF1R tyrosine kinase by regulating expression of the ligand IGF1. Additionally, the data in this paper reveal that specific targeting of IGF1R selectively impacts LMP1-positive cells. These findings suggest that therapies directed against IGF1R may specifically impair the growth of EBV-infected cells.

  • The stress granule protein G3BP1 recruits PKR to promote multiple innate immune antiviral responses [PublishAheadOfPrint]

  • Stress granules (SGs) are cytoplasmic storage sites containing translationally silenced mRNPs that can be released to resume translation after stress subsides. We previously showed that poliovirus 3C proteinase cleaves the SG-nucleating protein G3BP1, blocking the ability of cells to form SGs late in infection. Many other viruses also target G3BP1 and inhibit SG formation but reasons why these functions evolved are unclear. Previously, we also showed a link between G3BP1-induced SGs and PKR-mediated translational control, but the mechanism of PKR interplay with SG and antiviral consequences are unknown. Here we show that G3BP1 exhibits antiviral activity against several enteroviruses whereas truncated G3BP1 that cannot form SGs does not. G3BP1-induced SGs are linked to activation of innate immune transcriptional responses through NFB and JNK. The G3BP1-induced SGs also recruit PKR and other antiviral proteins. We show that the PxxP domain within G3BP1 is essential for the recruitment of PKR to SGs, for eIF2aalpha; phosphorylation driven by PKR, and for nucleating SGs of normal composition. We also show that deletion of the PxxP domain in G3BP1 compromises its antiviral activity. These findings tie PKR activation to its recruitment to SGs by G3BP1 and indicate G3BP1 promotes innate immune responses at both transcriptional and translational levels and integrates cellular stress responses and innate immunity.

    IMPORTANCE Stress granules appear during virus infection and their importance is not well understood. Previously it was assumed that they were nonfunctional artifacts associated with cellular stress. PKR is a well-known antiviral protein, however its regulation in cells is not well understood. Our work links cellular stress granules with activation of PKR and other innate immune pathways through the activity of G3BP1, a critical stress granule component. The ability of stress granules and G3BP1 to activate PKR and other innate immune transcriptional responses indicates that G3BP1 is an antiviral protein. This work helps to refine a longstanding paradigm indicating stress granules are inert structures, and explains why G3BP1 is subverted by many viruses to promote a productive infection.

  • Ataxia telangiectasia mutated (ATM) kinase mediates NF{kappa}B Ser 276 phosphorylation and IFN expression via the IRF7-RIG-I amplification loop in paramyxovirus virus infection [PublishAheadOfPrint]

  • RSV is a primary etiological agent of childhood lower respiratory tract disease. Molecular patterns induced by active infection trigger a coordinated retinoic acid-inducible gene (RIG)-I-toll like receptor (TLR) signaling response to induce inflammatory cytokines and anti-viral mucosal interferons. Recently we discovered a nuclear oxidative stress-sensitive pathway mediated by the DNA-damage response protein, ATM, in cytokine-induced NFB/RelA Ser 276 phosphorylation. Here we, observe that ATM silencing results in enhanced ssRNA replication of RSV and Sendai Viruses, due to decreased expression and secretion of type nndash;I and nndash;III interferons (IFNs), despite maintenance of IRF3-dependent IFN-stimulated genes (ISGs). In addition to enhanced oxidative stress, RSV replication enhances foci of phosphorylated histone 2AX variant (H2AX), Ser 1981 phosphorylation of ATM, and IKK/NEMO-dependent ATM nuclear export indicating activation of the DNA-damage response. ATM deficient cells show defective RSV-induced mitogen and stress-activated kinase (MSK)-1 Ser 376 phosphorylation and reduced RelA Ser 276 phosphorylation, whose formation is required for IRF7 expression. We observe that RelA binds inducibly binds the native IFN regulatory factor (IRF)-7 promoter in an ATM-dependent manner, and IRF7 inducibly binds to the endogenous retinoic acid inducible gene (RIG)-I promoter. Ectopic IRF7 expression restores RIG-I expression and type I/III IFN expression in ATM-silenced cells. We conclude that paramyxoviruses trigger the DNA-damage response, a pathway required for MSK1 activation of phospho Ser 276 RelA formation to trigger the IRF7-RIG-I amplification loop necessary for mucosal IFN production. These data provide molecular pathogenesis for defects in cellular innate immunity patients with homozygous ATM mutations.

    IMPORTANCE RNA virus infections trigger cellular response pathways to limit spread to adjacent tissues. This "innate immune response" is mediated by germline-encoded pattern recognition receptors that trigger activation of two, largely independent, intracellular NF-B and IRF3 transcription factors. Downstream, expression of protective anti-viral interferons is amplified by positive feedback loops mediated by inducible interferon response factors (IRFs) and retinoic acid inducible gene (RIG-I). Our results indicate that a nuclear oxidative stress and DNA-damage sensing factor, ATM, is required to mediate a cross-talk pathway between NF-B and IRF7 through mediating phosphorylation of NF-B. Our studies provide further information about the defects in cellular- and innate immunity in patients with inherited ATM mutations.

  • Role of Cdk1 in the p53-independent abrogation of the postmitotic checkpoint by human papillomavirus E6 [PublishAheadOfPrint]

  • Specific types of human papillomavirus (HPV) are strongly associated with the development of cervical carcinoma. The HPV E6 oncoprotein from HPV degrades p53 and abrogates cell cycle checkpoints. Nonetheless, functional p53 has been observed in cervical cancer. We have previously identified a p53-independent function of E6 in attenuating the postmitotic G1-like checkpoint that can lead to polyploidy, an early event during cervical carcinogenesis that predisposes cells to aneuploidy. How E6 promotes cell cycle progression in the presence of p53 and its target, p21, remains a mystery. In this study, we examined the expression of cell cycle-related genes in cells expressing wild-type and the mutant E6 that is defective in p53 degradation but competent in abrogating the postmitotic checkpoint. Our results demonstrated an increase in the steady-state levels of G1- and G2-related cyclins/Cdks in E6-expressing keratinocytes. Interestingly, only Cdk1 remained active in E6 mutant-expressing cells while bypassing the postmitotic checkpoint. Furthermore, the down-regulation of Cdk1 impaired the ability of both wild-type and mutant E6 to induce polyploidy. Our study thus demonstrated an important role for Cdk1, which binds p21 with lower affinity than Cdk2, in abrogating the postmitotic checkpoint in E6-expressing cells. We further show that E2F1 is important for E6 to up-regulate Cdk1. Moreover, reduced nuclear p21 localization in the E6 mutant-expressing cells was observed. These findings shed light on the mechanisms by which HPV induces genomic instability and hold promise for the identification of drug targets.

    Importance HPV infection is strongly associated with the development of cervical carcinoma. HPV encodes an E6 oncoprotein that degrades the tumor suppressor p53 and abrogates cell cycle checkpoints. Nonetheless, functional p53 has been observed in cervical cancer. We have recently demonstrated a p53-independent abrogation of the postmitotic checkpoint by HPV E6 that induces polyploidy. However, the mechanism is not known. In this study, we provide evidence that Cdk1 plays an important role in this process. Previously, Cdk2 was thought to be essential for the G1/S transition, while Cdk1 only compensated its function in the absence of Cdk2. Our studies have demonstrated a novel role of Cdk1 at the postmitotic G1-like checkpoint in the presence of Cdk2. These findings shed light on the mechanisms by which HPV induces genomic instability and hold promise for the identification of drug targets.

  • Delayed inflammatory and cell death responses are associated with reduced pathogenicity in Lujo virus-infected cynomolgus macaques [PublishAheadOfPrint]

  • To identify host factors associated with arenavirus virulence, we used a cynomolgus macaque model to evaluate the pathogenesis of Lujo virus (LUJV), a recently emerged arenavirus that caused an outbreak of severe viral hemorrhagic fever in southern Africa. In contrast to human cases, LUJV caused mild, non-lethal illness in macaques. We then compared this to opposed clinical outcomes during arenavirus infection, specifically to samples obtained from macaques infected with three highly pathogenic lines of Lassa virus (LASV), the causative agent of Lassa fever (LF). We assessed gene expression in peripheral blood mononuclear cells (PBMC), and determined genes that significantly changed expression relative to uninfected animals over the course of infection. We detected a 72-hour delay in induction of host responses to infection during LUJV infection compared to the animals infected with LASV. This included genes associated with inflammatory and antiviral responses, and was particularly apparent among groups of genes promoting cell death. We also observed early differential expression of a subset of genes specific to LUJV infection that accounts for the delayed inflammatory response. Cell-type enrichment analysis suggested that host response induction delay and LUJV-specific profile may be due to a different proportion of natural killer cells responding in LUJV infection compared to the LASV-infected animals. Together, these data indicate that delayed pro-inflammatory and pro-apoptotic host responses to arenavirus infection could ameliorate disease severity. This conclusion provides insight into the cellular and molecular mechanisms of arenaviral hemorrhagic fever, as well as suggests potential strategies for therapeutic development.

    Importance Old World arenaviruses are significant human pathogens that are often associated with high mortality. However, mechanisms underlying disease severity and virulence in arenavirus hemorrhagic fever are largely unknown, particularly regarding host responses that contribute to pathogenicity. This study describes a comparison between Lujo and Lassa virus infection in cynomolgus macaques. Lujo-infected macaques developed only mild illness, while Lassa-infected macaques developed severe illness consistent with Lassa fever. We determined that mild disease is associated with a delay in host expression of genes linked to virulence, such as those causing inflammation and cell death, and with distinct cell types that may mediate this delay. This is the first study to associate the timing and directionality of gene expression with arenaviral pathogenicity and disease outcome, and evokes new potential approaches for developing effective therapeutics for treating these deadly emerging pathogens.

  • A Human herpesvirus 6A encoded miRNA: A role in viral lytic replication [PublishAheadOfPrint]

  • Human herpesvirus 6A (HHV-6A), a member of the betaherpesvirus family, is associated with several human diseases. Like all herpesviruses, HHV-6A establishes a life-long, latent infection in its host. Reactivation of HHV-6A is frequent within the immuno-suppressed and immuno-compromised populations and results in lytic viral replication within multiple organs, often leading to severe disease. MicroRNAs (miRNAs) are key regulators of multiple cellular processes that regulate the translation of specific transcripts. miRNAs encoded by herpesviruses play important roles in modulating the host cell, thereby facilitating a suitable environment for productive viral infection and/or latency. Currently there are approximately 150 known human herpesvirus-encoded miRNAs, although miRNA(s) encoded by HHV-6A have yet to be reported. We hypothesized that HHV-6A, like other members of the human herpesvirus family, encodes miRNAs, which function to promote viral infection. We utilized deep sequencing of small RNA species isolated from cells harboring HHV-6A to identify five novel small non-coding RNA species that originate from the viral genome, one of which has the characteristics of a viral miRNA. These RNAs are expressed during productive infection of either BAC derived virus in Jjhan cells or wild type HHV-6A U1102 strain infections of HSB2 cells and are associated with the RISC machinery. Growth analyses of mutant viruses that lack each individual miRNA revealed that a viral miRNA candidate (miR-U86) targets the HHV-6A IE gene U86, thereby regulating lytic replication. The identification and biological characterization of this HHV-6A specific miRNA is the first step to defining how the virus regulates its life cycle.

    IMPORTANCE A majority of the human population is infected with human herpesvirus 6A (HHV-6A), a betaherpesvirus family member. Infections usually occur in young children and upon resolution, the virus remains in a latent state within the host. Importantly, during times of weakened immune responses, the virus can reactivate and is correlated with significant disease states. Viruses encode many different types of factors that both undermine the host antiviral response as well as regulate viral replication including small RNA species called microRNAs (miRNAs). Here we report that HHV-6A encodes at least one miRNA, which we named miR-U86. We have characterized the requirement of this viral miRNA and its impact on the viral lifecycle and found that it functions to regulate a viral protein important for efficient viral replication. Our data suggest that viral miRNAs are important for HHV-6A and that they may serve as an important therapeutic target to inhibit the virus.

  • Mutations within the pathogenic region of HSV-1 gK signal sequences alter cell surface expression and neurovirulence [PublishAheadOfPrint]

  • To investigate the role of the signal sequences of HSV-1 gK on virus replication and viral pathogenesis, we constructed recombinant viruses with and without mutations within the signal sequences of gK. These recombinant viruses expressed two additional copies of the mutated (MgK) or native form (NgK) of gK gene in place of the latency associated transcript (LAT) with a myc epitope tag, to facilitate detection, at their 3rrsquo; end. The replication of MgK virus was similar to that of NgK both in vitro and in vivo as well as in the trigeminal ganglia (TG) of latently-infected mice. The levels of gB and gK transcripts in the corneas, TG, and brains of infected mice on days 3 and 5 post infection were markedly virus- and time-dependent as well as tissue-specific. Mutation in the signal sequence of gK in MgK virus blocked cell surface expression of gK-myc in rabbit skin (RS) cells, increased LD50 and decreased corneal scarring in ocularly infected mice as compared with the NgK or revertant (RgK) virus. MgK and NgK viruses and not the RgK virus had reduced extent of explant reactivation at the lower dose of ocular infection but not at the higher dose. However, time of reactivation was not affected by overexpression of the different forms of gK. Taken together, these results strongly suggest that the 8mer peptide (ITAYGLVL) within the signal sequence of gK promotes cell surface expression of gK in infected cells and ocular pathogenesis in infected mice.

    Significance In this study we have shown for the first time that mutations within the signal sequence of gK blocked cell surface expression of inserted recombinant gK in vitro. Furthermore, this blockage in cell surface expression was correlated with higher LD50 and lower CS in vivo. Thus, these studies point to a key role for the 8mer within the signal sequence of gK in HSV-1-induced pathogenicity.

  • Analysis of the early immune response to infection by Infectious Bursal Disease Virus (IBDV) in chickens differing in their resistance to the disease [PublishAheadOfPrint]

  • Chicken whole genome gene expression arrays were used to analyse the host response to infection by Infectious Bursal Disease Virus (IBDV). Spleen and bursal tissue were examined from control and infected birds at 2, 3 and 4 days post-infection from two lines that differ in their resistance to IBDV infection. The host response was evaluated over this period and differences between susceptible and resistant chicken lines were examined. Anti-viral genes, including IFNA, IFNG, MX1, IFITM1, IFITM3 and IFITM5 were up-regulated in response to infection. Evaluation of this gene expression data has allowed us to predicted several genes as candidates for involvement in resistance to IBDV.

    IMPORTANCE Infectious bursal disease (IBD) is of economic importance to the poultry industry and thus is also important for food security. Vaccines are available but field strains of the virus are of increasing virulence. There is thus an urgent need to explore new control solutions, one of which would be to breed birds with greater resistance to IBD. A goal which is perhaps uniquely achievable with poultry, of all farm animal species, as the genetics of 85% of the 60 billion chickens produced worldwide each year is under the control of essentially two breeding companies. This is the most comprehensive study to try to identify global transcriptomic differences in the target organ of the virus between chicken lines that differ in resistance, and to predict candidate resistance genes.

  • Virion-independent transfer of replication competent HCV RNA between permissive cells [PublishAheadOfPrint]

  • In this study we show that replication-competent subgenomic HCV RNA can be transferred to permissive Huh7 cells leading to the establishment of viral RNA replication. Further, we show that these events are mediated by exosomes rather than infectious virus particles. If similar events occur in vivo, this could represent a novel, albeit inefficient, mechanism of viral spread and immune escape.

  • Timing of galectin-1 exposure differentially modulates Nipah virus entry and syncytia formation in endothelial cells [PublishAheadOfPrint]

  • Nipah virus (NiV) is a deadly emerging enveloped paramyxovirus that primarily targets human endothelial cells. Endothelial cells express the innate immune effector galectin-1 that we have previously shown can bind to specific N-glycans on the NiV envelope fusion glycoprotein (F). NiV-F mediates fusion of infected endothelial cells into syncytia, resulting in endothelial disruption and hemorrhage. Galectin-1 is an endogenous carbohydrate binding protein that binds to specific glycans on NiV-F to reduce endothelial cell fusion, an effect that may reduce pathophysiologic sequelae of NiV infection. However, galectins play multiple roles in regulating host-pathogen interactions; for example, galectins can promote attachment of HIV to T cells and macrophages and attachment of HSV-1 to keratinocytes, but can also inhibit influenza entry into airway epithelial cells. Using live Nipah virus, in the present study, we demonstrate that galectin-1 can enhance NiV attachment to and infection of primary human endothelial cells by bridging glycans on the viral envelope to host cell glycoproteins. In order to exhibit an enhancing effect, galectin-1 must be present during the initial phase of virus attachment; in contrast, addition of galectin-1 post-infection results in reduced production of progeny virus and syncytia formation. Thus, galectin-1 can have dual and opposing effects on NiV infection of human endothelial cells. While various roles for galectin family members in microbial-host interactions have been described, we report opposing effects of the same galectin family member on a specific virus, with the timing of exposure during the viral life cycle determining the outcome.

    Importance Nipah virus is an emerging pathogen that targets endothelial cells lining blood vessels; the high mortality rate (up to 70%) in Nipah virus infections results from destruction of these cells and resulting catastrophic hemorrhage. Host factors that promote or prevent Nipah virus infection are not well understood. Endogenous human lectins, such as galectin-1, can function as pattern recognition receptors to reduce infection and initiate immune responses; however, lectins can also be exploited by microbes to enhance infection of host cells. We found that galectin-1, which is made by inflamed endothelial cells, can both promote Nipah virus infection of endothelial cells by "bridging" the virus to the cell, as well as reduce production of progeny virus and reduce endothelial cell fusion and damage, depending on timing of galectin-1 exposure. This is the first report of spatiotemporal opposing effects of a host lectin for a virus in one type of host cell.

  • Interferon-induced Ifit proteins: Their role in viral pathogenesis [PublishAheadOfPrint]

  • A major component of the protective antiviral host defense is contributed by the intracellular actions of the proteins encoded by interferon-stimulated genes (ISG); amongst these are the Interferon-induced proteins with tetratricopeptide repeats (IFIT), consisting of four members in human and three in mouse. IFIT proteins do not have any known enzyme activity; instead, they inhibit virus replication by binding and regulating the functions of cellular and viral proteins and RNAs. Although all IFITs are comprised of multiple copies of the degenerate tetratricopeptide repeats, their distinct tertiary structures enable them to bind different partners and affect host-virus interactions differently. The recent use of Ifit knock-out mouse models has revealed novel antiviral functions of these proteins and new insights into the specificities of ISG actions. This article focuses on human and murine IFIT1 and IFIT2 by reviewing their mechanisms of action, their critical roles in protecting mice from viral pathogenesis and viral strategies to evade IFIT action.

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