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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]

  • Adenovirus Serotype 5 Vaccination Results in Suboptimal CD4 T Helper 1 Responses in Mice [Pathogenesis and Immunity]

  • Adenovirus serotype 5 (Ad5) is one of the most widely used viral vectors and is known to generate potent T cell responses. While many previous studies have characterized Ad5-induced CD8 T cell responses, there is a relative lack of detailed studies that have analyzed CD4 T cells elicited by Ad5 vaccination. Here, we immunized mice with Ad5 vectors encoding lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) and examined GP-specific CD4 T cell responses elicited by Ad5 vectors and compared them to those induced by an acute LCMV infection. In contrast to LCMV infection, where balanced CD4 T helper 1 (Th1) and T follicular helper (Tfh) responses were induced, Ad5 immunization resulted in a significantly reduced frequency of Th1 cells. CD4 T cells elicited by Ad5 vectors expressed decreased levels of Th1 markers, such as Tim3, SLAM, T-bet, and Ly6C, had smaller amounts of cytotoxic molecules like granzyme B, and produced less interferon gamma than CD4 T cells induced by LCMV infection. This defective CD4 Th1 response appeared to be intrinsic for Ad5 vectors and not a reflection of comparing a nonreplicating vector to a live viral infection, since immunization with a DNA vector expressing LCMV-GP generated efficient CD4 Th1 responses. Analysis at early time points (day 3 or 4) after immunization with Ad5 vectors revealed a defect in the expression of CD25 (interleukin-2 [IL-2] receptor alpha chain) on Ad5-elicited CD4 T cells, and administration of exogenous IL-2 following Ad5 immunization partially restored CD4 Th1 responses. These results suggest that impairment of Th1 commitment after Ad5 immunization could be due to reduced IL-2-mediated signaling.

    IMPORTANCE During viral infection, generating balanced responses of Th1 and Tfh cells is important to induce effective cell-mediated responses and provide optimal help for antibody responses. In this study, to investigate vaccine-induced CD4 T cell responses, we characterized CD4 T cells after immunization with Ad5 vectors expressing LCMV-GP in mice. Ad5 vectors led to altered effector differentiation of LCMV GP-specific CD4 T cells compared to that during LCMV infection. CD4 T cells following Ad5 immunization exhibited impaired Th1 lineage commitment, generating significantly decreased Th1 responses than those induced by LCMV infection. Our results suggest that suboptimal IL-2 signaling possibly plays a role in reduced Th1 development following Ad5 immunization.

  • Human Cytomegalovirus Requires Epidermal Growth Factor Receptor Signaling To Enter and Initiate the Early Steps in the Establishment of Latency in CD34+ Human Progenitor Cells [Virus-Cell Interactions]

  • The establishment of human cytomegalovirus (HCMV) latency and persistence relies on the successful infection of hematopoietic cells, which serve as sites of viral persistence and contribute to viral spread. Here, using blocking antibodies and pharmacological inhibitors, we document that HCMV activation of the epidermal growth factor receptor (EGFR) and downstream phosphatidylinositol 3-kinase (PI3K) mediates viral entry into CD34+ human progenitor cells (HPCs), resulting in distinct cellular trafficking and nuclear translocation of the virus compared to that in other immune cells, such as we have documented in monocytes. We argue that the EGFR allows HCMV to regulate the cellular functions of these replication-restricted cells via its signaling activity following viral binding. In addition to regulating HCMV entry/trafficking, EGFR signaling may also shape the early steps required for the successful establishment of viral latency in CD34+ cells, as pharmacological inhibition of EGFR increases the transcription of lytic IE1/IE2 mRNA while curbing the expression of latency-associated UL138 mRNA. EGFR signaling following infection of CD34+ HPCs may also contribute to changes in hematopoietic potential, as treatment with the EGFR kinase (EGFRK) inhibitor AG1478 alters the expression of the cellular hematopoietic cytokine interleukin 12 (IL-12) in HCMV-infected cells but not in mock-infected cells. These findings, along with our previous work with monocytes, suggest that EGFR likely serves as an important determinant of HCMV tropism for select subsets of hematopoietic cells. Moreover, our new data suggest that EGFR is a key receptor for efficient viral entry and that the ensuing signaling regulates important early events required for successful infection of CD34+ HPCs by HCMV.

    IMPORTANCE HCMV establishes lifelong persistence within the majority of the human population without causing overt pathogenesis in healthy individuals. Despite this, reactivation of HCMV from its latent reservoir in the bone marrow causes significant morbidity and mortality in immunologically compromised individuals, such as bone marrow and solid organ transplant patients. Lifelong persistent infection has also been linked with the development of various cardiovascular diseases in otherwise healthy individuals. Current HCMV therapeutics target lytic replication, but not the latent viral reservoir; thus, an understanding of the molecular basis for viral latency and persistence is paramount to controlling or eliminating HCMV infection. Here, we show that the viral signalosome activated by HCMV binding to its entry receptor, EGFR, in CD34+ HPCs initiates early events necessary for successful latent infection of this cell type. EGFR and associated signaling players may therefore represent promising targets for mitigating HCMV persistence.

  • Mechanism of Protein Kinase R Inhibition by Human Cytomegalovirus pTRS1 [Virus-Cell Interactions]

  • Double-stranded RNAs (dsRNA) produced during human cytomegalovirus (HCMV) infection activate the antiviral kinase protein kinase R (PKR), which potently inhibits virus replication. The HCMV pTRS1 and pIRS1 proteins antagonize PKR to promote HCMV protein synthesis and replication; however, the mechanism by which pTRS1 inhibits PKR is unclear. PKR activation occurs in a three-step cascade. First, binding to dsRNA triggers PKR homodimerizaton. PKR dimers then autophosphorylate, leading to a conformational shift that exposes the binding site for the PKR substrate eIF2aalpha;. Consistent with previous in vitro studies, we found that pTRS1 bound and inhibited PKR. pTRS1 binding to PKR was not mediated by an RNA intermediate, and mutations in the pTRS1 RNA binding domain did not affect PKR binding or inhibition. Rather, mutations that disrupted the pTRS1 interaction with PKR ablated the ability of pTRS1 to antagonize PKR activation by dsRNA. pTRS1 did not block PKR dimerization and could bind and inhibit a constitutively dimerized PKR kinase domain. In addition, pTRS1 binding to PKR inhibited PKR kinase activity. Single amino acid point mutations in the conserved eIF2aalpha; binding domain of PKR disrupted pTRS1 binding and rendered PKR resistant to inhibition by pTRS1. Consistent with a critical role for the conserved eIF2aalpha; contact site in PKR binding, pTRS1 bound an additional eIF2aalpha; kinase, heme-regulated inhibitor (HRI), and inhibited eIF2aalpha; phosphorylation in response to an HRI agonist. Together our data suggest that pTRS1 inhibits PKR by binding to conserved amino acids in the PKR eIF2aalpha; binding site and blocking PKR kinase activity.

    IMPORTANCE The antiviral kinase PKR plays a critical role in controlling HCMV replication. This study furthered our understanding of how HCMV evades inhibition by PKR and identified new strategies for how PKR activity might be restored during infection to limit HCMV disease.

  • IRAV (FLJ11286), an Interferon-Stimulated Gene with Antiviral Activity against Dengue Virus, Interacts with MOV10 [Pathogenesis and Immunity]

  • Dengue virus (DENV) is a member of the genus Flavivirus and can cause severe febrile illness. Here, we show that FLJ11286, which we refer to as IRAV, is induced by DENV in an interferon-dependent manner, displays antiviral activity against DENV, and localizes to the DENV replication complex. IRAV is an RNA binding protein and localizes to cytoplasmic processing bodies (P bodies) in uninfected cells, where it interacts with the MOV10 RISC complex RNA helicase, suggesting a role for IRAV in the processing of viral RNA. After DENV infection, IRAV, along with MOV10 and Xrn1, localizes to the DENV replication complex and associates with DENV proteins. Depletion of IRAV or MOV10 results in an increase in viral RNA. These data serve to characterize an interferon-stimulated gene with antiviral activity against DENV, as well as to propose a mechanism of activity involving the processing of viral RNA.

    IMPORTANCE Dengue virus, a member of the family Flaviviridae, can result in a life-threatening illness and has a significant impact on global health. Dengue virus has been shown to be particularly sensitive to the effects of type I interferon; however, little is known about the mechanisms by which interferon-stimulated genes function to inhibit viral replication. A better understanding of the interferon-mediated antiviral response to dengue virus may aid in the development of novel therapeutics. Here, we examine the influence of the interferon-stimulated gene IRAV (FLJ11286) on dengue virus replication. We show that IRAV associates with P bodies in uninfected cells and with the dengue virus replication complex after infection. IRAV also interacts with MOV10, depletion of which is associated with increased viral replication. Our results provide insight into a newly identified antiviral gene, as well as broadening our understanding of the innate immune response to dengue virus infection.

  • An Optimized Hepatitis C Virus E2 Glycoprotein Core Adopts a Functional Homodimer That Efficiently Blocks Virus Entry [Structure and Assembly]

  • The hepatitis C virus (HCV) envelope glycoprotein E2 is the major target of broadly neutralizing antibodies in vivo and is the focus of efforts in the rational design of a universal B cell vaccine against HCV. The E2 glycoprotein exhibits a high degree of amino acid variability which localizes to three discrete regions: hypervariable region 1 (HVR1), hypervariable region 2 (HVR2), and the intergenotypic variable region (igVR). All three variable regions contribute to immune evasion and/or isolate-specific structural variations, both important considerations for vaccine design. A high-resolution structural definition of the intact HCV envelope glycoprotein complex containing E1 and E2 remains to be elucidated, while crystallographic structures of a recombinant E2 ectodomain failed to resolve HVR1, HVR2, and a major neutralization determinant adjacent to HVR1. To obtain further information on E2, we characterized the role of all three variable regions in E2 ectodomain folding and function in the context of a recombinant ectodomain fragment (rE2). We report that removal of the variable regions accelerates binding to the major host cell receptor CD81 and that simultaneous deletion of HVR2 and the igVR is required to maintain wild-type CD81-binding characteristics. The removal of the variable regions also rescued the ability of rE2 to form a functional homodimer. We propose that the rE2 core provides novel insights into the role of the variable motifs in the higher-order assembly of the E2 ectodomain and may have implications for E1E2 structure on the virion surface.

    IMPORTANCE Hepatitis C virus (HCV) infection affects ~2% of the population globally, and no vaccine is available. HCV is a highly variable virus, and understanding the presentation of key antigenic sites at the virion surface is important for the design of a universal vaccine. This study investigates the role of three surface-exposed variable regions in E2 glycoprotein folding and function in the context of a recombinant soluble ectodomain. Our data demonstrate the variable motifs modulate binding of the E2 ectodomain to the major host cell receptor CD81 and have an impact on the formation of an E2 homodimer with high-affinity binding to CD81.

  • Shifting Clade Distribution, Reassortment, and Emergence of New Subtypes of Highly Pathogenic Avian Influenza A(H5) Viruses Collected from Vietnamese Poultry from 2012 to 2015 [Genetic Diversity and Evolution]

  • Whole-genome sequences of representative highly pathogenic avian influenza A(H5) viruses from Vietnam were generated, comprising samples from poultry outbreaks and active market surveillance collected from January 2012 to August 2015. Six hemagglutinin gene clades were characterized. Clade 1.1.2 was predominant in southern Mekong provinces throughout 2012 and 2013 but gradually disappeared and was not detected after April 2014. Clade 2.3.2.1c viruses spread rapidly during 2012 and were detected in the south and center of the country. A number of clade 1.1.2 and 2.3.2.1c interclade reassortant viruses were detected with different combinations of internal genes derived from 2.3.2.1a and 2.3.2.1b viruses, indicating extensive cocirculation. Although reassortment generated genetic diversity at the genotype level, there was relatively little genetic drift within the individual gene segments, suggesting genetic stasis over recent years. Antigenically, clade 1.1.2, 2.3.2.1a, 2.3.2.1b, and 2.3.2.1c viruses remained related to earlier viruses and WHO-recommended prepandemic vaccine strains representing these clades. Clade 7.2 viruses, although detected in only low numbers, were the exception, as indicated by introduction of a genetically and antigenically diverse strain in 2013. Clade 2.3.4.4 viruses (H5N1 and H5N6) were likely introduced in April 2014 and appeared to gain dominance across northern and central regions. Antigenic analyses of clade 2.3.4.4 viruses compared to existing clade 2.3.4 candidate vaccine viruses (CVV) indicated the need for an updated vaccine virus. A/Sichuan/26221/2014 (H5N6) virus was developed, and ferret antisera generated against this virus were demonstrated to inhibit some but not all clade 2.3.4.4 viruses, suggesting consideration of alternative clade 2.3.4.4 CVVs.

    IMPORTANCE Highly pathogenic avian influenza (HPAI) A(H5) viruses have circulated continuously in Vietnam since 2003, resulting in hundreds of poultry outbreaks and sporadic human infections. Despite a significant reduction in the number of human infections in recent years, poultry outbreaks continue to occur and the virus continues to diversify. Vaccination of poultry has been used as a means to control the spread and impact of the virus, but due to the diversity and changing distribution of antigenically distinct viruses, the utility of vaccines in the face of mismatched circulating strains remains questionable. This study assessed the putative amino acid changes in viruses leading to antigenic variability, underscoring the complexity of vaccine selection for both veterinary and public health purposes. Given the overlapping geographic distributions of multiple, antigenically distinct clades of HPAI A(H5) viruses in Vietnam, the vaccine efficacy of bivalent poultry vaccine formulations should be tested in the future.

  • Nonstructural Protein 4 of Porcine Reproductive and Respiratory Syndrome Virus Modulates Cell Surface Swine Leukocyte Antigen Class I Expression by Downregulating {beta}2-Microglobulin Transcription [Virus-Cell Interactions]

  • Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which has important impacts on the pig industry. PRRSV infection results in disruption of the swine leukocyte antigen class I (SLA-I) antigen presentation pathway. In this study, highly pathogenic PRRSV (HP-PRRSV) infection inhibited transcription of the bbeta;2-microglobulin (bbeta;2M) gene (B2M) and reduced cellular levels of bbeta;2M, which forms a heterotrimeric complex with the SLA-I heavy chain and a variable peptide and plays a critical role in SLA-I antigen presentation. HP-PRRSV nonstructural protein 4 (Nsp4) was involved in the downregulation of bbeta;2M expression. Exogenous expression of Nsp4 downregulated bbeta;2M expression at both the mRNA and the protein level and reduced SLA-I expression on the cell surface. Nsp4 bound to the porcine B2M promoter and inhibited its transcriptional activity. Domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter were identified as essential for the interaction between Nsp4 and B2M. These findings demonstrate a novel mechanism whereby HP-PRRSV may modulate the SLA-I antigen presentation pathway and provide new insights into the functions of HP-PRRSV Nsp4.

    IMPORTANCE PRRSV modulates the host response by disrupting the SLA-I antigen presentation pathway. We show that HP-PRRSV downregulates SLA-I expression on the cell surface via transcriptional inhibition of B2M expression by viral Nsp4. The interaction between domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter is essential for inhibiting B2M transcription. These observations reveal a novel mechanism whereby HP-PRRSV may modulate SLA-I antigen presentation and provide new insights into the functions of viral Nsp4.

  • In Vivo Imaging with Bioluminescent Enterovirus 71 Allows for Real-Time Visualization of Tissue Tropism and Viral Spread [Pathogenesis and Immunity]

  • Hand, foot, and mouth disease (HFMD) is a reemerging illness caused by a variety of enteroviruses. The main causative agents are enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and, most recently, coxsackievirus A6 (CVA6). Enterovirus infections can vary from asymptomatic infections to those with a mild fever and blisters on infected individuals' hands, feet, and throats to infections with severe neurological complications. Viral persistence for weeks postinfection (wpi) has also been documented by the demonstration of virus in children's stools. However, little is known about disease progression, viral spread, and tissue tropism of these viruses. These types of studies are limited because many recently developed mouse models mimic the severe neurological complications that occur in a small percentage of enterovirus infections. In the present study, we documented real-time EV71 infection in two different mouse strains by the use of in vivo imaging. Infection of BALB/c mice with a bioluminescent mouse-adapted EV71 construct (mEV71-NLuc) resulted in a lack of clinical signs of disease but in relatively high viral replication, as visualized by luminescence, for 2 wpi. In contrast, mEV71-NLuc infection of AG129 mice (alpha/beta and gamma interferon receptor deficient) showed rapid spread and long-term persistence of the virus in the brain. Interestingly, AG129 mice that survived infection maintained luminescence in the brain for up to 8 wpi. The results we present here will allow future studies on EV71 antiviral drug susceptibility, vaccine efficacy, transmissibility, and pathogenesis.

    IMPORTANCE We report here that a stable full-length enterovirus 71 (EV71) reporter construct was used to visualize real-time viral spread in AG129 and BALB/c mice. To our knowledge, this is the first report of in vivo imaging of infection with any member of the Picornaviridae family. The nanoluciferase (NLuc) gene, one of the smallest luciferase genes currently available, was shown to be stable in the EV71 genome for eight passages on rhabdomyosarcoma cells. Real-time visualization of EV71 infection in mice identified areas of tropism that would have been missed by traditional methods, including full characterization of EV71 replication in BALB/c mice. Additionally, the bioluminescent construct allowed for increased speed and sensitivity of cell culture assays and will allow future studies involving various degrees of enterovirus infection in mice, not just severe infections. Our data suggest that interferon plays an important role in controlling EV71 infection in the central nervous system of mice.

  • Disruption of Stress Granule Formation by the Multifunctional Cricket Paralysis Virus 1A Protein [Virus-Cell Interactions]

  • Stress granules (SGs) are cytosolic ribonucleoprotein aggregates that are induced during cellular stress. Several viruses modulate SG formation, suggesting that SGs have an impact on virus infection. However, the mechanisms and impact of modulating SG assembly in infected cells are not completely understood. In this study, we identify the dicistrovirus cricket paralysis virus 1A (CrPV-1A) protein that functions to inhibit SG assembly during infection. Moreover, besides inhibiting RNA interference, CrPV-1A also inhibits host transcription, which indirectly modulates SG assembly. Thus, CrPV-1A is a multifunctional protein. We identify a key R146A residue that is responsible for these effects, and mutant CrPV(R146A) virus infection is attenuated in Drosophila melanogaster S2 cells and adult fruit flies and results in increased SG formation. Treatment of CrPV(R146A)-infected cells with actinomycin D, which represses transcription, restores SG assembly suppression and viral yield. In summary, CrPV-1A modulates several cellular processes to generate a cellular environment that promotes viral translation and replication.

    IMPORTANCE RNA viruses encode a limited set of viral proteins to modulate an array of cellular processes in order to facilitate viral replication and inhibit antiviral defenses. In this study, we identified a viral protein, called CrPV-1A, within the dicistrovirus cricket paralysis virus that can inhibit host transcription, modulate viral translation, and block a cellular process called stress granule assembly. We also identified a specific amino acid within CrPV-1A that is important for these cellular processes and that mutant viruses containing mutations of CrPV-1A attenuate virus infection. We also demonstrate that the CrPV-1A protein can also modulate cellular processes in human cells, suggesting that the mode of action of CrPV-1A is conserved. We propose that CrPV-1A is a multifunctional, versatile protein that creates a cellular environment in virus-infected cells that permits productive virus infection.

  • Ribosome Profiling Reveals Translational Upregulation of Cellular Oxidative Phosphorylation mRNAs during Vaccinia Virus-Induced Host Shutoff [Cellular Response to Infection]

  • Vaccinia virus infection causes a host shutoff that is marked by global inhibition of host protein synthesis. Though the host shutoff may facilitate reallocation of cellular resources for viral replication and evasion of host antiviral immune responses, it poses a challenge for continuous synthesis of cellular proteins that are important for viral replication. It is, however, unclear whether and how certain cellular proteins may be selectively synthesized during the vaccinia virus-induced host shutoff. Using simultaneous RNA sequencing and ribosome profiling, two techniques quantifying genome-wide levels of mRNA and active protein translation, respectively, we analyzed the responses of host cells to vaccinia virus infection at both the transcriptional and translational levels. The analyses showed that cellular mRNA depletion played a dominant role in the shutoff of host protein synthesis. Though the cellular mRNAs were significantly reduced, the relative translation efficiency of a subset of cellular mRNAs increased, particularly those involved in oxidative phosphorylation that are responsible for cellular energy production. Further experiments demonstrated that the protein levels and activities of oxidative phosphorylation increased during vaccinia virus infection, while inhibition of the cellular oxidative phosphorylation function significantly suppressed vaccinia virus replication. Moreover, the short 5' untranslated region of the oxidative phosphorylation mRNAs contributed to the translational upregulation. These results provide evidence of a mechanism that couples translational control and energy metabolism, two processes that all viruses depend on host cells to provide, to support vaccinia virus replication during a host shutoff.

    IMPORTANCE Many viral infections cause global host protein synthesis shutoff. While host protein synthesis shutoff benefits the virus by relocating cellular resources to viral replication, it also poses a challenge to the maintenance of cellular functions necessary for viral replication if continuous protein synthesis is required. Here we measured the host mRNA translation rate during a vaccinia virus-induced host shutoff by analyzing total and actively translating mRNAs in a genome-wide manner. This study revealed that oxidative phosphorylation mRNAs were translationally upregulated during vaccinia virus-induced host protein synthesis shutoff. Oxidative phosphorylation is the major cellular energy-producing pathway, and we further showed that maintenance of its function is important for vaccinia virus replication. This study highlights the fact that vaccinia virus infection can enhance cellular energy production through translational upregulation in the context of an overall host protein synthesis shutoff to meet energy expenditure.

  • Infectious Bursal Disease Virus Subverts Autophagic Vacuoles To Promote Viral Maturation and Release [Cellular Response to Infection]

  • Autophagy functions as an intrinsic antiviral defense. However, some viruses can subvert or even enhance host autophagic machinery to increase viral replication and pathogenesis. The role of autophagy during avibirnavirus infection, especially late stage infection, remains unclear. In this study, infectious bursal disease virus (IBDV) was used to investigate the role of autophagy in avibirnavirus replication. We demonstrated IBDV induction of autophagy as a significant increase in puncta of LC3+ autophagosomes, endogenous levels of LC3-II, and ultrastructural characteristics typical of autophagosomes during the late stage of infection. Induction of autophagy enhances IBDV replication, whereas inhibition of autophagy impairs viral replication. We also demonstrated that IBDV infection induced autophagosome-lysosome fusion, but without active degradation of their contents. Moreover, inhibition of fusion or of lysosomal hydrolysis activity significantly reduced viral replication, indicating that virions utilized the low-pH environment of acidic organelles to facilitate viral maturation. Using immuno-transmission electron microscopy (TEM), we observed that a large number of intact IBDV virions were arranged in a lattice surrounded by p62 proteins, some of which lay between virions. Additionally, many virions were encapsulated within the vesicular membranes, with an obvious release stage observed by TEM. The autophagic endosomal pathway facilitates low-pH-mediated maturation of viral proteins and membrane-mediated release of progeny virions.

    IMPORTANCE IBDV is the most extensively studied virus in terms of molecular characteristics and pathogenesis; however, mechanisms underlying the IBDV life cycle require further exploration. The present study demonstrated that autophagy enhances viral replication at the late stage of infection, and the autophagy pathway facilitates IBDV replication complex function and virus assembly, which is critical to completion of the virus life cycle. Moreover, the virus hijacks the autophagic vacuoles to mature in an acidic environment and release progeny virions in a membrane-mediated cell-to-cell manner. This autophagic endosomal pathway is proposed as a new mechanism that facilitates IBDV maturation, release, and reinternalization. This report presents a concordance in exit strategies among some RNA and DNA viruses, which exploit autophagy pathway for their release from cells.

  • Interaction of the Small GTPase Cdc42 with Arginine Kinase Restricts White Spot Syndrome Virus in Shrimp [Pathogenesis and Immunity]

  • Many types of small GTPases are widely expressed in eukaryotes and have different functions. As a crucial member of the Rho GTPase family, Cdc42 serves a number of functions, such as regulating cell growth, migration, and cell movement. Several RNA viruses employ Cdc42-hijacking tactics in their target cell entry processes. However, the function of Cdc42 in shrimp antiviral immunity is not clear. In this study, we identified a Cdc42 protein in the kuruma shrimp (Marsupenaeus japonicus) and named it MjCdc42. MjCdc42 was upregulated in shrimp challenged by white spot syndrome virus (WSSV). The knockdown of MjCdc42 and injection of Cdc42 inhibitors increased the proliferation of WSSV. Further experiments determined that MjCdc42 interacted with an arginine kinase (MjAK). By analyzing the binding activity and enzyme activity of MjAK and its mutant, MjAK, we found that MjAK could enhance the replication of WSSV in shrimp. MjAK interacted with the envelope protein VP26 of WSSV. An inhibitor of AK activity, quercetin, could impair the function of MjAK in WSSV replication. Further study demonstrated that the binding of MjCdc42 and MjAK depends on Cys271 of MjAK and suppresses the WSSV replication-promoting effect of MjAK. By interacting with the active site of MjAK and suppressing its enzyme activity, MjCdc42 inhibits WSSV replication in shrimp. Our results demonstrate a new function of Cdc42 in the cellular defense against viral infection in addition to the regulation of actin and phagocytosis, which has been reported in previous studies.

    IMPORTANCE The interaction of Cdc42 with arginine kinase plays a crucial role in the host defense against WSSV infection. This study identifies a new mechanism of Cdc42 in innate immunity and enriches the knowledge of the antiviral innate immunity of invertebrates.

  • NS1 Protein Amino Acid Changes D189N and V194I Affect Interferon Responses, Thermosensitivity, and Virulence of Circulating H3N2 Human Influenza A Viruses [Pathogenesis and Immunity]

  • Influenza virus NS1 protein is a nonstructural, multifunctional protein that counteracts host innate immune responses, modulating virus pathogenesis. NS1 protein variability in subjects infected with H3N2 influenza A viruses (IAVs) during the 2010/2011 season was analyzed, and amino acid changes in residues 86, 189, and 194 were found. The consequences of these mutations for the NS1-mediated inhibition of IFN responses and the pathogenesis of the virus were evaluated, showing that NS1 mutations D189N and V194I impaired the ability of the NS1 protein to inhibit general gene expression, most probably because these mutations decreased the binding of NS1 to the cleavage and polyadenylation specificity factor 30 (CPSF30). A recombinant A/Puerto Rico/8/34 (PR8) H1N1 virus encoding the H3N2 NS1-D189N protein was slightly attenuated, whereas the virus encoding the H3N2 NS1-V194I protein was further attenuated in mice. The higher attenuation of this virus could not be explained by differences in the ability of the two NS1 proteins to counteract host innate immune responses, indicating that another factor must be responsible. In fact, we showed that the virus encoding the H3N2 NS1-V194I protein demonstrated a temperature-sensitive (ts) phenotype, providing a most likely explanation for the stronger attenuation observed. As far as we know, this is the first description of a mutation in NS1 residue 194 conferring a ts phenotype. These studies are relevant in order to identify new residues important for NS1 functions and in human influenza virus surveillance to assess mutations affecting the pathogenicity of circulating viruses.

    IMPORTANCE Influenza viral infections represent a serious public health problem, with influenza virus causing a contagious respiratory disease that is most effectively prevented through vaccination. The multifunctional nonstructural protein 1 (NS1) is the main viral factor counteracting the host antiviral response. Therefore, influenza virus surveillance to identify new mutations in the NS1 protein affecting the pathogenicity of the circulating viruses is highly important. In this work, we evaluated amino acid variability in the NS1 proteins from H3N2 human seasonal viruses and the effect of the mutations on innate immune responses and virus pathogenesis. NS1 mutations D189N and V194I impaired the ability of the NS1 protein to inhibit general gene expression, and recombinant viruses harboring these mutations were attenuated in a mouse model of influenza infection. Interestingly, a virus encoding the H3N2 NS1-V194I protein demonstrated a temperature-sensitive phenotype, further attenuating the virus in vivo.

  • Surveillance of Bat Coronaviruses in Kenya Identifies Relatives of Human Coronaviruses NL63 and 229E and Their Recombination History [Genetic Diversity and Evolution]

  • Bats harbor a large diversity of coronaviruses (CoVs), several of which are related to zoonotic pathogens that cause severe disease in humans. Our screening of bat samples collected in Kenya from 2007 to 2010 not only detected RNA from several novel CoVs but, more significantly, identified sequences that were closely related to human CoVs NL63 and 229E, suggesting that these two human viruses originate from bats. We also demonstrated that human CoV NL63 is a recombinant between NL63-like viruses circulating in Triaenops bats and 229E-like viruses circulating in Hipposideros bats, with the breakpoint located near 5' and 3' ends of the spike (S) protein gene. In addition, two further interspecies recombination events involving the S gene were identified, suggesting that this region may represent a recombination "hot spot" in CoV genomes. Finally, using a combination of phylogenetic and distance-based approaches, we showed that the genetic diversity of bat CoVs is primarily structured by host species and subsequently by geographic distances.

    IMPORTANCE Understanding the driving forces of cross-species virus transmission is central to understanding the nature of disease emergence. Previous studies have demonstrated that bats are the ultimate reservoir hosts for a number of coronaviruses (CoVs), including ancestors of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and human CoV 229E (HCoV-229E). However, the evolutionary pathways of bat CoVs remain elusive. We provide evidence for natural recombination between distantly related African bat coronaviruses associated with Triaenops afer and Hipposideros sp. bats that resulted in a NL63-like virus, an ancestor of the human pathogen HCoV-NL63. These results suggest that interspecies recombination may play an important role in CoV evolution and the emergence of novel CoVs with zoonotic potential.

  • On the Analysis of Intrahost and Interhost Viral Populations: Human Cytomegalovirus as a Case Study of Pitfalls and Expectations [Genetic Diversity and Evolution]

  • Intrahost and interhost assessments of viral diversity are often treated as measures of separate and distinct evolutionary processes, with numerous investigations reporting seemingly incompatible results between the two. For example, in human cytomegalovirus, the nucleotide diversity estimates are 10-fold higher for interhost data, while the number of segregating (i.e., polymorphic) sites is 6-fold lower. These results have been interpreted as demonstrating that sampled intrahost variants are strongly deleterious. In reality, however, these observations are fully consistent with standard population genetic expectations. Here, we analyze published intra- and interhost data sets within this framework, utilizing statistical inference tools to quantify the fitness effects of segregating mutations. Further, we utilize population level simulations to clarify expectations under common evolutionary models. Contrary to common claims in the literature, these results suggest that most observed polymorphisms are likely nearly neutral with regard to fitness and that standard population genetic models in fact well predict observed levels of both intra- and interhost variability.

    IMPORTANCE With the increasing number of evolutionary virology studies examining both intrahost and interhost patterns of genomic variation, a number of seemingly incompatible results have emerged, revolving around the far greater level of observed intrahost than interhost variation. This has led many authors to suggest that the great majority of sampled within-host polymorphisms are strongly deleterious. Here, we demonstrate that there is in fact no incompatibility of these results and, indeed, that the vast majority of sampled within-host variation is likely neutral. These results thus represent a major shift in the current view of observed viral variation.

  • Restricted TET2 Expression in Germinal Center Type B Cells Promotes Stringent Epstein-Barr Virus Latency [Genome Replication and Regulation of Viral Gene Expression]

  • Epstein-Barr virus (EBV) latently infects normal B cells and contributes to the development of certain human lymphomas. Newly infected B cells support a highly transforming form (type III) of viral latency; however, long-term EBV infection in immunocompetent hosts is limited to B cells with a more restricted form of latency (type I) in which most viral gene expression is silenced by promoter DNA methylation. How EBV converts latency type is unclear, although it is known that type I latency is associated with a germinal center (GC) B cell phenotype, and type III latency with an activated B cell (ABC) phenotype. In this study, we have examined whether expression of TET2, a cellular enzyme that initiates DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), regulates EBV latency type in B cells. We found that TET2 expression is inhibited in normal GC cells and GC type lymphomas. In contrast, TET2 is expressed in normal naive B cells and ABC type lymphomas. We also demonstrate that GC type cell lines have increased 5mC levels and reduced 5hmC levels in comparison to those of ABC type lines. Finally, we show that TET2 promotes the ability of the EBV transcription factor EBNA2 to convert EBV-infected cells from type I to type III latency. These findings demonstrate that TET2 expression is repressed in GC cells independent of EBV infection and suggest that TET2 promotes type III EBV latency in B cells with an ABC or naive phenotype by enhancing EBNA2 activation of methylated EBV promoters.

    IMPORTANCE EBV establishes several different types of viral latency in B cells. However, cellular factors that determine whether EBV enters the highly transforming type III latency, versus the more restricted type I latency, have not been well characterized. Here we show that TET2, a cellular enzyme that initiates DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), regulates EBV latency type in B cells by enhancing the ability of the viral transcription factor EBNA2 to activate methylated viral promoters that are expressed in type III (but not type I) latency. Furthermore, we demonstrate that (independent of EBV) TET2 is turned off in normal and malignant germinal center (GC) B cells but expressed in other B cell types. Thus, restricted TET2 expression in GC cells may promote type I EBV latency.

  • Human Adenovirus Type 37 Uses {alpha}V{beta}1 and {alpha}3{beta}1 Integrins for Infection of Human Corneal Cells [Virus-Cell Interactions]

  • Epidemic keratoconjunctivitis (EKC) is a severe, contagious ocular disease that affects 20 to 40 million individuals worldwide every year. EKC is mainly caused by six types of human adenovirus (HAdV): HAdV-8, -19, -37, -53, -54, and -56. Of these, HAdV-8, -19, and -37 use sialic acid-containing glycans as cellular receptors. aalpha;Vbbeta;3, aalpha;Vbbeta;5, and a few additional integrins facilitate entry and endosomal release of other HAdVs. With the exception of a few biochemical analyses indicating that HAdV-37 can interact physically with aalpha;Vbbeta;5, little is known about the integrins used by EKC-causing HAdVs. Here, we investigated the overall integrin expression on human corneal cells and found expression of aalpha;2, aalpha;3, aalpha;6, aalpha;V, bbeta;1, and bbeta;4 subunits in human corneal in situ epithelium and/or in a human corneal epithelial (HCE) cell line but no or less accessible expression of aalpha;4, aalpha;5, bbeta;3, or bbeta;5. We also identified the integrins used by HAdV-37 through a series of binding and infection competition experiments and different biochemical approaches. Together, our data suggest that HAdV-37 uses aalpha;Vbbeta;1 and aalpha;3bbeta;1 integrins for infection of human corneal epithelial cells. Furthermore, to confirm the relevance of these integrins in the HAdV-37 life cycle, we developed a corneal multilayer tissue system and found that HAdV-37 infection correlated well with the patterns of aalpha;V, aalpha;3, and bbeta;1 integrin expression. These results provide further insight into the tropism and pathogenesis of EKC-causing HAdVs and may be of importance for future development of new antiviral drugs.

    IMPORTANCE Keratitis is a hallmark of EKC, which is caused by six HAdV types (HAdV-8, -19, -37, -53, -54, and -56). HAdV-37 and some other HAdV types interact with integrin aalpha;Vbbeta;5 in order to enter nonocular human cells. In this study, we found that aalpha;Vbbeta;5 is not expressed on human corneal epithelial cells, thus proposing other host factors mediate corneal infection. Here, we first characterized integrin expression patterns on corneal tissue and corneal cells. Among the integrins identified, competition binding and infection experiments and biochemical assays pointed out aalpha;Vbbeta;1 and aalpha;3bbeta;1 to be of importance for HAdV-37 infection of corneal tissue. In the absence of a good animal model for EKC-causing HAdVs, we also developed an in vitro system with multilayer HCE cells and confirmed the relevance of the suggested integrins during HAdV-37 infection.

  • Mapping the Human Memory B Cell and Serum Neutralizing Antibody Responses to Dengue Virus Serotype 4 Infection and Vaccination [Virus-Cell Interactions]

  • The four dengue virus (DENV) serotypes are mosquito-borne flaviviruses responsible for dengue fever and dengue hemorrhagic fever. People exposed to DENV develop antibodies (Abs) that strongly neutralize the serotype responsible for infection. Historically, infection with DENV serotype 4 (DENV4) has been less common and less studied than infections with the other three serotypes. However, DENV4 has been responsible for recent large and sustained epidemics in Asia and Latin America. The neutralizing antibody responses and the epitopes targeted against DENV4 have not been characterized in human infection. In this study, we mapped and characterized epitopes on DENV4 recognized by neutralizing antibodies in people previously exposed to DENV4 infections or to a live attenuated DENV4 vaccine. To study the fine specificity of DENV4 neutralizing human antibodies, B cells from two people exposed to DENV4 were immortalized and screened to identify DENV-specific clones. Two human monoclonal antibodies (MAbs) that neutralized DENV4 were isolated, and their epitopes were finely mapped using recombinant viruses and alanine scan mutation array techniques. Both antibodies bound to quaternary structure epitopes near the hinge region between envelope protein domain I (EDI) and EDII. In parallel, to characterize the serum neutralizing antibody responses, convalescence-phase serum samples from people previously exposed to primary DENV4 natural infections or a monovalent DENV4 vaccine were analyzed. Natural infection and vaccination also induced serum-neutralizing antibodies that targeted similar epitope domains at the EDI/II hinge region. These studies defined a target of neutralizing antigenic site on DENV4 targeted by human antibodies following natural infection or vaccination.

    IMPORTANCE The four serotypes of dengue virus are the causative agents of dengue fever and dengue hemorrhagic fever. People exposed to primary DENV infections develop long-term neutralizing antibody responses, but these principally recognize only the infecting serotype. An effective vaccine against dengue should elicit long-lasting protective antibody responses to all four serotypes simultaneously. We and others have defined antigenic sites on the envelope (E) protein of viruses of dengue virus serotypes 1, 2, and 3 targeted by human neutralizing antibodies. The epitopes on DENV4 E protein targeted by the human neutralizing antibodies and the mechanisms of serotype 4 neutralization are poorly understood. Here, we report the properties of human antibodies that neutralize dengue virus serotype 4. People exposed to serotype 4 infections or a live attenuated serotype 4 vaccine developed neutralizing antibodies that bound to similar sites on the viral E protein. These studies have provided a foundation for developing and evaluating DENV4 vaccines.

  • The Autographa californica Multiple Nucleopolyhedrovirus ac83 Gene Contains a cis-Acting Element That Is Essential for Nucleocapsid Assembly [Structure and Assembly]

  • Baculoviridae is a family of insect-specific viruses that have a circular double-stranded DNA genome packaged within a rod-shaped capsid. The mechanism of baculovirus nucleocapsid assembly remains unclear. Previous studies have shown that deletion of the ac83 gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) blocks viral nucleocapsid assembly. Interestingly, the ac83-encoded protein Ac83 is not a component of the nucleocapsid, implying a particular role for ac83 in nucleocapsid assembly that may be independent of its protein product. To examine this possibility, Ac83 synthesis was disrupted by insertion of a chloramphenicol resistance gene into its coding sequence or by deleting its promoter and translation start codon. Both mutants produced progeny viruses normally, indicating that the Ac83 protein is not required for nucleocapsid assembly. Subsequently, complementation assays showed that the production of progeny viruses required the presence of ac83 in the AcMNPV genome instead of its presence in trans. Therefore, we reasoned that ac83 is involved in nucleocapsid assembly via an internal cis-acting element, which we named the nucleocapsid assembly-essential element (NAE). The NAE was identified to lie within nucleotides 1651 to 1850 of ac83 and had 8 conserved A/T-rich regions. Sequences homologous to the NAE were found only in alphabaculoviruses and have a conserved positional relationship with another essential cis-acting element that was recently identified. The identification of the NAE may help to connect the data of viral cis-acting elements and related proteins in the baculovirus nucleocapsid assembly, which is important for elucidating DNA-protein interaction events during this process.

    IMPORTANCE Virus nucleocapsid assembly usually requires specific cis-acting elements in the viral genome for various processes, such as the selection of the viral genome from the cellular nucleic acids, the cleavage of concatemeric viral genome replication intermediates, and the encapsidation of the viral genome into procapsids. In linear DNA viruses, such elements generally locate at the ends of the viral genome; however, most of these elements remain unidentified in circular DNA viruses (including baculovirus) due to their circular genomic conformation. Here, we identified a nucleocapsid assembly-essential element in the AcMNPV (the archetype of baculovirus) genome. This finding provides an important reference for studies of nucleocapsid assembly-related elements in baculoviruses and other circular DNA viruses. Moreover, as most of the previous studies of baculovirus nucleocapsid assembly have been focused on viral proteins, our study provides a novel entry point to investigate this mechanism via cis-acting elements in the viral genome.

  • Autographa californica Multiple Nucleopolyhedrovirus AC83 is a Per Os Infectivity Factor (PIF) Protein Required for Occlusion-Derived Virus (ODV) and Budded Virus Nucleocapsid Assembly as well as Assembly of the PIF Complex in ODV Envelopes [Structure and Assembly]

  • Baculovirus occlusion-derived virus (ODV) initiates infection of lepidopteran larval hosts by binding to the midgut epithelia, which is mediated by per os infectivity factors (PIFs). Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes seven PIF proteins, of which PIF1 to PIF4 form a core complex in ODV envelopes to which PIF0 and PIF6 loosely associate. Deletion of any pif gene results in ODV being unable to bind or enter midgut cells. AC83 also associates with the PIF complex, and this study further analyzed its role in oral infectivity to determine if it is a PIF protein. It had been proposed that AC83 possesses a chitin binding domain that enables transit through the peritrophic matrix; however, no chitin binding activity has ever been demonstrated. AC83 has been reported to be found only in the ODV envelopes, but in contrast, the Orgyia pseudotsugata MNPV AC83 homolog is associated with both ODV nucleocapsids and envelopes. In addition, unlike known pif genes, deletion of ac83 eliminates nucleocapsid formation. We propose a new model for AC83 function and show AC83 is associated with both ODV nucleocapsids and envelopes. We also further define the domain required for nucleocapsid assembly. The cysteine-rich region of AC83 is also shown not to be a chitin binding domain but a zinc finger domain required for the recruitment or assembly of the PIF complex to ODV envelopes. As such, AC83 has all the properties of a PIF protein and should be considered PIF8. In addition, pif7 (ac110) is reported as the 38th baculovirus core gene.

    IMPORTANCE ODV is essential for the per os infectivity of the baculovirus AcMNPV. To initiate infection, ODV binds to microvilli of lepidopteran midgut cells, a process which requires a group of seven virion envelope proteins called PIFs. In this study, we reexamined the function of AC83, a protein that copurifies with the ODV PIFs, to determine its role in the oral infection process. A zinc finger domain was identified and a new model for AC83 function was proposed. In contrast to previous studies, AC83 was found to be physically located in both the envelope and nucleocapsid of ODV. By deletion analysis, the AC83 domain required for nucleocapsid assembly was more finely delineated. We show that AC83 is required for PIF complex formation and conclude that it is a true per os infectivity factor and should be called PIF8.

  • Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis [Virus-Cell Interactions]

  • The human genome displays a rich fossil record of past gammaretrovirus infections, yet no current epidemic is evident, despite environmental exposure to viruses that infect human cells in vitro. Feline leukemia viruses (FeLVs) rank high on this list, but neither domestic nor workplace exposure has been associated with detectable serological responses. Nonspecific inactivation of gammaretroviruses by serum factors appears insufficient to explain these observations. To investigate further, we explored the susceptibilities of primary and established human cell lines to FeLV-B, the most likely zoonotic variant. Fully permissive infection was common in cancer-derived cell lines but was also a feature of nontransformed keratinocytes and lung fibroblasts. Cells of hematopoietic origin were generally less permissive and formed discrete groups on the basis of high or low intracellular protein expression and virion release. Potent repression was observed in primary human blood mononuclear cells and a subset of leukemia cell lines. However, the early steps of reverse transcription and integration appear to be unimpaired in nonpermissive cells. FeLV-B was subject to G-ggt;A hypermutation with a predominant APOBEC3G signature in partially permissive cells but was not mutated in permissive cells or in nonpermissive cells that block secondary viral spread. Distinct cellular barriers that protect primary human blood cells are likely to be important in protection against zoonotic infection with FeLV.

    IMPORTANCE Domestic exposure to gammaretroviruses such as feline leukemia viruses (FeLVs) occurs worldwide, but the basis of human resistance to infection remains incompletely understood. The potential threat is evident from the human genome sequence, which reveals many past epidemics of gammaretrovirus infection, and from recent cross-species jumps of gammaretroviruses from rodents to primates and marsupials. This study examined resistance to infection at the cellular level with the most prevalent human cell-tropic FeLV variant, FeLV-B. We found that blood cells are uniquely resistant to infection with FeLV-B due to the activity of cellular enzymes that mutate the viral genome. A second block, which appears to suppress viral gene expression after the viral genome has integrated into the host cell genome, was identified. Since cells derived from other normal human cell types are fully supportive of FeLV replication, innate resistance of blood cells could be critical in protecting against cross-species infection.

  • Mildly Acidic pH Triggers an Irreversible Conformational Change in the Fusion Domain of Herpes Simplex Virus 1 Glycoprotein B and Inactivation of Viral Entry [Virus-Cell Interactions]

  • Herpes simplex virus (HSV) entry into a subset of cells requires endocytosis and endosomal low pH. Preexposure of isolated virions to mildly acidic pH of 5 to 6 partially inactivates HSV infectivity in an irreversible manner. Acid inactivation is a hallmark of viruses that enter via low-pH pathways; this occurs by pretriggering conformational changes essential for fusion. The target and mechanism(s) of low-pH inactivation of HSV are unclear. Here, low-pH-treated HSV-1 was defective in fusion activity and yet retained normal levels of attachment to cell surface heparan sulfate and binding to nectin-1 receptor. Low-pH-triggered conformational changes in gB reported to date are reversible, despite irreversible low-pH inactivation. gB conformational changes and their reversibility were measured by antigenic analysis with a panel of monoclonal antibodies and by detecting changes in oligomeric conformation. Three-hour treatment of HSV-1 virions with pH 5 or multiple sequential treatments at pH 5 followed by neutral pH caused an irreversible ggt;2.5 log infectivity reduction. While changes in several gB antigenic sites were reversible, alteration of the H126 epitope was irreversible. gB oligomeric conformational change remained reversible under all conditions tested. Altogether, our results reveal that oligomeric alterations and fusion domain changes represent distinct conformational changes in gB, and the latter correlates with irreversible low-pH inactivation of HSV. We propose that conformational change in the gB fusion domain is important for activation of membrane fusion during viral entry and that in the absence of a host target membrane, this change results in irreversible inactivation of virions.

    IMPORTANCE HSV-1 is an important pathogen with a high seroprevalence throughout the human population. HSV infects cells via multiple pathways, including a low-pH route into epithelial cells, the primary portal into the host. HSV is inactivated by low-pH preexposure, and gB, a class III fusion protein, undergoes reversible conformational changes in response to low-pH exposure. Here, we show that low-pH inactivation of HSV is irreversible and due to a defect in virion fusion activity. We identified an irreversible change in the fusion domain of gB following multiple sequential low-pH exposures or following prolonged low-pH treatment. This change appears to be separable from the alteration in gB quaternary structure. Together, the results are consistent with a model by which low pH can have an activating or inactivating effect on HSV depending on the presence of a target membrane.

  • Potential Role for a {beta}-Catenin Coactivator (High-Mobility Group AT-Hook 1 Protein) during the Latency-Reactivation Cycle of Bovine Herpesvirus 1 [Virus-Cell Interactions]

  • The latency-related (LR) RNA encoded by bovine herpesvirus 1 (BoHV-1) is abundantly expressed in latently infected sensory neurons. Although the LR gene encodes several products, ORF2 appears to mediate important steps during the latency-reactivation cycle because a mutant virus containing stop codons at the amino terminus of ORF2 does not reactivate from latency in calves. We recently found that the Wnt/bbeta;-catenin signaling pathway is regulated during the BoHV-1 latency-reactivation cycle (Y. Liu, M. Hancock, A. Workman, A. Doster, and C. Jones, J Virol 90:3148nndash;3159, 2016). In the present study, a bbeta;-catenin coactivator, high-mobility group ATnndash;hook 1 protein (HMGA1), was detected in significantly more neurons in the trigeminal ganglia of latently infected calves than in those of uninfected calves. Consequently, we hypothesized that HMGA1 cooperates with ORF2 and bbeta;-catenin to maintain latency. In support of this hypothesis, coimmunoprecipitation studies demonstrated that ORF2 stably interacts with a complex containing bbeta;-catenin and/or HMGA1 in transfected mouse neuroblastoma (Neuro-2A) cells. Confocal microscopy provided evidence that ORF2 was relocalized by HMGA1 and bbeta;-catenin in Neuro-2A cells. ORF2 consistently enhanced the ability of HMGA1 to stimulate bbeta;-catenin-dependent transcription, suggesting that interactions between ORF2 and a complex containing bbeta;-catenin and HMGA1 have functional significance. An ORF2 stop codon mutant, an ORF2 nuclear localization mutant, or a mutant lacking the 5 protein kinase A or C phosphorylation sites interfered with its ability to stimulate bbeta;-catenin-dependent transcription. Since the canonical Wnt/bbeta;-catenin signaling pathway promotes neurogenesis (synapse formation and remodeling) and inhibits neurodegeneration, interactions between ORF2, HMGA1, and bbeta;-catenin may be important for certain aspects of the latency-reactivation cycle.

    IMPORTANCE The lifelong latency of bovine herpesvirus 1 (BoHV-1) requires that significant numbers of infected sensory neurons survive infection and maintain normal functions. Consequently, we hypothesize that viral products expressed during latency cooperate with neuronal factors to maintain latency. Our studies revealed that a bbeta;-catenin coactivator, high-mobility group ATnndash;hook 1 protein (HMGA1), was readily detected in a subset of trigeminal ganglion neurons in latently infected calves but not in uninfected calves. A viral protein (ORF2) expressed in latently infected neurons interacted with bbeta;-catenin and HMGA1 in transfected cells, which resulted in the nuclear localization of bbeta;-catenin. This interaction correlated with the ability of ORF2 to stimulate the coactivator functions of HMGA1. These findings are significant because the canonical Wnt/bbeta;-catenin signaling pathway promotes neurogenesis and inhibits neurodegeneration.

  • Feline Immunodeficiency Virus Cross-Species Transmission: Implications for Emergence of New Lentiviral Infections [Genetic Diversity and Evolution]

  • Owing to a complex history of host-parasite coevolution, lentiviruses exhibit a high degree of species specificity. Given the well-documented viral archeology of human immunodeficiency virus (HIV) emergence following human exposures to simian immunodeficiency virus (SIV), an understanding of processes that promote successful cross-species lentiviral transmissions is highly relevant. We previously reported natural cross-species transmission of a subtype of feline immunodeficiency virus, puma lentivirus A (PLVA), between bobcats (Lynx rufus) and mountain lions (Puma concolor) for a small number of animals in California and Florida. In this study, we investigate host-specific selection pressures, within-host viral fitness, and inter- versus intraspecies transmission patterns among a larger collection of PLV isolates from free-ranging bobcats and mountain lions. Analyses of proviral and viral RNA levels demonstrate that PLVA fitness is severely restricted in mountain lions compared to that in bobcats. We document evidence of diversifying selection in three of six PLVA genomes from mountain lions, but we did not detect selection among 20 PLVA isolates from bobcats. These findings support the hypothesis that PLVA is a bobcat-adapted virus which is less fit in mountain lions and under intense selection pressure in the novel host. Ancestral reconstruction of transmission events reveals that intraspecific PLVA transmission has occurred among panthers (Puma concolor coryi) in Florida following the initial cross-species infection from bobcats. In contrast, interspecific transmission from bobcats to mountain lions predominates in California. These findings document outcomes of cross-species lentiviral transmission events among felids that compare to the emergence of HIV from nonhuman primates.

    IMPORTANCE Cross-species transmission episodes can be singular, dead-end events or can result in viral replication and spread in the new species. The factors that determine which outcome will occur are complex, and the risk of new virus emergence is therefore difficult to predict. We used molecular techniques to evaluate the transmission, fitness, and adaptation of puma lentivirus A (PLVA) between bobcats and mountain lions in two geographic regions. Our findings illustrate that mountain lion exposure to PLVA is relatively common but does not routinely result in communicable infections in the new host. This is attributed to efficient species barriers that largely prevent lentiviral adaptation. However, the evolutionary capacity for lentiviruses to adapt to novel environments may ultimately overcome host restriction mechanisms over time and under certain ecological circumstances. This phenomenon provides a unique opportunity to examine cross-species transmission events leading to new lentiviral emergence.

  • Human CD4+ T Cell Responses to an Attenuated Tetravalent Dengue Vaccine Parallel Those Induced by Natural Infection in Magnitude, HLA Restriction, and Antigen Specificity [Vaccines and Antiviral Agents]

  • Dengue virus (DENV) is responsible for growing numbers of infections worldwide and has proven to be a significant challenge for vaccine development. We previously demonstrated that CD8+ T cell responses elicited by a dengue live attenuated virus (DLAV) vaccine resemble those observed after natural infection. In this study, we screened peripheral blood mononuclear cells (PBMCs) from donors vaccinated with a tetravalent DLAV vaccine (TV005) with pools of dengue virus-derived predicted major histocompatibility complex (MHC) class II binding peptides. The definition of CD4+ T cell responses after live vaccination is important because CD4+ T cells are known contributors to host immunity, including cytokine production, help for CD8+ T and B cells, and direct cytotoxicity against infected cells. While responses to all antigens were observed, DENV-specific CD4+ T cells were focused predominantly on the capsid and nonstructural NS3 and NS5 antigens. Importantly, CD4+ T cell responses in vaccinees were similar in magnitude and breadth to those after natural infection, recognized the same antigen hierarchy, and had similar profiles of HLA restriction. We conclude that TV005 vaccination has the capacity to elicit CD4+ cell responses closely mirroring those observed in a population associated with natural immunity.

    IMPORTANCE The development of effective vaccination strategies against dengue virus infection is of high global public health interest. Here we study the CD4 T cell responses elicited by a tetravalent live attenuated dengue vaccine and show that they resemble responses seen in humans naturally exposed to dengue virus. This is an important issue, since it is likely that optimal immunity induced by a vaccine requires induction of CD4+ responses against the same antigens as those recognized as dominant in natural infection. Detailed knowledge of the T cell response may further contribute to the identification of robust correlates of protection against dengue virus.

  • Channel-Inactivating Mutations and Their Revertant Mutants in the Envelope Protein of Infectious Bronchitis Virus [Structure and Assembly]

  • It has been shown previously in the severe acute respiratory syndrome coronavirus (SARS-CoV) that two point mutations, N15A and V25F, in the transmembrane domain (TMD) of the envelope (E) protein abolished channel activity and led to in vivo attenuation. Pathogenicity was recovered in mutants that also regained E protein channel activity. In particular, V25F was rapidly compensated by changes at multiple V25F-facing TMD residues located on a neighboring monomer, consistent with a recovery of oligomerization. Here, we show using infected cells that the same mutations, T16A and A26F, in the gamma-CoV infectious bronchitis virus (IBV) lead to, in principle, similar results. However, IBV E A26F did not abolish oligomer formation and was compensated by mutations at N- and C-terminal extramembrane domains (EMDs). The C-terminal EMD mutations clustered along an insertion sequence specific to gamma-CoVs. Nuclear magnetic resonance data are consistent with the presence of only one TMD in IBV E, suggesting that recovery of channel activity and fitness in these IBV E revertant mutants is through an allosteric interaction between EMDs and TMD. The present results are important for the development of IBV live attenuated vaccines when channel-inactivating mutations are introduced in the E protein.

    IMPORTANCE The ion channel activity of SARS-CoV E protein is a determinant of virulence, and abolishment of channel activity leads to viral attenuation. E deletion may be a strategy for generating live attenuated vaccines but can trigger undesirable compensatory mechanisms through modifications of other viral proteins to regain virulence. Therefore, a more suitable approach may be to introduce small but critical attenuating mutations. For this, the stability of attenuating mutations should be examined to understand the mechanisms of reversion. Here, we show that channel-inactivating mutations of the avian infectious bronchitis virus E protein introduced in a recombinant virus system are deficient in viral release and fitness and that revertant mutations also restored channel activity. Unexpectedly, most of the revertant mutations appeared at extramembrane domains, particularly along an insertion specific for gammacoronaviruses. Our structural data propose a single transmembrane domain in IBV E, suggesting an allosteric interaction between extramembrane and transmembrane domains.

  • Genesis and Dissemination of Highly Pathogenic H5N6 Avian Influenza Viruses [Genetic Diversity and Evolution]

  • Clade 2.3.4.4 highly pathogenic avian influenza viruses (H5Nx) have spread from Asia to other parts of the world. Since 2014, human infections with clade 2.3.4.4 highly pathogenic avian influenza H5N6 viruses have been continuously reported in China. To investigate the genesis of the virus, we analyzed 123 H5 or N6 environmental viruses sampled from live-poultry markets or farms from 2012 to 2015 in Mainland China. Our results indicated that clade 2.3.4.4 H5N2/N6/N8 viruses shared the same hemagglutinin gene as originated in early 2009. From 2012 to 2015, the genesis of highly pathogenic avian influenza H5N6 viruses occurred via two independent pathways. Three major reassortant H5N6 viruses (reassortants A, B, and C) were generated. Internal genes of reassortant A and B viruses and reassortant C viruses derived from clade 2.3.2.1c H5N1 and H9N2 viruses, respectively. Many mammalian adaption mutations and antigenic variations were detected among the three reassortant viruses. Considering their wide circulation and dynamic reassortment in poultry, we highly recommend close monitoring of the viruses in poultry and humans.

    IMPORTANCE Since 2014, clade 2.3.4.4 highly pathogenic avian influenza (H5Nx) viruses have caused many outbreaks in both wild and domestic birds globally. Severe human cases with novel H5N6 viruses in this group were also reported in China in 2014 and 2015. To investigate the genesis of the genetic diversity of these H5N6 viruses, we sequenced 123 H5 or N6 environmental viruses sampled from 2012 to 2015 in China. Sequence analysis indicated that three major reassortants of these H5N6 viruses had been generated by two independent evolutionary pathways. The H5N6 reassortant viruses had been detected in most provinces of southern China and neighboring countries. Considering the mammalian adaption mutations and antigenic variation detected, the spread of these viruses should be monitored carefully due to their pandemic potential.

  • Lineage A Betacoronavirus NS2 Proteins and the Homologous Torovirus Berne pp1a Carboxy-Terminal Domain Are Phosphodiesterases That Antagonize Activation of RNase L [Pathogenesis and Immunity]

  • Viruses in the family Coronaviridae, within the order Nidovirales, are etiologic agents of a range of human and animal diseases, including both mild and severe respiratory diseases in humans. These viruses encode conserved replicase and structural proteins as well as more diverse accessory proteins, encoded in the 3' ends of their genomes, that often act as host cell antagonists. We previously showed that 2',5'-phosphodiesterases (2',5'-PDEs) encoded by the prototypical Betacoronavirus, mouse hepatitis virus (MHV), and by Middle East respiratory syndrome-associated coronavirus antagonize the oligoadenylate-RNase L (OAS-RNase L) pathway. Here we report that additional coronavirus superfamily members, including lineage A betacoronaviruses and toroviruses infecting both humans and animals, encode 2',5'-PDEs capable of antagonizing RNase L. We used a chimeric MHV system (MHVMut) in which exogenous PDEs were expressed from an MHV backbone lacking the gene for a functional NS2 protein, the endogenous RNase L antagonist. With this system, we found that 2',5'-PDEs encoded by the human coronavirus HCoV-OC43 (OC43; an agent of the common cold), human enteric coronavirus (HECoV), equine coronavirus (ECoV), and equine torovirus Berne (BEV) are enzymatically active, rescue replication of MHVMut in bone marrow-derived macrophages, and inhibit RNase L-mediated rRNA degradation in these cells. Additionally, PDEs encoded by OC43 and BEV rescue MHVMut replication and restore pathogenesis in wild-type (WT) B6 mice. This finding expands the range of viruses known to encode antagonists of the potent OAS-RNase L antiviral pathway, highlighting its importance in a range of species as well as the selective pressures exerted on viruses to antagonize it.

    IMPORTANCE Viruses in the family Coronaviridae include important human and animal pathogens, including the recently emerged viruses severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and Middle East respiratory syndrome-associated coronavirus (MERS-CoV). We showed previously that two viruses within the genus Betacoronavirus, mouse hepatitis virus (MHV) and MERS-CoV, encode 2',5'-phosphodiesterases (2',5'-PDEs) that antagonize the OAS-RNase L pathway, and we report here that these proteins are furthermore conserved among additional coronavirus superfamily members, including lineage A betacoronaviruses and toroviruses, suggesting that they may play critical roles in pathogenesis. As there are no licensed vaccines or effective antivirals against human coronaviruses and few against those infecting animals, identifying viral proteins contributing to virulence can inform therapeutic development. Thus, this work demonstrates that a potent antagonist of host antiviral defenses is encoded by multiple and diverse viruses within the family Coronaviridae, presenting a possible broad-spectrum therapeutic target.

  • Zika Virus Methyltransferase: Structure and Functions for Drug Design Perspectives [Genome Replication and Regulation of Viral Gene Expression]

  • The Flavivirus Zika virus (ZIKV) is the causal agent of neurological disorders like microcephaly in newborns or Guillain-Barre syndrome. Its NS5 protein embeds a methyltransferase (MTase) domain involved in the formation of the viral mRNA cap. We investigated the structural and functional properties of the ZIKV MTase. We show that the ZIKV MTase can methylate RNA cap structures at the N-7 position of the cap, and at the 2'-O position on the ribose of the first nucleotide, yielding a cap-1 structure. In addition, the ZIKV MTase methylates the ribose 2'-O position of internal adenosines of RNA substrates. The crystal structure of the ZIKV MTase determined at a 2.01-AAring; resolution reveals a crystallographic homodimer. One chain is bound to the methyl donor (S-adenosyl-l-methionine [SAM]) and shows a high structural similarity to the dengue virus (DENV) MTase. The second chain lacks SAM and displays conformational changes in the aalpha;X aalpha;-helix contributing to the SAM and RNA binding. These conformational modifications reveal a possible molecular mechanism of the enzymatic turnover involving a conserved Ser/Arg motif. In the second chain, the SAM binding site accommodates a sulfate close to a glycerol that could serve as a basis for structure-based drug design. In addition, compounds known to inhibit the DENV MTase show similar inhibition potency on the ZIKV MTase. Altogether these results contribute to a better understanding of the ZIKV MTase, a central player in viral replication and host innate immune response, and lay the basis for the development of potential antiviral drugs.

    IMPORTANCE The Zika virus (ZIKV) is associated with microcephaly in newborns, and other neurological disorders such as Guillain-Barre syndrome. It is urgent to develop antiviral strategies inhibiting the viral replication. The ZIKV NS5 embeds a methyltransferase involved in the viral mRNA capping process, which is essential for viral replication and control of virus detection by innate immune mechanisms. We demonstrate that the ZIKV methyltransferase methylates the mRNA cap and adenosines located in RNA sequences. The structure of ZIKV methyltransferase shows high structural similarities to the dengue virus methyltransferase, but conformational specificities highlight the role of a conserved Ser/Arg motif, which participates in RNA and SAM recognition during the reaction turnover. In addition, the SAM binding site accommodates a sulfate and a glycerol, offering structural information to initiate structure-based drug design. Altogether, these results contribute to a better understanding of the Flavivirus methyltransferases, which are central players in the virus replication.

  • Binding of the Methyl Donor S-Adenosyl-L-Methionine to Middle East Respiratory Syndrome Coronavirus 2'-O-Methyltransferase nsp16 Promotes Recruitment of the Allosteric Activator nsp10 [Genome Replication and Regulation of Viral Gene Expression]

  • The Middle East respiratory syndrome coronavirus (MERS-CoV) nonstructural protein 16 (nsp16) is an S-adenosyl-l-methionine (SAM)-dependent 2'-O-methyltransferase (2'-O-MTase) that is thought to methylate the ribose 2'-OH of the first transcribed nucleotide (N1) of viral RNA cap structures. This 2'-O-MTase activity is regulated by nsp10. The 2'-O methylation prevents virus detection by cell innate immunity mechanisms and viral translation inhibition by the interferon-stimulated IFIT-1 protein. To unravel the regulation of nsp10/nsp16 2'-O-MTase activity, we used purified MERS-CoV nsp16 and nsp10. First, we showed that nsp16 recruited N7-methylated capped RNA and SAM. The SAM binding promotes the assembly of the enzymatically active nsp10/nsp16 complex that converted 7mGpppG (cap-0) into 7mGpppG2'Om (cap-1) RNA by 2'-OH methylation of N1 in a SAM-dependent manner. The subsequent release of SAH speeds up nsp10/nsp16 dissociation that stimulates the reaction turnover. Alanine mutagenesis and RNA binding assays allowed the identification of the nsp16 residues involved in RNA recognition forming the RNA binding groove (K46, K170, E203, D133, R38, Y47, and Y181) and the cap-0 binding site (Y30, Y132, and H174). Finally, we found that nsp10/nsp16 2'-O-MTase activity is sensitive to known MTase inhibitors, such as sinefungin and cap analogues. This characterization of the MERS-CoV 2'-O-MTase is a preliminary step toward the development of molecules to inhibit cap 2'-O methylation and to restore the host antiviral response.

    IMPORTANCE MERS-CoV codes for a cap 2'-O-methyltransferase that converts cap-0 into cap-1 structure in order to prevent virus detection by cell innate immunity mechanisms. We report the biochemical properties of MERS-CoV 2'O-methyltransferase, which is stimulated by nsp10 acting as an allosteric activator of the nsp16 2'-O-methyltransferase possibly through enhanced RNA binding affinity. In addition, we show that SAM promotes the formation of the active nsp10/nsp16 complex. Conversely, after cap methylation, the reaction turnover is speeded up by cap-1 RNA release and nsp10/nsp16 complex dissociation, at the low intracellular SAH concentration. These results suggest that SAM/SAH balance is a regulator of the 2'-O-methyltransferase activity and raises the possibility that SAH hydrolase inhibitors might interfere with CoV replication cycle. The enzymatic and RNA binding assays developed in this work were also used to identify nsp16 residues involved in cap-0 RNA recognition and to understand the action mode of known methyltransferase inhibitors.

  • Residues in the gp41 Ectodomain Regulate HIV-1 Envelope Glycoprotein Conformational Transitions Induced by gp120-Directed Inhibitors [Pathogenesis and Immunity]

  • Interactions between the gp120 and gp41 subunits of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer maintain the metastable unliganded form of the viral spike. Binding of gp120 to the receptor, CD4, changes the Env conformation to promote gp120 interaction with the second receptor, CCR5 or CXCR4. CD4 binding also induces the transformation of Env into the prehairpin intermediate, in which the gp41 heptad repeat 1 (HR1) coiled coil is assembled at the trimer axis. In nature, HIV-1 Envs must balance the requirements to maintain the noncovalent association of gp120 with gp41 and to evade the host antibody response with the need to respond to CD4 binding. Here we show that the gp41 HR1 region contributes to gp120 association with the unliganded Env trimer. Changes in particular amino acid residues in the gp41 HR1 region decreased the efficiency with which Env moved from the unliganded state. Thus, these gp41 changes decreased the sensitivity of HIV-1 to cold inactivation and ligands that require Env conformational changes to bind efficiently. Conversely, these gp41 changes increased HIV-1 sensitivity to small-molecule entry inhibitors that block Env conformational changes induced by CD4. Changes in particular gp41 HR1 amino acid residues can apparently affect the relative stability of the unliganded state and CD4-induced conformations. Thus, the gp41 HR1 region contributes to the association with gp120 and regulates Env transitions from the unliganded state to downstream conformations.

    IMPORTANCE The development of an efficient vaccine able to prevent HIV infection is a worldwide priority. Knowledge of the envelope glycoprotein structure and the conformational changes that occur after receptor engagement will help researchers to develop an immunogen able to elicit antibodies that block HIV-1 transmission. Here we identify residues in the HIV-1 transmembrane envelope glycoprotein that stabilize the unliganded state by modulating the transitions from the unliganded state to the CD4-bound state.

  • Severe Symptomatic Primary Human Cytomegalovirus Infection despite Effective Innate and Adaptive Immune Responses [Pathogenesis and Immunity]

  • Primary human cytomegalovirus (HCMV) infection usually goes unnoticed, causing mild or no symptoms in immunocompetent individuals. However, some rare severe clinical cases have been reported without investigation of host immune responses or viral virulence. In the present study, we investigate for the first time phenotypic and functional features, together with gene expression profiles in immunocompetent adults experiencing a severe primary HCMV infection. Twenty primary HCMV-infected patients (PHIP) were enrolled, as well as 26 HCMV-seronegative and 39 HCMV-seropositive healthy controls. PHIP had extensive lymphocytosis marked by massive expansion of natural killer (NK) and T cell compartments. Interestingly, PHIP mounted efficient innate and adaptive immune responses with a deep HCMV imprint, revealed mainly by the expansion of NKG2C+ NK cells, CD16+ V2(nndash;) T cells, and conventional HCMV-specific CD8+ T cells. The main effector lymphocytes were activated and displayed an early immune phenotype that developed toward a more mature differentiated status. We suggest that both massive lymphocytosis and excessive lymphocyte activation could contribute to massive cytokine production, known to mediate tissue damage observed in PHIP. Taken together, these findings bring new insights into the comprehensive understanding of immune mechanisms involved during primary HCMV infection in immunocompetent individuals.

    IMPORTANCE HCMV-specific immune responses have been extensively documented in immunocompromised patients and during in utero acquisition. While it usually goes unnoticed, some rare severe clinical cases of primary HCMV infection have been reported in immunocompetent patients. However, host immune responses or HCMV virulence in these patients has not so far been investigated. In the present study, we show massive expansion of NK and T cell compartments during the symptomatic stage of acute HCMV infection. The patients mounted efficient innate and adaptive immune responses with a deep HCMV imprint. The massive lymphocytosis could be the result of nonadapted or uncontrolled immune responses limiting the effectiveness of the specific responses mounted. Both massive lymphocytosis and excessive lymphocyte activation could contribute to massive cytokine production, known to mediate tissue damage. Furthermore, we cannot exclude a delayed immune response caused by immune escape established by HCMV strains.

  • CD63 Regulates Epstein-Barr Virus LMP1 Exosomal Packaging, Enhancement of Vesicle Production, and Noncanonical NF-{kappa}B Signaling [Transformation and Oncogenesis]

  • Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded oncoprotein that is packaged into small extracellular vesicles (EVs) called exosomes. Trafficking of LMP1 into multivesicular bodies (MVBs) alters the content and function of exosomes. LMP1-modified exosomes enhance the growth, migration, and invasion of malignant cells, demonstrating the capacity to manipulate the tumor microenvironment and enhance the progression of EBV-associated cancers. Despite the growing evidence surrounding the significance of LMP1-modified exosomes in cancer, very little is understood about the mechanisms that orchestrate LMP1 incorporation into these vesicles. Recently, LMP1 was shown to be copurified with CD63, a conserved tetraspanin protein enriched in late endosomal and lysosomal compartments. Here, we demonstrate the importance of CD63 presence for exosomal packaging of LMP1. Nanoparticle tracking analysis and gradient purification revealed an increase in extracellular vesicle secretion and exosomal proteins following LMP1 expression. Immunoisolation of CD63-positive exosomes exhibited accumulation of LMP1 in this vesicle population. Functionally, CRISPR/Cas9 knockout of CD63 resulted in a reduction of LMP1-induced particle secretion. Furthermore, LMP1 packaging was severely impaired in CD63 knockout cells, concomitant with a disruption in the perinuclear localization of LMP1. Importantly, LMP1 trafficking to lipid rafts and activation of NF-B and PI3K/Akt pathways remained intact following CD63 knockout, while mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and noncanonical NF-B activation were observed to be increased. These results suggest that CD63 is a critical player in LMP1 exosomal trafficking and LMP1-mediated enhancement of exosome production and may play further roles in limiting downstream LMP1 signaling.

    IMPORTANCE EBV is a ubiquitous gamma herpesvirus linked to malignancies such as nasopharyngeal carcinoma, Burkitt's lymphoma, and Hodgkin's lymphoma. In the context of cancer, EBV hijacks the exosomal pathway to modulate cell-to-cell signaling by secreting viral components such as an oncoprotein, LMP1, into host cell membrane-bound EVs. Trafficking of LMP1 into exosomes is associated with increased oncogenicity of these secreted vesicles. However, we have only a limited understanding of the mechanisms surrounding exosomal cargo packaging, including viral proteins. Here, we describe a role of LMP1 in EV production that requires CD63 and provide an extensive demonstration of CD63-mediated exosomal LMP1 release that is distinct from lipid raft trafficking. Finally, we present further evidence of the role of CD63 in limiting LMP1-induced noncanonical NF-B and ERK activation. Our findings have implications for future investigations of physiological and pathological mechanisms of exosome biogenesis, protein trafficking, and signal transduction, especially in viral-associated tumorigenesis.

  • Screening for Novel Small-Molecule Inhibitors Targeting the Assembly of Influenza Virus Polymerase Complex by a Bimolecular Luminescence Complementation-Based Reporter System [Vaccines and Antiviral Agents]

  • Influenza virus RNA-dependent RNA polymerase consists of three viral protein subunits: PA, PB1, and PB2. Protein-protein interactions (PPIs) of these subunits play pivotal roles in assembling the functional polymerase complex, which is essential for the replication and transcription of influenza virus RNA. Here we developed a highly specific and robust bimolecular luminescence complementation (BiLC) reporter system to facilitate the investigation of influenza virus polymerase complex formation. Furthermore, by combining computational modeling and the BiLC reporter assay, we identified several novel small-molecule compounds that selectively inhibited PB1-PB2 interaction. Function of one such lead compound was confirmed by its activity in suppressing influenza virus replication. In addition, our studies also revealed that PA plays a critical role in enhancing interactions between PB1 and PB2, which could be important in targeting sites for anti-influenza intervention. Collectively, these findings not only aid the development of novel inhibitors targeting the formation of influenza virus polymerase complex but also present a new tool to investigate the exquisite mechanism of PPIs.

    IMPORTANCE Formation of the functional influenza virus polymerase involves complex protein-protein interactions (PPIs) of PA, PB1, and PB2 subunits. In this work, we developed a novel BiLC assay system which is sensitive and specific to quantify both strong and weak PPIs between influenza virus polymerase subunits. More importantly, by combining in silico modeling and our BiLC assay, we identified a small molecule that can suppress influenza virus replication by disrupting the polymerase assembly. Thus, we developed an innovative method to investigate PPIs of multisubunit complexes effectively and to identify new molecules inhibiting influenza virus polymerase assembly.

  • The Structural Interface between HIV-1 Vif and Human APOBEC3H [Virus-Cell Interactions]

  • Human APOBEC3H (A3H) is a cytidine deaminase that inhibits HIV-1 replication. To evade this restriction, the HIV-1 Vif protein binds A3H and mediates its proteasomal degradation. To date, little information on the Vif-A3H interface has been available. To decipher how both proteins interact, we first mapped the Vif-binding site on A3H by functionally testing a large set of A3H mutants in single-cycle infectivity and replication assays. Our data show that the two A3H aalpha;-helixes aalpha;3 and aalpha;4 represent the Vif-binding site of A3H. We next used viral adaptation and a set of Vif mutants to identify novel, reciprocal Vif variants that rescued viral infectivity in the presence of two Vif-resistant A3H mutants. These A3H-Vif interaction points were used to generate the first A3H-Vif structure model, which revealed that the A3H helixes aalpha;3 and aalpha;4 interact with the Vif bbeta;-sheet (bbeta;2-bbeta;5). This model is in good agreement with previously reported Vif and A3H amino acids important for interaction. Based on the predicted A3H-Vif interface, we tested additional points of contact, which validated our model. Moreover, these experiments showed that the A3H and A3G binding sites on HIV-1 Vif are largely distinct, with both host proteins interacting with Vif bbeta;-strand 2. Taken together, this virus-host interface model explains previously reported data and will help to identify novel drug targets to combat HIV-1 infection.

    IMPORTANCE HIV-1 needs to overcome several intracellular restriction factors in order to replicate efficiently. The human APOBEC3 locus encodes seven proteins, of which A3D, A3F, A3G, and A3H restrict HIV-1. HIV encodes the Vif protein, which binds to the APOBEC3 proteins and leads to their proteasomal degradation. No HIV-1 Vif-APOBEC3 costructure exists to date despite extensive research. We and others previously generated HIV-1 Vif costructure models with A3G and A3F by mapping specific contact points between both proteins. Here, we applied a similar approach to HIV-1 Vif and A3H and successfully generated a Vif-A3H interaction model. Importantly, we find that the HIV-1 Vif-A3H interface is distinct from the Vif-A3G and Vif-A3F interfaces, with a small Vif region being important for recognition of both A3G and A3H. Our Vif-A3H structure model informs on how both proteins interact and could guide toward approaches to block the Vif-A3H interface to target HIV replication.

  • Association of Human Papillomavirus 16 E2 with Rad50-Interacting Protein 1 Enhances Viral DNA Replication [Virus-Cell Interactions]

  • Rad50-interacting protein 1 (Rint1) associates with the DNA damage response protein Rad50 during the transition from the S phase to the G2/M phase and functions in radiation-induced G2 checkpoint control. It has also been demonstrated that Rint1 is essential in vesicle trafficking from the Golgi apparatus to the endoplasmic reticulum (ER) through an interaction with Zeste-White 10 (ZW10). We have isolated a novel interaction between Rint1 and the human papillomavirus 16 (HPV16) transcription and replication factor E2. E2 binds to Rint1 within its ZW10 interaction domain, and we show that in the absence of E2, Rint1 is localized to the ER and associates with ZW10. E2 expression results in a disruption of the Rint1-ZW10 interaction and an accumulation of nuclear Rint1, coincident with a significant reduction in vesicle movement from the ER to the Golgi apparatus. Interestingly, nuclear Rint1 and members of the Mre11/Rad50/Nbs1 (MRN) complex were found in distinct E2 nuclear foci, which peaked during mid-S phase, indicating that the recruitment of Rint1 to E2 foci within the nucleus may also result in the recruitment of this DNA damage-sensing protein complex. We show that exogenous Rint1 expression enhances E2-dependent virus replication. Conversely, the overexpression of a truncated Rint1 protein that retains the E2 binding domain but not the Rad50 binding domain acts as a dominant negative inhibitor of E2-dependent HPV replication. Put together, these experiments demonstrate that the interaction between Rint1 and E2 has an important function in HPV replication.

    IMPORTANCE HPV infections are an important driver of many epithelial cancers, including those within the anogenital and oropharyngeal tracts. The HPV life cycle is tightly regulated and intimately linked to the differentiation of the epithelial cells that it infects. HPV replication factories formed in the nucleus are locations where viral DNA is copied to support virus persistence and amplification of infection. The recruitment of specific cellular protein complexes to these factories aids efficient and controlled viral replication. We have identified a novel HPV-host interaction that functions in the cellular response to DNA damage and cell cycle control. We show that the HPV E2 protein targets Rad50-interacting protein 1 (Rint1) to facilitate virus genome replication. These findings add to our understanding of how HPV replicates and the host cell pathways that are targeted by HPV to support virus replication. Understanding these pathways will allow further research into novel inhibitors of HPV genome replication.

  • Classic Spotlight, 1976 and 1977: Articles of Significant Interest Selected from the Journal of Virology Archives by the Editors [Editorial]

  • Herpes Simplex Virus 1 Ubiquitin-Specific Protease UL36 Abrogates NF-{kappa}B Activation in DNA Sensing Signal Pathway [Virus-Cell Interactions]

  • The DNA sensing pathway triggers innate immune responses against DNA virus infection, and NF-B signaling plays a critical role in establishing innate immunity. We report here that the herpes simplex virus 1 (HSV-1) ubiquitin-specific protease (UL36USP), which is a deubiquitinase (DUB), antagonizes NF-B activation, depending on its DUB activity. In this study, ectopically expressed UL36USP blocked promoter activation of beta interferon (IFN-bbeta;) and NF-B induced by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). UL36USP restricted NF-B activation mediated by overexpression of STING, TANK-binding kinase 1, IB kinase aalpha; (IKKaalpha;), and IKKbbeta;, but not p65. UL36USP was also shown to inhibit IFN-stimulatory DNA-induced IFN-bbeta; and NF-B activation under conditions of HSV-1 infection. Furthermore, UL36USP was demonstrated to deubiquitinate IBaalpha; and restrict its degradation and, finally, abrogate NF-B activation. More importantly, the recombinant HSV-1 lacking UL36USP DUB activity, denoted as C40A mutant HSV-1, failed to cleave polyubiquitin chains on IBaalpha;. For the first time, UL36USP was shown to dampen NF-B activation in the DNA sensing signal pathway to evade host antiviral innate immunity.

    IMPORTANCE It has been reported that double-stranded-DNA-mediated NF-B activation is critical for host antiviral responses. Viruses have established various strategies to evade the innate immune system. The N terminus of the HSV-1 UL36 gene-encoded protein contains the DUB domain and is conserved across all herpesviruses. This study demonstrates that UL36USP abrogates NF-B activation by cleaving polyubiquitin chains from IBaalpha; and therefore restricts proteasome-dependent degradation of IBaalpha; and that DUB activity is indispensable for this process. This study expands our understanding of the mechanisms utilized by HSV-1 to evade the host antiviral innate immune defense induced by NF-B signaling.

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    JVI Accepts: Articles Published Ahead of Print

  • Restriction of HCMV replication by ISG15, a host effector regulated by cGAS-STING dsDNA sensing [PublishAheadOfPrint]

  • Accumulation of the interferon-stimulated gene (ISG) 15 protein product, which is reversibly conjugated to numerous polypeptide targets, impacts the proteome and physiology of uninfected and infected cells. While many viruses, including human cytomegalovirus (HCMV) blunt host antiviral defenses by limiting ISG expression, the overall abundance of ISG15 monomer and protein conjugates rises in HCMV-infected cells. However, the molecular signals underlying ISG15 accumulation and whether the ISG15 polypeptide itself influences HCMV infection biology remain unknown. Here, we establish that the ISG15 gene product itself directly regulates HCMV replication and its accumulation restricts productive virus growth. Although ISG15 monomer and protein conjugate accumulation was induced in cells infected with UV-inactivated HCMV, they were subsequently reduced, but not eliminated, by an immediate-early (IE) or early (E) virus-encoded function(s). Instead, HCMV-induced ISG15 monomer and protein conjugate accumulation was dependent upon the double-stranded DNA (dsDNA) sensor cGAS, the innate immune adaptor STING, and interferon signaling. Significantly, dsDNA itself was sufficient to induce cGAS-, STING-, and interferon signaling-dependent ISG15 monomer and conjugate protein accumulation in uninfected cells. Accumulation of ISGylated proteins in uninfected cells treated with dsDNA was prevented by expressing the HCMV multifunctional IE1 transactivator. This demonstrates that expression of a single host interferon-stimulated gene, ISG15, restricts HCMV replication, and that IE1 is sufficient to blunt ISGylation in response to dsDNA sensing in uninfected cells. Moreover, it establishes that ISGylation modifies the proteomes of virus-infected and uninfected normal cells in response to cell intrinsic dsDNA sensing dependent upon cGAS-STING.

    IMPORTANCE By antagonizing type I interferon production and action, many viruses, including human cytomegalovirus (HCMV), evade host defenses. However, levels of the interferon-induced ISG15 protein, which is covalently conjugated to host and viral proteins, increases in HCMV-infected cells. How ISG15 accumulation is regulated and whether the ISG15 polypeptide influences HCMV replication remains unknown. This study establishes that ISG15 itself restricts HCMV replication and HCMV-induced ISG15 accumulation is triggered by host defenses that detect cytoplasmic double strand (ds) DNA. Remarkably, dsDNA triggered ISG15 accumulation even in uninfected cells and this was reduced by HCMV IE1 expression. This shows that ISG15 itself controls replication of HCMV, which causes life-threatening disease among the immunocompromised and is a significant source of congenital morbidity and mortality among newborns. Moreover, it demonstrates that ISG15 modifies the uninfected cell proteome in response to dsDNA, potentially impacting responses to DNA vaccines, gene therapy and autoimmune disease pathogenesis.

  • A glycosylation benchmark profile for HIV-1 envelope glycoprotein production based on eleven Env trimers. [PublishAheadOfPrint]

  • HIV-1 envelope glycoprotein (Env) glycosylation is important because individual glycans are components of multiple broadly neutralizing antibody epitopes, while shielding other sites that might otherwise be immunogenic. The glycosylation on Env is influenced by a variety of factors, including the genotype of the protein, the cell line used for its expression, and the details of the construct design. Here, we used a mass spectrometry-based approach to map the complete glycosylation profile at every site in multiple HIV-1 Env trimers, accomplishing two goals: 1) We determined which glycosylation sites contain conserved glycan profiles across many trimeric Envs. 2) We identified the variables that impact Env's glycosylation profile at sites with divergent glycosylation. Over half of the gp120 glycosylation sites on eleven different trimeric Env proteins have a conserved glycan profile, indicating that a native consensus glycosylation profile does indeed exist among trimers. We showed that some soluble gp120s and gp140s exhibit highly divergent glycosylation profiles compared to trimeric Env. We also assessed the impact of several variables on Env glycosylation: truncating the full-length Env; producing Env in CHO cells, instead of more virologically relevant T lymphocytes; and purifying Env with different chromatographic platforms: Ni-NTA, 2G12, or PGT151 affinity. This study provides the first consensus glycosylation profile of Env trimers, which should serve as a useful benchmark for HIV-1 vaccine developers. This study also defines the sites where glycosylation may be impacted when Env trimers are truncated or produced in CHO cells.

    IMPORTANCE: A protective HIV-1 vaccine will likely include a recombinant version of the viral envelope glycoprotein (Env). Env is highly glycosylated, yet vaccine developers have lacked guidance on how to assess whether their immunogens have optimal glycosylation. Important questions are still unanswered: 1) What is the "target" glycosylation profile, when the goal is to generate a natively glycosylated protein? 2) What variables exert the greatest influence on Env glycosylation? We identified numerous sites on Env where the glycosylation profile does not deviate in eleven different Env trimers, and we investigated the impact on the divergent glycosylation profiles of changing the genotype of the Env sequence, the construct design, the purification method, and the producer cell type. The data herein give vaccine developers a "glycosylation target" for their immunogens, and they show how protein production variables can impact Env glycosylation.

  • Maternal binding and neutralizing IgG responses targeting the C terminal region of the V3 loop are predictive of reduced peripartum HIV-1 transmission risk [PublishAheadOfPrint]

  • The development of an effective maternal HIV-1 vaccine that could synergize with antiretroviral therapy (ART) to eliminate pediatric HIV-1 infection will require the characterization of maternal immune responses capable of blocking transmission of autologous HIV viruses to the infant. We previously identified that maternal plasma antibody binding to linear epitopes within the variable loop 3 (V3) region of HIV envelope (Env) and neutralizing responses against easy to neutralize tier 1 viruses were associated with reduced risk of peripartum HIV infection in the historic U.S. Woman and Infant Transmission Study (WITS) cohort. Here, we defined the fine-specificity and function of the potentially-protective maternal V3-specific IgG antibodies associated with reduced peripartum HIV transmission risk in this cohort. The V3-specific IgG binding that predicted low risk of mother-to-child-transmission (MTCT) was dependent on the C-terminal flank of the V3 crown and particularly on amino acid position 317, a residue that has also been associated with breakthrough transmission in the RV144 vaccine trial. Remarkably, the fine-specificity of potentially-protective maternal plasma V3-specific tier 1 virus neutralizing responses was dependent on the same region in the V3 loop. Our findings suggest that MTCT risk is associated with neutralizing maternal IgG that target amino acid residues in the C-terminal region of the V3 loop crown, suggesting the importance of this region in immunogen design for maternal vaccines to prevent MTCT.

    IMPORTANCE Efforts to curb HIV-1 transmission in pediatric populations by antiretroviral therapy (ART) have been highly successful in both developed and developing countries. However, more than 150,000 infants continue to be infected each year likely due to a combination of late maternal HIV diagnosis, lack of ART access or adherence, and drug-resistant viral strains. Defining the fine-specificity of maternal humoral responses that partially protect against MTCT of HIV is required to inform the development of a maternal HIV vaccine that will enhance these responses during pregnancy. In this study, we identified amino acid residues targeted by potentially-protective maternal V3-specific IgG binding and neutralizing responses, localizing the potentially-protective response in the C terminal region of the V3 loop crown. Our findings have important implications in the design of maternal vaccination strategies that could synergize with ART during pregnancy to achieve the elimination of pediatric HIV infections.

  • The Long Noncoding RNA NEAT1 Exerts Anti-Hantaviral Effects by Acting as a Positive Feedback for RIG-I Signaling [PublishAheadOfPrint]

  • Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs (lncRNAs) play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA, NEAT1, as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus (HTNV) infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced interferon (IFN) bbeta; production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I-IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline-glutamine rich (SFPQ) by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections.

    IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs (lncRNAs) as key regulators of innate immunity; however, their roles in Hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon (IFN) responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I-IRF7 pathway in a time and dose-dependent manner, and promoted HTNV-induced IFN production by facilitating RIG-I and DDX60 expression. Intriguingly, NEAT1 relocated SFPQ and formed paraspeckles after HTNV infection, which might reverse inhibitive effects of SFPQ on the transcription of RIG-I and DDX60. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections.

  • Genotype-specific evolution of hepatitis E virus [PublishAheadOfPrint]

  • Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis globally. HEV comprises four genotypes with different geographic distributions and host ranges. We utilise this natural case-control study for investigating the evolution of zoonotic viruses compared to single host viruses, using 244 near full length HEV genomes. Genome wide estimates of dN/dS located a region of overlapping reading frames, which is subject to positive selection in genotypes 3 and 4. The open reading frames (ORFs) involved have functions related to host-pathogen interaction, so genotype specific evolution of these regions may reflect their fitness. Bayesian inference of evolutionary rates shows genotypes 3 and 4 have significantly elevated rates relative to genotypes 1 across all ORFs. Reconstructing phylogenies of zoonotic genotypes demonstrates significant intermingling of isolates between hosts. We speculate that the genotype specific differences may result from cyclical adaptation to different hosts in genotypes 3 and 4.

    Importance: Hepatitis E virus (HEV) is increasingly recognised as a pathogen which affects both the developing, and the developed world. While most often clinically mild, HEV can be severe or fatal in certain demographics, such as expectant mothers. Like many other viral pathogens, HEV has been grouped into several distinct genotypes. We show that most of the HEV genome is evolutionarily constrained. One locus of positive selection is unusual as it encodes two distinct protein products. We are the first to detect positive selection in this overlap region. Genotype 1, which only infects humans, appears to be evolving differently to genotypes 3 and 4, which infect multiple species, possibly because genotypes 3 and 4 are unable to achieve the same fitness due to repeated host jumps.

  • Low Pathogenic Influenza A Viruses in North American Diving Ducks Contribute to the Emergence of a Novel Highly Pathogenic Influenza A(H7N8) Virus [PublishAheadOfPrint]

  • Introductions of low pathogenic avian influenza (LPAI) viruses of subtypes H5 and H7 into poultry from wild birds have the potential to mutate to highly pathogenic avian influenza (HPAI), but such viruses' origins are often unclear. In January 2016, a novel H7N8 HPAI virus caused an outbreak in turkeys in Indiana, USA. To determine the virus's origin, we sequenced genomes of 441 wild birdmmdash;origin influenza A viruses (IAVs) from North America and subjected them to evolutionary analyses. Results showed that the H7N8 LPAI virus most likely circulated among diving ducks in the Mississippi flyway during autumn 2015 and was subsequently introduced to Indiana turkeys, in which it evolved high pathogenicity. Preceding the outbreak, an isolate with six gene segments (PB2, PB1, PA, HA, NA, and NS) sharing ggt;99% sequence identity with those of H7N8 turkey isolates was recovered from a diving duck sampled in Kentucky, USA. H4N8 IAVs from other diving ducks possessed five H7N8mmdash;like gene segments (PB2, PB1, NA, MP, and NS, ggt;98% sequence identity). Our findings suggest that viral gene constellations circulating among diving ducks can contribute towards the emergence of IAVs that affect poultry. Therefore, diving ducks may serve an important and understudied role in the maintenance, diversification, and transmission of IAVs in the wild bird reservoir.

    IMPORTANCE In January 2016, a novel H7N8 HPAI virus caused a disease outbreak in turkeys in Indiana, USA. Toward determining the origin of this virus, we sequenced and analyzed 441 wild birdmmdash;origin influenza strains isolated from wild birds inhabiting North America. We found that H7N8 LPAI virus most likely circulated among diving ducks in the Mississippi flyway during autumn 2015 and was subsequently introduced to Indiana turkeys, in which it evolved high pathogenicity. Our results suggest that viral gene constellations circulating among diving ducks can contribute towards the emergence of IAVs that affect poultry. Therefore diving ducks may serve an important and understudied role in the maintenance, diversification, and transmission of IAVs in the wild bird reservoir. Our study also highlights the importance of a coordinated, systematic, and collaborative surveillance for IAVs in both poultry and wild bird populations.

  • Endogenous brain lipids inhibit prion amyloid formation in vitro [PublishAheadOfPrint]

  • The normal cellular prion protein (PrPC) resides in detergent-resistant outer membrane lipid rafts in which conversion to the pathogenic misfolded form is believed to occur. Once misfolding occurs, the pathogenic isoform polymerizes into highly stable amyloid fibrils. In vitro assays have demonstrated an intimate association between prion conversion and lipids, specifically phosphatidylethanolamine, which is a critical cofactor in the formation of synthetic infectious prions. In the current work, we demonstrate an alternative inhibitory function of lipids in the prion conversion process as assessed in vitro by real-time, quaking induced conversion (RT-QuIC). Using an alcohol-based extraction technique, we removed the lipid content from chronic wasting disease (CWD)-infected white tailed deer brain homogenates and found lipid extraction enabled RT-QuIC detection of CWD prions in two logs greater concentration of brain sample. Conversely, addition of brain-derived lipid extracts to CWD-prion brain or lymph node samples inhibited amyloid formation in a dose-dependent manner. Subsequent lipid analysis demonstrated this inhibitory function was restricted to the polar lipid fraction in brain. We further investigated three phospholipids commonly found in lipid membranes, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol, and found all three similarly inhibited RT-QuIC. These results demonstrating polar-lipid, and specifically phospholipid, inhibition of prion-seeded amyloid formation highlight the diverse roles lipid constituents may play in the prion conversion process.

    IMPORTANCE Prion conversion is likely influenced by lipid interactions given the location of normal prion protein (PrPC) in lipid rafts and lipid cofactors generating infectious prions in in vitro models. Here, we use real-time quaking induced conversion (RT-QuIC) to demonstrate that endogenous brain polar lipids can inhibit prion seeded amyloid formation, suggesting, prion conversion is guided by an environment of pro-conversion and anti-conversion lipids. These experiments also highlight the applicability of RT-QuIC to identify potential therapeutic inhibitors of prion conversion.

  • Inhibition of HIV-1 Maturation via Small Molecule Targeting of the Amino-Terminal Domain in the Viral Capsid Protein [PublishAheadOfPrint]

  • The HIV-1 capsid protein is an attractive therapeutic target owing to its multifunctionality in virus replication and the high fitness cost of amino acid substitutions in capsid to HIV-1 infectivity. To date, small molecule inhibitors have been identified that inhibit HIV-1 capsid assembly and/or impair its function in target cells. Here we describe the mechanism of action of the previously reported capsid-targeting HIV-1 inhibitor, BI compound 1 (C1). We show that C1 acts during HIV-1 maturation to prevent assembly of a mature viral capsid. However, unlike the maturation inhibitor Bevirimat, C1 did not significantly affect the kinetics or fidelity of Gag processing. HIV-1 particles produced in the presence of C1 contained unstable capsids that lacked associated electron density and exhibited impairments in early postentry stages of infection, most notably reverse transcription. C1 inhibited assembly of recombinant HIV-1 CA in vitro and induced aberrant crosslinks in mutant HIV-1 particles capable of spontaneous intersubunit disulfide bonds at the interhexamer interface in the capsid lattice. Resistance to C1 was conferred by a single amino acid substitution within the compound-binding site in the N-terminal domain of the CA protein. Our results demonstrate that the binding site for C1 represents a new pharmacological vulnerability in the capsid assembly stage of the HIV-1 life cycle.

    IMPORTANCE The HIV-1 capsid protein is an attractive but unexploited target for clinical drug development. Prior studies have identified HIV-1 capsid-targeting compounds that display different mechanisms of action, which in part reflects the requirement for capsid function at both the efferent and afferent phases of viral replication. Here we show that one such compound, Compound 1, interferes with assembly of the conical viral capsid during virion maturation, and results in perturbations at a specific protein-protein interface in the capsid lattice. We also identify and characterize a mutation in the capsid protein that confers resistance to the inhibitor. This study reveals a novel mechanism by which a capsid-targeting small molecule can inhibit HIV-1 replication.

  • Human herpesvirus 6A (HHV-6A) exhibits restrictive propagation with limited activation of PKR-eIF2{alpha} stress pathway [PublishAheadOfPrint]

  • The eIF2aalpha; protein plays a critical role in the regulation of translation. The production of dsRNA during viral replication can activate protein kinase R (PKR) which phosphorylates the eIF2aalpha;, leading to inhibition of the initial step of translation. Many viruses have evolved gene products targeting PKR-eIF2a pathway, indicating its importance in antiviral defense. In the present study we focus on alternations of PKR-eIF2a pathway along HHV-6A infection while monitoring viral gene expression and infectious viral yields. We have found increased phosphorylated PKR as well as phosphorylated eIF2aalpha; coincidental with accumulation of the late gp82-105 viral protein. The level of total PKR was relatively constant, but it decreased by 144 hours post infection. The phosphorylation of eIF2a led to a moderate increase in ATF4 accumulation, indicating moderate inhibition of protein translation during HHV-6A infection. The overexpression of PKR led to decreased viral propagation coincidental with increased accumulation of phosphorylated PKR and phosphorylated eIF2a. Moreover, addition of dominant-negative PKR mutant resulted in a moderate increase in viral replication. HHV-6A exhibits relatively low propagation efficiency of progeny virus secreted into the culture media. This study suggests that the replicative strategy of HHV-6A involves a mild infection over lengthy life cycle in culture, while preventing severe activation of the PKR-eIF2aalpha; pathway.

    IMPORTANCE Human herpesvirus 6A (HHV-6A) and HHV-6B are common, wildly prevalent viruses, causing from mild to severe disease. Our study focuses on the PKR-eIF2aalpha; stress pathway that limits viral replication. The HHV-6 genome encodes multiple genes transcribed from the two strands predicting accumulation of dsRNAs which can activates PKR and inhibition of protein synthesis. We report that HHV-6A induced the accumulation of phosphorylated PKR, phosphorylated eIF2aalpha; and a moderate increase of the activating transcription factor 4 (ATF4), known to transcribe stress genes. Overexpression of PKR led to increased eIF2aalpha; phosphorylation and decreased viral replication whereas overexpression of dominant-negative PKR mutant resulted in a moderate increase in viral replication. These results suggest that HHV-6A replication strategy involves restricted activation of the PKR-eIF2aalpha; pathway, partial translation inhibition and lower yields of infectious virus. In essence, HHV-6A limits its own replication due to the inability to bypass the eIF2aalpha; phosphorylation.

  • The lateral organization of influenza virus proteins in the budozone region of the plasma membrane [PublishAheadOfPrint]

  • Influenza virus assembles and buds at the plasma membrane of virus-infected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some, like HA, NA, and M2 are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with the viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immuno-gold staining. The distribution of these proteins was examined individually and pair-wise using the Ripley K function, a type of nearest neighbor analysis. Individually HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly co-clustered in the plasma membrane; however, in the case of NA and M2 clustering depends upon the expression system used. Despite both being raft-resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly co-cluster but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the co-expression of other viral proteins. Similarly, M2 and NP occupy separate compartments but an association can be bridged by co-expression of M1.

    Importance The complement of influenza proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft like domains whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships between viral proteins in the plasma membrane. Some proteins such as HA and M2 inherently co-cluster within the membrane. Although M2 is mostly found on the periphery of regions of HA consistent with the proposed role of M2 in scission at the end of budding. The association between some pairs of influenza proteins, such as M2 and NP, appear to be brokered by additional influenza proteins, in this case M1. HA and NA, while both raft associated, reside in distinct domains, reflecting their distributions in the viral membrane.

  • BCA2/RABRING7 Interferes with HIV-1 Proviral Transcription by Enhancing the SUMOylation of I{kappa}B{alpha} [PublishAheadOfPrint]

  • BCA2/Rabring7 is a BST2 co-factor that promotes the lysosomal degradation of trapped HIV-1 virions, but also functions as a BST2-independent anti-HIV factor by targeting Gag for lysosomal degradation. Since many antiviral factors regulate the NF-B innate signaling pathway, we investigated whether BCA2 is also connected to this pro-inflammatory cascade. Here we show for the first time that BCA2 is induced by NF-B-activating pro-inflammatory cytokines, and that up-regulation of BCA2 provides a regulatory negative feedback on NF-B. Specifically, BCA2 serves as an E3 SUMO-ligase in the SUMOylation of IBaalpha;, which in turn enhances the sequestration of NF-B components in the cytoplasm. Since HIV-1 utilizes NF-B to promote proviral transcription, the BCA2-mediated inhibition of NF-B significantly decreases the transcriptional activity of HIV-1 (up to 4.4-fold in CD4+ T cells). Therefore, our findings indicate that BCA2 poses an additional barrier to HIV-1 infection: not only does BCA2 prevent assembly and release of nascent virions, but also significantly restricts HIV-1 transcription by inhibiting the NF-B pathway.

    IMPORTANCE Understanding the interactions between HIV-1 and its host cells is highly relevant for the design of new drugs aimed at eliminating HIV-1 from affected individuals. We have previously shown that BCA2, a co-factor of BST2 in the restriction of HIV-1, also prevents virion assembly in a BST2-independent manner. In this study we found that BCA2 negatively regulates the NF-B pathway nndash; a signaling cascade necessary for HIV-1 replication and infectivity nndash;, which in turn detrimentally affects proviral transcription and virus propagation. Thus, our results indicate that besides its already described functions as an antiviral factor, BCA2 poses an additional barrier to HIV-1 replication at the transcriptional level.

  • Relationship between measures of HIV reactivation and the decline of latent reservoir under latency-reversing agents [PublishAheadOfPrint]

  • Antiretroviral-free HIV remission requires substantial reduction of the number of latently infected cells and enhanced immune control of viremia. Latency-reversing agents (LRA) aim to eliminate latently infected cells by increasing the rate of reactivation of HIV transcription, which exposes these cells to killing by the immune system. As LRA are explored in clinical trials, it becomes increasingly important to assess the effect of increased HIV reactivation rate on the decline of latently infected cells, and estimate LRA efficacy in increasing virus reactivation. However, whether the extent of HIV reactivation is a good predictor of the rate of decline of the number of latently infected cells is dependent on a number of factors. Our modeling shows that mechanisms of maintenance and clearance of the reservoir, lifespan of cells with reactivated HIV and other factors may significantly impact the relationship between measures of HIV reactivation and the decline of the number of latently infected cells.

    The usual measures of HIV reactivation are the increase in cell-associated HIV RNA (CA RNA) and/or plasma HIV RNA soon after administration. We analyze two recent studies where CA RNA was used to estimate the impact of two novel LRAs, panobinostat and romidepsin. Both drugs increased the CA RNA level 3-4 fold in clinical trials. However, cells with panobinostat-reactivated HIV appeared long-lived (half-life ggt; month), suggesting that HIV reactivation rate increased approximately by 8%. With romidepsin, the lifespan of cells that reactivated HIV was short (2 days), suggesting that HIV reactivation rate may have doubled under treatment.

    Importance Long-lived latently infected cells that persist on antiretroviral treatment (ART) are thought to be the source of viral rebound soon after ART interruption. The elimination of latently infected cells is an important step to achieving antiretroviral-free HIV remission. Latency-reversing agents (LRA) aim to activate HIV expression in latently infected cells, which could lead to their death. Here we discuss the possible impact of the LRA on the reduction of the number of latently infected cells, depending on the mechanisms of their loss and self-renewal and on the lifespan of the cells that have activated HIV transcription by the LRAs.

  • Superiority in Rhesus Macaques of Targeting HIV-1 Env Gp140 to CD40 Versus LOX-1 in Combination with Replication Competent NYVAC-KC for Induction of Env-Specific Antibody and T Cell Responses [PublishAheadOfPrint]

  • We compared the HIV-1-specific immune responses generated by targeting HIV-1 envelope protein (Env gp140) to either CD40 or LOX-1, two endocytic receptors on dendritic cells (DCs), in Rhesus macaques primed with a poxvirus vector (NYVAC-KC) expressing Env gp140. The DC-targeting vaccines, humanized recombinant monoclonal antibodies fused to Env gp140, were administered as a boost with poly ICLC adjuvant either alone or co-administered with the NYVAC-KC vector. All the DC-targeting vaccine administrations with poly ICLC increased the low-level serum anti-Env IgG responses elicited by NYVAC-KC priming significantly more (up to P =0.01) than a group without poly ICLC. The responses were robust, cross-reactive, and contained antibodies specific to multiple epitopes within gp140 including the C1, C2, V1-3, C4, C5, and gp41 immuno-dominant regions. The DC-targeting vaccines also elicited modest serum Env-specific IgA responses. All groups gave serum neutralization activity limited to Tier 1 viruses and antibody dependent cytotoxicity responses (ADCC) after DC-targeting boosts. Furthermore, CD4+ and CD8+ T cell responses specific to multiple Env epitopes were strongly boosted by the DC-targeting vaccines + poly ICLC. Together, these results indicate that prime/boost immunization via NYVAC-KC and either aalpha;CD40.Env gp140/poly ICLC or aalpha;LOX-1.Env gp140/poly ICLC induced balanced antibody and T cell responses against HIV-1 Env. Co-administration of NYVAC-KC with the DC-targeting vaccines increased T cell responses, but had minimal effects on antibody responses except for suppressing serum IgA responses. Overall, compared to LOX-1, targeting Env to CD40 gave more robust T cell and serum antibody responses with broader epitope representation and greater durability.

    IMPORTANCE An effective vaccine to prevent HIV-1 infection does not yet exist. An approach to elicit strong protective antibody development is to direct virus protein antigens specifically to dendritic cells, which are now known to be the key cell type for controlling immunity. Here we have tested in non-human primates two prototype vaccines engineered to direct the HIV-1 coat protein Env to dendritic cells. These vaccines bind to either CD40 or LOX-1, two dendritic cell surface receptors with different functions and tissue distributions. We tested the vaccines described above in combination with attenuated virus vectors that express Env. Both vaccines, but especially that delivered via CD40, raised robust immunity against HIV-1 as measured by monitoring potentially protective antibody and T cell responses in the blood. The safety and efficacy of the CD40-targeted vaccine justifies further development for future human clinical trials.

  • Careers in Virology: Working at the Centers for Disease Control and Prevention [PublishAheadOfPrint]

  • As non-academic careers in science have become less and less "alternative," one field that has consistently attracted early-career virologists is public health research. The desire to make tangible contributions towards public health needs and better protect the public from infectious disease often motivate the transition. In this Career Gem, two academically-trained virologists offer insights into pursuing a research career in public health at the Centers for Disease Control and Prevention.

  • Inhibition of Vpx-mediated SAMHD1 and Vpr-mediated HLTF degradation by selective disruption of viral CRL4 (DCAF1) E3 ubiquitin ligase assembly [PublishAheadOfPrint]

  • The lentiviral accessory proteins Vpx and Vpr are known to utilize CRL4 (DCAF1) E3 ligase to induce the degradation of the host restriction factor SAMHD1 or transcriptional factor HLTF, respectively. Selective disruption of viral CRL4 (DCAF1) E3 ligase could be a promising antiviral strategy. Recently, we have determined that post-translational modification (neddylation) of Cullin-4 is required for the activation of Vpx-CRL4 (DCAF1) E3 ligase. However, the mechanism of Vpx/Vpr-CRL4 (DCAF1) E3 ligase assembly is still poorly understood. Here, we report that zinc coordination is an important regulator of Vpx-CRL4 E3 ligase assembly. Residues in a conserved zinc-binding motif of Vpx were essential for the recruitment of the CRL4 (DCAF1) E3 complex and Vpx-induced SAMHD1 degradation. Importantly, altering the intracellular zinc concentration by treatment with the zinc chelator, N,N,N,N-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN) potently blocked Vpx-mediated SAMHD1 degradation and inhibited wild-type SIVmac infection of myeloid cells, even in the presence of Vpx. TPEN selectively inhibited Vpx and DCAF1 binding, but not the Vpx-SAMHD1 interaction or Vpx virion packaging. Moreover, we have shown that zinc coordination is also important for the assembly of the HIV-1 Vpr-CRL4 E3 ligase. In particular, Vpr zinc-binding motif mutation or TPEN treatment efficiently inhibited Vpr-CRL4 (DCAF1) E3 ligase assembly and Vpr-mediated HTLF degradation or Vpr-induced G2 cell cycle arrest. Collectively, our study sheds light on a conserved strategy by the viral proteins Vpx and Vpr to recruit host CRL4 (DCAF1) E3 ligase, which represents a target for novel anti-HIV drug development.

    IMPORTANCE The Vpr and its paralog Vpx are accessory proteins encoded by different HIV/SIV lentiviruses. To facilitate viral replication, Vpx has evolved to induce SAMHD1 degradation and Vpr to mediate HLTF degradation. Both Vpx and Vpr perform their functions by recruiting CRL4 (DCAF1) E3 ligase. In this study, we demonstrate that the assembly of the Vpx- or Vpr-CRL4 E3 ligase requires a highly conserved zinc-binding motif. This motif is specifically required for the DCAF1 interaction but not for the interaction of Vpx or Vpr with their substrates. Selective disruption of Vpx- or Vpr-CRL4 E3 ligase function was achieved by zinc sequestration using N,N,N' -tetrakis-(2' -pyridylmethyl) ethylenediamine (TPEN). At the same time, zinc sequestration had no effect on zinc-dependent cellular protein functions. Therefore, information obtained from this study may be important for novel anti-HIV drug development.

  • The viral gene ORF79 encodes a repressor regulating induction of the lytic life cycle in the haloalkaliphilic virus {varphi}Ch1 [PublishAheadOfPrint]

  • In this study, we describe the construction of the first genetically modified mutant of a halovirus infecting haloalkaliphilic Archaea. By random choice we targeted ORF79, a so far uncharacterized viral gene of the haloalkaliphilic virus Ch1. We used a PEG-mediated transformation method to deliver a disruption cassette into a lysogenic strain of the haloalkaliphilic archaeon Natrialba magadii bearing Ch1 as provirus. This approach yielded mutant virus particles carrying a disrupted version of the ORF79. Disruption of ORF79 did not influence morphology of the mature virions. The mutant virus was able to infect cured strains of N. magadii resulting in a lysogenic, ORF79-disrupted strain. Analysis of this strain carrying the mutant virus revealed a repressor function of ORF79. In absence of gp79, onset of lysis as well as expression of viral proteins occurred prematurely compared to the wild-type strain. Constitutive expression of ORF79 in a cured strain of N. magadii reduced the plating efficiency of Ch1 by seven orders of magnitude. Overexpression of ORF79 in a lysogenic strain of N. magadii resulted in an inhibition of lysis and total absence of viral proteins as well as viral progeny. In further experiments, gp79 directly regulated the expression of the tail fiber protein ORF34 but did not influence the methyltransferase gene ORF94. Further, we describe the establishment of an inducible promoter for in vivo studies in N. magadii.

    IMPORTANCE Genetic analyses of haloalkaliphilic Archaea or haloviruses are only rarely reported. Therefore, only little insight into the in vivo roles of proteins and their functions has been gained so far. We used a reverse genetic approach to identify the function of a yet undescribed gene of Ch1. We provide evidence that gp79, a so far unknown protein of Ch1, acts as a repressor protein of the viral life cycle, affecting the transition from the lysogenic to the lytic state of the virus. Thus, repressor genes in other haloviruses could be identified by sequence homologies to gp79 in the future. Moreover, we describe the use of an inducible promoter of N. magadii. Our work provides valuable tools for the identification of other unknown viral genes by our approach as well as for functional studies of proteins by inducible expression.

  • TRIM25 Is Required for the Antiviral Activity of Zinc-finger Antiviral Protein (ZAP) [PublishAheadOfPrint]

  • Zinc-finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses by binding to viral mRNAs, and repressing the translation and/or promoting the degradation of target mRNA. In addition, ZAP regulates the expression of certain cellular genes. Here, we report that tripartite motif-containing protein 25 (TRIM25), a ubiquitin E3 ligase, is required for the antiviral activity of ZAP. Downregulation of endogenous TRIM25 abolished ZAP's antiviral activity. The E3 ligase activity of TRIM25 is required for this regulation. TRIM25 mediated ZAP ubiquitination, but the ubiquitination of ZAP itself did not seem to be required for its antiviral activity. Downregulation of endogenous ubiquitin or overexpression of deubiquitinase OTUB1 impaired ZAP's activity. We provide evidence indicating that TRIM25 modulates the target RNA binding activity of ZAP. These results uncover a mechanism by which the antiviral activity of ZAP is regulated.

    IMPORTANCE ZAP is a host antiviral factor that specifically inhibits the replication of certain viruses, including HIV-1, Sindbis virus and Ebola virus. ZAP binds directly to target mRNA, and represses the translation and promotes the degradation of target mRNA. While the mechanisms by which ZAP post-transcriptionally inhibits target RNA expression has been extensively studied, how its antiviral activity is regulated is not very clear. Here we report that TRIM25, a ubiquitin E3 ligase, is required for the antiviral activity of ZAP. Downregulation of endogenous TRIM25 remarkably abolished ZAP's activity. TRIM25 is required for ZAP optimal binding to target mRNA. These results help to better understand how the antiviral activity of ZAP is regulated.

  • JAK-STAT Signaling Pathways and Inhibitors Affect Reversion of Envelope Mutated HIV-1 [PublishAheadOfPrint]

  • HIV can spread by both cell-free and cell-to-cell transmission. Here, we show that numerous of the amino acid changes in Env that are close to the CD4 binding pocket can affect HIV replication. We generated a number of mutant viruses that were unable to infect T cells as cell-free viruses but were nevertheless able to infect certain T cell lines as cell-associated viruses, followed by reversion to wild type. However, the activation of JAK-STAT signaling pathways caused inhibition of such cell-to-cell infection as well as the reversion of multiple HIV Env mutants that displayed differences in ability to bind to the CD4 receptor. Specifically, two T cell activators, Interleukin-2 (IL2) and phorbol-12-myristate 13-acetate (PMA), both capable of activation of JAK-STAT pathways, were able to inhibit cell-to-cell viral transmission. In contrast, but consistent with the above result, a number of JAK-STAT-mTOR inhibitors actually promoted HIV-1 transmission and reversion. Hence, JAK-STAT signaling pathways may differentially affect the replication of a variety of HIV Env mutants in ways that differ from the role that these pathways play in the replication of wild type viruses.

    Importance: Specific alterations in HIV Env close to the CD4 binding site can differentially change the ability of HIV to mediate infection for cell-free and cell-associated viruses. However, such differences are dependent to some extent on the types of target cells used. JAK-STAT signaling pathways are able to play major roles in these processes. This work sheds new light on factors that can govern HIV infection of target cells.

  • Mutation of the 2nd Sialic Acid-Binding Site Resulting in Reduced Neuraminidase Activity Preceded Emergence of H7N9 Influenza A Virus [PublishAheadOfPrint]

  • The emergence of novel influenza A virus (IAV) H7N9 since 2013 has caused concerns about the ability of this virus to spread between humans. Analysis of the receptor-binding properties of the H7 protein of a human isolate revealed modestly increased binding to aalpha;2,6 sialosides and reduced, but still dominant, binding to aalpha;2,3-linked sialic acids (SIAs) compared to a closely related avian H7N9 virus from 2008. Here we show that the corresponding N9 neuraminidases display equal enzymatic activity on a soluble monovalent substrate and similar substrate specificity on a glycan array. In contrast, solid phase activity and binding assays demonstrated reduced specific activity and decreased binding of the novel N9 protein. Mutational analysis showed these differences to result from substitution T401A in the 2nd SIA-binding site, indicating that substrate binding via this site enhances NA catalytic activity. Substitution T401A in the novel N9 protein appears to functionally mimic the substitutions that are found in the 2nd SIA-binding site of NA proteins of avian-derived IAVs that became human pandemic viruses. Our phylogenetic analyses show that substitution T401A occurred prior to substitutions in HA causing the altered receptor-binding properties mentioned above. Hence, in contrast to the widespread assumption that such changes in NA are only obtained after acquisition of functional changes in HA, our data indicate that mutations in the 2nd SIA binding site may have enabled and even driven the acquisition of altered HA receptor-binding properties, and may have contributed to the spread of the novel H7N9 viruses.

    IMPORTANCE Novel H7N9 IAVs continue to cause human infections and pose an ongoing public health threat. Here, we show that their N9 protein displays reduced binding to and lower enzymatic activity against multivalent substrates, resulting from mutation of the 2nd sialic acid-binding site. This mutation preceded and may have driven the selection of substitutions in H7 that modify H7 receptor-binding properties. Of note, all animal IAVs that managed to cross the host species barrier and became human viruses carry mutated 2nd sialic acid-binding sites. Screening of animal IAVs to monitor their potential to cross the host species barrier should therefore not only focus on the HA protein, but also on the functional properties of NA.

  • Differential Disruption of Nucleocytoplasmic Trafficking Pathways by Rhinovirus 2A Proteases [PublishAheadOfPrint]

  • The RNA rhinoviruses (RV) encode 2A proteases (2Apro) that contribute essential polyprotein processing and host-cell shutoff functions during infection, including the cleavage of Phe/Gly-containing nucleoporin proteins (Nups) within nuclear pore complexes (NPC). Within the 3 RV species, multiple divergent genotypes encode diverse 2Apro sequences which act differentially on specific Nups. Since only subsets of Phe/Gly motifs, particularly those within Nup62, Nup98 and Nup153, are recognized by transport receptors (karyopherins) when trafficking large molecular cargos through the NPC, the processing preferences of individual 2Apro predict RV genotype-specific targeting of NPC pathways and cargos. To test this idea, transformed HeLa cells lines were created with fluorescent cargos (mCherry) for the importin aalpha;/bbeta;, transportin-1, and transportin-3 import pathways, and the Crm1-mediated export pathway. Live cell imaging of single cells expressing recombinant RV 2Apro (A16, A45, B04, B14, B52, C02, and C15) showed disruption of each pathway with measurably different efficiencies and reaction rates. The B04 and B52 proteases preferentially targeted Nups in the import pathways, while B04 and C15 proteases were more effective against the export pathway. Virus type-specific trends were also observed during infection of cells with A16, B04, B14, and B52 viruses or their chimeras, as measured by NF-KB (p65/Rel) translocation into the nucleus and the rates of virus-associated cytopathic effects. This study provides new tools for evaluating the host cell response to RV infections in real time, and suggests that differential 2Apro activities explain, in part, strain-dependent host responses and diverse RV disease phenotypes.

    IMPORTANCE Genetic variation among human rhinovirus types includes unexpected diversity in the viral protease genes (2Apro) which help these viruses achieve anti-host responses. When the enzyme activities of 7 different 2Apro were measured comparatively in transformed cells programed with fluorescent reporter systems and quantitative cell imaging, indeed, the cellular substrates, particularly in the nuclear pore complex, used by these proteases were attacked with different rates and affinities. The importance of this finding is that it provides a mechanistic explanation for how different types (strains) of rhinoviruses may elicit different cell responses which directly or indirectly lead to distinct disease phenotypes.

  • A murine herpesvirus closely related to ubiquitous human herpesviruses causes T-cell depletion [PublishAheadOfPrint]

  • Mouse models of human herpesvirus infections The human roseoloviruses HHV6A, HHV6B, and HHV7 comprise the Roseolovirus genus of the human Betaherpesvirinae subfamily. Infections with these viruses have been implicated in many diseases; however, it has been challenging to establish infections with Roseoloviruses as direct drivers of pathology because they are nearly ubiquitous and display species-specific tropism. Furthermore, controlled study of infection has been hampered by the lack of experimental models, and until now, a mouse roseolovirus has not been identified. Herein we describe a virus that causes severe thymic necrosis in neonatal mice, characterized by a loss of CD4+ T-cells. These phenotypes resemble those caused by the previously described mouse thymic virus (MTV), a putative herpesvirus that has not been molecularly characterized. By Next Generation sequencing of infected tissue homogenates, we assembled a contiguous 174Kb genome sequence encoding 128 unique predicted open reading frames (ORFs), many of which were most closely related to herpesvirus genes. Moreover, the structure of the virus genome and phylogenetic analysis of multiple genes strongly suggested that this virus is a betaherpesvirus more closely related to the roseoloviruses, HHV6A, HHV6B, and HHV7, than another murine betaherpesvirus, mouse cytomegalovirus (MCMV). As such, we have named this virus murine roseolovirus (MRV) because these data strongly suggest that MRV is a mouse homolog of HHV6A/HHV6B/HHV7.

    Importance: Herein we describe the complete genome sequence of a novel murine herpesvirus. By sequence and phylogenetic analyses, we show that it is a betaherpesvirus most closely related to the roseoloviruses, human herpesvirus 6A, 6B, and 7. These data combined with physiological similarities with human roseoloviruses collectively suggest that this virus is a murine roseolovirus (MRV), the first definitively described rodent roseolovirus, to our knowledge. Many biological and clinical ramifications of roseolovirus infection in humans have been hypothesized, but studies showing definitive causative relationships between infection and disease susceptibility are lacking. Here we show that MRV infects the thymus and causes T-cell depletion, suggesting that other roseoloviruses may have similar properties.

  • Differentiation-dependent LMP1 expression is required for efficient lytic EBV reactivation in epithelial cells [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV)-associated diseases of epithelial cells, including tumors such as nasopharyngeal carcinoma (NPC) that have latent infection, and oral hairy leukoplakia (OHL) lesions that have lytic infection, frequently express the viral latent membrane protein 1 (LMP1). In lytically-infected cells, LMP1 expression is activated by the BRLF1 (R) immediate-early (IE) protein. However, the mechanisms by which LMP1 expression is normally regulated in epithelial cells remain poorly understood, and its potential roles in regulating lytic reactivation in epithelial cells are as yet unexplored. We previously showed that the differentiation-dependent cellular transcription factors, KLF4 and BLIMP1, induce lytic EBV reactivation in epithelial cells by synergistically activating the two EBV immediate-early promoters (Zp and Rp). Here we show that epithelial cell differentiation also induces LMP1 expression. We demonstrate that KLF4 and BLIMP1 cooperatively induce the expression of LMP1, even in the absence of the EBV IE proteins BZLF1 (Z) and R, via activation of the two LMP1 promoters. Furthermore, we find that differentiation of NOKs-Akata cells by either methylcellulose suspension, or by organotypic culture, induces LMP1 expression prior to Z and R expression. We show that LMP1 enhances the lytic-inducing effects of epithelial cell differentiation, as well as TPA and sodium butyrate treatment, in EBV-infected epithelial cells by increasing expression of the Z and R proteins. Our results suggest that differentiation of epithelial cells activates a feed-forward loop in which KLF4 and BLIMP1 first activate LMP1 expression, and then cooperate with LMP1 to activate Z and R expression.

    IMPORTANCE The EBV protein LMP1 is expressed in EBV-associated epithelial cell diseases, regardless of whether these diseases are due to lytic infection (such as oral hairy leukoplakia), or latent infection (such as nasopharyngeal carcinoma). However, surprisingly little is known about how LMP1 expression is regulated in epithelial cells, and there are conflicting reports about whether it plays any role in regulating viral lytic reactivation. In this study, we show that epithelial cell differentiation induces LMP1 expression by increasing expression of two cellular transcription factors (KLF4 and BLIMP1) which cooperatively activate the two LMP1 promoters. We also demonstrate that LMP1 promotes efficient lytic reactivation in EBV-infected epithelial cells by enhancing expression of the Z and R proteins. Thus, in EBV-infected epithelial cells, LMP1 expression is promoted by differentiation and positively regulates lytic viral reactivation.

  • A Sequence-Independent, Unstructured IRES is Responsible for Internal Expression of the Coat Protein of Turnip Crinkle Virus [PublishAheadOfPrint]

  • To maximize the coding potential of viral genomes, internal ribosome entry sites (IRES) can be used to bypass the traditional requirement of a 5' cap and some/all of the associated translation initiation factors. Although viral IRES typically contain higher order RNA structure, an unstructured sequence of about 84-nt immediately upstream of the Turnip crinkle virus (TCV) coat protein (CP) ORF has been found to promote internal expression of the CP from the genomic (g)RNA both in vitro and in vivo. Absence of extensive RNA structure was predicted using RNA folding algorithms and confirmed by SHAPE structure probing. Analysis of the IRES region in vitro using both the TCV gRNA and reporter constructs did not reveal any sequence-specific elements but rather suggested that overall lack of structure was an important feature for IRES activity. The CP IRES is A-rich, independent of orientation, and is strongly conserved among viruses in the same genus. The IRES was dependent on eIF4G, but not eIF4E, for activity. Low levels of CP accumulated in vivo in the absence of detectable TCV subgenomic RNAs, strongly suggesting that the IRES was active in the gRNA in vivo. Since the TCV CP also serves as the viral silencing suppressor, early translation of the CP from the viral gRNA is likely important for countering host defenses. Cellular mRNA IRES also lack extensive RNA structures or sequence conservation suggesting that this viral IRES and cellular IRES may share a similar strategy for internal translation initiation.

    IMPORTANCE Cap-independent translation is a common strategy among positive-sense, single-stranded RNA viruses for bypassing the host cell requirement of a 5' cap structure. Viral IRES in general contain extensive secondary structure that is critical for activity. In contrast, we demonstrate that a region of viral RNA devoid of extensive secondary structure has IRES activity and produces low levels of viral coat protein in vitro and in vivo. Our findings may be applicable to cellular mRNA IRES that also have little or no sequences/structures in common.

  • Two white spot syndrome virus miRNAs target Dorsal to promote virus infection in shrimp Marsupenaeus japonicas [PublishAheadOfPrint]

  • In eukaryotes, microRNAs (miRNAs) serve as regulators of many biological processes, including virus infection. A miRNA can generally target diverse genes during virusmmdash;host interactions. However, regulation of gene expression by multiple miRNAs has not yet been extensively explored during virus infection. This study found that the Spz-Toll-Dorsal-ALF signaling pathway played a very important role in antiviral immunity in shrimp (Marsupenaeus japonicus) against invasion of white spot syndrome virus (WSSV). Dorsal, the central gene in Toll pathway, was targeted by two viral miRNAs (WSSV-miR-N13 and WSSV-miR-N23) during WSSV infection. Regulation of Dorsal expression by viral miRNAs suppressed the Spz-Toll-Dorsal-ALF signaling pathway in shrimp in vivo, leading to virus infection. Our study contributed novel insights into the viral miRNA-mediated Toll signaling pathway during virusmmdash;host interaction.

    Importance A miRNA can target diverse genes during virusmmdash;host interactions. However, regulation of gene expression by multiple miRNAs has not yet been extensively explored during virus infection. Results of this study indicated that shrimp Dorsal, the central gene in Toll pathway, was targeted by two viral miRNAs during infection with white spot syndrome virus. Regulation of Dorsal expression by viral miRNAs suppressed the Spz-Toll-Dorsal-ALF signaling pathway in shrimp in vivo, leading to virus infection. Our study provided a new insight into the viral miRNA-mediated Toll signaling pathway in virusmmdash;host interactions.

  • HIV-specific granzyme B, but not interferon-{gamma} secreting T Cells are associated with reduced viral reservoirs in early HIV infection [PublishAheadOfPrint]

  • A major barrier to an Human Immunodeficiency Virus Type 1 (HIV) infection cure is the establishment of a viral reservoir despite cART. It is unclear how HIV-specific CTLs influence the size of the reservoir in early HIV infection. 28 subjects with early HIV infection were recruited to receive cART and followed for 48 weeks. HIV reservoirs in peripheral CD4+ T cells measured by cell associated proviral DNA and viral outgrowth cultures were determined at baseline and 48 weeks of cART. At baseline, Granzyme B and IFN- ELISpot assays were performed with peptides spanning the HIV proteome. All subjects had detectable HIV-specific granzyme B and IFN- responses at baseline. The quantity and specificity of granzyme B responses did not correlate with IFN- responses. For Granzyme B, Tat/Rev was the most dominant whereas for IFN-, Gag predominated. HIV-specific granzyme B T cell responses negatively correlated with HIV proviral loads at baseline and 48 weeks, and with replication competent viral IUPM at baseline but not significantly at 48 weeks. Tat/Rev-, Env-, Gag- and Vif-specific granzyme B responses correlated most strongly with reservoir control. There was no correlation with HIV-specific IFN- responses with reservoir size at baseline or 48 weeks. The majority of granzyme B responses were contributed by CD8+ T cells. Thus, our findings suggest that the induction of potent granzyme B producing CTL to Tat, Rev, Env, Gag and Vif during early infection may be able to prevent the establishment of a large viral reservoir, thereby facilitating a reduced HIV burden.

    IMPORTANCE A major barrier to the cure of Human Immunodeficiency Virus Type 1 (HIV) infection is the establishment of a viral reservoir that must be significantly reduced or eradicated entirely to enable a cure. Combined antiretroviral therapy (cART) alone is unable to clear this viral reservoir. It has been shown that CD8+ cytotoxic T lymphocytes (CTLs) are important in controlling early HIV infection by reducing plasma viremia. However, it is not known if these HIV-specific CTLs influence the establishment of the viral reservoir in early HIV infection. We show that HIV-specific granzyme B targeting HIV Tat/rev, Env, Gag and Vif, but not IFN- responses are associated with reduced virus reservoirs at baseline and at 48 weeks of cART. These findings shed light on the nature of the effector CTL response that might limit reservoir size with implications on cure research and HIV vaccines.

  • A point mutation in the RNA-binding domain of human parainfluenza virus type 2 nucleoprotein elicits an abnormally enhanced polymerase activity [PublishAheadOfPrint]

  • The genome RNA of human parainfluenza virus type 2 (hPIV2) that acts as template for the polymerase complex is entirely encapsidated by the nucleoprotein (NP). Recently, the crystal structure of NP of PIV5, a virus closely related to hPIV2, was resolved in association with RNA. Ten amino acids that contact the bound RNA-binding were identified, and are strictly conserved between PIV5 and hPIV2 NP. Mutation of hPIV2 NP Q202 (that contacts a base rather than the RNA backbone) to some amino acids resulted in an over thirty-fold increase of polymerase activity as evidenced by a minireplicon assay, even though RNA-binding affinity was unaltered. Using various modified minireplicons, we found that enhanced reporter gene expression could be accounted for by increased minigenome replication, whereas mRNA synthesis itself was not affected by Q202 mutation. Moreover, the enhanced activities were still observed in minigenomes partially lacking the leader sequence and which were not of hexamer genome length. Unexpectedly, recombinant hPIV2 possessing the NP Q202A mutation could not be recovered from cDNA.

    IMPORTANCE We examined the importance of amino acids in the putative RNA-binding domain of hPIV2 NP for polymerase activity using minireplicons. Abnormally enhanced genome replication was observed by the substitution in NP Q202 position to various amino acids. Surprisingly, this mutation enabled polymerase to use minigenomes partially lacking the leader sequence and not of hexamer genome length. This mutation does not affect fundamental properties of NP, e.g., recognition of gene junctional and editing signals. However, the strongly enhanced polymerase activity may not be viable for infectious life-cycle. This report highlights the potential of the polymerase complex with point mutations in NP, and helps our detailed understanding of the molecular basis of gene expression.

  • HIV/AIDS vaccine candidates based on replication-competent recombinant poxvirus NYVAC-C-KC expressing trimeric gp140 and Gag-derived VLPs or lacking the viral molecule B19 that inhibits type I interferon activate relevant HIV-1-specific B and T cell immune functions in non-human primates [PublishAheadOfPrint]

  • The non-replicating attenuated poxvirus vector NYVAC expressing clade C(CN54) HIV-1 Env(gp120), Gag-Pol-Nef antigens (NYVAC-C) showed in phase I clinical trials limited immunogenicity. To enhance the capacity of the NYVAC vector to trigger broad humoral responses and a more balanced activation of CD4+ and CD8+ T cells, here we compared the HIV-1-specific immunogenicity elicited in non-human primates immunized with two replicating NYVAC vectors that have been modified by the insertion of K1L and C7L vaccinia viral host-range genes and express clade C(ZM96) trimeric HIV-1 gp140 protein or a Gag(ZM96)-Pol-Nef(CN54) polyprotein as Gag-derived virus-like particles (termed NYVAC-C-KC). Additionally, one NYVAC-C-KC vector was generated by deleting the viral gene B19R, an inhibitor of type I interferon response (NYVAC-C-KC-B19R). An immunization protocol mimicking the RV144 phase III clinical trial was used. Two groups of macaques received two doses of the corresponding NYVAC-C-KC vectors (weeks 0 and 4), and booster doses with NYVAC-C-KC vectors plus clade C HIV-1 gp120 protein (weeks 12 and 24). The two replicating NYVAC-C-KC vectors induced an enhanced and similar HIV-1-specific CD4+ and CD8+ T cell responses, similar levels of binding IgG antibodies, low levels of IgA antibodies, high levels of antibody-dependent cellular cytotoxicity responses and HIV-1-neutralizing antibodies. Small differences within the NYVAC-C-KC-B19R group were seen in the magnitude of CD4+ and CD8+ T cells, induction of some cytokines and in the neutralization of some HIV-1 isolates. Thus, replication-competent NYVAC-C-KC vectors acquired relevant immunological properties as vaccine candidates against HIV/AIDS, and the viral B19 molecule exerts some control of immune functions.

    IMPORTANCE It is of special importance to find a safe and effective HIV/AIDS vaccine that can induce strong and broad T cell and humoral immune responses correlating with HIV-1 protection. Here we developed novel replicating poxvirus NYVAC-based HIV/AIDS vaccine candidates expressing clade C HIV-1 antigens, with one of them lacking the vaccinia B19 protein, an inhibitor of type I interferon response. Immunization of non-human primates with these novel NYVAC-C-KC vectors and the protein component gp120 elicited a high level of T cell and humoral immune responses, with the vector containing the deletion in B19R inducing a trend to higher magnitude of CD4+ and CD8+ T cells and neutralization of some HIV-1 strains. These poxvirus vectors could be considered as HIV/AIDS vaccine candidates based on their activation of potential immune correlates of protection.

  • Toll-Like Receptor 7 Agonist GS-9620 Induces HIV Expression and HIV-Specific Immunity in Cells from HIV-Infected Individuals on Suppressive Antiretroviral Therapy [PublishAheadOfPrint]

  • Antiretroviral therapy can suppress HIV replication to undetectable levels but does not eliminate latent HIV, thus necessitating lifelong therapy. Recent efforts to target this persistent reservoir have focused on inducing the expression of latent HIV so that infected cells may be recognized and eliminated by the immune system. Toll like receptor (TLR) activation stimulates antiviral immunity and has been shown to induce HIV from latently infected cells. Activation of TLR7 leads to the production of several stimulatory cytokines, including type I interferons (IFNs). In this study, we show that the selective TLR7 agonist, GS-9620, induced HIV in peripheral blood mononuclear cells (PBMCs) from HIV-infected individuals on suppressive antiretroviral therapy. GS-9620 increased extracellular HIV RNA 1.5-2-fold through a mechanism that required type I IFN signaling. GS-9620 also activated HIV-specific T cells and enhanced antibody-mediated clearance of HIV-infected cells. Activation by GS-9620 in combination with HIV peptide stimulation increased CD8 T cell degranulation, production of intracellular cytokines, and cytolytic activity. T cell activation was again dependent on type I IFNs produced by plasmacytoid dendritic cells. GS-9620 induced phagocytic cell maturation and improved effector-mediated killing of HIV-infected CD4 T cells by the HIV envelope-specific broadly neutralizing antibody PGT121. Collectively, these data show that GS-9620 can activate HIV production and improve the effector functions that target latently infected cells. GS-9620 may effectively complement orthogonal therapies designed to stimulate antiviral immunity, such as therapeutic vaccines or broadly neutralizing antibodies. Clinical studies are underway to determine if GS-9620 can target HIV reservoirs.

    IMPORTANCE Though antiretroviral therapies effectively suppress viral replication, they do not eliminate integrated proviral DNA. This stable intermediate of viral infection is persistently maintained in reservoirs of latently infected cells. Consequently, lifelong therapy is required to maintain viral suppression. Ultimately, new therapies are needed that specifically target and eliminate the latent HIV reservoir. Toll-like receptor agonists are potent enhancers of innate antiviral immunity that can also improve the adaptive immune response. Here, we show that a highly selective TLR7 agonist, GS-9620, activated HIV from peripheral blood mononuclear cells isolated from HIV-infected individuals with suppressed infection. GS-9620 also improved immune effector functions that specifically targeted HIV-infected cells. Previously published studies on this compound in other chronic viral infections show that it can effectively induce immune activation at safe and tolerable clinical doses. Together, the results of these studies suggest that GS-9620 may be useful for treating HIV-infected individuals on suppressive antiretroviral therapy.

  • Progesterone-based contraceptives reduce adaptive immune responses and protection against sequential influenza A virus infections [PublishAheadOfPrint]

  • In addition to their intended use, progesterone (P4)-based contraceptives promote anti-inflammatory immune responses, yet their effects on the outcome of infectious diseases, including influenza A virus (IAV), are rarely evaluated. To evaluate their impact on immune responses to sequential IAV infections, adult female mice were treated with placebo or one of two progestins, P4 or levonorgestrel (LNG), and infected with mouse adapted (ma) H1N1 virus. Treatment with P4 or LNG reduced morbidity, but had no effect on pulmonary virus titers, during primary H1N1 infection as compared to placebo treatment. In serum and bronchoalveolar lavage fluid, total anti-IAV IgG and IgA titers and virus neutralizing antibody titers, but not hemagglutinin stalk antibody titers, were lower in progestin-treated mice as compared with placebo-treated mice. Females were challenged six weeks later with either a maH1N1 drift variant (maH1N1dv) or maH3N2 IAV. Protection following infection with the maH1N1dv was similar among all groups. In contrast, following challenge with maH3N2, progestin treatment reduced survival as well as numbers and activity of H1N1- and H3N2-specific memory CD8+ T cells, including tissue resident cells, compared with placebo treatment. In contrast to primary IAV infection, progestin treatment increased neutralizing and IgG antibody titers against both challenge viruses compared with placebo treatment. While the immunomodulatory properties of progestins protected naïve females against severe outcome from IAV infection, it made them more susceptible to secondary challenge with a heterologous IAV, despite improving their antibody responses against a secondary IAV infection. Taken together, the immunomodulatory effects of progestins differentially regulate the outcome of infection depending on exposure history.

    IMPORTANCE The impact of hormone-based contraceptives on the outcome of infectious diseases outside of the reproductive tract is rarely considered. Using a mouse model, we have made the novel observation that treatment with either progesterone or a synthetic analog found in hormonal contraceptives, levonorgestrel, impacts sequential influenza A virus infection, by modulating antibody responses and decreasing memory CD8+ T cells. Progestins reduced antibody responses during primary H1N1 virus infection, but increased antibody titers following a sequential infection with either a H1N1 drift variant or a H3N2 virus. Following challenge with a H3N2 virus, female mice treated with progestins experienced greater mortality with increased pulmonary inflammation and reduced numbers and activity of CD8+ T cell. This study suggests that progestins significantly affect adaptive immune responses to influenza A virus infection, with their effect on influence outcome depending on exposure history.

  • A bivalent heterologous DNA-virus-like particles prime-boost vaccine elicits broad protection against both groups 1 and 2 influenza A viruses [PublishAheadOfPrint]

  • Current seasonal influenza vaccines are efficacious when vaccine strains are matched with circulating strains. But they do not protect antigenic variants and newly emerging pandemic and outbreak strains. Thus there is a critical need for developing so-called "universal" vaccines that protect against all influenza viruses. In the present study we developed a bivalent heterologous DNA-virus-like particles (VLP) prime-boost vaccine strategy. We show that mice immunized with this vaccine were broadly protected against lethal challenge of group 1 (H1, H5 and H9) and group 2 (H3 and H7) viruses with 94% aggregate survival. To determine immune correlates of protection, we performed passive immunizations and in vitro assays. We show that this vaccine elicited antibody responses that bound HA from group 1 (H1, H2, H5, H6, H8, H9, H11 and H12) and group 2 (H3, H4, H7, H10, H14 and H15) and neutralized homologous and intrasubtypic H5 and H7 and heterosubtypic H1 viruses and HA-specific CD4 and CD8 T cell responses. As a result, passive immunization with immune sera fully protected mice from H5, H7 and H1 challenge; whereas with both immune sera and T cells completely survived from heterosubtypic H3 and H9 challenge. Thus it appears that neutralizing antibodies alone fully protect homologous and intrasubtypic H5 and H7; neutralizing and binding antibodies are sufficient to protect heterosubtypic H1; but against heterosubtypic H3 and H9 binding antibodies and T cells are required for complete survival. We believe that this vaccine regimen could potentially be a candidate for "universal" influenza vaccine.

    IMPORTANCE Influenza virus infection is global health problem. Current seasonal influenza vaccines are efficacious only when vaccine strains are matched with circulating strains. But they do not protect antigenic variants and newly emerging pandemic and outbreak strains. Because of this, there is an urgent need to develop so-called "universal" influenza vaccines that can protect against both current and future influenza strains. In the present study we developed a bivalent heterologous prime-boost vaccine strategy. We show that this bivalent vaccine regimen elicited broad binding and neutralizing antibody and T cell responses that conferred broad protection against diverse challenge viruses in mice, suggesting that this bivalent prime-boost strategy could practically be a candidate for "universal" influenza vaccine.

  • Identification of novel functions for hepatitis C virus envelope glycoprotein E1 in virus entry and assembly [PublishAheadOfPrint]

  • Hepatitis C Virus (HCV) envelope glycoprotein complex is composed of E1 and E2 subunits. E2 is the receptor-binding protein as well as the major target of neutralizing antibodies, whereas the functions of E1 remain poorly defined. Here, we took advantage of the recently published structure of the N-terminal region of E1 ectodomain to interrogate the functions of this glycoprotein by mutating residues within this 79 amino acid region in the context of an infectious clone. The phenotypes of the mutants were characterized to determine the effects of the mutations on virus entry, replication and assembly. Furthermore, biochemical approaches were also used to characterize the folding and assembly of E1E2 heterodimers. Thirteen out of nineteen mutations led to viral attenuation or inactivation. Interestingly, two attenuated mutants, T213A and I262A, were less dependent on claudin-1 for cellular entry in Huh-7 cells. Instead, these viruses relied on claudin-6, indicating a shift in receptor dependence for these two mutants in the target cell line. An unexpected phenotype was also observed for mutant D263A which was no longer infectious but still showed a good level of core protein secretion. Furthermore, genomic RNA was absent from these non-infectious viral particles, indicating that D263A mutation leads to the assembly and release of viral particles devoid of genomic RNA. Finally, a change in subcellular co-localization between HCV RNA and E1 was observed for D263A mutant. This unique observation highlights for the first time a crosstalk between HCV glycoprotein E1 and the genomic RNA during HCV morphogenesis.

    IMPORTANCE Hepatitis C virus (HCV) infection is a major public health problem worldwide. It encodes two envelope proteins, E1 and E2, which play a major role in the life cycle of this virus. E2 has been extensively characterized, whereas E1 remains poorly understood. Here, we investigated E1 functions by using site-directed mutagenesis in the context of the viral life cycle. Our results identify unique phenotypes. Unexpectedly, two mutants clearly showed a shift in receptor dependence for cell entry, highlighting a role for E1 in modulating HCV particle interaction with cellular receptor(s). More importantly, another mutant led to the assembly and release of viral particles devoid of genomic RNA. This unique phenotype was further characterized and we observed a change in subcellular co-localization between HCV RNA and E1. This unique observation highlights for the first time a crosstalk between a viral envelope protein and the genomic RNA during morphogenesis.

  • Japanese Encephalitis Virus NS5 Inhibits the Type I Interferon Production by Blocking the Nuclear Translocation of IRF3 and NF-{kappa}B [PublishAheadOfPrint]

  • The type I interferon (IFN) response is part of a first-line defense against viral infection. To initiate replication, viruses have developed powerful evasion strategies to counteract host IFN responses. In present study, we found that Japanese encephalitis virus (JEV) NS5 protein could inhibit double strand RNA (dsRNA)-induced IFN-bbeta; expression in a dose-dependent manner. Our data further demonstrated that JEV NS5 suppressed the activation of IFN transcriptional factors, IRF3 and NF-B. However, there was no defect in the phosphorylation of IRF3 and degradation of IB, an upstream inhibitor of NF-B, upon NS5 expression, indicating a direct inhibition of the nuclear localization of IRF3 and NF-B by NS5. Mechanically, NS5 was shown to interact with the nuclear transport proteins, KPNA2, KPNA3 and KPNA4, which competitively blocked the interaction of KPNA3 and KPNA4 with their cargo molecules, IRF3 and p65, a subunit of NF-B, and thus inhibited the nuclear translocation of IRF3 and NF-B. Furthermore, overexpression of KPNA3 and KPNA4 restored the activity of IRF3 and NF-B and increased the production of IFN-bbeta; in NS5-expressing or JEV-infected cells. Additionally, an up-regulated replication level of JEV was shown upon KPNA3 or KPNA4 overexpression. These results suggest that JEV NS5 inhibits the induction of type I IFN by targeting KPNA3 and KPNA4.

    IMPORTANCE JEV is the major cause of viral encephalitis in South and Southeast Asia with high mortality. However, the molecular mechanisms contributing to the severe pathogenesis are poorly understood. The ability of JEV to counteract the host innate immune response may be one of the potential mechanisms responsible for JEV virulence. Here, we demonstrate the ability of JEV NS5 to interfere with the dsRNA-induced nuclear translocation of IRF3 and NF-B by competitively inhibiting the interaction of IRF3 and NF-B with nuclear transport proteins. Via this mechanism, JEV NS5 suppresses the induction of type I IFN and antiviral response in host cells. These findings reveal a novel strategy for JEV to escape the host innate immune response and provide us new insight into the pathogenesis of JEV.

  • Impaired STING pathway in the human osteosarcoma U2OS cells contributes to the growth of ICP0-null mutant herpes simplex virus [PublishAheadOfPrint]

  • Human herpes simplex virus-1 (HSV-1) is a wide spread pathogen with 80% of the population being latently infected. To successfully evade the host the virus has evolved strategies to counteract antiviral responses, including the gene silencing and innate immunity machineries. The immediately early protein of the virus, Infected Cell Protein 0 (ICP0) plays central role in these processes. ICP0 blocks innate immunity and one mechanism is by degrading hostile factors with its intrinsic E3 ligase activity. ICP0 functions also as a promiscuous transactivator and it blocks repressor complexes to enable viral gene transcription. For these reasons the growth of a ICP0 virus is impaired in most cells except the human osteosarcoma cells U2OS and it is only partially impaired in the human osteosarcoma cells Saos-2. We found that the two human osteosarcoma cell lines that supported the growth of the ICP0 virus failed to activate innate immune responses upon treatment with 2rrsquo; 3rrsquo; -cGAMP, the natural agonist of STING (STimulator of INterferon Genes) or after infection with the ICP0 mutant virus. Innate immune responses were restored in these cells by transient expression of the STING protein but not after overexpression of the IFI16 protein. Restoration of STING expression resulted in suppression of ICP0 virus gene expression and a decrease in viral yields. Overexpression of IFI16 also suppressed ICP0 virus gene expression, albeit to a lesser extent than STING. These data suggest that the susceptibility of U2OS and Saos-2 cells to the ICP0 HSV-1 is in part due to an impaired STING pathway.

    IMPORTANCE The DNA sensor STING plays pivotal role in controlling HSV-1 infection both in cell culture and in mice. HSV-1 genome encodes for numerous proteins that are dedicated to combat host antiviral responses. The immediate-early protein of the virus ICP0 plays major role in this process as it targets hostile host proteins for degradation with its E3 ligase activity and it disrupts repressor complexes via protein-protein interaction to enable viral gene transcription. Therefore, the ICP0 HSV-1 virus is defective for growth in most cells, except the human osteosarcoma cell lines U2OS and Saos-2. We found that both cell lines that support ICP0 virus infection have defects in the STING DNA sensing pathway, which partially accounts for the rescue of the ICP0 virus growth. Restoration of STING expression in these cells rescued innate immunity and suppressed ICP0 virus infection. This study underscores the importance of STING in the control of HSV-1.

  • Atypical RNA Elements Modulate Translational Readthrough in Tobacco necrosis virus-D [PublishAheadOfPrint]

  • Tobacco necrosis virus-D (TNV-D) is a positive-strand RNA virus in the genus Betanecrovirus and family Tombusviridae. The production of its RNA-dependent RNA polymerase (RdRp), p82, is achieved by translational readthrough. This process is stimulated by an RNA structure that is positioned immediately downstream of the recoding site, termed the readthrough stem-loop (RTSL), and a sequence in the 3rrsquo; -untranslated region of the TNV-D genome, called the distal readthrough element (DRTE). Notably, a base pairing interaction between the RTSL and the DRTE, spanning ~3,000 nt, is required for enhancement of readthrough. Here, some of the structural features of the RTSL, as well as RNA sequences and structures that flank either the RTSL or DRTE, were investigated for their involvement in translational readthrough and virus infectivity. The results revealed that (i) the RTSL-DRTE interaction cannot be functionally replaced by stabilizing the RTSL structure, (ii) a novel tertiary RNA structure positioned just 3rrsquo; to the RTSL is required for optimal translational readthrough and virus infectivity, and (iii) these same activities also rely on an RNA stem-loop located immediately upstream of the DRTE. Functional counterparts for the RTSL-proximal structure may also be present in other tombusvirids. The identification of additional distinct RNA structures that modulate readthrough suggests that regulation of this process by genomic features may be more complex than previously appreciated. Possible roles for these novel RNA elements are discussed.

    IMPORTANCE The analysis of factors that affect recoding events in viruses is leading to an ever more complex picture of this important process. In this study, two new atypical RNA elements were shown to contribute to efficient translational readthrough of the TNV-D polymerase and to mediate robust viral genome accumulation in infections. One of the structures, located close to the recoding site, could have functional equivalents in related genera, while the other structure, positioned 3rrsquo; -proximally in the viral genome, is likely limited to betanecroviruses. Irrespective of their prevalence, the identification of these novel RNA elements adds to the current repertoire of viral genome-based modulators of translational readthrough and provides a notable example of the complexity of regulation of this process.

  • HIV-1 Vpu downmodulates ICAM-1 expression resulting in decreased killing of infected CD4+ T cells by NK cells [PublishAheadOfPrint]

  • HIV-1 Vpu is known to alter the expression of numerous cell surface molecules. Given the ever-increasing list of Vpu targets identified to date, we undertook a proteomic screen to discover novel cell membrane proteins modulated by this viral protein. Plasma membrane proteome isolates from Vpu-inducible T cells were subjected to SILAC-based mass spectrometry analysis and putative targets were validated by infection of primary CD4+ T cells. We report here that while Intercellular Adhesion Molecule (ICAM)-1 and ICAM-3 are upregulated by HIV-1 infection, expression of Vpu offsets this increase by downregulating these molecules from the cell surface. Specifically, we show that Vpu is sufficient to downregulate and deplete ICAM-1 in a manner requiring the Vpu transmembrane domain and a dual serine (S52,56) motif necessary for recruitment of the SCFbbeta;-TrCP E3 ubiquitin ligase. Vpu interacts with ICAM-1 to induce its proteasomal degradation. Interestingly, the E3 ubiquitin ligase component bbeta;-TrCP-1 is dispensable for ICAM-1 surface downregulation, yet is necessary for ICAM-1 degradation. Functionally, Vpu-mediated ICAM-1 downregulation lowers packaging of this adhesion molecule into virions, resulting in decreased infectivity. Importantly, while Vpu-mediated downregulation of ICAM-3 has a limited effect on the conjugation of NK cells to HIV-1-infected CD4+ T cells, downregulation of ICAM-1 by Vpu results in a reduced ability of NK cells to bind and kill infected T cells. Vpu-mediated ICAM-1 downregulation may therefore represent an evolutionary compromise in viral fitness by impeding the formation of cell-to-cell contacts between immune cells and infected T cells at the cost of decreased virion infectivity.

    IMPORTANCE The major barrier to eradicating HIV-1 infection is the establishment of treatment-resistant reservoirs early in infection. Vpu-mediated ICAM-1 downregulation may contribute to the evasion of cell-mediated immunity during acute infection to promote viral dissemination and the development of viral reservoirs. By aiding the immune system to clear infection prior to the development of reservoirs, novel treatments designed to disrupt Vpu-mediated ICAM-1 downregulation may be beneficial during acute infection or as a prophylactic treatment.

  • Targeting Vaccine-induced Extrafollicular Pathway of B cell Differentiation Improves Rabies Post-exposure Prophylaxis [PublishAheadOfPrint]

  • Vaccine-induced B cells differentiate along two pathways. The follicular pathway gives rise to germinal centers (GCs) that can take weeks to fully develop. The extrafollicular pathway gives rise to short-lived plasma cells (PCs) that can rapidly secrete protective antibodies within days of vaccination. Rabies virus (RABV) post-exposure prophylaxis (PEP) requires rapid vaccine-induced humoral immunity for protection. Therefore, we hypothesized that targeting extrafollicular B cell responses for activation would improve the speed and magnitude of RABV PEP. To test this hypothesis, we constructed, recovered, and characterized a recombinant RABV-based vaccine expressing murine B cell activating factor (BAFF) (rRABV-mBAFF). BAFF is an ideal molecule to improve early pathways of B cell activation as it links innate and adaptive immunity, promoting potent B cell responses. Indeed, rRABV-mBAFF induced a faster, higher antibody response in mice and enhanced survivorship in PEP settings compared to rRABV. Interestingly, rRABV-mBAFF and rRABV induced equivalent numbers of GC B cells, suggesting that rRABV-mBAFF augmented the extrafollicular B cell pathway. To confirm that rRABV-mBAFF modulated the extrafollicular pathway, we used a signaling lymphocytic activation molecule (SLAM)-associated protein (SAP)-deficient mouse model. In response to antigen, SAP-deficient mice form extrafollicular B cell responses but do not generate GCs. rRABV-mBAFF induced similar anti-RABV antibody responses in SAP-deficient and wild-type mice, demonstrating that BAFF modulated immunity through the extrafollicular and not the GC B cell pathway. Collectively, strategies that manipulate pathways of B cell activation may facilitate the development of a single-dose RABV vaccine that replaces current complicated and costly RABV PEP.

    IMPORTANCE Effective RABV PEP is currently resource- and cost-prohibitive in regions of the world where RABV is most prevalent. In order to diminish the requirements for rabies immunoglobulin (RIG) and multiple vaccinations for effective prevention of clinical rabies, a more rapidly protective vaccine is needed. This work presents a successful approach to rapidly generate antibody-secreting PCs in response to vaccination by targeting the extrafollicular B cell pathway. We demonstrate that the improved early antibody responses induced by rRABV-mBAFF confer improved protection against RABV in a PEP model. Significantly, activation of the early extrafollicular B cell pathway, such as that demonstrated herein, could improve the efficacy of vaccines targeting other pathogens against which rapid protection would decrease morbidity and mortality.

  • Reduced potency and incomplete neutralization of broadly neutralizing antibodies against cell-to-cell transmission of HIV-1 with transmitted founder Envs [PublishAheadOfPrint]

  • Broadly neutralizing antibodies (bNAbs) have been isolated from HIV-1 patients and can potently block infection of a wide spectrum of HIV-1 subtypes. These antibodies define common epitopes shared by many viral isolates. While bNAbs potently antagonize infection with cell-free virus, inhibition of HIV-1 transmission from infected to uninfected CD4+ T cells through virological synapses (VS), has been found to require greater amounts of antibody. In this study, we examined two well-studied molecular clones and two transmitted founder (T/F) viruses for their sensitivities to a panel of bNAbs in cell-free and cell-to-cell infection assays. We observed a relative resistance of cell-to-cell transmission to antibody neutralization that is reflected not only by reductions of antibody potency, but also by decreases in maximum neutralization capacity relative to cell-free infections. BNAbs targeting different epitopes exhibited incomplete neutralization against cell-associated virus with T/F Envs, which was not observed with cell-free form of the same virus. We further identified the membrane proximal internal tyrosine-based sorting motif as a determinant that can affect the incomplete neutralization of these T/F clones in cell-to-cell infection. These findings indicate that the signal that affects surface expression and/or internalization of Env from the plasma membrane can modulate the presentation of neutralizing epitopes on infected cells. These findings highlight that a fraction of virus can escape from high concentrations of antibody through cell-to-cell infection while maintaining sensitivity to neutralization in cell-free infection. The ability to fully inhibit cell-to-cell transmission may represent an important consideration in development of antibodies for treatment or prophylaxis.

    IMPORTANCE In recent years, isolation of new generation HIV-1 bNAbs has invigorated HIV vaccine research. These bNAbs display remarkable potency and breadth against cell-free virus, however, they exhibit diminished ability to block HIV-1 cell-to-cell transmission. The mechanism(s) by which HIV-1 resists neutralization when transmitting through VS remains uncertain. We examined a panel of bNAbs for their ability to neutralize HIV-1 T/F viruses in cell-to-cell infection assays. We find that some antibodies exhibit not only reduced potency but also decreased maximum neutralization capacity, or in vitro efficacy against cell-to-cell infection of HIV-1 with T/F Envs compared to cell-free infection of the same virus. We further identify the membrane proximal internal tyrosine-based sorting motif YXXL as a determinant that can affect the incomplete neutralization phenotype of these T/F clones. When the maximum neutralization capacity falls short of 100%, this can have a major impact on the ability of antibodies to halt viral replication.

  • The ATP-dependent RNA Helicase DDX3X modulates Herpes Simplex Virus Type 1 Gene Expression [PublishAheadOfPrint]

  • The human protein DDX3X is a DEAD-box ATP-dependent RNA helicase that regulates transcription, mRNA maturation, mRNA export and translation. DDX3X concomitantly modulates the replication of several RNA viruses and promotes innate immunity. We previously showed that herpes simplex virus type 1 (HSV-1), a human DNA virus, incorporates DDX3X into its mature particles and that DDX3X is required for optimal HSV-1 infectivity. Here, we show that viral gene expression, replication and propagation depend on optimal DDX3X protein levels. Surprisingly, DDX3X from incoming viral particles was not required for the early stages of the HSV-1 infection but the protein rather controlled the assembly of new viral particles. This was independent of the previously reported ability of DDX3X to stimulate interferon type I production. Instead, both the lack and overexpression of DDX3X disturbed viral gene transcription and thus subsequent genome replication. This suggests that in addition to its effect on RNA viruses, DDX3X impacts DNA viruses such as HSV-1 by an interferon-independent pathway.

    IMPORTANCE Viruses interact with a variety of cellular proteins to complete their life cycle. Among them is DDX3X, a RNA helicase that participates in most aspects of RNA biology, including transcription, splicing, nuclear export and translation. Several RNA viruses and a limited number of DNA viruses are known to manipulate DDX3X for their own benefit. In contrast, DDX3X is also known to promote interferon production to limit viral propagation. Here we show that DDX3X, which we previously identified in mature HSV-1 virions, stimulates HSV-1 gene expression and consequently virion assembly by a process that is independent of its ability to promote the interferon pathway.

  • Extreme mutation tolerance: Nearly half of the archaeal fusellovirus SSV1 genes are not required for virus function, including the minor capsid protein gene, vp3. [PublishAheadOfPrint]

  • Viruses infecting the Archaea harbor a tremendous amount of genetic diversity. This is especially true for the spindle-shaped viruses of the family Fuselloviridae, where ggt;90% of the viral genes do not have detectable homologs in public databases. This significantly limits our ability to elucidate the role of viral proteins in the infection cycle. To address this, we have developed genetic techniques to study the well-characterized fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1) which infects Sulfolobus solfataricus in volcanic hot springs at 80ddeg;C and pH 3.

    Here, we present a new comparative genome analysis and a thorough genetic analysis of SSV1 using both specific and random mutagenesis and thereby generate mutations in all ORFs. We demonstrate that almost half of the SSV1 genes are not essential for infectivity and the requirement for a particular gene correlates well with its degree of conservation within the Fuselloviridae. The major capsid gene vp1 is essential for SSV1 infectivity. However, the universally conserved minor capsid gene vp3 could be deleted without a loss in infectivity and results in virions with abnormal morphology.

    Importance: Most of the putative genes in the spindle-shaped archaeal hyperthermophile Fuselloviruses have no clearly similar sequences to characterized genes. In order to determine which of these SSV genes are important for function we disrupted all of the putative genes in the prototypical fusellovirus, SSV1. Surprisingly, about half of the genes could be disrupted without destroying virus function. Even deletions of one of the known structural protein genes that is present in all known Fuselloviruses, vp3, allow the production of infectious viruses. However, viruses lacking vp3 have abnormal shapes, indicating that the vp3 gene is important for virus structure. Identification of essential genes will allow focused research on minimal SSV genomes and further understanding of the structure of these unique, ubiquitous and extremely stable archaeal viruses.

  • A microRNA screen identifies the Wnt signaling pathway as a regulator of the interferon response during flavivirus infection. [PublishAheadOfPrint]

  • The impact of mosquito-borne flavivirus infections worldwide is significant, and many critical aspects of these viruses' biology, including virus-host interactions, host cell requirements for replication, and how virus-host interactions impact pathology, remain to be fully understood. The recent re-emergence and spread of flaviviruses, including dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV), highlights the importance of performing basic research on this important group of pathogens. MicroRNAs (miRNAs) are small, noncoding RNAs that modulate gene expression post-transcriptionally and have been demonstrated to regulate a broad range of cellular processes. Our research is focused on identifying pro- and anti-flaviviral miRNAs as a means of characterizing cellular pathways that support or limit viral replication. We have screened a library of known human miRNA mimics for their effect on replication of three flavivirusesmmdash;DENV, WNV, and Japanese encephalitis virus (JEV)mmdash;using a high content immunofluorescence screen. Several families of miRNAs were identified as inhibiting multiple flaviviruses, including the miR-34, miR-15, and miR-517 families. Members of the miR-34 family, which have been extensively characterized for their ability to repress Wnt/bbeta;-catenin signaling, demonstrated strong anti-flaviviral effects, and this inhibitory activity extended to other viruses, including ZIKV, alphaviruses, and herpesviruses. Previous research has suggested a possible link between the Wnt and type I interferon (IFN) signaling pathways. Therefore, we investigated the role of type I IFN induction in the antiviral effects of the miR-34 family and confirmed that these miRNAs potentiate IRF3 phosphorylation and translocation to the nucleus, induction of IFN-responsive genes, and release of type I IFN from transfected cells. We further demonstrate that the intersection between the Wnt and IFN signaling pathways occurs at the point of GSK3bbeta;-TBK1 binding, inducing TBK1 to phosphorylate IRF3 and initiate downstream IFN signaling. In this way, we have identified a novel cellular signaling network with a critical role in regulating replication of multiple virus families. These findings highlight the opportunities for using miRNAs as tools to discover and characterize unique cellular factors involved in supporting or limiting virus replication, opening up new avenues for antiviral research.

    Importance MicroRNAs are a class of small regulatory RNAs that modulate cellular processes through post-transcriptional repression of multiple transcripts. We hypothesized that individual miRNAs may be capable of inhibiting viral replication through their effects on host proteins or pathways. To test this, we performed a high content screen for miRNAs that inhibit replication of three medically relevant members of the flavivirus family: West Nile virus, Japanese encephalitis virus, and dengue virus-2. The results of this screen identify multiple miRNAs that inhibit one or more of these viruses. Extensive follow up on members of the miR-34 family of miRNAs, which are active against all three viruses as well as the closely related Zika virus, demonstrated that miR-34 functions through increasing the infected cell's ability to respond to infection through the interferon-based innate immune pathway. Our results not only add to the knowledge of how viruses interact with cellular pathways, but provide a basis for more extensive data mining by providing a comprehensive list of miRNAs capable of inhibiting flavivirus replication. Finally, the miRNAs themselves or cellular pathways identified as modulating virus infection may prove to be novel candidates for the development of therapeutic interventions.

  • Dinucleotide composition in animal RNA viruses is shaped more by virus family than host species [PublishAheadOfPrint]

  • Viruses use the cellular machinery of their hosts for replication. It has therefore been proposed that the nucleotide and dinucleotide composition of viruses should match that of their host species. If upheld, it may then be possible to use dinucleotide composition to predict the true host species of viruses sampled in metagenomic surveys. However, it is also clear that different taxonomic groups of viruses tend to have distinctive patterns of dinucleotide composition that may be independent of host species. To determine the relative strength of the effect of host versus virus family in shaping dinucleotide composition we performed a comparative analysis of 20 RNA virus families from 15 host groupings, spanning two animal phyla and more than 900 virus species. In particular, we determined the odds ratios for the 16 possible dinucleotides and performed a discriminant analysis to evaluate the capability of virus dinucleotide composition to predict the correct virus family or host taxon from which it was isolated. Notably, while 81% of the data analyzed here were predicted to the correct virus family, only 62% of these data were predicted to their correct subphylum/class host, and a mere 32% to their correct mammalian order. Similarly, dinucleotide composition has a weak predictive power for different hosts within individual virus families. We therefore conclude that dinucleotide composition is generally uniform within a virus family but less well reflects that of its host species. This has obvious implications for attempts to accurately predict host species from virus genome sequences alone.

    IMPORTANCE Determining the processes that shape virus genomes is central to understanding virus evolution and emergence. One question of particular importance is why nucleotide and dinucleotide frequencies differ so markedly between viruses? In particular, it is currently unclear whether host species or virus family has the biggest impact on dinucleotide frequencies, and whether dinucleotide composition can be used to accurately predict host species. Using a comparative analysis we show that dinucleotide composition has a strong phylogenetic association across different RNA virus families, such that dinucleotide composition can predict the family a virus sequence has been isolated from. Conversely, dinucleotide composition has a poorer predictive power for the different host species within a virus family and across different virus families, indicating that the host has a relatively small impact on the dinucleotide composition of a virus genome.

  • Computational prediction of the heterodimeric and higher order structure of gpE1/gpE2 envelope glycoproteins encoded by the hepatitis C virus (HCV). [PublishAheadOfPrint]

  • Despite the recent success of newly developed direct acting antivirals against Hepatitis C, the disease continues to be a global health threat due to lack of diagnosis of most carriers and the high cost of treatment. The heterodimer formed by the glycoproteins E1 and E2 within the hepatitis C virus (HCV) lipid envelope is a potential vaccine candidate and antiviral target. While the structure of E1/E2 has not yet been resolved, partial crystal structures of the E1 and E2 ectodomains have been determined. The unresolved parts of the structure are within the realm of what can be modeled with current computational modeling tools. Further, a variety of additional experimental data is available to support computational predictions of E1/E2 structure such as antibody binding studies, cryoEM, mutational analyses, peptide binding, linker-scanning mutagenesis and NMR studies. In accordance with these rich experimental data, we have built an in silico model of the full-length E1/E2 heterodimer. Our model supports that E1/E2 assembles into a trimer, which was previously suggested from a study by Falson and coworkers. Size exclusion chromatography and Western blotting data obtained using purified recombinant E1/E2 support our hypothesis. Our model suggests that during virus assembly the trimer of E1/E2 may further be assembled into a pentamer with 12 pentamers comprising a single HCV virion. We anticipate that this new model will provide a useful framework for HCV envelope structure and the development of antiviral strategies.

    Importance 150 million people have been estimated as infected with the Hepatitis C virus and many more are at risk for infection. A better understanding of the structure of the HCV viral envelope, which is responsible for attachment and fusion, could aid in the development of a vaccine and/or new treatments for this disease. We draw upon computational techniques to predict a full-length model of the E1/E2 heterodimer based on the partial crystal structures of the envelope glycoproteins E1 and E2. E1/E2 has been widely studied experimentally and this provides valuable data, which has assisted us in our modeling. Our proposed structure is used to suggest the organization of the HCV viral envelope. We also present new experimental data from size exclusion chromatography that supports our computational prediction of a trimeric oligomeric state of E1/E2.

  • Immunization with low doses of recombinant post-fusion or pre-fusion RSV F primes for vaccine-enhanced disease in the cotton rat model independent of the presence of a Th1-biasing (GLA-SE) or Th2-biasing (Alum) adjuvant [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) infection of children previously immunized with a non-live, formalin-inactivated (FI)-RSV vaccine was associated with serious enhanced respiratory disease (ERD). Consequently, detailed studies of potential ERD are a critical step in the development of non-live RSV vaccines targeting RSV-naïve children and infants. The fusion glycoprotein (F) of RSV in either its post- or pre-fusion conformation is a target for neutralizing antibodies and therefore an attractive antigen candidate for a pediatric RSV subunit vaccine. Here we report the evaluation of RSV post-F and pre-F in combination with GLA-SE and Alum adjuvants in the cotton rat model. Immunization with optimal doses of RSV F antigens in the presence of GLA-SE induced high titers of virus neutralizing antibodies, and conferred complete lung protection from virus challenge, with no ERD signs in the form of alveolitis. To mimic a waning immune response, and to assess priming for ERD under suboptimal conditions, an antigen dose de-escalation study was performed in the presence of either GLA-SE or Alum. At low RSV F doses, alveolitis-associated histopathology was unexpectedly observed with either adjuvant, at levels comparable to FI-RSV immunized controls. This occurred despite neutralizing antibody titers above the minimum levels required for protection, and with no/low virus replication in the lungs. These results emphasize the need to investigate a pediatric RSV vaccine candidate carefully for priming of ERD over a wide dose range, even in the presence of strong neutralizing activity, Th1 bias inducing adjuvant, and protection from virus replication in the lower respiratory tract.

    IMPORTANCE RSV disease is of great importance worldwide, with the highest burden of serious disease occurring upon primary infection in infants and children. FI-RSV-induced enhanced disease, observed in the 1960s, presented a major and ongoing obstacle for the development of non-live RSV vaccine candidates. The findings presented here underscore the need to evaluate a non-live RSV vaccine candidate during pre-clinical development over a wide dose range in the cotton rat RSV-enhanced disease model as suboptimal dosing of several RSV F subunit vaccine candidates led to the priming for ERD. These observations are relevant to the validity of the cotton rat model itself, and to safe development of non-live RSV vaccines for seronegative infants and children.

  • SARS Coronavirus Nucleocapsid Inhibits Type I Interferon Production by Interfering with TRIM25-Mediated RIG-I Ubiquitination [PublishAheadOfPrint]

  • Severe acute respiratory syndrome (SARS) is a respiratory disease caused by a coronavirus (SARS-CoV) that is characterized by atypical pneumonia. The nucleocapsid protein (N protein) of SARS-CoV plays an important role in inhibition of type I interferon (IFN) production via an unknown mechanism. In this study, the SARS-CoV N protein was found to bind to the SPRY domain of the tripartite motif protein 25 (TRIM25) E3 ubiquitin ligase, thereby interfering with the association between TRIM25 and retinoic acid-inducible gene I (RIG-I) and inhibiting TRIM25-mediated RIG-I ubiquitination and activation. Type I IFN production induced by poly I:C or Sendai virus (SeV) was suppressed by the SARS-CoV N protein. SARS-CoV replication was increased by over-expression of the full-length N protein but not N (1-361), which could not interact with TRIM25. These findings provide an insightful interpretation of the SARS-CoV-mediated host innate immune suppression caused by the N protein.

    Importance The SARS-CoV N protein is essential for the viral life cycle and plays a key role in the virus-host interaction. We demonstrated that the interaction between the C-terminus of the N protein and the SPRY domain of TRIM25 inhibited TRIM25-mediated RIG-I ubiquitination, which resulted in the inhibition of IFN production. We also found that the MERS-CoV N protein interacted with TRIM25 and inhibited RIG-I signaling. The outcomes of these findings indicate the function of the coronavirus N protein in modulating the host's initial innate immune response.

  • Patterns of cellular immunity associated with experimental infection with rDEN2{Delta}30 (Tonga/74) supports its suitability as a human DENV challenge strain [PublishAheadOfPrint]

  • A deletion variant of the DENV2 Tonga/74 strain lacking 30 nucleotides from its 3rrsquo; untranslated region (rDEN230) has previously been established for use in a controlled DENV human challenge model. To evaluate if this model is appropriate to derive correlates of protection for DENV vaccines based on cellular immunity, we wanted to compare how the cellular immune response to this challenge strain compares to the response induced by natural infection. To achieve this, we predicted HLA class I and class II restricted peptides from rDEN230 and used them, in an IFN-gamma ELISPOT assay, to interrogate CD8+ and CD4+ T cell responses in healthy volunteers infected with rDEN230. At the level of CD8 responses, vigorous ex vivo responses were detected in approximately 80% of donors. These responses were similar in terms of magnitude and numbers of epitopes recognized to previously reported responses observed in PBMC from donors from DENV hyper-endemic regions. The similarity extended to the immunodominance hierarchy of the DENV nonstructural proteins NS3, NS5, and NS1 being dominant in both donor cohorts. At the CD4 level, responses were less vigorous compared to natural DENV infection, and were more focused on nonstructural proteins. The epitopes recognized following rDEN230 infection and natural infection were largely overlapping for both CD8 (100%) and CD4 (85%) responses. Finally, rDEN230 induced stronger CD8 responses compared to other more attenuated DENV isolates.

    IMPORTANCE The lack of a known correlate of protection and the failure of a neutralizing antibody to correlate with protection against dengue have highlighted the need for human DENV challenge model to better evaluate the candidate live attenuated dengue vaccines. In this study we sought to characterize the immune profiling of rDEN230 infected subjects comparing them with subjects from areas where DENV is hyperendemic. Our data demonstrate that T cell responses to rDENV230 are largely similar to natural infection in terms of specificity, highlighting this virus as an appropriate human model for the T cell response to primary DENV2 infection.

  • Mutations during the adaptation of H9N2 avian influenza virus to the respiratory epithelium of pigs enhance the sialic acid binding activity and the virulence in mice [PublishAheadOfPrint]

  • The natural reservoir for influenza viruses is waterfowl from where they succeeded to cross the barrier to different mammalian species. We analyzed the adaptation of avian influenza viruses to a mammalian host by passaging an H9N2 strain three times in differentiated swine airway epithelial cells. Using precision-cut slices from the porcine lung to passage the parental virus, isolates from each of the three passages (P1-P3) were characterized by assessing growth curves and ciliostatic effects. The only difference noted was an increased growth kinetics of the P3 virus. Sequence analysis revealed four mutations: one each in the PB2 and NS1, and two in the HA protein. The HA mutations, A190V and T212I, were characterized by generating recombinant viruses containing either one or both amino acid exchanges. Whereas the parental virus recognized aalpha;2,3-linked sialic acids preferentially, the HA190 mutant bound to a broad spectrum of glycans with aalpha;2,6/8/9-linked sialic acids. The HA212 mutant alone differed only slightly from the parental virus; however, the combination of both mutations (HA190+HA212) increased the binding affinity to those glycans recognized by the HA190 mutant. Remarkably, only the HA double mutant showed a significantly increased pathogenicity in mice. By contrast, none of those mutations affected the ciliary activity of the epithelial cells which is characteristic for virulent swine influenza viruses. Taken together, our results indicate that shifts in the HA receptor affinity are just an early adaptation step of avian H9N2 strains; further mutational changes may be required to become virulent for pigs.

    IMPORTANCE Swine play an important role in the interspecies transmission of influenza viruses. Avian influenza A viruses (IAV) of the H9N2 subtype have successfully infected hosts from different species but not established a stable lineage. We have analyzed the adaptation of IAV-H9N2 virus to target cells of a new host by passaging the virus three times in differentiated porcine respiratory epithelial cells. Among the four mutations detected the two HA mutations were analyzed by generating recombinant viruses. Depending on the infection system used, the mutations differed in their phenotypic expression, e.g sialic acid binding activity, replication kinetics, plaque size, and pathogenicity in inbred mice. But none of the mutations affected the ciliary activity which serves as a virulence marker. Thus, early adaptative mutation enhance the replication kinetics but more mutations are required for IAV of the H9N2 subtype to become virulent.

  • Inactivated Recombinant Rabies Viruses Displaying the Canine Distemper Virus Glycoproteins Induce Protective Immunity Against Both Pathogens [PublishAheadOfPrint]

  • The development of multivalent vaccines is an attractive methodology for the simultaneous prevention of several infectious diseases in vulnerable populations. Canine distemper (CDV) and rabies (RABV) viruses both cause lethal disease in wild and domestic carnivores. While RABV vaccines are inactivated, the live-attenuated CDV vaccines retain residual virulence for highly susceptible wild life species. In the current study, we have developed recombinant bivalent vaccine candidates based on recombinant vaccine strain rabies virus particles, which concurrently display the protective CDV and RABV glycoprotein antigens. The recombinant viruses replicated to near wild type titers and the heterologous glycoproteins were efficiently expressed and incorporated in the viral particles. Immunization of ferrets with beta-propiolactone inactivated recombinant virus particles elicited protective RABV antibody titers, and animals immunized with a combination of CDV attachment and fusion protein-expressing recombinant viruses were protected from lethal CDV challenge. However, animals that were only immunized with a RABV expressing the attachment protein of the CDV vaccine strain Onderstepoort succumbed to the infection with a more recent wild type strain, indicating that immune responses to the more conserved fusion protein contribute to protection against heterologous CDV strains.

    IMPORTANCE Rabies and canine distemper virus (CDV) cause high mortality and death in many carnivores. While rabies vaccines are inactivated and thus have an excellent safety profile and high stability, live-attenuated CDV vaccines can retain residual virulence in highly susceptible species. Here we generated recombinant inactivated rabies viruses that carry one of the CDV glycoproteins on their surface. Ferrets immunized twice with a mix of recombinant rabies viruses carrying the CDV fusion and attachment glycoproteins resulted in protection from lethal CDV challenge, whereas all animals receiving recombinant rabies viruses carrying only the CDV attachment protein following the same immunization scheme died. Irrespective of the CDV antigens used, all animals developed protective titers against rabies virus, illustrating that a bivalent rabies-based vaccine against CDV induces protective immune responses against both pathogens.

  • M gene reassortment in H9N2 influenza virus promotes early infection and replication: contribution to rising virus prevalence in chickens in China [PublishAheadOfPrint]

  • Segment reassortment and base mutagenesis of influenza A viruses are the primary routes to the rapid evolution of high fitness virus genotypes. We recently described a predominant G57 genotype of avian H9N2 viruses that caused country-wide outbreaks in chickens in China during 2010-2013 which led to the zoonotic emergence of H7N9 viruses. One of the key features of the G57 genotype is the substitution of the earlier BJ/94-like M gene with the G1-like M gene of quail origin. We report here on the functional significance of the G1-like M gene in H9N2 viruses in conferring increased infection severity and infectivity in primary chicken embryonic fibroblasts and chickens. H9N2 virus housing the G1-like M gene, in place of BJ/94-like M gene, showed early surge in viral mRNA and vRNA transcription that were associated with enhanced viral protein production, and with early elevated release of progeny virus comprising largely spherical rather than filamentous virions. Importantly, H9N2 virus with G1-like M gene conferred extrapulmonary virus spread in chickens. Five highly represented signature amino acid residues (37A, 95K, 224N and 242N in M1 protein, and 21G in M2 protein) encoded by the prevalent G1-like M gene were demonstrated as prime contributors to enhanced infectivity. Therefore, the genetic evolution of M gene in H9N2 virus increases reproductive virus fitness, indicating its contribution to rising virus prevalence in chickens in China.

    Importance We recently described the circulation of a dominant genotype (G57) of H9N2 viruses in country-wide outbreaks in chickens in China, which was responsible through reassortment for the emergence of H7N9 viruses that cause severe human infections. A key feature of the G57 genotype H9N2 virus is the presence of quail origin G1-like M gene which had replaced the earlier BJ/94-like M gene. We found that H9N2 virus with G1-like M gene, but not BJ/94-like M gene, showed early surge in progeny virus production, more severe pathology and extrapulmonary virus spread in chickens. Five highly represented amino acid residues in M1 and M2 proteins derived from G1-like M gene were shown to mediate enhanced virus infectivity. These observations enhance what we currently know about the roles of reassortment and mutations on virus fitness and have implications for assessing the potential of variant influenza viruses that can cause rising prevalence in chickens.

  • Hepatitis B virus-encoded miRNA controls viral replication [PublishAheadOfPrint]

  • microRNAs (miRNAs) are a class of small single-stranded non-coding functional RNAs. Hepatitis B virus (HBV) is an enveloped DNA virus with virions and subviral forms of particles that lack a core. It was not known whether HBV encodes miRNAs. Here, we identified an HBV-encoded miRNA (called HBV-miR-3) by deep sequencing and northern blot. HBV-miR-3 is located at nts 373-393 of the HBV genome and was generated from 3.5Kb, 2,4Kb and 2.1Kb HBV in classic miRNA biogenesis (Drosha-Dicer dependent) manner. HBV-miR-3 was highly expressed in hepatoma cell lines with an integrated HBV genome and HBV (+) hepatoma tumors. In patients with HBV infection, HBV-miR-3 was released to circulation by exosome and HBV viron, and HBV-miR-3 expression was a positive correlation with HBV titers in the serum in the acute phase of patients with HBV infection. More interestingly, we found that HBV-miR-3 represses HBsAg, HBeAg and replication of HBV. HBV-miR-3 represses HBV replication by targeting the region of HBV 3.5 kb mRNA encoding HBV core protein (HBc) to reduce HBc protein and HBV pregenomic RNA (pgRNA), which in turn led to attenuate HBV replication. Overall, these results indicate that HBV encodes miRNA which may provide new potential biomarkers for clinical HBV infection and shed lights on a new mechanism of HBV replication regulation by which HBV-encoded miRNAs control self-replication via targeting viral transcripts.

    IMPORTANCE Hepatitis B is a liver infection caused by the hepatitis B virus (HBV), which can become a long-term, chronic infection and lead to cirrhosis or liver cancer. Hepatitis B virus (HBV) is a small DNA virus that belongs to the hepadnavirus family with virions and subviral forms of particles that lack a core. microRNA (miRNA), a small (~22 nt) non-coding RNA, is recently found to be an important regulator of gene expression. We found that HBV encodes miRNA (HBV-miR-3). More importantly, we revealed that HBV-miR-3 targets itself transcripts to attenuate HBV replication. This may contribute to explain that HBV infection leads to mild damage in live cells and the subsequent establishment/maintenance of its persistent infection. Our findings highlight a mechanism by which HBV-encoded miRNA controls the process of self-replication by regulating the virus itself during infection, and might provide new biomarkers for diagnostic and treatment of hepatitis B.

  • REPLICATION CAPACITY OF VIRUSES FROM ACUTE INFECTION DRIVES HIV-1 DISEASE PROGRESSION [PublishAheadOfPrint]

  • Viral genotype has been shown to play an important role in HIV pathogenesis following transmission. However, the viral phenotypic properties that contribute to disease progression remain unclear. Most studies have been limited to the evaluation of Gag function in the context of a recombinant virus backbone. Using this approach, important biological information may be lost making the evaluation of viruses obtained during acute infection, representing the transmitted virus, a more biologically relevant model. Here we evaluate the role of viral infectivity and replication capacity of viruses from acute infection on disease progression in women who seroconverted in the CAPRISA 004 tenofovir microbicide trial. We show that viral replication capacity but not viral infectivity correlates with set point viral load (Spearman r=0.346, p=0.045) and that replication capacity (HR=4.52, p=0.01) can predict CD4 decline independently of viral load (HR=2.9, p=0.004) or protective HLA alleles (HR=0.61, p=0.36). We further demonstrate that Gag-Pro is not the main driver of this association suggesting that additional properties of the transmitted virus play a role in disease progression. Finally, we find that although viruses from the tenofovir arm were two fold less infectious, they replicated at similar rates compared to viruses from the placebo arm. This indicates that the use of tenofovir gel did not select for viral variants with higher replication capacity. Overall, this study supports a strong influence of replication capacity in acute infection on disease progression, potentially driven by interaction of multiple genes rather than a dominant role of the major structural gene gag.

    IMPORTANCE HIV disease progression is known to differ between individuals and defining which fraction of this variation can be attributed to the virus is of importance both clinically and epidemiologically. In this study we show that the replication capacity of viruses isolated during acute infection predicts subsequent disease progression and drives CD4 decline independently of viral load. This provides further support for the hypothesis that the replication capacity of the transmitted virus determines the initial damage to the immune system, setting the pace for later disease progression. However, we did not find evidence that the major structural gene gag is driving this correlation, highlighting the importance of other genes in determining disease progression.

  • Recombinant Isfahan virus and vesicular stomatitis virus vaccine vectors provide durable, multivalent, single dose protection against lethal alphavirus challenge [PublishAheadOfPrint]

  • The demonstrated clinical efficacy of a recombinant vesicular stomatitis virus (rVSV) vaccine vector has stimulated the investigation of additional serologically distinct Vesiculovirus vectors as therapeutic and/or prophylactic vaccine vectors to combat emerging viral diseases. Amongst these viral threats are encephalitic alphaviruses, Venezuelan and eastern equine encephalitic viruses (VEEV, EEEV), which have demonstrated potential for natural disease outbreaks, yet no licensed vaccines are available in the event of an epidemic. Here we report rescue of recombinant Isfahan virus (rISFV) from genomic cDNA as a potential new vaccine vector platform. The rISFV genome was modified to attenuate virulence and engineered to express VEEV and EEEV E2/E1 surface glycoproteins as vaccine antigens. A single dose of the rISFV vaccine vectors elicited neutralizing antibody responses and protected mice from lethal VEEV and EEEV challenge at one month post-vaccination as well as lethal VEEV challenge at eight months post-vaccination. A mixture of rISFV vectors expressing VEEV and EEEV E2/E1 glycoproteins also provided durable, single-dose protection from lethal VEEV and EEEV challenge, demonstrating the potential for a multivalent vaccine formulation. These findings were paralleled in studies with an attenuated form of rVSV expressing VEEV E2/E1 glycoproteins. Both rVSV and rISFV vectors were attenuated using an approach which has demonstrated safety in human trials of an rVSV/HIV-1 vaccine. Vaccines based on either of these vaccine vector platforms may present a safe and effective approach to prevent alphavirus induced disease in humans.

    Importance: This work introduces rISFV as a novel vaccine vector platform that is serologically distinct and phylogenetically distant from VSV. The rISFV vector has been attenuated by an approach used for an rVSV vector that has demonstrated safety in clinical studies. The vaccine potential of the rISFV vector was investigated in a well-established alphavirus disease model. The findings indicate the feasibility of producing a safe, efficacious, multivalent vaccine against the encephalitic alphaviruses VEEV and EEEV, both of which can cause fatal disease. The work also demonstrates efficacy of an attenuated rVSV vector that has already demonstrated safety and immunogenicity in multiple HIV-1 Phase I clinical studies. The absence of serological cross-reactivity between rVSV and rISFV and their phylogenetic divergence within the Vesiculovirus genus indicates potential for two stand-alone vaccine vector platforms that could be used to target multiple bacterial and/or viral agents in successive immunization campaigns, or as heterologous prime-boost agents.

  • Neurovirulent murine coronavirus JHM.SD uses cellular zinc metalloproteases for virus entry and cell-cell fusion [PublishAheadOfPrint]

  • The coronavirus S protein requires cleavage by host cell proteases to mediate virus-cell and cell-cell fusion. Many strains of the murine coronavirus mouse hepatitis virus (MHV) have distinct, S-dependent organ and tissue tropisms despite using a common receptor, suggesting that they employ different cellular proteases for fusion. In support of this hypothesis, we found that inhibition of endosomal acidification only modestly decreased entry and overexpression of the cell surface protease TMPRSS2 greatly enhanced entry of the highly neurovirulent MHV strain JHM.SD relative to their effects on the reference strain A59. However, TMPRSS2 overexpression decreased MHV structural protein expression, release of infectious particles, and syncytia formation, and endogenous serine protease activity did not contribute greatly to infection. We therefore investigated the importance of other classes of cellular proteases and found that inhibition of MMP- and ADAM-family zinc metalloproteases markedly decreased both entry and cell-cell fusion. Suppression of virus by metalloprotease inhibition varied among tested cell lines and MHV S proteins, suggesting a role for metalloprotease use in strain-dependent tropism. We conclude that zinc metalloproteases must be considered potential contributors to coronavirus fusion.

    IMPORTANCE The family Coronaviridae includes viruses that cause two emerging diseases of humans, Severe Acute Respiratory Syndrome (SARS) Middle East Respiratory Syndrome (MERS), as well as a number of important animal pathogens. Because coronaviruses depend on host protease-mediated cleavage of their S proteins for entry, a number of protease inhibitors have been proposed as antiviral agents. However, it is unclear which proteases mediate in vivo infection: for example, SARS-CoV infection of cultured cells depends on endosomal acid pH-dependent proteases rather than on the cell-surface acid pH-independent serine protease TMPRSS2, but Zhou et al. (Antiviral Res 116:76-84, 2015, doi:10.1016/j.antiviral.2015.01.011) found that a serine protease inhibitor was more protective than a cathepsin inhibitor in SARS-CoV-infected mice. This paper explores the contributions of endosomal acidification and various proteases to coronavirus infection and identifies an unexpected class of proteases, the matrix metalloproteinase and A-Disintegrin-And-Metalloprotease (ADAM) families, as potential targets for anti-coronavirus therapy.

  • Different Expression of Interferon Stimulated Genes in Response to HIV-1 Infection in Dendritic Cells According to Their Maturation State. [PublishAheadOfPrint]

  • Dendritic cells (DCs) are professional antigen presenting cells whose functions are dependent on their degree of differentiation. In their immature state, DCs, capture pathogens and migrate to the lymph nodes. During this process DCs become resident mature cells specialized in antigen presentation. DCs are characterized by a highly limiting environment to HIV-1 replication due to the expression of restriction factors as SAMHD1 and APOBEC3G. However, uninfected DCs capture and transfer viral particles to CD4 lymphocytes through a trans-enhancement mechanism in which chemokines are involved. We analyzed changes in gene expression with whole-genome-microarray when immature (IDCs) or mature (MDCs) dendritic cells were productively infected using Vpx-loaded HIV-1 particles. Whereas productive HIV infection of IDCs induced expression of interferon stimulated genes (ISGs), such induction was not produced in MDCs in which a sharp decrease in ISG and CXCR3-binding chemokines was observed lessening trans-infection of CD4 lymphocytes. Similar patterns of gene expression were found when DCs were infected with HIV-2 that naturally express Vpx. Differences were also observed in conditions of restrictive HIV-1 infection, in the absence of Vpx. ISGs expression was not modified in IDCs whereas an increase of ISG and CXCR3-binding chemokines was observed in MDCs. Overall these results suggest that sensing and restriction of HIV-1 infection are different between IDCs and MDCs. We propose that restrictive infection results in increased virulence through different mechanisms. In IDC avoiding sensing and induction of ISGs whereas in MDC increased production of CXCR3-binding chemokines would result in lymphocyte attraction and enhanced infection at the immune synapse.

    IMPORTANCE In this work we describe for the first time the activation of a different genetic program during HIV-1 infection depending on the state of maturation of DCs. This represents a breakthrough in the understanding of the restriction to HIV-1 infection by DCs.

    The results show that infection of DCs by HIV-1, reprogram their gene expression pattern. In immature cells, productive HIV-1 infection activates IFN-related-genes involved in the control of viral replication thus inducing an antiviral state in surrounding cells. Paradoxically restriction of HIV-1 by SAMHD1 would result in lack of sensing and IFN activation thus favoring initial HIV-1 scape from innate immune response.

    In mature DCs restrictive infection results in HIV-1 sensing and induction of ISGs, in particular CXCR3-binding chemokines, that could favor the transmission of HIV to lymphocytes.

    Our data support the hypothesis that genetic DC reprograming by HIV-1 infection favors viral escape and dissemination thus increasing HIV-1 virulence.

  • Beta-propiolactone inactivation of coxsackievirus A16 induces structural alteration and surface modification of viral capsids [PublishAheadOfPrint]

  • Beta-propiolactone (BPL) is an inactivating agent widely used in the vaccine industry. However, its effects on vaccine protein antigens and its mechanisms of action remain poorly understood. Here we presented cryo-EM structures of BPL-treated coxsackievirus A16 (CVA16) mature virion and procapsid at resolutions of 3.9 AAring; and 6.5 AAring;, respectively. Notably, both particles were found to adopt an expanded conformation resembling the 135S-like uncoating intermediate, with characteristic features including an opened 2-fold channel, the externalization of the N-terminus of VP1 capsid protein as well as the absence of pocket factor. However, major neutralizing epitopes are very well preserved on these particles. Further biochemical analyses revealed that BPL treatment impairs the binding abilities of CVA16 particles to the attachment receptor heparan sulfate and to a conformation-dependent monoclonal antibody in a BPL dose-dependent manner, indicating that BPL is able to modify surface-exposed amino acid residues. Taken together, our results demonstrate that BPL treatment may induce alteration of the overall structure and surface properties of a non-enveloped viral capsid, thus revealing a novel mode of action of BPL.

    IMPORTANCE Beta-propiolactone (BPL) is commonly used as an inactivating reagent to produce viral vaccines. It is recognized that BPL inactivates viral infectivity through modification of viral nucleic acids. However, its effect on viral proteins remains largely unknown. Here, we present high-resolution cryo-EM structures of BPL-treated coxsackievirus A16 (CVA16) mature virion and procapsid, which reveals an expanded overall conformation and characteristic features that are typical for the 135S-like uncoating intermediate. We further show that BPL concentration affects the binding of inactivated CVA16 particles to its receptor/antibody. Thus, BPL treatment can alter overall structure and surface properties of viral capsids, which may lead to antigenic and immunogenic variations. Our findings provide important information for future development of BPL-inactivated vaccines.

  • Animal Models of Zika Virus Infection, Pathogenesis, and Immunity [PublishAheadOfPrint]

  • Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that now causes epidemics affecting millions of people on multiple continents. The virus has reached global attention because of some of its unusual epidemiological and clinical features including persistent infection in the male reproductive tract and sexual transmission, an ability to cross the placenta during pregnancy and infect the developing fetus to cause congenital malformations, and its association with Guillain-Barreeacute; syndrome in adults. This past year has witnessed an intensive effort by the global scientific community to understand the biology of ZIKV and to develop pathogenesis models for the rapid testing of possible countermeasures. Here, we review the recent advances, utility, and limitations of newly developed mouse and non-human primate models of ZIKV infection and pathogenesis.

  • Suppression of type I interferon signaling by E1A via RuvBL1/Pontin. [PublishAheadOfPrint]

  • Suppression of interferon signaling is of paramount importance to a virus. Interferon signaling will significantly reduce or halt the ability of a virus to replicate, therefore viruses have evolved sophisticated mechanisms that suppress activation of the interferon pathway or responsiveness of the infected cell to interferon. Adenovirus has multiple modes of inhibiting cellular response to interferon. Here we report that E1A, previously shown to regulate interferon signaling in multiple ways, inhibits interferon stimulated gene expression by modulating RuvBL1 function. RuvBL1 was previously shown to affect type I interferon signaling. E1A binds to RuvBL1 and is recruited to RuvBL1-regulated promoters in an interferon-dependent manner, preventing their activation. Depletion of RuvBL1 impairs adenovirus growth, but does not appear to significantly affect viral protein expression. Although RuvBL1 has been shown to play a role in cell growth, depletion of it had no effect on the ability of the virus to replicate its genome or drive cells into S-phase. E1A was found to bind to RuvBL1 via the C-terminus of E1A and this interaction was important for suppression of interferon stimulated gene transcriptional activation and recruitment of E1A to interferon-regulated promoters. Here we report the identification of RuvBL1 as a new target for adenovirus in its quest to suppress interferon response.

    IMPORTANCE For most viruses, suppression of the interferon signaling pathway is crucial to ensure a successful replicative cycle. Human adenovirus has evolved several different mechanisms that prevent activation of interferon or the ability of the cell to respond to interferon. The viral immediate early gene, E1A, was previously shown to affect interferon signaling in several different ways. Here we report a novel mechanism reliant on RuvBL1 that E1A uses to prevent activation of interferon stimulated gene expression following infection or interferon treatment. This adds to the growing knowledge of how viruses are able to inhibit interferon, and identifies a novel target used by adenovirus for modulation of cellular interferon pathway.

  • Innate Immune Response of Bat and Human Cells to Filoviruses: Commonalities and Distinctions [PublishAheadOfPrint]

  • Marburg (MARV) and Ebola (EBOV) viruses are zoonotic pathogens that cause severe hemorrhagic fever in humans. The natural reservoir of MARV is the Egyptian rousette bat (Rousettus aegyptiacus); that of EBOV is unknown but believed to be another bat species. The Egyptian rousette develops subclinical productive infection with MARV but is refractory to EBOV. Interaction of filoviruses with hosts is greatly affected by the viral interferon (IFN)-inhibiting domains (IID). Our study was aimed at characterization of innate immune responses to filoviruses and the role of filovirus IID in bat and human cells. The study demonstrated that EBOV and MARV replicate to similar levels in all tested cell lines, indicating that permissiveness for EBOV at cell and organism levels do not necessarily correlate. Filoviruses, particularly MARV, induced a potent innate immune response in rousette cells, which was generally stronger than in human cells. Both EBOV VP35 and VP24 IID were found to suppress the innate immune response in rousette cells, but only VP35 IID appeared to promote virus replication. Along with IFNaalpha; and IFNbbeta;, IFN was demonstrated to control filovirus infection in bat cells but not in human cells suggesting host species specificity of the antiviral effect. The antiviral effects of bat IFNs appeared not to correlate with induction of IFN-stimulated genes 54 and 56, which was detected in human cells ectopically expressing bat IFNaalpha; and IFNbbeta;. As bat IFN induced the type I IFN pathway, its antiviral effect is likely to be partially induced via llsquo;cross talk'.

    IMPORTANCE Bats serve as reservoirs for multiple emerging viruses including filoviruses, henipaviruses, lyssaviruses, and zoonotic coronaviruses. Although there is no evidence for symptomatic disease caused by either Marburg (MARV) or Ebola (EBOV) viruses in bats, spillover of these viruses into human populations causes deadly outbreaks. The reason for the lack of symptomatic disease in bats infected with filoviruses remains unknown. The outcome of a virus-host interaction depends on the ability of the host immune system to suppress viral replication and the ability of a virus to counteract the host defenses. Our study is a comparative analysis of the host innate immune response to either MARV or EBOV infection in bat and human cells and the role of viral interferon-inhibiting domains in the host innate immune responses. The data are useful for understanding the interactions of filoviruses with natural and accidental hosts and for identification of factors that influence filovirus evolution.

  • The role of the PB2 627-domain in influenza A virus polymerase function [PublishAheadOfPrint]

  • The RNA genome of influenza A viruses is transcribed and replicated by the viral RNA-dependent RNA polymerase composed of the subunits PA, PB1 and PB2. High-resolution structural data revealed that the polymerase assembles into a central polymerase core and several auxiliary highly flexible, protruding domains. The auxiliary PB2 cap-binding and the PA endonuclease domains are both involved in cap-snatching, but the role of the auxiliary PB2 627-domain, implicated in host range restriction of influenza A viruses, is still poorly understood. Here we use structure-guided truncations of the PB2 subunit to show that a PB2 subunit lacking the 627-domain accumulates in the cell nucleus and assembles into a heterotrimeric polymerase with PB1 and PA. Furthermore, we show that a recombinant viral polymerase lacking the PB2 627-domain is able to carry out cap-snatching, cap-dependent transcription initiation as well as cap-independent ApG dinucleotide extension in vitro, indicating that the PB2 627-domain of the influenza virus RNA polymerase is not involved in core catalytic functions of the polymerase. However, in a cellular context, the 627-domain is essential for both transcription and replication. In particular, we show that the PB2 627-domain is essential for the accumulation of the cRNA replicative intermediate in infected cells. Together, these results further our understanding of the role of the PB2 627-domain in transcription and replication of the influenza virus RNA genome.

    IMPORTANCE Influenza A viruses are a major global health threat, not only causing disease in both humans and birds, but also placing significant strains on economies worldwide. Avian influenza A virus polymerases typically do not function efficiently in mammalian hosts and require adaptive mutations to restore polymerase activity. These adaptations include mutations in the 627-domain of the PB2 subunit of the viral polymerase, but it still remains to be established how these mutations enable host adaptation on a molecular level. In this study we characterise the role of the 627-domain in polymerase function and offer insights into the replication mechanism of influenza A viruses.

  • Transgenic CRISPR/Cas9-mediated viral gene targeting for antiviral therapy of Bombyx mori nucleopolyhedrovirus (BmNPV) [PublishAheadOfPrint]

  • We developed a novel anti-viral strategy by combining transposon-based transgenesis and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system for direct cleavage of the Bombyx mori nucleopolyhedrovirus (BmNPV) genome DNA to promote virus clearance in silkworms. We demonstrate that transgenic silkworms constitutively-expressing Cas9 and guide RNAs (gRNAs) targeting the BmNPV immediate early-1 (ie-1) gene and me53 gene effectively induce target-specific cleavage and subsequent mutagenesis, especially large segment deletions (~ 7 kilobase-pairs [kb]) in BmNPV genomes and thus exhibit a robust suppression of BmNPV proliferation. Transgenic animals exhibited higher and inheritable resistance to BmNPV infection when compared to wild-type animals. Our approach will not only contribute to modern sericulture but also shed light on future anti-viral therapy.

    Importance Pathogen genome targeting has shown its potential in anti-viral research. However, transgenic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system-mediated viral genome targeting has not been reported as an antiviral strategy in a natural animal host of a virus. Our data provide an effective approach against Bombyx mori nucleopolyhedrovirus (BmNPV) infection in a real-world biological system and demonstrate the potential of transgenic CRISPR/Cas9 systems in anti-viral research in other species.

  • Lack of ADCC breadth of human non-neutralizing anti-HIV-1 antibodies [PublishAheadOfPrint]

  • Anti-HIV-1 non-neutralizing antibodies (nnAbs) capable of antibody-dependent cellular cytotoxicity (ADCC) have been identified as a protective immune correlate in the RV144 vaccine efficacy trial. Broadly neutralizing antibodies (bNAbs) also mediate ADCC in cell culture and rely on their Fc region for optimal efficacy in animal models. Here, we selected 9 monoclonal nnAbs and 5 potent bNAbs, targeting various epitopes and conformations of the gp120/41 complex, and analyzed the potency of the two types of antibodies to bind and eliminate HIV-1-infected cells in culture. Regardless of their neutralizing activity, most of the selected antibodies recognized and killed cells infected with two laboratory-adapted HIV-1 strains. Some nnAbs also bound bystander cells that may have captured viral proteins. However, in contrast to the bNAbs, the nnAbs poorly bound to reactivated infected cells from 8 HIV-positive individuals and did not mediate effective ADCC against those cells. The nnAbs also inefficiently recognize cells infected with 8 different transmitted founder (T/F) isolates. Addition of a synthetic CD4 mimetic enhanced the binding and killing efficacy of some of the nnAbs in an epitope-dependent manner, without reaching the levels achieved by the most potent bNAbs. Overall, our data reveal important qualitative and quantitative differences between nnAbs and bNAbs in their ADCC capacity and strongly suggest that the breadth of recognition of HIV-1 by nnAbs is narrow.

    Importance Most of the anti-HIV antibodies generated by infected individuals do not display potent neutralizing activities. These non-neutralizing antibodies (nnAbs) with antibody-dependent cellular cytotoxicity (ADCC) have been identified as a protective immune correlate in the RV144 vaccine efficacy trial. However, in primate models, the nnAbs do not protect against SHIV acquisition. Thus, the role of nnAbs with ADCC activity in protecting from infection remains debatable. In contrast, broadly neutralizing antibodies (bNAbs) neutralize a large array of viral strains and mediate ADCC in cell culture. We analyzed the capacity of 9 nnAbs and 5 bNAbs to eliminate infected cells. We selected 18 HIV-1 strains, including virus reactivated from the reservoir of HIV+ individuals and transmitted-founder isolates. We report that the nnAbs poorly bind to cells infected with primary HIV-1 strains and do not mediate potent ADCC. Overall, our data show that the breadth of recognition of HIV-1 by nnAbs is narrow.

  • Parvovirus Expresses a Small Noncoding RNA that Plays an Essential Role in Virus Replication [PublishAheadOfPrint]

  • Human bocavirus 1 (HBoV1) belongs to species Primate bocaparvovirus of the genus Bocaparvovirus of the Parvoviridae family. HBoV1 causes acute respiratory tract infections in young children and has a selective tropism for the apical surface of well-differentiated human airway epithelia (HAE). In this study, we identify an additional HBoV1 gene, bocavirus-transcribed small non-coding RNA (BocaSR) within the 3rrsquo; non-coding region (nt 5199-5338) of the viral genome of positive sense. BocaSR is transcribed by RNA polymerase III (Pol III) from an intragenic promoter at similar levels to that of the capsid protein-coding mRNA and is essential for replication of the viral DNA in both transfected HEK293 and infected HAE cells. Mechanistically, we show that BocaSR regulates the expression of HBoV1-encoded non-structural proteins NS1, NS2, NS3, and NP1, but not NS4. BocaSR is similar to the adenovirus-associated type I (VAI) RNA in terms of both nucleotide sequence and secondary structure, but differs from it in that its regulation of viral protein expression is independent of RNA-activated protein kinase (PKR) regulation. Notably, BocaSR accumulates in the viral DNA replication centers within the nucleus and likely plays a direct role in replication of the viral DNA. Our findings reveal BocaSR to be a novel viral noncoding RNA that coordinates the expression of viral proteins and regulates replication of viral DNA within the nucleus. Thus, BocaSR may be a target for anti-viral therapies for HBoV and may also have utility in the production of recombinant HBoV vectors.

    Significance Human bocavirus 1 (HBoV1) is pathogenic to humans, causing acute respiratory tract infections in young children. In this study, we identify a novel HBoV1 gene that lies in the 3rrsquo; non-coding region of the viral positive sense genome and is transcribed by RNA polymerase III into a noncoding RNA of 140 nts. This bocavirus-transcribed small RNA (BocaSR) diverges from both adenovirus-associated (VA) RNAs and Epstein-Barr virus-encoded small RNAs (EBERs) with respect to RNA sequence, representing a third species of this kind of Pol III-dependent viral noncoding RNA and the first noncoding RNA identified in autonomous parvoviruses. Unlike the VA RNAs, BocaSR localizes to the viral DNA replication centers of the nucleus and is essential for expression of viral nonstructural proteins independent of RNA-activated protein kinase R and replication of HBoV1 genomes. The identification of BocaSR and its role in virus DNA replication reveals potential avenues for developing anti-viral therapies.

  • Impact of type 1 interferon on the safety and immunogenicity of an experimental live-attenuated herpes simplex virus type 1 vaccine in mice. [PublishAheadOfPrint]

  • Viral fitness dictates virulence and capacity to evade host immune defenses. Understanding the biological underpinnings of such features is essential for rational vaccine development. We have previously shown that the live-attenuated herpes simplex virus type 1 (HSV-1) 0NLS mutant is sensitive to inhibition by interferon bbeta; in vitro and functions as a highly efficacious experimental vaccine. Here, we characterize the host immune response and in vivo pathogenesis of HSV-1 0NLS relative to its fully virulent parental strain in C57BL/6 mice. Additionally, we explore the role of type 1 interferon (IFNaalpha;/bbeta;) signaling on virulence and immunogenicity of HSV-1 0NLS and uncover a probable sex bias in the induction of IFNaalpha;/bbeta; in the cornea during HSV-1 infection. Our data show that HSV-1 0NLS lacks neurovirulence even in highly immunocompromised mice lacking the IFNaalpha;/bbeta; receptor. These studies support the translational viability of the HSV-1 0NLS vaccine strain by demonstrating that while it is comparable to a virulent parental strain in terms of immunogenicity, HSV-1 0NLS does not induce significant tissue pathology.

    IMPORTANCE HSV-1 is a common human pathogen associated with a variety of clinical presentations ranging in severity from periodic "cold sores" to lethal encephalitis. Despite the consistent failures of HSV subunit vaccines in clinical trials spanning the past 28 years, opposition to live-attenuated HSV vaccines predicated on unfounded safety concerns currently limits their widespread acceptance. Herein, we demonstrate that a live-attenuated HSV-1 vaccine has great translational potential.

  • Monoclonal Antibodies to V2, V3, the CD4-binding site and gp41 HIV-1 Mediate Phagocytosis in a Dose-dependent Manner. [PublishAheadOfPrint]

  • In light of weak or absent neutralizing activity mediated by anti-V2 monoclonal Abs (mAbs), we tested whether they can mediate Ab-dependent cellular phagocytosis (ADCP) which is an important element of anti-HIV-1 immunity. We tested six anti-V2 mAbs and compared them with 21 mAbs specific for V3, the CD4-binding site (CD4bs), and gp41 derived from HIV-1 chronically infected individuals and produced by hybridoma cells. ADCP activity was measured by flow cytometry using uptake by THP-1 monocytic cells of fluorescent beads coated with gp120, gp41, BG505 SOSIP.664 or BG505 DS-SOSIP.664 complexed with mAbs. The ADCP activity measured by the area under the curve showed significantly higher activity of anti-gp41 mAbs compared to three other groups of mAbs tested using beads coated with monomeric gp41 or gp120; anti-V2 mAbs were dominant over anti-V3 and anti-CD4bs against clade C gp120ZM109. ADCP mediated by V2 and V3 mAbs was positive against stabilized DS-SOSIP.664 trimer but negligible against SOSIP.664 targets, suggesting a closed envelope conformation better exposes the variable loops. Two IgG3 mAbs against V2 and V3 regions displayed dominant ADCP activity over a panel of IgG1 mAbs. This superior ADCP activity was confirmed when two of three recombinant IgG3 anti-V2 mAbs were compared to IgG1 counterparts. The study demonstrated dominant ADCP activity of anti-gp41 against monomers but not trimers with some higher activity of anti-V2 mAbs over anti-V3 and anti-CD4bs mAbs. The ability to mediate ADCP suggests a mechanism by which anti-HIV-1 envelope Abs can contribute to protective efficacy.

    IMPORTANCE Anti-V2 antibodies (Abs) correlated with reduced risk of HIV-1 infection in recipients of the RV144 vaccine suggesting that they play a protective role, but a mechanism providing such protection remains to be determined. The rare and weak neutralizing activities of anti-V2 mAbs prompted us to study Fc-mediated activities. We compared anti-V2 mAbs with other mAbs specific for V3, CD4bs and gp41 for Ab-dependent cellular phagocytosis (ADCP) activity, implicated in protective immunity. The anti-V2 mAbs displayed stronger activity compared to other anti-gp120 mAbs when screened against one of two gp120s and against DS-SOSIP which mimics the native trimer. Anti-gp41 mAbs were superior when targeting monomeric gp41, but were comparable against trimers which may not adequately expose gp41 epitopes. While anti-envelope mAbs in general mediated ADCP, anti-V2 mAbs displayed some dominance compared to other mAbs. Our demonstration that anti-V2 mAbs mediate ADCP suggests a functional mechanism for their contribution to protective efficacy.

  • The pseudorabies virus glycoprotein gE/gI complex suppresses type I interferon production by plasmacytoid dendritic cells [PublishAheadOfPrint]

  • Plasmacytoid dendritic cells (pDC) play a central role in the antiviral immune response, both in the innate response and in shaping the adaptive response, mainly because of their ability to produce massive amounts of type I interferon (TI-IFN). Here, we report that cells infected with the live attenuated Bartha vaccine strain of porcine alphaherpesvirus pseudorabies virus trigger a dramatically increased TI-IFN response by porcine primary pDC compared to cells infected with wild type PRV strains (Becker and Kaplan). Since Bartha is one of relatively few examples of a highly successful alphaherpesvirus vaccine, identifying factors that may contribute to its efficacy may provide insights for the rational design of other alphaherpesvirus vaccines. The Bartha vaccine genome displays several mutations compared to the genome of wild type PRV strains, including a large deletion in the US (unique short) region, encompassing the glycoprotein E (gE), gI, US9 and US2 genes. Using recombinant PRV Becker strains harboring the entire Bartha US deletion or single mutations in the four affected US genes, we demonstrated that the absence of the viral gE/gI complex contributes to the observed increased IFN-aalpha; response. Furthermore, we showed that the absence of gE leads to an enhanced ERK1/2 phosphorylation in pDC, which correlates with a higher TI-IFN production by pDC. In conclusion, the PRV Bartha vaccine strain triggers strongly increased TI-IFN production by porcine pDC. Our data further indicate that the gE/gI glycoprotein complex suppresses TI-IFN production by pDC, which represents the first alphaherpesvirus factor that suppresses pDC activity.

    Importance Several alphaherpesviruses, including herpes simpex virus, still lack effective vaccines. However, the highly successful Bartha vaccine has contributed substantially to eradication of the porcine alphaherpesvirus pseudorabies virus (PRV) in several countries. The impact of Bartha on the immune response is still poorly understood. Type I interferon (TI-IFN) producing plasmacytoid dendritic cells (pDC) may play an important role in vaccine development. Here, we show that Bartha elicits a dramatically increased type I interferon (TI-IFN) response in primary porcine pDC compared to wild type strains. In addition, we found that the gE/gI complex, which is absent in Bartha, inhibits the pDC TI-IFN response. This is the first description of an immune cell type that is differentially affected by Bartha versus wild type PRV, and is the first report describing an alphaherpesvirus protein that inhibits the TI-IFN response by pDC. These data may therefore contribute to the rational design of other alphaherpesvirus vaccines.

  • Nucleic and amino acid sequences support structure-based viral classification [PublishAheadOfPrint]

  • Viral capsids ensure viral genome integrity by protecting the enclosed nucleic acids. Interactions between the genome, capsid and between individual capsid proteins (i.e. "capsid architecture") are intimate and expected to be characterized by strong evolutionary conservation. For this reason, a capsid structure-based viral classification has been proposed as a way to bring order to the viral universe. The seeming lack of sufficient sequence similarity to reproduce this classification has made it difficult to reject structural convergence as the basis for the classification. We reinvestigate whether the structure-based classification, for viral coat proteins making icosahedral virus capsids, is in fact supported by previously undetected sequence similarity. Since codon choices can influence nascent protein folding cotranslationally, we have searched for both amino acid and nucleotide sequence similarity. To demonstrate the sensitivity of the approach, we identify a candidate gene for the Pandoravirus capsid protein. We show that the structure-based classification is strongly supported by amino acid and also nucleotide sequence similarity, suggesting that similarities are due to common descent. The correspondence between structure-based and sequence-based analyses of the same proteins shown here allow them to be used in future analyses of the relationship between linear sequence information and macromolecular function, as well as between linear sequence and protein folds.

    IMPORTANCE Viral capsids protect nucleic acid genomes which in turn encode capsid proteins. This tight coupling of protein shell and nucleic acids, together with strong functional constraints on capsid protein folding and architecture, leads to the hypothesis that capsid protein coding nucleotide sequences may retain signatures of ancient viral evolution. We have been able to show that this is indeed the case, using the major capsid proteins of viruses forming icosahedral capsids. Importantly, we detect similarity at nucleotide level between capsid protein coding regions from viruses infecting cells belonging to all three domains of life, reproducing a previously established structure-based classification of icosahedral viral capsids.

  • TNF{alpha} Induced Recruitment of Inflammatory Mononuclear Cells Leads to Inflammation and Altered Brain Development in MCMV Infected Newborn Mice [PublishAheadOfPrint]

  • Congenital human cytomegalovirus (HCMV) infection is a significant cause of abnormal neurodevelopment and long term neurological sequelae in infants and children. Resident cell populations of the developing brain have been suggested to be more susceptible to virus-induced cytopathology, a pathway thought to contribute to the clinical outcomes following intrauterine HCMV infection. However, recent findings in a newborn mouse model of this infection in the developing brain have indicated that elevated levels of proinflammatory mediators leading to mononuclear cell activation and recruitment could underlie the abnormal neurodevelopment. In this study, we demonstrated that treatment with TNFaalpha; neutralizing antibodies decreased the frequency of CD45+/Ly6C hi/CD11b+/CCR2+ activated myeloid mononuclear cells (MMC) and the levels of proinflammatory cytokines in the blood and the brains of murine CMV infected mice. This treatment also normalized neurodevelopment in infected mice without significantly impacting the level of virus replication. These results indicate that TNFaalpha; is a major component of the inflammatory response associated with altered neurodevelopment that follows murine CMV infection of the developing brain and that a subset of peripheral blood myeloid mononuclear cells represent a key effector cell population in this model of virus-induced inflammatory disease of the developing brain.

    IMPORTANCE Congenital human cytomegalovirus (HCMV) is the most common viral infection of the developing human fetus and can result in neurodevelopmental sequelae. Mechanisms of disease leading to neurodevelopmental deficits in infected infants remain undefined but postulated pathways include loss of neuronal progenitor cells, damage to the developing vascular system of the brain, and altered cellular positioning. Direct virus-mediated cytopathic effects cannot explain the phenotypes of brain damage in most infected infants. Using a mouse model that recapitulates characteristics of the brain infection described in human infants, we have shown that TNFaalpha; plays a key role in brain inflammation, including recruitment of inflammatory mononuclear cells. Neutralization of TNFaalpha; normalized neurodevelopmental abnormalities in infected mice providing evidence that virus-induced inflammation is a major component of disease in the developing brain. These results suggest that interventions limiting inflammation associated with this infection could potentially improve the neurologic outcome of infants infected in-utero with HCMV.

  • MMP-9 Facilitates Hepatitis B Virus Replication through Binding with IFNAR1 to Repress IFN/JAK/STAT Signaling [PublishAheadOfPrint]

  • Hepatitis B virus (HBV) infection may cause acute hepatitis B (AHB), chronic hepatitis B (CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). However, the mechanisms by which HBV evades host immunity and maintains chronic infection are largely unknown. Here, we revealed that matrix metalloproteinase-9 (MMP-9) is activated in peripheral blood mononuclear cells (PBMCs) of HBV-infected patients, and HBV stimulates MMP-9 expression in macrophages and PBMCs isolated from healthy individuals. MMP-9 plays important roles in the breakdown of extracellular matrix and in the facilitation of tumor progression, invasion, metastasis and angiogenesis. MMP-9 also regulates respiratory syncytial virus (RSV) replication, but the mechanism underlying such regulation is unknown. We further demonstrated that MMP-9 facilitates HBV replication by repressing interferon/Janus kinase/signal transducers and activators of transcription (IFN/JAK/STAT) pathway, IFN action, STAT1/2 phosphorylation and IFN-stimulated genes (ISGs) expression. Moreover, MMP-9 binds to type I IFN receptor 1 (IFNAR1) and facilitates IFNAR1 phosphorylation, ubiquitination, sub-cellular distribution and degradation to interfere the binding of IFANR1 with IFN-aalpha;. Thus, we identified a novel positive feedback regulation loop between HBV replication and MMP-9 production. On one hand, HBV activates MMP-9 in infected patients and leukocytes. On the other hand, MMP-9 facilitates HBV replication through repressing IFN/JAK/STAT signaling, IFNAR1 function, and IFN-aalpha; action. Therefore, HBV may take the advantage of MMP-9 function to establish or maintain chronic infection.

    Importance Hepatitis B virus (HBV) infection may cause chronic hepatitis B (CHB) and hepatocellular carcinoma (HCC). However, the mechanisms by which HBV maintains chronic infection are largely unknown. Matrix metalloproteinase-9 (MMP-9) plays important roles in the facilitation of tumor progression, invasion, metastasis and angiogenesis. However, the effects of MMP-9 on HBV replication and pathogenesis are not known. This study reveals that MMP-9 expression is activated in patients with CHB and HBV stimulates MMP-9 production in PBMCs and macrophages. More interestingly, MMP-9 in turn promotes HBV replication through suppressing IFN-aalpha; action. Moreover, MMP-9 interacts with type I interferon receptor 1 (IFNAR1) to disturb the binding of IFN-aalpha; with IFNAR1 and facilitate phosphorylation, ubiquitination, sub-cellular distribution and degradation of IFNAR1. Therefore, these results discover a novel role of MMP-9 in viral replication, and reveal a new mechanism by which HBV evades host immunity to maintain persistent infection.

  • The FACT complex promotes avian leukosis virus DNA integration [PublishAheadOfPrint]

  • All retroviruses need to integrate a DNA copy of their genome into the host chromatin. Cellular proteins regulating and targeting lentiviral and gammaretroviral integration in infected cells have been discovered, but the factors that mediate alpharetroviral avian leukosis virus (ALV) integration are unknown. Here, we have identified the FACT protein complex, which consists of SSRP1 and Spt16, as a principal cellular binding partner of ALV integrase (IN). Biochemical experiments with purified recombinant proteins show that SSRP1 and Spt16 are able to individually bind ALV IN, but only the FACT complex effectively stimulates ALV integration activity in vitro. Likewise, in infected cells, the FACT complex promotes ALV integration activity with proviral integration frequency varying directly with cellular expression levels of the FACT complex. An increase in 2-LTR circles in the depleted FACT complex cell line indicates that this complex regulates the ALV life cycle at the level of integration. This regulation is shown to be specific to ALV, as disruption of the FACT complex did not inhibit either lentiviral or gammaretroviral integration in infected cells.

    IMPORTANCE The majority of human gene therapy approaches utilize HIV-1 or MLV based vectors, which preferentially integrate near genes and regulatory regions; thus, insertional mutagenesis is a substantial risk. In contrast, ALV integrates more randomly throughout the genome, which decreases the risks of deleterious integration. Understanding how ALV integration is regulated could facilitate the development of ALV-based vectors for use in human gene therapy. Here we show that the FACT complex directly binds and regulates ALV integration efficiency in vitro and in infected cells.

  • Conserved interaction of lentiviral Vif molecules with HIV-1 Gag and differential effects of species-specific Vif on virus production [PublishAheadOfPrint]

  • Open reading frame virion infectivity factor (Vif) is conserved among most lentiviruses. Vif molecules contribute to viral replication by inactivating host anti-viral factors, the APOBEC3 cytidine deaminases. However, various species of lentiviral Vif proteins have evolved different strategies for overcoming host APOBEC3. Whether different species of lentiviral Vif proteins still preserve certain common features has not been reported. Here, we show for the first time that diverse lentiviral Vif molecules maintain the ability to interact with the HIV-1 Gag precursor (Pr55Gag) polyprotein. Surprisingly, BIV Vif, but not HIV-1 Vif, interfered with HIV-1 production and viral infectivity even in the absence of APOBEC3. Further analysis revealed that BIV Vif demonstrated an enhanced interaction with Pr55Gag when compared to HIV-1 Vif, and BIV Vif defective for the Pr55Gag interaction lost its ability to inhibit HIV-1. The C-terminal region of CA and the p2 region of Pr55Gag, which are important for virus assembly and maturation, were involved in the interaction. Transduction of CD4+ T cells with BIV Vif blocked HIV-1 replication. Thus, the conserved Vif-Pr55Gag interaction provides a potential target for the future development of anti-viral strategies.

    IMPORTANCE The conserved Vif accessory proteins of primate lentiviruses HIV-1, SIV, and BIV all form ubiquitin ligase complexes to target host antiviral APOBEC3 proteins for degradation, with different cellular requirements and using different molecular mechanisms. Here we demonstrate that BIV Vif can interfere with HIV-1 Gag maturation and suppress HIV-1 replication through interaction with the precursor of the Gag (Pr55Gag) of HIV-1 in virus-producing cells. Moreover, the HIV-1 and SIV Vif proteins are conserved in terms of their interactions with HIV-1 Pr55Gag, although HIV-1 Vif proteins bind Pr55Gag less efficiently than do those of BIV Vif. Our research not only sheds new light on this feature of these conserved lentiviral Vif proteins but also provides a formerly unrecognized target for the development of anti-viral strategies. Since increasing the Vif-Pr55Gag interaction could potentially suppress virus proliferation, this approach could offer a new strategy for the development of HIV inhibitors.

  • Molecular analysis of Arthrobacter myovirus vB_ArtM-ArV1: we blame it on the tail [PublishAheadOfPrint]

  • This is the first report on a myophage that infects Arthrobacter. A novel virus vB_ArtM-ArV1 (ArV1) was isolated from soil using Arthrobacter sp. 68b strain for phage propagation. Transmission electron microscopy showed its resemblance to members of the family Myoviridae: ArV1 has an isometric head (~74 nm in diameter) and a contractile non-flexible tail (~192 nm). Phylogenetic and comparative sequence analyses, however, revealed that ArV1 has more genes in common with phages from the family Siphoviridae than it does with any myovirus characterized to date. The genome of ArV1 is a linear, circularly permuted, double-stranded DNA molecule (71,200 bp) with a GC content of 61.6 %. The genome codes for 101 ORFs, yet contains no tRNA genes. More than 50% of ArV1 genes encode unique proteins that either have no reliable identity to database entries, or have homologues only in Arthrobacter phages, both sipho- and myoviruses. Using bioinformatics approaches, 13 ArV1 structural genes were identified, including those coding for head, tail, tail fiber, and baseplate proteins. A further 6 ArV1 ORFs were annotated as putative structural proteins based on the results of proteomic analysis. Phylogenetic analysis based on the alignment of four conserved virion proteins revealed that Arthrobacter myophages form a discreet clade that seems to occupy a somewhat intermediate position between myo- and siphoviruses. Thus, the data presented here will help to advance our understanding of genetic diversity and evolution of phages that constitute the order Caudovirales.

    Importance Bacteriophages, which likely have originated in the early Precambrian Era, represent the most numerous population on the planet. Approximately 95% of known phages are tailed viruses that comprise three families: Podoviridae (short tail), Siphoviridae (long noncontractile tail), and Myoviridae (contractile tail). Based on the current hypothesis, myophages, which may have evolved from siphophages, are thought to have first emerged among Gram-negative bacteria, whereas only later - among Gram-positive bacteria. The results on the molecular characterization of a myophage vB_ArtM-ArV1 presented here conform to the aforementioned hypothesis since, at a glance, bacteriophage vB_ArtM-ArV1 appears to be a siphovirus that possesses a seemingly functional contractile tail. Our work demonstrates that such "chimeric" myophages are of cosmopolitan nature, and are likely characteristic to ecologically important soil bacterium Arthrobacter.

  • Functional analysis of HCV envelope protein E1 using a trans-complementation system reveals a dual role of a putative fusion peptide of E1 in both HCV entry and morphogenesis [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) is an enveloped RNA virus belonging to the Flaviviridae family. It mainly infects human hepatocytes and causes chronic liver diseases including cirrhosis and cancer. HCV encodes two envelope proteins E1 and E2 that form a heterodimer and mediate virus entry. While E2 has been extensively studied, less has been done so for E1, and its role in HCV life cycle still needs to be elucidated. Here we developed a new cell culture model for HCV infection based on trans-complementation of E1. The virus production of HCV genome lacking the E1-encoding sequence can be efficiently rescued by ectopic expression of E1 in trans. The resulting virus, designated as HCVE1, can propagate in the package cells expressing E1, but only results in single-cycle infection in naïve cells. By using the HCVE1 system we explored the role of a putative fusion peptide (FP) of E1in HCV infection. Interestingly, we found that FP not only contributes to HCV entry as previously reported, but may also be involved in virus morphogenesis. Finally, we identified amino acid residues in FP that are critical for biological functions of E1. In summary, our work not only provides a new cell culture model for studying HCV, but also shed some insights into understanding the role of E1 in HCV life cycle.

    Importance Hepatitis C virus (HCV), an enveloped RNA virus, encodes two envelope proteins E1 and E2 that form a heterodimeric complex to mediate virus entry. Compared to E2, the biological functions of E1 in virus life cycle are not adequately investigated. Here we developed a new cell culture model for single-cycle HCV infection based on trans-complementation of E1. The HCV genome lacking the E1-encoding sequence can be efficiently rescued for virus production by ectopic expression of E1 in trans. This new model renders a unique system to dissect functional domains and motifs in E1. Using this system, we found that a putative fusion peptide in E1 is a multi-functional structural element contributing to both HCV entry and morphogenesis. Our work has provided a new cell culture model to study HCV and shed insights into understanding the biological roles of E1 in HCV life cycle.

  • Recombinant adenoviruses displaying matrix 2 ectodomain epitopes (M2e) on their fiber proteins as universal influenza vaccines [PublishAheadOfPrint]

  • Influenza is a zoonotic disease that poses severe threats to public health and the global economy. Re-emerging influenza pandemics highlight the demand for universal influenza vaccines. We developed a novel virus platform, AdC68-F3M2e, by introducing three conserved M2e epitopes into the HI loop of the chimpanzee adenovirus (AdV) fiber protein. The M2e epitopes were expressed sufficiently on the AdV virion surface without affecting fiber trimerization. Additionally, one recombinant adenovirus, AdC68-F3M2e(H1-H5-H7), induced robust M2e-specific antibody responses in BALB/c mice after two sequential vaccinations and conferred efficient protection against homologous and heterologous IV challenges. We find that AdV with tandem M2e epitopes in fiber is a potential strategy for IV prevention.

    Importance Influenza epidemics and pandemics severely threaten public health. Universal influenza vaccines have increasingly attracted interest in recent years. Here, we describe a new strategy that incorporates triple M2e epitopes into the fiber protein of chimpanzee adenovirus 68. We optimized the process of inserting foreign genes into the AdC68 structural protein by one-step isothermal assembly, and demonstrated that this 225bp HI loop insertion could be well tolerated. Furthermore, two doses of adjuvant-free fiber-modified AdC68 could confer sufficient protection against homologous and heterologous influenza infections in mice. Our results show that AdC68-F3M2e could be pursued as a novel universal influenza vaccine.

  • Influence of the Envelope Gp120 Phe 43 Cavity on HIV-1 Sensitivity to ADCC Responses [PublishAheadOfPrint]

  • HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cellular-mediated cytotoxicity (ADCC). HIV-1 has evolved sophisticated mechanisms to avoid exposure of Env ADCC epitopes by downregulating CD4 and by limiting the overall amount of Env on the cell surface. In HIV-1, substitution of large residues such as histidine or tryptophan for serine 375 (S375H/W) in the gp120 Phe 43 cavity, where Phe 43 of CD4 contacts gp120, results in the spontaneous sampling of an Env conformation closer to CD4-bound state. While residue S375 is well-conserved in the majority of group M HIV-1 isolates, CRF01_AE strains have a naturally-occurring histidine at this position (H375). Interestingly, CRF01_AE is the predominant circulating strain in Thailand where the RV144 trial took place. In this trial, which resulted in a modest degree of protection, ADCC responses were identified as being part of the correlate of protection. Here we investigate the influence of the Phe 43 cavity on ADCC responses. Filling this cavity with a histidine or tryptophan residue in Envs with a natural serine residue at this position (S375H/W) increased the susceptibility of HIV-1-infected cells to ADCC. Conversely, replacing His 375 by a serine residue (H375S) within HIV-1 CRF01_AE decreased the efficiency of the ADCC response. Our results raise the intriguing possibility that the presence of His 375 in the circulating strain where the RV144 trial was held contributed to the observed vaccine efficacy.

    IMPORTANCE HIV-1-infected cells presenting Env in the CD4-bound conformation on their surface are preferentially targeted by ADCC mediated by HIV+ sera. Here we show that the gp120 Phe 43 cavity modulates the propensity of Env to sample this conformation and therefore affects the susceptibility of infected cells to ADCC. CRF01_AE HIV-1 strains have an unusual Phe 43 cavity-filling His 375 residue, which increases Env propensity to sample the CD4-bound conformation, thereby increasing susceptibility to ADCC.

  • The V3-loop of HIV-1 Env determines viral susceptibility to IFITM3 impairment of viral infectivity [PublishAheadOfPrint]

  • Interferon inducible transmembrane proteins (IFITMs) inhibit a broad spectrum of viruses including HIV-1. IFITM proteins deter HIV-1 entry when expressed in target cells and also impair HIV-1 infectivity when expressed in virus producer cells. However, little is known about how viruses resist IFITM inhibition. In this study, we have investigated the susceptibilities of different primary isolates of HIV-1 to the inhibition of viral infectivity by IFITMs. Our results demonstrate that the infectivity of different HIV-1 primary isolates including transmitted founder viruses is diminished by IFITM3 to various levels, with strain AD8-1 exhibiting strong resistance. Further mutagenesis studies revealed that HIV-1 Env, the V3-loop sequence in particular, determines the extent of inhibition of the viral infectivity by IFITM3. The IFITM3-sensitive Env proteins are also more susceptible to neutralization by soluble CD4 or the 17b antibody compared to the IFITM3-resistant Env proteins. Together, our study suggests that the propensity of HIV-1 Env to sample CD4-bound-like conformations modulates viral sensitivity to IFITM3 inhibition.

    IMPORTANCE Results of our study have revealed the key features of HIV-1 envelope protein that are associated with viral resistance to the IFITM3 protein. IFITM proteins are important effectors in interferon-mediated antiviral defence. A variety of viruses are inhibited by IFITMs at viral entry step. Although it is known that envelope proteins of several different viruses resist IFITM inhibition, the detailed mechanisms are not fully understood. Taking advantage of the fact that envelope proteins of different HIV-1 strains exhibit different degrees of resistance to IFITM3 and that these HIV-1 envelope proteins share the same domain structure and similar sequences, we have performed mutagenesis studies and determined the key role of the V3 loop in this viral resistance phenotype. We were also able to associate viral resistance to IFITM3 inhibition with the susceptibility of HIV-1 to the inhibition by soluble CD4 and the 17b antibody that recognizes CD4-binding induced epitopes.

  • Azacytidine treatment inhibits the progression of Herpes Stromal Keratitis by enhancing regulatory T cell function [PublishAheadOfPrint]

  • Ocular infection with Herpes Simplex Virus type 1 (HSV-1) sets off an inflammatory reaction in the cornea which leads to both virus clearance as well as chronic lesions that are orchestrated by CD4 T cells. Approaches that enhance the function of regulatory T cells (Treg) and dampen effector T cells can be effective to limit Stromal Keratitis (SK) lesion severity. In this report, we have explored the novel approach of inhibiting DNA methyltransferase activity using 5-Azacytidine (cytosine analog) to limit HSV-1 induced ocular lesions. We show that therapy begun after infection when virus was no longer actively replicating resulted in the pronounced reduction in lesion severity with markedly diminished numbers of T cells and non-lymphoid inflammatory cells along with reduced cytokine mediators. The remaining inflammatory reactions had a change in ratio of CD4 Foxp3+Treg to effector Th1 CD4 T cells in ocular lesions and lymphoid tissues with Treg becoming predominant over the effectors. In addition, compared to controls, Treg from Aza treated mice showed more suppressor activity in vitro and expressed higher levels of activation molecules. Additionally, cells induced in vitro in the presence of Aza showed epigenetic differences in the Treg Specific Demethylated Region (TSDR) of Foxp3 and were more stable when exposed to inflammatory cytokines. Our results show that therapy with Aza is an effective means of controlling a virus-induced inflammatory reaction and may act mainly by the effects on Treg.

    Importance: HSV-1 infection has been shown to initiate an inflammatory reaction in the cornea leading to tissue damage and loss of vision. The inflammatory reaction is orchestrated by IFN-gamma secreting Th1 cells and regulatory T cells play a protective role. Hence, novel therapeutics that can rebalance the ratio of regulatory T cells to effectors is a relevant issue. This study opens up a new avenue in treating HSV induced SK lesions by increasing the stability and function of regulatory T cells using DNA Methyltransferase inhibitor-5-Azacytidine (Aza). Aza increased the function of regulatory T cells leading to its enhanced suppressive activity and diminished lesions. Hence, therapy with Aza which acts mainly by the effects on Treg can be an effective means to control virus induced inflammatory lesions.

  • Overexpression of IL-7 extends the humoral immune response induced by rabies vaccination [PublishAheadOfPrint]

  • Rabies continues to present a public health threat in most countries of the world. The most efficient way to prevent and control rabies is to implement vaccination programmes for domestic animals. However, traditional inactivated vaccines used in animals are costly and have a relatively low efficiency, which impedes their extensive use in developing countries. There is therefore an urgent need to develop single-dose and long-lasting rabies vaccines. However, little information is available regarding the mechanisms underlying immunological memory, which can broaden humoral responses following rabies vaccination. In this study, a recombinant rabies virus (RABV) that expressed murine interleukin-7 (IL-7), referred to here as rLBNSE-IL7, was constructed, and its effectiveness was evaluated in a mouse model. rLBNSE-IL7 induced higher rates of T follicular helper (Tfh) cells and germinal center (GC) B cells from draining lymph nodes (LNs) than the parent virus rLBNSE. Interestingly, rLBNSE-IL7 improved the percentages of long-lived memory B cells (Bmem) in the draining LNs and plasma cells (PCs) in the bone marrow (BM) for up to 360 days post immunization (dpi). As a result of the long-lived PCs, it also generated prolonged virus-neutralizing antibodies (VNA), resulting in better protection against a lethal challenge than rLBNSE. Moreover, consistent with the increased numbers of Bmem and PCs after boost with rLBNSE, rLBNSE-IL7-immunized mice promptly produced a more potent secondary anti-RABV neutralising antibody response than rLBNSE-immunized mice. Overall, our data suggest that overexpressing IL-7 improved the induction of long-lasting primary and secondary antibody responses post RABV immunization.

    Importance Extending humoral immune responses using adjuvants is an important method to develop long-lasting and efficient vaccines against rabies. However, little information is currently available regarding prolonged immunological memory post-RABV vaccination. In this study, a novel rabies vaccine that expressed murine IL-7 was developed. This vaccine enhanced the numbers of Tfh cells and the GC responses, resulting in up-regulated quantity of Bmem and PCs. Moreover, we found the long-lived PCs that were elicited by the IL-7-expressing recombinant virus (rLBNSE-IL7) were able to sustain VNA levels much longer than the parent virus rLBNSE. Upon re-exposure to the pathogen, the longevous Bmem that were maintained higher numbers for up to 360 dpi by rLBNSE-IL7 compared to rLBNSE could differentiate into antibody-secreting cells, resulting in a rapid and potent secondary production of VNAs. These results suggest that the expression of IL-7 is beneficial to induce potent and long-lasting humoral immune responses.

  • The measles virus receptor SLAMF1 can mediate particle endocytosis [PublishAheadOfPrint]

  • The signalling lymphocyte activation molecule F1 (SLAMF1) is both a microbial sensor and entry receptor for Measles virus (MeV). Herein, we describe a new role for SLAMF1 to mediate MeV endocytosis that is in contrast with the alternative, and generally accepted, model that MeV genome enters cells only after fusion at the cell surface. We demonstrated that MeV engagement of SLAMF1 induces dramatic but transient morphological changes, most prominently in the formation of membrane blebs, which were shown to co-localise with incoming viral particles, and rearrangement of the actin cytoskeleton in infected cells. MeV infection was dependent on these dynamic cytoskeletal changes as well as fluid uptake through a macropinocytosis-like pathway as chemical inhibition of these processes inhibited entry. Moreover, we identified a role for the RhoA-ROCK-myosin-II signalling axis in this MeV internalisation process, highlighting a novel role for this recently characterised pathway in virus entry. Our study shows that MeV can hijack a microbial sensor normally involved in bacterial phagocytosis to drive endocytosis using a complex pathway that shares features with canonical viral macropinocytosis, phagocytosis and mechanotransduction. This uptake pathway is specific to SLAMF1-positive cells and occurs within 60 minutes of viral attachment. Measles remains a significant cause of mortality in human populations and this research sheds a new light on the very first steps of infection of this important pathogen.

    IMPORTANCE Measles causes a significant disease in humans and is estimated to have killed over 200 million people since records began. According to current World Health Organisation statistics it still kills over 100,000 people a year, mostly children in the developing world. The causative agent, measles virus is a small enveloped RNA virus that infects a broad range of cells during infection. In particular, immune cells are infected via interactions between glycoproteins found on the surface of the virus and SLAMF1, the immune cell receptor. In this research study we have investigated the steps governing entry of measles virus into SLAMF1-positive cells and identified endocytic uptake of viral particles. This research will impact on our understanding of morbillivirus-related immunosuppression as well as the application of measles as an oncolytic therapeutic.

  • The Envelope Gene of Transmitted HIV-1 Resists a Late IFN{gamma}-Induced Block [PublishAheadOfPrint]

  • Type I interferon (IFN) signaling engenders an antiviral state that likely plays an important role in constraining HIV-1 transmission and contributes to defining subsequent AIDS pathogenesis. Type II IFN (IFN) also induces an antiviral state but is often primarily considered to be an immunomodulatory cytokine. We report that IFN stimulation can induce an antiviral state that can be both distinct from that of type I interferon, and can potently inhibit HIV-1 in primary CD4+ T cells and a number of human cell lines. Strikingly, we find that transmitted/founder (TF) HIV-1 viruses can resist a late block that is induced by type II IFN, and the use of chimeric IFN- sensitive/resistant viruses indicates that interferon-resistance maps to the env gene. Simultaneously, in vitro evolution also revealed that just a single amino acid substitution in envelope can confer substantial resistance to IFN-mediated inhibition. Thus, the env gene of transmitted HIV-1 confers resistance to a late block that is phenotypically distinct from those previously described to be resisted by env, and is therefore mediated by unknown IFN-stimulated factor(s) in human CD4+ T cells and cell lines. This important unidentified block could play a key role in constraining HIV-1 transmission.

    IMPORTANCE The human immune system can hinder invading pathogens through interferon (IFN) signaling. One consequence of this signaling is that cells enter an 'antiviral state', increasing the levels of hundreds of defenses that can inhibit the replication and spread of viruses. The majority of HIV-1 infections result from a single virus particle (the transmitted/founder) that makes it past these defenses and colonizes the host. Thus, the founder virus is hypothesized to be a relatively interferon-resistant entity. Here we show that certain HIV-1 envelope genes have the unanticipated ability to resist specific human defenses mediated by different types of interferons. Strikingly, the envelope gene from a founder HIV-1 virus is far better at evading these defenses than the corresponding gene from a common HIV-1 lab strain. Thus, these defenses could play a role in constraining the transmission of HIV-1, and may select for transmitted viruses that are resistant to this IFN-mediated inhibition.

  • Glycosylation of the HA protein of H5N1 virus increases its virulence in mice by exacerbating the host immune response [PublishAheadOfPrint]

  • The highly pathogenic avian influenza (HPAI) H5N1 viruses continue to circulate in nature and threaten public health. Although several viral determinants and host factors that influence the virulence of HPAI H5N1 viruses in mammals have been identified, the detailed molecular mechanism remains poorly defined and requires further clarification. In our previous studies, we characterized two naturally isolated HPAI H5N1 viruses that had similar viral genomes but differed substantially in their lethality in mice. Here, we explored the molecular determinants and potential mechanism for this difference in virulence. By using reverse genetics, we found that a single amino acid at position 158 of the hemagglutinin (HA) protein substantially affected the systemic replication and pathogenicity of these H5N1 influenza viruses in mice. We further found that the G158N mutation introduced an N-linked glycosylation at positions 158nndash;160 of the HA protein and that this N-linked glycosylation enhanced viral productivity in infected mammalian cells and induced stronger host immune and inflammatory responses to viral infection. These findings further our understanding of the determinants of pathogenicity of H5N1 viruses in mammals.

    Importance Highly pathogenic avian influenza (HPAI) H5N1 viruses continue to evolve in nature and threaten human health. Key mutations in the virus hemagglutinin (HA) protein or reassortment with other pandemic viruses endow HPAI H5N1 viruses with the potential for aerosol transmissibility in mammals. A thorough understanding of the pathogenic mechanisms of these viruses will help us to develop more effective control strategies; however such mechanisms and virulent determinants for H5N1 influenza viruses have not been fully elucidated. Here, we identified glycosylation at positions 158nndash;160 of the HA protein of two naturally occurring H5N1 viruses as an important virulence determinant. This glycosylation event enhanced viral productivity, exacerbated the host response, and thereby contributed to the high pathogenicity of H5N1 virus in mice.

  • Dynamic modulation of expression of lentiviral restriction factors in primary CD4+ T cells following SIV infection [PublishAheadOfPrint]

  • Although multiple restriction factors have been shown to inhibit HIV/SIV replication, little is known about their expression in vivo. Expression of 45 confirmed and putative HIV/SIV restriction factors was analyzed in CD4+ T cells from peripheral blood and jejunum in rhesus macaques, revealing distinct expression patterns in naïve and memory subsets. In both peripheral blood and jejunum, memory CD4+ T cells expressed higher levels of multiple restriction factors compared with naïve cells. However, relative to their expression in peripheral blood CD4+ T cells, jejunal CCR5+ CD4+ T cells exhibited significantly lower expression of multiple restriction factors, including APOBEC3G, MX2, and TRIM25, which may contribute to the exquisite susceptibility of these cells to SIV infection. In vitro stimulation with anti-CD3/CD28 antibodies or type I interferon resulted in upregulation of distinct subsets of multiple restriction factors. After infection of rhesus macaques with SIVmac239, expression of most confirmed and putative restriction factors substantially increased in all CD4+ T cell memory subsets at the peak of acute infection. Jejunal CCR5+ CD4+ T cells exhibited the highest levels of SIV RNA, corresponding to the lower restriction factor expression in this subset relative to peripheral blood prior to infection. These results illustrate the dynamic modulation of confirmed and putative restriction factor expression by memory differentiation, stimulation, tissue microenvironment and SIV infection, and suggest that differential expression of restriction factors may play a key role in modulating the susceptibility of different populations of CD4+ T cells to lentiviral infection.

    IMPORTANCE Restriction factors are genes that have evolved to provide intrinsic defense against viruses. HIV and simian immunodeficiency virus (SIV) target CD4+ T cells. The baseline level of expression in vivo and degree to which expression of restriction factors is modulated by conditions such as CD4+ T cell differentiation, stimulation, tissue location, or SIV infection are currently poorly understood. Here we measured the expression of 45 confirmed and putative restriction factors in primary CD4+ T cells from rhesus macaques under various conditions, finding dynamic changes in each state. Most dramatically, in acute SIV infection, the expression of almost all target genes analyzed increased. These are the first measurements of many of these confirmed and putative restriction factors in primary cells or during the early events after SIV infection and suggest that the level of expression of restriction factors may contribute to the differential susceptibility of CD4+ T cells to SIV infection.

  • Sex-dependent intestinal replication of an enteric virus [PublishAheadOfPrint]

  • Coxsackievirus is an enteric virus that initiates infection in the gastrointestinal tract before disseminating to peripheral tissues to cause disease, but intestinal factors that influence viral replication are understudied. Furthermore, a sex bias for severe sequelae from coxsackievirus infections has been observed in humans. While mouse models mimicking human pathogenesis have been well characterized, many of these experiments use intraperitoneal injection of coxsackievirus to infect mice, bypassing the intestine. In light of recent studies identifying intestinal factors, such as the microbiota, that alter enteric viral replication, we sought to investigate coxsackievirus replication within the intestine. Here we orally infected mice with coxsackievirus B3 (CVB3) and found that CVB3 replication in the intestine is sex-dependent. CVB3 replicated efficiently in the intestine of male mice, but not female mice. Additionally, we found that the type 1 interferon response and sex hormones can alter both viral replication and lethality. Overall these data suggest that sex and the immune response play a vital role in CVB3 replication in the intestine and should be considered in light of the sex bias observed in human disease.

    IMPORTANCE Sex bias in severe sequelae from enteric viral infections have been observed. Since viruses have evolved to achieve optimal fitness in their environmental niche, it is imperative to study viruses at the site of initial replication. Here we used an oral inoculation system for CVB3, which follows the natural route of infection in the gastrointestinal tract. We found that sex can influence the replication of CVB3 in the intestine. Additionally the type 1 interferon response and sex hormones alter both CVB3 intestinal replication and lethality. Overall this work highlights the fact that sex should be considered when investigating enteric viral replication and pathogenesis.

  • Virion structure of black queen cell virus, a common honeybee pathogen [PublishAheadOfPrint]

  • Viral diseases are a major threat to honeybee (Apis mellifera) populations world-wide and therefore an important factor in reliable crop pollination and food security. Black queen cell virus (BQCV) is the etiological agent of a fatal disease of honeybee queen larvae and pupae. The virus belongs to the genus Triatovirus from the family Dicistroviridae that is part of the order Picornavirales. Here we present a crystal structure of BQCV determined to a resolution of 3.4 AAring;. The virion is formed by sixty copies of each of the major capsid proteins VP1, VP2, and VP3, however there is no density corresponding to a 75-residue-long minor capsid protein VP4 encoded by the BQCV genome. We show that the VP4 subunits are present in the crystallized virions that are infectious. This aspect of the BQCV virion is similar to that of the previously characterized triatoma virus and supports the recent establishment of the separate genus Triatovirus within the family Dicistroviridae. The C-terminus of VP1 and CD loops of capsid proteins VP1 and VP3 of BQCV form 34-AAring;-tall finger-like protrusions at the virion surface. The protrusions are larger than those of related dicistroviruses.

    IMPORTANCE The western honeybee is the most important pollinator of all, and is required to sustain the agricultural production and biodiversity of wild flowering plants. However, honeybee populations worldwide are suffering from virus infections that cause colony losses. One of the most common, and least known, honeybee pathogens is black queen cell virus (BQCV), which at high titers causes queen larvae and pupae to turn black and die. Here we present the three-dimensional virion structure of BQCV determined by X-ray crystallography. The structure of BQCV reveals large protrusions on the virion surface. Capsid protein VP1 of BQCV does not contain a hydrophobic pocket. Therefore, the BQCV virion structure provides evidence that capsid-binding antiviral compounds that can prevent the replication of vertebrate picornaviruses may be ineffective against honeybee virus infections.

  • Sterol-binding by the tombusviral replication proteins is essential for replication in yeast and plants [PublishAheadOfPrint]

  • Membranous structures derived from various organelles are important for replication of plus-stranded RNA viruses. Although the important roles of co-opted host proteins in RNA virus replication have been appreciated for a decade, the equally important functions of cellular lipids in virus replication are gaining full attention only recently. Previous works with Tomato bushy stunt tombusvirus (TBSV) in model host yeast have revealed essential roles for phosphatidylethanolamine and sterols in viral replication. To further our understanding of the role of sterols in tombusvirus replication, in this work we show that the TBSV p33 and p92 replication proteins could bind to sterols in vitro. The sterol-binding by p33 is supported by cholesterol recognition/amino acid consensus (CRAC) and CARC-like sequences within the two trans-membrane domains of p33. Mutagenesis of the critical Y amino acids within the CRAC and CARC sequences blocked TBSV replication in yeast and plant cells. We also show the enrichment of sterols in the detergent-resistant membrane (DRM) fractions obtained from yeast and plant cells replicating TBSV. The DRMs could support viral RNA synthesis both on the endogenous and exogenous templates. A lipidomic approach showed the lack of enhancement of sterol levels in yeast and plant cells replicating TBSV. The obtained data support that the TBSV replication proteins are associated with sterol-rich detergent-resistant membranes in yeast and plant cells. Altogether, the obtained and the previously published results support the local enrichment of sterols around the viral replication proteins that is critical for TBSV replication.

    Significance: One intriguing aspect of viral infections is their dependence on efficient subcellular assembly platforms serving replication, virion assembly or virus egress via budding out of infected cells. These assembly platforms might involve sterol-rich membrane microdomains, which are heterogeneous and highly dynamic nanoscale structures usurped by various viruses. Here, the authors demonstrate that TBSV p33 and p92 replication proteins can bind to sterol in vitro. Mutagenesis analysis of p33 within the CRAC and CARC sequences involved in sterol-binding shows the important connection between the ability of p33 to bind to sterol and to support TBSV replication in yeast and plant cells. Altogether, the obtained results further strengthen the model that cellular sterols are essential as pro-viral lipids during viral replication.

  • Human rhinovirus diversity and evolution: How strange the change from Major to minor [PublishAheadOfPrint]

  • Rhinoviruses are the most common cause of the common cold. Their many distinct lineages fall into "major" and "minor" groups that use different cell-surface receptors to enter host cells. Minor-group rhinoviruses are more immunogenic in laboratory studies, although their patterns of transmission and their cold symptoms are broadly similar to those of the major group. Here we present evolutionary evidence that minor-group viruses are also more immunogenic in humans. A key finding is that rates of amino-acid substitution at exposed sites in the capsid proteins VP2, VP3 and VP1 tend to be elevated in minor-group relative to major-group viruses, while rates at buried sites show no consistent differences. A reanalysis of historical virus-watch data also indicates higher immunogenicity of minor-group viruses, consistent with our findings about evolutionary rates at amino-acid positions most directly exposed to immune surveillance. The increased immunogenicity and speed of evolution in minor-group lineages may contribute to the very large numbers of rhinovirus serotypes that coexist while differing in virulence.

    IMPORTANCE Most colds are caused by rhinoviruses (RV). Those caused by a subset known as the minor-group members of the RV-A species are correlated with inception and aggravation of asthma in at-risk populations. Genetically, minor-group viruses are similar to the major-group RV-A viruses from which they were derived, although they tend to elicit stronger immune responses. Differences in their rates and patterns of molecular evolution should be highly relevant to their epidemiology. All RV-A viruses show high rates of amino-acid substitution in the capsid proteins at exposed sites not previously identified as immunogenic, and this acceleration is significantly greater in minor-group viruses. These findings will inform future studies of the recently discovered RV-C species, which also appear to exacerbate asthma in adults and children. In addition, these findings draw attention to the difficult problem of explaining the long-term coexistence of many serotypes of major and minor-group RVs.

  • Differences in glycoprotein complex (GPC) receptor binding site accessibility prompt poor cross-reactivity of neutralizing antibodies between closely related arenaviruses [PublishAheadOfPrint]

  • The glycoprotein complex (GPC) of arenaviruses, composed of stable signal peptide (SSP), GP1, and GP2, is the only antigen correlated with antibody-mediated neutralization. However, despite strong cross-reactivity of convalescent antisera between related arenavirus species, weak or no cross-neutralization occurs. Two closely related Clade B viruses, Machupo virus (MACV) and Juniiacute;n virus (JUNV), have near identical overall GPC architecture and share a host receptor, transferrin receptor 1. Given structural and functional similarities of the GP1 receptor binding site (RBS) of these viruses and recent demonstration that the RBS is an important target for neutralizing antibodies, it is not clear how these viruses avoid cross-neutralization. To address this, MACV/JUNV chimeric GPCs were assessed for interaction with a group of aalpha;-JUNV GPC monoclonal antibodies (mAbs) and mouse antisera against JUNV or MACV GPC. All six mAbs targeted GP1, with those that neutralized JUNV GPC-pseudovirions competing with each other for RBS binding. However, these mAbs were unable to bind to a chimeric GPC composed of JUNV GP1 containing a small disulfide bonded loop (loop 10) unique to MACV GPC, suggesting that this loop may block mAbs interaction with the GP1 RBS. Consistent with this loop causing interference, mouse anti-JUNV GPC antisera that solely neutralized pseudovirions bearing autologous GP1 provided enhanced neutralization of MACV GPC when this loop was removed. Our studies provide evidence that loop 10, which is unique to MACV GP1, is an important impediment to binding of neutralizing antibodies and contributes to the poor cross-neutralization of aalpha;-JUNV antisera against MACV.

    IMPORTANCE Multiple New World arenaviruses can cause severe disease in humans, and some geographic overlap exists among these viruses (1). A vaccine that protects against a broad range of New World arenaviruses is desirable for purposes of simplicity, cost, and broad protection against multiple NIAID-assigned Category A Priority Pathogens. In this study, we sought to better understand how closely related arenaviruses elude cross-species neutralization by investigating the structural bases of antibody binding and avoidance. In our studies, we found that neutralizing antibodies against two New World arenaviruses, Machupo virus (MACV) and Juniiacute;n virus (JUNV), bound to the envelope glycoprotein 1 (GP1) with JUNV monoclonal antibodies targeting the receptor-binding site (RBS). We further show that altered structures surrounding the RBS pocket in MACV GP1 impede access of JUNV-elicited antibodies.

  • Rhinovirus C, Asthma, and Cell Surface Expression of Virus Receptor, CDHR3 [PublishAheadOfPrint]

  • Human rhinoviruses of the A, B, and C species are defined agents of the common cold. But more than that, the RV-A and RV-C are the dominant cause of hospitalization-category infections in young children, especially those with asthma. The RV-C use of cadherin-related family member 3 (CDHR3) as its cellular receptor, creates a direct phenotypic link between human genetics ("G" vs "A" alleles cause Cys529 vs Tyr529 protein variants) and the efficiency with which RV-C can infect cells. With a lower cell surface display density, the human-specific Cys529 variant apparently confers partial protection from the severest virus-induced asthma episodes. Selective pressure favoring the Cys529 codon may have co-emerged with the evolution of the RV-C, and helped shape modern human genomics against the virus-susceptible, albeit ancestral Tyr529.

  • Temperature-sensitive Lesion in the N-terminal Domain of the Rotavirus Polymerase Affects its Intracellular Localization and Enzymatic Activity [PublishAheadOfPrint]

  • Temperature-sensitive (ts) mutants of simian rotavirus (RV) strain SA11 have been previously created to investigate the functions of viral proteins during replication. One mutant, SA11-tsC, maps to the gene encoding the VP1 polymerase and shows diminished growth and RNA synthesis at 39ddeg;C as compared to 31ddeg;C. In the current study, we sequenced all 11 genes of SA11-tsC, confirming the presence of an L138P mutation in the VP1 N-terminal domain and identifying 52 additional mutations in four other viral proteins (VP7, VP4, NSP2, and NSP1). To investigate whether the L138P mutation induces a ts phenotype in VP1 outside of the SA11-tsC genetic context, we employed ectopic expression systems. Specifically, we tested whether the L138P mutation affected the capacity of VP1 to localize to viroplasms, which are the sites of RV RNA synthesis, by expressing the mutant as a GFP-fusion protein (VP1L138P:GFP) (i) in WT SA11-infected cells or (ii) in uninfected cells along with the viroplasm-forming proteins NSP2 and NSP5. We found that VP1L138P:GFP localized to viroplasms and interacted with NSP2 and/or NSP5 at 31ddeg;C but not at 39ddeg;C. Next, we tested the enzymatic activity of a recombinant mutant polymerase (rVP1L138P) in vitro and found that it synthesized less RNA at 39ddeg;C versus 31ddeg;C, as well as less RNA than the control at all temperatures. Together, these results provide a mechanistic basis for the ts phenotype of SA11-tsC and raise important questions about the role of leucine 138 in supporting key protein interactions and catalytic function of the VP1 polymerase.

    IMPORTANCE RVs cause diarrhea in the young of many animal species, including humans. Despite their medical and economic importance, gaps in knowledge exist about how these viruses replicate inside of host cells. Previously, a mutant simian RV (SA11-tsC) that replicates worse at higher temperatures was identified. This virus has an amino acid mutation in VP1, which is the enzyme responsible for copying the viral RNA genome. The mutation is located in a poorly understood region of the polymerase called the N-terminal domain. In this study, we determined that the mutation reduced the capacity of VP1 to properly localize within infected cells at high temperatures as well as reduced the capacity of the enzyme to copy viral RNA in a test tube. The results of this study explain the temperature sensitivity of SA11-tsC and shed new light on functional protein-protein interaction sites of VP1.

  • The PTPN14 tumour suppressor is a degradation target of Human Papillomavirus E7 [PublishAheadOfPrint]

  • Activation of signalling pathways ensuring cell growth are essential for the proliferative competence of human papillomavirus (HPV)-infected cells. Tyrosine kinases and phosphatases are key regulators of cellular growth control pathways. A recently identified potential cellular target of HPV E7 is the cytoplasmic protein tyrosine phosphatase PTPN14, which is a potential tumour suppressor and is linked to the control of the Hippo and Wnt/beta-catenin signalling pathways. In this study we show that the E7 proteins of both high-risk and low-risk mucosal HPV types can interact with PTPN14. This interaction is independent of pRb and involves residues in the carboxyl-terminal region of E7. We also show that high-risk E7 induces proteasome-mediated degradation of PTPN14 in cells derived from cervical tumours. This degradation appears to be independent of cullin-1 or cullin-2, but most likely involves the UBR4/p600 ubiquitin ligase. The degree to which E7 down-regulates PTPN14 would suggest that this interaction is important for the viral life cycle, and potentially also in the development of malignancy. In support of this we find that over-expression of PTPN14 decreases the ability of HPV-16 E7 to cooperate with activated EJ-ras in primary cell transformation assays.

    IMPORTANCE: This study links HPV E7 to the deregulation of protein tyrosine phosphatase signalling pathways. PTPN14 is classified as a potential tumour suppressor protein and here we show that it is very susceptible to HPV E7 induced-proteasome mediated degradation. Intriguingly, this appears to use a mechanism that is different from that employed by E7 to target pRb. Therefore, this study has important implications for our understanding the molecular basis for E7 function, but also sheds important light on the potential role of PTPN14 as a tumour suppressor.

  • Herpes Simplex Virus Type 1 UL24 Abrogates DNA Sensing Signal Pathway by inhibiting NF-{kappa}B Activation [PublishAheadOfPrint]

  • Cyclic GMP-AMP synthase (cGAS) is a newly identified DNA sensor that recognizes foreign DNA, including the genome of herpes simplex virus 1 (HSV-1). Upon binding of viral DNA, cGAS produces cyclic GMP-AMP, which interacts with and activates stimulator of interferon genes (STING) to trigger the transcription of anti-viral genes such as type I interferons and production of inflammatory cytokines. HSV-1 UL24 is widely conserved among the herpesviruses family and is essential for efficient viral replication. In this study, we found that ectopically expressed UL24 could inhibit cGAS-STING mediated promoter activation of IFN-bbeta; and IL-6, and UL24 also inhibited interferon stimulatory DNA mediated interferon-bbeta; (IFN-bbeta;) and interleukin-6 (IL-6) production during HSV-1 infection. Furthermore, UL24 selectively blocked nuclear factor B (NF-B) but not IFN regulatory factor 3 promoter activation. Coimmunoprecipitation analysis demonstrated that UL24 bound to endogenous NF-B subunits p65 and p50 in HSV-1 infected cells, UL24 was also found to bind their Rel homology domain (RHD) respectively. Furthermore, UL24 reduced TNF-aalpha; mediated nuclear translocation of p65 and p50. Finally, mutational analysis revealed that the UL24 (74-134)aa is responsible for inhibiting cGAS-STING mediated NF-B promoter activity. For the first time, UL24 was shown to play an important role in immune evasion during HSV-1 infection.

    IMPORTANCE NF-B is a critical component of the innate immune response and is strongly induced downstream of most PRRs, leading to the production of IFN-bbeta; as well as a number of inflammatory chemokines and interleukins. To establish a persistent infection, viruses have evolved various mechanisms to counteract the host NF-B pathway. In the present study, for the first time, HSV-1 UL24 was demonstrated to inhibit the activation of NF-B in DNA sensing signal pathway via binding to the RHD of NF-B subunit p65 and p50 and abolishing their nuclear translocation.

  • Characterizing HIV-1 Splicing Using Next Generation Sequencing [PublishAheadOfPrint]

  • Full-length HIV-1 RNA serves as the genome or as an mRNA, or this RNA undergoes splicing using four donors and ten acceptors to create over 50 physiologically relevant transcripts in two size classes (1.8 kb and 4 kb). We developed an assay using Primer ID-tagged deep sequencing to quantify HIV-1 splicing. Using the NL4-3 lab strain we found that A5 (env/nef) is the most commonly used acceptor (about 50%) with A3 (tat) the least used (about 3%). Two small exons are made when a splice to acceptor A1 or A2 is followed by activation of donor D2 or D3, and the high-level use of D2 and D3 dramatically reduces the amount of vif and vpr transcripts. We observed distinct patterns of temperature sensitivity of spicing to acceptors A1 and A2. In addition, disruption of a conserved structure proximal to A1 caused a ten-fold reduction in all transcripts that utilized A1. Analysis of a panel of subtype B transmitted/founder viruses showed that splicing patterns are conserved, but with surprising variability of usage. A subtype C isolate was similar, while an SIV isolate showed significant differences. We also observed trans-splicing from a downstream donor on one transcript to an upstream acceptor on a different transcript, which we detected in 0.3% of 1.8 kb RNA reads. There were several examples of splicing suppression when the env intron was retained in the 4 kb size class. These results demonstrate the utility of this assay and identify new examples of HIV-1 splicing regulation.

    IMPORTANCE During HIV-1 replication over 50 conserved spliced RNA variants are generated. The splicing assay described here uses new developments in deep sequencing technology combined with Primer ID-tagged cDNA primers to efficiently quantify HIV-1 splicing at a depth that allows even low frequency splice variants to be monitored. We have used this assay to examine several features of HIV-1 splicing and to identify new examples of different mechanisms of regulation of these splicing patterns. This splicing assay can be used to explore in detail how HIV-1 splicing is regulated and, with moderate throughput, could be used to screen for structural elements, small molecules, and host factors that alter these relatively conserved splicing patterns.

  • Hierarchical and redundant roles of activating Fc{gamma}Rs in protection against influenza disease by M2e-specific IgG1 and IgG2a antibodies [PublishAheadOfPrint]

  • The ectodomain of matrix protein 2 is a universal influenza A vaccine candidate that provides protection through antibody-dependent effector mechanisms. Here we compared the functional engagement of Fc Receptor family members by two M2e-specific monoclonal antibodies: mAb 37 (IgG1) and mAb 65 (IgG2a), which recognize a similar epitope in M2e with similar affinity. Binding of mAb 65 to influenza A virus-infected cells triggered all three activating mouse Fc receptors in vitro, whereas mAb 37 only activated FcRIII. Passive transfer of mAb 37 or mAb 65 in wild type, Fcer1g-/-, Fcgr3-/- and Fcgr1-/- Fcgr3-/- BALB/c mice revealed the importance of these receptors for protection against influenza A virus challenge, with a clear requirement of FcRIII for IgG1 mAb 37. We also report that FcRIV contributes to protection by M2e-specific IgG2a antibodies.

    IMPORTANCE There is increased awareness that protection by antibodies directed against viral antigens is also mediated by the Fc domain of these antibodies. These Fc-mediated effector functions are often missed in clinical assays, which are used for example to define correlates of protection induced by vaccines. The use of antibodies to prevent and treat infectious diseases is on the rise, and has proven a promising approach in our battle against newly emerging viral infections. It is now also realized that Fc receptors significantly enhance the in vivo protective effect of broadly neutralizing antibodies directed against the conserved parts of the influenza virus hemagglutinin. We show here that two M2e-specific monoclonal antibodies with close to identical antigen- binding specificity and affinity have a very different in vivo protective potential that is controlled by their capacity to interact with activating Fc receptors.

  • A cytoplasmic RNA virus alters the function of the cell splicing protein SRSF2 [PublishAheadOfPrint]

  • To replicate efficiently, viruses must create favorable cell conditions and overcome cell antiviral responses. We previously reported that the reovirus protein mmu;2 from strain T1L, but not strain T3D, represses one antiviral response: interferon-aalpha;/bbeta; (IFN-bbeta;) signaling. We report here that T1L but not T3D mmu;2 localizes to nuclear speckles where it forms a complex with the mRNA splicing factor SRSF2 and alters its sub-nuclear localization. Reovirus replicates in cytoplasmic viral factories and there is no evidence that reovirus genomic or messenger RNAs are spliced, suggesting that T1L mmu;2 might target splicing of cell RNAs. Indeed, RNA-seq revealed that reovirus T1L but not T3D infection alters the splicing of transcripts for host genes involved in mRNA post-transcriptional modifications. Moreover, depletion of SRSF2 enhanced reovirus replication and cytopathic effect, suggesting that T1L mmu;2 modulation of splicing benefits the virus. This provides the first report of viral antagonism of the splicing factor SRSF2 and identifies the viral protein that determines strain-specific differences in cell RNA splicing.

    IMPORTANCE Efficient viral replication requires that the virus create favorable cell conditions. Many viruses accomplish this by repressing specific antiviral responses. Here we demonstrate that some mammalian reoviruses, RNA viruses that replicate strictly in the cytoplasm, express a protein variant that localizes to nuclear speckles where it targets a cell mRNA splicing factor. Infection with a reovirus strain that targets this splicing factor alters splicing of cell mRNAs involved in the maturation of many other cell mRNAs. Depletion of this cell splicing factor enhances reovirus replication and cytopathic effect. Results provide the first evidence of viral antagonism of this splicing factor and suggest that downstream consequences to the cell are global and benefit the virus.

  • HIV controllers exhibit enhanced frequencies of MHC class II tetramer+ Gag-specific CD4+ T cells in chronic clade C HIV-1 infection [PublishAheadOfPrint]

  • Immune control of viral infections is heavily dependent on helper CD4+ T cell function. However, understanding the contribution of HIV-specific CD4+ T cell responses to immune protection against HIV-1, particularly in clade C infection remains incomplete. Recently, MHC class II tetramers have emerged as a powerful tool for interrogating antigen specific CD4+ T cells without relying on effector functions. Here, we defined the MHC class II alleles for immunodominant Gag CD4+ T cell epitopes in clade C virus infection, constructed MHC class II tetramers, and then used these to define the magnitude, function, and relation to viral load of HIV-specific CD4+ T cell responses in a cohort of untreated HIV clade C infected persons. We observed significantly higher frequencies of MHC class II tetramer+ CD4+ T cells in HIV controllers compared to progressors (p=0.0001) and these expanded Gag-specific CD4+ T cells in HIV controllers showed higher expression of the cytolytic proteins, Granzymes A and B. Importantly, targeting of the immunodominant Gag41 peptide in the context of HLA class II DRB1*1101 was associated with HIV control (r=-0.5, p=0.02). These data identify an association between HIV-specific CD4+ T cell targeting of immunodominant Gag epitopes and immune control, particularly the contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infection. Furthermore, these results highlight the advantage of class II tetramers in evaluating HIV-specific CD4+ T cell responses in natural infections.

    IMPORTANCE Increasing evidence suggests that virus-specific CD4+ T cells contribute to immune-mediated control of clade B HIV-1 infection. Yet, there remains a relative paucity of data regarding the role of HIV-specific CD4+ T cells in shaping adaptive immune responses in clade C infection, which is responsible for majority of HIV infections worldwide. Understanding the contribution of HIV-specific CD4+ T cell responses in clade C infection is particularly important for developing vaccines that would be efficacious in sub-Saharan Africa, where clade C infection is dominant. Here, we employed MHC class II tetramers designed to immunodominant Gag epitopes and used them to characterize CD4+ T cell responses in HIV-1 clade C infection. Our results demonstrate an association between the frequency of HIV-specific CD4+ T cell responses targeting an immunodominant DRB1*11-Gag41 complex and HIV viral control, highlighting the important contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infections.

  • Klebsiella phage {Phi}K64-1 encodes multiple depolymerases for multiple host capsular types [PublishAheadOfPrint]

  • The genome of the multi-host bacteriophage K64-1, capable of infecting Klebsiella capsular types K1, K11, K21, K25, K30, K35, K64, and K69, as well as new capsular types KN4 and KN5 was analyzed and revealed that 11 genes (S1-1, S1-2, S1-3, S2-1, S2-2, S2-3, S2-4, S2-5, S2-6, S2-7, and S2-8) encode proteins with amino acid sequence similarity to tail fibers/spikes or lyases. S2-5 previously was shown to encode a K64 capsule depolymerase (K64dep). Specific capsule-degrading activities of an additional 8 putative capsule depolymerases (S2-4 against K1; S1-1 against K11; S1-3 against K21; S2-2 against K25; S2-6 against K30/K69; S2-3 against K35; S1-2 against KN4; S2-1 against KN5) was demonstrated by expression and purification of the recombinant proteins. Consistent with the capsular type-specific depolymerization activity of these gene products, phage mutant of S1-2, S2-2, S2-3, or S2-6 lost infectivity for KN4, K25, K35, or K30/K69, respectively, indicating that capsule depolymerase is crucial for infecting specific hosts. In conclusion, we identified 9 functional capsule depolymerase-encoding genes in a bacteriophage and correlated activities of the gene products to all 10 hosts of this phage, providing an example of type-specific host infection mechanisms in a multi-host bacteriophage.

    IMPORTANCE We currently identified 8 novel capsule depolymerases in a multi-host Klebsiella bacteriophage and correlated activities of the gene products to all hosts of this phage, providing an example of carriage of multiple depolymerases in a phage with a wide capsular type host spectrum. Besides, we also established a recombineering system for modification of Klebsiella bacteriophage genomes and demonstrated the importance of capsule depolymerase for infecting specific hosts. Based on the powerful tool for modification of phage genome, further studies can be conducted to improve the understanding of mechanistic details of Klebsiella phage infection. Furthermore, the newly identified capsule depolymerases will be of great value for applications in capsular typing.

  • Herpes Simplex Virus 1 Abrogates cGAS/STING-Mediated Cytosolic DNA-sensing Pathway via Its Virion Host Shutoff Protein UL41 [PublishAheadOfPrint]

  • Cyclic GMP-AMP synthase (cGAS) is a key DNA sensor capable of detecting microbial DNA and activating the adaptor protein stimulator of interferon genes (STING), leading to interferon (IFN) production and host antiviral responses. Cells exhibited reduced type I IFN production in response to cytosolic DNA in the absence of cGAS. Although cGAS/STING-mediated DNA-sensing signal is crucial for host defense against many viruses, especial for DNA viruses, few viral components have been identified to specifically target this signaling pathway. Herpes Simplex Virus 1 (HSV-1) is a DNA virus that has evolved multiple strategies to evade host immune responses. In the present study, we found that HSV-1 tegument protein UL41 was involved in counteracting the cGAS/STING-mediated DNA-sensing pathway. Our results showed that wild-type (WT) HSV-1 infection could inhibit immunostimulatory DNA induced activation of IFN signaling pathway compared with the UL41-null mutant virus (R2621), and ectopic expression of UL41 decreased cGAS/STING-mediated IFN-bbeta; promoter activation and IFN-bbeta; production. Further study indicated that UL41 reduced the accumulation of cGAS to abrogate host recognition of viral DNA. In addition, stably knockdown of cGAS facilitated the replication of R2621, but not WT HSV-1. For the first time, HSV-1 UL41 was demonstrated to evade cGAS/STING-mediated DNA-sensing pathway by degrading cGAS via its RNase activity.

    IMPORTANCE HSV-1 is well known for its ability to evade host antiviral responses and establish a lifelong latent infection whereas triggering reactivation and lytic infection under stress. Currently, whether HSV-1 evades the cytosolic DNA sensing and signaling are still poorly understood. In the present study, we found that tegument protein UL41 targeted cGAS/STING-mediated cellular DNA sensing pathway by selectively degrading cGAS mRNA. Knockdown of endogenous cGAS could facilitate the replication of R2621 but not WT HSV-1. Furthermore, UL41 is shown for the first time to act directly on cGAS. Findings in this study could provide new insights into the host-virus interaction and help develop new antiviral approaches against HSV-1.

  • Human Papillomavirus 16 E6 Up-regulates APOBEC3B via the TEAD Transcription Factor [PublishAheadOfPrint]

  • The cytidine deaminase APOBEC3B (A3B) underlies the genetic heterogeneity of several human cancers including cervical cancer, which is caused by human papillomavirus (HPV) infection. We previously identified a region within the A3B promoter that is activated by the viral protein HPV16 E6 in human keratinocytes. Here, we discovered three sites recognized by the TEAD family of transcription factors within this region of the A3B promoter. Reporter assays in HEK293 cells showed that exogenously expressed TEAD4 induced A3B promoter activation through binding to these sites. Normal immortalized human keratinocytes expressing E6 (NIKS-E6) displayed increased levels of TEAD1/4 protein compared to parental NIKS. A series of E6 mutants revealed that E6-mediated degradation of p53 was important for increasing TEAD4 levels. Knockdown of TEADs in NIKS-E6 significantly reduced A3B mRNA levels, whereas ectopic expression of TEAD4 in NIKS increased A3B mRNA levels. Finally, chromatin immunoprecipitation assays demonstrated increased levels of TEAD4 binding to the A3B promoter in NIKS-E6 compared to NIKS. Collectively, these results indicate that E6 induces up-regulation of A3B through increased levels of TEADs, highlighting the importance of the TEAD-A3B axis in carcinogenesis.

    IMPORTANCE The expression of APOBEC3B (A3B), a cellular DNA cytidine deaminase, is up-regulated in various human cancers and leaves characteristic, signature mutations in cancer genomes, suggesting that it plays a prominent role in carcinogenesis. Viral oncoproteins encoded by human papillomavirus (HPV) and polyomavirus have been reported to induce A3B expression, implying the involvement of A3B up-regulation in virus-associated carcinogenesis. However, the molecular mechanisms causing A3B up-regulation remain unclear. Here, we demonstrate that exogenous expression of the cellular transcription factor TEAD activates the A3B promoter. Further, the HPV oncoprotein E6 increases the levels of endogenous TEAD1/4 protein, thereby leading to A3B up-regulation. Since increased levels of TEAD4 are frequently observed in many cancers, an understanding of the direct link between TEAD and A3B up-regulation is of broad oncological interest.

  • TCR Clonotype-specific Differences in Inhibitory Activity of HIV-1 Cytotoxic T Cell Clones is Not Mediated by TCR Alone [PublishAheadOfPrint]

  • Functional analysis of T cell responses in HIV-infected individuals has indicated that virus-specific CD8+ T cells with superior antiviral efficacy are well represented in HIV-1 controllers but are rare or absent in HIV-1 progessors. To define the role of individual TCR clonotypes in differential antiviral CD8+ T cell function, we performed detailed functional and mass cytometric cluster analysis of multiple CD8+ T cell clones recognizing the identical HLA-B*2705-restricted HIV-1 epitope KK10 (KRWIILGLNK). Effective and ineffective CD8+ T cell clones segregated based on responses to HIV-1-infected and peptide-loaded target cells. Following cognate peptide stimulation, effective HIV-specific clones displayed significantly more rapid TCR signal propagation, more efficient initial lytic granule release and more sustained non-lytic cytokine and chemokine secretion compared to ineffective clones. To evaluate TCR clonotype contribution to CD8+ T cell function, we cloned the TCR aalpha; and bbeta; chain genes from one effective and two ineffective CD8+ T cell clones from an elite controller into TCR-expressing lentivectors. We show that Jurkat/MA cells and primary CD8+ T cells transduced with lentivirus expressing TCR from one of the ineffective clones exhibited comparable activation by cognate peptide as the effective clonotype, and comparable inhibition of in vitro HIV-1 infection, respectively. Taken together, these data suggest that the potent antiviral capacity of some HIV-specific CD8+ T cells is a consequence of factors in addition to TCR sequence that modulate functionality and contribute to the increased antiviral capacity of HIV-specific CD8+ T cells in elite controllers to inhibit HIV infection.

    IMPORTANCE The greater ex vivo antiviral inhibitory activity of CD8+ T cells from elite controllers as compared to those from HIV-1 progessors supports the crucial role of effective HIV-specific CD8+ T cells in controlling HIV-1 replication. The contribution of TCR clonotype to inhibitory potency was investigated by delineating the responsiveness of effective and ineffective CD8+ T cell clones recognizing the identical HLA-B*2705-restricted HIV-1 Gag-derived peptide, KK10 (KRWIILGLNK). KK10-stimulated "effective" CD8+ T cell clones displayed significantly more rapid TCR signal propagation, initial lytic granule release and sustained cytokine and chemokine secretion compared to "ineffective" CD8+ T cell clones. However, TCRs cloned from an effective and one of two ineffective clones conferred primary CD8+ T cells with the equivalent potent capacity to inhibit HIV-1 infection. Taken together, these data suggest that other factors aside from intrinsic TCR:peptide-MHC complex reactivity can contribute to the potent antiviral capacity of some HIV-specific CD8+ T cell clones.

  • Viral RNA at two stages of reovirus infection is required for the induction of necroptosis [PublishAheadOfPrint]

  • Necroptosis, a regulated form of necrotic cell death requires the activation of the RIP3 kinase. Here, we identify that infection of host cells with reovirus can result in necroptosis. We find that necroptosis requires sensing of the genomic RNA within incoming virus particles via cytoplasmic RNA sensors to produce type I IFN. While these events that occur prior to de novo synthesis of viral RNA are required for induction of necroptosis, they are not sufficient. Induction of necroptosis also requires late stages of reovirus infection. Specifically, efficient synthesis of dsRNA within infected cells is required for necroptosis. These data indicate that viral RNA interfaces with host components at two different stages of infection to induce necroptosis. This work provides new molecular details about events in the viral replication cycle that contribute to the induction of necroptosis following infection with an RNA virus.

    IMPORTANCE An appreciation of how cell death pathways are regulated following viral infection may reveal strategies to limit tissue destruction and prevent the onset of disease. Cell death following virus infection can occur by apoptosis or a regulated form of necrosis, known as necroptosis. Apoptotic cells are typically disposed of without activating the immune system. In contrast, necroptotic cells alert the immune system, resulting in inflammation and tissue damage. While apoptosis following virus infection has been extensively investigated, how necroptosis is unleashed following virus infection is only understood for a small group of viruses. Here, using mammalian reovirus, we highlight the molecular mechanism by which infection with a dsRNA virus results in necroptosis.

  • Role of Litopenaeus vannamei Yin Yang 1 in the regulation of the white spot syndrome virus immediate-early gene ie1 [PublishAheadOfPrint]

  • Yin Yang 1 (YY1) is a multifunctional zinc-finger transcription factor that regulates many key cellular processes. In this study, we report the cloning of YY1 from shrimp, Litopenaeus vannamei, (LvYY1). The study shows that LvYY1 is ubiquitously expressed in shrimp tissues, and knockdown of LvYY1 expression by dsRNA injection in WSSV-infected shrimp reduced both mRNA levels of the WSSV immediate-early gene, ie1, as well as overall copy numbers of the WSSV genome. The cumulative mortality of infected shrimp also declined with LvYY1 dsRNA injection. Using an insect cell model, we observed that LvYY1 activates ie1 expression, and a mutation introduced into the ie1 promoter subsequently repressed this capability. Moreover, reporter assay results suggested that LvYY1 is involved in basal transcriptional regulation via an interaction with L. vannamei TATA-binding protein, (LvTBP). Electrophoretic mobility shift assay (EMSA) results further indicated that LvYY1 binds to a YY1-binding site in the region between -119 to -126 in the ie1 promoter. Chromatin immunoprecipitation analysis also confirmed that LvYY1 binds to the ie1 promoter in WSSV-infected shrimp. Taken together, these results indicate that WSSV uses host LvYY1 to enhance ie1 expression via a YY1-binding site and the TATA box in the ie1 promoter, thereby facilitating lytic activation and viral replication.

    IMPORTANCE WSSV has long been a scourge of the shrimp industry, and remains a serious global threat. Thus, there is a pressing need to understand how the interactions between WSSV and its host drive infection, lytic development, pathogenesis, and mortality. Our successful cloning of L. vannamei YY1 (LvYY1) led to the elucidation of a critical viral-host interaction between LvYY1 and the WSSV immediate-early gene, ie1. We observed that LvYY1 regulates ie1 expression via a consensus YY1-binding site and TATA box. LvYY1 was also found to interact with L. vannamei TATA-binding protein (LvTBP), which may have an effect on basal transcription. Knockdown of LvYY1 expression inhibited ie1 transcription, and subsequently reduced viral DNA replication and decreased cumulative mortality in WSSV-infected shrimp. These findings are expected to contribute to future studies involving WSSV host interactions.

  • SUN2 silencing impairs CD4 T cell proliferation and alters sensitivity to HIV-1 infection independently of Cyclophilin A [PublishAheadOfPrint]

  • Linker of nucleoskeleton and cytoskeleton (LINC) complexes connect the nucleus to the cytoskeleton in eukaryotic cells. We previously reported that overexpression of SUN2, an inner nuclear membrane protein and LINC complex component, inhibits HIV infection between reverse transcription and nuclear import in a capsid-specific manner. We also reported that SUN2 silencing does not modulate HIV infection in several cell lines. Silencing of SUN2 was recently reported to decrease HIV infection of CD4 T cells, an effect suggested to result from modulation of CypA-dependent steps of HIV infection. We confirm here that HIV infection of primary CD4 T cells is compromised in the absence of endogenous SUN2, and we extend these findings to additional viral strains. However, we find that CypA is not required for the decreased infection observed in SUN2 silenced cells, and conversely, that endogenous SUN2 is not required for the well-documented positive modulation of HIV infection by CypA. In contrast, CD4 T cells lacking SUN2 exhibit a considerable defect in proliferative capacity, and display reduced levels of activation markers and decreased viability. Additionally, SUN2-silenced CD4 T cells that do become infected support reduced levels of viral protein expression. Our results demonstrate that SUN2 is required for optimal activation and proliferation of primary CD4 T cells, and suggest that disruption of these processes explains the contribution of endogenous SUN2 to HIV infection in primary lymphocytes.

    IMPORTANCE Linker of nucleoskeleton and cytoskeleton (LINC) complexes connect the nucleus to the cytoskeleton. We previously reported that overexpression of the LINC complex protein SUN2 inhibits HIV infection, by targeting the viral capsid and blocking infection before the virus enters the nucleus. A recent report showed that depletion of endogenous SUN2 in primary CD4 T cells results in decreased HIV infection, and that this involves Cyclophilin A (CypA), a host protein that interacts with the capsid of HIV to promote infection. We confirm that HIV infection is reduced in CD4 T cells lacking SUN2, but we find no role for CypA. Instead, SUN2 silencing results in CD4 T cells with decreased viability and much lower proliferation rates. Our results show that SUN2 is required for optimal CD4 T cell activation and proliferation, and explain the reduced level of HIV infection in the absence of SUN2.

  • Ultrasensitive HIV-1 p24 detects single infected cells and differences in reservoir induction by latency-reversal agents [PublishAheadOfPrint]

  • The existence of HIV reservoirs in infected individuals under cART represents a major obstacle towards cure. Viral reservoirs are assessed by quantification of HIV nucleic acids, which does not discriminate between infectious and defective viruses, or by viral outgrowth assays, which requires large number of cells and long-term cultures. Here, we used an ultrasensitive p24 digital assay, which we report to be 1000 fold more sensitive than classical ELISA in the quantification of HIV-1 Gag p24 production in HIV-infected individuals samples. Results from ultrasensitive p24 were compared to conventional viral RNA RT-qPCR based assays, and outgrowth assays readout by flow cytometry. Using serial dilutions and flow-based single cell sorting, we show that viral proteins produced by a single infected cell can be detected by ultrasensitive p24. This unique sensitivity allowed the early (as soon as day 1 in 49% of cases) and more efficient detection and quantification of p24 in PHA-stimulated CD4+ T cells from individuals under effective cART. When testing seven different classes of latency reversal agents (LRA) in resting CD4+ T cells from HIV-infected individuals, ultrasensitive p24 revealed differences in the extent of HIV reactivation. Of note, HIV RNA production was infrequently accompanied by p24 protein production (19%). Among the drugs tested, prostratin showed a superior capacity in inducing viral protein production. In summary, the ultrasensitive p24 assay allows the detection and quantification of p24 produced by single infected-CD4+ T cells and provides a unique tool to assess early reactivation of infectious virus from reservoirs in HIV-infected individuals.

    IMPORTANCE The persistence of HIV reservoirs in infected individuals under effective antiretroviral treatment represents a major obstacle towards cure. Different methods to estimate HIV reservoirs exist, but there is currently no optimal assay to measure HIV reservoirs in HIV-eradication interventions. In the present study we report an ultrasensitive digital ELISA platform for quantification of the HIV-1 protein p24. This method was employed to assess the early reactivation of infectious virus from reservoirs in HIV-1 infected individuals.

    We found that viral proteins produced by a single infected cell can be detected by ultrasensitive p24. This unprecedented resolution showed major advantages in comparison to other techniques currently used to assess viral replication in reactivation studies. In addition, such highly sensitive assays allow discrimination of drug-induced reactivation of productive HIV based on protein expression. The present study heralds new opportunities to evaluate the HIV reservoir and the efficacy of drugs used to target it.

  • Characterization of HCV envelope diversification from acute to chronic infection using SMRT sequencing within a sexually-transmitted hepatitis C virus cluster [PublishAheadOfPrint]

  • In contrast to other available next generation sequencing platforms, Pacbio Single Molecule, Real-Time (SMRT) sequencing has the advantage of generating long reads, albeit with a relatively higher error rate in unprocessed data. Using this platform we longitudinally sampled and sequenced the hepatitis C virus (HCV) envelope genome region (1680 nt) from individuals belonging to a cluster of sexually-transmitted cases. All five subjects were HIV-1 coinfected and infected with a closely related strain of HCV genotype 4d. In total 50 samples were analyzed using SMRT sequencing. By using 7 passes of circular consensus sequencing the error rate was reduced to 0.37% and the median number of sequences was 612 per sample. Further reduction of insertions was achieved by aligning against a sample-specific reference sequence. However, in vitro recombination during PCR amplification could not be excluded. Phylogenetic analysis supported close relationships among HCV sequences from the four male subjects and the subsequent transmission from one subject to his female partner. Transmission was characterized by a strong genetic bottleneck. Viral genetic diversity was low during acute infection, increased upon progression to chronicity, but subsequently fluctuated during chronic infection, caused by alternate detection of distinct co-existing lineages. SMRT sequencing combines long reads with sufficient depth for many phylogenetic analyses, and can therefore provide insights into within-host HCV evolutionary dynamics without the need for haplotype reconstruction using statistical algorithms.

    IMPORTANCE Next generation sequencing has revolutionized the study of genetically variable RNA virus populations, but for phylogenetic and evolutionary analyses longer sequences than generated by most available platforms are desired, while minimizing the intrinsic error rate. Here, we demonstrate for the first time that Pacbio SMRT sequencing technology can be used to generate full-length HCV envelope sequences at the single molecule level, providing a dataset with large sequencing depth for characterization of intra-host viral dynamics. Selecting consensus reads derived from at least 7 full circular consensus sequencing rounds significantly reduced the intrinsic high error rate of this method. We used this method to genetically characterize a unique transmission cluster of sexually transmitted HCV infections, providing insight in the distinct evolutionary pathways in each patient over time, identifying the transmission-associated genetic bottleneck, as well as fluctuations in viral genetic diversity over time, accompanied by dynamic shifts in viral subpopulations.

  • A conserved glycine residue is required for proper functioning of a baculovirus VP39 protein [PublishAheadOfPrint]

  • The baculovirus VP39 is a major nucleocapsid protein essential for viral propagation. However, the critical domains or residues of the VP39 protein have not yet been identified. Here, we performed mutagenesis experiments of Bombyx mori nucleopolyhedrovirus (BmNPV) using 5-bromo-2rrsquo; -deoxyuridine and isolated a BmNPV mutant that produced fewer occlusion bodies than the wild-type virus. This mutant also produced fewer infectious budded viruses (BVs), when compared with the wild-type virus in both cultured cells and B. mori larvae. Marker rescue experiments using genomic libraries identified a single nucleotide mutation in the vp39 gene. This mutation resulted in an amino acid substitution at glycine 276 (Gly-276) to serine, which was required for all the defective phenotypes observed in the mutant. Sequence comparison revealed that this residue is completely conserved among VP39 proteins of sequenced alphabaculoviruses, betabaculoviruses, and gammabaculoviruses. Although early viral gene expression was not significantly affected, expression of a late gene, vcath, was reduced. In addition, both of the very late genes were markedly downregulated in cells infected with this mutant. Western blot and qPCR analyses revealed that BVs produced from cells infected with this mutant contained lower amounts of VP39 protein and viral genomic DNA than those from wild-type virus-infected cells. Combined with the results of transmission electron microscopy, VP39 Gly-276 can be concluded to be essential for correct nucleocapsid assembly, viral DNA packaging, and viral gene expression, especially of very late genes.

    IMPORTANCE The major nucleocapsid protein gene vp39 is one of the most well-known baculovirus genes. Although several viral and host proteins that interact with the VP39 protein have been identified, the functionally important domains or residues of this protein remain unknown. The present study revealed that the glycine residue at 276, which is completely conserved among sequenced alphabaculoviruses, betabaculoviruses, and gammabaculoviruses, is important for the VP39 function, i.e., structural assembly of nucleocapsids and viral DNA packaging. Moreover, our results provide evidence for the link between nucleocapsid formation and transcription of viral very late genes.

  • Exosome mediated intercellular communication between hepatitis C virus infected hepatocytes and hepatic stellate cells [PublishAheadOfPrint]

  • Fibrogenic pathways in the liver are principally regulated by activation of hepatic stellate cells (HSC). Fibrosis is associated with chronic hepatitis C virus (HCV) infection, although the mechanism is poorly understood. HSC comprise the major population of the non-parenchymal cells in the liver. Since HCV does not replicate in HSC, we hypothesized that exosomes secreted from HCV-infected hepatocytes activate HSC. Primary or immortalized human hepatic stellate cells (LX2) were exposed to exosomes derived from HCV-infected hepatocytes (HCV-exo) and the expression of fibrosis related genes was examined. Our results demonstrated that HCV-exo internalized to HSC and increased expression of profibrotic markers. Further analysis suggested that HCV-exo carry miR-19a and targets SOCS3 in HSC, which in turn activates STAT3 mediated TGF-bbeta; signaling pathway and enhances fibrosis marker genes. The higher expression of miR-19a in exosomes was also observed from HCV-infected hepatocytes and in serum of chronic HCV patients with fibrosis as compared to healthy volunteers and non-HCV related liver disease patients with fibrosis. Together, our results demonstrated that the miR-19a carried through the exosomes from HCV-infected hepatocytes activates HSC by modulating SOCS-STAT3 axis. Our results implicated a novel mechanism of exosome mediated intercellular communication in the activation of HSC for liver fibrosis in HCV infection.

    IMPORTANCE HCV associated liver fibrosis is a critical step for end stage liver disease progression. However, the molecular mechanisms for hepatic stellate cell activation by HCV-infected hepatocytes are underexplored. Here, we provide a role of miR-19a carried through the exosomes for intercellular communication between HCV-infected hepatocytes and HSC in fibrogenic activation. Furthermore, we demonstrated the role of exosomal miR-19a in activation of STAT3-TGF-bbeta; pathway in HSC. This study contributes to the understanding of intercellular communication in the pathogenesis of liver disease during HCV infection.

  • Efficient Vpu-mediated tetherin antagonism by an HIV-1 group O strain [PublishAheadOfPrint]

  • Simian immunodeficiency viruses (SIVs) use their Nef proteins to counteract the restriction factor tetherin. However, a deletion in human tetherin prevents antagonism by the Nef proteins of SIVcpz and SIVgor, which represent the ape precursors of human immunodeficiency virus type 1 (HIV-1). To promote virus release from infected cells, pandemic HIV-1 group M strains evolved Vpu as tetherin antagonist, while the Nef protein of less widespread HIV-1 group O strains acquired the ability to target a region adjacent to this deletion. Here, we identified an unusual HIV-1 group O strain (RBF206) that evolved Vpu as an effective antagonist of human tetherin. While both RBF206 Vpu and Nef exert anti-tetherin activity in transient transfection assays, mainly Vpu promotes RBF206 release in infected CD4+ T cells. Although mutations distinct from the adaptive changes observed in group M Vpus were critical for the acquisition of its anti-tetherin activity, the RBF206 O-Vpu potently suppresses NF-B activation and reduces CD4 cell surface expression. Interestingly, the RBF206 Vpu counteracts tetherin in a largely species-independent manner degrading both the long and short isoform of human tetherin. Downmodulation of CD4, but not counteraction of tetherin, by RBF206 Vpu was dependent on the cellular ubiquitin ligase machinery. Our data present a first example of an HIV-1 group O Vpu that efficiently antagonizes human tetherin and suggest that counteraction by O-Nefs may be suboptimal.

    IMPORTANCE: Previous studies showed that HIV-1 groups M and O evolved two alternative strategies to counteract the human ortholog of the restriction factor tetherin. While HIV-1 group M switched from Nef to Vpu due to a deletion in the cytoplasmic domain of human tetherin, HIV-1 group O, which lacks Vpu-mediated anti-tetherin activity, acquired a Nef protein that is able to target a region adjacent to the deletion. Here, we report an unusual exception, identifying a strain of HIV-1 group O (RBF206), whose Vpu protein evolved an effective antagonism of human tetherin. Interestingly, the adaptive changes in RBF206 Vpu are distinct from those found in M-Vpus and mediate efficient counteraction of both the long and short isoform of this restriction factor. Our results further illustrate the enormous flexibility of HIV-1 in counteracting human defense mechanisms.

  • Chikungunya, Influenza, Nipah, and Semliki Forest Chimeric Viruses with Vesicular Stomatitis Virus: Actions in the Brain. [PublishAheadOfPrint]

  • Recombinant vesicular stomatitis virus (VSV)-based chimeric viruses that include genes from other viruses show promise as vaccines and oncolytic viruses. However, the critical safety concern is the neurotropic nature conveyed by the VSV glycoprotein. VSVs that include the VSV glycoprotein gene, even in most recombinant attenuated strains, can still show substantial adverse or lethal actions in the brain. Here we test 4 chimeric viruses in the brain including those in which glycoprotein genes from Nipah, chikungunya, or influenza H5N1 viruses were substituted for the VSV glycoprotein gene. We also test a virus-like vesicle (VLV) in which the VSV glycoprotein gene is expressed from a replicon encoding the non-structural proteins of Semliki Forest virus. VSVG-CHIKV, VSVG-H5N1, and VLVs were all safe in the adult mouse brain, as were VSVG viruses expressing either the Nipah F or G glycoproteins. In contrast, a complementing pair of VSVG viruses expressing Nipah G and F glycoproteins were lethal within the brain within a surprisingly rapid time frame of 2 days. Intranasal inoculation in postnatal day 14 mice with VSVG-CHIKV or VLV evoked no adverse response, whereas VSVG-H5N1 by this route was lethal in most mice. A key immune mechanism underlying the safety of VSVG-CHIKV, VSVG-H5N1, and VLV in the adult brain was the type 1 interferon response; all three were lethal in the brains of adult mice lacking the interferon receptor, suggesting that the viruses can infect, replicate, and spread in brain cells if not blocked by interferon stimulated genes within the brain.

    IMPORTANCE Vesicular stomatitis virus (VSV) shows considerable promise both as a vaccine vector, and as an oncolytic virus. The greatest limitation of VSV is that is a highly neurotropic, and can be lethal within the brain. The neurotropism can be mostly attributed to the VSV G glycoprotein. Here we test 4 chimeric viruses of VSV with glycoprotein genes from Nipah, Chikungunya, influenza, and non-structural genes from Semliki Forest Virus. Two of the four, VSVG-CHIKV and VLV, show substantially attenuated neurotropism and were safe in the normal adult mouse brain. VSVG-H5N1 was safe in the adult brain, but lethal in the younger brain. VSVG NipahF+G was even more neurotropic than wild type VSV, evoking a rapid lethal response in the adult brain. These results suggest that while chimeric VSVs show promise, each must be tested with both intranasal and intracranial administration to ensure the absence of lethal neurotropism.

  • MAVS is essential for primary CD4+ T cell immunity but not for recall T cell responses following an attenuated West Nile virus infection [PublishAheadOfPrint]

  • The use of pathogen recognition receptor (PRR) agonists and the molecular mechanisms involved have been the major focus of research in individual vaccine development. West Nile virus (WNV) nonstructural (NS) 4B-P38G mutant has several features for an ideal vaccine candidate, including significantly reduced neuroinvasiveness, induction of strong adaptive immunity, and protection of mice from wild-type (WT) WNV infection. Here, we determined the role of mitochondrial antiviral-signaling (MAVS), the adaptor protein for RIG-I like receptor in regulating host immunity against the NS4B-P38G vaccine. We found that Mavs-/- mice were more susceptible to NS4B-P38G priming than WT mice. Mavs-/- mice had a transiently reduced production of antiviral cytokines, and an impaired CD4+ T cell response in peripheral organs. However, antibody and CD8+ T cell responses were minimally affected. NS4B-P38G induced lower type I interferon (IFN), IFN stimulating gene, and proinflammatory cytokine responses in Mavs-/- dendritic cells, and subsequently compromised antigen presenting capacity for CD4+ T cells. Interestingly, Mavs-/- mice surviving NS4B-P38G priming were all protected from a lethal WT WNV challenge. NS4B-P38G- primed Mavs-/- mice exhibited equivalent levels of protective CD4+ T cell recall response, a modestly reduced WNV-specific IgM production, but more robust CD8+ T cell recall response. Taken together, our results suggest that MAVS is essential for boosting optimal primary CD4+ T cell responses upon NS4B-P38G vaccination, yet is dispensable for host protection and recall T cell responses during secondary WT WNV infection.

    IMPORTANCE The production of innate cytokines induced by the recognition of pathogen recognition receptor (PRR)s via their cognate ligands are critical for enhancing antigen presenting cell functions and influencing T cell responses during microbial infection. The use of PRR agonists and the underlying molecular mechanisms have been the major focus in individual vaccine development. Here, we determined the role of mitochondrial antiviral-signaling (MAVS), the adaptor protein for RIG-I like receptor in regulating host immunity against the live attenuated West Nile virus (WNV) vaccine strain- the nonstructural (NS) 4B-P38G mutant. We found that MAVS is important for boosting optimal primary CD4+ T cell response during NS4B-P38G vaccination. However, MAVS is dispensable for memory T cell development and host protection during secondary wild-type WNV infection. Overall, these results may be utilized as a paradigm to aid in the rational development of other efficacious live attenuated flavivirus vaccines.

  • Expression of Ifnlr1 on intestinal epithelial cells is critical to the antiviral effects of IFN-lambda against norovirus and reovirus [PublishAheadOfPrint]

  • Interferon-lambda (IFN-) has potent antiviral effects against multiple enteric viral pathogens, including norovirus and rotavirus, in both preventing and curing infection. Because the intestine includes a diverse array of cell types, however, the cell(s) upon which IFN- acts to exert its antiviral effects are unclear. Here, we sought to identify IFN- responsive cells by generation of mice with lineage-specific deletion of the receptor for IFN-, Ifnlr1. We found that expression of IFNLR1 on intestinal epithelial cells (IECs) in the small intestine and colon is required for enteric IFN- antiviral activity. IEC Ifnlr1 expression also determines the efficacy of IFN- in resolving persistent murine norovirus (MNoV) infection and regulates fecal shedding and viral titers in tissue. Expression of Ifnlr1 by IECs is thus necessary for the response to both endogenous and exogenous IFN-. We further demonstrate that IEC Ifnlr1 expression is required for the sterilizing innate immune effects of IFN- by extending these findings in Rag1-deficient mice. Finally, we assessed whether our findings pertained to multiple viral pathogens by infecting mice specifically lacking IEC Ifnlr1 expression with reovirus. These mice phenocopied Ifnlr1-null animals, exhibiting increased intestinal tissue titers and enhanced reovirus fecal shedding. IECs are thus the critical cell type responding to IFN- to control multiple enteric viruses. This is the first genetic evidence that supports an essential role for IECs in IFN--mediated control of enteric viral infection, and these findings thus provide insight into the mechanism of IFN--mediated antiviral activity.

    IMPORTANCE Human noroviruses (HNoVs) are the leading cause of epidemic gastroenteritis worldwide. Type III interferons (IFN-) control enteric viral infections in the gut, and have been shown to cure mouse norovirus, a small animal model for HNoVs. Using a genetic approach with conditional knock-out mice, we identified intestinal epithelial cells (IECs) as the dominant IFN--responsive cells in control of enteric virus infection in vivo. Upon murine norovirus or reovirus infection, Ifnlr1 depletion in IECs largely recapitulated the phenotype seen in Ifnlr1-/- mice of higher intestinal tissue viral titers and increased viral shedding in the stool. Moreover, IFN--mediated sterilizing immunity against murine norovirus requires the capacity of IECs to respond to IFN-. These findings clarify the mechanism of action of this cytokine, and emphasize the therapeutic potential of IFN- for treating mucosal viral infections.

  • Neutralization of Diverse Human Cytomegalovirus Strains Conferred by Antibodies Targets Viral gH/gL/pUL128-131 Pentameric Complex [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection and developing a prophylactic vaccine is of high priority to public health. We recently reported a replication-defective human cytomegalovirus with restored pentameric complex gH/gL/pUL128-131 for prevention of congenital HCMV infection. While the quantity of vaccine-induced antibody responses can be measured in a viral neutralization assay, assessing the quality of such responses, including the ability of vaccine-induced antibodies to cross neutralize the field strains of HCMV, remains a challenge. In this study, with a panel of neutralizing antibodies from three healthy human donors with natural HCMV infection or a vaccinated animal, we mapped eight sites on the dominant viral neutralizing antigen nndash; the pentameric complex of glycoprotein H (gH), gL and pUL128-130-131. By evaluating the site-specific antibodies in vaccine immune sera, we demonstrated that vaccination elicited functional antiviral antibodies to multiple neutralizing sites in rhesus macaques, with quality attributes comparable to CMV hyperimmune globulin. Furthermore, these immune sera showed antiviral activities against a panel of genetically distinct HCMV clinical isolates. These results highlighted the importance of understanding the quality of vaccine-induced antibody responses, which includes not only the neutralizing potency in key cell-types, but also the ability to protect against the genetically diverse field-strains.

    Significance Statement HCMV is the leading cause of congenital viral infection, and development of a preventive vaccine is a high public health priority. To understand the strain coverage of vaccine-induced immune responses in comparison with natural immunity, we used a panel of broadly neutralizing antibodies to identify the immunogenic sites of a dominant viral antigen mmdash; the pentameric complex. We further demonstrated that following vaccination of a replication-defective virus with the restored pentameric complex, rhesus macaques can develop broad neutralizing antibodies targeting multiple immunogenic sites of the pentameric complex. Such analyses of site-specific antibody responses are imperative to our assessment of the quality of vaccine-induced immunity in clinical studies.

  • Studies on the interaction of tumor-derived HD5 alpha defensins with adenoviruses and implications for oncolytic adenovirus therapy [PublishAheadOfPrint]

  • Defensins are small anti-microbial peptides capable of neutralizing human adenovirus (HAdV) in vitro by binding capsid proteins and blocking endosomal escape of virus. In humans, the alpha defensin HD5 is produced by specialized epithelial cells of the gastro-intestinal and genito-urinary tracts. Here we demonstrate that HD5 is also expressed as an active, secreted peptide by epithelial ovarian and lung cancer cells in situ, in patient biopsies. This finding triggered us to study the role of HD5 in infection and spread of replication-competent, oncolytic HAdV type 3 virus. HAdV-3 produces large amounts of penton-dodecahedra (PtDd), virus-like particles during replication. We have previously shown that PtDd are involved in opening epithelial junctions thus facilitating lateral spread of de novo produced virions. Here we describe a second function of PtDd, namely the blocking of HD5. A central tool to prove that viral PtDd neutralize HD5 and support spread of progeny virus was a HAdV-3 mutant virus that was disabled for forming PtDd (mut-Ad3GFP). We demonstrated that viral spread of mut-Ad3GFP was blocked by synthetic HD5 whereas that of the wild-type form (wt-Ad3GFP) was only minimally impacted. In human colon cancer Caco-2 cells, induction of cellular HD5 expression by fibroblast growth factor 9 (FGF9) significantly inhibited viral spread and progeny virus production for mut-Ad3GFP but not for wt-Ad3GFP. Finally, the ectopic expression of HD5 in tumor cells diminished the in vivo oncolytic activity of mut-Ad3GFP but not wt-Ad3GFP. These data suggest a new mechanism of HAdV-3 to overcome innate antiviral host responses. Our study has implications for oncolytic adenovirus therapy.

    IMPORTANCE Previously, it has been reported that human defensin HD5 inactivates specific human adenoviruses by binding to capsid proteins and blocking endosomal escape of virus. The central new findings described in our manuscript are: i) the discovery of a new mechanism that human adenovirus serotype 3 uses to overcome innate antiviral host responses mediate by human defensin HD5. This mechanism is based on the capacity of Ad3 to produce subviral penton-dodecahedral particles that act as decoys for HD5 and thus prevent the inactivation of virus progeny produced upon replication, ii) the demonstration that ectopic HD5 expression in cancer cells decreases the oncolytic efficacy of a serotype 5 based adenovirus vector and iii) the demonstration that epithelial ovarian and lung cancers express HD5. The study improves our understanding on how adenoviruses establish infection in epithelial tissues and has implications for cancer therapy with oncolytic adenoviruses.

  • LMP1 and LMP2A collaborate to promote Epstein-Barr virus (EBV)-induced B cell lymphomas in a cord blood-humanized mouse model but are not essential [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) infection is associated with B cell lymphomas in humans. The ability of EBV to convert human B cells into long-lived lymphoblastoid cell lines (LCLs) in vitro requires the collaborative effects of EBNA2 (which hijacks notch signaling), LMP1 (which mimics CD40 signaling), and EBNA 3A/3C (which inhibit oncogene-induced senescence and apoptosis). However, we recently showed that an LMP1-deleted EBV mutant induces B cell lymphomas in a newly developed cord blood-humanized mouse model that allows EBV-infected B cells to interact with CD4 T cells (the major source of CD40 ligand). Here we examined whether the EBV LMP2A protein, which mimics constitutively active B cell receptor signaling, is required for EBV-induced lymphomas in this model. We find that deletion of LMP2A delays the onset of EBV-induced lymphomas, but does not affect the tumor phenotype or the number of tumors. Simultaneous deletion of both LMP1 and LMP2A results in fewer tumors, and a further delay in tumor onset. Nevertheless, the double LMP1/LMP2A mutant induces lymphomas in approximately half of the infected animals. These results indicate that neither LMP1 nor LMP2A is absolutely essential for the ability of EBV to induce B cell lymphomas in the cord blood-humanized mouse model, although simultaneous loss of both LMP1/LMP2A decreases the proportion of animals developing tumors and increases the time to tumor onset. Thus, either LMP1 or LMP2A expression may be sufficient to promote early-onset EBV-induced tumors in this model.

    IMPORTANCE EBV causes human lymphomas, but few models are available for dissecting how EBV causes lymphomas in vivo in the context of a host immune response. We recently used a newly developed cord blood-humanized mouse model to show that EBV can cooperate with human CD4 T cells to cause B cell lymphomas even when a major viral transforming protein, LMP1, is deleted. Here we examined whether the EBV protein LMP2A, which mimics B cell receptor signaling, is required for EBV-induced lymphomas in this model. We find that deletion of LMP2A alone has little effect on the ability of EBV to cause lymphomas, but delays tumor onset. Deletion of both LMP1 and LMP2A results in a smaller number of lymphomas in infected animals, with an even more delayed tumor onset. These results suggest that LMP1 and LMP2A collaborate to promote early-onset lymphomas in this model, but neither is absolutely essential.

  • Adenovirus modulates Toll-like receptor 4 signaling by reprogramming ORP1L-VAP protein contacts for cholesterol transport from endosomes to the endoplasmic reticulum [PublishAheadOfPrint]

  • Human adenoviruses generally cause mild self-limiting infections but can lead to serious disease and even be fatal in high-risk individuals, underscoring the importance of understanding how the virus counteracts host defense mechanisms. This study had two goals. First, determine the molecular basis of cholesterol homeostatic responses induced by the early region 3 membrane protein RIDaalpha; via its direct interaction with the sterol-binding protein ORP1L. Second, determine how this interaction regulates innate immunity to adenovirus. ORP1L is known to form highly dynamic contacts with endoplasmic reticulum-resident VAP proteins that regulate late endosome function under regulation of Rab7-GTP. Our studies have demonstrated that ORP1L-VAP complexes also support transport of LDL-derived cholesterol from endosomes to endoplasmic reticulum where it is converted to cholesteryl esters stored in lipid droplets when ORP1L was bound to RIDaalpha;. The virally-induced mechanism counteracted defects in the predominant cholesterol transport pathway regulated by the late endosomal membrane protein NPC1 arising during early stages of viral infection. However, unlike NPC1, RIDaalpha; did not reconstitute transport to endoplasmic reticulum pools that regulate SREBP transcription factors. RIDaalpha;-induced lipid trafficking also attenuated pro-inflammatory signaling by Toll-like receptor 4 that has a central role in Ad pathogenesis, and which is known to be tightly regulated by cholesterol-rich "lipid rafts". Collectively these data show that RIDaalpha; utilizes ORP1L in a way that is distinct from its normal function in uninfected cells to fine-tune lipid raft cholesterol that regulates innate immunity to adenovirus in endosomes.

    IMPORTANCE Early region 3 proteins encoded by human adenoviruses that attenuate immune-mediated pathology have been a particularly rich source of information regarding intracellular protein trafficking. Our studies with the early region 3-encoded RIDaalpha; protein also provide fundamental new information regarding mechanisms of non-vesicular lipid transport, and the flow of molecular information at membrane contacts between different organelles. We describe a new pathway that delivers cholesterol from endosomes to the endoplasmic reticulum, where it is esterified and stored in lipid droplets. Although lipid droplets are attracting renewed interest from the standpoint of normal physiology and human diseases including those resulting from viral infections, experimental model systems for evaluating how and why they accumulate are still limited. Our studies also reveal an intriguing relationship between lipid droplets and innate immunity that may represent a new paradigm for viruses utilizing these organelles.

  • Phyloepidemiological analysis reveals that viral divergence led to the paucity of SIVmus/gsn/mon infections in wild populations [PublishAheadOfPrint]

  • Human immunodeficiency virus subtype-1 (HIV-1) is the result of cross-species transmission of simian immunodeficiency virus from chimpanzees (SIVcpz). SIVcpz is a chimeric virus which shares common ancestors with viruses infecting red capped mangabeys and a subset of guenon species. The epidemiology of SIV infection in hominoids is characterized by low prevalence and uneven geographical distribution. Surveys in Cameroon indicated that two closely related members of the guenon species subset, mustached guenons and greater spot-nosed guenons, infected with SIVmus and SIVgsn respectively, have also low rates of SIV infections in their populations. Compared to other monkeys, including red capped mangabeys and closely related guenon species, such an epidemiology is unusual.

    By intensifying sampling of geographically distinct populations of moustached and greater spot-nosed guenons in Gabon, and including large sample sets of mona guenons in Cameroon, we add strong support that the paucity of SIV infections in wild populations is a general feature of this monophyletic group of viruses. Furthermore, comparative phylogenetic analysis reveals that this phenotype is a feature of this group of viruses infecting phylogenetically dispirate hosts, suggesting that this epidemiological phenotype results from infection with these HIV-1 related viruses rather than a common host factor. Thus, these HIV-1 related viruses, SIVcpz and the guenon viruses which share a common ancestor with part of the SIVcpz genome, have a distinct epidemiology to that found in other African primate species.

    IMPORTANCE Stable virus-host relationships are established over multiple generations. The prevalence of viral infections in any given host is determined by various factors. Stable virus-host relationships of viruses that are able to cause persistent infections and exist with high incidences of viral infections are generally characterized by a lack of morbidity prior to host reproduction. Such is the case for cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infection in humans.

    SIV infections of most African primate species also satisfy these criteria, being found at a high prevalence, with rare cases of clinical disease. By contrast, SIVcpz, the ancestor of HIV-1 infection in humans, has a different epidemiology and it has been reported that these animals suffer from an AIDS-like disease in the wild. Here we conclusively demonstrate that viruses which are closely related to SIVcpz and infect a subset of guenon monkeys show an epidemiology resembling that of chimpanzees.

  • Melanin or melanin-like substance interacts with the N-terminal portion of prion protein and inhibits abnormal prion protein formation in prion-infected cells [PublishAheadOfPrint]

  • Prion diseases are progressive fatal neurodegenerative illnesses caused by the accumulation of transmissible abnormal prion protein (PrP). To find treatments for prion diseases, we searched for substances from natural resources that inhibit abnormal PrP formation in prion-infected cells. We found that high-molecular-weight components from insect cuticle extracts reduced abnormal PrP levels. The chemical nature of these components was consistent with that of melanin. In fact, synthetic melanin produced from tyrosine or 3-hydroxy-l-tyrosine inhibited abnormal PrP formation. Melanin did not modify cellular or cell-surface PrP levels, nor did it modify lipid raft or cellular cholesterol levels. Neither did it enhance autophagy or lysosomal function. Melanin was capable of interacting with PrP at two N-terminal domains. Specifically, it strongly interacted with the PrP 23nndash;50 region including a positively charged amino acid cluster and weakly interacted with the PrP 51nndash;90 octarepeat peptide region. However, the in vitro and in vivo data were inconsistent with those of prion-infected cells. Abnormal PrP formation in protein misfolding cyclic amplification was not inhibited by melanin. Survival after prion infection was not significantly altered in albino mice or exogenously melanin-injected mice compared with that in control mice. These data suggest that melanin, a main determinant of skin color, is not likely to modify prion disease pathogenesis even though racial differences in the incidence of human prion diseases have been reported. Thus, the findings identify an interaction between melanin and the N-terminus of PrP, but the pathophysiological roles of the PrPnndash;melanin interaction remain unclear. (248)

    IMPORTANCE The N-terminal region of PrP is reportedly important for neuroprotection, neurotoxicity, and abnormal PrP formation, as this region is bound by many factors such as metal ions, lipids, nucleic acids, anti-prion compounds, and several proteins including abnormal PrP in prion disease and the Abbeta; oligomer in Alzheimer's disease. In the present study, melanin, a main determinant of skin color, was newly found to interact with this N-terminal region and inhibits abnormal PrP formation in prion-infected cells. However, the data for prion infection in mice lacking melanin production suggest that melanin is not associated with the prion disease mechanism, although the incidence of prion disease is reportedly much higher in white people than in black people. Thus, the roles of the PrPnndash;melanin interaction remain to be further elucidated, but melanin might be a useful competitive tool for evaluating the functions of other ligands at the N-terminal region. (147)

  • Identification of a Continuous Neutralizing Epitope within UL128 of Human Cytomegalovirus [PublishAheadOfPrint]

  • As human cytomegalovirus (HCMV) is the most common infectious cause of fetal anomalies during pregnancy, developing a vaccine that prevents HCMV infection is considered a global health priority. Although HCMV immune correlates of protection are only poorly defined, neutralizing antibodies (NAb) targeting the envelope pentamer complex (PC) composed of subunits gH, gL, UL128, UL130, and UL131A are thought to contribute in preventing HCMV infection. Here, we describe a continuous target sequence within UL128 that is recognized by a previously isolated potent PC-specific NAb, termed 13B5. By using peptide based scanning procedures we identified a 13 amino acid long target sequence at the UL128 C-terminus that binds the 13B5 antibody with similar affinity as purified PC. In addition, the 13B5 binding site is universally conserved in HCMV, contains a previously described UL128/gL interaction site, and interferes with 13B5 neutralizing function, indicating that the 13B5 epitope sequence is located within the PC at a site of critical importance for HCMV neutralization. Vaccination of mice with peptides containing the 13B5 target sequence resulted in robust stimulation of binding antibodies, and in a subset of immunized animals in induction of detectable NAb, supporting that the identified 13B5 target sequence constitutes a PC-specific neutralizing epitope. These findings provide evidence for the discovery of a continuous neutralizing epitope within the UL128 subunit of the PC that could be an important target of humoral immune responses that are involved in the protection against congenital HCMV infection.

    IMPORTANCE. Neutralizing antibodies (NAb) targeting the human cytomegalovirus (HCMV) envelope pentamer complex (PC) are thought to be important in preventing HCMV transmission from the mother to the fetus, thereby mitigating severe developmental disabilities in newborns. However, the epitope sequences within the PC that are recognized by these potentially protective antibody responses are only poorly defined. Here, we provide evidence for the existence of a highly conserved, continuous PC-specific epitope sequence that appears to be located within the PC at a subunit interaction site of critical importance for HCMV neutralization. These discoveries provide insights into a continuous PC-specific neutralizing epitope, which could be an important target for a vaccine formulation to interfere with congenital HCMV infection.

  • Sensitive next generation sequencing method reveals deep genetic diversity of HIV-1 in the Democratic Republic of the Congo [PublishAheadOfPrint]

  • As the epidemiological epicentre of the human immunodeficiency virus (HIV) pandemic, the Democratic Republic of the Congo (DRC) is a reservoir of circulating HIV strains exhibiting high levels of diversity and recombination. In this study, we characterized HIV specimens collected in two rural areas of the DRC between 2001 and 2003 to identify rare strains of HIV. The env gp41 region was sequenced and characterized for 172 HIV-positive specimens. The env sequences were predominantly subtype A (43.02%), but 7 other subtypes (33.14%), 20 circulating recombinant forms (CRFs: 11.63%), and 20 unclassified (11.63%) sequences were also found. Of the rare and unclassified subtypes, 18 specimens were selected for next generation sequencing (NGS) by a modified HIV-SMART method to obtain full genome sequences. NGS produced 14 new complete genomes, which included pure subtypes C (n=2), D (n=1), F1 (n=1), H (n=3), and J (n=1). The two Cs and one of the H genomes branched basal to their respective subtype branches but had no evidence of recombination. The remaining 6 genomes were complex recombinants of 2 or more subtypes, including A1, F, G, H, J, K, and unclassified fragments, including one CRF25 isolate, which branched basal to all CRF25 references. Notably, all recombinant H fragments branched basal to the H clade. Spatial-geographical analysis indicated that the diverse sequences identified here did not expand globally. The full- and sub-genomic sequences identified in our study population significantly increase the documented diversity of the continually evolving HIV-1 pandemic.

    Importance: Very little is known about the ancestral HIV-1 strains that founded the global pandemic, and very few complete genome sequences are available from patients in the Congo Basin where HIV-1 expanded early in the global pandemic. By sequencing a sub-genomic fragment of the HIV-1 envelope from study participants in the DRC, we identified rare variants for complete genome sequencing. The basal branching of some of the complete genome sequences we recovered suggests that these strains are more closely related to ancestral HIV-1 sequences than to previously reported strains and is evidence that the local diversification of HIV in the DRC continues to outpace the diversity of global strains decades after the emergence of the pandemic.

  • Vaccine efficacy of an inactivated, chimeric HA H9/H5N2 avian influenza virus and its suitability for the marker vaccine strategy [PublishAheadOfPrint]

  • In order to produce a dual effective vaccine against H9 and H5 avian influenza viruses that aligns with the DIVA (differentiating infected from vaccinated animals) strategy, we generated a chimeric H9/H5N2 recombinant vaccine which expressed the whole HA1 region of A/CK/Korea/04163/04 H9N2 and HA2 region of recent HPAI A/MD/Korea/W452/14 H5N8 viruses. The Chimeric H9/H5N2 virus showed similar in vitro and in vivo growth properties and virulence to the LPAI H9 influenza virus. An inactivated vaccine based on this chimeric virus induced serum neutralizing (SN) antibodies against both H9 and H5 viruses, but only induced cross-reactive hemmaglutination inhibition (HI) antibody against H9 viruses. Thus, this suggests its compatibility for use in the DIVA strategy against H5 strains. Further, the chimeric H9/H5N2 recombinant vaccine protected immunized chickens against lethal challenge by HPAI H5N8 viruses, and significantly attenuated virus shedding after both H9N2 and HPAI H5N8 infection. In mice, serological analyses confirmed that HA1 and HA2 stalk-specific antibody responses were induced by vaccination and that the DIVA principle could be employed through use of an HI assay against H5 viruses. Further, each HA1 and HA2 stalk-specific antibody response was sufficient to inhibit viral replication and protect the chimeric virus-immunized mice from lethal challenge with both mouse-adapted H9N2 and wild-type HPAI H5N1 viruses, although differences in the vaccine efficacy against homologous H9 virus (HA1-head domain immune mediated protection) and heterosubtypic H5 virus (HA2 stalk domain immune mediated protection) were observed.

    Taken together, these results demonstrate that the novel chimeric H9/H5N2 recombinant virus is a low pathogenic virus and this chimeric vaccine is suitable for a DIVA vaccine with broad-spectrum neutralizing antibody against H5 avian influenza viruses.

    Importance Current influenza virus killed vaccines predominantly induce anti-hemagglutinin (HA) antibodies which commonly strain-specific in that the antibodies have potent neutralizing activity against homologous strains, but do not cross-react with the HA of other influenza subtypes. In contrast, the HA2 stalk domain is relatively well conserved among subtypes and recently broadly-neutralizing antibodies against this domain have been isolated. Therefore, in light of the need for a vaccine strain that applies the DIVA strategy utilizing an HI assay and induces broad cross-protectivity against H5N1 and H9N2 viruses, we generated a novel chimeric H9/H5N1 virus that expresses the entire HA1 portion from H9N2 and the HA2 region of heterosubtypic H5N8. The chimeric H9/H5N2 recombinant vaccine protected immunized hosts against from the lethal challenge of H9N2 and HPAI H5N1 viruses with significantly attenuated virus shedding in immunized hosts. Therefore, this chimeric vaccine strategy is suitable as a DIVA vaccine against H5 avian influenza viruses.

  • Bromodomain Containing 4 (BRD4) Couples NF{kappa}B/RelA With Airway Inflammation And The IRF-RIG-I Amplification Loop In Respiratory Syncytial Virus Infection [PublishAheadOfPrint]

  • The airway mucosa expresses protective interferon (IFN) and inflammatory cytokines in response to Respiratory Syncytial Virus (RSV) infection. In this study, we examine the role of bromodomain containing 4 (BRD4) in mediating this innate immune response in human small airway epithelial cells. We observe that RSV induces BRD4 to complex with NFB/RelA. BRD4 is functionally required for expression of the NFB-dependent inflammatory gene regulatory network (GRN), including the IFN Response Factor (IRFs)-1 and -7 that mediate a cross-talk pathway for RIG-I upregulation. Mechanistically, BRD4 is required for CDK9 recruitment and phospho-Ser 2 CTD RNA Pol II formation on the promoters of IRF1, IRF7 and RIG-I producing their enhanced expression by transcriptional elongation. We also find that BRD4 independently regulates CDK9/phospho-Ser 2 CTD RNA Pol II recruitment to the IRF3-dependent IFN-stimulated genes (ISGs). In vivo, poly(I:C)-induced neutrophilia and mucosal chemokine production is blocked by a small molecule BRD4 bromodomain inhibitor. Similarly, BRD4 inhibition reduces RSV-induced neutrophilia, mucosal CXC chemokine expression, activation of the IRF7-RIG-I auto-amplification loop, mucosal IFN expression and airway obstruction. RSV infection activates BRD4 acetyltransferase activity on Histone H3 Lys(K) 122, demonstrating that RSV infection activates BRD4 in vivo. These data validate BRD4 as a major effector of RSV-induced inflammation and disease. BRD4 is required for coupling NFB to expression of inflammatory genes, the IRF-RIG-I auto-amplification pathway and independently facilitates anti-viral ISG expression. BRD4 inhibition may be a strategy to reduce exuberant viral-induced mucosal airway inflammation.

    IMPORTANCE 2.1 M children annually in the US require medical attention for RSV infections. A first line of defense is the expression of the innate gene network by infected epithelial cells. Expression of the innate response requires the recruitment of transcriptional elongation factors to rapidly induce innate response genes through an unknown mechanism. We discovered that RSV infection induces a complex of bromodomain containing 4 (BRD4) with NFB and cyclin dependent kinase 9 (CDK9). BRD4 is required for stable CDK9 binding, phospho-Ser 2 RNA Pol II formation and histone acetyltransferase activity. Inhibition of BRD4 blocks TLR3-dependent neutrophilia and RSV-induced inflammation demonstrating its importance in the mucosal innate response in vivo. Our study shows that BRD4 plays a central role in inflammation and activation of the IRF7-RIG-I amplification loop vital for mucosal interferon expression. BRD4 inhibition may be a strategy for modulating exuberant mucosal airway inflammation.

  • Ovine herpesvirus 2 glycoproteins B, H, and L are sufficient for, and viral glycoprotein Ov8 can enhance, cell-cell membrane fusion [PublishAheadOfPrint]

  • Ovine herpesvirus 2 (OvHV-2) is a gammaherpesvirus in the genus Macavirus that is carried asymptomatically by sheep. Infection of poorly adapted animals with OvHV-2 results in sheep-associated malignant catarrhal fever, a fatal disease characterized by lymphoproliferation and vasculitis. There is no treatment or vaccine for the disease and no cell culture system to propagate the virus. The lack of cell culture has hindered studies of OvHV-2 biology including its entry mechanism. As an alternative method to study OvHV-2 glycoproteins responsible for membrane fusion as a part of the entry mechanism, we developed a virus-free cell-to-cell membrane fusion assay to identify the minimum required OvHV-2 glycoproteins to induce membrane fusion. OvHV-2 glycoproteins gB, gH and gL were able to induce membrane fusion together but not when expressed individually. Additionally, open reading frame Ov8, unique to OvHV-2, was found to encode a transmembrane glycoprotein that can significantly enhance membrane fusion. Thus, the OvHV-2 glycoproteins gB, gH and gL are sufficient to induce membrane fusion while glycoprotein Ov8 plays an enhancing role by an unknown mechanism.

    IMPORTANCE Herpesviruses enter cells via attachment of the virion to the cellular surface and fusion of the viral envelope with cellular membranes. Virus-cell membrane fusion is an important step for a successful viral infection. Elucidating the roles of viral glycoproteins responsible for membrane fusion is critical toward understanding viral entry. Entry of ovine herpesvirus 2 (OvHV-2), the causative agent of sheep associated-malignant catarrhal fever, which is one of the leading causes of deaths in bison and other ungulates, has not been well-studied due to the lack of a cell culture system to propagate the virus. The identification of OvHV-2 glycoproteins that mediate membrane fusion may help identify viral and/or cellular factors involved in OvHV-2 cell tropism and will advance investigation of cellular factors necessary for virus-cell membrane fusion. We found that OvHV-2 glycoproteins B, H, and L are sufficient for, and viral glycoprotein Ov8 can significantly enhance, cell-cell membrane fusion.

  • A point mutation in the N-terminal amphiphathic helix {alpha}0 in NS3 promotes HCV assembly by altering core localization to ER and facilitating virus budding [PublishAheadOfPrint]

  • The assembly of hepatitis C virus (HCV), a complicated process in which many viral and cellular factors are involved, has not been thoroughly deciphered. NS3 is a multifunctional protein that contains an N-terminal amphipathic aalpha; helix (denoted helix aalpha;0), whichis crucial for the membrane association and stability of NS3 protein, followed by a serine protease domain and a C-terminal helicase/NTPase domain. NS3 participates in HCV assembly likely through its C-terminal helicase domain where all reported adaptive mutations enhancing virion assembly reside. In this study, we reported that the N-terminal helix aalpha;0 of NS3 may contribute to HCV assembly. We identified a single mutation from methionine to threonine at amino acid position 21 in the helix aalpha;0 (denoted M21T), which significantly promoted viral production while had no apparent effect on NS3's membrane association and protease activity. Subsequent analyses demonstrated that M21T mutation did not affect HCV genome replication but rather promoted virion assembly. Further study revealed a shift in the subcellular localization of core protein from lipid droplets to endoplasmic reticulum. Finally, we showed that M21T increased the co-localization of core proteins and viral envelope proteins, leading to a more efficient envelopment of viral nucleocapsids. Collectively, our study revealed a new function of NS3 helix aalpha;0 and will shed light on the understanding of the role of NS3 in HCV virion morphogenesis.

    IMPORTANCE HCV NS3 protein possesses the protease activity in its N-terminal domain and the helicase activity in its C-terminal domain. The role of NS3 in virus assembly has been mainly attributed to its helicase domain because all adaptive mutations enhancing progeny virus production are all found to be within this C-terminal domain. Our study identified, for the first time to our knowledge, an adaptive mutation within the N-terminal helix aalpha;0 domain of NS3 that significantly enhanced virus assembly while had no effect on viral genome replication. The mechanistic studies suggested that this mutation promoted the relocation of core proteins from LD to ER, leading to a more efficient envelopment of viral nucleocapsids. Our results revealed a possible new function of helix aalpha;0 in HCV life cycle, and provided new clues to understanding the molecular mechanisms for the action of NS3 in HCV assembly.

  • 5 -Phospho-RNA acceptor specificity of GDP polyribonucleotidyltransferase of vesicular stomatitis virus in mRNA capping [PublishAheadOfPrint]

  • The GDP polyribonucleotidyltransferase (PRNTase) domain of the multifunctional L protein of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus, catalyzes the transfer of 5rrsquo; -phospho-RNA (pRNA) from 5rrsquo; -triphospho-RNA (pppRNA) to GDP via a covalent enzymenndash;pRNA intermediate to generate a 5rrsquo; -cap structure (GpppA). Here, using an improved oligo-RNA capping assay with the VSV L protein, we showed that the Michaelis constants for GDP and pppAACAG (VSV mRNA-start sequence) are 0.03 and 0.4 mmu;M, respectively. A competition assay between GDP and GDP analogues in the GpppA formation and pRNA transfer assay using GDP analogues as pRNA acceptors indicated that the PRNTase domain recognizes the C2-amino group, but not C6-oxo group, N1-hydrogen, or N7-nitrogen, of GDP for the cap formation. 2,6-Diaminopurine-riboside (DAP), 7-deazaguanosine (7-deaza-G), and 7-methylguanosine (m7G) diphosphates efficiently accepted pRNA, resulting in the formation of DAPpppA, 7-deaza-GpppA, and m7GpppA (cap 0), respectively. Furthermore, either the 2rrsquo; - or 3rrsquo; -hydroxyl group of GDP was found to be required for efficient pRNA transfer. A 5rrsquo; -diphosphate form of antiviral ribavirin weakly inhibited the GpppA formation, but did not act as a pRNA acceptor. These results indicate that the PRNTase domain has a unique guanosine-binding mode different from that of eukaryotic mRNA capping enzyme, guanylyltransferase.

    IMPORTANCE mRNAs of nonsegmented negative strand (NNS) RNA viruses, such as VSV, possess a fully methylated cap structure, which is required for mRNA stability, efficient translation, and evasion of anti-viral innate immunity in host cells. GDP polyribonucleotidyltransferase (PRNTase) is an unconventional mRNA capping enzyme of NNS RNA viruses that is distinct from eukaryotic mRNA capping enzyme, guanylyltransferase. In this study, we studied the pRNA acceptor specificity of VSV PRNTase using various GDP analogues and identified chemical groups of GDP as essential for the substrate activity. The findings presented here are useful not only for understanding the mechanism of the substrate recognition with PRNTase, but also for designing anti-viral agents targeting this enzyme.

  • Subcellular localization of HIV-1 gag-pol mRNAs regulates sites of virion assembly [PublishAheadOfPrint]

  • HIV-1 full-length, unspliced RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing full-length viral RNA abundance or distribution had little to no net effect on Gag assembly competency when provided in trans. By contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNA-dependent disruption of Gag trafficking required either of two cis-acting RNA regulatory elements; the 5rrsquo; packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE) that regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM.

    IMPORTANCE The spatial distribution of viral messenger RNAs (mRNAs) within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency virus type 1 (HIV-1) virus particles at the plasma membrane (PM). Artificially tethering viral mRNAs encoding Gag capsid proteins (gag-pol mRNAs) to distinct non-PM subcellular locales such as cytoplasmic vesicles or the actin cytoskeleton markedly alters Gag subcellular distribution, relocates sites of assembly, and reduces net virus particle production. These observations support a model for native HIV-1 assembly wherein HIV-1 gag-pol mRNA localization helps to confine interactions between Gag, viral RNAs, and host determinants in order to ensure virion production at the right place and right time. Direct perturbation of HIV-1 mRNA subcellular localization could represent a novel antiviral strategy.

  • GRP78 is an important host-factor for Japanese encephalitis virus entry and replication in mammalian cells [PublishAheadOfPrint]

  • Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is the leading cause of viral encephalitis in South-East Asia with potential to become a global pathogen. Here we identify the Glucose regulated protein 78 (GRP78) as an important host protein for virus entry and replication. Using the plasma membrane fractions from mouse neuronal (Neuro2a) cells, mass spectroscopy analysis identified GRP78 as a protein interacting with recombinant JEV envelope protein domain III. GRP78 was found to express on the plasma membrane of Neuro2a, mouse primary neurons, and human epithelial Huh-7 cells. Antibodies against GRP78 significantly inhibited JEV entry in all three cell types suggesting an important role of the protein in virus entry. Depletion of GRP78 by siRNA significantly blocked JEV entry into Neuro2a cells, further supporting its role in virus uptake. Immunofluorescence studies showed extensive co-localization of GRP78 with JEV envelope protein in virus-infected cells. This interaction was also confirmed by immunoprecipitation studies. Additionally, GRP78 was shown to have an important role in JEV replication, as treatment of cells post virus-entry with Subtilase cytotoxin that specifically cleaved GRP78, led to a substantial reduction in viral RNA replication and protein synthesis resulting in significantly reduced extracellular virus titers. Our results indicate that GRP78, an endoplasmic reticulum chaperon of the HSP70 family, is a novel host factor involved at multiple steps of the JEV life-cycle and could be a potential therapeutic target.

    IMPORTANCE Recent years have seen a rapid spread of mosquito-borne diseases caused by flaviviruses. This virus family includes West Nile, Dengue, Japanese encephalitis, and Zika viruses that are a major threat to public health with potential to become global pathogens. JEV is the major cause of viral encephalitis in several parts of South-East Asia affecting a predominantly paediatric population with a high mortality rate. This study is focussed on identification of crucial host factors that could be targeted to cripple virus infection and ultimately lead to development of effective anti-virals. We have identified a cellular protein GRP78 that plays a dual role in virus entry and virus replication, two crucial steps of the virus life cycle, and thus is a novel host factor that could be a potential therapeutic target.

  • Structural insight into African Swine Fever virus A179L mediated inhibition of apoptosis [PublishAheadOfPrint]

  • Programmed cell death is a tightly controlled process critical for the removal of damaged or infected cells. Pro- and anti-apoptotic proteins of the Bcl-2 family are pivotal mediators of this process. African Swine Fever virus (ASFV) is a large DNA virus, the only member of the Asfarviridae family, and harbors A179L, a putative Bcl-2 like protein. A179L has been shown to bind to several pro-apoptotic Bcl-2 proteins, however the hierarchy of binding and the structural basis for apoptosis inhibition are currently not understood. We systematically evaluated the ability of A179L to bind pro-apoptotic Bcl-2 family members, and show that A179L is the first anti-apoptotic Bcl-2 protein to bind to all major death inducing mammalian Bcl-2 proteins. We then defined the structural basis for apoptosis inhibition of A179L by determining crystal structures of A179L bound to both Bid and Bax BH3 motifs. Our findings provide a mechanistic understanding for the potent anti-apoptotic activity of A179L by identifying it as the first pan pro-death Bcl-2 binder, and serve as a platform for more detailed investigations into the role of A179L during ASFV infection.

    IMPORTANCE Numerous viruses have acquired strategies to subvert apoptosis by encoding proteins capable of sequestering pro-apoptotic host proteins. African Swine Fever virus (ASFV), a large DNA virus and the only member of the Asfarviridae family, encodes the protein A179L that functions to prevent apoptosis. We show that A179L is unusual amongst anti-apoptotic Bcl-2 proteins in being able to physically bind to all core death inducing mammalian Bcl-2 proteins. Currently, little is known regarding the molecular interactions between A179L and the pro-apoptotic Bcl-2 members. Using crystal structures of A179L bound to two of the identified pro-apoptotic Bcl-2 proteins, Bid and Bax, we now provide a 3D view of how A179L sequesters host pro-apoptotic proteins, which is crucial for subverting premature host cell apoptosis.

  • Heat shock protein 90 ensures efficient mumps virus replication by assisting with viral polymerase complex formation [PublishAheadOfPrint]

  • Paramyxoviral RNAs are synthesized by a viral RNA-dependent RNA polymerase (RdRp) consisting of the large (L) protein and its cofactor phosphoprotein (P protein). The L protein is a multifunctional protein that catalyzes RNA synthesis, mRNA capping and mRNA polyadenylation. Growing evidence shows that the stability of several paramyxovirus L proteins is regulated by heat shock protein 90 (Hsp90). In this study, we demonstrated that Hsp90 activity was important for mumps virus (MuV) replication. The Hsp90 activity was required for the L protein stability and activity, because an Hsp90-specific inhibitor, 17-AAG, destabilized the MuV L protein and suppressed the viral RNA synthesis. However, once the L protein formed a mature polymerase complex with the P protein, the Hsp90 activity was no longer required for the stability and activity of the L protein. When the Hsp90 activity was inhibited, the MuV L protein was degraded through the CHIP (C-terminus of Hsp70-interacting protein)-mediated proteasomal pathway. High concentrations of 17-AAG showed strong cytotoxicity to certain cell types, but combined use of an Hsp70 inhibitor, VER155008, potentiated degradation of the L protein, allowing a sufficient reduction of 17-AAG concentration to block the MuV replication with minimum cytotoxicity. Regulation of the L protein by Hsp90 and Hsp70 chaperones was also demonstrated for another paramyxovirus, the measles virus. Collectively, our data show that the Hsp90/Hsp70 chaperone machinery assists in the maturation of the paramyxovirus L protein, and thereby in the formation of a mature RdRp complex and efficient viral replication.

    IMPORTANCE Heat shock protein 90 (Hsp90) is nearly universally required for viral protein homeostasis. Here, we report that Hsp90 activity is required for efficient propagation of mumps virus (MuV). Hsp90 functions in the maintenance of the catalytic subunit of viral polymerase, the large (L) protein, prior to formation of a mature polymerase complex with the polymerase cofactor of L, phosphoprotein. Hsp70 collaborates with Hsp90 to regulate biogenesis of the MuV L protein. The functions of these chaperones on the viral polymerase may be common among paramyxoviruses, because the L protein of measles virus is also similarly regulated. Our data provide important insights into the molecular mechanisms of paramyxovirus polymerase maturation as well as a basis for the development of novel antiviral drugs.

  • Epstein-Barr Virus encoded Latent Membrane Protein-1 upregulates glucose transporter-1 transcription via the mTORC1/NF-{kappa}B signaling pathways [PublishAheadOfPrint]

  • Accumulating evidence indicates that oncogenic viral protein exerts a crucial role in activating aerobic glycolysis during tumorigenesis, but the underlying mechanisms are largely undefined. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a trans-membrane protein with potent cell signaling properties and has tumorigenic transformation property. Activation of NF-B is a major signaling pathway mediating many downstream transformation properties of LMP1. Here we report that activation of mTORC1 by LMP1 is a key modulator for activation of NF-B signaling to mediate aerobic glycolysis. NF-B activation is involved in the LMP1-induced upregulation of glucose transporter-1 (Glut-1) transcription and growth of nasopharyngeal carcinoma (NPC) cells. Blocking the activity of mTORC1 signaling effectively suppressed LMP1-induced NF-B activation and Glut-1 transcription. Interfering NF-B signaling had no effect on mTORC1 activity but effectively altered Glut-1 transcription. Luciferase promoter assay of Glut-1 also confirmed that Glut-1 is a direct target gene of NF-B signaling. Furthermore, we demonstrated that the C-terminal activating region (CTAR) 2 of LMP1 is the key domain involved in mTORC1 activation, mainly through IKKbbeta;-mediated phosphorylation of TSC2 at Ser939. Depletion of Glut-1 effectively led to suppression of aerobic glycolysis, inhibition of cell proliferation, colony formation, and attenuation of tumorigenic growth property of LMP1-expressing nasopharyngeal epithelial (NPE) cells. These findings suggest that targeting the signaling axis of mTORC1/NF-B/Glut-1 represents a novel therapeutic target against NPC.

    Importance: Aerobic glycolysis is one of the hallmarks of cancer including NPC. Recent studies suggest a role of LMP1 in mediating aerobic glycolysis. LMP1 expression is common in NPC. The delineation of essential signaling pathways induced by LMP1 in aerobic glycolysis contributes to the understanding of NPC pathogenesis. This current study provides evidences that LMP1 upregulates Glut-1 transcription to control aerobic glycolysis and tumorigenic growth of NPC cells through mTORC1/NF-B signaling. Our results reveal novel therapeutic targets against mTORC1/NF-B/Glut-1 signaling axis in the treatment of EBV-infected NPC.

  • HCMV Specific CD4+ T Cells are poly-functional and can respond to HCMV Infected Dendritic Cells in vitro. [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) infection and periodic re-activation is generally well controlled by the HCMV-specific T cell response in healthy people. While the CD8+ T cell response to HCMV has been extensively studied, the HCMV-specific CD4+ T cell effector response is not as well understood, especially in the context of direct interactions with HCMV infected cells. We screened the IFN and IL-10 response to 6 HCMV peptide pools (selected as the most frequently responded to in our previous studies: pp65, pp71, IE1, IE2, gB and US3) in 84 donors, aged 23 nndash; 74 years. Predominantly the HCMV specific CD4+ T cell response to pp65, IE1, IE2 and gB was Th1 biased with neither loss nor accumulation of these responses with increasing age. A larger proportion of donors produced an IL-10 response to pp71 and US3 but the IFN response was still dominant. CD4+ T cells specific to the HCMV proteins studied were predominantly effector memory cells and produced both cytotoxic (CD107a expression) and cytokine (MIP1bbeta; secretion) effector responses. Importantly, when we measured the CD4+ T cell response to CMV infected Dendritic Cells in vitro, we observed that the CD4+ T cells produced a range of cytotoxic and secretory effector functions, despite the presence of CMV encoded immune evasion molecules. CD4+ T cell responses to HCMV infected dendritic cells were sufficient to control the dissemination of virus in an in vitro assay. Together the results show that HCMV-specific CD4+ T cell responses are highly functional even from elderly individuals and are directly anti-viral.

    IMPORTANCE Human cytomegalovirus (HCMV) infection is carried for a lifetime and in healthy people is kept under control by the immune system. HCMV has evolved many mechanisms to evade the immune response, possibly explaining why the virus is never eliminated during the hosts' lifetime. Dysfunction of immune cells associated with long-term carriage of HCMV has been linked with poor responses to new pathogens and vaccines when older. In this study we have investigated the response of a subset of immune cells (CD4+ T cell) to HCMV proteins in healthy donors of all ages demonstrating that the functionality of the CD4+ T cells is maintained. We have also shown that CD4+ T cells produce effector functions in response to HCMV infected cells and can prevent virus spread. Our work demonstrates that these HCMV-specific immune cells retain many important functions and help to prevent deleterious HCMV disease in healthy older people.

  • Domain organization and evolution of the highly divergent 5 coding region of genomes of arteriviruses including the novel possum nidovirus [PublishAheadOfPrint]

  • In five experimentally characterized arterivirus species, the 5rrsquo; -end genome coding region comprises most divergent nonstructural proteins (nsp) 1 and 2 that include papain-like proteases (PLPs) and other poorly characterized domains. They are involved in regulation of transcription, polyprotein processing, and virus-host interaction. Here, we present results of bioinformatics analysis of this region of 14 arterivirus species, including that of the most distantly related wobbly possum disease virus (WPDV), determined by a modified 5rrsquo; RACE protocol. By combining profile-profile comparisons and phylogeny reconstruction, we identified an association of the four distinct domain layouts of nsp1-nsp2 with major phylogenetic lineages, implicating domain gain, including duplication, and loss into the early nsp1 evolution. Specifically, WPDV encodes highly divergent homologs of PLP1a, PLP1b, PLP1c and PLP2, with PLP1a lacking the catalytic Cys residue, but does not encode nsp1 ZnF and llsquo;Nuclease' domains, conserved in other arteriviruses. Unexpectedly, our analysis revealed that the only catalytically active nsp1 PLP of equine arteritis virus (EAV), known as PLP1b, is most similar to PLP1c and thus is likely to be a PLP1b paralog. In all non-WPDV arteriviruses, PLP1b/1c and PLP1a show contrasting patterns of conservation, with the N- and C-terminal subdomains, respectively, being enriched with conserved residues, indicative of different functional specialization. The least conserved HVR domain of nsp2 has its size varied five-fold and includes up to four copies of novel PxPxPR motif that is potentially recognized by SH3-domain-containing proteins. Apparently, only EAV lacks the signal that directs -2 ribosomal frameshifting in nsp2 region.

    IMPORTANCE Arteriviruses comprise a family of mammalian enveloped positive-stranded RNA viruses that include some of the most economically important pathogens of swine. Most of our knowledge about this family has been obtained through characterization of viruses from five species: Equine artheritis virus, Simian hemorrhagic fever virus, Lactate dehydrogenase virus, Porcine respiratory and reproductive syndrome virus -1 and -2. Here we present results of comparative genomics analyses of viruses from all known 14 arterivirus species, including the most distantly related WPDV, whose genome sequencing was completed in this study. Our analysis was focused on the multi-functional 5rrsquo; -end genome coding region that encodes multidomain nonstructural proteins 1 and 2. Using diverse bioinformatics techniques we identified many patterns of evolutionary conservation that are specific for members of distinct arterivirus species, characterized and novel, or their groups. They are likely associated with structural and functional determinants important for virus replication and virus-host interaction.

  • Cytokines Elevated in HIV Elite Controllers Reduce HIV Replication in vitro and Modulate HIV Restriction Factor Expression [PublishAheadOfPrint]

  • A subset of HIV infected individuals termed elite controllers (ECs) maintain CD4+ T cell counts and control viral replication in the absence of antiretroviral therapy (ART). Systemic cytokine responses may differentiate ECs from subjects with uncontrolled viral replication or those who require ART to suppress viral replication. We measured 87 cytokines in four groups of women: 73 EC, 42 with pharmacologically suppressed viremia (ART), 42 with uncontrolled viral replication (noncontrollers, NC), and 48 HIV uninfected (NEG) subjects. Four cytokines were elevated in ECs but not NCs or ART subjects: CCL14, CCL21, CCL27, and XCL1. In addition, median SDF-1 levels were 43% higher in ECs than NCs. The combination of the five cytokines suppressed R5 and X4 virus replication in resting CD4+ T cells, and individually SDF-1bbeta;, CCL14 and CCL27 suppressed R5 virus replication, while SDF-1bbeta;, CCL21, and CCL14 suppressed X4 virus replication. Functional studies revealed that the combination of the five cytokines up-regulated CD69 and CCR5 and down-regulated CXCR4 and CCR7 on CD4+ T cells. The CD69 and CXCR4 effects were driven by SDF-1, while CCL21 down-regulated CCR7. The combination of the EC-associated cytokines induced expression of the anti-HIV host restriction factors IFITM1 and IFITM2 and suppressed expression of RNase L and SAMHD1. These results identify a set of cytokines that are elevated in ECs and define its effects on cellular activation, HIV co-receptor expression, and innate restriction factor expression. This cytokine pattern may be a signature characteristic of HIV-1 elite control, potentially important for HIV therapeutic and curative strategies.

    IMPORTANCE Approximately 1% of people infected with HIV control virus replication without taking antiviral medications. These subjects, termed elite controllers (ECs), are known to have stronger immune responses targeting HIV than the typical HIV-infected subject, but the exact mechanisms of how their immune responses control infection are not known. In this study we identified five soluble immune signaling molecules (cytokines) in the blood that were higher in ECs than in subjects with typical chronic HIV infection. We demonstrated that these cytokines can activate CD4+ T cells, the target cells for HIV infection. Furthermore, these five EC-associated cytokines could change expression of intrinsic resistance factors, or molecules inside the target cell that fight HIV infection. This study is significant in that it identified cytokines elevated in subjects with a "good" immune response against HIV and defined potential mechanisms as to how these cytokines could induce resistance to the virus in target cells.

  • The Viral Bcl-2 Homologs of Kaposi's Sarcoma-Associated Herpesvirus and Rhesus Rhadinovirus Share an Essential Role for Viral Replication [PublishAheadOfPrint]

  • KS-Bcl-2 is a Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded viral Bcl-2 homolog (vBcl-2), which has apoptosis and autophagy inhibiting activity when expressed in transfected cells. However, little is known about its function during viral infection. As KS-Bcl-2 is expressed during the lytic replication cycle, we used constitutively lytic and inducible-lytic KSHV mutants to investigate the role of KS-Bcl-2 during the lytic cycle. We show that KSHV cannot complete the lytic replication cycle and produce infectious progeny in the absence of KS-Bcl-2, indicating that the protein is essential for KSHV replication. Substitution of the KS-Bcl-2 coding sequence, ORF16, by sequences encoding a potent cellular apoptosis and autophagy inhibitor, Bcl-XL, or the cytomegalovirus mitochondrial inhibitor of apoptosis, vMIA, did not rescue KSHV replication, suggesting that KS-Bcl-2 has a function that goes beyond apoptosis and autophagy inhibition. Strikingly, the vBcl-2 proteins of the related 2-herpesviruses murine herpesvirus 68 and herpesvirus saimiri did not rescue the replication of a KS-Bcl-2 deletion mutant, but the rhesus rhadinovirus (RRV) vBcl-2 did. Deletion of ORF16 from the RRV genome abrogated viral replication, but substitution by KSHV ORF16 rescued RRV replication, indicating that the essential vBcl-2 function is conserved between these two primate rhadinoviruses. We further show that the KSHV and RRV Bcl-2 homologs localize to mitochondria and the nucleus of infected cells. Deletion of 17 amino acids from the N-terminus of KS-Bcl-2 abrogates nuclear localization and KSHV replication, suggesting that KS-Bcl-2 might execute its essential function in the nucleus of infected cells.

    IMPORTANCE Several viruses express proteins homologous to cellular Bcl-2. Viral Bcl-2 proteins have functions similar to cellular Bcl-2: they can inhibit apoptosis, a form of programmed cell death, and autophagy, a self-degradative process for the disposal of dysfunctional or unwanted components. This study shows that the vBcl-2 proteins of KSHV and RRV differ from other vBcl-2 proteins in that they are essential for viral replication. The essential function is separate from the apoptosis and autophagy inhibiting activity but correlates with an unusual localization within the cell nucleus, suggesting that these proteins exert a novel function in the nucleus.

  • Matrix Metalloproteinase Activity in Infections by an Encephalitic Virus, Mouse Adenovirus Type 1 [PublishAheadOfPrint]

  • Mouse adenovirus type 1 (MAV-1) infection causes encephalitis in susceptible strains of mice and alters the permeability of infected brains to small molecules, which indicates disruption of the blood-brain barrier (BBB). In pathologic conditions, matrix metalloproteinases (MMPs) can disrupt the BBB through their proteolytic activity on basement membrane and tight junction proteins. We examined whether MAV-1 infection alters MMP activity in vivo and in vitro. Infected MAV-1-susceptible SJL mice had higher MMP2 and MMP9 activity in brains, measured by gelatin zymography, than mock infected mice. Infected MAV-1-resistant BALB/c mice had MMP activity levels equivalent to mock infection. Primary SJL mouse brain endothelial cells (a target of MAV-1 in vivo) infected ex vivo with MAV-1 had no difference in activity of secreted MMP2 or MMP9 from mock cells. We showed for the first time that astrocytes and microglia are also infected in vivo by MAV-1. Infected mixed primary cultures of astrocytes and microglia had higher levels of MMP2 and MMP9 activity than mock infection. These results indicate that increased MMP activity in the brains of MAV-1-infected susceptible mice may be due to MMP activity produced by endothelial cells, astrocytes, and microglia, which in turn may contribute to BBB disruption and encephalitis in susceptible mice.

    IMPORTANCE RNA and DNA viruses can cause encephalitis; in some cases this is accompanied by MMP-mediated disruption of the BBB. Activated MMPs degrade extracellular matrix and cleave tight junction proteins and cytokines, modulating their function. MAV-1 infection of susceptible mice is a tractable small animal model for encephalitis, and the virus causes disruption of the BBB. We showed that MAV-1 infection increases enzymatic activity of two key MMPs known to be secreted and activated in neuroinflammation, MMP2 and MMP9, in brains of susceptible mice. MAV-1 infects endothelial cells, astrocytes, and microglia, cell types in the neurovascular unit that can secrete MMPs. Ex vivo MAV-1 infection of these cell types caused higher MMP activity than mock infection, suggesting that they may contribute to higher MMP activity seen in vivo. To our knowledge, this provides the first evidence of an encephalitic DNA virus in its natural host causing increased MMP activity in brains.

  • Generation of a genetically stable high-fidelity influenza vaccine strain [PublishAheadOfPrint]

  • Vaccination is considered the most effective preventive means for influenza control. The development of a master virus with high growth and genetic stability, which may be used for the preparation of vaccine viruses by gene reassortment, is crucial for the enhancement of vaccine performance and efficiency of production. Here, we describe the generation of a high-fidelity and high-growth influenza vaccine master virus strain with a single V43I amino acid change in the PB1 polymerase of the high-growth PR8 master virus. The PB1-V43I mutation was introduced to increase replication fidelity, in order to design an H1N1 vaccine strain with low error rate. The PR8-PB1-V43I virus exhibited good replication compared with the parent PR8 virus. In order to compare the efficiency of egg-adaptation and occurrence of gene mutations leading to antigenic alterations, we constructed genetic 6:2 genetic reassortant viruses between A(H1N1)pdm09 and the PR8-PB1-V43I viruses; HA and NA were from the A(H1N1)pdm09 virus and the other genes from the PR8 virus. Mutations responsible for egg-adaptation mutations occurred in the HA of the PB1-V43I reassortant virus during serial egg passages; however, in contrast, antigenic mutations were introduced into the HA gene of the 6:2 reassortant virus possessing the wild-type PB1. This study shows that the mutant PR8 virus possessing the PB1 with V43I substitution may be utilized as a master virus for the generation of high-growth vaccine viruses with high polymerase fidelity, low error rates of gene replication, and reduced antigenic diversity during viral propagation in eggs, for vaccine production.

    IMPORTANCE Vaccination represents the most effective prophylactic option against influenza. The threat of emergence of influenza pandemics necessitates the ability to generate vaccine viruses rapidly. However, as the influenza virus exhibits a high mutation rate, vaccines must be updated to ensure a good match of the HA and NA antigens between vaccine and the circulating strain. Here, we generated a genetically stable master virus of A/Puerto Rico/8/1934(H1N1)-backbone encoding an engineered high-fidelity viral polymerase. Importantly, following the application of the high-fidelity PR8 backbone, no mutation resulting in antigenic change was introduced into the HA gene during propagation of the A(H1N1)pdm09 candidate vaccine virus. The low error rate of the present vaccine virus should decrease the risk of generation of mutant viruses with increased virulence. Therefore, our findings are expected to be useful for the development of pre-pandemic vaccines and live attenuated vaccines with higher safety relative to present candidate vaccines.

  • Unravelling the armour of a killer: Evasion of host defence's by African swine fever virus [PublishAheadOfPrint]

  • African swine fever is an acute haemorrhagic disease of pigs. Extensive recent spread in the Russian Federation and Eastern Europe has increased the risk to global pig production. The virus is a large DNA virus and is the only member of the Asfarviridae family. In pigs the virus replicates predominantly in macrophages. We review how the virus overcomes the barriers to replication in the macrophage and the virus mechanism to inhibit key host defence pathways.

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