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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]

  • Identification of a Functionally Relevant Adeno-Associated Virus Rep68 Oligomeric Interface [Genome Replication and Regulation of Viral Gene Expression]

  • The life cycle of the human parvovirus adeno-associated virus (AAV) is orchestrated by four Rep proteins. The large Rep proteins, Rep78 and Rep68, are remarkably multifunctional and display a range of biochemical activities, including DNA binding, nicking, and unwinding. Functionally, Rep78 and Rep68 are involved in transcriptional regulation, DNA replication, and genomic integration. Structurally, the Rep proteins share an AAA+ domain characteristic of superfamily 3 helicases, with the large Rep proteins additionally containing an N-terminal origin-binding domain (OBD) that specifically binds and nicks DNA. The combination of these domains, coupled with dynamic oligomerization properties, is the basis for the remarkable multifunctionality displayed by Rep68 and Rep78 during the AAV life cycle. In this report, we describe an oligomeric interface formed by Rep68 and demonstrate how disruption of this interface has drastic effects on both the oligomerization and functionality of the Rep proteins. Our results support a role for the four-helix bundle in the helicase domain of Rep68 as a bona fide oligomerization domain (OD). We have identified key residues in the OD that are critical for the stabilization of the Rep68-Rep68 interface; mutation of these key residues disrupts the enzymatic activities of Rep68, including DNA binding and nicking, and compromises viral DNA replication and transcriptional regulation of the viral promoters. Taken together, our data contribute to our understanding of the dynamic and substrate-responsive Rep78/68 oligomerization that is instrumental in the regulation of the DNA transitions that take place during the AAV life cycle.

    IMPORTANCE The limited genome size of small viruses has driven the evolution of highly multifunctional proteins that integrate different domains and enzymatic activities within a single polypeptide. The Rep68 protein from adeno-associated virus (AAV) combines a DNA binding and endonuclease domain with a helicase-ATPase domain, which together support DNA replication, transcriptional regulation, and site-specific integration. The coordination of the enzymatic activities of Rep68 remains poorly understood; however, Rep68 oligomerization and Rep68-DNA interactions have been suggested to play a crucial role. We investigated the determinants of Rep68 oligomerization and identified a hydrophobic interface necessary for Rep68 activity during the AAV life cycle. Our results provide new insights into the molecular mechanisms underlying the regulation of the versatile Rep proteins. Efficient production of AAV-based gene therapy vectors requires optimal Rep expression levels, and studies such as the one presented here could contribute to further optimization of AAV production schemes.

  • Structural and Biochemical Analyses of Swine Major Histocompatibility Complex Class I Complexes and Prediction of the Epitope Map of Important Influenza A Virus Strains [Structure and Assembly]

  • The lack of a peptide-swine leukocyte antigen class I (pSLA I) complex structure presents difficulties for the study of swine cytotoxic T lymphocyte (CTL) immunity and molecule vaccine development to eliminate important swine viral diseases, such as influenza A virus (IAV). Here, after cloning and comparing 28 SLA I allelic genes from Chinese Heishan pigs, pSLA-3*hs0202 was crystalized and solved. SLA-3*hs0202 binding with sbbeta;2m and a KMNTQFTAV (hemagglutinin [HA]-KMN9) peptide from the 2009 pandemic swine H1N1 strain clearly displayed two distinct conformations with HA-KMN9 peptides in the structures, which are believed to be beneficial to stimulate a broad spectrum of CTL immune responses. Notably, we found that different HA-KMN9 conformations are caused, not only by the flexibility of the side chains of residues in the peptide-binding groove (PBG), but also by the skewing of aalpha;1 and aalpha;2 helixes forming the PBG. In addition, alanine scanning and circular-dichroism (CD) spectra confirmed that the B, D, and F pockets play critical biochemical roles in determining the peptide-binding motif of SLA-3*hs0202. Based on biochemical parameters and comparisons to similar pockets in other known major histocompatibility complex class I (MHC-I) structures, the fundamental motif for SLA-3*hs0202 was determined to be X-(M/A/R)-(N/Q/R/F)-X-X-X-X-X-(V/I) by refolding in vitro and multiple mutant peptides. Finally, 28 SLA-3*hs0202-restricted epitope candidates were identified from important IAV strains, and two of them have been found in humans as HLA-A*0201-specific IAV epitopes. Structural and biochemical illumination of pSLA-3*hs0202 can benefit vaccine development to control IAV in swine.

    IMPORTANCE We crystalized and solved the first SLA-3 structure, SLA-3*hs0202, and found that it could present the same IAV peptide with two distinct conformations. Unlike previous findings showing that variable peptide conformations are caused only by the flexibility of the side chains in the groove, the skewing of the aalpha;1 and aalpha;2 helixes is important in the different peptide conformations in SLA-3*hs0202. We also determined the fundamental motif for SLA-3*hs0202 to be X-(M/A/R)-(N/Q/R/F)-X-X-X-X-X-(V/I) based on a series of structural and biochemical analyses, and 28 SLA-3*hs0202-restricted epitope candidates were identified from important IAV strains. We believe our structure and analyses of pSLA-3*hs0202 can benefit vaccine development to control IAV in swine.

  • HSP90 Chaperoning in Addition to Phosphoprotein Required for Folding but Not for Supporting Enzymatic Activities of Measles and Nipah Virus L Polymerases [Virus-Cell Interactions]

  • Nonsegmented negative-stranded RNA viruses, or members of the order Mononegavirales, share a conserved gene order and the use of elaborate transcription and replication machinery made up of at least four molecular partners. These partners have coevolved with the acquisition of the permanent encapsidation of the entire genome by the nucleoprotein (N) and the use of this N-RNA complex as a template for the viral polymerase composed of the phosphoprotein (P) and the large enzymatic protein (L). Not only is P required for polymerase function, but it also stabilizes the L protein through an unknown underlying molecular mechanism. By using NVP-AUY922 and/or 17-dimethylaminoethylamino-17-demethoxygeldanamycin as specific inhibitors of cellular heat shock protein 90 (HSP90), we found that efficient chaperoning of L by HSP90 requires P in the measles, Nipah, and vesicular stomatitis viruses. While the production of P remains unchanged in the presence of HSP90 inhibitors, the production of soluble and functional L requires both P and HSP90 activity. Measles virus P can bind the N terminus of L in the absence of HSP90 activity. Both HSP90 and P are required for the folding of L, as evidenced by a luciferase reporter insert fused within measles virus L. HSP90 acts as a true chaperon; its activity is transient and dispensable for the activity of measles and Nipah virus polymerases of virion origin. That the cellular chaperoning of a viral polymerase into a soluble functional enzyme requires the assistance of another viral protein constitutes a new paradigm that seems to be conserved within the Mononegavirales order.

    IMPORTANCE Viruses are obligate intracellular parasites that require a cellular environment for their replication. Some viruses particularly depend on the cellular chaperoning apparatus. We report here that for measles virus, successful chaperoning of the viral L polymerase mediated by heat shock protein 90 (HSP90) requires the presence of the viral phosphoprotein (P). Indeed, while P protein binds to the N terminus of L independently of HSP90 activity, both HSP90 and P are required to produce stable, soluble, folded, and functional L proteins. Once formed, the mature P+L complex no longer requires HSP90 to exert its polymerase functions. Such a new paradigm for the maturation of a viral polymerase appears to be conserved in several members of the Mononegavirales order, including the Nipah and vesicular stomatitis viruses.

  • Complete Unique Genome Sequence, Expression Profile, and Salivary Gland Tissue Tropism of the Herpesvirus 7 Homolog in Pigtailed Macaques [Genetic Diversity and Evolution]

  • Human herpesvirus 6A (HHV-6A), HHV-6B, and HHV-7 are classified as roseoloviruses and are highly prevalent in the human population. Roseolovirus reactivation in an immunocompromised host can cause severe pathologies. While the pathogenic potential of HHV-7 is unclear, it can reactivate HHV-6 from latency and thus contributes to severe pathological conditions associated with HHV-6. Because of the ubiquitous nature of roseoloviruses, their roles in such interactions and the resulting pathological consequences have been difficult to study. Furthermore, the lack of a relevant animal model for HHV-7 infection has hindered a better understanding of its contribution to roseolovirus-associated diseases. Using next-generation sequencing analysis, we characterized the unique genome of an uncultured novel pigtailed macaque roseolovirus. Detailed genomic analysis revealed the presence of gene homologs to all 84 known HHV-7 open reading frames. Phylogenetic analysis confirmed that the virus is a macaque homolog of HHV-7, which we have provisionally named Macaca nemestrina herpesvirus 7 (MneHV7). Using high-throughput RNA sequencing, we observed that the salivary gland tissue samples from nine different macaques had distinct MneHV7 gene expression patterns and that the overall number of viral transcripts correlated with viral loads in parotid gland tissue and saliva. Immunohistochemistry staining confirmed that, like HHV-7, MneHV7 exhibits a natural tropism for salivary gland ductal cells. We also observed staining for MneHV7 in peripheral nerve ganglia present in salivary gland tissues, suggesting that HHV-7 may also have a tropism for the peripheral nervous system. Our data demonstrate that MneHV7-infected macaques represent a relevant animal model that may help clarify the causality between roseolovirus reactivation and diseases.

    IMPORTANCE Human herpesvirus 6A (HHV-6A), HHV-6B, and HHV-7 are classified as roseoloviruses. We have recently discovered that pigtailed macaques are naturally infected with viral homologs of HHV-6 and HHV-7, which we provisionally named MneHV6 and MneHV7, respectively. In this study, we confirm that MneHV7 is genetically and biologically similar to its human counterpart, HHV-7. We determined the complete unique MneHV7 genome sequence and provide a comprehensive annotation of all genes. We also characterized viral transcription profiles in salivary glands from naturally infected macaques. We show that broad transcriptional activity across most of the viral genome is associated with high viral loads in infected parotid glands and that late viral protein expression is detected in salivary duct cells and peripheral nerve ganglia. Our study provides new insights into the natural behavior of an extremely prevalent virus and establishes a basis for subsequent investigations of the mechanisms that cause HHV-7 reactivation and associated disease.

  • Identification and Characterization of a Ribose 2'-O-Methyltransferase Encoded by the Ronivirus Branch of Nidovirales [Virus-Cell Interactions]

  • The order Nidovirales currently comprises four virus families: Arteriviridae, Coronaviridae (divided into the subfamilies Coronavirinae and Torovirinae), Roniviridae, and the recently recognized Mesoniviridae. RNA cap formation and methylation have been best studied for coronaviruses, with emphasis on the identification and characterization of two virus-encoded methyltransferases (MTases) involved in RNA capping, a guanine-N7-MTase and a ribose-2'-O-MTase. Although bioinformatics analyses suggest that these MTases may also be encoded by other nidoviruses with large genomes, such as toroviruses and roniviruses, no experimental evidence has been reported thus far. In this study, we show that a ronivirus, gill-associated virus (GAV), encodes the 2'-O-MTase activity, although we could not detect 2'-O-MTase activity for the homologous protein of a torovirus, equine torovirus, which is more closely related to coronaviruses. Like the coronavirus 2'-O-MTase, the roniviral 2'-O-MTase harbors a catalytic K-D-K-E tetrad that is conserved among 2'-O-MTases and can target only the N7-methylated cap structure of adenylate-primed RNA substrates. However, in contrast with the coronavirus protein, roniviral 2'-O-MTase does not require a protein cofactor for stimulation of its activity and differs in its preference for several biochemical parameters, such as reaction temperature and pH. Furthermore, the ronivirus 2'-O-MTase can be targeted by MTase inhibitors. These results extend our current understanding of nidovirus RNA cap formation and methylation beyond the coronavirus family.

    IMPORTANCE Methylation of the 5'-cap structure of viral RNAs plays important roles in genome replication and evasion of innate recognition of viral RNAs by cellular sensors. It is known that coronavirus nsp14 acts as an N7-(guanine)-methyltransferase (MTase) and nsp16 as a 2'-O-MTase, which are involved in the modification of RNA cap structure. However, these enzymatic activities have not been shown for any other nidoviruses beyond coronaviruses in the order Nidovirales. In this study, we identified a 2'-O-methyltransferase encoded by ronivirus that shows common and unique features in comparison with that of coronaviruses. Ronivirus 2'-O-MTase does not need a protein cofactor for MTase activity, whereas coronavirus nsp16 needs the stimulating factor nsp10 for its full activity. The conserved K-D-K-E catalytic tetrad is identified in ronivirus 2'-O-MTase. These results extend our understanding of nidovirus RNA capping and methylation beyond coronaviruses and also strengthen the evolutionary and functional links between roniviruses and coronaviruses.

  • Bile Acids Act as Soluble Host Restriction Factors Limiting Cytomegalovirus Replication in Hepatocytes [Virus-Cell Interactions]

  • The liver constitutes a prime site of cytomegalovirus (CMV) replication and latency. Hepatocytes produce, secrete, and recycle a chemically diverse set of bile acids, with the result that interactions between bile acids and cytomegalovirus inevitably occur. Here we determined the impact of naturally occurring bile acids on mouse CMV (MCMV) replication. In primary mouse hepatocytes, physiological concentrations of taurochenodeoxycholic acid (TCDC), glycochenodeoxycholic acid, and to a lesser extent taurocholic acid significantly reduced MCMV-induced gene expression and diminished the generation of virus progeny, while several other bile acids did not exert antiviral effects. The anticytomegalovirus activity required active import of bile acids via the sodium-taurocholate-cotransporting polypeptide (NTCP) and was consistently observed in hepatocytes but not in fibroblasts. Under conditions in which alpha interferon (IFN-aalpha;) lacks antiviral activity, physiological TCDC concentrations were similarly effective as IFN-. A detailed investigation of distinct steps of the viral life cycle revealed that TCDC deregulates viral transcription and diminishes global translation in infected cells.

    IMPORTANCE Cytomegaloviruses are members of the Betaherpesvirinae subfamily. Primary infection leads to latency, from which cytomegaloviruses can reactivate under immunocompromised conditions and cause severe disease manifestations, including hepatitis. The present study describes an unanticipated antiviral activity of conjugated bile acids on MCMV replication in hepatocytes. Bile acids negatively influence viral transcription and exhibit a global effect on translation. Our data identify bile acids as site-specific soluble host restriction factors against MCMV, which may allow rational design of anticytomegalovirus drugs using bile acids as lead compounds.

  • Initiation of Antiretroviral Therapy Restores CD4+ T Memory Stem Cell Homeostasis in Simian Immunodeficiency Virus-Infected Macaques [Pathogenesis and Immunity]

  • Treatment of human immunodeficiency virus (HIV) infection with antiretroviral therapy (ART) has significantly improved prognosis. Unfortunately, interruption of ART almost invariably results in viral rebound, attributed to a pool of long-lived, latently infected cells. Based on their longevity and proliferative potential, CD4+ T memory stem cells (TSCM) have been proposed as an important site of HIV persistence. In a previous study, we found that in simian immunodeficiency virus (SIV)-infected rhesus macaques (RM), CD4+ TSCM are preserved in number but show (i) a decrease in the frequency of CCR5+ cells, (ii) an expansion of the fraction of proliferating Ki-67+ cells, and (iii) high levels of SIV DNA. To understand the impact of ART on both CD4+ TSCM homeostasis and virus persistence, we conducted a longitudinal analysis of these cells in the blood and lymph nodes of 25 SIV-infected RM. We found that ART induced a significant restoration of CD4+ CCR5+ TSCM both in blood and in lymph nodes and a reduction in the fraction of proliferating CD4+ Ki-67+ TSCM in blood (but not lymph nodes). Importantly, we found that the level of SIV DNA in CD4+ transitional memory (TTM) and effector memory (TEM) T cells declined ~100-fold after ART in both blood and lymph nodes, while the level of SIV DNA in CD4+ TSCM and central memory T cells (TCM-) did not significantly change. These data suggest that ART is effective at partially restoring CD4+ TSCM homeostasis, and the observed stable level of virus in TSCM supports the hypothesis that these cells are a critical contributor to SIV persistence.

    IMPORTANCE Understanding the roles of various CD4+ T cell memory subsets in immune homeostasis and HIV/SIV persistence during antiretroviral therapy (ART) is critical to effectively treat and cure HIV infection. T memory stem cells (TSCM) are a unique memory T cell subset with enhanced self-renewal capacity and the ability to differentiate into other memory T cell subsets, such as central and transitional memory T cells (TCM and TTM, respectively). CD4+ TSCM are disrupted but not depleted during pathogenic SIV infection. We find that ART is partially effective at restoring CD4+ TSCM homeostasis and that SIV DNA harbored within this subset contracts more slowly than virus harbored in shorter-lived subsets, such as TTM and effector memory (TEM). Because of their ability to persist long-term in an individual, understanding the dynamics of virally infected CD4+ TSCM during suppressive ART is important for future therapeutic interventions aimed at modulating immune activation and purging the HIV reservoir.

  • HIV-1 Vpu Antagonizes CD317/Tetherin by Adaptor Protein-1-Mediated Exclusion from Virus Assembly Sites [Virus-Cell Interactions]

  • The host cell restriction factor CD317/tetherin traps virions at the surface of producer cells to prevent their release. The HIV-1 accessory protein Vpu antagonizes this restriction. Vpu reduces the cell surface density of the restriction factor and targets it for degradation; however, these activities are dispensable for enhancing particle release. Instead, Vpu has been suggested to antagonize CD317/tetherin by preventing recycling of internalized CD317/tetherin to the cell surface, blocking anterograde transport of newly synthesized CD317/tetherin, and/or displacing the restriction factor from virus assembly sites at the plasma membrane. At the molecular level, antagonism relies on the physical interaction of Vpu with CD317/tetherin. Recent findings suggested that phosphorylation of a diserine motif enables Vpu to bind to adaptor protein 1 (AP-1) trafficking complexes via two independent interaction motifs and to couple CD317/tetherin to the endocytic machinery. Here, we used a panel of Vpu proteins with specific mutations in individual interaction motifs to define which interactions are required for antagonism of CD317/tetherin. Impairing recycling or anterograde transport of CD317/tetherin to the plasma membrane was insufficient for antagonism. In contrast, excluding CD317/tetherin from HIV-1 assembly sites depended on Vpu motifs for interaction with AP-1 and CD317/tetherin and correlated with antagonism of the particle release restriction. Consistently, interference with AP-1 function or its expression blocked these Vpu activities. Our results define displacement from HIV-1 assembly sites as active principle of CD317/tetherin antagonism by Vpu and support a role of tripartite complexes between Vpu, AP-1, and CD317/tetherin in this process.

    IMPORTANCE CD317/tetherin poses an intrinsic barrier to human immunodeficiency virus type 1 (HIV-1) replication in human cells by trapping virus particles at the surface of producer cells and thereby preventing their release. The viral protein Vpu antagonizes this restriction, and molecular interactions with the restriction factor and adaptor protein complex 1 (AP-1) were suggested to mediate this activity. Vpu modulates intracellular trafficking of CD317/tetherin and excludes the restriction factor from HIV-1 assembly sites at the plasma membrane, but the relative contribution of these effects to antagonism remain elusive. Using a panel of Vpu mutants, as well as interference with AP-1 function and expression, we show here that Vpu antagonizes CD317/tetherin by blocking its recruitment to viral assembly sites in an AP-1-dependent manner. These results refine our understanding of the molecular mechanisms of CD317/tetherin antagonism and suggest complexes of Vpu with the restriction factor and AP-1 as targets for potential therapeutic intervention.

  • Arteriviruses, Pegiviruses, and Lentiviruses Are Common among Wild African Monkeys [Genetic Diversity and Evolution]

  • Nonhuman primates (NHPs) are a historically important source of zoonotic viruses and are a gold-standard model for research on many human pathogens. However, with the exception of simian immunodeficiency virus (SIV) (family Retroviridae), the blood-borne viruses harbored by these animals in the wild remain incompletely characterized. Here, we report the discovery and characterization of two novel simian pegiviruses (family Flaviviridae) and two novel simian arteriviruses (family Arteriviridae) in wild African green monkeys from Zambia (malbroucks [Chlorocebus cynosuros]) and South Africa (vervet monkeys [Chlorocebus pygerythrus]). We examine several aspects of infection, including viral load, genetic diversity, evolution, and geographic distribution, as well as host factors such as age, sex, and plasma cytokines. In combination with previous efforts to characterize blood-borne RNA viruses in wild primates across sub-Saharan Africa, these discoveries demonstrate that in addition to SIV, simian pegiviruses and simian arteriviruses are widespread and prevalent among many African cercopithecoid (i.e., Old World) monkeys.

    IMPORTANCE Primates are an important source of viruses that infect humans and serve as an important laboratory model of human virus infection. Here, we discover two new viruses in African green monkeys from Zambia and South Africa. In combination with previous virus discovery efforts, this finding suggests that these virus types are widespread among African monkeys. Our analysis suggests that one of these virus types, the simian arteriviruses, may have the potential to jump between different primate species and cause disease. In contrast, the other virus type, the pegiviruses, are thought to reduce the disease caused by human immunodeficiency virus (HIV) in humans. However, we did not observe a similar protective effect in SIV-infected African monkeys coinfected with pegiviruses, possibly because SIV causes little to no disease in these hosts.

  • p53 Is a Host Cell Regulator during Herpes Simplex Encephalitis [Pathogenesis and Immunity]

  • p53 is a critical host cell factor in the cellular response to a broad range of stress factors. We recently reported that p53 is required for efficient herpes simplex virus 1 (HSV-1) replication in cell culture. However, a defined role for p53 in HSV-1 replication and pathogenesis in vivo remains elusive. In this study, we examined the effects of p53 on HSV-1 infection in vivo using p53-deficient mice. Following intracranial inoculation, p53 knockout reduced viral replication in the brains of mice and led to significantly reduced rates of mortality due to herpes simplex encephalitis. These results suggest that p53 is an important host cell regulator of HSV-1 replication and pathogenesis in the central nervous system (CNS).

    IMPORTANCE HSV-1 causes sporadic cases of encephalitis, which, even with antiviral therapy, can result in severe neurological defects and even death. Many host cell factors involved in the regulation of CNS HSV-1 infection have been investigated using genetically modified mice. However, most of these factors are immunological regulators and act via immunological pathways in order to restrict CNS HSV-1 infection. They therefore provide limited information on intrinsic host cell regulators that may be involved in the facilitation of CNS HSV-1 infection. Here we demonstrate that a host cell protein, p53, which has generally been considered a host cell restriction factor for various viral infections, is required for efficient HSV-1 replication and pathogenesis in the CNS of mice. This is the first report showing that p53 positively regulates viral replication and pathogenesis in vivo and provides insights into its molecular mechanism, which may suggest novel clinical treatment options for herpes simplex encephalitis.

  • CD47 Plays a Role as a Negative Regulator in Inducing Protective Immune Responses to Vaccination against Influenza Virus [Vaccines and Antiviral Agents]

  • An integrin-associated protein CD47, which is a ligand for the inhibitory receptor signal regulatory protein aalpha;, is expressed on B and T cells, as well as on most innate immune cells. However, the roles of CD47 in the immune responses to viral infection or vaccination remain unknown. We investigated the role of CD47 in inducing humoral immune responses after intranasal infection with virus or immunization with influenza virus-like particles (VLPs). Virus infection or vaccination with VLPs containing hemagglutinin from A/PR8/34 influenza virus induced higher levels of antigen-specific IgG2c isotype dominant antibodies in CD47-deficient (CD47KO) mice than in wild-type (WT) mice. CD47KO mice with vaccination showed greater protective efficacy against lethal challenge, as evidenced by no loss in body weight and reduced lung viral titers compared to WT mice. In addition, inflammatory responses which include cytokine production, leukocyte infiltrates, and gamma interferon-producing CD4+ T cells, as well as an anti-inflammatory cytokine (interleukin-10), were reduced in the lungs of vaccinated CD47KO mice after challenge with influenza virus. Analysis of lymphocytes indicated that GL7+ germinal center B cells were induced at higher levels in the draining lymph nodes of CD47KO mice compared to those in WT mice. Notably, CD47KO mice exhibited significant increases in the numbers of antigen-specific memory B cells in spleens and plasma cells in bone marrow despite their lower levels of background IgG antibodies. These results suggest that CD47 plays a role as a negative regulator in inducing protective immune responses to influenza vaccination.

    IMPORTANCE Molecular mechanisms that control B cell activation to produce protective antibodies upon viral vaccination remain poorly understood. The CD47 molecule is known to be a ligand for the inhibitory receptor signal regulatory protein aalpha; and expressed on the surfaces of most immune cell types. CD47 was previously demonstrated to play an important role in modulating the migration of monocytes, neutrophils, polymorphonuclear neutrophils, and dendritic cells into the inflamed tissues. The results of this study demonstrate new roles of CD47 in negatively regulating the induction of protective IgG antibodies, germinal center B cells, and plasma cells secreting antigen-specific antibodies, as well as macrophages, upon influenza vaccination and challenge. As a consequence, vaccinated CD47-deficient mice demonstrated better control of influenza viral infection and enhanced protection. This study provides insights into understanding the regulatory functions of CD47 in inducing adaptive immunity to vaccination.

  • A Novel Open and Infectious Form of Echovirus 1 [Virus-Cell Interactions]

  • One of the hallmarks of enterovirus genome delivery is the formation of an uncoating intermediate particle. Based on previous studies of mostly heated picornavirus particles, intermediate particles were shown to have externalized the innermost capsid protein (VP4) and exposed the N terminus of VP1 and to have reduced infectivity. Here, in addition to the native and intact particle type, we have identified another type of infectious echovirus 1 (E1) particle population during infection. Our results show that E1 is slightly altered during entry, which leads to the broadening of the major virion peak in the sucrose gradient. In contrast, CsCl gradient separation revealed that in addition to the light intact and empty particles, a dense particle peak appeared during infection in cells. When the broad peak from the sucrose gradient was subjected to a CsCl gradient, it revealed light and dense particles, further suggesting that the shoulder represents the dense particle. The dense particle was permeable to SYBR green II, it still contained most of its VP4, and it was able to bind to its receptor aalpha;2bbeta;1 integrin and showed high infectivity. A thermal assay further showed that the aalpha;2bbeta;1 integrin binding domain (I-domain) stabilized the virus particle. Finally, heating E1 particles to superphysiological temperatures produced more fragile particles with aberrant ultrastructural appearances, suggesting that they are distinct from the dense E1 particles. These results describe a more open and highly infectious E1 particle that is naturally produced during infection and may represent a novel form of an uncoating intermediate.

    IMPORTANCE In this paper, we have characterized a possible uncoating intermediate particle of E1 that is produced in cells during infection. Before releasing their genome into the host cytosol, enteroviruses go through structural changes in their capsid, forming an uncoating intermediate particle. It was shown previously that structural changes can be induced by receptor interactions and, in addition, by heating the native virion to superphysiological temperatures. Here, we demonstrate that an altered, still infectious E1 particle is found during infection. This particle has a more open structure, and it cannot be formed by heating. It still contains the VP4 protein and is able to bind to its receptor and cause infection. Moreover, we show that in contrast to some other enteroviruses, the receptor-virion interaction has a stabilizing effect on E1. This paper highlights the differences between enterovirus species and further increases our understanding of various uncoating forms of enteroviruses.

  • Mosaic H5 Hemagglutinin Provides Broad Humoral and Cellular Immune Responses against Influenza Viruses [Vaccines and Antiviral Agents]

  • The most effective way to prevent influenza virus infection is via vaccination. However, the constant mutation of influenza viruses due to antigenic drift and shift compromises vaccine efficacy. This represents a major challenge to the development of a cross-protective vaccine that can protect against circulating viral antigenic diversity. Using the modified vaccinia Ankara (MVA) virus, we had previously generated a recombinant vaccine against highly pathogenic avian influenza virus (H5N1) based on an in silico mosaic approach. This MVA-H5M construct protected mice against multiple clades of H5N1 and H1N1 viruses. We have now further characterized the immune responses using immunodepletion of T cells and passive serum transfer, and these studies indicate that antibodies are the main contributors in homosubtypic protection (H5N1 clades). Compared to a MVA construct expressing hemagglutinin (HA) from influenza virus A/VN/1203/04 (MVA-HA), the MVA-H5M vaccine markedly increased and broadened B cell and T cell responses against H5N1 virus. The MVA-H5M also provided effective protection with no morbidity against H5N1 challenge, whereas MVA-HA-vaccinated mice showed clinical signs and experienced significant weight loss. In addition, MVA-H5M induced CD8+ T cell responses that play a major role in heterosubtypic protection (H1N1). Finally, expression of the H5M gene as either a DNA vaccine or a subunit protein protected mice against H5N1 challenge, indicating the effectiveness of the mosaic sequence without viral vectors for the development of a universal influenza vaccine.

    IMPORTANCE Influenza viruses infect up to one billion people around the globe each year and are responsible for 300,000 to 500,000 deaths annually. Vaccines are still the main intervention to prevent infection, but they fail to provide effective protection against heterologous strains of viruses. We developed broadly reactive H5N1 vaccine based on an in silico mosaic approach and previously demonstrated that modified vaccinia Ankara expressing an H5 mosaic hemagglutinin prevented infection with multiple clades of H5N1 and limited severe disease after H1N1 infection. Further characterization revealed that antibody responses and T cells are main contributors to protection against H5N1 and H1N1 viruses, respectively. The vaccine also broadens both T cell and B cell responses compared to native H5 vaccine from influenza virus A/Vietnam/1203/04. Finally, delivering the H5 mosaic as a DNA vaccine or as a purified protein demonstrated effective protection similar to the viral vector approach.

  • Uukuniemi Virus as a Tick-Borne Virus Model [Virus-Cell Interactions]

  • In the last decade, novel tick-borne pathogenic phleboviruses in the family Bunyaviridae, all closely related to Uukuniemi virus (UUKV), have emerged on different continents. To reproduce the tick-mammal switch in vitro, we first established a reverse genetics system to rescue UUKV with a genome close to that of the authentic virus isolated from the Ixodes ricinus tick reservoir. The IRE/CTVM19 and IRE/CTVM20 cell lines, both derived from I. ricinus, were susceptible to the virus rescued from plasmid DNAs and supported production of the virus over many weeks, indicating that infection was persistent. The glycoprotein GC was mainly highly mannosylated on tick cell-derived viral progeny. The second envelope viral protein, GN, carried mostly N-glycans not recognized by the classical glycosidases peptide-N-glycosidase F (PNGase F) and endoglycosidase H (Endo H). Treatment with bbeta;-mercaptoethanol did not impact the apparent molecular weight of GN. On viruses originating from mammalian BHK-21 cells, GN glycosylations were exclusively sensitive to PNGase F, and the electrophoretic mobility of the protein was substantially slower after the reduction of disulfide bonds. Furthermore, the amount of viral nucleoprotein per focus forming unit differed markedly whether viruses were produced in tick or BHK-21 cells, suggesting a higher infectivity for tick cell-derived viruses. Together, our results indicate that UUKV particles derived from vector tick cells have glycosylation and structural specificities that may influence the initial infection in mammalian hosts. This study also highlights the importance of working with viruses originating from arthropod vector cells in investigations of the cell biology of arbovirus transmission and entry into mammalian hosts.

    IMPORTANCE Tick-borne phleboviruses represent a growing threat to humans globally. Although ticks are important vectors of infectious emerging diseases, previous studies have mainly involved virus stocks produced in mammalian cells. This limitation tends to minimize the importance of host alternation in virus transmission to humans and initial infection at the molecular level. With this study, we have developed an in vitro tick cell-based model that allows production of the tick-borne Uukuniemi virus to high titers. Using this system, we found that virions derived from tick cells have specific structural properties and N-glycans that may enhance virus infectivity for mammalian cells. By shedding light on molecular aspects of tick-derived viral particles, our data illustrate the importance of considering the host switch in studying early virus-mammalian receptor/cell interactions. The information gained here lays the basis for future research on not only tick-borne phleboviruses but also all viruses and other pathogens transmitted by ticks.

  • Small-Molecule Fusion Inhibitors Bind the pH-Sensing Stable Signal Peptide-GP2 Subunit Interface of the Lassa Virus Envelope Glycoprotein [Vaccines and Antiviral Agents]

  • Arenavirus species are responsible for severe life-threatening hemorrhagic fevers in western Africa and South America. Without effective antiviral therapies or vaccines, these viruses pose serious public health and biodefense concerns. Chemically distinct small-molecule inhibitors of arenavirus entry have recently been identified and shown to act on the arenavirus envelope glycoprotein (GPC) to prevent membrane fusion. In the tripartite GPC complex, pH-dependent membrane fusion is triggered through a poorly understood interaction between the stable signal peptide (SSP) and the transmembrane fusion subunit GP2, and our genetic studies have suggested that these small-molecule inhibitors act at this interface to antagonize fusion activation. Here, we have designed and synthesized photoaffinity derivatives of the 4-acyl-1,6-dialkylpiperazin-2-one class of fusion inhibitors and demonstrate specific labeling of both the SSP and GP2 subunits in a native-like Lassa virus (LASV) GPC trimer expressed in insect cells. Photoaddition is competed by the parental inhibitor and other chemically distinct compounds active against LASV, but not those specific to New World arenaviruses. These studies provide direct physical evidence that these inhibitors bind at the SSP-GP2 interface. We also find that GPC containing the uncleaved GP1-GP2 precursor is not susceptible to photo-cross-linking, suggesting that proteolytic maturation is accompanied by conformational changes at this site. Detailed mapping of residues modified by the photoaffinity adducts may provide insight to guide the further development of these promising lead compounds as potential therapeutic agents to treat Lassa hemorrhagic fever.

    IMPORTANCE Hemorrhagic fever arenaviruses cause lethal infections in humans and, in the absence of licensed vaccines or specific antiviral therapies, are recognized to pose significant threats to public health and biodefense. Lead small-molecule inhibitors that target the arenavirus envelope glycoprotein (GPC) have recently been identified and shown to block GPC-mediated fusion of the viral and cellular endosomal membranes, thereby preventing virus entry into the host cell. Genetic studies suggest that these inhibitors act through a unique pH-sensing intersubunit interface in GPC, but atomic-level structural information is unavailable. In this report, we utilize novel photoreactive fusion inhibitors and photoaffinity labeling to obtain direct physical evidence for inhibitor binding at this critical interface in Lassa virus GPC. Future identification of modified residues at the inhibitor-binding site will help elucidate the molecular basis for fusion activation and its inhibition and guide the development of effective therapies to treat arenaviral hemorrhagic fevers.

  • Cell-to-Cell Contact and Nectin-4 Govern Spread of Measles Virus from Primary Human Myeloid Cells to Primary Human Airway Epithelial Cells [Virus-Cell Interactions]

  • Measles is a highly contagious, acute viral illness. Immune cells within the airways are likely first targets of infection, and these cells traffic measles virus (MeV) to lymph nodes for amplification and subsequent systemic dissemination. Infected immune cells are thought to return MeV to the airways; however, the mechanisms responsible for virus transfer to pulmonary epithelial cells are poorly understood. To investigate this process, we collected blood from human donors and generated primary myeloid cells, specifically, monocyte-derived macrophages (MDMs) and dendritic cells (DCs). MDMs and DCs were infected with MeV and then applied to primary cultures of well-differentiated airway epithelial cells from human donors (HAE). Consistent with previous results obtained with free virus, infected MDMs or DCs were incapable of transferring MeV to HAE when applied to the apical surface. Likewise, infected MDMs or DCs applied to the basolateral surface of HAE grown on small-pore (0.4-mmu;m) support membranes did not transfer virus. In contrast, infected MDMs and DCs applied to the basolateral surface of HAE grown on large-pore (3.0-mmu;m) membranes successfully transferred MeV. Confocal microscopy demonstrated that MDMs and DCs are capable of penetrating large-pore membranes but not small-pore membranes. Further, by using a nectin-4 blocking antibody or recombinant MeV unable to enter cells through nectin-4, we demonstrated formally that transfer from immune cells to HAE occurs in a nectin-4-dependent manner. Thus, both infected MDMs and DCs rely on cell-to-cell contacts and nectin-4 to efficiently deliver MeV to the basolateral surface of HAE.

    IMPORTANCE Measles virus spreads rapidly and efficiently in human airway epithelial cells. This rapid spread is based on cell-to-cell contact rather than on particle release and reentry. Here we posit that MeV transfer from infected immune cells to epithelial cells also occurs by cell-to-cell contact rather than through cell-free particles. In addition, we sought to determine which immune cells transfer MeV infectivity to the human airway epithelium. Our studies are based on two types of human primary cells: (i) myeloid cells generated from donated blood and (ii) well-differentiated airway epithelial cells derived from donor lungs. We show that different types of myeloid cells, i.e., monocyte-derived macrophages and dendritic cells, transfer infection to airway epithelial cells. Furthermore, cell-to-cell contact is an important component of successful MeV transfer. Our studies elucidate a mechanism by which the most contagious human respiratory virus is delivered to the airway epithelium.

  • CD8+ T Cell Breadth and Ex Vivo Virus Inhibition Capacity Distinguish between Viremic Controllers with and without Protective HLA Class I Alleles [Cellular Response to Infection]

  • The mechanisms of viral control and loss of viral control in chronically infected individuals with or without protective HLA class I alleles are not fully understood. We therefore characterized longitudinally the immunological and virological features that may explain divergence in disease outcome in 70 HIV-1 C-clade-infected antiretroviral therapy (ART)-naive South African adults, 35 of whom possessed protective HLA class I alleles. We demonstrate that, over 5 years of longitudinal study, 35% of individuals with protective HLA class I alleles lost viral control compared to none of the individuals without protective HLA class I alleles (P = 0.06). Sustained HIV-1 control in patients with protective HLA class I alleles was characteristically related to the breadth of HIV-1 CD8+ T cell responses against Gag and enhanced ability of CD8+ T cells to suppress viral replication ex vivo. In some cases, loss of virological control was associated with reduction in the total breadth of CD8+ T cell responses in the absence of differences in HIV-1-specific CD8+ T cell polyfunctionality or proliferation. In contrast, viremic controllers without protective HLA class I alleles possessed reduced breadth of HIV-1-specific CD8+ T cell responses characterized by reduced ability to suppress viral replication ex vivo. These data suggest that the control of HIV-1 in individuals with protective HLA class I alleles may be driven by broad CD8+ T cell responses with potent viral inhibitory capacity while control among individuals without protective HLA class I alleles may be more durable and mediated by CD8+ T cell-independent mechanisms.

    IMPORTANCE Host mechanisms of natural HIV-1 control are not fully understood. In a longitudinal study of antiretroviral therapy (ART)-naive individuals, we show that those with protective HLA class I alleles subsequently experienced virologic failure compared to those without protective alleles. Among individuals with protective HLA class I alleles, viremic control was associated with broad CD8+ T cells that targeted the Gag protein, and CD8+ T cells from these individuals exhibited superior virus inhibition capacity. In individuals without protective HLA class I alleles, HIV-1-specific CD8+ T cell responses were narrow and poorly inhibited virus replication. These results suggest that broad, highly functional cytotoxic T cells (cytotoxic T lymphocytes [CTLs]) against the HIV-1 Gag protein are associated with control among those with protective HLA class I alleles and that loss of these responses eventually leads to viremia. A subset of individuals appears to have alternative, non-CTL mechanisms of viral control. These controllers may hold the key to an effective HIV vaccine.

  • ISG12a Restricts Hepatitis C Virus Infection through the Ubiquitination-Dependent Degradation Pathway [Cellular Response to Infection]

  • Interferons (IFNs) restrict various kinds of viral infection via induction of hundreds of IFN-stimulated genes (ISGs), while the functions of the majority of ISGs are broadly unclear. Here, we show that a high-IFN-inducible gene, ISG12a (also known as IFI27), exhibits a nonapoptotic antiviral effect on hepatitis C virus (HCV) infection. Viral NS5A protein is targeted specifically by ISG12a, which mediates NS5A degradation via a ubiquitination-dependent proteasomal pathway. K374R mutation in NS5A domain III abrogates ISG12a-induced ubiquitination and degradation of NS5A. S-phase kinase-associated protein 2 (SKP2) is identified as an ubiquitin E3 ligase for NS5A. ISG12a functions as a crucial adaptor that promotes SKP2 to interact with and degrade viral protein. Moreover, the antiviral effect of ISG12a is dependent on the E3 ligase activity of SKP2. These findings uncover an intriguing mechanism by which ISG12a restricts viral infection and provide clues for understanding the actions of innate immunity.

    IMPORTANCE Upon virus invasion, IFNs induce numerous ISGs to control viral spread, while the functions of the majority of ISGs are broadly unclear. The present study shows a novel antiviral mechanism of ISGs and elucidated that ISG12a recruits an E3 ligase, SKP2, for ubiquitination and degradation of viral protein and restricts viral infection. These findings provide important insights into exploring the working principles of innate immunity.

  • Identification of Diverse Mycoviruses through Metatranscriptomics Characterization of the Viromes of Five Major Fungal Plant Pathogens [Genetic Diversity and Evolution]

  • Mycoviruses can have a marked effect on natural fungal communities and influence plant health and productivity. However, a comprehensive picture of mycoviral diversity is still lacking. To characterize the viromes of five widely dispersed plant-pathogenic fungi, Colletotrichum truncatum, Macrophomina phaseolina, Diaporthe longicolla, Rhizoctonia solani, and Sclerotinia sclerotiorum, a high-throughput sequencing-based metatranscriptomic approach was used to detect viral sequences. Total RNA and double-stranded RNA (dsRNA) from mycelia and RNA from samples enriched for virus particles were sequenced. Sequence data were assembled de novo, and contigs with predicted amino acid sequence similarities to viruses in the nonredundant protein database were selected. The analysis identified 72 partial or complete genome segments representing 66 previously undescribed mycoviruses. Using primers specific for each viral contig, at least one fungal isolate was identified that contained each virus. The novel mycoviruses showed affinity with 15 distinct lineages: Barnaviridae, Benyviridae, Chrysoviridae, Endornaviridae, Fusariviridae, Hypoviridae, Mononegavirales, Narnaviridae, Ophioviridae, Ourmiavirus, Partitiviridae, Tombusviridae, Totiviridae, Tymoviridae, and Virgaviridae. More than half of the viral sequences were predicted to be members of the Mitovirus genus in the family Narnaviridae, which replicate within mitochondria. Five viral sequences showed strong affinity with three families (Benyviridae, Ophioviridae, and Virgaviridae) that previously contained no mycovirus species. The genomic information provides insight into the diversity and taxonomy of mycoviruses and coevolution of mycoviruses and their fungal hosts.

    IMPORTANCE Plant-pathogenic fungi reduce crop yields, which affects food security worldwide. Plant host resistance is considered a sustainable disease management option but may often be incomplete or lacking for some crops to certain fungal pathogens or strains. In addition, the rising issues of fungicide resistance demand alternative strategies to reduce the negative impacts of fungal pathogens. Those fungus-infecting viruses (mycoviruses) that attenuate fungal virulence may be welcome additions for mitigation of plant diseases. By high-throughput sequencing of the RNAs from 275 isolates of five fungal plant pathogens, 66 previously undescribed mycoviruses were identified. In addition to identifying new potential biological control agents, these results expand the grand view of the diversity of mycoviruses and provide possible insights into the importance of intracellular and extracellular transmission in fungus-virus coevolution.

  • Both cis and trans Activities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication [Genome Replication and Regulation of Viral Gene Expression]

  • The Picornaviridae is a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a coordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided in trans (i.e., via expression from a separate RNA molecule), while others are required in cis (i.e., expressed from the template RNA molecule). In vitro studies have suggested that multiple copies of the RNA-dependent RNA polymerase (RdRp) 3D are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically active 3D molecules and those that build a replication complex. We report a novel nonenzymatic cis-acting function of 3D that is essential for viral-genome replication. Using an FMDV replicon in complementation experiments, our data demonstrate that this cis-acting role of 3D is distinct from the catalytic activity, which is predominantly trans acting. Immunofluorescence studies suggest that both cis- and trans-acting 3D molecules localize to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts in cis with RNA stem-loops that are essential for viral RNA replication. This study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further.

    IMPORTANCE Foot-and-mouth disease virus (FMDV) is an important animal pathogen responsible for foot-and-mouth disease. The disease is endemic in many parts of the world with outbreaks within livestock resulting in major economic losses. Propagation of the viral genome occurs within replication complexes, and understanding this process can facilitate the development of novel therapeutic strategies. Many of the nonstructural proteins involved in replication possess multiple functions in the viral life cycle, some of which can be supplied to the replication complex from a separate genome (i.e., in trans) while others must originate from the template (i.e., in cis). Here, we present an analysis of cis and trans activities of the RNA-dependent RNA polymerase 3D. We demonstrate a novel cis-acting role of 3D in replication. Our data suggest that this role is distinct from its enzymatic functions and requires interaction with the viral genome. Our data further the understanding of genome replication of this important pathogen.

  • Measurements of Intrahost Viral Diversity Are Extremely Sensitive to Systematic Errors in Variant Calling [Genetic Diversity and Evolution]

  • With next-generation sequencing technologies, it is now feasible to efficiently sequence patient-derived virus populations at a depth of coverage sufficient to detect rare variants. However, each sequencing platform has characteristic error profiles, and sample collection, target amplification, and library preparation are additional processes whereby errors are introduced and propagated. Many studies account for these errors by using ad hoc quality thresholds and/or previously published statistical algorithms. Despite common usage, the majority of these approaches have not been validated under conditions that characterize many studies of intrahost diversity. Here, we use defined populations of influenza virus to mimic the diversity and titer typically found in patient-derived samples. We identified single-nucleotide variants using two commonly employed variant callers, DeepSNV and LoFreq. We found that the accuracy of these variant callers was lower than expected and exquisitely sensitive to the input titer. Small reductions in specificity had a significant impact on the number of minority variants identified and subsequent measures of diversity. We were able to increase the specificity of DeepSNV to ggt;99.95% by applying an empirically validated set of quality thresholds. When applied to a set of influenza virus samples from a household-based cohort study, these changes resulted in a 10-fold reduction in measurements of viral diversity. We have made our sequence data and analysis code available so that others may improve on our work and use our data set to benchmark their own bioinformatics pipelines. Our work demonstrates that inadequate quality control and validation can lead to significant overestimation of intrahost diversity.

    IMPORTANCE Advances in sequencing technology have made it feasible to sequence patient-derived viral samples at a level sufficient for detection of rare mutations. These high-throughput, cost-effective methods are revolutionizing the study of within-host viral diversity. However, the techniques are error prone, and the methods commonly used to control for these errors have not been validated under the conditions that characterize patient-derived samples. Here, we show that these conditions affect measurements of viral diversity. We found that the accuracy of previously benchmarked analysis pipelines was greatly reduced under patient-derived conditions. By carefully validating our sequencing analysis using known control samples, we were able to identify biases in our method and to improve our accuracy to acceptable levels. Application of our modified pipeline to a set of influenza virus samples from a cohort study provided a realistic picture of intrahost diversity and suggested the need for rigorous quality control in such studies.

  • Dynein Regulators Are Important for Ecotropic Murine Leukemia Virus Infection [Virus-Cell Interactions]

  • During the early steps of infection, retroviruses must direct the movement of the viral genome into the nucleus to complete their replication cycle. This process is mediated by cellular proteins that interact first with the reverse transcription complex and later with the preintegration complex (PIC), allowing it to reach and enter the nucleus. For simple retroviruses, such as murine leukemia virus (MLV), the identities of the cellular proteins involved in trafficking of the PIC in infection are unknown. To identify cellular proteins that interact with the MLV PIC, we developed a replication-competent MLV in which the integrase protein was tagged with a FLAG epitope. Using a combination of immunoprecipitation and mass spectrometry, we established that the microtubule motor dynein regulator DCTN2/p50/dynamitin interacts with the MLV preintegration complex early in infection, suggesting a direct interaction between the incoming viral particles and the dynein complex regulators. Further experiments showed that RNA interference (RNAi)-mediated silencing of either DCTN2/p50/dynamitin or another dynein regulator, NudEL, profoundly reduced the efficiency of infection by ecotropic, but not amphotropic, MLV reporters. We propose that the cytoplasmic dynein regulators are a critical component of the host machinery needed for infection by the retroviruses entering the cell via the ecotropic envelope pathway.

    IMPORTANCE Retroviruses must access the chromatin of host cells to integrate the viral DNA, but before this crucial event, they must reach the nucleus. The movement through the cytoplasmmmdash;a crowded environment where diffusion is slowmmdash;is thought to utilize retrograde transport along the microtubule network by the dynein complex. Different viruses use different components of this multisubunit complex. We found that the preintegration complex of murine leukemia virus (MLV) interacts with the dynein complex and that regulators of this complex are essential for infection. Our study provides the first insight into the requirements for retrograde transport of the MLV preintegration complex.

  • Stability of HIV Frameshift Site RNA Correlates with Frameshift Efficiency and Decreased Virus Infectivity [Structure and Assembly]

  • Human immunodeficiency virus (HIV) replication is strongly dependent upon a programmed ribosomal frameshift. Here we investigate the relationships between the thermodynamic stability of the HIV type 1 (HIV-1) RNA frameshift site stem-loop, frameshift efficiency, and infectivity, using pseudotyped HIV-1 and HEK293T cells. The data reveal a strong correlation between frameshift efficiency and local, but not overall, RNA thermodynamic stability. Mutations that modestly increase the local stability of the frameshift site RNA stem-loop structure increase frameshift efficiency 2-fold to 3-fold in cells. Thus, frameshift efficiency is determined by the strength of the thermodynamic barrier encountered by the ribosome. These data agree with previous in vitro measurements, suggesting that there are no virus- or host-specific factors that modulate frameshifting. The data also indicate that there are no sequence-specific requirements for the frameshift site stem-loop. A linear correlation between Gag-polymerase (Gag-Pol) levels in cells and levels in virions supports the idea of a stochastic virion assembly mechanism. We further demonstrate that the surrounding genomic RNA secondary structure influences frameshift efficiency and that a mutation that commonly arises in response to protease inhibitor therapy creates a functional but inefficient secondary slippery site. Finally, HIV-1 mutants with enhanced frameshift efficiencies are significantly less infectious, suggesting that compounds that increase frameshift efficiency by as little as 2-fold may be effective at suppressing HIV-1 replication.

    IMPORTANCE HIV, like many retroviruses, utilizes a nndash;1 programmed ribosomal frameshift to generate viral enzymes in the form of a Gag-Pol polyprotein precursor. Thus, frameshifting is essential for viral replication. Here, we utilized a panel of mutant HIV strains to demonstrate that in cells, frameshifting efficiency is correlated with the stability of the local thermodynamic barrier to ribosomal translocation. Increasing the stability of the frameshift site RNA increases the frameshift efficiency 2-fold to 3-fold. Mutant viruses with increased frameshift efficiencies have significantly reduced infectivity. These data suggest that this effect might be exploited in the development of novel antiviral strategies.

  • Capsid-CPSF6 Interaction Is Dispensable for HIV-1 Replication in Primary Cells but Is Selected during Virus Passage In Vivo [Virus-Cell Interactions]

  • Cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a host factor that interacts with the HIV-1 capsid (CA) protein, is implicated in diverse functions during the early part of the HIV-1 life cycle, including uncoating, nuclear entry, and integration targeting. Preservation of CA binding to CPSF6 in vivo suggests that this interaction is fine-tuned for efficient HIV-1 replication in physiologically relevant settings. Nevertheless, this possibility has not been formally examined. To assess the requirement for optimal CPSF6-CA binding during infection of primary cells and in vivo, we utilized a novel CA mutation, A77V, that significantly reduced CA binding to CPSF6. The A77V mutation rendered HIV-1 largely independent from TNPO3, NUP358, and NUP153 for infection and altered the integration site preference of HIV-1 without any discernible effects during the late steps of the virus life cycle. Surprisingly, the A77V mutant virus maintained the ability to replicate in monocyte-derived macrophages, primary CD4+ T cells, and humanized mice at a level comparable to that for the wild-type (WT) virus. Nonetheless, revertant viruses that restored the WT CA sequence and hence CA binding to CPSF6 emerged in three out of four A77V-infected animals. These results suggest that the optimal interaction of CA with CPSF6, though not absolutely essential for HIV-1 replication in physiologically relevant settings, confers a significant fitness advantage to the virus and thus is strictly conserved among naturally circulating HIV-1 strains.

    IMPORTANCE CPSF6 interacts with the HIV-1 capsid (CA) protein and has been implicated in nuclear entry and integration targeting. Preservation of CPSF6-CA binding across various HIV-1 strains suggested that the optimal interaction between CA and CPSF6 is critical during HIV-1 replication in vivo. Here, we identified a novel HIV-1 capsid mutant that reduces binding to CPSF6, is largely independent from the known cofactors for nuclear entry, and alters integration site preference. Despite these changes, virus carrying this mutation replicated in humanized mice at levels indistinguishable from those of the wild-type virus. However, in the majority of the animals, the mutant virus reverted back to the wild-type sequence, hence restoring the wild-type level of CA-CPSF6 interactions. These results suggest that optimal binding of CA to CPSF6 is not absolutely essential for HIV-1 replication in vivo but provides a fitness advantage that leads to the widespread usage of CPSF6 by HIV-1 in vivo.

  • A Role of Influenza Virus Exposure History in Determining Pandemic Susceptibility and CD8+ T Cell Responses [Pathogenesis and Immunity]

  • Novel influenza viruses often cause differential infection patterns across different age groups, an effect that is defined as heterogeneous demographic susceptibility. This occurred during the A/H2N2 pandemic, when children experienced higher influenza attack rates than adults. Since the recognition of conserved epitopes across influenza subtypes by CD8+ cytotoxic T lymphocytes (CTLs) limit influenza disease, we hypothesized that conservation of CTL antigenic peptides (Ag-p) in viruses circulating before the pH2N2-1957 may have resulted in differential CTL immunity. We compared viruses isolated in the years preceding the pandemic (1941 to 1957) to which children and adults were exposed to viruses circulating decades earlier (1918 to 1940), which could infect adults only. Consistent with phylogenetic models, influenza viruses circulating from 1941 to 1957, which infected children, shared with pH2N2 the majority (~89%) of the CTL peptides within the most immunogenic nucleoprotein, matrix 1, and polymerase basic 1, thus providing evidence for minimal pH2N2 CTL escape in children. Our study, however, identified potential CTL immune evasion from pH2N2 irrespective of age, within HLA-A*03:01+ individuals for PB1471-L473V/N476I variants and HLA-B*15:01+ population for NP404nndash;414-V408I mutant. Further experiments using the murine model of B-cell-deficient mice showed that multiple influenza infections resulted in superior protection from influenza-induced morbidity, coinciding with accumulation of tissue-resident memory CD8+ T cells in the lung. Our study suggests that protection against H2N2-1957 pandemic influenza was most likely linked to the number of influenza virus infections prior to the pandemic challenge rather than differential preexisting CTL immunity. Thus, the regimen of a CTL-based vaccine/vaccine-component may benefit from periodic boosting to achieve fully protective, asymptomatic influenza infection.

    IMPORTANCE Due to a lack of cross-reactive neutralizing antibodies, children are particularly susceptible to influenza infections caused by novel viral strains. Preexisting T cell immunity directed at conserved viral regions, however, can provide protection against influenza viruses, promote rapid recovery and better clinical outcomes. When we asked whether high susceptibility of children (compared to adults) to the pandemic H2N2 influenza strain was associated with immune evasion from T-cell immunity, we found high conservation within T-cell antigenic regions in pandemic H2N2. However, the number of influenza infections prior to the challenge was linked to protective, asymptomatic infections and establishment of tissue-resident memory T cells. Our study supports development of vaccines that prime and boost T cells to elicit cross-strain protective T cells, especially tissue-resident memory T cells, for lifelong immunity against distinct influenza viruses.

  • Visualization and Sequencing of Membrane Remodeling Leading to Influenza Virus Fusion [Structure and Assembly]

  • Protein-mediated membrane fusion is an essential step in many fundamental biological events, including enveloped virus infection. The nature of protein and membrane intermediates and the sequence of membrane remodeling during these essential processes remain poorly understood. Here we used cryo-electron tomography (cryo-ET) to image the interplay between influenza virus and vesicles with a range of lipid compositions. By following the population kinetics of membrane fusion intermediates imaged by cryo-ET, we found that membrane remodeling commenced with the hemagglutinin fusion protein spikes grappling onto the target membrane, followed by localized target membrane dimpling as local clusters of hemagglutinin started to undergo conformational refolding. The local dimples then transitioned to extended, tightly apposed contact zones where the two proximal membrane leaflets were in most cases indistinguishable from each other, suggesting significant dehydration and possible intermingling of the lipid head groups. Increasing the content of fusion-enhancing cholesterol or bis-monoacylglycerophosphate in the target membrane led to an increase in extended contact zone formation. Interestingly, hemifused intermediates were found to be extremely rare in the influenza virus fusion system studied here, most likely reflecting the instability of this state and its rapid conversion to postfusion complexes, which increased in population over time. By tracking the populations of fusion complexes over time, the architecture and sequence of membrane reorganization leading to efficient enveloped virus fusion were thus resolved.

    IMPORTANCE Enveloped viruses employ specialized surface proteins to mediate fusion of cellular and viral membranes that results in the formation of pores through which the viral genetic material is delivered to the cell. For influenza virus, the trimeric hemagglutinin (HA) glycoprotein spike mediates host cell attachment and membrane fusion. While structures of a subset of conformations and parts of the fusion machinery have been characterized, the nature and sequence of membrane deformations during fusion have largely eluded characterization. Building upon studies that focused on early stages of HA-mediated membrane remodeling, here cryo-electron tomography (cryo-ET) was used to image the three-dimensional organization of intact influenza virions at different stages of fusion with liposomes, leading all the way to completion of the fusion reaction. By monitoring the evolution of fusion intermediate populations over the course of acid-induced fusion, we identified the progression of membrane reorganization that leads to efficient fusion by an enveloped virus.

  • Generating Bona Fide Mammalian Prions with Internal Deletions [Prions]

  • Mammalian prions are PrP proteins with altered structures causing transmissible fatal neurodegenerative diseases. They are self-perpetuating through formation of beta-sheet-rich assemblies that seed conformational change of cellular PrP. Pathological PrP usually forms an insoluble protease-resistant core exhibiting beta-sheet structures but no more alpha-helical content, loosing the three alpha-helices contained in the correctly folded PrP. The lack of a high-resolution prion structure makes it difficult to understand the dynamics of conversion and to identify elements of the protein involved in this process. To determine whether completeness of residues within the protease-resistant domain is required for prions, we performed serial deletions in the helix H2 C terminus of ovine PrP, since this region has previously shown some tolerance to sequence changes without preventing prion replication. Deletions of either four or five residues essentially preserved the overall PrP structure and mutant PrP expressed in RK13 cells were efficiently converted into bona fide prions upon challenge by three different prion strains. Remarkably, deletions in PrP facilitated the replication of two strains that otherwise do not replicate in this cellular context. Prions with internal deletion were self-propagating and de novo infectious for naive homologous and wild-type PrP-expressing cells. Moreover, they caused transmissible spongiform encephalopathies in mice, with similar biochemical signatures and neuropathologies other than the original strains. Prion convertibility and transfer of strain-specific information are thus preserved despite shortening of an alpha-helix in PrP and removal of residues within prions. These findings provide new insights into sequence/structure/infectivity relationship for prions.

    IMPORTANCE Prions are misfolded PrP proteins that convert the normal protein into a replicate of their own abnormal form. They are responsible for invariably fatal neurodegenerative disorders. Other aggregation-prone proteins appear to have a prion-like mode of expansion in brains, such as in Alzheimer's or Parkinson's diseases. To date, the resolution of prion structure remains elusive. Thus, to genetically define the landscape of regions critical for prion conversion, we tested the effect of short deletions. We found that, surprisingly, removal of a portion of PrP, the C terminus of alpha-helix H2, did not hamper prion formation but generated infectious agents with an internal deletion that showed characteristics essentially similar to those of original infecting strains. Thus, we demonstrate that completeness of the residues inside prions is not necessary for maintaining infectivity and the main strain-specific information, while reporting one of the few if not the only bona fide prions with an internal deletion.

  • Effects of MF59 Adjuvant on Induction of Isotype-Switched IgG Antibodies and Protection after Immunization with T-Dependent Influenza Virus Vaccine in the Absence of CD4+ T Cells [Vaccines and Antiviral Agents]

  • CD4+ T cells play a central role in orchestrating adaptive immunity. To better understand the roles of CD4+ T cells in the effects of adjuvants, we investigated the efficacy of a T-dependent influenza virus split vaccine with MF59 or alum in CD4 knockout (CD4KO) and wild-type (WT) mice. CD4+ T cells were required for the induction of IgG antibody responses to the split vaccine and the effects of alum adjuvant. In contrast, MF59 was found to be highly effective in raising isotype-switched IgG antibodies to a T-dependent influenza virus split vaccine in CD4KO mice or CD4-depleted WT mice equivalent to those in intact WT mice, thus overcoming the deficiency of CD4+ T cells in helping B cells and inducing immunity against influenza virus. Vaccination with the MF59-adjuvanted influenza virus vaccine was able to induce protective CD8+ T cells and long-lived antibody-secreting cells in CD4KO mice. The effects of MF59 adjuvant in CD4KO mice might be associated with uric acid, inflammatory cytokines, and the recruitment of multiple immune cells at the injection site, but their cellularity and phenotypes were different from those in WT mice. These findings suggest a new paradigm of CD4-independent adjuvant mechanisms, providing the rationales to improve vaccine efficacy in infants, the elderly, immunocompromised patients, as well as healthy adults.

    IMPORTANCE MF59-adjuvanted influenza vaccines were licensed for human vaccination, but the detailed mechanisms are not fully elucidated. CD4+ T cells are required to induce antibody isotype switching and long-term memory responses. In contrast, we discovered that MF59 was highly effective in inducing isotype-switched IgG antibodies and long-term protective immune responses to a T-dependent influenza vaccine independent of CD4+ T cells. These findings are highly significant for the following reasons: (i) MF59 can overcome a defect of CD4+ T cells in inducing protective immunity to vaccination with a T-dependent influenza virus vaccine; (ii) a CD4-independent pathway can be an alternative mechanism for certain adjuvants such as MF59; and (iii) this study has significant implications for improving vaccine efficacies in young children, the elderly, and immunocompromised populations.

  • Targeted Mutagenesis of Guinea Pig Cytomegalovirus Using CRISPR/Cas9-Mediated Gene Editing [Genome Replication and Regulation of Viral Gene Expression]

  • The cytomegaloviruses (CMVs) are among the most genetically complex mammalian viruses, with viral genomes that often exceed 230 kbp. Manipulation of cytomegalovirus genomes is largely performed using infectious bacterial artificial chromosomes (BACs), which necessitates the maintenance of the viral genome in Escherichia coli and successful reconstitution of virus from permissive cells after transfection of the BAC. Here we describe an alternative strategy for the mutagenesis of guinea pig cytomegalovirus that utilizes clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing to introduce targeted mutations to the viral genome. Transient transfection and drug selection were used to restrict lytic replication of guinea pig cytomegalovirus to cells that express Cas9 and virus-specific guide RNA. The result was highly efficient editing of the viral genome that introduced targeted insertion or deletion mutations to nonessential viral genes. Cotransfection of multiple virus-specific guide RNAs or a homology repair template was used for targeted, markerless deletions of viral sequence or to introduce exogenous sequence by homology-driven repair. As CRISPR/Cas9 mutagenesis occurs directly in infected cells, this methodology avoids selective pressures that may occur during propagation of the viral genome in bacteria and may facilitate genetic manipulation of low-passage or clinical CMV isolates.

    IMPORTANCE The cytomegalovirus genome is complex, and viral adaptations to cell culture have complicated the study of infection in vivo. Recombineering of viral bacterial artificial chromosomes enabled the study of recombinant cytomegaloviruses. Here we report the development of an alternative approach using CRISPR/Cas9-based mutagenesis in guinea pig cytomegalovirus, a small-animal model of congenital cytomegalovirus disease. CRISPR/Cas9 mutagenesis can introduce the same types of mutations to the viral genome as bacterial artificial chromosome recombineering but does so directly in virus-infected cells. CRISPR/Cas9 mutagenesis is not dependent on a bacterial intermediate, and defined viral mutants can be recovered after a limited number of viral genome replications, minimizing the risk of spontaneous mutation.

  • HIV-1-Specific Chimeric Antigen Receptors Based on Broadly Neutralizing Antibodies [Pathogenesis and Immunity]

  • Although the use of chimeric antigen receptors (CARs) based on single-chain antibodies for gene immunotherapy of cancers is increasing due to promising recent results, the earliest CAR therapeutic trials were done for HIV-1 infection in the late 1990s. This approach utilized a CAR based on human CD4 as a binding domain and was abandoned for a lack of efficacy. The growing number of HIV-1 broadly neutralizing antibodies (BNAbs) offers the opportunity to generate novel CARs that may be more active and revisit this modality for HIV-1 immunotherapy. We used sequences from seven well-defined BNAbs varying in binding sites and generated single-chain-antibody-based CARs. These CARs included 10E8, 3BNC117, PG9, PGT126, PGT128, VRC01, and X5. Each novel CAR exhibited conformationally relevant expression on the surface of transduced cells, mediated specific proliferation and killing in response to HIV-1-infected cells, and conferred potent antiviral activity (reduction of viral replication in log10 units) to transduced CD8+ T lymphocytes. The antiviral activity of these CARs was reproducible but varied according to the strain of virus. These findings indicated that BNAbs are excellent candidates for developing novel CARs to consider for the immunotherapeutic treatment of HIV-1.

    IMPORTANCE While chimeric antigen receptors (CARs) using single-chain antibodies as binding domains are growing in popularity for gene immunotherapy of cancers, the earliest human trials of CARs were done for HIV-1 infection. However, those trials failed, and the approach was abandoned for HIV-1. The only tested CAR against HIV-1 was based on the use of CD4 as the binding domain. The growing availability of HIV-1 broadly neutralizing antibodies (BNAbs) affords the opportunity to revisit gene immunotherapy for HIV-1 using novel CARs based on single-chain antibodies. Here we construct and test a panel of seven novel CARs based on diverse BNAb types and show that all these CARs are functional against HIV-1.

  • Erratum for Metrick and Heldwein, Novel Structure and Unexpected RNA-Binding Ability of the C-Terminal Domain of Herpes Simplex Virus 1 Tegument Protein UL21 [Errata]

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    JVI Accepts: Articles Published Ahead of Print

  • The PB2 subunit of the influenza A virus RNA polymerase is imported into the mitochondrial matrix [PublishAheadOfPrint]

  • The PB2 subunit of the RNA polymerase complex of seasonal human influenza A viruses has been shown to localise to the mitochondria. Various roles including the regulation of apoptosis and innate immune responses to viral infection have been proposed for mitochondrial PB2. In particular, PB2 has been shown to inhibit interferon expression by associating with the mitochondrial antiviral signalling (MAVS) protein, which acts downstream of RIG-I and MDA-5 in the interferon induction pathway. However, in spite of a growing body of literature on the potential roles of mitochondrial PB2, the exact location of PB2 in mitochondria has not been determined. Here, we use Enhanced Ascorbate Peroxidase (APEX) tagged PB2 proteins and electron microscopy to study the localisation of PB2 in mitochondria. We find that PB2 is imported into mitochondria where it localises to the mitochondrial matrix. We also demonstrate that MAVS is not required for the import of PB2 into mitochondria by showing that PB2 associates with mitochondria in MAVS knockout mouse embryo fibroblasts. Instead, we find that amino acid residue 9 in the N-terminal mitochondrial targeting sequence is a determinant of the mitochondrial import of PB2, differentiating the localisation of PB2 of human from that of avian influenza A virus strains. We also show that a virus encoding non-mitochondrial PB2 is attenuated in MEFs compared with an isogenic virus encoding mitochondrial PB2, in a MAVS-independent manner, suggesting a role for PB2 within the mitochondrial matrix. This work extends our understanding of the interplay between influenza virus and mitochondria.

    IMPORTANCE The PB2 subunit of the influenza virus RNA polymerase is a major determinant of viral pathogenicity. However, the molecular mechanisms of how PB2 determines pathogenicity remain poorly understood. PB2 associates with mitochondria and inhibits the function of the mitochondrial antiviral signalling protein MAVS, implicating PB2 in the regulation of innate immune responses. We find that PB2 is imported into the mitochondrial matrix and show that amino acid residue 9 is a determinant of mitochondrial import. Asparagine or threonine that is present in over 99% of all human seasonal influenza virus pre-2009 H1N1, H2N2 and H3N2 strains is compatible with mitochondrial import while an aspartic acid that is present in over 95% of all avian influenza viruses is not, resulting in a clear distinction between human-adapted and avian influenza viruses. These findings provide insights into the interplay between influenza virus and mitochondria and suggest mechanisms by which PB2 could affect pathogenicity.

  • A Single Mutation in the VP1 of Enterovirus 71 is Responsible for Increased Virulence and Neurotropism in Adult Interferon Deficient Mice [PublishAheadOfPrint]

  • Hand, foot, and mouth disease (HFMD) has spread throughout the Asia-Pacific region, affecting millions of young children who develop symptoms ranging from painful blisters around their mouth and hands to neurological complications. Many members of the Enterovirus genus (family: Picornaviridae) cause HFMD. Enterovirus 71 (EV71) is one of the primary causative agents and has been linked to severe disease. Vaccine efficacy and pathogenesis studies for EV71 have been limited because there is a lack of suitable animal models. Previously, we generated a mouse adapted EV71 (mEV71) capable of infecting 12 week-old interferon receptor deficient AG129 mice, and used this model to evaluate the efficacy of candidate HFMD vaccines. Herein, we present data investigating the genetic correlates of EV71 adaptation, as well as characterize the virus's tissue tropism in mice. Using reverse genetics, a VP1 mutation (K244E) was found to be necessary for mEV71 virulence in adult mice. Another VP1 mutation (H37R) was required for mEV71 recovery on rhabdomyosarcoma (RD) cells. Viral loads determined by real time RT-PCR confirmed the presence of mEV71 in serum and multiple organs of mice. Histological analysis revealed signs of meningitis and encephalitis, characteristic of severe human disease. The further description of this model has provided insight into EV71 pathogenesis and demonstrates the importance of the VP1 region in facilitating mEV71 adaptation.

    IMPORTANCE EV71 is a re-emerging pathogen with little known about the genetic determinants involved with pathogenesis. The absence of animal models has contributed to this lack in knowledge. Data presented here improve upon the existing animal models by characterizing a mouse adapted strain of EV71. We determined that a VP1 mutation (K244E) was needed for EV71 virulence in adult AG129 mice. While this mutation was found previously for EV71 adaptation in 5 day-old Balb/c mice, neurotropic disease did not develop. Using interferon deficient mice, we raised the age of susceptibility beyond 6 weeks-old and provided clear evidence that our model mimics severe human infections. This model can be exploited to identify determinants of EV71 virulence and reveal molecular mechanisms that control the virus-host interaction, especially those associated with neurotropic disease. Furthermore, these data provide useful information regarding the importance of the VP1, specifically position 244, on host adaptation and tissue dissemination.

  • Concurrence of iridovirus, polyomavirus and a unique member of a new group of fish papillomaviruses in lymphocystis disease affected gilthead seabream [PublishAheadOfPrint]

  • Lymphocystis disease is a geographically widespread disease affecting more than 150 different species of marine and freshwater fish. The disease, provoked by the iridovirus lymphocystis disease virus (LCDV), is characterized by the appearance of papilloma-like lesions on the skin of affected animals that usually self-resolve over time. Development of the disease is usually associated with several environmental factors and, more frequently, with stress conditions provoked by the intensive culture conditions present in fish farms. In gilthead seabream (Sparus aurata), an economically important cultured fish species in the Mediterranean area, a distinct LCDV has been identified but not completely characterized yet. We have used direct sequencing of the virome of lymphocystis lesions from affected S. aurata to obtain the complete genome of a new LCDV-Sa species that is the largest vertebrate iridovirus sequenced to date. Importantly, this approach allowed us to assemble the full-length circular genome sequence of two previously unknown viruses belonging to the papilloma- and polyomaviruses termed Sparus aurata papillomavirus 1 (SaPV1) and Sparus aurata polyomavirus 1 (SaPyV1), respectively. Epidemiological surveys showed that lymphocystis disease was frequently associated with the concurrent appearance of one or both of the new viruses. SaPV1 has unique characteristics such as an intron within the L1 gene and, as the first member of the Papillomaviridae family described in fish, provides evidence for a more ancient origin of this family than previously thought.

    Importance Lymphocystis disease affects marine and freshwater fish species worldwide. It is characterized by the appearance of papilloma-like lesions on the skin that contain heavily enlarged cells (lymphocysts). The causative agent is the lymphocystis disease virus (LCDV), a large icosahedral virus of the family Iridoviridae. In the Mediterranean area the gilthead seabream (Sparus aurata), an important farmed fish, is frequently affected. Using next generation sequencing, we have identified within lymphocystis lesions the concurrent presence of a further LCDV species (LCDV-Sa) as well as two novel viruses. These are members of polyoma- and papillomavirus families and here we report them to be frequently associated with the presence of lymphocysts in affected fish. Because papillomaviruses had not been described in fish before, these findings support a more ancient origin of this virus family than previously thought and evolutionary implications are discussed.

  • Interaction between Herpes Simplex Virus 1 Tegument Proteins UL51 and UL14 and Its Role in Virion Morphogenesis [PublishAheadOfPrint]

  • To investigate the molecular mechanism(s) by which herpes simplex virus 1 (HSV-1) tegument protein UL51 promotes viral replication, we screened for viral proteins that interact with UL51 in infected cells. Affinity purification of tagged-UL51 in HSV-1-infected Vero cells was coupled with immunoblotting of the purified UL51 complexes with various antibodies to HSV-1 virion proteins. Subsequent analyses revealed that UL51 interacted with another tegument protein, UL14, in infected cells. Mutational analyses of UL51 showed that UL51 amino acid residues Leu-111, Ile-119 and Tyr-123 were required for interaction with UL14 in HSV-1-infected cells. Alanine substitutions of these UL51 amino acid residues reduced viral replication and produced an accumulation of unenveloped and partially enveloped nucleocapsids in the cytoplasm at levels comparable to UL51-null, UL14-null and UL51/UL14-double null mutations. In addition, although UL51 and UL14 co-localized at juxtanuclear domains in HSV-1-infected cells, the amino acid substitutions in UL51 produced aberrant localization of UL51 and UL14. The effects of these substitutions on localization of UL51 and UL14 were similar to that of the UL51-null and UL14-null mutations on localization of UL14 and UL51, respectively. These results suggested that the interaction between UL51 and UL14 was required for proper localization of these viral proteins in infected cells and that the UL51-UL14 complex regulated final viral envelopment for efficient viral replication.

    IMPORTANCE Herpesviruses contain a unique virion structure designated the tegument, which is a protein layer between the nucleocapsid and the envelope. HSV-1 has dozens of viral proteins in the tegument, which are thought to facilitate viral envelopment by interacting with other virion components. However, although numerous interactions among virion proteins have been reported, data on how these interactions facilitate viral envelopment is limited. In this study, we have presented data showing that the interaction of HSV-1 tegument proteins UL51 and UL14 promoted viral final envelopment for efficient viral replication. In particular, prevention of this interaction induced aberrant accumulation of partially enveloped capsids in the cytoplasm, suggesting that the UL51-UL14 complex acted in the envelopment process but not in an upstream event, such as transport of capsids to the site for envelopment. This is the first report showing that an interaction between HSV-1 tegument proteins directly regulated final virion envelopment.

  • An Ebola virus-like particle-based reporter system enables evaluation of antiviral drugs in vivo under non-BSL-4 conditions [PublishAheadOfPrint]

  • Ebola virus (EBOV) is a highly contagious lethal pathogen. As a biosafety level four (BSL-4) agent, however, EBOV is restricted to costly BSL-4 labs for experimentation, thus significantly impeding the evaluation of EBOV vaccines and drugs. Here, we reported an EBOV-like particle (EBOVLP) based luciferase reporter system that enables the evaluation of anti-EBOV agents in vitro and in vivo outside the BSL-4 facilities. Co-transfection of HEK293T cells with four plasmids encoding the VP40, NP, and GP proteins of EBOV, and the firefly luciferase (Fluc), respectively, resulted in the production of Fluc-containing filamentous particles that morphologically resemble authentic EBOV. The reporter EBOVLP was capable of delivering Fluc into various cultured cells in a GP-dependent manner, and recognized by a conformation-dependent anti-EBOV monoclonal antibody (mAb). Significantly, inoculation of mice with the reporter EBOVLP led to the delivery of Fluc protein into target cells and rapid generation of intense bioluminescence signals that can be blocked by the administration of EBOV neutralizing mAbs. This BSL-4-free reporter system should facilitate high-throughput screening for anti-EBOV drugs targeting viral entry and efficacy testing of candidate vaccines.

    IMPORTANCE Ebola virus (EBOV) researches were limited in costly biosafety level four (BSL-4) facilities due to the lack of animal model independent of BSL-4 lab. In this study, we revealed that a firefly luciferase bearing EBOV-like particle (EBOVLP) with typical filamentous EBOV morphology was capable of delivering the reporter protein into murine target cells both in vitro and in vivo. Moreover, we demonstrated that the reporter delivery could be inhibited both in vitro and in vivo by a known anti-EBOV protective monoclonal antibody 13C6. Our work provides a BSL-4-free system that can facilitate the in vivo evaluation of anti-EBOV antibodies, drugs and vaccines. The system may also be useful for mechanistic study of the viral entry process.

  • Hepatitis B virus polymerase localizes to the mitochondria and its terminal protein domain contains the mitochondrial-targeting signal [PublishAheadOfPrint]

  • To understand subcellular sites of hepatitis B virus (HBV) replication, we visualized core (Cp), polymerase (Pol) and pregenomic RNA (pgRNA) in infected cells. Interestingly, we found that majority of the Pol localizes to the mitochondria in cells undergoing viral replication. The mitochondrial localization of Pol was independent of both the cell-type and other viral components indicating that Pol contains an intrinsic mitochondrial targeting signal (MTS). Neither Cp nor pgRNA localized to the mitochondria during active replication, suggesting a role of Pol at the mitochondria other than DNA synthesis. The Pol of duck hepatitis B virus (DHBV) also localized to the mitochondria. This result indicates that localization of Pol to mitochondria is likely a feature of all hepadnaviruses. To map the MTS within HBV Pol, we generated a series of Pol-GFP fusions and found that a stretch spanning a.a.141-160 of Pol was sufficient to target GFP to the mitochondria. Surprisingly, deleting a.a.141-160 in full-length Pol did not fully ablate Pol's mitochondrial localization suggesting that additional sequences are involved in mitochondrial targeting. Only by deleting the N-terminal 160 amino acids in full length Pol was mitochondrial localization ablated. Crucial residues for pgRNA packaging are contained within a.a.141-160 indicating a multi-functional role of this region of Pol in the viral life cycle. Our studies show an unexpected Pol trafficking behavior that is uncoupled from its role in viral DNA synthesis.

    Importance Chronic infection by HBV is a serious health concern. Existing therapies for chronically infected individuals are not curative, underscoring the need for a better understanding of the viral life cycle to develop better antiviral therapies. To date, the most thoroughly studied function of Pol is to package the pgRNA and reverse transcribe it to double-stranded DNA within capsids. This study provides evidence for mitochondrial localization of Pol and defines the MTS. Recent findings have implicated a non-reverse transcription role for Pol in evading host innate immune responses. Mitochondria play an important role in controlling cellular metabolism, apoptosis and innate immunity. Pol could be altering one or more of these host mitochondrial functions to gain a replicative advantage and persist in chronically infected individuals.

  • Epstein-Barr Virus Oncoprotein LMP1 Mediates Epigenetic Changes in Host Gene Expression through PARP1 [PublishAheadOfPrint]

  • The latent infection of Epstein-Barr virus (EBV) is associated with 1% of human cancer incidence. Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification catalyzed by poly(ADP-ribose) polymerases (PARPs) that mediates EBV replication during latency. In this study, we detail the mechanisms that drive cellular PARylation during latent EBV infection and the effects of PARylation on host gene expression and cellular function. EBV-infected B cells had higher PAR levels compared to EBV-negative B cells. Moreover, cellular PAR levels were up to two-fold greater in Type III compared to Type I latently-infected EBV B cells. We identified a positive association between expression of the EBV-genome-encoded latency membrane protein 1 (LMP1) and PAR levels that was dependent upon PARP1. PARP1 regulates gene expression by numerous mechanisms, including modifying chromatin structure and altering the function of chromatin-modifying enzymes. Since LMP1 is essential in establishing EBV latency and promoting tumorigenesis, we explored the model that disruption in cellular PARylation, driven by LMP1 expression, subsequently promotes epigenetic alterations to elicit changes in host gene expression. PARP1 inhibition resulted in the accumulation of the repressive histone mark H3K27me3 at a subset of LMP1-regulated genes. Inhibition of PARP1, or abrogation of PARP1 expression, also suppressed the expression of LMP1-activated genes and LMP1-mediated cellular transformation, demonstrating an essential role for PARP1 activity in LMP1-induced gene expression and cellular transformation associated with LMP1. In summary, we identified a novel mechanism by which LMP1 drives expression of host tumor-promoting genes by blocking generation of the inhibitory histone modification H3K27me3 through PARP1 activation.

    Importance Epstein-Barr virus (EBV) is causally linked to several malignancies and is responsible for 1% of cancer incidence worldwide. The EBV-encoded protein LMP1 is essential for promoting viral tumorigenesis by aberrant activation of several well-known intracellular signaling pathways. We have identified and defined an additional novel molecular mechanism by which LMP1 regulates the expression of tumor-promoting host genes. We found that LMP1 activates the cellular protein PARP1, leading to a decrease in a repressive histone modification, accompanied by induction in expression of multiple cancer-related genes. PARP1 inhibition or depletion led to a decrease in LMP1-induced cellular transformation. Therefore, targeting PARP1 activity may be an effective treatment for EBV-associated malignancies.

  • Response of Three Different Viruses to Interferon Priming and Dithiothreitol Treatment of Avian Cells. [PublishAheadOfPrint]

  • We have previously shown that the replication of avian reovirus in chicken cells is much more resistant to interferon than the replication of vesicular stomatitis virus or vaccinia virus. In this study we have investigated the role that the double-stranded RNA (dsRNA)-activated protein kinase (PKR) plays in the sensitivity of these three viruses towards the antiviral action of chicken interferon. Our data suggest that while interferon priming of avian cells blocks vaccinia virus replication by promoting PKR activation, the replication of vesicular stomatitis virus appears to be blocked at a pre-translational step. Our data further suggest that the replication of avian reovirus in chicken cells is quite resistant to interferon priming because this virus uses strategies to downregulate PKR activation and also because translation of avian reovirus mRNAs is more resistant to phosphorylation of the alpha subunit of initiation factor eIF2 than translation of their cellular counterparts. Our results further reveal that the avian reovirus protein sigmaA is able to prevent PKR activation, and that this function is dependent on its double-stranded RNA-binding activity. Finally, this study demonstrates that vaccinia virus and avian reovirus, but not vesicular stomatitis virus, express/induce factors that counteract the ability of dithiothreitol to promote eIF2 phosphorylation. Our data demonstrate that each of the three different viruses used in this study elicits distinct responses to interferon and to dithiothreitol-induced eIF2 phosphorylation when infecting avian cells.

    IMPORTANCE Type I interferons constitute the first barrier of defense against viral infections, and one of the best characterized anti-viral strategies is mediated by the double-stranded RNA-activated protein kinase R (PKR). The results of this study revealed that IFN priming of avian cells has little effect on avian reovirus (ARV) replication, but drastically diminishes the replication of vaccinia virus (VV) and vesicular stomatitis virus (VSV) by PKR-dependent and nndash;independent mechanisms, respectively. Our data also demonstrate that the dsRNA-binding ability of ARV protein sigmaA plays a key role in the resistance of ARV towards IFN by preventing PKR activation. Our findings will contribute to improve the current understanding of the interaction of viruses with the host's innate immune system. Finally, it would be of interest to uncover the mechanisms that allow avian reovirus transcripts to be efficiently translated under conditions (moderate eIF2 phosphorylation) that block the synthesis of cellular proteins.

  • An RNA Element that Facilitates Programmed Ribosomal Readthrough in Turnip Crinkle Virus Adopts Multiple Conformations [PublishAheadOfPrint]

  • Ribosome recoding is used by RNA viruses for translational readthrough or frameshifting past termination codons for synthesis of extension products. Recoding sites along with downstream Recoding Stimulatory Elements (RSEs) have long been studied in reporter constructs as these fragments alone mediate customary levels of recoding and are thus assumed to contain complete instructions for establishment of the proper ratio of termination to recoding. RSEs from the Tombusviridae and Luteoviridae are thought to be exceptions, as they contain a long-distance RNA:RNA connection with the 3rrsquo; end. This interaction has been suggested to substitute for pseudoknots thought to be missing in Tombusvirid RSEs. We provide evidence that the phylogenetically conserved RSE of carmovirus Turnip crinkle virus (TCV) adopts an alternative, smaller structure that extends an upstream conserved hairpin and this alternative structure is the predominant form of the RSE within nascent viral RNA in plant cells and when RNA is synthesized in vitro. The TCV RSE also contains an internal pseudoknot along with the long-distance interaction, and the pseudoknot is not compatible with the phylogenetically conserved structure. Conserved residues just past the recoding site are important for recoding, and these residues are also conserved in RSEs of gamma retroviruses. Our data demonstrate the dynamic nature of the TCV RSE, and suggest that studies using reporter constructs may not be effectively recapitulating RSE-mediated recoding within viral genomes.

    Importance Ribosome recoding is used by RNA viruses for ribosomes to extend translation past termination codons for synthesis of longer products. Recoding sites and a downstream Recoding Stimulatory Element (RSE) mediate expected levels of recoding when excised and placed in reporter constructs, and thus are assumed to contain complete instructions for establishment of the proper ratio of termination to recoding. We provide evidence that most of the TCV RSE adopts an alternative structure that extends an upstream conserved hairpin, and this alternative structure, and not the phylogenetically conserved structure, is the predominant form of the RSE in RNA synthesized in vitro and in plant cells. The TCV RSE also contains an internal pseudoknot that is not compatible with the phylogenetically conserved structure and an RNA bridge to the 3rrsquo; end. These data suggest that the TCV RSE is structurally dynamic and that multiple conformations are likely required to regulate ribosomal readthrough.

  • Guinea pig prion protein supports rapid propagation of bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease prions [PublishAheadOfPrint]

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

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

  • Complementary effects of IL-15 and IFN-{alpha} induce immunity in HBV transgenic mice [PublishAheadOfPrint]

  • In chronic hepatitis B (CHB) failure of HBV control is associated with T cell dysfunction. HBV transgenic mice mirror many features of the human disease, including T-cell unresponsiveness, and thus represent an appropriate model to test novel therapeutic strategies. To date the tolerant state of CD8+ T cells in these animals could only be altered by strong immunogens or immunization with HBV antigen-pulsed dendritic cells, however, the induced effectors were unable to suppress viral gene expression or replication. Because of their known stimulatory properties, this study explored the therapeutic potential of a liver-directed gene transfer of IFN-aalpha; and IL-15 in a murine model of CHB using AAV delivery. Their combination resulted not only in a reduction of the viral load in the liver and the induction of an antibody response but gave rise to functional and specific CD8+ immunity. Furthermore, when transferring splenic and intrahepatic lymphocytes from IFN-aalpha;/IL-15-treated animals to new HBV carriers, partial antiviral immunity was achieved. Contrary to previous observations made using either cytokine alone, a markedly attenuated PD-L1 induction in hepatic tissue was observed upon co-administration. An initial study with CHB patient samples also gave promising results. Hence, synergizing the effects of two stimulating cytokines, IL-15 and IFN-aalpha;, demonstrated a potent approach to significantly enhance the CD8+ T cell response in a state of immune hyporesponsiveness and may be useful for treating chronic viral infections and neoplastic conditions.

    Impact: With 350 million people affected worldwide and 600,000 annual deaths due to HBV-induced liver cirrhosis and/or hepatocellular carcinoma, chronic hepatitis B (CHB) is a major health problem. However, current treatment options are costly, not very effective and/or need to be administered for life. The unprecedented efficacy of the strategy described in our paper may offer an alternative and is relevant for a broad spectrum of readers because of its clear translational importance to other chronic viral infections in which a hyporesponsive antigen-specific T cell repertoire prevents clearance of the pathogen.

  • The molecular determinants directing HIV-1 Gag assembly to the virus-containing compartments in primary macrophages [PublishAheadOfPrint]

  • The subcellular sites of HIV-1 assembly, determined by the localization of the structural protein Gag, vary in a cell-type-dependent manner. In T cells and transformed cell lines used as model systems, HIV-1 assembles at the plasma membrane (PM). The binding and localization of HIV-1 Gag to the PM is mediated by the interaction between the matrix (MA) domain, specifically the highly basic region, and a PM-specific acidic phospholipid, phosphatidylinositiol-4,5-bisphosphate [PI(4,5)P2]. In primary macrophages, prominent accumulation of assembling or assembled particles is found in the virus-containing compartments (VCCs), which largely consist of convoluted invaginations of the PM. To elucidate the molecular mechanism of HIV-1 Gag targeting to the VCCs, we examined the impact of overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV), which depletes cellular PI(4,5)P2, in primary macrophages. We found that the VCC localization and virus release of HIV-1 are severely impaired upon 5ptaseIV overexpression, suggesting an important role for the MA-PI(4,5)P2 interaction in HIV-1 assembly in primary macrophages. However, our analysis of HIV-1 Gag derivatives with MA changes showed that this interaction contributes to Gag membrane binding but is dispensable for specific targeting of Gag to the VCCs per se. We further determined that deletion of the NC domain abolishes VCC-specific localization of HIV-1 Gag. Notably, HIV-1 Gag localized to the VCCs efficiently when the NC domain was replaced with a leucine zipper dimerization motif, which promotes Gag multimerization. Altogether, our data revealed that targeting of HIV-1 Gag to the VCCs requires NC-dependent multimerization.

    IMPORTANCE In T cells and model cell lines, HIV-1 Gag localizes to the PM in a manner dependent on the MA-PI(4,5)P2 interaction. On the other hand, in primary macrophages, HIV-1 Gag localizes to convoluted intracellular membrane structures termed virus-containing compartments (VCCs). Although these compartments have been known over decades, and despite the implication of viruses in VCCs in virus reservoir maintenance and spread, the viral determinant(s) that promote Gag targeting to VCCs are unknown. In this study, we found that the MA-PI(4,5)P2 interaction facilitates efficient Gag membrane binding in macrophages, but is not essential for Gag targeting to VCCs. Rather, our results revealed that NC-dependent multimerization promotes VCC targeting. Our findings highlight the differential roles played by MA and NC in HIV-1 Gag membrane binding and targeting and suggest a multimerization-dependent mechanism for Gag trafficking in primary macrophages similar to that for Gag localization to uropods in polarized T cells.

  • Deep-sequencing of distinct preparations of the live-attenuated VZV vaccine reveals a conserved core of attenuating SNPs [PublishAheadOfPrint]

  • The continued success of the live-attenuated varicella zoster virus vaccine in preventing varicella and herpes zoster is well documented, as are many of the mutations that contribute to the attenuation of the vOka virus for replication in skin. At least three different preparations of vOKa are marketed. Here, we show using deep sequencing of seven batches of vOka vaccine (including ZostaVax, VariVax, VarilRix and the Oka/Biken working seed) from three different manufacturers (VariVax, GSK and Biken) that 137 SNP mutations are present in all vaccine batches. This includes six sites at which the vaccine allele is fixed or near-fixation which we speculate are likely to be important for attenuation. We additionally show that despite differences in the vaccine populations between preparations, batch to batch variation is minimal, as is the number and frequency of mutations unique to individual batches. This suggests that the vaccine manufacturing processes are not introducing new mutations and notwithstanding the mixture of variants present, VZV live vaccines are extremely stable.

    Importance The continued success of vaccinations to prevent chickenpox and shingles, combined with the extremely low incidence of adverse reactions, is testament to the quality of these vaccines. The vaccine itself is comprised of a heterogeneous live-attenuated virus population and thus requires deep sequencing technologies to explore the differences and similarities in the virus populations between different preparations and batches of the vaccines. Our data demonstrate minimal variation between batches, an important safety feature, and provide new insights into the extent of the mutations present in this attenuated virus.

  • Varicella-Zoster Virus Activates CREB and Inhibition of the pCREB-p300/CBP- Interaction Inhibits Viral Replication in vitro and Skin Pathogenesis in vivo. [PublishAheadOfPrint]

  • Varicella zoster virus (VZV) is an aalpha;-herpesvirus that causes varicella upon primary infection and zoster upon reactivation from latency in sensory ganglion neurons. The replication of herpesviruses requires manipulation of cell signaling pathways. Notably, CREB, a factor involved in the regulation of several cellular processes, is activated upon infection of T cells with VZV. Here we report that VZV infection also induced CREB phosphorylation in fibroblasts and that XX-650-23, a newly identified inhibitor of the pCREB interaction with p300/CBP, restricted cell-cell spread of VZV in vitro. CREB phosphorylation did not require the viral ORF47 or ORF 66 kinases encoded by VZV. Evaluating the biological relevance of these observations during VZV infection of human skin xenografts in the SCID mouse model of VZV pathogenesis showed both that pCREB was upregulated in infected skin and that treatment with XX-650-23 reduced infectious virus production and limited lesion formation compared to vehicle control. Thus, processes of CREB activation and p300/CBP binding are important for VZV skin infection and may be targeted for antiviral drug development.

    IMPORTANCE Varicella zoster virus (VZV) is a common pathogen that causes chickenpox and shingles. As with all herpesviruses, the infection is acquired for life and the virus can periodically reactivate from latency. Although VZV infection is usually benign with few or no deleterious consequences, infection can be life-threatening in immunocompromised patients. Otherwise healthy elderly individuals who develop zoster as a consequence of viral reactivation are at risk for post-herpetic neuralgia (PHN), a painful and long lasting complication. Current vaccines use a live attenuated virus that is usually safe but cannot be given to many immuno-deficient patients and retains the capacity to establish latency and reactivate, causing zoster. Antiviral drugs are effective against severe VZV infections but have little impact on PHN. A better understanding of virus-host cell interactions is relevant for developing improved therapies to safely interfere with cellular processes that are crucial for VZV pathogenesis.

  • Exocytosis of varicella-zoster virions involves a convergence of endosomal and autophagy pathways [PublishAheadOfPrint]

  • Varicella-zoster virus (VZV) is an extremely cell-associated herpesvirus, with limited egress of viral particles. The induction of autophagy in VZV-infected monolayers is easily detectable; inhibition of autophagy leads to decreased VZV glycoprotein biosynthesis and diminished viral titers. To explain how autophagic flux could exert a proviral effect on the VZV infectious cycle, we postulated that the VZV exocytosis pathway following secondary envelopment may converge with the autophagy pathway. This hypothesis depended on known similarities between VZV gE and autophagy-related (Atg) Atg9/Atg16L1 trafficking pathways. Investigations were carried out with highly purified fractions of VZ virions. When the virion fraction was tested for the presence of autophagy and endosomal proteins, microtubule-associated protein 1 light chain (MAP1LC3B) and Ras-like GTPase 11 (Rab11) were detected. By 2D and 3D imaging after immunolabeling, both proteins also colocalized with VZV gE in a proportion of cytoplasmic vesicles. When purified VZ virions were enumerated after immuno-electron microscopy, gold beads were detected on viruses following incubation with antibodies to VZV gE (~100%), Rab11 (50%) and LC3B (30 %). Examination of numerous electron micrographs demonstrated that enveloped virions were housed in single-membraned vesicles; viral particles were not observed in autophagosomes. Taken together, our data suggested that some viral particles after secondary envelopment accumulated in a heterogeneous population of single-membraned vesicular compartments, which were decorated with components from both the endocytic pathway (Rab11) and the autophagy pathway (LC3B). The latter cytoplasmic viral vesicles resembled an amphisome.

    IMPORTANCE: VZV infection leads to increased autophagic flux, while inhibition of autophagy leads to a marked reduction in virus spread. In this investigation of the proviral role of autophagy, we found evidence for an intersection of viral exocytosis and autophagy pathways. Specifically, both LC3-II and Rab11 proteins copurified with some infectious VZV particles. The results suggested that a subpopulation of VZV particles were carried to the cell surface in single-walled vesicles with attributes of an amphisome, an organelle formed from fusion of an endosome and an autophagosome. Our results also addressed interpretation of autophagy/xenophagy results with mutated herpes simplex virus lacking its ICP34.5 neurovirulence gene. The VZV genome lacks an ICP34.5 ortholog, yet we found no evidence of VZV particles housed in a double-membraned autophagosome. In other words, xenophagy, a degradative process documented after infection with HSV 34.5, was not observed in VZV infected cells.

  • Herpes simplex virus type 1 interaction with myeloid cells in vivo [PublishAheadOfPrint]

  • Herpes simplex virus type 1 (HSV-1) enters mice via olfactory epithelial cells, then colonizes the trigeminal ganglia (TG). Most TG nerve endings are subepithelial, so this colonization implies subepithelial viral spread, where myeloid cells provide an important line of defence. The outcome of myeloid cell infection by HSV-1 in vitro depends on their differentiation state; the outcome in vivo is unknown. Epithelial HSV-1 commonly infected myeloid cells, and cre-lox virus marking showed nose and lung infections passing through lysM+ and CD11c+ cells. By contrast subcapsular sinus macrophages (SSM) exposed to lymph-borne HSV-1 were permissive only when type 1 interferon (IFN-I) signaling was blocked; normally their infection was suppressed. Thus the myeloid infection outcome helped to determine HSV-1 distribution: subepithelial myeloid cells provided a route of spread from the olfactory epithelium to TG neurons, while SSM blocked systemic spread.

    Importance Herpes simplex virus type 1 (HSV-1) infects most people and can cause severe disease. This reflects its persistence in nerve cells that connect to the mouth, nose, eye and face. Established infection seems impossible to clear. Therefore we must understand how it starts. This is hard in humans, but mice show HSV-1 entry via the nose then spread to its preferred nerve cells. We show that this spread proceeds in part via myeloid cells, which normally function in host defence. Myeloid infection was productive in some settings, but was efficiently suppressed by interferon in others. Therefore interferon acting on myeloid cells can stop HSV-1 spread and enhancing this defence offers a way to improve infection control.

  • Unmasking stem-specific neutralizing epitopes by abolishing N-linked glycosylation sites of influenza hemagglutinin proteins for vaccine design [PublishAheadOfPrint]

  • Influenza hemagglutinin (HA) protein consists of two componentsmmdash;a globular head and stem region that are folded within six disulfide bondsmmdash;plus several N-linked glycans that produce a homo-trimeric complex structure. While N-linked glycosylation sites on the globular head are variable among different strains and different subtypes, N-linked glycosylation sites in the stem region are mostly well-conserved among various influenza virus strains. Targeting highly conserved HA stem regions has been proposed as a useful strategy for designing universal influenza vaccines. Since the HA stem region is constituted by a HA1 N-terminal part and a full HA2 part, we expressed a series of recombinant HA mutant proteins with deleted N-linked glycosylation sites in the HA1-stem and HA2-stem regions of H5N1 and pH1N1 viruses. Unmasking N-glycans in the HA2-stem region (H5 N484A and H1 N503A) were found to elicit more potent neutralizing antibody titers against homologous, heterologous and heterosubtypic viruses. Unmasking the HA2-stem N-glycans of H5HA but not H1HA resulted in more CR6261-like and FI6v3-like antibodies and also correlated with the increase of cell fusion inhibition activity in antisera. Only H5 N484A HA2-stem mutant protein immunization increased the numbers of antibody-secreting cells, the germinal center B cells, and the memory B cells targeting the stem helix A epitopes in splenoctyes. Unmasking the HA2-stem N-glycans of H5HA mutant proteins show a significantly improvement in the protection against homologous virus challenges, but to a less degree for the protection against heterosubtypic pH1N1 virus challenges. These results may provide useful information for designing more effective influenza vaccines.

    Significance N-linked glycosylation sites in the stem region of influenza hemagglutinin (HA) proteins are mostly well-conserved among various influenza virus strains. Targeting highly conserved HA stem regions has been proposed as a useful strategy for designing universal influenza vaccines. Our studies indicate that unmasking the HA2-stem N-glycans of recombinant HA proteins from H5N1 and pH1N1 viruses induced more potent neutralizing antibody titers against homologous and heterosubtypic viruses. However, only the immunization with the H5N1 HA2-stem mutant protein can refocus B antibody responses to the helix A epitope for inducing more CR6261-like/FI6v3-like and fusion inhibition antibodies in antisera, resulting in a significant improvement for the protection against lethal H5N1 virus challenges. These results may provide useful information for designing more effective influenza vaccines.

  • Induction of heterologous Tier 2 HIV-1 neutralizing and cross-reactive V1/V2-specific antibodies in rabbits by prime-boost immunization [PublishAheadOfPrint]

  • Poxvirus prime-protein boost used in the RV144 trial remains the only immunization strategy shown to elicit a modest level of protection against HIV-1 acquisition in humans. Although neutralizing antibodies (Nab) were generated, they were against sensitive viruses, not the more resistant "Tier 2" isolates that dominate circulating strains. Instead, risk reduction correlated with antibodies recognizing epitopes in the V1/V2 region of HIV-1 envelope glycoprotein (Env). Here, we examined if Tier 2-virus Nab and V1/V2-specific non-Nab could be elicited by a poxvirus prime-gp120 boost strategy in a rabbit model. We studied two Clade B Envs that differ in multiple parameters, including their tissue origin, neutralization sensitivity, and the presence of the N197 (N7) glycan that was previously shown to modulate the exposure of conserved epitopes on Env. We demonstrate that immunized rabbits generated cross-reactive neutralizing activities against ggt;50% of Tier 2 global HIV-1 isolates tested. Some of these activities were directed against the CD4 binding site (CD4bs). These rabbits also generated antibodies that recognized protein scaffolds bearing V1/V2 sequences from diverse HIV-1 isolates and mediated antibody-dependent cellular cytotoxicity. However, there are subtle differences in the specificity and the response rate of V1/V2-specific antibodies between animals immunized with different Env, with or without the N7 glycan. These findings demonstrate that antibody responses that have been correlated with protection against HIV-1 acquisition in humans can be elicited in a preclinical model by a poxvirus prime-gp120 boost strategy and that improvements may be achievable by optimizing the nature of the priming and boosting immunogens.

    IMPORTANCE The only vaccine approach shown to elicit any protective efficacy against HIV-1 acquisition is based on a poxvirus prime-protein boost regimen (RV144 Thai trial). Reduction of risk was associated with non-neutralizing antibodies targeting the V1/V2 loops of the envelope protein gp120. However, the modest efficacy (31.2%) achieved in this trial highlights the need to examine approaches and factors that may improve vaccine-induced responses, including cross-reactive neutralizing activities. We show here that rabbits immunized with a novel recombinant vaccinia prime-gp120 protein boost regimen generated antibodies that recognize protein scaffolds bearing V1/V2 sequences from diverse HIV-1 isolates and mediated antibody-dependent cellular cytotoxicity. Importantly, immunized rabbits also showed neutralizing activities against heterologous Tier 2 HIV-1 isolates. These findings may inform the design of prime-boost immunization approaches and help improve the protective efficacy of candidate HIV-1 vaccines.

  • Non-human TRIM5 variants enhance recognition of HIV-1-infected cells by CD8+ T cells [PublishAheadOfPrint]

  • Tripartite motif-containing protein 5 (TRIM5) restricts human immunodeficiency virus type-1 (HIV-1) in a species-specific manner by uncoating viral particles while activating early innate responses. Although the contribution of TRIM5 proteins to cellular immunity has not yet been studied, their interactions with the incoming viral capsid and the cellular proteasome led us to hypothesize a role for them. Here, we investigate whether the expression of two non-human TRIM5 orthologs, rhesus TRIM5aalpha; (RhT5) and TRIM-cyclophilin A (TCyp), both of which are potent restrictors of HIV-1, could enhance immune recognition of infected cells by CD8+ T cells. We illustrate how TRIM5 restriction improves CD8+ T cellnndash;mediated HIV-1 inhibition. Moreover, when TRIM5 activity was blocked by the non-immunosuppressive analog of cyclosporin A, SmBz-CsA, we found a significant reduction in CD107a/MIP1bbeta; expression in HIV-1nndash;specific CD8+ T cells. This finding underscores the direct link between TRIM5 restriction and activation of CD8+ T-cell responses. Interestingly, cells expressing RhT5 induced stronger CD8+ T-cell responses through the specific recognition of the HIV-1 capsid by the immune system. The underlying mechanism of this process may involve TRIM5-specific capsid recruitment to cellular proteasomes and increase peptide availability for loading and presentation of HLA class I antigens. In summary, we identified a novel function for non-human TRIM5 variants in cellular immunity. We hypothesise that TRIM5 can couple innate viral sensing and CD8+ T-cell activation to increase species barriers against retrovirus infection.

    IMPORTANCE New therapeutics to tackle HIV-1 infection should aim to combine rapid innate viral sensing and cellular immune recognition. Such strategies could prevent seeding of the viral reservoir and the immune damage that occurs during acute infection. The non-human TRIM5 variants, rhesus TRIM5aalpha; (RhT5) and TRIM-cyclophilin A (TCyp), are attractive candidates owing to their potency in sensing HIV-1 and blocking its activity. Here, we show that expression of RhT5 and TCyp in HIV-1nndash;infected cells improves CD8+ T cellnndash;mediated inhibition through the direct activation of HIV-1nndash;specific CD8+ T-cell responses. We found that the potency in CD8+ activation was stronger for RhT5 variants and capsid-specific CD8+ T-cells in a mechanism that relies on TRIM5-dependent particle recruitment to cellular proteasomes. This novel mechanism couples innate viral sensing with cellular immunity in a single protein and could be exploited to develop innovative therapeutics for control of HIV-1 infection.

  • The relationship between vaccine-induced antibody capture of infectious virus and infection outcomes following low-dose, repeated rectal challenge with SIVmac251 [PublishAheadOfPrint]

  • Antibodies are known to enhance in vitro infection by human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). We measured the ability of antibodies induced by ALVAC-SIV/gp120 vaccination, given with alum or MF59 adjuvant, to capture infectious SIVmac251 and determined the association between capture and infection outcomes following low-dose, repeated rectal challenge of rhesus macaques. We found that capture correlated with the number of transmitted/founder (T/F) variants that established infection, such that animals whose plasma captured more virus were infected with a higher number of T/F strains. Capture also correlated with results of Env binding assays, indicating that greater immunogenicity resulted in greater capture. Although vaccination elicited negligible neutralizing activity against the challenge strain (50% inhibitory dilutions ggt;1/80 in all cases), animals with low capture and whose plasma, at a fixed dilution, inhibited a higher fraction of virus, were infected at a lower rate than animals with high capture and low neutralization (p = 0.039); only animals with the low capture/high neutralization response profile were protected when compared with unvaccinated control animals (p = 0.026). In a sieve analysis, high and low capture were distinguishable on the basis of polymorphisms in the V1 loop of Env at amino acids 144 and 145. Our results indicate that vaccine-induced antibody that binds to and captures infectious virus but does not inhibit its infectivity may enhance the likelihood of infection following rectal challenge with SIVmac251. Higher immunogenicity resulting in better antibody capture but similar anti-infectivity may not improve vaccine efficacy.

    Importance Vaccines generally prevent viral infections by eliciting antibodies that inhibit virus infectivity. However, antibodies, including those induced by vaccination, have the potential to enhance, rather than prevent infection. We measured the ability of vaccine-induced antibodies to capture infectious simian immunodeficiency virus (SIV) and explored the relationship between virus capture and infection outcomes. We found that capture correlated with the number of SIV variants that established infection, such that animals whose plasma captured more virus were infected with a higher number of unique strains. In addition, animals whose sera had high capture but weak anti-infectivity activity were infected at a higher rate than were animals with low capture and stronger anti-infectivity activity. These results suggest that vaccines that induce antibodies that bind to and capture infectious virus but don't inhibit virus infectivity will not be effective in preventing infection.

  • Characterization of Viral Populations using Circular Sequencing [PublishAheadOfPrint]

  • With the enormous sizes viral populations reach, many variants are at too low a frequency to be detected by conventional next generation sequencing methods. Circular Sequencing (CirSeq) is a method by which the error rate of NGS is decreased so that even low frequency viral variants can be accurately detected. The ability to visualize almost the entire genetic makeup of a viral swarm has implications in epidemiology, viral evolution and vaccine design. Here we discuss experimental planning, analysis, and recent insights using CirSeq.

  • HIV-1 Vpr Inhibits Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication by Inducing MiR-942-5p and Activating NF-{kappa}B Signaling [PublishAheadOfPrint]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) infection is required for the development of several AIDS-related malignancies, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). The high incidence of AIDS-KS has been ascribed to the interaction of KSHV and HIV-1. We have previously shown that HIV-1 secreted proteins, Tat and Nef, regulate KSHV lifecycle, and synergize with KSHV oncogenes to promote angiogenesis and tumorigenesis. Here, we examined the regulation of KSHV latency by HIV-1 viral protein R (Vpr). We found that soluble Vpr inhibits the expression of KSHV lytic transcripts and proteins, as well as viral particle production by activating NF-B signaling following internalization into PEL cells. By analyzing the expression profiles of microRNAs combined with target search by bioinformatics and luciferase reporter analyses, we identified a Vpr-upregulated cellular microRNA (miRNA), miR-942-5p, that directly targeted IBaalpha;. Suppression of miR-942-5p relieved the expression of IBaalpha; and reduced Vpr inhibition of KSHV lytic replication while overexpression of miR-942-5p enhanced Vpr inhibition of KSHV lytic replication. Our findings collectively illustrate that, by activating NF-B signaling through upregulating a cellular miRNA to target IBaalpha;, internalized HIV-1 Vpr inhibits KSHV lytic replication. These results have demonstrated an essential role of Vpr in the lifecycle of KSHV.

    IMPORTANCE Co-infection by HIV-1 promotes the aggressive growth of Kaposi's sarcoma-associated herpesvirus (KSHV)-related malignancies, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). In this study, we have shown that soluble HIV-1 Vpr inhibits KSHV lytic replication by activating NF-B signaling following internalization into PEL cells. Mechanistic studies revealed that a cellular microRNA upregulated by Vpr, miR-942-5p, directly targeted IBaalpha;. Suppression of miR-942-5p relieved IBaalpha; expression and reduced Vpr inhibition of KSHV replication while overexpression of miR-942-5p enhanced Vpr inhibition of KSHV replication. These results indicate that, by activating NF-B signaling through upregulating a cellular miRNA to target IBaalpha;, internalized Vpr inhibits KSHV lytic replication. This work illustrates a molecular mechanism by which HIV-1 secreted regulatory protein Vpr regulates KSHV latency and pathogenesis of AIDS-related malignancies.

  • HCMV can procure deoxyribonucleotides for viral DNA replication in the absence of retinoblastoma protein phosphorylation. [PublishAheadOfPrint]

  • Viral DNA replication requires deoxyribonucleotides (dNTPs). These molecules, which are found at low levels in non-cycling cells, are generated by either salvage pathways or through de novo synthesis. Nucleotide synthesis utilizes the activity of a series of nucleotide biosynthetic enzymes (NBEs) whose expression is repressed in non-cycling cells by complexes between the E2F transcription factors and the retinoblastoma (Rb) tumor suppressor. Rb-E2F complexes are dissociated and NBE expression is activated during cell cycle transit by Cyclin-dependent kinase (Cdk) -mediated Rb phosphorylation. The DNA virus Human Cytomegalovirus (HCMV) encodes a v-Cdk (the UL97 protein) that phosphorylates Rb, induces the expression of cellular NBEs, and is required for efficient viral DNA synthesis. A long-held hypothesis proposed that viral proteins with functionally similar Rb-inactivating activities as UL97 facilitated viral DNA replication in part by inducing the de novo production of dNTPs. However, we found that dNTPs were limiting even in cells infected with wild type HCMV in which UL97 is expressed and Rb is phosphorylated. Furthermore, we revealed that both de novo and salvage pathway enzymes contribute to viral DNA replication during HCMV infection, and that Rb phosphorylation by cellular Cdks does not correct the viral DNA replication defect observed in cells infected with a UL97 deficient virus. We conclude that HCMV can obtain dNTPs in the absence of Rb phosphorylation and that UL97 can contribute to the efficiency of DNA replication in an Rb-phosphorylation independent manner.

    IMPORTANCE Transforming viral oncoproteins such as Adenovirus E1A and Papillomavirus E7 inactivate Rb. The standard hypothesis for how Rb inactivation facilitates infection with these viruses is through an increase in the enzymes required for DNA synthesis, which include nucleotide biosynthetic enzymes. However, HCMV UL97, which functionally mimics these viral oncoproteins through phosphorylation of Rb, fails to induce the production of non-limiting amounts of dNTPs. This finding challenges the paradigm of the role of Rb inactivation during DNA virus infection and uncovers the existence of an alternative mechanism by which UL97 contributes to HCMV DNA synthesis. The ineffectiveness of the UL97 inhibitor Maribavir in clinical trials might be better explained with a fuller understanding of the role of UL97 during infection. Furthermore, as the nucleoside analog ganciclovir is the current drug of choice for treating HCMV, knowing the provenance of the dNTPs incorporated into viral DNA may help inform antiviral therapeutic regimens.

  • Viral Macro Domains Reverse Protein ADP-ribosylation [PublishAheadOfPrint]

  • ADP-ribosylation is a post-translational protein modification in which ADP-ribose is transferred from nicotinamide dinucleotide (NAD+) to specific acceptors to regulate a wide variety of cellular processes. The Macro domain is an ancient and highly evolutionary conserved protein domain widely distributed throughout all kingdoms of life, including viruses. The human TARG1/C6orf130, MacroD1 and MacroD2 proteins can reverse ADP-ribosylation by acting on ADP-ribosylated substrates through the hydrolytic activity of their Macro domain. Here, we report that the Macro domain from hepatitis E virus (HEV) serves as an ADP-ribose-protein hydrolase to remove mono-ADP-ribose (MAR, de-MARylation) and poly(ADP-ribose) chains (PAR, de-PARylation) from mono- and poly(ADP)-ribosylated proteins, respectively. The presence of the HEV helicase in cis dramatically increases the binding of the Macro domain to poly(ADP-ribose) and stimulates the de-PARylation activity. Abrogation of the latter dramatically decreases replication of a HEV sub-genomic replicon. The deMARylation activity is present in all three pathogenic (+)ssRNA virus families which carry a Macro domain: Coronaviridae (severe acute respiratory syndrome coronavirus, human coronavirus 229E), Togaviridae (venezuelan equine encephalitis virus), and Hepeviridae (HEV), indicating that it might be a significant tropism and/or pathogenic determinant.

    Importance Protein ADP-ribosylation is a covalent post-translational modification regulating cellular protein activities in a dynamic fashion to modulate and coordinate a variety of cellular processes. Three viral families, Coronaviridae, Togaviridae, and Hepeviridae possess Macro domains embedded in their polyproteins. Here we show that viral Macro domains reverse cellular ADP-ribosylation, potentially cutting the signal of a viral infection in the cell. Various Poly(ADP-ribose) polymerases which are notorious guardians of cellular integrity are de-modified by Macro domains from members of these virus families. In the case of hepatitis E virus, the adjacent viral helicase domain dramatically increases the binding of the Macro domain to PAR and simulates the de-modification activity.

  • The interferon-stimulated gene IFITM3 restricts infection and pathogenesis of arthritogenic and encephalitic alphaviruses [PublishAheadOfPrint]

  • Host cells respond to viral infections by producing type I interferon (IFN), which induces the expression of hundreds of interferon stimulated genes (ISGs). Although ISGs mediate a protective state against many pathogens, the antiviral functions of the majority of these genes have not been identified. IFITM3 is a small transmembrane ISG that restricts a broad range of viruses including orthomyxoviruses, flaviviruses, filoviruses, and coronaviruses. Here, we show that alphavirus infection is increased in Ifitm3-/- and Ifitm locus deletion (Ifitm-del) fibroblasts, and reciprocally, reduced in fibroblasts trans-complemented with Ifitm3. Mechanistic studies showed that Ifitm3 did not affect viral binding or entry, but inhibited pH-dependent fusion. In a murine model of chikungunya virus arthritis, Ifitm3-/- mice sustained greater joint swelling in the ipsilateral ankle at days 3 and 7 post infection, and this correlated with higher levels of pro-inflammatory cytokines and viral burden. Flow cytometric analysis suggested that Ifitm3-/- macrophages from the spleen were infected at greater levels than observed in wild-type (WT) mice, results that were supported by experiments with Ifitm3-/- bone marrow derived macrophages. Ifitm3-/- mice also were more susceptible than WT mice to lethal alphavirus infection with Venezuelan equine encephalitis virus, and this was associated with greater viral burden in multiple organs. Collectively, our data define an antiviral role for Ifitm3 in restricting infection of multiple alphaviruses.

    IMPORTANCE The interferon-induced transmembrane protein 3 (IFITM3) inhibits infection of multiple families of viruses in cell culture. Compared to other viruses, much less is known about the antiviral effect of IFITM3 on alphaviruses. In this study, we characterized the antiviral activity of mouse Ifitm3 against arthritogenic and encephalitic alphaviruses using cells and animals with a targeted gene deletion of Ifitm3 as well as deficient cells trans-complemented with Ifitm3. Based on extensive virological analysis, we demonstrate greater levels of alphavirus infection and disease pathogenesis when Ifitm3 expression is absent. Our data establish an inhibitory role for Ifitm3 in controlling infection of alphaviruses.

  • MORC3, a component of PML Nuclear Bodies, has a role in restricting herpes simplex virus type 1 and human cytomegalovirus [PublishAheadOfPrint]

  • We previously reported that MORC3, a protein associated with promyelocytic leukemia nuclear bodies (PML NBs), is a target of HSV-1 ICP0 mediated degradation. Since it is well known that certain other components of the PML NB complex play an important role during an intrinsic immune response to HSV-1, and are also degraded or inactivated by ICP0, we further investigate here the role of MORC3 during HSV-1 infection. We demonstrate that MORC3 has antiviral activity during HSV-1 infection and that this antiviral role is counteracted by ICP0. In addition, MORC3's antiviral role extends to wild type (wt) HCMV infection as its plaque forming efficiency increased in MORC3 depleted cells. We found that MORC3 is recruited to sites associated with HSV-1 genomes after their entry into the nucleus of an infected cell, and in wt infections this is followed by its association with ICP0 foci prior to its degradation. The RING finger domain of ICP0 was required for degradation of MORC3 and we confirmed that no other HSV-1 protein is required for the loss of MORC3. We also found that MORC3 is required for fully efficient recruitment of PML, Sp100, hDaxx and H2AX to sites associated with HSV-1 genomes entering the host cell nucleus. This study further unravels the intricate ways in which HSV-1 has evolved to counteract the host immune response and reveals a novel function for MORC3 during the host intrinsic immune response.

    IMPORTANCE Herpesviruses have devised ways to manipulate the host intrinsic immune response to promote their own survival and persistence within the human population. One way in which this is achieved is through degradation or functional inactivation of PML nuclear body (PML NB) proteins which are recruited to viral genomes in order to repress viral transcription. Because MORC3 associates with PML NBs in uninfected cells, and is a target for HSV-1 mediated degradation, we investigated the role of MORC3 during HSV-1 infection. We found that MORC3 is also recruited to viral HSV-1 genomes and importantly it contributes to the fully efficient recruitment of PML, hDaxx, Sp100 and H2AX to these sites. Depletion of MORC3 resulted in an increase in ICP0-null HSV-1 and wt HCMV replication and plaque formation, and therefore this study reveals that MORC3 is an antiviral factor which plays an important role during HSV-1 and HCMV infection.

  • MHC-bound HIV Peptides Identified from Various Cell Types Reveal Common Nested Peptides and Novel T Cell Responses [PublishAheadOfPrint]

  • Despite the critical role of epitope presentation for immune recognition we still lack a comprehensive definition of HIV peptides presented by HIV-infected cells. Here we identified 107 MHC-bound HIV peptides directly from the surface of live HIV-transfected 293T cells, HIV-infected B cell lines and primary CD4 T cells expressing a variety of HLA. The majority of peptides was 8-12aa long and mostly derived from Gag and Pol. The analysis of the total MHC-peptidome and of HLA-A02-bound peptides identified new non-canonical HIV peptides of up to 16 aa that could not be predicted by HLA anchor scanning and revealed an heterogeneous surface peptidome. Nested sets of surface HIV peptides included optimal and extended HIV epitopes, peptides partly overlapping or distinct from known epitope, revealing new immune responses in HIV-infected persons. Surprisingly in all three cell types a majority of Gag peptides derived from p15 rather than from the most immunogenic p24. The cytosolic degradation of peptide precursors in corresponding cells confirmed the generation of identified surface nested peptides. Cytosolic degradation revealed peptides commonly produced in all cell types and displayed by various HLAs, peptides commonly produced by all cell and selectively displayed by specific HLAs, and peptides produced in only one cell type. Importantly we identified areas of proteins leading to common presentation of non-canonical peptides by several cell types with distinct HLA. These peptides may benefit the design of immunogens focusing T cell responses toward relevant markers of HIV infection in the context of HLA diversity.

    Importance The recognition of HIV-infected cells by immune T cells relies on the presentation of HIV-derived peptides by diverse HLA molecules at the surface of cells. The landscape of HIV peptides displayed by HIV-infected cells is not well defined. Considering the diversity of HLA molecules in the human population it is critical for vaccine design to identify HIV peptides that may be displayed despite the HLA diversity. We identified 107 HIV peptides directly from the surface of three cell types infected with HIV. They corresponded to nested sets of HIV peptides of canonical and novel non-canonical lengths not predictable by the presence of HLA anchors. Importantly we identified areas of HIV proteins leading to presentation of non-canonical peptides by several cell types with distinct HLA. Including such peptides in vaccine immunogen may help to focus immune responses toward common markers of HIV infection in the context of HLA diversity.

  • Cryo-electron microscopy reconstruction and stability studies of Wild-Type and R432A Variant of AAV2 Reveals Capsid Structural Stability is a Major Factor in Genome Packaging [PublishAheadOfPrint]

  • The Adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at ~5.0 AAring; (medium) and also 3.8 and 3.7 AAring; (high) resolution, respectively. The high resolution structures showed that removal of the arginine side-chain in AAV2-R432A eliminated hydrogen bonding interactions resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis towards the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ~10ddeg; C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. Additionally, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the bbeta;A strand region under the icosahedral 2-fold axis rather than anti-parallel to the bbeta;B strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency.

    IMPORTANCE The mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid-Rep interaction. Understanding this mechanism may aid in the improvement of AAV packaging efficiency, which is currently ~1:10 (10%) genome packaged to empty capsid in vector preparations. This report identifies regions of the AAV capsid that play roles in genome packaging and that may be important for Rep recognition. It also demonstrates the need to maintain capsid stability for the success of this process. This information is important for efforts to improve AAV genome packaging and will also inform the engineering of AAV capsid variants for improved tropism, specific tissue targeting, and host antibody escape by defining amino acids that cannot be altered without detriment to infectious vector production.

  • Human cathelicidin compensates for the role of apolipoproteins in the infectious particle formation of HCV [PublishAheadOfPrint]

  • Exchangeable apolipoproteins (ApoA, C and E) have been shown to redundantly participate in the formation of infectious hepatitis C virus (HCV) particles during the assembly process, although their precise role in the viral life cycle is not well understood. Recently, it has been shown that the exogenous expression of only short sequences containing amphipathic aalpha;-helices from various apolipoproteins is sufficient to restore the formation of infectious HCV particles in ApoB and ApoE double-gene knockout Huh7 (BE-KO) cells. In this study, through the expression of a small library of human secretory proteins containing amphipathic aalpha;-helix structures, we identified human cathelicidin hCAP18/LL-37 (CAMP), the only known member of the cathelicidin family of antimicrobial peptides (AMPs) in humans and expressed mainly in bone marrow and leukocytes. We showed that CAMP is able to rescue the infectious particle formation of HCV in BE-KO cells. In addition, we revealed that the LL-37 domain in CAMP containing amphipathic aalpha;-helices is crucial for the compensation of infectivity in BE-KO cells, and the expression of CAMP in non-hepatic 293T cells expressing Claudin 1 and miR-122 confers complete propagation of HCV. These results suggest the possibility of extra-hepatic propagation of HCV in cells with low or no expression of apolipoproteins but expressing secretory proteins containing amphipathic aalpha;-helices such as CAMP.

    IMPORTANCE Various exchangeable apolipoproteins play a pivotal role in the formation of infectious HCV during the assembly of the viral particle, and amphipathic aalpha;-helix motifs in the apolipoproteins have been shown to be a key factor. To the best of our knowledge, we have identified for the first time human cathelicidin CAMP as a cellular protein that can compensate the role of apolipoproteins in the life cycle of HCV. We have also identified the domain in CAMP that contains amphipathic aalpha;-helices crucial for the compensation, and show that the expression of CAMP in non-hepatic cells expressing Claudin 1 and miR-122 confers complete propagation of HCV. We speculate that low levels of HCV propagation might be possible in the extrahepatic tissues expressing secretory proteins containing amphipathic aalpha;-helices without expression of apolipoproteins.

  • Assessing host-virus co-divergence for close relatives of Merkel cell polyomavirus infecting African great apes [PublishAheadOfPrint]

  • It has long been hypothesized that polyomaviruses (PyV; family Polyomaviridae) co-diverged with their animal hosts. In contrast, recent analyses suggested co-divergence may only marginally influence the evolution of PyV. We re-assess this question by focusing on a single lineage of PyV infecting hominine hosts, the Merkel cell polyomavirus (MCPyV) lineage. By characterizing their genetic diversity in seven African great ape taxa, we show that these viruses exhibit very strong host-specificity. Reconciliation analyses identify more co-divergence than non co-divergence events. In addition, we find that a number of host and PyV divergence events are synchronous. Collectively, our results support co-divergence as the dominant process at play during the evolution of the MCPyV lineage. More generally, it adds to the growing body of evidence suggesting an ancient and stable association of PyV and their animal hosts.

    Importance The processes involved in viral evolution and the interaction of viruses with their hosts are of great scientific interest and public health relevance. It has long been thought that the genetic diversity of double stranded DNA viruses was generated over long periods of time, similar to typical host evolutionary timescales. This was also hypothesized for polyomaviruses (family Polyomaviridae), a group comprising several human pathogens, but this remains a point of controversy. Here we investigate this question by focusing on a single lineage of polyomaviruses that infect both humans and their closest relatives, the African great apes. We show that these viruses exhibit considerable host-specificity and that their evolution largely mirrors that of their hosts, suggesting co-divergence with their hosts played a major role in their diversification. Our results provide statistical evidence in favor of an association of polyomaviruses and their hosts over millions of years.

  • Reversion of Cold-adapted Live Attenuated Influenza Vaccine into a Pathogenic Virus [PublishAheadOfPrint]

  • The only licensed live attenuated influenza A vaccines (LAIVs) in the United States (FluMistrreg;) are created using internal protein coding gene segments from the cold-adapted temperature sensitive master donor virus A/Ann Arbor/6/1960 and HA/NA gene segments from circulating viruses. During serial passage of A/Ann Arbor/6/1960 at low temperatures to select the desired attenuating phenotypes, multiple cold-adaptive mutations and temperature-sensitive mutations arose. A substantial amount of scientific and clinical evidence has proven that FluMist is safe and effective. Nevertheless, no study has been conducted specifically to determine if the attenuating temperature sensitive phenotype can revert, and if so, the type of substitutions that will emerge (i.e., compensatory substitutions versus reversion of existing attenuating mutations). Serial passage of the monovalent FluMist 2009 H1N1 pandemic vaccine at increasing temperatures in vitro generated a variant that replicated efficiently at higher temperatures. Sequencing of the variant identified seven nonsynonymous mutations including PB1-E51K, PB1-I171V, PA-N350K, PA-L366I, NP-N125Y, NP-V186I, and NS2-G63E. None occurred at positions previously reported to affect temperature sensitivity of influenza A viruses. Synthetic genomics technology was used to synthesize the whole genome of the virus, and the role of individual mutations was characterized by assessing their effects on RNA polymerase activity and virus replication kinetics at various temperatures. The revertant also regained virulence and caused significant disease in mice, with severity comparable to that caused by a wild type 2009 H1N1 pandemic virus.

    IMPORTANCE The live attenuated influenza vaccine FluMistrreg; has been proven safe and effective and are widely used in the USA. The phenotype and genotype of the vaccine virus are believed to be very stable and mutants that cause disease in animals or humans have never been reported. By propagating the virus under well-controlled laboratory conditions, we found that the FluMist vaccine backbone could regain virulence to cause severe disease in mice. The identification of the responsible substitutions and elucidation of the underlying mechanisms provide unique insights on the attenuation of influenza virus, which is important to basic research on vaccines, attenuation reversion, and replication. In addition, this study suggests that the safety of LAIVs should be closely monitored after mass vaccination and novel strategies to continue to improve LAIV vaccine safety should be investigated.

  • To conquer the host, Influenza virus is packing it in: interferon-antagonistic strategies beyond NS1 [PublishAheadOfPrint]

  • The non-structural protein NS1 is well established as a virulence factor of influenza A virus counteracting induction of the antiviral type I interferon system. Recent studies now show that viral structural proteins, their derivatives, and even the genome itself are also contributing to keeping the host defense under control. Here, we will summarize the current knowledge on these NS1-independent interferon escape strategies.

  • High Throughput siRNA Screening Identifies Phosphatidylinositol 3-kinase Class II Alpha as Important for Production of Human Cytomegalovirus Virions [PublishAheadOfPrint]

  • High throughput siRNA screening is a useful methodology to identify cellular factors required for virus replication. Here we utilized a high throughput siRNA screen based on detection of a viral antigen by microscopy to interrogate cellular protein kinases and phosphatases for their importance during human cytomegalovirus (HCMV) replication, and identified the Class II Phosphatidylinositol 3-kinase PI3K-C2A as being involved in HCMV replication. Confirming this observation, infected cells treated with either pooled or individual siRNAs targeting PI3K-C2A mRNA produced approximately 10-fold less infectious virus compared to controls. Western blotting and quantitative PCR analysis of infected cells treated with siRNAs indicated that depletion of PI3K-C2A slightly reduced accumulation of late, but not immediate-early or early, viral antigens and had no appreciable effect of viral DNA synthesis. Analysis of siRNA treated cells by electron microscopy and western blotting indicated that PI3K-C2A was not required for production of viral capsids, but did lead to increased numbers of enveloped capsids in the cytoplasm that had undergone secondary envelopment and reduction of viral particles exiting the cell. Therefore, PI3K-C2A is a factor important for HCMV replication and has a role in production of HCMV virions.

    IMPORTANCE There is limited information about the cellular factors required for human cytomegalovirus (HCMV) replication. Therefore, to identify proteins involved in HCMV replication we developed a methodology to conduct a high throughput siRNA screen in HCMV infected cells. From our screening data we focused our studies on the top "hit" from our screen, the lipid kinase phosphatidylinositol 3-kinase Class II Alpha (PI3K-C2A), as its role in HCMV replication was unknown. Interestingly, we found that PI3K-C2A is important for the production of HCMV virions and is involved in virion production after secondary envelopment of viral capsids, the encapsidation of HCMV capsids by a lipid bilayer that occurs before virions exit the cell.

  • Myristoylation of the arenavirus envelope glycoprotein stable signal peptide is critical for membrane fusion but dispensable for virion morphogenesis [PublishAheadOfPrint]

  • Arenaviruses are responsible for severe and often-fatal hemorrhagic disease. In the absence of effective antiviral therapies and vaccines, these viruses pose serious threats to public health and biodefense. Arenaviruses enter the host cell by fusion of the viral and endosomal membranes, a process mediated by the virus envelope glycoprotein GPC. Unlike other class I viral fusion proteins, GPC retains its stable signal peptide (SSP) as an essential third subunit in the mature complex. SSP spans the membrane twice and is myristoylated at its cytoplasmic N terminus. Mutations that abolish SSP myristoylation have been shown to reduce pH-induced cell-cell fusion activity of ectopically expressed GPC to ~20% of wild-type levels. In order to examine the role of SSP myristoylation in the context of the intact virus, we used reverse genetics to generate Juniiacute;n viruses (Candid#1 isolate) in which the critical glycine-2 residue in SSP was either replaced by alanine (G2A) or deleted (G2). These mutant viruses produced smaller foci of infection in Vero cells and showed a ~5-fold reduction in specific infectivity, commensurate with the defect in cell-cell fusion. However, virus assembly and GPC incorporation into budded virions were unaffected. Our findings suggest that the myristate moiety is cryptically disposed in the prefusion GPC complex, and may function late in the fusion process to promote merger of the viral and cellular membranes.

    IMPORTANCE Hemorrhagic fever arenaviruses pose significant threats to public health and biodefense. Arenavirus entry into the host cell is promoted by the virus envelope glycoprotein GPC. Unlike other viral envelope glycoproteins, GPC contains a myristoylated stable signal peptide (SSP) as an essential third subunit. Myristoylation has been shown to be important for the membrane-fusion activity of recombinantly expressed GPC. Here, we use reverse genetics to study the role of SSP myristoylation in the context of the intact virion. We find that non-myristoylated GPC mutants of the Candid#1 strain of Juniiacute;n virus display a commensurate deficiency in their infectivity, albeit without additional defects in virion assembly and budding. These results suggest that SSP myristoylation may function late in the fusion process to facilitate merger of the viral and cellular membranes. Antiviral agents that target this novel aspect of GPC membrane fusion may be useful in the treatment of arenavirus hemorrhagic fevers.

  • Human papillomavirus entry: hiding in a bubble [PublishAheadOfPrint]

  • Incoming human papillomavirus (HPV) utilize vesicular transport to traffic from the plasma membrane to the trans-Golgi network. Following nuclear envelope breakdown during mitosis, the viral DNA associates with condensed chromosomes utilizing spindle microtubules for delivery. Most intriguingly, the viral DNA resides in a transport vesicle until mitosis is completed and the nuclear envelope has reformed. This finding provides support for the transient existence of nuclear membrane-bound vesicles. Due to their transient nature it also points to the existence of a cell pathway for the disposal of vesicles ending up fortuitously or purposefully in the nucleus.

  • Biochemical and structural insights into nairoviral deISGylases preference for interferon-stimulated-gene-product 15 originating from certain species [PublishAheadOfPrint]

  • The regulation of the interferon (IFN) type 1 response has been shown to rely on post-translational modification by ubiquitin (Ub) and Ub-like Interferon-stimulated gene product 15 (ISG15) to stabilize, or activate, a variety of IFN1 signaling and downstream effector proteins. Unlike Ub that is almost perfectly conserved among eukaryotes, ISG15 is highly divergent, even among mammals. Since, zoonotic viruses rely on viral proteins to recognize, or cleave, ISG15 conjugates in order to evade, or suppress, innate immunity the impact of ISG15 biodiversity plays on deISGylating proteases of the ovarian tumor familily (vOTU) from nairoviruses was evaluated. The enzymatic activity of vOTUs originating from the Crimean Congo hemorrhagic fever virus, Erve virus, and Nairobi sheep disease virus vOTUs were tested against human, mouse, shrew, sheep, bat, and camel ISG15s, which are mammalian species known to be infected by nairoviruses. This along with investigation of binding by isothermal titration calorimetry illustrated significant differences in the ability of nairovirus deISGylases to accommodate certain species of ISG15. To investigate the molecular underpinnings regarding species preferences of these vOTUs, a structure was determined to 2.5 AAring; for a complex of Erve virus vOTU protease and a mouse ISG15 domain. This structure revealed the molecular basis of Erve virus vOTU's preference for ISG15 over Ub and the first structural insight into a non-human ISG15. This structure also revealed key interactions, or lack thereof, surrounding three amino acids that may drive a viral deISgylase to prefer an ISG15 from one species over that of another.

    IMPORTANCE Viral ovarian tumor domain proteases (vOTUs) are among the two principle classes of viral proteases observed to reverse post-translational modification of host proteins by ubiquitin and interferon stimulated gene product 15 (ISG15), subsequently facilitating down-regulation of IFN1 signaling pathways. Unlike ubiquitin, the amino acid sequence of ISG15 is notably divergent from species to species. We illustrate that vOTUs have clear preferences for ISG15s from certain species. In addition, these observations are related to the molecular insights acquired in the first X-ray structure of the vOTU from the Erve nairovirus in complex with the first structurally resolved non-human ISG15. This information implication of certain amino acids that drive the preference of vOTUs for ISG15 from certain species.

  • Single Site Glycoprotein Mutants Inhibit a Late Event in Sindbis Virus Assembly [PublishAheadOfPrint]

  • A panel of Sindbis virus mutants that were suspected to have deficiencies in one or more aspects of their replication cycles was examined in Baby Hamster Kidney (BHK) cells. These included an amino acid deletion (H230) and substitution (H230A) in the Sindbis glycoprotein E1_H230 and similar mutants in E2_G209 (G209A, G209D, and G209). Neither H230 mutation produced measurable titer, but repeated passaging of the H230A in BHK cells produced a second site compensatory mutant (V231I) that partially rescued both H230 mutants. Electron micrograph (EM) images of these mutants showed assembled viral nucleocapsids but no completed, mature virions. EM of the compensatory mutant strains showed complete virus particles, but these now formed paracrystalline arrays. Neither E2_G209 substitution mutant had any effect on virus production; however, the deletion mutant ( G209) showed a very low titer when grown at 37ddeg;C and no titer when grown at 28ddeg;C. When the deletion mutant grown at 28ddeg;C was examined by EM, partially budded virions were observed at the cell surface. S-35 labeling of this mutant confirmed the presence of mutant virus protein in the transfected BHK cell lysate. We conclude that H230 is essential for the assembly of complete infectious Sindbis virions and that the presence of an amino acid at E2 position 209 is required for complete budding of Sindbis virus particles although several different amino acids can be at this location without affecting titer.

    Importance: Our data show the importance of single site mutations at E1_H230 and E2_G209 in Sindbis virus's glycoproteins. These sites have been shown to affect assembly and antibody binding in previous studies. Our data indicate that mutation of one histidine residue in E1 is detrimental to the assembly of Sindbis virus particles in baby hamster kidney cells. Repeated passaging led to a second site substitution that partially restores titer although EM shows a still altered phenotype. Substitutions at position G209 in E2 have no effect on titer, but deletion of this residue greatly reduces titer and again prevents assembly. When this mutant is grown at lowered temperature, virus particles bud from the host cell, but budding arrests before the progeny virus escape. These results allow us to conclude that these sites have essential roles in assembly, and E2_G209 shows us a new viral egress phenotype.

  • Varying Roles of Herpes Simplex Virus 1 ICP0 and Vhs in Loss of Cellular IFI16 in Different Cell Types [PublishAheadOfPrint]

  • The HSV-1 ICP0 protein is an E3 ubiquitin ligase that promotes the degradation of several host cell proteins. Most studies have found that ICP0 promotes the loss of IFI16 in infected cells, but one study reported that ICP0 was not necessary or sufficient for loss of IFI16 in a tumor-derived cell line. Therefore, in this study we examined the requirement for ICP0 in promoting the loss of IFI16 in several normal and tumor-derived cell lines. HSV-1 infection resulted in an observable decrease of IFI16 protein levels in normal human foreskin fibroblasts (HFF), normal oral keratinocytes (NOK), and HeLa cells, but not in U2OS cells. During infection with an ICP0-null virus, we observed a reduced loss of IFI16 in HFF and NOK cells, but not in HeLa cells. Ectopic expression of ICP0 from a transfected plasmid was sufficient to promote the loss of IFI16 in HFF and NOKs. In the absence of ICP0, we observed a delayed reduction of IFI16 protein that correlated with a reduction in the steady state levels of IFI16 mRNA. In addition, we show that the ICP0-independent loss of IFI16 in HeLa cells is dependent on the activity of the viral virion host shutoff (vhs) tegument protein. Together these results demonstrate that HSV-1 promotes the loss of IFI16 through two mechanisms: 1) by ICP0-dependent degradation of IFI16, and 2) vhs-dependent turnover of IFI16 mRNA. In addition, this study highlights a potential intrinsic difference between normal and tumor-derived cells for the activities of IFI16 and HSV-1 ICP0.

    Importance HSV-1 is a ubiquitous virus that establishes a lifetime persistent infection in humans. The relative success of HSV-1 as a pathogen is, in part, dependent on the expression of viral proteins that counteract host intrinsic defense mechanisms and that modulate immune responses during viral infection. In this study we examine the relative roles of two viral gene products for their ability to promote loss of the antiviral IFI16 DNA sensor. We demonstrate that the viral immediate-early ICP0 protein plays a dominant role in the loss of IFI16 in normal, but not tumor-derived, human cell lines. In contrast, viral vhs-mediated loss of IFI16 by mRNA destabilization is revealed to be dominant in tumor-derived cells where ICP0 is non-functional. Together, these results contribute to our understanding of how HSV-1 modulates IFI16 protein levels and highlights cell type dependent differences between normal and tumor-derived cells.

  • Derivation and characterization of pathogenic transmitted/founder molecular clones from SIVsmE660 and SIVmac251 following mucosal infection [PublishAheadOfPrint]

  • Currently available simian immunodeficiency virus (SIV) infectious molecular clones (IMCs) and isolates used in non-human primate (NHP) models of AIDS were originally derived from infected macaques during chronic infection or end stage disease and may not authentically recapitulate features of transmitted/founder (T/F) genomes that are of particular interest in transmission, pathogenesis, prevention, and treatment studies. We therefore generated and characterized T/F IMCs from genetically and biologically heterogeneous challenge stocks of SIVmac251 and SIVsmE660. Single genome amplification (SGA) was used to identify full-length T/F genomes present in plasma during acute infection resulting from atraumatic rectal inoculation of Indian rhesus macaques with low doses of SIVmac251 or SIVsmE660. All 8 T/F clones yielded viruses that were infectious and replication competent in vitro, with replication kinetics similar to the widely-used chronic-infection derived IMCs SIVmac239 and SIVsmE543. Phenotypically, the new T/F virus strains exhibited a range of neutralization sensitivity profiles. Four T/F virus strains were inoculated into rhesus macaques and each exhibited typical SIV replication kinetics. The SIVsm T/F viruses were sensitive to TRIM5aalpha; restriction. All T/F viruses were pathogenic in rhesus macaques resulting in progressive CD4+ T cell loss in gastrointestinal tissues, peripheral blood and lymphatic tissues. Animals developed pathological immune activation, lymphoid tissue damage including fibrosis, and clinically significant immunodeficiency leading to AIDS defining clinical endpoints. These T/F clones represent a new molecular platform for the analysis of virus transmission and immunopathogenesis and for the generation of novel "bar-coded" challenge viruses and next-generation simian-human immunodeficiency viruses that may advance the HIV/AIDS vaccine agenda.

    Importance Statement Nonhuman primate research has relied on only a few infectious molecular clones for a myriad of diverse research projects including pathogenesis, preclinical vaccine evaluations, transmission, and host vs pathogen interactions. With new data suggesting a selected phenotype of the virus that causes infection (i.e. the transmitted/founder virus), we sought to generate and characterize infectious molecular clones from two widely used simian immunodeficiency virus lineages (SIVmac251 and SIVsmE660). Although the exact requirements necessary to be a T/F virus is not yet fully understood, we generated cloned viruses with all the necessary characteristic of a successful T/F virus. Cloned viruses revealed typical acute and set point viral load dynamics with pathological immune activation, lymphoid tissue damage progressing to significant immunodeficiency and AIDS defining clinical endpoints in some animals. These T/F clones represent a new molecular platform for studies requiring authentic T/F viruses.

  • Human papillomavirus E2 protein: linking replication, transcription, and RNA processing. [PublishAheadOfPrint]

  • The human papillomavirus (HPV) life cycle is tightly linked to differentiation of the infected epithelium. This means that viral proteins must exert control over epithelial gene expression in order to optimize viral production. The HPV E2 protein controls replication, transcription and viral genome partitioning during the viral infectious life cycle. It consists of a nucleic acid-binding domain and a protein-protein interaction domain separated by a flexible serine and arginine-rich hinge region. Over the last few years, mounting evidence has uncovered an important new role for E2 in viral and cellular RNA processing. This Gem discusses the role of E2 in controlling the epithelial cellular environment and how E2 might act to coordinate late events in the viral replication cycle.

  • Identification of a new benzimidazole derivative as an antiviral against hepatitis C virus [PublishAheadOfPrint]

  • Aminoquinolines and piperazines, linked or not, have been used successfully to treat malaria and some molecules of this family also exhibit antiviral properties. Here we tested several derivatives of 4-aminoquinolines and piperazines for their activity against hepatitis C virus (HCV). We screened eleven molecules from three different families of compounds, and we identified anti-HCV activity in cell culture for six of them. Of these, we selected a compound (B5) that is currently ending clinical phase I evaluation for neurodegenerative diseases. In hepatoma cells, B5 inhibited HCV infection in a pangenotypic and dose-dependent manner, and its antiviral activity was confirmed in primary hepatocytes. B5 also inhibited infection by pseudoparticles expressing HCV envelope glycoproteins E1 and E2, and we demonstrated that it affects a post-attachment stage of the entry step. Virus with resistance to B5 was selected by sequential passage in the presence of the drug, and reverse genetics experiments indicated that resistance was mainly conferred by a single mutation in the putative fusion peptide of E1 envelope glycoprotein (F291I). Furthermore, analyses of the effect of other closely related compounds on B5 resistant mutant, suggest that B5 shares a mode of action with other 4-aminoquinolines based molecules. Finally, mice with humanized liver that were treated with B5 showed a delay in the kinetics of the viral infection. In conclusion, B5 is a novel interesting anti-HCV molecule that could be used to decipher the early steps of the HCV life cycle.

    IMPORTANCE In the last four years, HCV therapy has been profoundly improved with the approval of direct-acting antivirals in the clinical practice. Nevertheless, the high costs of these drugs limit access to therapy in most countries. The present study reports the identification and characterization of a compound (B5) that inhibits HCV propagation in cell culture and is currently ending clinical phase I evaluation for neurodegenerative diseases. This molecule inhibits the HCV life cycle by blocking virus entry. Interestingly, after selection of drug-resistant virus, a resistance mutation was identified in the putative fusion peptide of E1 envelope glycoprotein, indicating that B5 could be used to further investigate the fusion mechanism. Furthermore, mice with humanized liver treated with B5 showed a delay in the kinetics of the viral infection. In conclusion, B5 is a novel interesting anti-HCV molecule that could be used to decipher the early steps of the HCV life cycle.

  • Poliovirus Polymerase Leu420 Facilitates RNA Recombination and Ribavirin Resistance [PublishAheadOfPrint]

  • RNA recombination is important in the formation of picornavirus species groups and the ongoing evolution of viruses within species groups. In this study we examined the structure and function of poliovirus polymerase, 3Dpol, as it relates to RNA recombination. Recombination occurs when nascent RNA products exchange one viral RNA template for another during RNA replication. Because recombination is a natural aspect of picornavirus replication, we hypothesized that some features of 3Dpol may exist, in part, to facilitate RNA recombination. Furthermore, we reasoned that alanine substitution mutations that disrupt 3Dpol-RNA interactions within the polymerase elongation complex might increase and/or decrease magnitudes of recombination. We found that an L420A mutation in 3Dpol decreased the frequency of RNA recombination whereas alanine substitutions at other sites in 3Dpol increased the frequency of recombination. The 3Dpol Leu420 side chain interacts with a ribose in the nascent RNA product three nucleotides from the active site of the polymerase. Notably, the L420A mutation that reduced recombination also rendered virus more susceptible to inhibition by ribavirin, coincident with the accumulation of ribavirin-induced G-ggt;A and C-ggt;U mutations in viral RNA. We conclude that 3Dpol Leu420 is critically important for RNA recombination and that RNA recombination contributes to ribavirin resistance.

    IMPACT Recombination contributes to the formation of picornavirus species groups and the emergence of circulating vaccine-derived polioviruses (cVDPVs). The recombinant viruses that arise in nature are occasionally more fit than either parental strain, especially when the two partners in recombination are closely related; i.e., members of characteristic species groups such as enterovirus species groups A-H or rhinovirus species groups A-C. Our study shows that RNA recombination requires conserved features of the viral polymerase. Furthermore, a polymerase mutation that disables recombination renders virus more susceptible to the antiviral drug ribavirin, suggesting that recombination contributes to ribavirin resistance. Elucidating the molecular mechanisms of RNA replication and recombination may help mankind achieve and maintain poliovirus eradication.

  • Infection of common marmosets with GB virus B chimeric virus encoding for the major nonstructural proteins NS2-4A of hepatitis C virus [PublishAheadOfPrint]

  • Lack of immunocompetent small primate models has been an obstacle for developing HCV vaccines and affordable antiviral drugs. In this study, HCV/GBV-B chimeric virus (HCV NS2-4A chimera) carrying the major nonstructural proteins NS2 to 4A was produced and used to infect common marmosets, since HCV NS2-4A proteins are critical protease and major antigens. Seven marmosets were inoculated intra-hepatically with HCV NS2-4A chimeric RNA for primary infection or intravenously injected with chimera-containing serum for passage infection. Three animals used as the controls were injected with PBS or GBV-B, respectively. Six of seven HCV NS2-4A chimera-infected marmosets exhibited consistent viremia, and one showed transient viremia during the course of follow-up detection. All these six infected animals with persistent circulating viremia presented characteristics typical of viral hepatitis, including viral RNA and proteins in hepatocytes and histopathological changes in liver tissues. Viremia was consistently detected for 5 to 54 weeks of follow-up. FK506 immunosuppression facilitated the establishment of chimera persistent infection in marmosets. An animal with chimera infection spontaneously cleared the virus in blood seven weeks following the first inoculation, but viral RNA persistence, low-level viral protein and mild necroinflammation remained in liver tissue. The specific antibody and T cell response to HCV NS3 in this viremia-resolved marmoset was boosted by re-challenging, but no viremia was detected during 57 weeks of follow-up. The chimera-infected marmosets described can be used as a suitable small primate animal model for studying novel antiviral drugs and T-cell based vaccines against HCV infection.

    Importance HCV infection causes approximately 70% of chronic hepatitis and frequently associates with primary liver cancer globally. Chimpanzees have been used as a reliable primate model for HCV infection but ethical considerations have restricted their utility in biomedical researches. GB virus B (GBV-B) is a flavivirus related to HCV. It can infect common marmosets, a New World small primate, and induces viral hepatitis similar to HCV infection in humans. To minimize differences between GBV-B and HCV, we generated HCV NS2-4A/GBV-B chimeric viruses and established a chimera-infected marmoset model. HCV NS2-4A chimera-infected marmosets provide a small animal model for evaluating novel antiviral drugs targeting HCV NS3-4A protease and T-cell based HCV vaccines.

  • UNUSUAL FEATURES OF SODIUM TAUROCHOLATE COTRANSPORTING POLYPEPTIDE AS A HEPATITIS B VIRUS RECEPTOR [PublishAheadOfPrint]

  • Cell culture (cc)-derived hepatitis B virus (HBV) can infect differentiated HepaRG cells, but efficient infection requires addition of polyethylene glycol (PEG) during inoculation. Identification of sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor enabled ccHBV infection of NTCP reconstituted HepG2 cells, although very little hepatitis B surface antigen (HBsAg) is produced. We found infection by patient serum-derived HBV (sHBV), which required purification of viral particles through ultracentrifugation or PEG precipitation, was PEG independent and much more efficient in HepaRG than HepG2/NTCP cells. In contrast to hepatitis B e antigen (HBeAg), HBsAg was not a reliable marker of productive sHBV infection at early time points. Low HBsAg/HBeAg ratio by ccHBV infected HepG2/NTCP cells was attributable to dimethylsulfoxide in culture medium, NTCP overexpression, and HBV genotype D. HepG2/NTCP cells released more viral antigens than HepG2 cells following HBV genome delivery by adeno-associated virus, and stable expression of NTCP in a ccHBV producing cell line increased viral mRNAs, proteins, replicative DNA, and covalently closed circular DNA. NTCP protein expression in HepG2/NTCP cells, despite being driven by the CMV promoter, was markedly increased by dimethylsulfoxide treatment. This at least partly explains ability of DMSO to promote ccHBV infection in such cell lines. In conclusion, NTCP appeared inefficient to mediate infection by serum-derived HBV. It could promote HBV RNA transcription while inhibiting HBsAg secretion. Efficient PEG-independent sHBV infection of HepaRG cells permits comparative studies of diverse clinical HBV isolates, and will help identify additional factors on virion surface promoting attachment to hepatocytes.

    Importance Currently in vitro infection with hepatitis B virus (HBV) depends on cell culture-derived HBV inoculated in the presence of polyethylene glycol. We found patient serum-derived HBV could efficiently infect differentiated HepaRG cells independent of polyethylene glycol, which represents a more physiological infection system. Serum-derived HBV has poor infectivity in HepG2 cells reconstituted with sodium taurocholate cotransporting polypeptide (NTCP), the currently accepted HBV receptor. Moreover, HepG2/NTCP cells secreted very little hepatitis B surface antigen following infection with cell culture-derived HBV, which was attributed to NTCP overexpression, genotype D virus, and dimethylsulfoxide added to culture medium. NTCP could promote HBV RNA transcription, protein expression, and DNA replication in HepG2 cells stably transfected with HBV DNA, while dimethylsulfoxide could increase NTCP protein level despite transcriptional control by a CMV promoter. Therefore, this study revealed several unusual features of NTCP as an HBV receptor and established conditions for efficient serum virus infection in vitro.

  • The Specificity, Size and Frequency of Spaces That Characterize the Mechanism of Bulk Transepithelial Transport of Prions in the Nasal Cavity of Hamsters and Mice [PublishAheadOfPrint]

  • Inhalation of infected brain homogenate results in transepithelial transport of prions across the nasal mucosa of hamsters, some of which occurs rapidly in relatively large amounts between cells (A.E. Kincaid, K.F. Hudson, M.W. Richey, J.C. Bartz. J. Virol 86:12731-12740, 2012, doi:10.1128/JVI.01930-12). Bulk transepithelial transport in the nasal cavity has not been studied to date. Here we characterize the frequency, size and specificity of intercellular spaces that mediate bulk transport of inhaled prions between cells of mice or hamsters following extranasal inoculation with mock-infected brain homogenate, different strains of prion-infected brain homogenate, or brain homogenate mixed with India ink. Infected or mock-infected inoculum was identified within lymphatic vessels of the lamina propria and in spaces ggt; 5 mmu;m between a small number of cells of the nasal mucosa in ggt; 90% of animals from 5 to 60 min after inhalation. The width of the spaces between cells, the amount of the inoculum within the lumen of lymphatic vessels and the timing of the transport indicate that this type of transport was taking place through preexisting spaces in the nasal cavity that were orders of magnitude wider than what is normally reported for paracellular transport. The indiscriminate rapid bulk transport of brain homogenate in the nasal cavity results in immediate entry into nasal cavity lymphatics following inhalation. This novel mechanism may underlie the recent report of the early detection of prions in blood following inhalation and has implications for horizontal prion transmission.

    Importance The results of these studies demonstrate that the nasal mucosa of mice and hamsters is not an absolute anatomical barrier to inhaled prion-infected, or uninfected brain homogenate. Relatively large amounts of infected and uninfected brain homogenate rapidly cross the nasal mucosa and enter the lumen of lymphatic vessels following inhalation. These bulk transepithelial transport events were relatively rare but present in ggt;90% of animals 5 to 60 min following inhalation. This novel mechanism of bulk transepithelial transport was seen in experimental and control hamsters and mice, indicating it was not species-specific or in response to prion exposure. The indiscriminate bulk intercellular transport of inhaled pathogens across the nasal mucosa followed by entry into the lymphatic system may be a mechanism that underlies the entry and spread of other toxins and pathogens in olfactory-driven animals.

  • Porcine Epidemic Diarrhea Virus Infection Inhibits Interferon Signaling by Targeted Degradation of STAT1 [PublishAheadOfPrint]

  • Porcine epidemic diarrhea virus (PEDV) is a worldwide-distributed alphacoronavirus, but the pathogenesis of PEDV infection is not fully characterized. During virus infection, the type I interferon (IFN) is a key mediator of innate antiviral responses. Most coronaviruses develop some strategy for at least partially circumventing the IFN response by limiting the production of IFN and by delaying the activation of IFN response. However, the molecular mechanisms by which PEDV antagonizes antiviral effects of interferon have not been fully characterized. Especially, how PEDV impacts on IFN signaling components has yet to be elucidated. In this current study, we observed that PEDV was relatively resistant to IFN-I treatment. Western blot data showed that STAT1 expression was markedly reduced in PEDV-infected cells, which was not due to the inhibition of STAT1 transcription. STAT1 downregulation was blocked by proteasome inhibitor but not by autophagy inhibitor, strongly implicating the ubiquitin-proteasome targeting degradation system. Since PEDV infection-induced STAT1 degradation was evident in cells pretreated with the general tyrosine kinase inhibitor, we conclude that STAT1 degradation is IFN signaling pathway-independent. Furthermore, we described that PEDV-induced STAT1 degradation inhibits IFNaalpha; signal transduction pathways. Pharmacologically inhibiting STAT1 degradation rescued host ability to suppress virus replication. Collectively, these data show that PEDV is capable of subverting the type I interferon response by inducing STAT1 degradation.

    IMPORTANCE In this study, we show that PEDV is resistant to the antiviral effect of IFN. The molecular mechanism is that PEDV infection degrades STAT1 in a proteasome-dependent manner. This PEDV infection-induced STAT1 degradation contributes to favoring PEDV replication. Our findings provide a new mechanism evolved by PEDV to circumvent host antiviral response.

  • Structure-activity relationships of the Human Immunodeficiency Virus Type 1 maturation inhibitor PF-46396 [PublishAheadOfPrint]

  • HIV-1 maturation inhibitors are a novel class of antiretroviral compounds, which consist of two structurally distinct chemical classes; betulinic acid derivatives and the pyridone-based compound PF-46396. It is currently believed that both classes act by a similar mode of action to generate aberrant non-infectious particles via inhibition of CA-SP1 cleavage during Gag proteolytic processing. In this study we utilized a series of novel analogues, with decreasing similarity to PF-46396, to determine the chemical groups within PF-46396 that contribute to antiviral activity, Gag binding and the relationship between these essential properties. A spectrum of antiviral activity (active, intermediate, inactive) was observed across the analogue series with respect to CA-SP1 cleavage and HIV-1 (NL4-3) replication kinetics in Jurkat T cells. We demonstrate that selected inactive analogues are incorporated into WT immature particles and that one inactive analogue is capable of interfering with PF-46396 inhibition of CA-SP1 cleavage. Mutations that confer PF-46396 resistance can impose a defective phenotype on HIV-1 that can be rescued in a compound-dependent manner. Some inactive analogues retained the capacity to rescue PF-46396-dependent mutants (SP1-A3V, SP1-A3T, CA-P157S), implying that they can also interact with mutant Gag. The structure-activity relationships observed in this study demonstrate that (i) the tert-butyl group is essential for antiviral activity, but not an absolute requirement for Gag binding, (ii) the trifluromethyl group is optimal but not essential for antiviral activity and (iii) the 2-aminoindan group is important for antiviral activity and Gag binding but not essential as its replacement is tolerated.

    Importance Combinations of antiretroviral drugs successfully treat HIV/AIDS patients, however drug resistance problems make development of new mechanistic drug classes an ongoing priority. HIV-1 maturation inhibitors are novel as they target the Gag protein, specifically by inhibiting CA-SP1 proteolytic cleavage. Lack of high resolution structural information of the CA-SP1 target in Gag has hindered our understanding of the inhibitor binding pocket and maturation inhibitor mode of action. Therefore, we utilized analogues of the maturation inhibitor PF-46396, as chemical tools to determine the chemical components of PF-46396 that contribute to antiviral activity and Gag binding and the relationship between these essential properties. This is the first study to report structure-activity relationships of the maturation inhibitor PF-46396. PF-46396 is chemically distinct from betulinic acid derived maturation inhibitors; therefore our data provide a foundation of knowledge that will aid our understanding of how structurally distinct maturation inhibitors act by a similar mode of action.

  • A Highly Conserved gp120 Inner Domain Residue Modulates Env Conformation and Trimer Stability [PublishAheadOfPrint]

  • Previous studies have shown that highly-conserved residues in the inner domain of the gp120 are required for HIV-1 envelope glycoproteins (Env) transitions to the CD4-bound conformation. Moreover, W69, a highly conserved residue located at the interface between Layer 1 and Layer 2 of the inner domain, was recently shown to be important for efficient Env recognition by CD4-induced (CD4i) antibodies capable of potent antibody-dependent cellular cytotoxicity. We evaluated the contribution of the hydrophobicity of W69 on conformational changes of Env by replacing it with a series of residues with aliphatic or aromatic side chains of decreasing chain length. We have found that the hydrophobicity of residue 69 is important for Env processing, CD4 binding and its transition to the CD4-bound conformation. The most deleterious effect was observed when W69 was replaced by alanine or glycine residues. However, the functions lost due to W69 mutations could be progressively restored with amino acids of increasing aliphatic chain length and fully recovered with residue bearing an aromatic ring.

    Interestingly, poor CD4 binding of W69A could be fully restored by introducing a compensatory mutation within Layer 2 (S115W). Structural studies of HIV-1 gp120 coree W69A/S115W mutant bound to the CD4 peptide mimetic M48U1 and Fab of anti-Cluster A antibody N60-i3 revealed no perturbations to the overall structure of the double mutant as compared to the wild type protein but identified higher mobility within the interface between Layer 1 and Layer 2, the bridging sheet region and the CD4 binding site.

    Importance HIV-1 Env transitions to the CD4-bound conformation are required for viral entry. Previous studies identified a highly-conserved residue of the inner domain, W69, as being involved in these conformational transitions. Here, we show that W69, located at the interface between the gp120 and the gp41 in the PGT151-bound trimer, plays a critical role in the inter-protomer signaling induced by CD4 binding. This new information might be useful in immunogen design.

  • The molecular determinants of antibody recognition and antigenic drift in the H3 hemagglutinin of swine influenza A virus [PublishAheadOfPrint]

  • Influenza A virus (IAV) of the H3 subtype is an important respiratory pathogen that affects both humans and swine. Vaccination to induce neutralizing antibodies against the surface glycoprotein hemagglutinin (HA) is the primary method used to control disease. However, due to antigenic drift, vaccine strains must be periodically updated. Six of the 7 positions previously identified in human seasonal H3 (positions 145, 155, 156, 158, 159, 189, 193) were also indicated in swine H3 antigenic evolution. To experimentally test the effect on virus antigenicity of these 7 positions, substitutions were introduced into the HA of an isogenic swine-lineage virus. We tested the antigenic effect of these introduced substitutions by using hemagglutination inhibition (HI) data with monovalent swine antisera and antigenic cartography to evaluate the antigenic phenotype of the mutant viruses. Combinations of substitutions within the antigenic motif caused significant changes in antigenicity. One virus mutant that varied at only two positions relative to wild-type had a ggt;4-fold reduction in HI titers compared to homologous antisera. Potential changes in pathogenesis and transmission of the double mutant were evaluated in pigs. Although the double mutant had comparable virus shedding titers and transmissibility, it caused significantly lower percentage of lung lesions. Elucidating the antigenic effects of specific amino acid substitutions at these sites in swine H3 IAV have important implications for understanding IAV virus evolution within pigs as well as for improved vaccine development and control strategies in swine.

    IMPORTANCE A key component of influenza virus evolution is antigenic drift mediated by the accumulation of amino acid substitutions in the hemagglutinin (HA) protein, resulting in escape from prior immunity generated by natural infection or vaccination. Understanding which amino acid positions of the HA contribute to the ability of the virus to avoid prior immunity is important for understanding antigenic evolution and informs vaccine efficacy predictions based on the genetic sequence data from currently circulating strains. Following our previous work characterizing antigenic phenotypes of contemporary wild-type swine H3 influenza viruses, we experimentally validated that substitutions at 6 amino acid positions in the HA protein have major effects on antigenicity. An improved understanding of the antigenic diversity of swine influenza will facilitate a rational approach for selecting more effective vaccine components to control circulation of influenza in pigs, and reduce the potential for zoonotic viruses to emerge.

  • Genetic lineage and reassortment of influenza C viruses circulating between 1947 and 2014 [PublishAheadOfPrint]

  • Since influenza C virus was first isolated in 1947, the virus has been only occasionally isolated by cell culture; there are only four strains for which complete genome sequences are registered. Here, we analyzed a total of 106 complete genomes ranging from the first isolate from 1947 to recent isolates from 2014 to determine the genetic lineages of influenza C virus, the reassortment events, and the rates of nucleotide substitution. The results showed that there are six lineages named C/Taylor, C/Mississippi, C/Aichi, C/Yamagata, C/Kanagawa, and C/Sao Paulo. They contain both antigenic and genetic lineages of the hemagglutinin-esterase (HE) gene, and the internal genes PB2, PB1, P3, NP, M, and NS are divided into two major lineages, a C/Mississippi/80-related lineage and a C/Yamagata/81-related lineage. Reassortment events were found over the entire period of 68 years. Several outbreaks of influenza C virus between 1990 and 2014 in Japan consisted of reassortant viruses, suggesting that the genomic constellation is related to influenza C virus epidemics. The nucleotide sequences were highly homologous to each other. The minimum percentage identity between viruses ranged from 91.1% for the HE gene to 96.1% for the M gene, and the rate of nucleotide substitution for the HE gene was the highest at 5.20 x 10-4 substitutions/site/year. These results indicate that reassortment is an important factor that increases the genetic diversity of influenza C virus, resulting in its ability to prevail in humans.

    IMPORTANCE Influenza C virus is a pathogen that causes acute respiratory illness in children and results in hospitalization of infants. We previously demonstrated that periodic epidemics of this virus occurred in Japan between 1996 and 2014 and that replacement of the dominant antigenic group occurred every several years as a result of selection by herd immunity. However, the antigenicity of the HE glycoprotein is highly stable, and antigenic drift has not occurred for at least 30 years. Here, we analyzed a total of 106 complete genomes spanning 68 years for the first time, and we found that influenza C viruses are circulating worldwide while undergoing reassortment as well as selection by herd immunity, resulting in an increased ability to prevail in humans. The results presented in this study contribute to the understanding of the evolution, including reassortment events, underlying influenza C virus epidemics.

  • Regulatory Interaction between the Cellular Restriction Factor IFI16 and Viral pp65 (pUL83) Modulates Viral Gene Expression and IFI16 Protein Stability [PublishAheadOfPrint]

  • A key player in the intrinsic resistance against human cytomegalovirus (HCMV) is the interferon--inducible protein 16 (IFI16), which behaves as a viral DNA sensor in the first hours post infection and as a repressor of viral gene transcription in the later stages. Previous studies on HCMV replication demonstrated that IFI16 binds to the viral protein kinase pUL97, undergoes phosphorylation and relocalizes to the cytoplasm of infected cells. In this study, we demonstrate that the tegument protein pp65 (pUL83) recruits IFI16 to the promoter of the UL54 gene and downregulates viral replication as shown by use of the HCMV mutant v65Stop, which lacks pp65 expression. Interestingly, at late time-points of HCMV infection, IFI16 is stabilized by its interaction with pp65, which stood in contrast to IFI16 degradation, observed in herpes simplex virus (HSV-1)-infected cells. Moreover, we found that its translocation to the cytoplasm, in addition to pUL97, strictly depends on pp65, as demonstrated with the HCMV mutant RV-VM1, which expresses a form of pp65 unable to translocate into the cytoplasm. Thus, these data reveal a dual role for pp65: during early infection, it modulates IFI16 activity at the promoter of immediate-early and early genes; subsequently, it delocalizes IFI16 from the nucleus into the cytoplasm, thereby stabilizing and protecting it from degradation. Overall, these data identify a novel activity of the pp65/IFI16 interactome involved in the regulation of UL54 gene expression and IFI16 stability during early and late phases of HCMV replication.

    IMPORTANCE The DNA sensor IFI16, a member of the PYHIN protein, restricts human cytomegalovirus (HCMV) replication by impairing viral DNA synthesis. Using a mutant virus lacking the tegument protein pp65 (v65Stop), we demonstrate that pp65 recruits IFI16 to the early UL54 gene promoter. As a putative counteraction towards its restriction activity, pp65 supports the nucleo-cytoplasmic export of IFI16, which was demonstrated with the viral mutant RV-VM1 expressing a nuclearly retented pp65. These data reveal a dual role of pp65 in IFI16 regulation: in the early phase of HCMV infection, it contributes to viral evasion from IFI16 restriction activity, while at later time points, it promotes the nuclear delocalization of IFI16, thereby stabilizing and protecting it from degradation. In the present work, we further clarify the mechanisms HCMV relies on to overcome intracellular innate immune restriction and provide new insights into the relevance of DNA-sensing restriction factor IFI16 during HCMV infection.

  • Prevailing PA mutation K356R in avian influenza H9N2 virus increases mammalian replication and pathogenicity [PublishAheadOfPrint]

  • Adaptation of viral polymerase complex comprising PB1, PB2 and PA is necessary for efficient influenza A virus replication in new host species. We found that mutation PA-K356R has become predominant since 2014 in avian H9N2 viruses in China as with seasonal human H1N1 viruses. The same mutation is also found in most human isolates of emergent avian H7N9 and H10N8 viruses whose six internal gene segments are derived from the H9N2 virus. We further demonstrated the mammalian adaptive functionality of PA-K356R mutation. Avian H9N2 virus with PA-K356R mutation in human A549 cells showed increased nuclear accumulation of PA, and raised viral polymerase activity that resulted in elevated viral transcription and virus output. The same mutant virus in mice also enhanced virus replication and caused lethal infection. In addition, combined mutations of PA-K356R with PB2-E627K, a well-known mammalian adaptive marker, in H9N2 virus showed further cooperative increase in virus production and severity of infection in vitro and in vivo. In summary, PA-K356R behaves as a novel mammalian tropism mutation which along with other mutations such as PB2-E627K might render avian H9N2 viruses adapted for human infection.

    Importance Mutations of polymerase complex (PB1, PB2 and PA) of influenza A virus are necessary for viral adaptation to new hosts. This study reports on a novel and predominant mammalian adaptive mutation PA-K356R in avian H9N2 viruses and human isolates of emergent H7N9 and H10N8 viruses. We found that PA-356R in H9N2 virus causes significant increase in virus replication and severity of infection in human cells and mice, and that PA-K356R cooperates with PB2-E627K mutation, a well characterized human adaptive marker, to exacerbate mammalian infection in vitro and in vivo. Therefore, PA-K356R mutation is a significant adaptation in H9N2 viruses and related H7N9 and H10N8 reassortants towards human infectivity.

  • Virion structure of Israeli acute bee paralysis virus [PublishAheadOfPrint]

  • The pollination services provided by the western honeybee (Apis mellifera) are critical for the agricultural production and diversity of wild flowering plants. However, honeybees suffer from environmental pollution, habitat loss, and pathogens, including viruses that can cause fatal diseases. Israeli acute bee paralysis virus (IAPV) from the family Dicistroviridae has been shown to cause colony collapse disorder in the United States. Here we present the IAPV virion structure determined to a resolution of 4.0 AAring; and the structure of a pentamer of capsid protein protomers at a resolution of 2.7 AAring;. IAPV has major capsid proteins VP1 and VP3 with non-canonical jellyroll bbeta;-barrel folds composed of only seven instead of eight bbeta;-strands, as is the rule for proteins of other viruses with the same fold. The maturation of dicistroviruses is connected to the cleavage of precursor capsid protein VP0 into subunits VP3 and VP4. We show that a putative catalytic site formed by residues asp-asp-phe of VP1 is optimally positioned to perform the cleavage. Furthermore, unlike many picornaviruses, IAPV does not contain a hydrophobic pocket in capsid protein VP1 that could be targeted by capsid-binding antiviral compounds.

    Importance Honeybee pollination is required for agricultural production and to sustain the biodiversity of wild flora. However, honeybee populations in Europe and North America are under pressure from pathogens including viruses that cause colony losses. Viruses from the family Dicistroviridae can cause honeybee infections that are lethal not only to individual honeybees, but to whole colonies. Here we present the virion structure of an Aparavirus Israeli acute bee paralysis virus (IAPV), a member of a complex of closely related viruses that are distributed worldwide. IAPV exhibits unique structural features not observed in other picorna-like viruses. Capsid protein VP1 of IAPV does not contain a hydrophobic pocket, implying that capsid-binding antiviral compounds that can prevent the replication of vertebrate picornaviruses may be ineffective against honeybee virus infections.

  • Mapping Murine Corneal Neovascularization and Weight Loss Virulence Determinants in the HSV-1 Genome and the Detection of an Epistatic Interaction between the UL and IRS/US Regions. [PublishAheadOfPrint]

  • Herpes simplex virus type 1 (HSV-1) most commonly causes recrudescent labial ulcers, however it is also the leading cause of infectious blindness in developed countries. Previous research in animal models has demonstrated that the severity of HSV-1 ocular disease is influenced by three main factors; host innate immunity, host immune response, and viral strain. We have previously shown that mixed infection with two avirulent HSV-1 strains (OD4 and CJ994) results in recombinants with a wide range of ocular disease phenotype severity. Recently, we developed a quantitative trait locus (QTL) based computational approach (vQTLmap) to identify viral SNPs predicted to influence the severity of the ocular disease phenotypes. We have now applied vQTLmap to identify HSV-1 single nucleotide polymorphisms (SNPs) associated with corneal neovascularization and mean peak percentage weight loss (MPWL) using 65 HSV-1 OD4-CJ994 recombinants. The vQTLmap analysis using Random Forest for neovascularization identified phenotypically meaningful nonsynonymous SNPs in the ICP4, UL41 (VHS), UL42, UL46 (VP11/12), UL47 (VP13/14), UL48 (VP22), US3, US4 (gG), US6 (gD), and US7 (gI) coding regions. The ICP4 gene was previously identified as a corneal neovascularization determinant, validating the vQTLmap method. Further analysis detected an epistatic interaction for neovascularization between a segment of the UL region and a segment of the IRS/US region. Ridge regression was used to identify MPWL associated nonsynonymous SNPs in the UL1 (gL), UL2, UL4, UL49 (VP22), UL50, and ICP4 coding regions. The data provide additional insights into virulence gene and epistatic interaction discovery in HSV-1.

    Importance Herpes simplex virus type 1 (HSV-1) typically causes recurrent cold sores, however it is also the leading source of infectious blindness in developed countries. Corneal neovascularization is critical for the progression of blinding ocular disease, and weight loss is a measure of infection severity. Previous HSV-1 animal virulence studies have shown the severity of ocular disease is partially due to viral strain. In the current study, we used a recently described computational quantitative trait locus (QTL) approach in conjunction with 65 HSV-1 recombinants to identify viral single nucleotide polymorphisms (SNPs) involved in neovascularization and weight loss. Neovascularization SNPs were identified in the ICP4, VHS, UL42, VP11/12, VP13/14, VP22, gG, US3, gD, and gI genes. Further analysis revealed an epistatic interaction between the UL and US regions. MPWL associated SNPs were detected in the UL1 (gL), UL2, UL4, VP22, UL50, and ICP4 genes. This approach will facilitate future HSV virulence studies.

  • Roles of the Rabies Virus Phosphoprotein Isoforms in Pathogenesis [PublishAheadOfPrint]

  • Rabies virus (RABV) P gene mRNA encodes five in-frame start codons, resulting in expression of full-length P protein (P1) and N-terminally truncated P proteins (tPs) designated as P2, P3, P4, and P5. Despite the fact that some tPs are known as IFN antagonists, the importance of tPs in the pathogenesis of RABV is still unclear. In this study, to examine whether tPs contribute to the pathogenesis, we exploited a reverse genetics approach to generate CE(NiP)P2-5, a mutant of pathogenic CE(NiP), in which the P gene was mutated by replacing all of the start codons (AUG) for tPs with AUA. We confirmed that, while CE(NiP) expresses detectable levels of P2 and P3, CE(NiP)P2-5 has impaired ability to express these tPs. After intramuscular inoculation, CE(NiP)P2-5 caused significantly lower morbidity and mortality rates in mice than did CE(NiP), indicating that tPs play a critical role in RABV neuroinvasiveness. Further examinations revealed that this less neuroinvasive phenotype of CE(NiP)P2-5 correlates with its impaired ability to replicate in muscle cells, indicative of the importance of tPs in viral replication in muscle cells. We also demonstrated that CE(NiP)P2-5 infection induced a higher level of Ifn-bbeta; gene expression in muscle cells than did CE(NiP) infection, consistent with the results of an IFN-bbeta; promoter reporter assay suggesting that all tPs function to antagonize IFN induction in muscle cells. Taken together, our findings strongly suggest that tPs promote viral replication in muscle cells through their IFN antagonist activities and thereby support infection of peripheral nerves.

    IMPORTANCE Despite the fact that previous studies have demonstrated that P2 and P3 of RABV have IFN antagonist activities, the actual importance of tPs in the pathogenesis has remained unclear. Here we provide the first evidence that tPs contribute to the pathogenesis of RABV, especially its neuroinvasiveness. Our results also show the mechanism underlying the neuroinvasiveness driven by tPs, highlighting the importance of their IFN antagonist activities, which support viral replication in muscle cells.

  • An endogenous gibbon ape leukemia virus (GALV) identified in a rodent (Melomys burtoni subsp.) from Wallacea (Indonesia) [PublishAheadOfPrint]

  • Gibbon ape leukemia virus (GALV) and koala retrovirus (KoRV) most likely originated from a cross-species transmission of an ancestral retrovirus into koalas and gibbons via one or more intermediate as yet unknown hosts. A highly similar virus to GALV has been identified in an Australian native rodent (Melomys burtoni) after extensive screening of Australian wildlife. GALV-like viruses have also been discovered in several Southeast Asian species although screening has not been extensive and viruses discovered to date are only distantly related to GALV. We therefore screened 26 Southeast Asian rodent species for KoRV- and GALV-like sequences, using hybridization capture and high-throughput sequencing, in the attempt to identify potential GALV and KoRV hosts. Only the individuals belonging to a newly discovered subspecies of Melomys burtoni from Indonesia were positive yielding an endogenous provirus very closely related to a strain of GALV. The sequence of the critical receptor domain for GALV infection in the Indonesian M. burtoni subsp. was consistent with the susceptibility of the species to GALV infection. The second record of a GALV in M. burtoni provides further evidence that M. burtoni, and potentially other lineages within the widespread subfamily Murinae, may play a role in the spread of GALV-like viruses. The discovery of a GALV in the most western part of the Australo-Papuan distribution of M. burtoni, specifically in a transitional zone between Asia and Australia (Wallacea), may be relevant to the cross-species transmission to gibbons in Southeast Asia and broadens the known distribution of GALVs in wild rodents.

    IMPORTANCE Gibbon ape leukemia virus (GALV) and the koala retrovirus (KoRV) are very closely related, yet their hosts are neither closely related nor overlap geographically. Direct cross-species infection between koalas and gibbons is unlikely. Therefore, GALV and KoRV may have arisen via a cross-species transfer from an intermediate host that overlaps in range with both gibbons and koalas. Using hybridization capture and high-throughput sequencing, we have screened a wide range of rodent candidate hosts from Southeast Asia for KoRV- and GALV-like sequences. Only a Melomys burtoni subspecies from Wallacea (Indonesia) was positive for GALV. We report the genome sequence of this newly identified GALV, the critical domain for infection of its potential cellular receptor and its phylogenetic relationships with the other previously characterized GALVs. We hypothesize that Melomys burtoni, and potentially related lineages with an Australo-Papuan distribution, may have played a key role in cross-species transmission to other taxa.

  • CD25+FoxP3+ memory CD4 T cells are frequent targets of HIV infection in vivo [PublishAheadOfPrint]

  • Interleukin 2 (IL2) signaling through the IL2 receptor alpha chain+ (CD25) facilitates HIV replication in vitro and facilitates homeostatic proliferation of CD25+FoxP3+CD4+ T cells. CD25+FoxP3+CD4+ T cells may therefore constitute a suitable subset for HIV infection and plasma virion production.

    CD25+FoxP3+CD4+ T cell frequencies, absolute numbers and the expression of CCR5 and cell cycle marker Ki67 were studied in peripheral blood from HIV+ and HIV- study volunteers. Different memory CD4+ T cell subsets were then sorted for quantification of cell-associated HIV-DNA and phylogenetic analyses of the highly variable EnvV1V3 region in comparison to plasma-derived virus sequences.

    In HIV+ subjects, 51% (median) of CD25+FoxP3+CD4+ T cells expressed the HIV co-receptor CCR5. Very high frequencies of Ki67+ cells were detected in CD25+FoxP3+ (median, 27.6%) in comparison to memory CD25-FoxP3- memory CD4+ T cells (median, 4.1%, pllt;0.0001). HIV-DNA content was 15-fold higher in CD25+FoxP3+ compared to CD25-FoxP3- memory CD4+ T cells (p=0.003). EnvV1V3 sequences derived from CD25+FoxP3+ memory CD4+ T cells did not preferentially cluster with plasma-derived sequences. Quasi-identical cell-plasma-sequence pairs were rare and their proportion further decreased with the estimated HIV infection duration.

    These data suggest that specific cellular characteristics of CD25+FoxP3+ memory CD4+ T cell might facilitate efficient HIV infection in vivo and passage of HIV DNA to cell progeny in the absence of active viral replication. Contribution of this cell population to plasma virion production remains unclear.

    Importance: Despite recent advances in the understanding of AIDS virus pathogenesis, it is incompletely understood, which cell subsets support HIV infection and replication in vivo. In vitro, the IL2 signaling pathway and IL2 dependent cell cycle induction are essential for HIV infection of stimulated T cells. CD25+FoxP3+ memory CD4 T cells - often referred to as regulatory CD4 T cells nndash; depend on IL2 signaling for homeostatic proliferation in vivo. Our results show that CD25+FoxP3+ memory CD4+ T cells often express the HIV co-receptor CCR5, are significantly more proliferative and contain more HIV-DNA compared to CD25-FoxP3- memory CD4 T cell subsets. The specific cellular characteristics of CD25+FoxP3+ memory CD4+ T cell probably facilitate efficient HIV infection in vivo and passage of HIV DNA to cell progeny in the absence of active viral replication. However contribution of this cell subset to plasma viremia remains unclear.

  • The interferon-stimulated gene IFITM3 restricts West Nile virus infection and pathogenesis [PublishAheadOfPrint]

  • The interferon induced transmembrane protein (IFITM) family of proteins inhibit infection of several different enveloped viruses in cell culture by virtue of their ability to restrict entry and fusion from late endosomes. As few studies have evaluated the importance of IFITM3 in vivo in restricting viral pathogenesis, we investigated its significance as an antiviral gene against West Nile virus (WNV), an encephalitic flavivirus, in cells and mice. Ifitm3-/- mice were more vulnerable to lethal WNV infection, and this was associated with greater virus accumulation in peripheral organs and central nervous system tissues. As no difference in viral burden in the brain or spinal cord was observed after direct intracranial inoculation, Ifitm3 likely functions as an antiviral protein in non-neuronal cells. Consistent with this, Ifitm3-/- fibroblasts but not dendritic cells resulted in higher yields of WNV in multi-step growth analyses. Moreover, trans-complementation experiments showed that Ifitm3 inhibited WNV infection independently of Ifitm1, Ifitm2, Ifitm5, and Ifitm6. Beyond a direct effect on viral infection in cells, analysis of the immune response in WNV-infected Ifitm3-/- mice showed decreases in the total number of B cells, CD4+ T cells, and antigen-specific CD8+ T cells. Finally, bone marrow chimera experiments demonstrated that Ifitm3 functioned in both radioresistant and radiosensitive cells, as higher levels of WNV were observed in the brain only when Ifitm3 was absent from both compartments. Our analyses suggest that Ifitm3 restricts WNV pathogenesis likely through multiple mechanisms including the direct control of infection in subsets of cells.

    IMPORTANCE As part of the mammalian host response to viral infections, hundreds of interferon-stimulated genes (ISGs) are induced. The inhibitory activity of individual ISGs varies depending on the specific cell type and viral pathogen. Among ISGs, the interferon-induced transmembrane proteins (IFITM) genes have been reported to inhibit multiple families of viruses in cell culture. However, few reports have evaluated the impact of IFITM genes on viral pathogenesis in vivo. In this study, we characterized the antiviral activity of Ifitm3 against West Nile virus (WNV), an encephalitic flavivirus, using mice with a targeted gene deletion of Ifitm3. Based on extensive virological and immunological analyses, we determined that Ifitm3 protects mice from WNV-induced mortality by restricting virus accumulation in peripheral organs and subsequently, in central nervous system tissues. Our data suggest that Ifitm3 restricts WNV pathogenesis by multiple mechanisms and functions in part, by controlling infection in different cell types.

  • Genome-wide screening of retroviral envelope genes in the nine-banded armadillo (Dasypus novemcinctus; Xenarthra) reveals an unfixed chimeric endogenous betaretrovirus using the ASCT2 receptor [PublishAheadOfPrint]

  • Retroviruses enter host cells through interaction of their Envelope (Env) protein with a cell surface receptor, which triggers fusion of viral and cellular membranes. The sodium-dependent neutral amino-acid transporter ASCT2 is the common receptor of the large RD114 retrovirus interference group, whose members display frequent env recombination events. Germline retrovirus infections have led to numerous inherited endogenous retroviruses (ERVs) in vertebrate genomes, which provide useful insights into the co-evolutionary history of retroviruses and their hosts. Rare ERV-derived genes display conserved viral functions, as illustrated by the fusogenic syncytin env genes involved in placentation. Here, we searched for functional env genes within the nine-banded armadillo (Dasypus novemcinctus) genome and identified dasy-env1.1, which clusters with RD114 interference group envs and with two syncytin genes sharing ASCT2 receptor usage. Using ex vivo pseudotyping and cell-cell fusion assays, we demonstrated that the Dasy-Env1.1 protein is fusogenic and can use both human and armadillo ASCT2 as a receptor. This gammaretroviral env gene belongs to a provirus with betaretrovirus-like features, suggesting acquisition through recombination. Provirus insertion was found in several Dasypus species, where it has not reached fixation, whereas related family members integrated before diversification of the genus Dasypus ggt;12 Mya. This newly described ERV lineage is potentially useful as a population genetic marker. Our results extend usage of ASCT2 as a retrovirus receptor to the mammalian clade Xenarthra, and suggest that acquisition of an ASCT2-interacting env gene is a major selective force driving the emergence of numerous chimeric viruses in vertebrates.

    IMPORTANCE: Retroviral infection is initiated by binding of the viral envelope glycoprotein to a host cell receptor(s), triggering membrane fusion. Ancient germline infections have generated numerous endogenous retroviruses (ERVs) in nearly all vertebrate genomes. Here we report a previously uncharacterized ERV lineage from the genome of a xenarthran species, the nine-banded armadillo (Dasypus novemcinctus). It entered the Dasypus genus ggt;12 Mya, with one element inserted more recently in some Dasypus species, where it could serve as a useful marker of population genetics. This element exhibits an env gene, acquired by recombination events, with conserved viral fusogenic properties through binding to ASCT2, a receptor used by a wide range of recombinant retroviruses infecting other vertebrate orders. This designates the ASCT2 transporter as a successful receptor for ERV endogenization, and suggests that ASCT2-binding env acquisition events have favored the emergence of numerous chimeric viruses in a wide range of species.

  • Comprehensive characterization of the HIV-1 molecular epidemiology and demographic history in the Brazilian region most heavily affected by AIDS [PublishAheadOfPrint]

  • High incidence of AIDS cases and dominance of HIV-1 subtype C infections are two features that distinguish HIV-1 epidemic in the two southernmost Brazilian states (RS and SC) from the epidemic in other parts of the country. Nevertheless, studies on HIV molecular epidemiology were mainly conducted in capital cities and more comprehensive understanding of factors driven this unique epidemic in Brazil is necessary. Blood samples were collected from 13 municipalities in Brazilian southern region. HIV-1 env and pol genes were submitted to phylogenetic analyses for assignment of subtype and viral population phylodynamics were reconstructed applying Skygrid and logistic coalescent models in a Bayesian analysis. High prevalence of subtype C was observed in all sampled locations, however increased frequency of recombinant strains was found in RS, with evidences for new CRFs. In SC state subtype B and C epidemics were associated to distinct exposure groups. Though logistic models estimated similar growth rates for HIV-1C and B, Skygrid plot reveals the former epidemic to expand for longer time. Our results highlight a consistent expansion of HIV-1C in south Brazil and also discuss how the heterosexual and MSM transmission chains might have impacted in the current prevalence of HIV-1 subtypes in the region.

    Importance: AIDS epidemic in south Brazil is expanding fast, but the circumstances driven this condition are not well known. High prevalence of HIV-1 subtype C was reported in the capital cities of the region, contrasting with the subtype B dominance in the rest of the country. This study sought to comparatively investigate HIV-1 subtype B and C epidemic by sampling several cities in the two states with the highest AIDS incidence in Brazil. Our analyses showed distinct epidemic growth curves for the two epidemics and we also found evidences suggesting that separate transmission chains can be impacting in the viral phylodynamic and in the emergence of new recombinant forms.

  • THE VIRAL FGARAT HOMOLOG ORF75 OF RHESUS MONKEY RHADINOVIRUS EFFECTS PROTEASOMAL DEGRADATION OF THE ND10 COMPONENTS SP100 AND PML [PublishAheadOfPrint]

  • Nuclear domain 10 (ND10) components restrict herpesviral infection, and herpesviruses antagonize this restriction by a variety of strategies, including degradation or relocalization of ND10 proteins. The rhesus monkey rhadinovirus (RRV) shares many key biological features with the closely related Kaposi's sarcoma-associated human Herpesvirus 8 (KSHV) and readily infects cells of both human and rhesus monkey origin. We used the CRISPR:Cas9 technique to generate knockout (ko) cells for each of the four ND10 components PML, SP100, DAXX, and ATRX. These ko cells were analyzed with regard to permissiveness for RRV infection. In addition, we analyzed the fate of the individual ND10 components in infected cells by immunofluorescence and Western blot. Knock-out of the ND10 component DAXX markedly increased RRV infection, while knockout of PML or SP100 had a less pronounced effect. In line with these observations, RRV infection resulted in rapid degradation of SP100, followed by degradation of PML and loss of ND10 structures, whereas protein levels of ATRX and DAXX remained constant. Notably, inhibition of the proteasome but not inhibition of de-novo gene expression prevented loss of SP100 and PML in cells that do not support lytic replication, compatible with proteasomal degradation of these ND10 components through the action of a viral tegument protein. Expression of the RRV FGARAT homolog ORF75 was sufficient to effect loss of SP100 and PML in transfected or transduced cells, implicating ORF75 as the viral effector protein.

    IMPORTANCE Our findings highlight the antiviral role of ND10 and its individual components and further establish the viral FGARAT homologs of the gamma-herpesviruses as important viral effectors that counteract ND10-instituted intrinsic immunity. Surprisingly, even closely related viruses like KSHV and RRV evolved to use different strategies to evade ND10-mediated restriction. RRV first targets SP100 for degradation and then PML with a delayed kinetic, which clearly differs from other gamma-herpesviruses. Despite efficient degradation of these two major ND10 components, RRV is still restricted by DAXX, another abundant ND10 component, as evidenced by a marked increase in RRV infection and replication upon knockout of DAXX. Taken together, our findings substantiate PML, SP100, and DAXX as key antiviral proteins, in that the former two are targeted for degradation by RRV, and the latter still potently restricts replication of RRV.

  • The MHC II HLA-DR{alpha} is Downregulated by KSHV encoded lytic transactivator RTA and MARCH8 [PublishAheadOfPrint]

  • Kaposi's-sarcoma associated herpesvirus (KSHV) maintains two modes of life cycle, latency and lytic phase. To evade the attack of cell host immune system, KSHV switches from lytic to latency, a phase where only a few of viral proteins are expressed. The mechanism by which KSHV evades the attack of the immune system and establishes latency has not been fully understood. MHC-II molecules are key componments of the immune system defense mechanism against viral infections. Here we report that the HLA-DRaalpha;, a member of the MHC II molecules, was downregulated by the replication and trancription activator (RTA) protein encoded by KSHV ORF50, an important regulator of the viral life cycle. RTA not only downregulated HLA-DRaalpha; at the protein level through direct binding and degradation through the proteasome pathway, but also indirectly down regulated the protein level of HLA-DR aalpha; by enchancing the expression of MARCH8, a member of the membrane-associated RING-CH (MARCH) proteins. Our findings indicate that KSHV RTA facilitates evasion of the virus from the immune system through manipulation of HLA-DRaalpha;.

    Importance Kaposi's sarcoma-associated herpesvirus has a causal role in a number of human cancers and its persistence in infected cells is controlled by the host's immune system. The mechanism by which KSHV evades an attack by immune system has not been well understood. This work represents studies which identifies a novel mechanism by which the virus can facilitate evasion of an immune system. We now show that RTA, the replication and transcription activator encoded by KSHV (ORF50) can function as an E3 ligase to degrade HLA-DRaalpha;. It can directly bind and induce degradation of the HLA-DRaalpha; through the ubiquitin-proteasomal degradation pathway. In addition to the direct regulation of HLA-DRaalpha;, RTA can also indirectly down-regulate the level of HLA-DRaalpha; protein by upregulating transcription of MARCH8. Increased MARCH8 results in the downregulation of HLA-DRaalpha;. Furthermore we also demonstrated that expression of HLA-DRaalpha; was impaired in KSHV de novo infection.

  • Flow Cytometric and Cytokine ELISpot Approaches to Characterize the Cell-mediated Immune Response in Ferrets Following Influenza Virus Infection [PublishAheadOfPrint]

  • Influenza virus infections represent a significant socioeconomic and public health burden worldwide. Although ferrets are considered by many to be ideal for modeling human responses to influenza infection and vaccination, efforts to understand the cellular immune response have been severely hampered by a paucity of standardized procedures and reagents. In this report, we developed flow cytometric and T cell ELISpot approaches to characterize the leukocyte composition and antigen-specific T cell response within key lymphoid tissues following influenza virus infection in ferrets. Through a newly designed and implemented set of serological reagents, we used multi-parameter flow cytometry to directly quantify the frequency of CD4+ and CD8+ T cells, Ig+ B cells, CD11b+ myeloid-derived cells and MHC class II+ APCs both prior to and after intranasal infection with A/California/04/09 (H1N1). We found that the leukocyte composition was altered 10 days post-infection, with notable gains in the frequency of T cells and myeloid cells within the draining lymph node. Furthermore, these studies revealed that the antigen-specificity of influenza-reactive CD4 and CD8 T cells was very broad, with recognition of the viral HA, NA, M1, NS1 and NP proteins and that total reactivity to influenza post-infection represented approximately 0.1% of the circulating PBMCs. Finally, we observed distinct patterns of reactivity between individual animals, suggesting heterogeneity at the MHC locus in ferrets within commercial populations, a finding of considerable interest in efforts to move the ferret model forward for influenza vaccine and challenge studies.

    IMPORTANCE Ferrets are an ideal animal model to study transmission, disease and vaccine efficacies of respiratory viruses because of their close anatomical and physiological resemblances to humans. However, a lack of reagents has limited our understanding of the cell-mediated immune response following infection and vaccination. In this study, we used cross-reactive and ferret-specific antibodies to study the leukocyte composition and antigen specific CD4 and CD8 T cell responses following influenza A/California/04/09 (H1N1) virus infection. These studies revealed strikingly distinct patterns of reactivity between CD4 and CD8 T cells, which was overlayed with differences in protein-specific responses between individual animals. Our results provide a first, in-depth look at the T cell repertoire in response to influenza infection and suggest there is considerable heterogeneity at the MHC locus, akin to humans and an area of intense research interest.

  • Kynurenine Reduces Memory CD4 T-cell Survival by Interfering with IL-2 Signaling early During HIV-1 Infection [PublishAheadOfPrint]

  • Early HIV-1 infection is characterized by enhanced tryptophan catabolism, which contributes to immune suppression and disease progression. However, mechanism by which kynurenine, a tryptophan-related metabolite, induces immune suppression remains poorly understood. Herein, we showed that the increased production of kynurenine correlates with defective IL-2 signaling in memory CD4 T-cells from HIV-infected subjects. Defective IL-2 signaling in these subjects, which drives reduced protection from Fas-mediated apoptosis, was also associated with memory CD4 T-cell loss. Treatment of memory CD4 T-cells with plasma concentration of kynurenine inhibits IL-2 signaling through the production of reactive oxygen species. We further showed that IL-2 signaling in memory CD4 T-cells was improved by the antioxidant N-acetylcysteine. Early initiation of antiretroviral therapy restored IL-2 response in memory CD4 T-cells by reducing ROS and kynurenine production. Study findings provide a kynurenine-dependant mechanism though IL-2 signaling for the reduced CD4 T cell survival that can be reversed by early treatment initiation in HIV-1 infection.

    IMPORTANCE The persistence of functional memory CD4 T-cells represents the basis for a long-lasting immune protection in individuals after exposure to HIV-1. Unfortunately, primary HIV-1 infection results in the massive loss of these cells within weeks of infection which is mainly driven by inflammation and massive infection by the virus. These new findings show that the enhanced production of kynurenine, a metabolite related to the tryptophan catabolism, also impairs memory CD4 T-cell survival and interferes with IL-2 signaling early during HIV-1 infection.

  • Immunization with Attenuated Equine Herpesvirus 1 Strain KyA Induces Innate Immune Responses that Protect Mice from Lethal Challenge [PublishAheadOfPrint]

  • Equine herpesvirus 1 (EHV-1) is a major pathogen affecting equines worldwide and causes respiratory disease, abortion, and in some cases, neurological disease. EHV-1 strain KyA is attenuated in the mouse and equine, whereas wild-type strain RacL11 induces severe inflammation of the lung, causing infected mice to succumb at 4 to 6 days post-infection. Our previous results showed that KyA immunization protected CBA mice from pathogenic RacL11 challenge at 2 and 4 weeks post-immunization, and that the KyA infection elicited protective humoral and cell-mediated immune responses. To investigate the protective mechanisms of KyA by innate immune responses, KyA-immunized mice were challenged with RacL11 at various times post-vaccination. KyA immunization protected mice from RacL11 challenge at 1 to 7 days post-immunization. Immunized mice lost less than 10% of their body weight and rapidly regained weight. Lung virus titers in KyA-immunized mice were 1,000-fold lower at 2 days post-RacL11 challenge than titers in lungs of non-immunized mice, indicative of accelerated virus clearance. Affymetrix microarray analysis revealed that IFN- and 16 antiviral interferon-stimulated genes (ISGs) were upregulated 3.1 to 48.2-fold at 8 h post-challenge in the lungs of RacL11-challenged mice that had been immunized with KyA. Murine IFN- inhibited EHV-1 infection of murine alveolar macrophage cells and protected mice against lethal EHV-1 challenge, suggesting that IFN- expression is important in mediating protection elicited by KyA immunization. These results suggest that EHV-1 KyA may be used as a live attenuated EHV-1 vaccine as well as a prophylactic agent in horses.

    IMPORTANCE Viral infection of cells initiates a signal cascade of events that ultimately attempts to limit viral replication and prevent infection through the expression of host antiviral proteins. Here, we show that EHV-1 KyA immunization effectively protected CBA mice from pathogenic RacL11 challenge at 1 to 7 days post-vaccination, and increased expression of IFN- and 16 antiviral interferon-stimulated genes (ISG). The administration of IFN- blocked EHV-1 replication in murine alveolar macrophages and mouse lungs and protected mice from lethal challenge. To our knowledge, this is the first report of an attenuated EHV-1 vaccine that protects the animal at 1 to 7 days post-immunization by innate immune responses. Our findings suggested that IFN- serves as a novel prophylactic agent and may offer new strategies for the development of anti-EHV-1 agents in the equine.

  • In vivo examination of mouse APOBEC3- and human APOBEC3A- and APOBEC3G-mediated restriction of parvovirus and herpesvirus infection in mouse models [PublishAheadOfPrint]

  • APOBEC3 knockout and human APOBEC3A and 3G transgenic mice were tested for their ability to be infected by the herpesviruses Herpes Simplex Virus 1 and Murine Herpes Virus 68, and the parvovirus Minute Virus of Mice (MVM). Knockout, APOBEC3A and APOBEC3G transgenic and wild type mice were equally infected by the herpesviruses while APOBEC3A but not mouse APOBEC3 conferred resistance to MVM. No viruses showed evidence of cytidine deamination by mouse or human APOBEC3s. These data suggest that in vitro studies implicating APOBEC3 proteins in virus resistance may not reflect their role in vivo.

    IMPORTANCE It is well-established that APOBEC3 proteins in different species are a critical component of the host anti-retroviral defense. Whether these proteins also function to inhibit other viruses is not clear. There have been a number of in vitro studies suggesting that different APOBEC3 proteins restrict herpesviruses and parvoviruses, among others, but whether they also work in vivo has not been demonstrated. Our studies looking at the role of mouse and human APOBEC3 proteins in transgenic and knockout mouse models of viral infection suggest that these restriction factors are not broadly anti-viral and demonstrate the importance of testing their activity in vivo.

  • The Evolutionary Histories of Antiretroviral Proteins SERINC3 and SERINC5 Do Not Support an Evolutionary Arms Race in Primates [PublishAheadOfPrint]

  • Molecular evolutionary arms races between viruses and their hosts are important drivers of adaptation. These Red Queen dynamics have been frequently observed in primate retroviruses and their antagonists, host restriction factor genes, such as APOBEC3F/G, TRIM5-aalpha;, SAMHD1, and BST-2. Host restriction factors have experienced some of the most intense and pervasive adaptive evolution documented in primates. Recently, two novel host factors, SERINC3 and SERINC5, were identified as the targets of HIV-1 Nef, a protein crucial for the optimal infectivity of virus particles. Here, we compared the evolutionary fingerprints of SERINC3 and SERINC5 to those of other primate restriction factors and to a set of other genes with diverse functions. SERINC genes evolved in a manner distinct from the canonical arms race dynamics seen in the other restriction factors. Despite their antiviral activity against HIV-1 and other retroviruses, SERINC3 and SERINC5 have a relatively uneventful evolutionary history in primates.

    Importance Restriction factors are host proteins that block viral infection and replication. Many viruses, like HIV-1 and related retroviruses, evolved accessory proteins to counteract these restriction factors. The importance of these interactions is evidenced by the intense adaptive selection pressures that dominates the evolutionary histories of both the host and viral genes involved in this so-called arms race. The dynamics of these arms races can point to mechanisms by which these viral infections can be prevented. Two human genes were recently identified as targets of an HIV-1 accessory protein important for viral infectivity: SERINC3 and SERINC5. Unexpectedly, we found that these SERINC genes, unlike other host restriction factors, show no evidence of a recent evolutionary arms race with viral pathogens.

  • The influenza A virus genotype determines the anti-viral function of NF-{kappa}B [PublishAheadOfPrint]

  • The role of NF-B for influenza A virus (IAV) infection does not reveal a coherent picture, as pro- and also anti-viral functions of this transcription factor have been described. To address this issue, we used CRISPR-Cas9-mediated genome engineering to generate murine MLE-15 cells lacking two essential components of the NF-B pathway. Cells devoid of either the central NF-B essential modulator (NEMO) scaffold protein and thus defect in IB kinase (IKK) activation or cells not expressing the NF-B DNA-binding and transactivation subunit p65 were tested for propagation of the SC35 virus, which has an avian host range and its mouse-adapted variant SC35M. While NF-B was not relevant for replication of SC35M, the absence of NF-B activity increased replication of the non-adapted SC35. This anti-viral effect of NF-B was most prominent upon infection of cells with low virus titers as they usually occur during the initiation phase of IAV infection. Defect NF-B signaling resulted in diminished IAV-triggered phosphorylation of IRF3 and expression of the anti-viral IFNbbeta; gene. To identify the viral proteins responsible for NF-B-dependency, reassortant viruses were generated by reverse genetics. SC35 viruses containing the SC35M segment encoding neuraminidase (NA) were completely inert to the inhibitory effect of NF-B, emphasizing the importance of the viral genotype for susceptibility to the anti-viral functions of NF-B.

    IMPORTANCE This study addresses two different issues: First, we investigated the role of the host cell transcription factor NF-B for IAV replication by genetic manipulation of IAVs by reverse genetics combined with targeted genome engineering of host cells using CRISPR-Cas9. The analysis of these two highly defined genetic systems indicated that the IAV genotype can influence whether NF-B displays an anti-viral function, and thus might in part explain incoherent results from the literature. Second, we found that perturbation of NF-B function greatly improved the growth of a non-adapted IAV, suggesting that NF-B may contribute to the maintenance of the host species barrier.

  • Structure-Function Analysis of the {Phi}X174 DNA Piloting Protein using Length Altering Mutations [PublishAheadOfPrint]

  • Although the X174 H protein is monomeric during procapsid morphogenesis, ten proteins oligomerize to form a DNA translocating conduit (H-tube) for penetration. However, the timing and location of H-tube formation is unknown. The H-tube's highly repetitive primary and quaternary structures made it amenable to a genetic analysis using in-frame insertions and deletions. Length-altered proteins were characterized for their ability to perform the protein's three known functions: participation in particle assembly, genome translocation, and stimulation of viral protein synthesis. Insertion mutants were viable. Theoretically, these proteins would produce an assembled tube exceeding the capsid's internal diameter, suggesting that virions do not contain a fully assembled tube. Lengthened proteins were also used to test the biological significance of the crystal structure. Particles containing H proteins of two different lengths were significantly less infectious than both parents, indicating an inability to pilot DNA. Shortened H proteins were not fully functional. Although they could still stimulate viral protein synthesis, they were either not incorporated into virions or, if incorporated, failed to pilot the genome. Mutant proteins that failed to incorporate contained deletions within an 85 amino acid segment, suggesting the existence of an incorporation domain. The revertants of shortened H protein mutants fell into two classes. The first class duplicated sequences neighboring the deletion, restoring wild-type length but not wild-type sequence. The second class suppressed an incorporation defect, allowing the use of the shortened protein.

    IMPORTANCE The H-tube crystal structure represents the first high-resolution structure of a virally encoded DNA translocating conduit. It has similarities with other viral proteins through which DNA must travel, such as the aalpha;-helical barrel domains of P22 portal proteins and T7 proteins that form tail tube extensions during infection. Thus, the H protein serves as a paradigm for the assembly and function of long aalpha;-helical supramolecular structures and nanotubes. Highly repetitive in primary and quaternary structure, they are amenable to structure-function analyses using inframe insertions and deletions as presented herein.

  • Distinct particle morphologies revealed through comparative parallel analyses of retrovirus-like particles [PublishAheadOfPrint]

  • The Gag protein is the main retroviral structural protein, and its expression alone is usually sufficient for production of virus-like particles (VLPs). In this study, we sought to investigate nndash; in parallel comparative analyses nndash; Gag cellular distribution, VLP size, and basic morphological features using Gag expression constructs (Gag or Gag-YFP) created from all representative retroviral genera: alpharetrovirus, betaretrovirus, deltaretrovirus, epsilonretrovirus, gammaretrovirus, lentivirus, and spumaretrovirus. We analyzed Gag cellular distribution by confocal microscopy, VLP budding by thin-section transmission electron microscopy (TEM), and general morphological features of the VLPs by cryogenic transmission electron microscopy (cryo-TEM). Punctate Gag was observed near the plasma membrane for all Gag constructs tested except for the representative beta- and epsilonretrovirus Gag proteins. This is the first report of epsilonretrovirus Gag localizing to the nucleus of HeLa cells. While VLPs were not produced by the representative beta- and epsilonretrovirus Gag proteins, the other Gag proteins produced VLPs as confirmed by TEM, and morphological differences were observed by cryo-TEM. In particular, we observed deltaretrovirus-like particles with flat regions of electron density that did not follow viral membrane curvature, lentivirus-like particles with a narrow range and consistent electron density, suggesting a tightly packed Gag lattice, and spumaretrovirus-like particles with large envelope protein spikes and no visible electron density associated with a Gag lattice. Taken together, these parallel comparative analyses demonstrate for the first time the distinct morphological features that exist among retrovirus-like particles. Investigation of these differences will provide greater insights into the retroviral assembly pathway.

    Importance: Comparative analysis among retroviruses has been critically important in enhancing our understanding of retroviral replication and pathogenesis nndash; including that of important human pathogens such as human T-cell leukemia virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1). Here in this study, parallel comparative analyses have been used to study Gag expression and virus-like particle morphology among representative retroviruses in the known retroviral genera. Distinct differences were observed, which enhances current knowledge of the retroviral assembly pathway.

  • Establishment and Reversal of HIV-1 Latency in Naïve and Central Memory CD4+ T Cells in Vitro [PublishAheadOfPrint]

  • The latent HIV-1 reservoir primarily resides in resting CD4+ T cells which is a heterogeneous population composed of both naïve (TN) and memory cells. In HIV-1-infected individuals, viral DNA has been detected in both naïve and memory CD4+ T cell subsets, although the frequency of HIV-1 DNA is typically higher in memory cells, particularly in the central memory (TCM) cell subset. TN and TCM cells are distinct cell populations distinguished by many phenotypic and physiological differences. In this study we used a primary cell model of HIV-1 latency that utilizes direct infection of highly purified TN and TCM cells to address differences in the establishment and reversal of HIV-1 latency. Consistent with what is seen in vivo, we found that HIV-1 infected TN cells less efficiently than TCM cells. However, when the infected TN cells were treated with latency reversing agents, including anti-CD3/CD28 antibodies, phorbol-myristate-acetate/phytohaemagglutinin and prostratin, as much, if not more, extracellular virion-associated HIV-1 RNA was produced per infected TN cell than infected TCM cell. There were no major differences in the genomic distribution of HIV-1 integration sites between TN and TCM cells that accounted for these observed differences. We observed decay of the latent HIV-1 cells in both T cell subsets after exposure to each of the latency reversing agents. Collectively, these data highlight significant differences in the establishment and reversal of HIV-1 latency in TN and TCM CD4+ T cells, and suggest that each subset should be independently studied in pre-clinical and clinical studies.

    IMPORTANCE The latent HIV-1 reservoir is frequently described as residing within resting memory CD4+ T cells. This is largely due to the consistent finding that memory CD4+ T cells, specifically the central (TCM) and transitional memory compartments, harbor the highest levels of HIV-1 DNA in individuals on suppressive therapy. This has yielded little research into the contribution of CD4+ naïve (TN) cells to the latent reservoir. In this study, we show that although TN cells harbor significantly lower levels of HIV-1 DNA, following latency reversal, they produced as much, if not more, virions than did the TCM cells. This suggests that latently infected TN cells may be a major source of virus following treatment interruption or failure. These findings highlight the need for a better understanding of the establishment and reversal of HIV-1 latency in TN cells when evaluating therapeutic approaches to eliminate the latent reservoir.

  • Development of Potent Antiviral Drugs Inspired by Viral Hexameric DNA Packaging Motors with Revolving Mechanism [PublishAheadOfPrint]

  • The intracellular parasitic nature of viruses and the emergence of antiviral drug resistance necessitate development of new potent antiviral drugs. Recently, a method has been described for developing potent inhibitory drugs by targeting biological machines with high stoichiometry and a sequential action mechanism. Inspired by this finding, we reviewed the development of antiviral drugs targeting viral DNA packaging motors. Inhibiting multi-subunit targets with sequential action resembles breaking one bulb in a series of Christmas lights, which turns off the entire string. Indeed, studies on viral DNA packaging might lead to the development of new antiviral drugs. Recent elucidation of the mechanism of the viral dsDNA packaging motor with sequential one-way revolving will promote the development of potent antiviral drugs with high specificity and efficiency. Traditionally, biomotors were classified into two categories: linear and rotation motion. Recently discovered was a third type of biomotor, including the viral DNA packaging motor, that use a revolving mechanism without rotation. By analogy, rotation resembles the Earth rotating on its own axis, while revolving resembles the Earth revolving around the Sun (see animations: http://nanobio.uky.edu/movie.html). Herein, we review the structures of viral dsDNA packaging motors, the stoichiometry of the motor components, and the motion mechanism of the motors. All viral dsDNA packaging motors contain a high stoichiometry machine composed of multiple components that work cooperatively and sequentially. Thus, it is an ideal target for potent drug development based on the power function of the stoichiometry of target complexes that work sequentially.

  • Human Th17 cells lack HIV-inhibitory RNases and are highly permissive to productive HIV infection [PublishAheadOfPrint]

  • Human Immunodeficiency Virus (HIV) infects and depletes CD4+ T cells, but subsets of CD4+ T cells vary in their susceptibility and permissiveness to infection. For example, HIV preferentially depletes IL-17-producing "T helper 17" (Th17) cells and "T follicular helper" (Tfh) cells. The preferential loss of Th17 cells during the acute phase of infection impairs the integrity of the gut mucosal barrier, which drives chronic immune activation - a key determinant of disease progression. The preferential loss of Th17 cells has been attributed to high CD4, CCR5, and CXCR4 expression. Here we show that Th17 cells also exhibit heightened permissiveness to productive HIV infection. Primary human CD4+ T cells were sorted, activated in Th17- or Th0-polarizing conditions and infected, then analyzed by flow cytometry. Th17-polarizing cytokines increased HIV infection, and HIV infection was disproportionately higher among Th17 cells compared with IL-17- or IFN+ cells, even upon infecting with a replication-defective HIV vector with a pseudotype envelope. Further, Th17-polarized cells produced more viral capsid protein. Our data also reveal that Th17-polarized cells have diminished expression of Ribonuclease A superfamily proteins, and report for the first time that RNase6 inhibits HIV. Thus, our findings link Th17 polarization to increased HIV replication.

    IMPORTANCE Our study compares the intracellular replicative capacity of several different HIV isolates among different T cell subsets, providing a link between the differentiation of Th17 cells and HIV replication. Th17 cells are of key importance in mucosal integrity, and in the immune response to certain pathogens. Based on our findings and the work of others, we propose a model in which HIV replication is favored by the intracellular environment of two CD4+ T cell subsets that share several requirements for their differentiation: Th17 and Tfh cells.

    Characterizing cells that support high levels of viral replication (rather than becoming latently infected or undergoing cell death) informs the search for new therapeutics aimed at manipulating intracellular signaling pathways and/or transcriptional factors that affect HIV replication.

  • cGAS-STING Signaling Regulates Initial Innate Control of Cytomegalovirus Infection [PublishAheadOfPrint]

  • Several innate sensing pathways contribute to control of early cytomegalovirus (CMV) infection, leading to a multi-phasic type I interferon (IFN-I) response that limits viral replication and promotes host defenses. Toll-like receptor (TLR)-dependent pathways induce IFN-I production in CMV-infected plasmacytoid dendritic cells, however the initial burst of IFN-I that occurs within the first few hours in vivo is TLR-independent and emanates from stromal cells. Here we show that primary human endothelial cells mount robust IFN-I responses to human CMV that are dependent upon cyclic GMP-AMP synthase (cGAS), STING and IRF3 signaling. Disruption of STING expression in endothelial cells by CRISPR-Cas9 revealed it is essential for the induction of IFN-I and restricting CMV replication. Consistently, STING was necessary to mount the first phase of IFN-I production and curb CMV replication in infected mice. Thus, DNA sensing through STING is critical for primary detection of both human and mouse CMV in non-hematopoietic cells, and drives the initial wave of IFN-I that is key for controlling early viral replication in vivo.

    Importance: Cytomegalovirus (CMV) is one of the most common viral pathogens, with a majority of people contracting the virus in their lifetime. Although acute infection is mostly asymptomatic in healthy persons, significant pathology is observed in immune compromised individuals, and chronic CMV infection may exacerbate a myriad of inflammatory conditions. Here we show that primary human endothelial cells mount robust IFN-I responses against CMV via a cGAS/STING/IRF3 pathway. Disruption of STING expression by CRISPR revealed an essential role in eliciting IFN-I responses and restricting CMV replication. Consistently, in mice STING is necessary for the first-phase of IFN-I production that limits early CMV replication. Our results demonstrate a pivotal role for the cGAS-STING pathway in the initial detection of CMV infection.

  • Adeno-Associated Virus Vectors Efficiently Transduce Mouse and Rabbit Sensory Neurons Co-Infected with HSV-1 Following Peripheral Inoculation [PublishAheadOfPrint]

  • Following infection of epithelial tissues, Herpes Simplex Virus type 1 (HSV-1) virions travel via axonal transport to sensory ganglia and establish a lifelong latent infection within neurons. Recent studies have revealed that, following intraganglionic or intrathecal injection, recombinant adeno-associated virus (rAAV) vectors can also infect sensory neurons and are capable of stable, long-term transgene expression. We sought to determine if application of rAAV to peripheral nerve termini at the epithelial surface would allow rAAV to traffic to sensory ganglia in a manner similar to HSV. We hypothesized that footpad or ocular inoculation with rAAV8 would result in transduction of dorsal root ganglia (DRG) or trigeminal ganglia (TG), respectively. To test this, we inoculated the footpads of mice with varying amounts of rAAV as well as rAAV capsid mutants. We demonstrate that this method of inoculation can achieve transduction of ggt;90% of the sensory neurons in the DRG that innervate the footpad. Similarly, we show that corneal inoculation with rAAV vectors in the rabbit efficiently transduces ggt;70% of the TG neurons in the optic tract. Finally, we demonstrate that co-infection of mouse footpads or rabbit eyes with rAAV vectors and HSV-1 results in co-localization in nearly all of the HSV-1-positive neurons. These results suggest that rAAV is a useful tool for the study of HSV-1 infection and may provide a means to deliver therapeutic cargos for the treatment of HSV or sensory ganglia dysfunctions.

    IMPORTANCE Adeno-associated virus (AAV) has been shown to transduce dorsal root ganglia sensory neurons following direct intraganglionic sciatic nerve injection, intraperitoneal and intravenous injection, as well as intrathecal injection. We sought to determine if rAAV vectors would be delivered to the same sensory neurons that herpes simplex virus (HSV-1) infects when applied peripherally, at an epithelial surface that has been treated to expose the underlying sensory nerve termini. For this study, we chose two well-established HSV-1 infection models: mouse footpad and rabbit ocular infection. The results presented here provide the first description of AAV vectors transducing neurons following delivery at the skin/epithelium/eye. The ability of AAV to co-transduce HSV-1 infected neurons both in the mouse and rabbit opens the opportunity to experimentally explore and disrupt host and viral proteins that are integral to the establishment of HSV-1 latency, to the maintenance of latency and to reactivation from latency in vivo.

  • BZLF1 Attenuates Transmission of Inflammatory Paracrine Senescence in Epstein-Barr Virus-Infected Cells by Downregulating TNF{alpha} [PublishAheadOfPrint]

  • Recent studies have shown that inflammatory responses trigger and transmit senescence to neighboring cells and activate the senescence-associated secretory phenotype (SASP). Latent Epstein-Barr virus (EBV) infection induces increased secretion of several inflammatory factors, whereas lytic infections evade the antiviral inflammatory response. However, the changes and roles of the inflammatory microenvironment during the switch between EBV lifecycles remain unknown. In the present study, we demonstrate that latent EBV infection in EBV-positive cells triggers SASP in neighboring epithelial cells. By contrast, lytic EBV infection abolishes this phenotype, BZLF1 attenuates the transmission of paracrine senescence during lytic EBV infection by down-regulating TNFaalpha; secretion. A mutant BZLF1 protein, BZLF1207-210, that cannot inhibit TNFaalpha; secretion while maintaining viral transcription, fails to block paracrine senescence, whereas a neutralizing antibody against TNFaalpha; is sufficient to restore its inhibition. Furthermore, latent EBV infection induces oxidative stress in neighboring cells, while BZLF1-mediated downregulation of TNFaalpha; reduces reactive oxygen species (ROS) levels in neighboring cells, and ROS scavengers alleviate paracrine senescence. These results suggest that lytic EBV infection attenuates the transmission of inflammatory paracrine senescence through BZLF1-downregulating TNFaalpha; secretion and alters the inflammatory microenvironment to allow virus propagation and persistence.

    IMPORTANCE The senescence-associated secretory phenotype (SASP), an important tumorigenic process, is triggered and transmitted by inflammatory factors. The different life cycles of Epstein-Barr virus (EBV) infection in EBV-positive cells employ distinct strategies to modulate the inflammatory response and senescence. The elevation of inflammatory factors during latent EBV infection promotes SASP in uninfected cells. By contrast, during the viral lytic cycle, BZLF1 suppresses the production of TNFaalpha;, resulting in the attenuation of paracrine inflammatory senescence. This finding indicates that EBV evades inflammatory senescence during lytic infection and switches from facilitating tumor-promoting SASP to generating a virus-propagating microenvironment, thereby facilitating viral spread in EBV-associated diseases.

  • HSV-2 Virion Host Shutoff Endoribonuclease Activity is Required to Disrupt Stress Granule Formation [PublishAheadOfPrint]

  • We previously established that cells infected with HSV-2 are disrupted in their ability to form stress granules (SGs) in response to oxidative stress and that this disruption is mediated by virion host shutoff protein (vhs), a virion associated endoribonuclease. Here, we test the requirement for vhs endoribonuclease activity in disruption of SG formation. We analyzed the ability of HSV-2 vhs carrying the point mutation D215N, which ablates its endoribonuclease activity, to disrupt SG formation in both transfected and infected cells. We present evidence that ablation of vhs endoribonuclease activity results in defects in vhs-mediated disruption of SG formation. Furthermore, we demonstrate that preformed SGs can be disassembled by HSV-2 infection in a manner that requires vhs endoribonuclease activity and that, befitting this ability to promote SG disassembly, vhs is able to localize to SGs. Taken together, these data indicate that endoribonuclease activity must be maintained in order for vhs to disrupt SG formation. We propose a model whereby vhs-mediated destruction of SG mRNA promotes SG disassembly and may also prevent SG assembly.

    IMPORTANCE Stress granules (SGs) are transient cytoplasmic structures that form when a cell is exposed to stress. SGs are emerging as potential barriers to viral infection, necessitating a more thorough understanding of their basic biology. We identified virion host shutoff protein (vhs) as a herpes simplex virus 2 (HSV-2) protein capable of disrupting SG formation. As mRNA is a central component of SGs and the best-characterized activity of vhs is as an endoribonuclease specific for mRNA in vivo, we investigated the requirement for vhs endoribonuclease activity in disruption of SG formation. Our studies demonstrate that endoribonuclease activity is required for vhs to disrupt SG formation and, more specifically, that SG disassembly can be driven by vhs endoribonuclease activity. Notably, during the course of these studies we discovered that there is an ordered departure of SG components during their disassembly and furthermore, that vhs itself has the capacity to localize to SGs.

  • Mutagenesis of Paramyxovirus Hemagglutinin-Neuraminidase Membrane Proximal Stalk Region Influences Stability, Receptor Binding and Neuraminidase Activity [PublishAheadOfPrint]

  • Paramyxoviridae consist of a large family of enveloped, negative sense, non-segmented single stranded RNA viruses that account for a significant number of human and animal diseases. The fusion process for nearly all paramyxoviruses involves the mixing of the host cell plasma membrane and the virus envelope in a pH-independent fashion. Fusion is orchestrated via the concerted action of two surface glycoproteins: an attachment protein called hemagglutinin-neuraminidase (HN; also called H or G depending on virus type and substrate) which acts as a receptor binding protein and a fusion (F) protein which undergoes a major irreversible refolding process to merge the two membranes. Recent biochemical evidence suggests that receptor binding by HN is dispensable for cell-cell fusion. However, factors that influence the stability and/or conformation of the HN four helix bundle (4HB) stalk have not been studied. Here, we used oxidative cross linking as well as functional assays to investigate the role of the structurally unresolved membrane proximal stalk region (MPSR) (residues 37-58) of HN in the context of headless and full length HN membrane fusion promotion. Our data suggest that the receptor binding head serves to stabilize the stalk to regulate fusion. Moreover, we found that the MPSR of HN modulates receptor binding and neuraminidase activity without a corresponding regulation of F-triggering.

    IMPORTANCE Paramyxoviruses require two viral membrane glycoproteins, the attachment protein variously called HN, H, or G and the fusion protein (F) to couple host receptor recognition to virus-cell fusion. The HN protein has a globular head that is attached to a membrane anchored flexible stalk of ~80 residues and has three activities: receptor binding, neuraminidase, and fusion activation. In this report, we have identified the functional significance of the membrane proximal stalk region (MPSR) (HN, residues 37-56) of the paramyxovirus parainfluenza virus (PIV5), a region of the HN stalk that has not had its structure determined by X-ray crystallography. Our data suggest that the MPSR influences receptor binding and neuraminidase activity via an indirect mechanism. Moreover, the receptor binding head group stabilizes the 4HB stalk as part of the general mechanism to fine-tune F-activation.

  • Opposing Roles of Double-Stranded RNA Effector Pathways and Viral Defense Proteins Revealed with CRISPR/Cas9 Knock-Out Cell Lines and Vaccinia Virus Mutants [PublishAheadOfPrint]

  • Vaccinia virus (VACV) decapping enzymes and cellular exoribonuclease Xrn1 catalyze successive steps in mRNA degradation and prevent dsRNA accumulation, whereas the viral E3 protein can bind dsRNA. We showed that dsRNA and E3 co-localized within cytoplasmic viral factories in cells infected with a decappng enzyme mutant as well as wild-type VACV and they co-precipitated with antibody. An E3 deletion mutant induced PKR and eIF2aalpha; phosphorylation earlier and more strongly than a decapping enzyme mutant, even though less dsRNA was made, leading to more profound effects on viral gene expression. Human Hap1 and A549 cells were genetically modified by CRISPR/Cas9 to determine whether the same pathways restrict E3 and decapping mutants. The E3 mutant replicated in PKR knock-out (KO) Hap1 cells in which RNase L is intrinsically inactive but only in PKR+RNase L double KO (DKO) A549 cells, indicating that both pathways decreased replication equivalently and no additional dsRNA pathway was crucial. In contrast, replication of the decapping enzyme mutant increased significantly (though less than wild-type virus) in DKO A549 cells but not in DKO Hap1 cells where a smaller increase in viral protein synthesis occurred. Xrn1 KO A549 cells were viable but non-permissive for VACV; however wild-type and mutant viruses replicated in triple KO cells in which RNase L and PKR were also inactivated. Since KO of PKR and RNase L were sufficient to enable VACV replication in the absence of E3 or Xrn1, the poor replication of the decapping mutant particularly in HAP1 DKO cells indicated additional translational defects.

    IMPORTANCE Viruses have evolved ways of preventing or counteracting the cascade of antiviral responses that double-stranded (ds) RNA triggers in host cells. We showed that the dsRNA produced in excess in cells infected with a vaccinia virus (VACV) decapping enzyme mutant and by wild-type virus co-localized with the viral E3 protein in cytoplasmic viral factories. Novel human cell lines defective in either or both protein kinase R and RNase L dsRNA effector pathways and/or the cellular 5rrsquo; exonuclease Xrn1 were prepared by CRISPR/Cas9 gene editing. Inactivation of both pathways was necessary and sufficient to allow full replication of the E3 mutant and reverse the defect cause by inactivation of Xrn1, whereas the decapping enzyme mutant still exhibited defects in gene expression. The study provided new insights into functions of the VACV proteins and the well-characterized panel of CRISPR/Cas9 modified human cell lines should have broad applicability for studying innate dsRNA pathways.

  • Phosphorylation of the Brome Mosaic Virus Capsid Regulates the Timing of Viral Infection [PublishAheadOfPrint]

  • The four Brome mosaic virus (BMV) RNAs are encapsidated in three distinct virions that have different disassembly rates in infection. The mechanism for differential release of BMV RNAs from virions is unknown, since 180 copies of the same coat protein (CP) encapsidates each of the BMV genomic RNAs. Using mass spectrometry, we found that the BMV CP contains a complex pattern of post-translational modifications. Treatment with phosphatase was found to not significantly affect the stability of the virions containing RNA1, but significantly impacted the stability of the virions that encapsidated BMV RNA2 and RNA3/4. CryoEM reconstruction revealed dramatic structural changes in the capsid and the encapsidated RNA. A phosphomimetic mutation in the flexible N-terminal arm of the CP increased BMV RNA replication and virion production. The degree of phosphorylation affected CP-RNA interaction to modulate interaction with the encapsidated RNA, the release of three of the BMV RNAs. CLIP-seq experiments showed that phosphorylation of the BMV CP can impact binding to RNAs in the virions, including sequences that contained regulatory motifs for BMV RNA gene expression and replication. Phosphatase-treated virions affected the timing of CP expression and viral RNA replication in plants. The degree of phosphorylation decreased when the plant hosts were grown at elevated temperature. These results show that phosphorylation of the capsid modulates BMV infection.

    Importance: How icosahedral viruses regulate the release of viral RNA into the host is not well understood. The selective release of viral RNA can regulate the timing of replication and gene expression. Brome mosaic virus (BMV) is an RNA virus with its three genomic RNAs encapsidated in separate virions. Through proteomic structural, and biochemical analyses this work shows that post-translational modifications, specifically phosphorylation, on the capsid protein regulates capsid-RNA interaction, the stability of the virions, and affected viral gene expression. Mutational analysis confirmed that changes in modification affected virion stability and the timing of viral infection. The mechanism for modification of the virion has striking parallels to the regulation of chromatin packaging by nucleosomes.

  • Distinct Viral Lineages from Fish and Amphibians Reveal the Complex Evolutionary History of Hepadnaviruses [PublishAheadOfPrint]

  • Hepadnaviruses (HBVs) are the only animal viruses that replicate their DNA by reverse transcription of an RNA intermediate. Until recently, the host range of hepadnaviruses was limited to mammals and birds. We obtained and analyzed the first amphibian HBV genome, as well as several prototype fish HBVs that allow the first comprehensive comparative genomic analysis of hepadnaviruses from four classes of vertebrates. Bluegill hepadnavirus (BGHBV) was characterized from in-house viral metagenomic sequencing. The African cichlid hepadnavirus (ACHBV) and the Tibetan frog hepadnavirus (TFHBV) were discovered using in silico analyses of the whole-genome shotgun and transcriptome Shotgun assembly databases. Residues in the hydrophobic base of the capsid (core) proteins, designated motif I, II and III, are highly conserved, suggesting that structural constraints for proper capsid folding are key to capsid protein evolution. Surface proteins in all vertebrate HBVs contain similar predicted membrane topology, characterized by three transmembrane domains. Most striking was that the BGHBV, ACHBV, and the previously described white sucker hepadnavirus did not form a fish-specific monophyletic group in the phylogenetic analysis of all three hepadnaviral genes. Notably, BGHBV was more closely related to the mammalian hepadnaviruses, indicating that cross-species transmission events have played a major role in viral evolution. Evidence of cross-species transmission was also observed with TFHBV. Hence, these data indicate that the evolutionary history of the hepadnaviruses is more complex than previously realized and combines both virus-host co-divergence over millions of years and host species jumping.

    IMPORTANCE Hepadnaviruses are responsible for significant disease in humans (hepatitis B virus) and have been reported from a diverse range of vertebrates as both exogenous and endogenous viruses. We report the full length genome of a novel hepadnavirus from a fish and the first hepadnavirus genome from an amphibian. The novel fish hepadnavirus, sampled from bluegill, was more closely related to mammalian hepadnaviruses than to other fish viruses. This phylogenetic pattern reveals that although hepadnaviruses have likely been associated with vertebrates for hundreds of millions of years, they have also been characterized by species jumping across wide phylogenetic distances.

  • CD4-induced antibodies promote association of the HIV-1 envelope glycoprotein with CD4-binding site antibodies [PublishAheadOfPrint]

  • The HIV-1 envelope glycoprotein (Env) is a trimer of gp120/gp41 heterodimers that mediates viral entry. Env binds cellular CD4, an association which stabilizes a conformation favorable to its subsequent association with a coreceptor, typically CCR5 or CXCR4. The CD4- and coreceptor-binding sites serve as epitopes for two classes of HIV-1 neutralizing antibodies nndash; CD4 binding site (CD4bs) and CD4-induced (CD4i) antibodies, respectively. Here we observed that, at a fixed total concentration, mixtures of the CD4i antibodies (E51 or 412d) and the CD4bs antibody VRC01 neutralized the HIV-1 isolates 89.6, ADA, SG3, and SA32 more efficiently than either antibody alone. We found that E51, and to a lesser extent 412d and 17b, promoted association of four CD4bs antibodies to the Env trimer, but not to monomeric gp120. We further demonstrated that the binding of the sulfotyrosine binding pocket by CCR5mim2-Ig was sufficient for promoting CD4bs antibody binding to Env. Interestingly, the relationship is not reciprocal: CD4bs antibodies were not as efficient as CD4-Ig at promoting E51 or 412d binding to Env trimer. Consistent with these observations, CD4-Ig, but none of the CD4bs antibodies tested, substantially increased HIV-1 infection of a CD4-negative, CCR5-positive cell line. We conclude that the ability of CD4i antibodies to promote VRC01 association with Env trimers accounts for the increase potency of VRC01 and CD4i antibody mixtures. Our data further suggest that potent CD4bs antibodies avoid inducing Env conformations that bind CD4i antibodies or CCR5.

    IMPORTANCE STATEMENT Potent HIV-1 neutralizing antibodies can prevent viral transmission and suppress an ongoing infection. Here we show that CD4-induced (CD4i) antibodies, which recognize the conserved coreceptor-binding site of the HIV-1 envelope glycoprotein (Env), can increase the association of Env with potent broadly neutralizing antibodies that recognize the CD4-binding site (CD4bs antibodies). We further show that, unlike soluble forms of CD4, CD4bs antibodies poorly induce envelope glycoprotein conformations that efficiently bind CCR5. This study provides insight into the properties of potent CD4bs antibodies and suggests that, under some conditions, CD4i antibodies can improve their potency. These observations may be helpful to the development of vaccines designed to elicit specific antibody classes.

  • ICAM-1 Binding Rhinoviruses A89 and B14 Uncoat in Different Endosomal Compartments [PublishAheadOfPrint]

  • Human rhinovirus (HRV) -A89 and HRV-B14 bind to and are internalized by intercellular adhesion molecule 1 (ICAM-1); as demonstrated earlier, the RNA genome of HRV-B14 penetrates into the cytoplasm from endosomal compartments of the lysosomal pathway. Here we show, by immunofluorescence microscopy, that HRV-A89 but not HRV-B14 colocalizes with transferrin in the endocytic recycling compartment (ERC). Applying drugs differentially interfering with endosomal recycling and with the pathway to lysosomes, we demonstrate that these two major group HRVs productively uncoat in distinct endosomal compartments. Overexpression of constitutively active (Rab11-GTP) and dominant-negative (Rab11-GDP) mutants revealed that uncoating of HRV-A89 depends on functional Rab-11. Thus, two ICAM-1 binding HRVs are routed into distinct endosomal compartments for productive uncoating.

    IMPORTANCE Based on similarity of their RNA genomic sequences the more than 150 currently known common cold virus serotypes were classified as species A, B, and C. The majority of HRV-A and all HRV-B use ICAM-1 for cell attachment and entry. Our results highlight important differences of two ICAM-1 binding HRVs with respect to their intracellular trafficking and productive uncoating; they demonstrate that serotypes belonging to species A and B respectively, but entering the cell via the same receptor, direct the endocytosis machinery to ferry them along distinct pathways towards different endocytic compartments for uncoating.

  • Analysis of the Cis and Trans Requirements for DNA Replication at the Right End Hairpin of the Human Bocavirus 1 Genome [PublishAheadOfPrint]

  • Parvoviruses are single-stranded DNA viruses that use the palindromic structures at the ends of the viral genome for their replication. The mechanism of parvovirus replication has been studied mostly in Dependoparvovirus adeno-associated virus 2 (AAV2) and Protoparvovirus minute virus of mice (MVM). Here, we used human bocavirus 1 (HBoV1) to understand the replication mechanism of Bocaparvovirus. HBoV1 is pathogenic to humans, causing acute respiratory tract infections, especially in young children under 2 years old. By using the duplex replicative form of the HBoV1 genome in human embryonic kidney (HEK) 293 cells, we identified the HBoV1 minimal replication origin at the right-end hairpin (OriR). Mutagenesis analyses confirmed the putative NS1 binding and nicking sites within the OriR. Of note, unlike the large non-structural protein (Rep78/68 or NS1) of other parvoviruses, HBoV1 NS1 did not specifically bind OriR in vitro, indicating that other viral and cellular components or the oligomerization of NS1 are required for the NS1 binding to the OriR. In vivo studies demonstrated that residues responsible for NS1 binding and nicking are within the origin-binding domain. Further analysis identified that the small non-structural protein NP1 is required for HBoV1 DNA replication at OriR. The NP1 and other viral non-structural proteins (NS1-4) colocalized within the viral DNA replication centers in both OriR-transfected cells and virus-infected cells, highlighting a direct involvement of the NP1 in viral DNA replication at OriR. Overall, our study revealed characteristics of HBoV1 DNA replication at OriR, suggesting novel characteristics of autonomous parvovirus DNA replication.

    IMPORTANCE Human bocavirus 1 (HBoV1) causes acute respiratory tract infections in young children. The duplex HBoV1 genome replicates in HEK293 cells, and produces progeny virions that are infectious in well-differentiated airway epithelial cells. Recombinant AAV2 vector pseudotyped with HBoV1 capsid has been developed to efficiently deliver the cystic fibrosis transmembrane conductance regulator gene to human airway epithelia. Here, we identified both cis-acting elements and trans-acting proteins that are required for HBoV1 DNA replication at the right-end hairpin in HEK293 cells. We localized the minimal replication origin to a 46-nt sequence in the right-end hairpin, which contains both NS1 nicking and binding sites. The identification of these essential elements of HBoV1 DNA replication acting both in cis and in trans will provide guidance to develop antiviral strategies targeting viral DNA replication at the right-end hairpin, and to design next generation recombinant HBoV1 vectors, a promising tool for gene therapy of lung diseases.

  • DC-SIGN and L-SIGN are attachment factors that promote infection of target cells by human metapneumovirus in the presence or absence of cellular glycosaminoglycans. [PublishAheadOfPrint]

  • It is well established that glycosaminoglcyans (GAGs) function as attachment factors for human metapneumovirus (HMPV), concentrating virions at the cell-surface to promote interaction with other receptors for virus entry and infection. There is increasing evidence to suggest that multiple receptors may exhibit the capacity to promote infectious entry of HMPV into host cells, however definitive identification of specific transmembrane receptors for HMPV attachment and entry is complicated by the widespread expression of cell surface GAGs. pgsA745 Chinese Hamster Ovary (CHO) cells are deficient in expression of cell-surface GAGs and resistant to HMPV infection. Herein, we demonstrate that expression of the Ca2+-dependent C-type lectin receptors (CLRs) DC-SIGN (CD209L) or L-SIGN (CD209L) rendered pgsA745 cells permissive to HMPV infection. Unlike infection of parental CHO cells, HMPV infection of pgsA745 cells expressing DC-SIGN or L-SIGN was dynamin-dependent and inhibited by mannan, but not by pre-treatment with bacterial heparinase. Parental CHO cells expressing DC-SIGN/L-SIGN also showed enhanced susceptibility to dynamin-dependent HMPV infection, confirming CLRs can promote HMPV infection in the presence or absence of GAGs. Comparison of pgsA745 cells expressing wild-type or endocytosis-defective mutants of DC/L-SIGN indicated that the endocytic function of CLRs was not essential, but could contribute to HMPV infection of GAG-deficient cells. Together, these studies confirm a role for CLR as attachment factors and entry receptors for HMPV infection. Moreover, they define an experimental system that can be exploited to identify transmembrane receptors and entry pathways where permissivity to HMPV infection can be rescued following expression of a single cell-surface receptor.

    IMPORTANCE On the surface of CHO cells, glycosaminoglycans (GAGs) function as the major attachment factor for human metapneumoviruses (HMPV), promoting dynamin-independent infection. Consistent with this, GAG-deficient pgaA745 CHO cells are resistant to HMPV. However, expression of DC-SIGN or L-SIGN rendered pgsA745 cells permissive to dynamin-dependent infection by HMPV, although the endocytic function of DC/L-SIGN was not essential for, but could contribute to, enhanced infection. These studies provide direct evidence implicating DC/L-SIGN as alternate attachment factors for HMPV attachment, promoting dynamin-dependent infection via other unknown receptors in the absence of GAGs. Moreover, they describe a unique experimental system for the assessment of putative attachment and entry receptors for HMPV.

  • miR-155 is necessary for efficient gammaherpesvirus reactivation from latency, but not for latency establishment. [PublishAheadOfPrint]

  • MicroRNA-155 (miR-155) has been shown to play significant roles in the immune response, including in the formation of germinal centers (GC) and the development and maturation of T follicular helper cells (Tfh). There is in vitro evidence to support a critical role for cellular miR-155 and viral miR-155 homologues in the establishment of gammaherpesvirus latency in B cells. We sought to determine the contribution of miR-155 to the establishment and maintenance of latency in vivo using murine gammaherpesvirus (MHV-68) infection. MHV-68-infected mice deficient in miR-155 exhibited decreases in GC B cells and Tfh cells. However, the frequency of spleen cells harboring latent MHV-68 genomes was the same in both miR-155 deficient and WT mice. Similar latent loads were also observed in mixed bone marrow chimeric mice, where B cell-extrinsic effects of miR-155 deficiency were normalized. Interestingly, we observed markedly lower efficiency of reactivation from latency in miR-155 deficient cells, indicating an important role for miR-155 in this process. These in vivo data complement previous in vitro studies and reach the conclusion that miR-155 is not necessary for the establishment or maintenance of gammaherpesvirus latency, but does affect reactivation efficiency.

    IMPORTANCE: Gammaherpesvirus infection leads to severe disease in immunosuppressed populations. MiR-155 has been shown to play important roles in many pathological processes, including tumorigenesis and diseases caused by an overly aggressive immune response. Our work provides valuable in vivo data showing that miR-155 is dispensable for gammaherpesvirus latency, but it is critical for reactivation from latency, which is a crucial step in the viral life cycle.

  • Cell-free versus cell-to-cell infection by HIV-1 and HTLV-1: exploring the link among viral source, viral trafficking and viral replication [PublishAheadOfPrint]

  • HIV-1 and HTLV-1 are complex retroviruses mainly infecting CD4+ T lymphocytes. In addition, antigen-presenting cells such as dendritic cells (DCs) are targeted in vivo by both viruses, although to a lesser extend. Interaction of HIV-1 with DCs plays a key role in viral dissemination from the mucosa to CD4+ T lymphocytes present in lymphoid organs. While similar mechanisms may occur for HTLV-1 as well, most HTLV-1 data were obtained from T-cell studies, and little is known regarding the trafficking of this virus in DCs. We first compared the efficiency of cell-free versus cell-associated viral sources of both retroviruses at infecting DCs. We showed that both HIV-1 and HTLV-1 cell-free particles are poorly efficient at productively infecting DCs, except if DC-SIGN has been engaged. Furthermore, while SAMHD1 accounts for restriction of cell-free HIV-1 infection, it is not involved in HTLV-1 restriction. In addition, cell-free viruses mainly lead to a non-productive DC infection leading to trans-infection of T-cell a process important for HIV-1 spread but not for that of HTLV-1. Finally, we show that T:DC cell-to-cell transfer implies viral trafficking in vesicles that may both increase productive infection of DCs ("cis-infection") and allow viral escape from immune surveillance. Altogether, these observations allowed us to draw a model on HTLV-1 and HIV-1 trafficking in DCs.

  • Pestivirus Npro directly interacts with interferon regulatory factor 3 (IRF3) monomer and dimer [PublishAheadOfPrint]

  • Interferon regulatory factor 3 (IRF3) is a transcription factor involved in the activation of type I interferon (IFN-aalpha;/bbeta;) in response to viral infection. Upon viral infection, IRF3 monomer is activated into a phosphorylated dimer, which induces the transcription of interferon genes in the nucleus. Viruses have evolved several ways to target IRF3 in order to subvert the innate immune response. Pestiviruses such as classical swine fever virus (CSFV) target IRF3 for ubiquitination and subsequent proteasomal degradation. This is mediated by the viral protein, Npro that interacts with IRF3, but the molecular details for this interaction are largely unknown. We use recombinant Npro and IRF3 proteins and show that Npro interacts with IRF3 directly without additional proteins, and forms a soluble 1:1 complex. The full-length IRF3, but not merely either of the individual domains, is required for this interaction. The interaction between Npro and IRF3 is not dependent on the activation state of IRF3, since Npro binds to a constitutively active form of IRF3 in the presence of its transcriptional coactivator CREB-binding protein (CBP). The results indicate that the Npro binding site on IRF3 encompasses a region unperturbed by the phosphorylation and subsequent activation of IRF3, which include the dimer interface and CBP binding site.

    IMPORTANCE The pestivirus N-terminal protease, Npro, is essential for evading the host's immune system by facilitating the degradation of interferon regulatory factor 3 (IRF3). However, the nature of Npro interaction with IRF3, including IRF3 species (inactive monomer vs. activated dimer) that Npro targets for degradation, is largely unknown. We show that classical swine fever virus Npro and porcine IRF3 directly interact in solution and that full length IRF3 is required for interaction with Npro. Additionally, Npro interacts with a constitutively active form of IRF3 bound to its transcriptional co-factor, the CREB-binding protein. This is the first study to demonstrate that Npro is able to bind both inactive IRF3 monomer and activated IRF3 dimer and thus likely targets both IRF3 species for ubiquitination and proteasomal degradation.

  • A Novel Non-Replication Competent Cytomegalovirus Capsid Mutant Vaccine Strategy Is Effective in Reducing Congenital Infection [PublishAheadOfPrint]

  • Congenital cytomegalovirus (CMV) is a leading cause of mental retardation and deafness in newborns. The guinea pig is the only small animal model for congenital CMV. A novel CMV vaccine was investigated as an intervention strategy against congenital guinea pig cytomegalovirus (GPCMV). In this disabled infectious single cycle (DISC) vaccine strategy, a GPCMV mutant lacked the ability to express an essential capsid gene (UL85 homolog, GP85), except when grown on a complementing cell line. In vaccinated animals, the GP85 mutant virus (GP85 DISC) induced an antibody response to important glycoprotein complexes considered neutralizing target antigens (gB, gH/gL/gO and gM/gN). The vaccine also generated a T cell response to the pp65 homolog (GP83), determined via a newly established guinea pig interferon gamma ELISPOT assay. In a congenital protection study, GP85 DISC vaccinated animals and non-vaccinated control group were challenged during pregnancy with wild type GPCMV (10^5 pfu). Animals went to term and viral load in target organs of pups analyzed. Based on live pup births in the vaccinated and control groups (94.1% vs 63.6%) the vaccine was successful in reducing mortality (P=0.0002). Additionally, pups from the vaccinated group had reduced CMV transmission with 23.5% infected target organs compared to 75.9% in the control group .Overall, these preliminary studies indicate that a DISC CMV vaccine strategy has the ability to induce an immune response similar to that of natural virus infection but has the increased safety of a non-replication competent virus, which makes this approach attractive as a CMV vaccine strategy.

    IMPORTANCE Congenital CMV is a leading cause of mental retardation and deafness in newborns. An effective vaccine against CMV still remains an elusive goal despite over fifty years of CMV research. The guinea pig, with a placenta structure similar to humans, is the only small animal model for congenital CMV and recapitulates disease symptoms (eg. deafness) in newborn pups. In this report, a novel vaccine strategy against congenital guinea pig cytomegalovirus (GPCMV) was developed, characterized and tested for efficacy. This disabled infectious single cycle (DISC) vaccine strategy induced a neutralizing antibody, or T cell response to important target antigens. In a congenital protection study, animals were protected against CMV in comparison to the non-vaccinated group (52% reduction of transmission). This novel vaccine was more effective than previously tested gB based vaccines and most other strategies live virus vaccines. Overall, the DISC vaccine is a safe and promising approach against congenital CMV.

  • Protein-protein interactions suggest novel activities of human cytomegalovirus tegument protein pUL103 [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) is an enveloped double-stranded DNA virus that causes severe disease in newborns and immunocompromised patients. During infection, the host cell endosecretory system is remodeled to form the cytoplasmic virion assembly complex (cVAC). We and others previously identified the conserved, multifunctional HCMV virion tegument protein pUL103 as important for cVAC biogenesis and efficient secondary envelopment. To help define its mechanisms of action and predict additional functions, we used two complementary methods, co-immunoprecipitation (co-IP) and BioID, to identify viral and cellular proteins that interact with pUL103. By using the two methods in parallel and applying stringent selection criteria, we identified potentially high value interactions of pUL103 with 13 HCMV and 18 cellular proteins. Detection of the previously identified pUL103-pUL71 interaction, as well as verification of several interactions by reverse co-IP, support the specificity of our screening process. As might be expected for a tegument protein, interactions were identified that suggest distinct roles for pUL103 across the arc of lytic infection, including interactions with proteins involved in cellular antiviral responses, nuclear activities, and biogenesis and transport of cytoplasmic vesicles. Further analysis of some of these interactions expand our understanding of the multifunctional repertoire of pUL103; we detected HCMV pUL103 in nuclei of infected cells, and identified an ALIX-binding domain within the pUL103 sequence.

    IMPORTANCE Human cytomegalovirus (HCMV) is able to reconfigure the host cell machinery to establish a virion production factory, the cytoplasmic virion assembly complex (cVAC). cVAC biogenesis and operation represent targets for development of novel HCMV antivirals. We previously showed that the HCMV tegument protein, pUL103, is required for cVAC biogenesis. Using pUL103 as bait, we investigated viral and cellular protein-protein interactions to identify and understand the range of pUL103 functions. We found that pUL103 interacts with cellular antiviral defense systems and proteins involved in organelle biogenesis and transport of cytoplasmic vesicles, and is present in infected cell nuclei. These results expand our understanding of the functional repertoire of pUL103 to include activities that extend from the earliest stages of infection through virion assembly and egress.

  • The K1 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) augments viral lytic replication. [PublishAheadOfPrint]

  • The K1 gene of Kaposi's sarcoma-associated herpesvirus (KSHV) is encoded by the first open reading frame (ORF) of the viral genome. To investigate the role of the K1 gene during the KSHV lifecycle, we constructed a set of recombinant viruses that contained either wild-type K1, a deleted K1 ORF (KSHVK1), stop codons within the K1 ORF (KSHV-K15XSTOP) or a revertant K1 virus (KSHV-K1REV). We report that the KSHVK1 and KSHV-K15XSTOP recombinant viruses displayed significantly reduced lytic replication compared to WT KSHV and KSHV-K1REV viruses upon reactivation from latency. Additionally, the KSHVK1 and KSHV-K15XSTOP recombinant viruses also yielded lower amounts of infectious progeny upon reactivation compared to WT KSHV and KSHV-K1REV virus infected cells. Upon reactivation from latency, WT KSHV and the KSHV-K1REV infected cells displayed activated Akt kinase as evidenced by its phosphorylation, while cells infected with viruses deleted for K1 showed reduced phosphorylation and activation of Akt kinase. Overall, our results suggest that K1 plays an important role during the KSHV lifecycle.

    IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of three human malignancies, KSHV K1 is a signaling protein that has been shown to be involved in cellular transformation and to activate the PI3K/Akt/mTOR pathway. In order to investigate the role of the K1 protein in the lifecycle of KSHV, we constructed recombinant viruses that were deficient for K1. We found that K1 deletion viruses displayed reduced lytic replication compared to WT virus and also yielded lower amounts of infectious progeny. We report that K1 plays an important role in the lifecycle of KSHV.

  • Characterization of a novel conformational GII.4 norovirus epitope: Implications in norovirus-host interactions [PublishAheadOfPrint]

  • Human noroviruses (NoVs) are the main etiological agents of acute gastroenteritis worldwide. While NoVs are highly diverse (more than 30 genotypes have been detected in humans), during the last 40 years most outbreaks and epidemics have been caused by GII.4 genotype strains, raising questions about their persistence in the population. Among other potential explanations, immune evasion is considered to be a main driver for their success. In order to study antibody recognition and evasion in detail, we have analyzed a conformational epitope recognized by a monoclonal antibody (3C3G3) by phage display, site-directed mutagenesis and surface plasmon resonance. Our results show that the predicted epitope is composed of eleven amino acids within the P domain: P245, E247, I389, Q390, R397, R435, G443, Y444, P445, N446, and D448. Only two of them, R397 and D448, differ from the homologous variant (GII.4 Den-Haag_2006b) and from a previous variant (GII.4 VA387_1996) which is not recognized by the antibody. A double mutant derived from the VA387_1996 variant containing both changes Q396R and N447D is recognized by the 3C3G3 monoclonal antibody, confirming the participation of these two sites in the epitope recognized by this antibody. Furthermore, a single change, Q396R, is able to modify the HBGA recognition pattern. These results provide evidence that the epitope recognized by the 3C3G3 antibody is involved in the virus-host interactions both at the immunological, as well as at the receptor levels.

    IMPORTANCE Human noroviruses are the main cause of viral diarrhea worldwide in people of all ages. Noroviruses can infect individuals who had been previously exposed to the same or different norovirus genotypes. Norovirus genotype GII.4 has been reported to be the most prevalent during the last 40 years. In the present study, we describe a novel viral epitope identified by a monoclonal antibody and located within the highly diverse P domain of the capsid protein. The evolution of this epitope along sequential GII.4 variants has allowed noroviruses to evade previously elicited antibodies, thus explaining how the GII.4 genotype can persist over long periods, re-infecting the population. Our results also show that this epitope participates in the recognition of host receptors which have evolved over time as well.

  • Recapitulating Cross-Species Transmission of SIVcpz to Humans Using Humanized-BLT Mice [PublishAheadOfPrint]

  • The origins of HIV-1 have been widely accepted to be the consequence of simian immunodeficiency viruses from wild chimpanzees (SIVcpz) crossing over to humans. However, there has not been any in vivo study of SIVcpz infection of humans. Also, it remains largely unknown why only specific SIVcpz strains have achieved cross-species transmission and what transmission risk might exist for those SIVcpz strains that have not been found to infect humans. Closing this knowledge gap is essential for better understanding cross-species transmission and predicting the likelihood of additional cross-species transmissions of SIV into humans. Here we show hu-BLT mice are susceptible to all studied strains of SIVcpz, including the inferred ancestral viruses of pandemic and non-pandemic HIV-1 groups M (SIVcpzMB897) and N (SIVcpzEK505), also strains that have not been found in humans (SIVcpzMT145 and SIVcpzBF1167). Importantly, the ability of SIVcpz to cross the interspecies barrier to infect humanized mice correlates with their phylogenetic distance to pandemic HIV-1. We also identified mutations of SIVcpzMB897 (Env G411R aamp; G413R) and SIVcpzBF1167 (Env H280Q aamp; Q380R) at 14 weeks post inoculation. Together, our results have recapitulated the events of SIVcpz cross-species transmission to humans and identified mutations that occurred during the first 16 weeks of infection, providing in vivo experimental evidence that the origins of HIV-1 are the consequence of SIVcpz crossing over to humans. This study also revealed that SIVcpz viruses whose inferred descendants have not been found in humans still have the potential to cause HIV-1 like zoonosis.

    IMPORTANCE It is believed that the origins of HIV-1 are the consequence of SIV viruses from wild chimpanzees crossing over to humans. However, the origins of HIV-1 have been linked back to only specific SIVcpz strains. There have been no experiments that directly test the in vivo cross-species transmissibility of SIVcpz strains to humans. This is the first in vivo study of SIVcpz cross-species transmission. With the humanized-BLT mouse model, we have provided in vivo experimental evidence of multiple SIVcpz strains crossing over to humans and identified several important mutations of divergent SIVcpz strains after long-term replication in human cells. We also found the cross-species transmission barrier of SIVcpz to humans correlates with their phylogenetic distance to pandemic HIV-1 group M. Importantly, this work provides evidence that SIVcpz viruses, whose inferred descendants have not been found in humans, still have the potential to cause a future HIV-1 like zoonotic outbreak.

  • LANA E3 Ubiquitin-Ligase Activity Impacts Gammaherpesvirus-Driven Germinal Center B Cell Proliferation [PublishAheadOfPrint]

  • Viruses have evolved mechanisms to hijack components of cellular E3 ubiquitin-ligases, thus modulating the ubiquitination pathway. However, the biological relevance of such mechanisms for viral pathogenesis in vivo remains largely unknown. Here, we utilized murid herpesvirus-4 (MuHV-4) infection of mice as a model system to address the role of latency-associated nuclear antigen (mLANA) E3 ligase activity in gammaherpesvirus latent infection. We show that specific mutations in the mLANA SOCS-box (V199A, V199A/L202A or P203A/P206A) disrupted mLANA's ability to recruit ElonginC and Cullin5, thereby impairing the formation of the EC5SmLANA complex and mLANA's E3 ligase activity on host NF-B and Myc. Although these mutations resulted in considerably reduced mLANA binding to viral terminal repeat DNA as assessed by EMSA, the mutations did not disrupt mLANA's ability to mediate episome persistence. In vivo, MuHV-4 recombinant viruses bearing these mLANA SOCS-box mutations exhibited a deficit in latency amplification in germinal center (GC) B cells. These findings demonstrate that the E3 ligase activity of mLANA contributes to gammaherpesvirus-driven GC B cell proliferation. Hence, pharmacological inhibition of viral E3 ligase activity through targeting SOCS-box motifs is a putative strategy to control gammaherpesvirus-driven lymphoproliferation and associated disease.

    IMPORTANCE The gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) cause life-long persistent infection and exert causative roles in several human malignancies. Colonization of B cells is crucial for virus persistence, and access to the B cell compartment is gained by virus-driven proliferation in germinal center (GC) B cells. Infection of B cells is predominantly latent, with the viral genome persisting as a multi-copy episome, and expressing only a small subset of viral genes. Here, we focused on latency-associated nuclear antigen (mLANA) encoded by murid herpesvirus-4 (MuHV-4), which exhibits homology in sequence, structure and function to KSHV LANA (kLANA), thereby allowing the study of LANA-mediated pathogenesis in mice. Our experiments show that mLANA's E3 ubiquitin-ligase activity is necessary for efficient expansion of latency in GC B cells, suggesting that the development of pharmacological inhibitors of LANA E3 ubiquitin-ligase activity may allow strategies to interfere with gammaherpesvirus-driven lymphoproliferation and associated disease.

  • Macrophages in progressive HIV/SIV infections [PublishAheadOfPrint]

  • The cells that are targeted by primate lentiviruses (HIV and SIV) are of intense interest given the renewed effort to identify potential cures for HIV. These viruses have been reported to infect multiple cell lineages of hematopoietic origin including all phenotypic and functional CD4 T cell subsets. The two most commonly reported cell types that become infected in vivo are memory CD4 T cells and tissue-resident macrophages. Though viral infection of CD4 T cells is routinely detected in both HIV-infected humans and SIV-infected Asian macaques, significant viral infection of macrophages is only routinely observed in animal models wherein CD4 T cells are almost entirely depleted. Here we review the roles of macrophages in lentiviral disease progression, evidence that macrophages support viral replication in vivo, animal models where macrophage-mediated replication of SIV is thought to occur, how the virus can interact with macrophages in vivo, pathologies thought to be attributed to viral replication within macrophages, how viral replication in macrophages might contribute to the asymptomatic phase of HIV/SIV, and whether macrophages represent a long-lived reservoir for the virus.

  • Antiviral activity of myticin C peptide from mussel: an ancient defence against herpesviruses [PublishAheadOfPrint]

  • Little is known about the antiviral response in molluscs. As in other invertebrates, the interferon signalling pathways have not been identified, and in fact, there is a debate on whether invertebrates possess an antiviral immunity similar to that of vertebrates. In marine bivalves, due to their filtering activity, the interaction with putative pathogens, including viruses, is very high, suggesting that they should have mechanisms to address these infections. In this study, we confirmed that constitutively expressed molecules in naïve mussels confer resistance in oysters to Ostreid herpesvirus 1 (OsHV-1) when oyster haemocytes are incubated with mussel haemolymph. Using a proteomic approach, myticin C peptides were identified in both mussel haemolymph and haemocytes. Myticins, antimicrobial peptides that have been previously characterized, were constitutively expressed in a fraction of mussel haemocytes and showed antiviral activity against OsHV-1, suggesting that these molecules could be responsible of the antiviral activity of mussel haemolymph. For the first time, a molecule from a bivalve has shown antiviral activity against a virus affecting molluscs. Moreover, modified myticin C peptides showed antiviral activity against human herpes simplex viruses types 1 (HSV-1) and 2 (HSV-2). In summary, our work sheds light on the invertebrate antiviral immune response with the identification of a molecule with potential biotechnological applications.

    IMPORTANCE Several bioactive molecules have been identified and isolated from marine invertebrates that have potential pharmaceutical or industrial applications. Myticin C, an antimicrobial peptide from the Mediterranean mussel (Mytilus galloprovincialis) that was identified by proteomic techniques in both mussel haemolymph and haemocytes, showed potential as an antiviral agent against Ostreid herpesvirus 1 (OsHV-1), which represents a major threat to the oyster farming sector. Both haemolymph from mussels and a myticin C peptide inhibited OsHV-1 replication in oyster haemocytes. Additionally, a modified peptide derived from myticin C also showed antiviral activity against the human herpesviruses HSV-1 and HSV-2. Therefore, myticin C is an example of the biotechnological and therapeutic potential of molluscs.

  • Mitochondrial p53 contributes to reovirus-induced neuronal apoptosis and CNS injury in a mouse model of viral encephalitis [PublishAheadOfPrint]

  • The tumor suppressor p53 plays a critical part in determining cell fate both as a regulator of the transcription of several pro-apoptotic genes and through its binding interactions with Bcl-2 family proteins at the mitochondria. We now demonstrate that p53 protein levels increase in infected brains during reovirus encephalitis. This increase occurs in the cytoplasm of reovirus-infected neurons and is associated with the activation of caspase 3. Increased levels of p53 in reovirus-infected brains are not associated with increased expression of p53 mRNA suggesting that p53 regulation occurs at the protein level. Increased levels of p53 are also not associated with increased expression of p53-regulated, pro-apoptotic genes. In contrast, upregulated p53 accumulates in the mitochondria. Previous reports have demonstrated that the binding of p53 to Bak at the mitochondria causes Bak activation and results in apoptosis. We now show that Bak is activated, and that activated Bak is bound to p53 during reovirus encephalitis. In addition, survival is enhanced in reovirus infected Bak-/- mice compared to controls, demonstrating a role for Bak in reovirus pathogenesis. Inhibition of the mitochondrial translocation of p53with pifithrin mmu; prevents the formation of p53/Bak complexes following reovirus infection of ex vivo brain slice cultures and results in decreased apoptosis and tissue injury. These results suggest that the mitochondrial localization of p53 regulates reovirus-induced pathogensis in the CNS through its interactions with Bak.

    IMPORTANCE There are virtually no specific treatments of proven efficacy for virus-induced neuroinvasive diseases. A better understanding of the pathogenesis of virus-induced CNS injury is crucial for the rational development of novel therapies. Our studies demonstrate that p53 is activated in the brain following reovirus infection and may provide a therapeutic target for virus-induced CNS disease.

  • Repair of a Mutation Disrupting the Guinea Pig Cytomegalovirus Pentameric Complex Acquired During Fibroblast Passage Restores Pathogenesis in Immune Suppressed Guinea Pigs and in the Context of Congenital Infection [PublishAheadOfPrint]

  • Guinea pig cytomegalovirus (GPCMV) provides a valuable model for congenital cytomegalovirus transmission. Salivary gland (SG)-passaged stocks of GPCMV are pathogenic, while tissue culture (TC) passage in fibroblasts results in attenuation. Non-pathogenic TC-derived virus N13R10 (cloned as a bacterial artificial chromosome) has a 4-bp deletion that disrupts GP129, which encodes a subunit of the GPCMV pentameric complex (PC) believed to govern viral entry into select cell types, and GP130, an overlapping ORF of unknown function. To determine if this deletion contributes to attenuation of N13R10, markerless gene transfer in E. coli was used to construct virus r129, a variant of N13R10 in which the 4-bp deletion is repaired. Virions from r129 were found to contain GP129 as well as two other PC subunit proteins, GP131 and GP133, whereas these three PC subunits were absent from N13R10 virions. Replication of r129 in fibroblasts appeared unaltered compared to N13R10. However, following experimental challenge of immune compromised guinea pigs, r129 induced significant weight loss, longer duration of viremia, and dramatically higher (up to 1.5 x 106-fold) viral loads in blood and end-organs compared to N13R10. In pregnant guinea pigs, challenge with doses gge;5 x 106 pfu of r129 virus resulted in levels of maternal viremia, congenital transmission, pup viral loads, IUGR, and pup mortality comparable to that induced by pathogenic SG virus, although higher doses of r129 were required. These results suggest that the GP129/GP130 mutation is a significant contributor to attenuation of N13R10, likely by abrogating expression of a functional PC.

    Importance Tissue culture adaptation of cytomegaloviruses rapidly selects for mutations, deletions and rearrangements in the genome, particularly for viruses passaged in fibroblast cells. Some of these mutations are focused in the region of the genome encoding components of the pentameric complex (PC), in particular homologs of HCMV UL128, UL130 and UL131A. These mutations can attenuate the course of infection when the virus when re-introduced into animals for vaccine and pathogenesis studies. This study demonstrates that a deletion that arose during the process of tissue culture passage can be repaired, with subsequent restoration of pathogenicity, using BAC-based mutagenesis. Restoration of pathogenicity by repair of a frame-shift mutation in GPCMV gene GP129 using this approach provides a valuable genetic platform for future studies using the guinea pig model of congenital CMV infection.

  • Evidence that receptor destruction by the Sendai virus hemagglutinin-neuraminidase protein is responsible for homologous interference. [PublishAheadOfPrint]

  • Receptor destruction has been considered as one of the mechanisms of homologous Sendai virus (SeV) interference. However, direct evidence of receptor destruction upon virus infection and its relevance to interference is missing. To investigate a precise mechanism of homologous interference, we established SeV persistently infected cells. The persistently infected cells inhibited superinfection by homologous SeV but supported replication of human parainfluenza virus type 2 (hPIV2) and influenza A virus (IAV). We confirmed that SeV particles could not attach or penetrate the infected cells, and that the hemagglutinin-neuraminidase (HN) protein of SeV was involved in the interference. Lectin blot assays showed that aalpha;2,3 linked sialic acids were specifically reduced in the SeV infected cells, but aalpha;2,6 linked sialic acids had not changed. As infection of IAV removed both aalpha;2,3 and aalpha;2,6 linked sialic acids, especially aalpha;2,3 linked sialic acids, IAV infected cells inhibited superinfection of SeV. These results provide concrete evidence that destruction of the specific SeV receptor, aalpha;2,3 linked sialic acids is relevant to homologous interference by SeV.

    IMPORTANCE Viral interference is a classically observed phenomenon but the precise mechanism is not clear. Using SeV interference, we provided concrete evidence that reduction of aalpha;2,3 linked sialic acids receptor by the HN of SeV was closely related with viral interference. Since SeV infection resulted in decrease of only aalpha;2,3 linked sialic acids, IAV, which also utilized aalpha;2,6 linked sialic acids to initiate infection, superinfected the SeV infected cells. In contrast, SeV could not superinfect the IAV infected cells because both aalpha;2,3 and aalpha;2,6 linked sialic acids were removed. These results indicate that receptor destruction critically contributes to viral interference.

  • Changes in HIV-1 Capsid stability induced by common CTL-driven viral sequence mutations [PublishAheadOfPrint]

  • HIV-1-infected individuals encoding for protective HLA class I alleles exhibit better control of viremia and slower disease progression. Viral control in these individuals has been associated with strong and potent HIV-1-specific CTL responses restricted by protective HLA alleles, but control of viremia also occurs in the presence of selected CTL-escape mutations. CTL escape mutations restricted by protective HLA class I molecules are frequently located in the conserved p24 Gag sequence of HIV-1 that encodes for the conical capsid core, and have been suggested to reduce viral replication capacity. In this study the consequences of well-described CTL-associated p24 Gag sequence mutations for HIV-1 capsid stability were assessed using a Cyclosporine A (CsA) washout assay. The frequently occurring HLA-B57- and HLA-B27-associated CTL escape mutations T242N and R264K resulted in delayed capsid uncoating, suggesting modulation of capsid stability. The described compensatory mutations L268M and S173A observed in R264K viruses reconstituted capsid uncoating half time. Interestingly, capsid stability was correlated to infectivity. Taken together, these data demonstrate that CTL-driven escape mutations within p24 Gag restricted by protective HLA class I alleles have a significant impact on capsid stability that might contribute to the persistent control of viral replication observed despite viral escape from CTL responses.

    IMPORTANCE Sequence mutations within p24 Gag selected by CTL-responses restricted by protective HLA class I alleles have been associated with reduced viral fitness. However the precise mechanisms underlying the reduced viral replication capacity and lower viral loads associated with these mutations remain unclear. Here we demonstrate that dominant HLA-B27-associated CTL-escape mutations within HIV-1 capsid are leading to enhanced capsid rigidity, providing a possible mechanism for the reduced viral fitness of these variants.

  • Spatial-temporal patterns of viral amplification and interference initiated by a single infected cell [PublishAheadOfPrint]

  • When viruses infect their host cells they can make defective virus-like particles along with intact virus. Cells co-infected with virus and defective particles often exhibit interference of virus growth caused by the competition for resources by defective genomes. Recent reports of the co-existence and co-transmission in vivo of such defective interfering particles (DIPs), across epidemiological length and time scales, suggest a role in viral pathogenesis, but it is not known how DIPs impact infection spread, even under controlled culture conditions. Using fluorescence microscopy, we quantified co-infections of vesicular stomatitis virus (VSV) expressing a fluorescent reporter protein and its DIPs on BHK-21 host-cell monolayers. We found viral gene expression was more delayed, infections spread more slowly, and patterns of spread became more "patchy" for higher DIP inputs to the initial cell. To examine how infection spread might depend on the behavior of the initial co-infected cell we built a computational model, adapting a cellular automata (CA) approach to incorporate for the first time-kinetic data of virus growth. Specifically, changes in observed patterns of infection spread could be directly linked to previous high-throughput single-cell measures of virus-DIP co-infection. The CA model also provided testable hypotheses on the spatial-temporal distribution of the DIPs, which remain governed by their predator-prey interaction. More generally, this work offers a data-driven computational modeling approach to better understand how single infected cells impact the multi-round spread of virus infections across cell populations.

    IMPORTANCE Defective interfering particles (DIPs) compete with intact virus, depleting host-cell resources that are essential for virus growth and infection spread. However, it is not known how such competition, strong or weak, ultimately impacts how infections spread and cause disease. Here we address this unmet need by developing an integrated experimental-computational approach, which sheds new light on how infections spread. We anticipate our approach will also be useful in the development of DIPs as therapeutic agents to manage the spread of viral infections.

  • A role for the {alpha}V integrin subunit in Varicella Zoster Virus-mediated fusion and infection. [PublishAheadOfPrint]

  • Varicella zoster virus (VZV) is an alphaherpesvirus that causes varicella and herpes zoster. Membrane fusion is essential for VZV entry and the distinctive syncytia formation in VZV infected skin and neuronal tissue. Herpesvirus fusion is mediated by a complex of glycoproteins gB and gH-gL, which are necessary and sufficient for VZV to induce membrane fusion. However, the cellular requirements of fusion are poorly understood. Integrins have been implicated to facilitate entry of several human herpesviruses, but their role in VZV entry has not yet been explored. To determine the involvement of integrins in VZV fusion, a quantitative cell-cell fusion assay was developed using a VZV-permissive melanoma cell line. The cells constitutively expressed a reporter protein and short hairpin RNAs (shRNAs) to knockdown expression of integrin subunits shown to be expressed in these cells by RNA sequencing. The aalpha;V integrin subunit was identified to mediate VZV gB/gH-gL fusion as its knockdown by shRNAs reduced fusion levels to 60% of control cells. A comparable reduction in fusion levels was observed when an anti-aalpha;V antibody specific to its extracellular domain was tested in the fusion assay, confirming that the domain was important for VZV fusion. In addition, reduced spread was observed in aalpha;V knockdown cells infected with the VZV pOka strain relative to the control cells. This was demonstrated by reductions in plaque size, replication kinetics and virion entry in the aalpha;V subunit knockdown cells. Thus, the aalpha;V integrin subunit is important for VZV gB/gH-gL fusion and infection.

    IMPORTANCE Varicella Zoster Virus (VZV) is a highly infectious pathogen that causes chicken pox and shingles. A common complication of shingles is the excruciating painful condition called postherpetic neuralgia that has proven difficult to treat. While a vaccine is now available, it is not recommended for immune compromised individuals and its efficacy decreases with the recipient's age. These limitations highlight the need for new therapies. This study examines the role of integrins in membrane fusion mediated by VZV glycoproteins, gB and gH-gL, a required process for VZV infection. This knowledge will further the understanding of VZV entry and provide insight into the development of better therapies.

  • Pandemic-seasonal H1N1 reassortants recovered from patient material display a phenotype similar to the seasonal parent. [PublishAheadOfPrint]

  • We have previously shown that eleven patients became naturally co-infected with seasonal H1N1 (A/H1N1) and pandemic H1N1 (pdm/H1N1) during the Southern hemisphere winter of 2009 in New Zealand. Reassortment of influenza A viruses is readily observed during co-infection of host animals and in vitro, however, reports of reassortment occurring naturally in humans are rare. Using clinical specimen material, we show reassortment between the two co-infecting viruses occurred with high likelihood directly in one of the previously identified patients. Despite the lack of spread of these reassortants in the community, we did not find them to be attenuated in several model systems for viral replication and virus transmission: multistep growth curves in differentiated human bronchial epithelial cells revealed no growth deficiency in six recovered reassortants when compared to A/H1N1 and pdm/H1N1 isolates. Two reassortant viruses were assessed in ferrets and showed transmission to aerosol contacts. This study demonstrates that influenza virus reassortants can arise in naturally co-infected patients.

    IMPORTANCE Reassortment of influenza A viruses is an important driver of virus evolution, but little has been done to address humans as hosts for the generation of novel influenza viruses. We show here that multiple reassortant viruses were generated during natural co-infection of a patient with pandemic H1N1 (2009) and seasonal H1N1 influenza A viruses. Though apparently fit in model systems, these reassortants did not become established in the wider population, presumably due to herd immunity against their seasonal H1 antigen.

  • Structure and genome release mechanism of human cardiovirus Saffold virus-3 [PublishAheadOfPrint]

  • In order to initiate an infection, viruses need to deliver their genomes into cells. This involves uncoating the genome and transporting it to the cytoplasm. The process of genome delivery is not well understood for non-enveloped viruses. We address this gap in our current knowledge by studying the uncoating of the non-enveloped human cardiovirus Saffold virus-3 (SAFV-3) of the family Picornaviridae. SAFVs cause diseases ranging from gastrointestinal disorders to meningitis. We present a structure of a native SAFV-3 virion determined to 2.5 AAring; by X-ray crystallography and an 11 AAring;-resolution cryo-electron microscopy reconstruction of an "altered" particle that is primed for genome release. The altered particles are expanded relative to the native virus and contain pores in the capsid that might serve as channels for the release of VP4 subunits, N-termini of VP1, and the RNA genome. Unlike in the related enteroviruses, pores in SAFV-3 are located roughly between the icosahedral threefold and fivefold axes at an interface formed by two VP1 and one VP3 subunit. Furthermore, in native conditions many cardioviruses contain a disulfide bond formed by cysteins that are separated by just one residue. The disulfide bond is located in a surface loop of VP3. We determined the structure of the SAFV-3 virion in which the disulfide bonds are reduced. Disruption of the bond had minimal effect on the structure of the loop, but it increased the stability and decreased the infectivity of the virus. Therefore, compounds specifically disrupting or binding to the disulfide bond might limit SAFV infection.

    IMPORTANCE A capsid assembled from viral proteins protects the virus genome during transmission from one cell to another. However, when a virus enters a cell the virus genome has to be released from the capsid in order to initiate infection. This process is not well understood for non-enveloped viruses. We address this gap in our current knowledge by studying the genome release of human Saffold virus-3. Saffold viruses cause diseases ranging from gastrointestinal disorders to meningitis. We show that before the genome is released, the Saffold virus-3 particle expands and holes form in the previously compact capsid. These holes serve as channels for the release of the genome and small capsid proteins VP4 that in related enteroviruses facilitate subsequent transport of the virus genome into the cell cytoplasm.

  • Role of Conserved E2 Residue W420 in Receptor Binding and Hepatitis C Virus Infection [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) enters cells via interactions with several host factors, a key one being that between the viral E2 envelope glycoprotein and the CD81 receptor. We previously identified the E2 tryptophan 420 (W420) as an essential CD81-binding residue. However, the importance of W420 in the context of the native virion is unknown as these earlier studies predate the infectious HCV cell-culture (HCVcc) system. Here, we introduced four separate mutations (F, Y, A or R) at position 420 within the infectious JFH-1 HCVcc genome and characterized their effects on the viral cycle. Whilst all mutations reduced E2-CD81 binding, only two (W420A and W420R) reduced HCVcc infectivity. Further analyses of mutants with hydrophobic residues (F or Y) found that interactions with receptors SR-BI as well as CD81 were modulated which in-turn determined the viral uptake route. Both mutant viruses were significantly less dependent on SR-BI, and its lipid-transfer activity, for virus entry. Furthermore, they were resistant to the drug erlotinib that targets EGFR (a host co-factor for HCV entry) and also blocks SR-BI dependent HDL-mediated enhancement of virus entry. Together, our data indicate a model where alteration at position 420 causes a subtle change in E2 conformation that prevents interaction with SR-BI and increases accessibility to the CD81 binding site in-turn favoring a particular internalization route. They further show that a hydrophobic residue with a strong preference for tryptophan at position 420 is important, both functionally and structurally, to provide an additional hydrophobic anchor to stabilize the E2-CD81 interaction.

    IMPORTANCE Hepatitis C virus (HCV) is a leading cause of liver disease causing up to 500000 deaths annually. The first step in the viral life-cycle is the entry process. This study investigates the role of a highly conserved residue, tryptophan 420 of the viral glycoprotein E2 in this process. We analyzed the effect of changing this residue in the virus and confirmed that this region is important for binding to the CD81 receptor. Furthermore, alteration of this residue modulated the interaction with the SR-BI receptor and changes to these key interactions were found to affect the virus internalization route involving the host co-factor, EGFR. Our results also show that the nature of the amino acid at this position is important functionally and structurally to provide an anchor-point to stabilize the E2-CD81 interaction.

  • Virion structure of iflavirus slow bee paralysis virus at 2.6A resolution [PublishAheadOfPrint]

  • The western honeybee (Apis mellifera) is the most important commercial insect pollinator. However, bees are under pressure from habitat loss, environmental stress and pathogens, including viruses that can cause lethal epidemics. Slow bee paralysis virus (SBPV) belongs to the Iflaviridae family of non-enveloped single-stranded RNA viruses. Here we present the structure of the SBPV virion determined from two crystal forms to resolutions of 3.4 AAring; and 2.6 AAring;. The overall structure of the virion resembles that of picornaviruses with the three major capsid proteins VP1-3 organized into a pseudo-T3 icosahedral capsid. However, the SBPV capsid protein VP3 contains a C-terminal globular domain that has not been observed in other viruses from the order Picornavirales. The protruding (P)-domains form "crowns" on the virion surface around each fivefold axis in one of the crystal forms. However, the P-domains are shifted 36 AAring; towards the threefold axis in the other crystal form. Furthermore, the P-domain contains the ser-his-asp triad within a surface patch of eight conserved residues that constitutes a putative catalytic or receptor-binding site. The movements of the domain might be required for efficient substrate cleavage or receptor binding during virus cell entry. In addition, capsid protein VP2 contains an RGD sequence that is exposed on the virion surface, indicating that integrins might be cellular receptors of SBPV.

    IMPORTANCE Pollination by honeybees is needed to sustain agricultural productivity as well as the biodiversity of wild flora. However, honeybee populations in Europe and North America have been declining since the 1950s. Honeybee viruses from the Iflaviridae family are among the major causes of honeybee colony mortality. We determined the virion structure of an Iflavirus, slow bee paralysis virus (SBPV). SBPV exhibits unique structural features not observed in other picorna-like viruses. The SBPV capsid protein VP3 has a large C-terminal domain, five of which form highly prominent protruding "crowns" on the virion surface. However, the domains can change their positions depending on the conditions of the environment. The domain includes a putative catalytic or receptor binding site that might be important for SBPV cell entry.

  • Arabidopsis RNA polymerases IV and V are required to establish H3K9 methylation, but not cytosine methylation, on geminivirus chromatin [PublishAheadOfPrint]

  • In plants, RNA-directed DNA methylation (RdDM) employs small RNAs to target enzymes that methylate cytosine residues. Cytosine methylation and dimethylation of histone 3 lysine 9 (H3K9me2) are often linked. Together they condition an epigenetic defense that results in chromatin compaction and transcriptional silencing of transposons and viral chromatin. Canonical RdDM (Pol IV-RdDM), involving RNA polymerases IV and V (Pol IV and Pol V), was believed necessary to establish cytosine methylation, which in turn could recruit H3K9 methyltransferases. However, recent studies have revealed that a pathway involving Pol II and RNA-dependent RNA polymerase 6 (RDR6) (RDR6-RdDM) is likely responsible for establishing cytosine methylation at naïve loci, while Pol IV-RdDM acts to reinforce and maintain it. We used the geminivirus Beet curly top virus (BCTV) as a model to examine the roles of Pol IV and Pol V in establishing repressive viral chromatin methylation. As geminivirus chromatin is formed de novo in infected cells, these viruses are unique models for processes involved in the establishment of epigenetic marks. We confirm that Pol IV and Pol V are not needed to establish viral DNA methylation, but are essential for its amplification. Remarkably, however, both Pol IV and Pol V are required for deposition of H3K9me2 on viral chromatin. Our findings suggest that cytosine methylation alone is not sufficient to trigger de novo deposition of H3K9me2, and further that Pol IV-RdDM is responsible for recruiting H3K9 methyltransferases to viral chromatin.

    IMPORTANCE In plants, RNA-directed DNA methylation (RdDM) uses small RNAs to target cytosine methylation, which is often linked to histone 3 lysine 9 dimethylation (H3K9me2). These epigenetic marks silence transposable elements and DNA virus genomes, but how they are established is not well understood. Canonical RdDM (Pol IV-RdDM), involving RNA polymerases IV and V (Pol IV and Pol V), was thought to establish cytosine methylation that in turn could recruit H3K9 methyltransferases, but recent studies compel a re-evaluation of this view. We used Beet curly top virus (BCTV) to investigate the roles of Pol IV and Pol V in chromatin methylation. We found that both are needed to amplify, but not to establish, DNA methylation. However, both are required for deposition of H3K9me2. Our findings suggest that cytosine methylation is not sufficient to recruit H3K9 methyltransferases to naïve viral chromatin, and further that Pol IV-RdDM is responsible.

  • Infectious bronchitis coronavirus limits interferon production by inducing a host shutoff that requires accessory protein 5b. [PublishAheadOfPrint]

  • During infection of their host cell, viruses often inhibit production of host proteins, a process which is referred to as host shutoff. By doing this, viruses limit production of antiviral proteins and increase production capacity for viral proteins. Coronaviruses from the Alpha- and Betacoronavirus genera, such as severe acute respiratory syndrome coronavirus (SARS-CoV) establish host shutoff via their non-structural protein 1 (nsp1). The genomes of Gamma- and Deltacoronaviruses however do not encode nsp1, and it has been suggested that these viruses do not induce host shutoff. Here we show that infectious bronchitis Gammacoronavirus (IBV) does induce host shutoff and we find that its accessory protein 5b is indispensable for this function. Importantly, we found that 5b-null viruses, unlike wild type viruses, induce production of high concentrations of type I interferon protein in vitro, indicating that host shutoff by IBV plays an important role in antagonizing the host's innate immune response. Altogether we demonstrate that 5b is a functional equivalent of nsp1 thereby answering the long-standing question whether lack of nsp1 in Gammacoronaviruses is compensated for by another viral protein. As such, our study is a significant step forward in the understanding of coronavirus biology and closes a gap in the understanding of some IBV virulence strategies.

    IMPORTANCE Many viruses inhibit protein synthesis of their host cell to enhance virus replication and antagonize anti-viral defense mechanisms. This process is referred to as llsquo;host-shutoff'. We have studied gene expression and protein synthesis in chicken cells infected with the important poultry pathogen, infectious bronchitis virus (IBV). We show that IBV inhibits synthesis of host proteins, including that of type I interferon, a key component of the antiviral response. The IBV-induced host shutoff however, does not require degradation of host RNA. Furthermore, we demonstrate that accessory protein 5b of IBV plays a crucial role in the onset of the host shutoff. Our findings suggest that inhibition of host protein synthesis is a common feature of coronaviruses and primarily serves to inhibit the antiviral response of the host.

  • Antiretroviral therapy in SIV-infected sooty mangabeys: implications for AIDS pathogenesis [PublishAheadOfPrint]

  • Simian immunodeficiency virus (SIV)-infected sooty mangabeys (SMs) do not develop AIDS despite high levels of viremia. Key factors involved in the benign course of SIV infection in SMs are the absence of chronic immune activation and low levels of infection of CD4+ central memory (TCM) and stem cell memory (TSCM) T-cells. To better understand the role of virus replication in determining the main features of SIV infection in SMs, we treated twelve SMs with a potent antiretroviral therapy (ART) regimen for 2-12 months. We observed that ART suppressed viremia to llt;60 copies/ml of plasma in 10 out of 12 animals, and induced a variable decrease in the level of cell-associated SIV-DNA in peripheral blood (average fold-change: 0.9, 1.1, 1.5 and 3.7 for CD4+ transitional memory, TTM, TCM, effector memory, TEM, and TSCM, respectively). ART-treated SIV-infected SMs showed (i) increased percentage of circulating CD4+ TCM; (ii) increased levels of CD4+ T-cells in the rectal mucosa, and (iii) significant decline in the frequency of HLA-DR+CD8+ T-cells in blood and rectal mucosa. In addition, we observed that ART interruption resulted in rapid viral rebound in all SIV-infected SMs, indicating that the virus reservoir persists for at least a year under ART despite lower infection of CD4+ TCM and TSCM when compared to pathogenic SIV infection of macaques. Overall these data indicate that ART induces specific immunological changes in SIV-infected SMs, thus suggesting that virus replication impacts on the immune function even in the context of this clinically benign infection.

    IMPORTANCE Studies of natural, non-pathogenic Simian Immunodeficiency Virus (SIV) infection of African monkeys have provided important insights into the mechanisms responsible progression to AIDS during pathogenic HIV infection of humans and SIV infection of Asian macaques. In this study, we treated for the first time SIV-infected sooty mangabeys, a natural host for the infection, with a potent antiretroviral therapy (ART) regimen for a period ranging between 2 and 12 months and monitored in detail how suppression of virus replication impacts the main virological and immunological features of this non-pathogenic infection. The observed findings provide novel information on both the pathogenesis of residual immunological disease under ART during pathogenic infection and the mechanisms involved in virus persistence during primate lentiviral infections.

  • Assembly of replication-incompetent African horse sickness virus particles: rational design of vaccines for all serotypes [PublishAheadOfPrint]

  • African horse sickness virus, an orbivirus in the Reoviridae family, with nine different serotypes causes devastating disease in equids. The virion particle is composed of seven proteins, organized in three concentric layers, an outer made of VP2 and VP5, a VP7 middle layer and inner layer of VP3 that encloses a replicase complex of VP1, VP4 and VP6 and a genome of 10 double-stranded RNA segments. In this study, we sought to develop highly efficacious vaccine candidates against all AHSV serotypes, taking into account not only immunogenic and safety properties, but also virus productivity and stability parameters, which are essential criteria for vaccine candidates. To achieve this goal, we first established highly efficient reverse genetics (RG) system for AHSV1 and subsequently, VP6-defective AHSV1 strain in combination with in trans complementation of VP6. This was then used to generate defective particles of all nine serotypes, which required exchange of two to five RNA segments to achieve equivalent titers of particles. In the VP6-complementary cells, all reassortant defective viruses could be amplified and propagated to high titers, but were totally incompetent in any other cells. Furthermore, these replication-incompetent AHSV particles were demonstrated to be highly protective against homologous virulent virus challenges in type I interferon receptor knock-out mice. Thus, these defective viruses have potential for the development of safe and stable vaccine candidates. The RG system also provides a powerful tool for the study of the role of individual AHSV proteins in virus assembly, morphogenesis and pathogenesis.

    IMPORTANCE African horse sickness virus is transmitted by biting midges and causes African horse sickness in equids, reaching up to 95% mortality in naïve horses. Therefore, the development of efficient vaccines is extremely important due to major economic losses in the equine industry. Through establishing a highly efficient RG system, replication-deficient viruses of all nine AHSV serotypes have been generated. These defective viruses achieved high titers in a VP6-complementing cell line, but failed to propagate in wild-type mammalian or insect cells. Importantly, these vaccine candidate strains showed strong protective efficacy against AHSV infection in an IFNARnndash;/nndash; mouse model.

  • Complex interplay between HIV-1 Capsid and MX2-independent IFN{alpha}-induced antiviral factors [PublishAheadOfPrint]

  • Type I interferons (IFNs), including IFNaalpha; upregulate an array of interferon-stimulated genes (ISGs) and potently suppress HIV-1 infectivity in CD4+ T cells, monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs). Recently, we and others identified the ISG myxovirus resistance 2 (MX2) to block HIV-1 nuclear entry. However, additional antiviral blocks exist upstream of nuclear import, but the ISGs that suppress infection e.g. prior to (or during) reverse transcription remain to be defined. Here we show that HIV-1 CA mutants such as N74D or A105T, which both allow escape from inhibition by MX2 and the truncated version of the cleavage and polyadenylation specific factor 6 (CPSF6), as well as the cyclophilin A (CypA)-binding loop mutant P90A, each exhibit increased sensitivity to IFNaalpha;-mediated inhibition. Using CRISPR/Cas9 technology, we demonstrate that the IFNaalpha;-hypersensitivity of these mutants in THP-1 cells is independent of MX2 or CPSF6. As expected, CypA depletion had no additional effect on the behavior of the P90A mutant, but modestly increased the IFNaalpha;-sensitivity of wild type virus. Interestingly, the infectivity of wild type or P90A virus could be rescued from the MX2-independent IFNaalpha;-induced blocks in THP-1 cells by treatment with cyclosporine (Cs), or its non-immunosuppressive analogue SDZ-NIM811, indicating that Cs-sensitive host cell cyclophilins other than CypA contribute to the activity of IFNaalpha;-induced blocks. We propose that cellular interactions with incoming HIV-1 capsids help shielding the virus from recognition by antiviral effector mechanisms. Thus, the CA protein is a fulcrum for the dynamic interplay between cell-encoded functions that inhibit or promote HIV-1 infection.

    Importance Human immunodeficiency virus type-1 (HIV-1) is the causative of the acquired immunodeficiency syndrome (AIDS). During acute HIV-1 infection, numerous pro-inflammatory cytokines are produced, including type I interferons (IFNs). IFNs can limit HIV-1 replication by inducing the expression of a set of antiviral genes that inhibit HIV-1 at multiple steps in its life cycle, including the post-entry steps of reverse transcription and nuclear import. This is observed in cultured cell systems, as well as in clinical trials in HIV-1 infected patients. The identities of the cellular antiviral factors, their viral targets and the underpinning mechanisms are largely unknown. We show here that the HIV-1 Capsid protein plays a central role in protecting the virus from IFN-induced inhibitors that block early post-entry steps of infection. We further show that host cell cyclophilins play an important role in regulating these processes, thus highlighting the complex interplay between antiviral effector mechanisms and viral survival.

  • Mapping of Ebolavirus Neutralization by Monoclonal Antibodies in the ZMapp Cocktail Using Cryo-Electron Tomography and Studies of Cellular Entry [PublishAheadOfPrint]

  • ZMapp, a cocktail of three monoclonal antibodies (mAbs; c2G4, c4G7 and c13C6) against the ebolavirus (EBOV) glycoprotein (GP), shows promise for combatting outbreaks of EBOV, as occurred in West Africa in 2014. Prior studies showed that Fabs from these mAbs bind a soluble EBOV GP ectodomain, and that mAbs c2G4 and c4G7, but not c13C6, neutralize infections in cell cultures. Using cryo-electron tomography, we extended these findings by characterizing the structures of c2G4, c4G7 and c13C6 IgGs bound to native, full-length GP from the West African 2014 isolate embedded in filamentous viral-like particles (VLPs). As with the isolated ectodomain, c13C6 bound to the glycan cap, while c2G4 and c4G7 bound to the base region of membrane-bound GP. The tomographic data suggest that all three mAbs bind with high occupancy, and that the base-binding antibodies can potentially bridge neighboring GP spikes. Functional studies indicated that c2G4 and c4G7, but not c13C6, competitively inhibit entry of VLPs bearing EBOV GP into the host cell cytoplasm, without blocking trafficking of VLPs to NPC1+ endolysosomes, where EBOV fuses. Moreover, c2G4 and c4G7 bind to and can block entry mediated by the primed (19 kDa) form of GP without impeding binding of the C-loop of NPC1, the endolysosomal receptor for EBOV. The most likely mode of action of c2G4 and c4G7 is, therefore, by inhibiting conformational changes in primed, NPC1-bound GP that initiate fusion between the viral and target membranes, similar to the action of certain broadly neutralizing antibodies against influenza HA and HIV Env.

    IMPORTANCE The recent West African outbreak of ebolavirus caused the deaths of over 11,000 individuals. Hence there is an urgent need to be prepared with vaccines and therapeutics for similar future disasters. ZMapp, a cocktail of three mAbs directed against the ebolavirus glycoprotein, is a promising anti-ebolavirus therapeutic. Using cryo-electron tomography we provide structural information on how each of the mAbs in this cocktail binds to the ebolavirus glycoprotein as it is displayedmmdash;embedded in the membrane and present at high densitymmdash;on filamentous viral-like particles that recapitulate the surface structure and entry functions of ebolavirus. Moreover, after confirming that two of the mAbs bind to the same region in the base of the glycoprotein, we show that they competitively block the entry function of the glycoprotein and that they can do so after the glycoprotein is proteolytically-primed and bound to its intracellular receptor, Niemann-Pick C1. These findings should inform future developments of ebolavirus therapeutics.

  • Co-infection with human CMV genetic variants in transplant recipients and its impact on anti-viral T cell immunity [PublishAheadOfPrint]

  • Reconstitution of T cell immunity is absolutely critical for the effective control of virus-associated infectious complications in hematopoietic stem cell transplant (HSCT) recipients. Co-infection with genetic variants of human cytomegalovirus (CMV) in transplant recipients has been linked to clinical disease manifestation, however how these genetic variants impact on T cell immune reconstitution remains poorly understood. Here we have evaluated dynamic changes in the emergence of genetic variants of CMV in HSCT recipients and correlated these changes with reconstitution of anti-viral T cell responses. Analysis of single nucleotide polymorphisms within sequences encoding HLA class I-restricted CMV epitopes from the immediate early 1 gene of CMV revealed that co-infection with genetically distinct variants of CMV was detected in 52% of patients. However in spite of exposure to multiple viral variants, the T cell responses in these patients were preferentially directed to a limited repertoire of HLA class I-restricted CMV epitopes, either conserved, variant or cross-reactive. More importantly, we also demonstrate that long-term control of CMV infection after HSCT is primarily mediated through the efficient induction of a stable anti-viral T cell immunity irrespective of the nature of the antigenic target. These observations provide important insights for the future design of anti-viral T cell-based immunotherapeutic strategies for transplant recipients emphasising the critical impact of robust immune reconstitution for efficient control of viral infection.

    IMPORTANCE Infection and disease caused by human Cytomegalovirus (CMV) remains a significant burden in patients undergoing haematopoietic stem cell transplantation (HSCT).The establishment of efficient immunological control, primarily mediated by cytotoxic T cells plays a critical role in preventing CMV-associated disease in transplant recipients. Recent evidence has also begun to investigate the impact genetic variation in CMV has upon disease outcome in transplant recipients. In this study we sought to investigate the role T cell immunity plays in recognising and controlling genetic variants of CMV. We demonstrate that while a significant proportion of HSCT recipients may be exposed to multiple genetic variants of CMV, this does not necessarily lead to immune control mediated via recognition of this genetic variation. Rather immune control is associated with the efficient establishment of a stable immune response predominantly directed against immunodominant conserved T cell epitopes.

  • RNA-binding of Ebola virus VP30 is essential for activating viral transcription [PublishAheadOfPrint]

  • The template for Ebola virus (EBOV) transcription and replication is the helical viral nucleocapsid composed of the viral (-) RNA genome, which is complexed by the nucleoprotein, NP, VP35, the polymerase L, VP24, and VP30. While viral replication is exerted by the polymerase L and its cofactor VP35, EBOV mRNA synthesis is regulated by the viral nucleocapsid protein VP30, an essential EBOV-specific transcription factor. VP30 is a homohexameric phosphoprotein containing a nonconventional zinc finger. The transcriptional support activity of VP30 is strongly influenced by its phosphorylation state. We studied here how RNA binding contributed to VP30's function in transcriptional activation. Using a novel mobility shift assay and the 3rrsquo; -terminal 154 nucleotides of the EBOV genome as standard RNA substrate, we detected that RNA binding of VP30 was severely impaired by VP30 mutations that (i) destroy the protein's capability to form homohexamers, (ii) disrupt the integrity of its zinc-finger domain, (iii) mimic its fully phosphorylated state, or (iv) alter the putative RNA binding region. RNA binding of the mutant VP30 proteins correlated strongly with their transcriptional support activity. Furthermore, we showed that the interaction between VP30 and the polymerase cofactor VP35 is RNA-dependent, while formation of VP30 homohexamers and VP35 homotetramers are not. Our data indicate that RNA binding of VP30 is essential for its transcriptional support activity and stabilizes complexes of VP35/L polymerase with the (-) RNA template to favor productive transcriptional initiation in the presence of termination-active RNA secondary structures.

    IMPORTANCE Ebola virus causes severe fevers with unusually high case fatality rates. The recent outbreak of Ebola virus in West Africa claimed more than 11,000 lives and threatened to destabilize a whole region because of its dramatic effects on the public health systems. It is currently not completely understood how Ebola virus manages to balance viral transcription and replication in the infected cells. This study shows that transcriptional support activity of the Ebola virus transcription factor VP30 is highly correlated with its ability to bind viral RNA. The interaction between VP30 and VP35, the Ebola virus polymerase cofactor, is dependent on the presence of RNA as well. Our data contribute to the understanding of the dynamic interplay between nucleocapsid proteins and the viral RNA template in order to promote viral RNA synthesis.

  • P3N-PIPO, a Frameshift Product from P3, Pleiotropically Determines the Virulence of Clover Yellow Vein Virus in both Resistant and Susceptible Peas [PublishAheadOfPrint]

  • Peas carrying the cyv1 recessive resistance gene are resistant to clover yellow vein virus (ClYVV) isolates No. 30 and 90-1 (Cl-No.30 and Cl-90-1), but can be infected by a derivative of Cl-90-1 (Cl-90-1 Br2). The main determinant for the breaking of cyv1 resistance by Cl-90-1 Br2 is P3N-PIPO produced from the P3 gene via transcriptional slippage, and the higher level of P3N-PIPO produced by Cl-90-1 Br2 than by Cl-No.30 contributes to the breaking. Here we show that P3N-PIPO is also a major virulence determinant in susceptible peas that possess another resistance gene, Cyn1, which does not inhibit systemic infection with ClYVV but causes hypersensitive reactionnndash;like lethal systemic cell death. We previously assumed that the susceptible pea cultivar PI 226564 has a weak allele of Cyn1. Cl-No.30 did not induce cell death but Cl-90-1 Br2 killed the plants. Our results suggest that P3N-PIPO is recognized by Cyn1 and induces cell death. Unexpectedly, heterologously strongly expressed P3N-PIPO of Cl-No.30 appears to be recognized by Cyn1 in PI 226564. P3N-PIPO accumulation from the P3 gene of Cl-No.30 was significantly lower than that from Cl-90-1 Br2 in a Nicotiana benthamiana transient assay. Therefore, Cyn1-mediated cell death also appears to be determined by the level of P3N-PIPO. The more efficiently a ClYVV isolate broke cyv1 resistance, the more it induced cell death systemically (resulting in a loss of environment for virus accumulation) in susceptible peas carrying Cyn1, suggesting that antagonistic pleiotropy of P3N-PIPO controls the resistance breaking of ClYVV.

    IMPORTANCE Control of plant viral disease has relied on the use of resistant cultivars; however, emerging mutant viruses have broken many types of resistance. Recently, we revealed that Cl-90-1 Br2 breaks the recessive resistance conferred by cyv1, mainly by accumulating a higher level of P3N-PIPO than the non-breaking isolate Cl-No.30. Here, we show that a susceptible pea line recognized the increased P3N-PIPO amount produced by Cl-90-1 Br2 and activated the salicylic-acid-mediated defense pathway, inducing lethal systemic cell death. We found a gradation of virulence among ClYVV isolates in cyv1 pea and two susceptible peas. This study suggests a trade-off between breaking of recessive resistance (cyv1) and host viability; the latter is presumably regulated by the dominant Cyn1 gene, which may impose evolutionary constraints upon P3N-PIPO for overcoming resistance. We propose a working model of the host strategy to sustain the durability of resistance and control fast-evolving viruses.

  • 1H NMR metabolomics of plasma unveils liver dysfunction in dengue patients [PublishAheadOfPrint]

  • Dengue, due to its global burden, is the most important arthropod-born flavivirus disease and early detection lowers fatality rates to below 1%. Since the metabolic resources crucial for viral replication are provided by host cells, detecting changes in the metabolic profile associated with disease pathogenesis could help identifying markers of prognostic and diagnostic importance. We applied 1H NMR exploratory metabolomics to study longitudinal changes in plasma metabolites in a cohort in Recife, Brazil. To gain statistical power, we used innovative paired multivariate analyzes to discriminate individuals with dengue fever (DF; mild) and dengue hemorrhagic fever (DHF; severe) presenting primary and secondary infection and subjects with non-specific infection (ND). Our results showed that a decrease in plasma LDL and VLDL discriminated dengue-infected from ND subjects and also those subjects with severe infection presented an even decrease in lipoproteins concentration when compared to subjects with mild infection. These results add to the ongoing discussion that manipulation of lipid metabolism is crucial for DENV replication and infection. In addition, a decrease in plasma glutamine was characteristic of DENV infection and disease severity and an increase in plasma acetate discriminated subjects with DF and DHF from ND-subjects. Several other metabolites showed to be altered in DENV infection and the implications of these alterations are discussed. We hypothesize that these changes in plasma metabolome are suggestive of liver dysfunction and could provide insights into the underlying molecular mechanisms of dengue pathogenesis and could help discriminating individuals at risk to develop severe infection and to predict disease outcome.

    IMPORTANCE Dengue virus infection, due to its global burden, is the most important mosquito-born viral disease. There is no specific treatment for dengue disease and early detection lowers fatality rates to below 1%. In this study we observed the effects of dengue virus infection on the profile of small molecules in the blood of patients with mild and severe infection. Variations in the profile of these small molecules reflect the replication of dengue virus in different tissues and the extent of tissue damage during infection. The results of this study showed that the molecules that changed the most were VLDL and LDL lipoproteins and amino acids. We proposed that these changes reflect liver dysfunction and also that they can be used to discriminate subjects with mild and severe dengue infection.

  • Homology-based identification of a mutation in the coronavirus RNA-dependent RNA polymerase that confers resistance to multiple mutagens [PublishAheadOfPrint]

  • Positive-sense (+) RNA viruses encode RNA-dependent RNA polymerases (RdRps) essential for genomic replication. With the exception of the large nidoviruses, such as coronaviruses (CoVs), RNA viruses lack proofreading and thus are dependent on RdRps to control nucleotide selectivity and fidelity. CoVs encode a proofreading exonuclease in nonstructural protein 14 (nsp14-ExoN), which confers a greater-than 10-fold increase in fidelity compared to other RNA viruses. It is unknown to what extent the CoV polymerase (nsp12-RdRp) participates in replication fidelity. We sought to determine whether homology modeling could identify putative determinants of nucleotide selectivity and fidelity in CoV RdRps. We modeled the CoV murine hepatitis virus (MHV) nsp12-RdRp structure and superimposed it with solved picornaviral RdRp structures. Fidelity-altering mutations previously identified in coxsackie virus B3 (CVB3) were mapped onto the nsp12-RdRp model structure and then engineered into the MHV genome with [nsp14-ExoN(+)] or without [nsp14-ExoN(-)] ExoN activity. Using this method we identified two mutations conferring resistance to the mutagen 5-fluorouracil (5-FU): nsp12-M611F and nsp12-V553I. For nsp12-V553I we also demonstrate resistance to the mutagen 5-azacytidine (5-AZC) and decreased accumulation of mutations. Resistance to 5-FU, and decreased number of genomic mutations, was effectively masked by nsp14-ExoN proofreading activity. These results indicate that nsp12-RdRp likely functions in fidelity regulation and that, despite low sequence conservation, some determinants of RdRp nucleotide selectivity are conserved across RNA viruses. These results also indicate that, with regards to nucleotide selectivity, nsp14-ExoN is epistatic to nsp12-RdRp, consistent with its proposed role in a multi-protein replicase/proofreading complex.

    IMPORTANCE RNA viruses have evolutionarily fine-tuned replication fidelity to balance requirements for genetic stability and diversity. Responsibility for replication fidelity in RNA viruses has been attributed to the RNA-dependent RNA polymerases, with mutations in RdRps for multiple RNA viruses shown to alter fidelity and attenuate virus replication and virulence. Coronaviruses (CoVs) are the only known RNA viruses to encode a proofreading exonuclease (nsp14-ExoN), as well as other replicase proteins involved in regulation of fidelity. This report shows that the CoV RdRp (nsp12) likely functions in replication fidelity, that residue determinants of CoV RdRp nucleotide selectivity map to similar structural regions of other unrelated RNA viral polymerases, and that for CoVs, the proofreading activity of the nsp14-ExoN is epistatic to the function of the RdRp in fidelity.

  • A recombinant respiratory syncytial virus vaccine candidate attenuated by a low-fusion F protein is immunogenic and protective against challenge in cotton rats [PublishAheadOfPrint]

  • Although respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants, a safe and effective vaccine is not yet available. Live-attenuated vaccines (LAVs) are the most advanced vaccine candidates in RSV-naïve infants. However, designing an LAV with appropriate attenuation, yet sufficient immunogenicity has proven challenging. Here, we implemented reverse genetics to address these obstacles with a multifaceted LAV design that combined the codon deoptimization of genes for non-structural proteins NS1 and NS2 (dNS); deletion of the small hydrophobic protein (SH) gene; and replacement of the wild-type fusion (F) protein gene with a low-fusion RSV subgroup B F consensus sequence of the Buenos Aires clade (BAF). This vaccine candidate RSV-A2-dNS-SH-BAF named "DB1" was attenuated in two models of primary human airway epithelial cells and in the upper and lower airways of cotton rats. DB1 was also highly immunogenic in cotton rats and elicited broadly neutralizing antibodies against a diverse panel of recombinant RSV strains. When vaccinated cotton rats were challenged with wild-type RSV A, DB1 reduced viral titers in the upper and lower airways by 3.8 log10 total PFU and 2.7 log10 PFU/g tissue respectively compared to unvaccinated animals (P llt; 0.0001). DB1 was thus attenuated, highly immunogenic, and protective against RSV challenge in cotton rats. DB1 is the first RSV LAV to incorporate a low-fusion F protein as a strategy to attenuate viral replication and preserve immunogenicity.

    IMPORTANCE RSV is a leading cause of infant hospitalizations and deaths. The development of an effective vaccine for this high-risk population is therefore a public health priority. Although live-attenuated vaccines have been safely administered to RSV-naïve infants, strategies to balance vaccine attenuation with immunogenicity have been elusive. Here, we introduced a novel strategy to attenuate a recombinant RSV vaccine by incorporating a low-fusion, subgroup B F protein in the genetic background of codon-deoptimized non-structural protein genes and a deleted small hydrophobic protein gene. The resultant vaccine candidate "DB1" was attenuated, highly immunogenic, and protective against RSV challenge in cotton rats.

  • Identification and Characterization of Influenza Virus Entry Inhibitors Through Dual-Myxovirus High-Throughput Screening [PublishAheadOfPrint]

  • Influenza A virus (IAV) infections cause major morbidity and mortality, generating an urgent need for novel antiviral therapeutics. We recently established a dual-myxovirus high-throughput screening protocol, that combines a fully replication-competent IAV-WSN and a respiratory syncytial virus reporter strain for the simultaneous identification of IAV-specific, paramyxovirus-specific, and broad-spectrum inhibitors. Here, this protocol was applied to a screening campaign, assessing a diverse chemical library with over 142,000 entries. Focusing on IAV-specific hits, we obtained a hit rate of 0.03% after cytotoxicity testing and counterscreening. Three chemically distinct hit classes with nanomolar potency and favorable cytotoxicity profiles were selected. Time-of-addition, minigenome, and viral entry studies demonstrated that these classes block hemagglutinin (HA)-mediated membrane fusion. Antiviral activity extends to an insolate of the 2009 pandemic and, in one case, another group 1 subtype. Target identification through biolayer interferometry confirmed binding of all hits to HA. Resistance profiling revealed two distinct escape mechanisms: primary-type resistance associated with reduced compound binding and secondary resistance with unaltered binding. Secondary resistance was mediated, unusually, through two different pairs of cooperative mutations, each combining a mutation eliminating the membrane-proximal stalk N-glycan with a membrane-distal change in HA1 or HA2. Chemical synthesis of an analog library combined with in silico docking extracted a docking pose of the hit classes. Chemical interrogation spotlights IAV HA as a major druggable target for small-molecule inhibition. Our study identifies novel chemical scaffolds with high developmental potential, outlines diverse routes of IAV escape from entry inhibition, and establishes a path towards structure-aided lead development.

    Importance This study is one of the first to apply a fully replication-competent third generation IAV reporter strain to a large-scale HTS drug discovery campaign, allowing multi-cycle infection and screening in physiologically relevant human respiratory cells. A large number of potential druggable targets was thus chemically interrogated, but mechanistic characterization, positive target identification, and resistance profiling demonstrated that three chemically promising and structurally distinct hit classes selected for further analysis all block HA-mediated membrane fusion. Viral escape from inhibition could be achieved through primary and secondary resistance mechanisms. In silico docking predicted compound binding into a microdomain located at the membrane-distal site of the prefusion HA stalk that was also previously suggested as target site for chemically unrelated HA inhibitors. This study identifies an unexpected chemodominance of the HA stalk microdomain for small-molecule inhibitors in IAV inhibitor screening campaigns and highlights a novel mechanism of cooperative resistance to IAV entry blockers.

  • HSV capsid localization to ESCRT-VPS4 complexes in the presence and absence of the large tegument protein UL36p. [PublishAheadOfPrint]

  • UL36p (VP1/2) is the largest protein encoded by HSV-1 and resides in the innermost layer of tegument, the complex protein layer between the capsid and envelope. UL36p performs multiple functions in the HSV life cycle, including a critical but unknown role in capsid cytoplasmic envelopment. We tested whether UL36p is essential for envelopment because it is required to engage capsids with the cellular ESCRT/Vps4 apparatus. A GFP-fused form of the dominant negative ATPase Vps4-EQ was used to irreversibly tag ESCRT-envelopment sites during infection by UL36p-expressing and UL36-null HSV strains. Using fluorescence microscopy and scanning electron microscopy we quantitated capsid/Vps4-EQ colocalization and examined the ultrastructure of the corresponding viral assembly intermediates. We found that loss of UL36p resulted in a two-thirds reduction in the efficiency of capsid/Vps4-EQ association, but the remaining UL36p-null capsids were still able to engage with the ESCRT envelopment apparatus. It appears that, although UL36p helps to couple HSV capsids to the ESCRT pathway, this is likely not the sole reason for its absolute requirement in envelopment.

    Importance Envelopment of the HSV capsid is essential for the assembly of an infectious virion, and requires the complex interplay of a large number of viral and cellular proteins. Critical to envelope assembly is the virally encoded protein UL36p, whose function is unknown. Here we test the hypothesis that UL36p is essential for the recruitment of cellular ESCRT complexes, also known to be required for envelopment.

  • Two Polypyrimidine Tracts in Intron 4 of the Major Immediate-Early (MIE) Gene Are Critical for the Gene Expression Switching from IE1 to IE2 and for Replication of Human Cytomegalovirus [PublishAheadOfPrint]

  • The human cytomegalovirus (HCMV) major immediate-early (MIE) gene is essential for viral replication. The most abundant products encoded by the MIE gene include IE1 and IE2. Genes of IE1 and IE2 share the MIE promoter (MIEP), first 3 exons and first 2 introns. IE1 is expressed earlier than IE2 after CMV infection or MIE gene transfection. In this study, we identified 2 polypyrimidine (Py) tracts in intron 4 (between exons 4 and 5) that are responsible for transcriptional switching from IE1 to IE2. The first Py is important and the second one essential for the splicing and expression of IE2. In searching for the mechanisms of MIE gene switching from IE1 to IE2, we found that the second Py was required for the IE2's 4th intron to bind to a splicing factor such as U2AF65, as determined by an RNA electrophoretic mobility shift assay and a ChIP assay, while the first Py enhanced the binding of U2AF65 with the intron. An HCMV BACmid with the second Py mutated failed to produce any virus, while the HCMV with the first Py mutated replicated with a defective phenotype. Furthermore, we designed a small RNA (scRNAPy) that is complementary to the intron RNA covering the two Pys. The scRNAPy interfered with the interaction of U2AF65 with the intron and repressed the IE2 expression. Therefore, our studies implied that IE2 gene splicing might be an anti-CMV target.

    Importance CMV is a ubiquitous herpesvirus and a significant cause of disease and death in the immunocompromised and elderly. Insights into its gene regulation will provide clues in designing anti-CMV strategies. The MIE gene is one of the earliest genes of CMV and is essential for CMV replication. It is known that the MIE gene needs to be spliced to produce more than 2 proteins; however, how MIE gene splicing is regulated remains elusive. In the present studies, we identified 2 Pys in intron 4 and found that the first Py is important and the second is required for the splicing and expression of IE2. We further investigated the mechanisms of gene switching from IE1 to IE2 and found that the 2 Pys are responsible for U2AF65rrsquo; binding with intron 4. Therefore, the Pys in intron 4 are the cis-elements that determine the fate of IE2 splicing. Furthermore, we found that a small RNA that is complementary to the intron 4 repressed IE2 expression. Hence, we provide the first piece of evidence for a unique mechanism of MIE gene regulation at the splicing level.

  • Phosphorylation of human metapneumovirus M2-1 protein upregulates viral replication and pathogenesis [PublishAheadOfPrint]

  • Human metapneumovirus (hMPV) is a major causative agent of upper and lower respiratory tract infections in infants, the elderly, and immunocompromised individuals worldwide. Like all pneumoviruses, hMPV encodes the zinc binding protein M2-1 that plays important regulatory roles in RNA synthesis. The M2-1 protein is phosphorylated but the specific role(s) of the phosphorylation in viral replication and pathogenesis remains unknown. In this study, we found that hMPV M2-1 is phosphorylated at amino acid residues S57 and S60. Subsequent mutagenesis found that phosphorylation is not essential for zinc binding activity and oligomerization whereas inhibition of zinc binding activity abolished the phosphorylation and oligomerization of the M2-1 protein. Using reverse genetics, recombinant hMPVs lacking either one or both phosphorylation sites in the M2-1 protein were recovered. These recombinant viruses had a significant decrease in both genomic RNA replication and mRNA transcription. In addition, these recombinant viruses were highly attenuated in cell culture and cotton rats. Importantly, rhMPVs lacking phosphorylation sites in the M2-1 protein triggered high levels of neutralizing antibody and provided complete protection against challenge with wildtype hMPV. Collectively, these data demonstrated that phosphorylation of the M2-1 protein upregulates hMPV RNA synthesis, replication and pathogenesis in vivo.

    Importance The pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the leading causes of acute respiratory tract infection in infants and children. Currently, there is no antiviral or vaccine to combat these diseases. All known pneumoviruses encode a zinc binding protein, M2-1, which is a transcriptional anti-termination factor. In this work, we found that phosphorylation of M2-1 is essential for virus replication and pathogenesis in vivo. Recombinant hMPVs lacking phosphorylation sites in M2-1 exhibited limited replication in the upper and lower respiratory tract and triggered strong protective immunity in cotton rats. This work highlights the important role of M2-1 phosphorylation in viral replication and that inhibition of M2-1 phosphorylation may serve as a novel approach to develop live attenuated vaccines, as well as antiviral drugs for pneumoviruses.

  • Time-dependent rate phenomenon in viruses [PublishAheadOfPrint]

  • Among the most fundamental questions in viral evolutionary biology are how fast viruses evolve and how their rates vary among viruses and fluctuate through time. Traditionally, viruses are loosely classed into two groups: slow-evolving DNA viruses and fast-evolving RNA viruses. As viral evolutionary rate estimates become more available, it appears that the rates are negatively correlated with the measurement timescales, and that the boundary between the rates of DNA and RNA viruses might not be as clear as previously thought. In this study, we collected 396 viral evolutionary rate estimates across almost all viral genome types and replication strategies, and examined their rate dynamics. We showed that the time-dependent rate phenomenon exists across multiple levels of viral taxonomy, from the Baltimore classification viral groups to genera. We also showed that, by taking the rate-decay dynamics into account, a clear division between the rates of DNA and RNA as well as reverse-transcribing viruses could be recovered. Surprisingly, despite large differences in their biology, our analyses suggested that the rate-decay speed is independent of viral types, and thus it might be useful for better estimation of the evolutionary timescale of any virus. To illustrate this, we used our model to re-estimate evolutionary timescales of extant lentiviruses, which were previously suggested to be very young by standard phylogenetic analyses. Our analyses suggested that they are millions of years old, consistent with paleovirological evidence, and therefore for the first time, reconciled molecular analyses of ancient and extant viruses.

    IMPORTANCE This work provides direct evidence that viral evolutionary rate estimates decay with their measurement timescales, and that the rate-decay speeds do not differ significantly among viruses despite the vast differences in their molecular features. With the rate-decay dynamics adjusted for, the division between the rates of dsDNA, ssRNA, and ssDNA/reverse-transcribing viruses could be seen more clearly than before. Our results provide a guideline for further improvement of molecular clock. As a demonstration of this, we used our model to re-estimate the timescales of modern lentiviruses, which were previously thought to be very young, to be millions of years old. This result matches the estimate from paleovirological analyses, thus bridging the gap between ancient and extant viral evolutionary studies.

  • Suppression of IRG-1 improves immune lung injury after RSV infection by reducing ROS production [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) infection is a common cause of lower respiratory tract illness in infants and children. RSV is a negative sense, single strand RNA (ssRNA) virus that mainly infects airway epithelial cells. Accumulating evidence indicates that reactive oxygen species (ROS) production is a major factor for pulmonary inflammation and tissue damage of RSV disease. We investigated immune responsive gene-1 (IRG1) expression during RSV infection since IRG1 has been shown to mediate innate immune response to intracellular bacterial pathogens by modulating ROS and itaconic acid production. We found that RSV infection induced IRG1expression in human A549 cells and in the lung tissues of RSV-infected mice. RSV infection or IRG1 overexpression promoted ROS production. Accordingly, knockdown of IRG1 induction blocked RSV-induced ROS production and proinflammatory cytokine gene expression. Finally, we showed that suppression of IRG1 induction reduced immune cell infiltration and prevented lung injury in RSV-infected mice. These results therefore link IRG1 induction to ROS production and immune lung injury after RSV infection.

    Importance Respiratory syncytial virus (RSV) infection is among the most common cause of childhood diseases. Recent studies identify ROS production as a contributing factor to RSV disease. We investigated the cause of ROS production and identified IRG1 as a critical factor linking ROS production to immune lung injury after RSV infection. We found that IRG1 was induced in A549 alveolar epithelial cells and in mouse lungs after RSV infection. Importantly, suppression of IRG1 induction reduced inflammatory cell infiltration and lung injury in mice. This study links IRG1 induction to oxidative damage and RSV disease. It also uncovers a potential therapeutic target in reducing RSV-caused lung injury.

  • Mutagenesis of S-adenosyl-L-methionine-binding Residues in Coronavirus nsp14 N7-Methyltransferase Demonstrates Differing Requirements for Genome Translation and Resistance to Innate Immunity [PublishAheadOfPrint]

  • Eukaryotic mRNAs possess a methylated 5rrsquo; -guanosine cap that is required for RNA stability, efficient translation, and protection from cell-intrinsic defenses. Many viruses use 5rrsquo; caps or other mechanisms to mimic a cap structure to limit detection of viral RNAs by intracellular innate sensors and to direct efficient translation of viral proteins. The coronavirus (CoV) nonstructural protein 14 (nsp14) is a multifunctional protein with N7-methyltransferase (N7-MTase) activity. The highly conserved S-adenosyl-L-methionine (SAM)-binding residues of the DxG motif are required for nsp14 N7-MTase activity in vitro. However, the requirement for CoV N7-MTase activity and the importance of the SAM-binding residues during viral replication have not been determined. Here, we engineered mutations in murine hepatitis virus (MHV) nsp14 N7-MTase at residues D330 and G332 and determined the effects of these mutations on viral replication, sensitivity to mutagen, inhibition by type I interferon, and translation efficiency. Virus encoding a G332A substitution in nsp14 displayed delayed replication kinetics and decreased peak titers relative to WT MHV. In addition, replication of nsp14 G332A virus was diminished following treatment of cells with interferon-bbeta;, and nsp14 G332A genomes were translated less efficiently both in vitro and during viral infection. In contrast, alanine substitution of MHV nsp14 D330 did not affect viral replication, sensitivity to mutagen, or inhibition by interferon-bbeta; compared to WT MHV. Our results demonstrate that the conserved MHV N7-MTase SAM-binding site residues are not required for MHV viability and suggest that the determinants of CoV N7-MTase activity differ in vitro and during virus infection.

    IMPORTANCE Human coronaviruses, most notably SARS-CoV and MERS-CoV, cause severe and lethal human disease. Since specific antiviral therapies are not available for the treatment of human coronavirus infections, it is essential to understand the functions of conserved CoV proteins in viral replication. Here, we show that alanine substitution of G332 in the N7-MTase domain of nsp14 impairs viral replication, enhances sensitivity to the innate immune response, and reduces viral RNA translation efficiency. Our data support the idea that coronavirus RNA capping could be targeted for development of antiviral therapeutics.

  • Human T-Lymphotropic Virus (HTLV)-1-Induced Overexpression of Activated Leukocyte Cell Adhesion Molecule (ALCAM) Facilitates Trafficking of Infected Lymphocytes through the Blood-Brain Barrier [PublishAheadOfPrint]

  • Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). This disease develops upon infiltration of HTLV-1-infected lymphocytes into the central nervous system, mostly the thoracic spinal cord. The central nervous system is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. In this report, we study the role of Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166), a member of the immunoglobulin superfamily, in the crossing of the BBB by HTLV-1-infected lymphocytes. We demonstrated that ALCAM is overexpressed on the surface of HTLV-1-infected lymphocytes, both in chronically-infected cell lines and primary infected CD4+ T lymphocytes. ALCAM overexpression results from the activation of the canonical NF-B pathway by the viral transactivator Tax. In contrast, staining of spinal cord sections of HAM/TSP patients shows that ALCAM expression is not altered on the BBB endothelium in the context of HTLV-1 infection. ALCAM blockade or downregulation of ALCAM levels significantly reduced the migration of HTLV-1-infected lymphocytes across a monolayer of human BBB endothelial cells. This study suggests a potential role for ALCAM in HAM/TSP pathogenesis.

    Importance Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). This disease is the consequence of the infiltration of HTLV-1-infected lymphocytes into the central nervous system (CNS), mostly the thoracic spinal cord. The central nervous system is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. The mechanism of migration of lymphocytes into the CNS is unclear. Here, we show that the viral transactivator Tax increases Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166) expression. This molecule facilitates the migration of lymphocytes across the BBB endothelium. Targeting this molecule could be of interest in preventing or reducing the development of HAM/TSP.

  • Mutants at the 2-fold interface of AAV2 structural proteins suggest a role in viral transcription for AAV capsids [PublishAheadOfPrint]

  • We previously reported that an amino acid substitution, Y704A, near the 2 fold interface of AAV was defective for transcription of the packaged genome (Salganik et al, 2014, J Virol, 88:1071). In this report we have characterized the defect in 6 additional capsid mutants located in a region approximately 30AAring; in diameter on the surface of the AAV2 capsid near the 2-fold interface. These mutants, which are highly conserved among primate serotypes, displayed a severe defect (3-6 logs) in infectivity. All of the mutants accumulated significant levels of uncoated DNA in the nucleus, but none of the mutants were able to accumulate significant amounts of genomic mRNA post infection. In addition, wild type capsids that were bound to the conformational antibody A20, which is known to bind the capsid surface in the region of the mutants, were also defective for transcription. In all cases, the mutant virus particles, as well as the antibody bound wild type capsids, were able to enter the cell, travel to the nucleus, uncoat and synthesize a second strand, but were unable to transcribe their genomes. Taken together, the phenotype of these mutants provides compelling evidence that the AAV capsid plays a role in transcription of its genome, and the mutants map this functional region on the surface of the capsid near the 2 fold interface. This appears to be the first example of a viral structural protein that is also involved in the transcription of the viral genome it delivers to the nucleus.

    IMPORTANCE Many viruses package enzymes within their capsids that assist in expressing their genomes post infection, e.g., retroviruses. A number of non-enveloped viruses, including AAV, carry protease that are needed for capsid maturation or for capsid modification during infection. We describe here what appears to be the first example of a non-enveloped viral capsid that appears to have a role in promoting transcription. A total of six mutants at the AAV capsid 2 fold interface were shown to have a severe defect in expressing their genomes, and the defect was at the level of mRNA accumulation. This suggests that AAV capsids have a novel role in promoting transcription of the genomes they have packaged. Since wt virions could not complement the mutant viruses, and the mutant viruses did not effectively inhibit wt gene expression, our results suggest that the capsid exerts its effect on transcription in cis.

  • Vaccine-elicited mucosal and systemic antibody responses are associated with reduced simian immunodeficiency viremia in infant rhesus macaques [PublishAheadOfPrint]

  • Despite significant progress in reducing peri-partum mother-to-child transmission (MTCT) of HIV with antiretroviral therapy (ART), continued access to ART throughout the breastfeeding period is still a limiting factor, and breast milk exposure to HIV accounts for up to 44% of MTCT. As abstinence from breastfeeding is not recommended, alternative means are needed to prevent MTCT of HIV. We have previously shown that oral vaccination at birth with live attenuated Mycobacterium tuberculosis (Mtb) strains expressing SIV genes safely induces persistent SIV-specific cellular and humoral immune responses both systemically and at the oral and intestinal mucosa. Here we tested the ability of oral Mtb vaccine strains expressing SIV Env and Gag proteins, followed by systemic heterologous (MVA-SIV Env/Gag/Pol) boosting, to protect neonatal macaques against oral SIV challenge. While vaccination did not protect infant macaques against oral SIV acquisition, a subset of immunized animals had significantly lower peak viremia which inversely correlated with pre-challenge SIV Env-specific salivary and intestinal IgA responses and higher avidity SIV Env-specific IgG in plasma. These controller animals also maintained CD4+ T cell populations better and showed reduced tissue pathology compared to non-controller animals. We show that infants vaccinated at birth can develop vaccine-induced SIV specific IgA and IgG antibodies and cellular immune responses within weeks of life. Our data further suggest that affinity maturation of vaccine-induced plasma antibodies and induction of mucosal IgA responses at potential SIV entry sites are associated with better control of viral replication, thereby likely reducing SIV morbidity.

    IMPORTANCE Despite significant progress in reducing peri-partum mother-to-child transmission (MTCT) of HIV with antiretroviral therapy (ART), continued access to ART throughout the breastfeeding period is still a limiting factor. Breast milk exposure to HIV accounts for up to 44% of MTCT. Alternative measures, in addition to ART, are needed to achieve the goal of an AIDS-free generation. Pediatric HIV vaccines constitute a core component of such efforts.

    The results of our pediatric vaccine study highlight the potential importance of vaccine-elicited mucosal Env-specific IgA responses in combination with high-avidity systemic Env-specific IgG in protection against oral SIV transmission and control of viral replication in infant macaques. The induction of potent mucosal IgA antibodies by our vaccine is remarkable considering the age-dependent development of mucosal IgA responses post-birth. A deeper understanding of postnatal immune development may inform the design of improved vaccine strategies to enhance systemic and mucosal SIV/HIV antibody responses.

  • Ebola virus does not induce stress granule formation during infection and sequesters stress granule proteins within viral inclusions [PublishAheadOfPrint]

  • A hallmark of Ebola virus (EBOV) infection is the formation of viral inclusions in the cytoplasm of infected cells. These viral inclusions contain the EBOV nucleocapsids and are sites of viral replication and nucleocapsid maturation. Although there is growing evidence that viral inclusions create a protected environment that fosters EBOV replication, little is known about their role in the host response to infection. The cellular stress response is an effective antiviral strategy that leads to stress granule (SG) formation and translational arrest mediated by the phosphorylation of the translation initiation factor eIF2aalpha;. Here, we show that selected SG proteins are sequestered within EBOV inclusions where they form distinct granules that colocalize with viral RNA. These inclusion-bound (IB) granules are functionally and structurally different from canonical SGs. Formation of IB granules does not indicate translational arrest in the infected cells. We further show that EBOV does not induce formation of canonical SGs or eIF2aalpha; phosphorylation at any time post infection but is unable to fully inhibit SG formation induced by different exogenous stressors, including sodium arsenite, heat, and hippuristanol. Despite the sequestration of SG marker proteins into IB granules, canonical SGs are unable to form within inclusions, which we propose might be mediated by a novel function of VP35, which disrupts SG formation. This function is independent of VP35's RNA binding activity. Further studies aim to reveal the mechanism for SG protein sequestration and precise function within inclusions.

    Importance Although progress has been made developing antiviral therapeutics and vaccines against the highly pathogenic Ebola virus (EBOV), the cellular mechanisms involved in EBOV infection are still largely unknown. To better understand these intracellular events, we investigated the cellular stress response, an antiviral pathway manipulated by many viruses. We show that EBOV does not induce formation of stress granules (SGs) in infected cells and is therefore unrestricted by their concomitant translational arrest. We identified SG proteins sequestered within viral inclusions, which did not impair protein translation. We further show that EBOV is unable to block SG formation triggered by exogenous stress early in infection. These findings provide insight into potential targets of therapeutic intervention. Additionally, we identified a novel function of the interferon antagonist VP35, which is able to disrupt SG formation.

  • Using the E4orf6-based E3 ubiquitin ligase as a tool to analyze the evolution of adenoviruses [PublishAheadOfPrint]

  • E4orf6 proteins from all human adenoviruses form Cullin-based ubiquitin ligase complexes that, in association with E1B55K, target cellular proteins for degradation. While most are assembled with Cul5, a few utilize Cul2. BC-box motifs enable all these E4orf6 proteins to assemble ligase complexes with Elongins B and C. We also identified a Cul2-box motif used for Cul2 selection in all Cul2-based complexes. With this information we set out to determine if other adenoviruses also possess the ability to form the ligase complex, and if so to predict Cullin usage. Here we report that all adenoviruses known to encode an E4orf6-like protein (mastadenoviruses and atadenoviruses) maintain the potential to form the ligase complex. We could accurately predict Cullin usage in E4orf6 products of mastadenoviruses and all but one atadenovirus. Interestingly in non-human primate adenoviruses, we found a clear segregation of Cullin binding, with Cul5 utilized by viruses infecting great apes and Cul2 by Old/New World monkey viruses, suggesting that a switch from Cul2 to Cul5 binding occurred during the period when great apes diverged from monkeys. We also suggest based on analysis of Cullin selection that the majority of human adenoviruses, which exhibit a broader tropism for the eye and respiratory tract, exhibit Cul5 specificity and resemble viruses infecting great apes, whereas those that infect the gastro-intestinal tract may have originated from monkey viruses that share Cul2 specificity. Finally, aviadenoviruses also appear to contain E4orf6 genes that contain a conserved xCxC motif followed by, in most cases, a BC-box motif.

    Importance Two early adenoviral proteins, E4orf6 and E1B55K form a ubiquitin ligase complex with cellular proteins to ubiquitinate specific substrates leading to their degradation by the proteasome. In a study with representatives of each human adenovirus species we (and others) have previously discovered that some viruses use Cul2 to form the complex while others use Cul5. In the present study we have expanded our analyses to all sequenced adenoviruses and found that E4orf6 genes from all mast- and atadenoviruses contain the motifs necessary to form the ligase complex. We found a clear separation in Cullin specificity between adenoviruses of great apes and Old/New World monkeys, lending support for a monkey origin for human viruses of species Human mastadenovirus A, F and G. We have also identified previously unrecognized E4orf6 genes in the aviadenoviruses that contain motifs permitting formation of the ubiquitin ligase.

  • Nonstructural 3 protein of hepatitis C virus modulates tribbles homolog 3/Akt signaling pathway for persistent viral infection [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) infection often causes chronic hepatitis, liver cirrhosis, and ultimately hepatocellular carcinoma. However, the mechanisms underlying HCV-induced liver pathogenesis are still not fully understood. By RNA-Seq analysis, we recently identified host genes that were significantly differentially expressed in cell culture-grown HCV (HCVcc)-infected cells. Of these, tribbles homolog 3 (TRIB3) was selected for further characterization. TRIB3 is initially identified as a binding partner of protein kinase B (PKB, also known as Akt). TRIB3 blocks phosphorylation of Akt and induces apoptosis under ER stress conditions. HCV has been shown to enhance Akt phosphorylation for its own propagation. In the present study, we demonstrated that both mRNA and protein levels of TRIB3 were increased in the context of HCV replication. We further showed that promoter activity of TRIB3 was increased by HCV-induced ER stress. Silencing of TRIB3 resulted in increase of RNA and protein levels of HCV, whereas overexpression of TRIB3 decreased HCV replication. By employing HCV pseudoparticle entry assay, we further showed that TRIB3 was a negative host factor involved in HCV entry. Both in vitro binding and immunoprecipitation assays demonstrated that HCV NS3 specifically interacted with TRIB3. Consequently, TRIB3 and Akt association was disrupted by HCV NS3 and thus TRIB3-Akt signaling was impaired in HCV-infected cells. Moreover, HCV modulated TRIB3 to promote ERK phosphorylation, AP-1 activity, and cell migration. Collectively, these data indicate that HCV exploits the TRIB3-Akt signaling pathway to promote persistent viral infection and may contribute to HCV-mediated pathogenesis.

    IMPORTANCE TRIB3 is a pseudokinase protein which acts as an adaptor in signaling pathway for important cellular processes. So far, functional involvement of TRIB3 in virus-infected cells has not been demonstrated yet. We showed that both mRNA and protein expression levels of TRIB3 were increased in the context of HCV RNA replication. Gene silencing of TRIB3 increased HCV RNA and protein levels and thus overexpression of TRIB3 decreased HCV replication. TRIB3 is known to promote apoptosis by negatively regulating Akt signaling pathway under ER stress conditions. Most importantly, we demonstrated that TRIB3-Akt signaling pathway was disrupted by NS3 in HCV-infected cells. These data provide evidence that HCV modulates TRIB3-Akt signaling pathway to establish persistent viral infection.

  • Single-molecule sequencing reveals complex genomic variation of hepatitis B virus during 15 years of chronic infection following liver transplantation. [PublishAheadOfPrint]

  • Chronic hepatitis B (CHB) is prevalent worldwide. The infectious agent, hepatitis B virus (HBV) replicates via an RNA intermediate and is error-prone, leading to rapid generation of closely related but not identical viral variants, including those that can escape host immune responses and antiviral treatments. The complexity of CHB can be further enhanced by the presence of HBV variants with large deletions in the genome, generated via splicing (spHBV). Although spHBV variants are incapable of autonomous replication, their replication is rescued by wild-type HBV. SpHBV variants have been shown to enhance wild-type virus replication, and their prevalence increases with liver disease progression. Single-molecule deep sequencing was performed on whole HBV genomes extracted from longitudinal samples of a post-liver transplant CHB subject, collected over a 15-year period that included the liver explant. By employing novel bioinformatics methods, this analysis showed a complex dynamics of the viral population across a period of changing treatment regimens. The spHBV detected in the liver explant remained present post-transplantation, along with emergence of a highly diverse novel spHBV population as well as variants with multiple deletions in the preS genes. The identification of novel mutations outside the HBV reverse transcriptase gene that co-occur with known drug resistant mutations, highlight the relevance of using full genome deep sequencing and support the hypothesis that drug resistance involves interactions across the full-length HBV genome.

    IMPORTANCE Single-molecule sequencing allowed characterising, in unprecedented detail, the evolution of HBV populations and offered unique insights into the dynamics of defective and spHBV variants following liver transplantation and complex treatment regimes. This analysis also showed rapid adaptation of HBV populations to treatment regimens with evolving drug resistance phenotypes and evidence of purifying selection across the whole genome. Finally, the new open source bioinformatics tools are freely available, with the capacity to easily identify potential spliced variants from deep sequencing data.

  • Dengue virus subverts host innate immunity by targeting adaptor protein MAVS [PublishAheadOfPrint]

  • Dengue virus (DENV) is the most common mosquito-borne virus infecting humans and is currently a serious global health challenge. To establish infection in its host cells, DENV must subvert the production and/or antiviral effects of interferon (IFN). The aim of this study was to understand the mechanisms by which DENV suppresses IFN production. We determined that DENV NS4A interacts with mitochondrial antiviral signaling protein (MAVS), which has previously been found to activate NF-B and IFN regulatory factor 3 (IRF3), thus inducing type I IFN in the mitochondria-associated endoplasmic reticulum membranes (MAM). We further demonstrated that NS4A is associated with the N-terminal CARD-like domain (CL) and the C-terminal transmembrane domain (TM) of MAVS. This association prevented the binding of MAVS to RIG-I, resulting in repression of RIG-I-induced IRF3 activation and, consequently, abrogation of IFN production. Collectively, our findings illustrate a new molecular mechanism by which DENV evades the host immune system and suggest new targets for anti-DENV strategies.

    IMPORTANCE Type I interferon (IFN) constitutes the first line of host defense against invading viruses. To successfully establish infection, dengue virus (DENV) must counteract either the production or the function of IFN. The mechanism by which DENV suppresses IFN production is poorly understood and characterized. In this study, we demonstrate that the DENV NS4A protein plays an important role in suppressing interferon production through binding MAVS and disrupting the RIG-I-MAVS interaction in the mitochondria-associated endoplasmic reticulum membranes (MAM). Our study reveals MAVS as a novel host target of NS4A and provides a molecular mechanism for DENV's evasion of the host innate immune response. These findings have important implications for understanding the pathogenesis of DENV and may provide new insights into using NS4A as a therapeutic and/or prevention target.

  • Interaction Between Non-Structural Proteins NS4B And NS5A Is Essential For Proper NS5A Localization And Hepatitis C Virus RNA Replication [PublishAheadOfPrint]

  • The hepatitis C virus NS5A protein is tethered to cellular membranes via an amphipathic amino-terminal helix that is inserted in plane into the outer ER derived membrane leaflet. The charged face of the helix faces the cytoplasm and may contribute to interactions involved in replicase assembly and function. Using an aggressive charge flip mutagenesis strategy we identified a number of essential residues for replication on the charged face of NS5A anchor, and identified a double charge face mutant that is lethal for RNA replication but generates suppressor mutations in the carboxy terminal helix of the NS4B protein. This suppressor restores RNA replication of the NS5A helix double flip mutant, and, interestingly, seems to function by restoring the proper localization of NS5A to the viral replicase. These data add to our understanding of the complex organization and assembly of the viral replicase via NS4B-NS5A interactions.

    IMPORTANCE The information about the functional role of cytosolic face of NS5A anchoring helix remains obscure. In this study we show that, while the hydrophobic face of the NS5A anchor helix mediates membrane association, the polar cytosolic face of the helix plays key role during HCV replication by mediating interaction of NS5A with other HCV nonstructural proteins via NS4B. Such interaction determines the subcellular localization of NS5A by engaging NS5A into HCV replication process during the formation of functional HCV replication complex. Thus, collectively it can be stated, that the current study findings provide further information about the interactions between the HCV nonstructural proteins during HCV RNA replication and provide the platform to gain more insights about the molecular architecture of HCV replication complexes.

  • Tegument protein ORF45 plays an essential role in virion morphogenesis of murine gammaherpesvirus-68 [PublishAheadOfPrint]

  • Tegument proteins play critical roles in herpesvirus morphogenesis. ORF45 is a conserved tegument protein of gammaherpesviruses, however, its role in virion morphogenesis is largely unknown. In this work, we determined the ultrastructural localization of murine gammaherpesvirus-68 (MHV-68) ORF45 and found that it was incorporated into virions around the site of host-derived vesicles. Notably, absence of ORF45 inhibited nucleocapsid egress and blocked cytoplasmic virion maturation, demonstrating that ORF45 is essential for MHV-68 virion morphogenesis.

  • Critical role of the PA-X C-terminal domain of influenza A virus on its subcellular localization and shutoff activity [PublishAheadOfPrint]

  • PA-X is a recently identified influenza virus protein, which is composed of PA N-terminal 191 amino acids and unique C-terminal 41 or 61 residues. We and others showed that PA-X has a strong ability to suppress host protein synthesis via host mRNA decay, which is mediated by endonuclease activity in its N-terminal domain. However, the mechanism of host mRNA degradation, especially where and how PA-X targets mRNAs has not been analyzed. In this study, we determined the localization of PA-X and the role of the C-terminal unique region on shutoff activity. Quantitative subcellular localization analysis revealed that PA-X was located equally in both cytoplasm and nucleus. By characterizing a series of PA-X C-terminal deletion mutants, we found that the first 9 amino acids were sufficient for nuclear localization, but an additional 6 residues were required to induce the maximum shutoff activity observed with intact PA-X. Importantly, forced nuclear localization of the PA-X C-terminal deletion mutant enhanced shutoff activity, highlighting the ability of nuclear PA-X to degrade host mRNAs more efficiently. However, PA-X also inhibited luciferase expression from transfected mRNAs synthesized in vitro, suggesting that PA-X also degrades mRNAs in the cytoplasm. Among the basic amino acids in the PA-X C-terminal region, 3 residues, 195K, 198K, and 199R were identified as key residues for inducing host shutoff and nuclear localization. Overall, our data indicate a critical role for the 15 residues in the PA-X C-terminal domain in degrading mRNAs in both the cytoplasm and nucleus.

    IMPORTANCE Influenza A viruses express PA-X proteins to suppress global host gene expression, including host antiviral genes to allow efficient viral replication in infected cells. However, little is known about how PA-X induces host shutoff. In this study, we showed that PA-X localized equally in both the cytoplasm and nucleus of the cells, but the nuclear localization of PA-X mediated by its C-terminal region has a significant impact on shutoff activity. Three basic residues at the C-terminal region play a critical role in nuclear localization, but additional basic residues were required for maximum shutoff activity. Our findings indicate that PA-X targets and degrades mRNAs in both the nucleus and cytoplasm, and that the first 15 residues of the PA-X unique C-terminal region play a critical role in shutoff activity.

  • Release of infectious hepatitis C virus from Huh7 cells occurs via a trans-Golgi network to endosome pathway independent of VLDL [PublishAheadOfPrint]

  • The release of infectious hepatitis C virus (HCV) particles from infected cells remains poorly characterized. We previously demonstrated that virus release is dependent on the endosome sorting complex required for transport (ESCRT). Here, we show a critical role of TGN-endosome trafficking during the assembly, but principally the secretion of infectious virus. This was demonstrated by both siRNA mediated silencing of TGN-associated adaptor proteins, and a panel of dominant negative (DN) Rab GTPases involved in TGN-endosome trafficking steps. Importantly, interfering with factors critical for HCV release did not have a concomitant effect on secretion of either triglyceride, ApoB or ApoE, indicating that particles are likely released from Huh7 cells via pathways distinct to that of VLDL. Finally we show that HCV NS2 perturbs trans-Golgi network (TGN) architecture, redistributing TGN membranes to closely associate with HCV core residing on lipid droplets. These findings support the notion that HCV hijacks TGN-endosome trafficking to facilitate particle assembly and release. Moreover, whist essential for assembly and infectivity, the trafficking of mature virions is seemingly independent of host lipoproteins.

    Importance. The mechanisms by which infectious hepatitis C virus particles are assembled and released from the cell are poorly understood. We show that the virus subverts host cell trafficking pathways to effect the release of virus particles and disrupts the structure of the Golgi apparatus, a key cellular organelle involved in secretion. In addition we demonstrate that the mechanisms used by the virus to exit the cell are distinct from those used by the cell to release lipoproteins, suggesting that the virus effects an unique modification to cellular trafficking pathways.

  • Human Cytomegalovirus pUL93 Links Nucleocapsid Maturation and Nuclear Egress [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) pUL93 and pUL77 are both essential for virus growth but their functions in the virus life cycle remain mostly unresolved. Homologs of pUL93 and pUL77 in herpes simplex virus (HSV-1) and pseudorabies virus (PRV) are known to interact to form a complex at capsid vertices known as the capsid vertex-specific component (CVSC), which likely stabilizes nucleocapsids during virus maturation and also aids in nuclear egress. In herpesviruses, nucleocapsids assemble and partially mature in nuclear replication compartments and then travel to the inner nuclear membrane (INM) for nuclear egress. The factors governing the recruitment of nucleocapsids to the INM are not known. Kinetic analysis of pUL93 demonstrates that this protein is expressed late during infection and localizes primarily to the nucleus of infected cells. pUL93 associates with both virions and capsids, and interacts with the components of the nuclear egress complex (NEC), namely pUL50, pUL53, and pUL97, during infection. Also, multiple regions in pUL93 can independently interact with pUL77, which has been shown to help retain viral DNA during capsid assembly. These studies, combined with our earlier report of an essential role of pUL93 in viral DNA packaging, indicate that pUL93 may serve as an important link between nucleocapsid maturation and nuclear egress.

    IMPORTANCE Human cytomegalovirus (HCMV) causes life-threatening disease and disability in immunocompromised patients and congenitally infected newborns. In this study, we investigated the functions of HCMV essential tegument protein pUL93 and determined that it interacts with the components of the nuclear egress complex, namely pUL50, pUL53, and pUL97. We also found that pUL93 specifically interacts with pUL77, which helps retain viral DNA during capsid assembly. Together, our data point towards an important role of pUL93 in linking virus maturation to nuclear egress. In addition to expanding our knowledge of the process of HCMV maturation, information from these studies will also be utilized to develop new antiviral therapies.

  • MAVS expressed by hematopoietic cells is critical for control of West Nile virus infection and pathogenesis [PublishAheadOfPrint]

  • West Nile virus (WNV) is the most important cause of epidemic encephalitis in North America. Innate immune responses, which are critical for control of WNV infection, are initiated by signaling through pathogen recognition receptors, RIG-I and MDA5, and their downstream adaptor molecule, MAVS. Here we show that a deficiency of MAVS in hematopoietic cells resulted in increased mortality and delayed WNV clearance from the brain. In Mavs-/- mice, a dysregulated immune response was detected, characterized by a massive influx of macrophages and virus-specific T cells into the infected brain. These T cells were polyfunctional and lysed peptide-pulsed target cells in vitro. However, virus-specific T cells in the brains of infected Mavs-/- mice exhibited lower functional avidity than those in wild-type animals, and even virus-specific memory T cells generated by prior immunization could not protect Mavs-/- mice from WNV-induced lethal disease. Concomitant with ineffective virus clearance, macrophage numbers were increased in the Mavs-/- brain and both macrophages and microglia exhibited an activated phenotype. Microarray analyses of leukocytes in the infected Mavs-/- brain showed a preferential expression of genes associated with activation and inflammation. Together, these results demonstrate a critical role for MAVS in hematopoietic cells in augmenting the kinetics of WNV clearance and thereby preventing a dysregulated and pathogenic immune response.

    IMPORTANCE West Nile virus (WNV) is the most important cause of mosquito-transmitted encephalitis in the United States. The innate immune response is known to be critical for protection in infected mice. Here, we show that expression of MAVS, a key adaptor molecule in the RIG-I-like receptor RNA sensing pathway, in hematopoietic cells is critical for protection from lethal WNV infection. In the absence of MAVS, there is a massive infiltration of myeloid cells and virus-specific T cells into the brain and over-exuberant production of pro-inflammatory cytokines. These results demonstrate the important role that MAVS expression in hematopoietic cells has in regulating the inflammatory response in the WNV-infected brain.

  • Viral evasion and manipulation of host RNA quality control pathways [PublishAheadOfPrint]

  • Viruses have evolved diverse strategies to maximize the functional and coding capacities of their genetic material. Individual viral RNAs are often used as substrates for both replication and translation and can contain multiple, sometimes overlapping, open reading frames. Further, viral RNAs engage in a wide variety of interactions with both host and viral proteins to modify the activities of important cellular factors and direct their own trafficking, packaging, localization, stability, and translation. However, adaptations increasing the information density of small viral genomes can have unintended consequences. In particular, viral RNAs have developed features that mark them as potential targets of host RNA quality control pathways. This review focuses on ways in which viral RNAs run afoul of cellular the mRNA quality control and decay machinery, as well as strategies developed by viruses to circumvent or exploit cellular mRNA surveillance.

  • Glutathione depletion is linked with Th2 polarization in mice with a retrovirus-induced immunodeficiency syndrome, MAIDS: role of pro-glutathione molecules as immunotherapeutics [PublishAheadOfPrint]

  • LP-BM5 murine leukemia virus injection causes murine AIDS, a disease characterized by many dysfunctions of immunocompetent cells. To establish whether the disease is characterized by glutathione imbalance, reduced glutathione (GSH) and cysteine were quantified in different organs. A marked redox imbalance, consisting in GSH and/or cysteine depletion, was found in the lymphoid organs, such as the spleen and lymph nodes. Moreover, a significant decrease in cysteine and GSH levels was measured in pancreas and brain respectively at 5 weeks post infection. Th2 immune response was predominant at all the times investigated as revealed by the expression of Th1/Th2 cytokines. Furthermore, an investigation of the activation status of peritoneal macrophages showed that the expression of genetic markers of alternative activation, namely, Fizz1, Ym1, and Arginase1, was induced. Conversely, expression of iNOS, a marker of classical activation of macrophages, was detected only when Th1 cytokines were more expressed. In vitro studies revealed that during the very early phases of infection, GSH depletion and down-regulation of interleukin-12 p40 mRNA were correlated with the level of the infecting dose of LP-BM5 used to infect macrophages. Treatment of LP-BM5-infected mice with I-152, a N-acetyl-cysteine supplier, restored GSH/cysteine levels in the organs, reduced the expression of alternatively activated macrophage markers, and increased IFN- production while decreased Th2 cytokines, such as IL-4 and IL-5. Our findings thus establish a link between GSH deficiency and Th1/Th2 disequilibrium in LP-BM5 infection, and I-152 can be used to restore the GSH level and a balanced Th1/Th2 response in infected mice.

    IMPORTANCE This study represents the first report of association between Th2 polarization and alteration of the redox state in LP-BM5 infection. Moreover, it is shown the evidence that LP-BM5 infection causes a decrease in thiol content of peritoneal macrophages which can influence IL-12 production.

    The restoration of GSH levels by GSH-replenishing molecules can represent a new therapeutic avenue to fight this retroviral infection re-establishing the Th1/Th2 balance. The immunotherapy based on the use of pro-GSH molecules would permit to more effectively combat LP-BM5 infection and probably all those viral infections characterized by GSH deficiency and Th1/Th2 imbalance.

  • A Genome-Wide RNAi Screen Identifies a Role for Wnt/Beta-Catenin Signaling During Rift Valley Fever Virus Infection [PublishAheadOfPrint]

  • Rift Valley fever virus (RVFV) is an arbovirus within the Bunyaviridae family capable of causing serious morbidity and mortality in humans and livestock. To identify host factors involved in bunyavirus replication, we employed genome-wide RNA interference (RNAi) screening and identified 381 genes whose knockdown reduced infection. The Wnt pathway was the most represented pathway when gene hits were functionally clustered. With further investigation, we found that RVFV infection activated Wnt signaling, was enhanced when Wnt signaling was pre-activated, was reduced with knockdown of bbeta;-catenin, and was blocked using Wnt signaling inhibitors. Similar results were found using distantly related bunyaviruses La Crosse virus and California encephalitis virus, suggesting a conserved role for Wnt signaling in bunyaviral infection. We propose a model where bunyaviruses activate Wnt responsive genes to regulate optimal cell cycle conditions needed to promote efficient viral replication. The findings in this study should aid in the design of efficacious host-directed anti-viral therapeutics.

    IMPORTANCE: RVFV is a mosquito-borne bunyavirus that is endemic to Africa but has demonstrated a capacity for emergence in new territories (e.g. Arabian Peninsula). As a zoonotic pathogen that primarily affects livestock, RVFV can also cause lethal hemorrhagic fever and encephalitis in humans. Currently, there are no treatments or fully-licensed vaccines for this virus. Using high-throughput RNAi screening, we identified canonical Wnt signaling as an important host pathway regulating RVFV infection. The beneficial role of Wnt signaling was observed for RVFV, along with other disparate bunyaviruses, indicating a conserved bunyaviral replication mechanism involving Wnt signaling. These studies supplement our knowledge of the fundamental mechanisms of bunyavirus infection and provide new avenues for countermeasure development against pathogenic bunyaviruses.

  • Deep Sequencing of the HIV-1 env Gene Reveals Discrete X4 Lineages and Linkage Disequilibrium Between X4 and R5 Viruses In the V1/V2 and V3 Variable Regions [PublishAheadOfPrint]

  • HIV-1 requires the CD4 receptor and a co-receptor (CCR5 (R5 phenotype] or CXCR4 [X4 phenotype]) to enter cells. Co-receptor tropism can be assessed by either phenotypic or genotypic analysis, the latter using bioinformatics algorithms to predict tropism based on the env V3 sequence. We used the Primer ID sequencing strategy with the MiSeq sequencing platform to reveal the structure of viral populations in the V1/V2 and C2/V3 regions of the HIV-1 env gene in 30 late stage and 6 early stage subjects. We also used end-point dilution PCR followed by cloning of env genes to create pseudotyped virus to explore the link between genotypic predictions and phenotypic assessment of coreceptor usage. We found out that the most stringently sequence-based calls of X4 variants (Geno2Pheno false positive rate [FPR] lle; 2%) formed distinct lineages within the viral population, and these were detected in 24 of 30 late-stage samples (80%), significantly higher than has been seen previously using other approaches. Non-X4 lineages were not skewed toward lower FPR scores in X4-containing populations. Phenotypic assays showed that variants with intermediate FPR (2nndash;20%) could be either X4/dual or R5 variants, although the X4 variants made up only about 25% of the lineages with an FPR of less than 10%, and these carried a distinctive sequence change. Phylogenetic analysis of both V1/V2 and C2/V3 regions showed evidence of recombination within but very little between the X4 and R5 lineages, suggesting these populations are genetically isolated.

    IMPORTANCE Primer ID sequencing provides a novel approach to study genetic structures of viral populations. X4 variants may be more prevalent than previously reported when assessed using next generation sequencing (NGS) and with greater depth of sampling than SGA. Phylogenetic analysis to identify lineages of sequences with intermediate FPR values may provide additional information for accurately predicting X4 variants using V3 sequences. Limited recombination occurs between X4 and R5 lineages, suggesting X4 and R5 variants are genetically isolated and may be replicating in different cell types, or that X4/R5 recombinants have reduced fitness.

  • Notwithstanding circumstantial alibis, cytotoxic T cells can be major killers of HIV-1 infected cells. [PublishAheadOfPrint]

  • Several experiments suggest that in the chronic phase of HIV-1 infection CD8+cytotoxic T lymphocytes (CTL) contribute very little to the death of productively infected cells. First, the expected life span of productively infected cells is fairly long, i.e., about one day. Second, this life span is hardly affected by the depletion of CD8+T cells. Third, the rate at which mutants escaping a CTL response take over the viral population tends to be slow. Our main result is that all these observations are perfectly compatible with killing rates that are much faster than one per day once we invoke the fact that infected cells proceed through an eclipse phase of about one day before they start producing virus. Assuming that the major protective effect of CTL is cytolytic, we demonstrate that mathematical models with an eclipse phase account for the data when the killing is fast, and when it varies over the life cycle of infected cells. Considering the steady state corresponding to the chronic phase of the infection, we find that the rate of immune escape, and the rate at which the viral load increases following CD8+T cell depletion, should reflect the viral replication rate, . A meta analysis of previous data shows that viral replication rates during chronic infection vary between 0.5lle;lle;1 day-1. Balancing such fast viral replication requires killing rates that are several times larger than , implying that most productively infected cells would die by cytolytic effects.

    Importance Most current data suggest that Cytotoxic T cells (CTL) mediate their control of HIV-1 infection by non-lytic mechanisms, i.e., data suggest that CTL hardly kill. This interpretation of these data has been based upon the general mathematical model for HIV infection. Because this model ignores the eclipse phase between the infection of a target cell and the start of viral production by that cell, we re-analyze the same data sets with novel models that do account for the eclipse phase. We find that the data are perfectly consistent with lytic control by CTL, and predict that most productively infected cells are killed by CTL. Because that the killing rate should balance the viral replication rate, we estimate both parameters from a large set of published experiments depleting CD8+T cells in SIV infected monkeys. This confirms that the killing rate can be much faster than is currently appreciated.

  • Transformation by Polyomavirus Middle T Antigen Involves A Unique Bimodal Interaction with the Hippo Effector YAP [PublishAheadOfPrint]

  • Murine polyomavirus has repeatedly provided insights into tumorigenesis, revealing such key control mechanisms as tyrosine phosphorylation and PI3K signaling. We recently demonstrated that polyomavirus ST antigen binds YAP, a major effector of Hippo signaling, to regulate differentiation. Here we characterize YAP as a target of MT antigen important for transformation. Through a surface including residues R103 and D182, wild-type MT binds to the YAP WW domains. Mutation of either R103 or D182 of MT abrogates YAP binding without affecting binding to other signaling molecules or the strength of PI3K or Ras signaling. Either genetic abrogation of YAP binding to MT or silencing of YAP via shRNA reduced MT transformation, suggesting that YAP makes a positive contribution to the transformed phenotype. MT targets YAP both by activating signaling pathways that affect it and by binding to it. MT signaling, whether from wild-type or YAP-binding mutant MT, promoted YAP phosphorylation at S127 and S381/397(YAP2/YAP1). Consistent with the known functions of these phosphorylated serines, MT signaling leads to loss of YAP from the nucleus and degradation. Binding of YAP to MT brings it together with PP2A leading to YAP's dephosphorylation in the MT complex. It also leads to YAP's enrichment in membranes. Taken together, these results indicate that YAP promotes MT transformation via mechanisms that may depart from YAP's canonical oncogenic transcriptional activation functions.

    IMPORTANCE The highly conserved Hippo/YAP pathway is important for tissue development and homeostasis. Increasingly, changes in this pathway are being associated with cancer. Middle T (MT) is the primary polyomavirus oncogene responsible for tumor formation. In this study we show that MT signaling promotes YAP phosphorylation, loss from the nucleus and increased turn-over. Notably MT genetics demonstrate that YAP binding to MT is important for transformation. Because MT also binds PP2A, the YAP bound to MT is dephosphorylated, stabilized and localized to membranes. Taken together, these results indicate that YAP promotes MT transformation via mechanisms that depart from YAP's canonical oncogenic transcriptional activation functions.

  • Functional incompatibility between the generic NF-{kappa}B motif and a subtype-specific Sp1III element drives the formation of HIV-1 subtype C viral promoter [PublishAheadOfPrint]

  • Of the various genetic subtypes of HIV-1, HIV-2 and SIV, only in subtype C of HIV-1, a genetically variant NF-B binding site is found at the core of the viral promoter in association with a subtype-specific Sp1III motif. How the subtype-associated variations in the core transcription factor binding sites (TFBS) influence gene expression from the viral promoter has not been examined previously. Using panels of infectious viral molecular clones, we demonstrate that subtype-specific NF-B and Sp1III motifs have evolved for optimal gene expression, and neither of the motifs can be substituted by a corresponding TFBS variant.The variant NF-B motif binds NF-B with an affinity two-fold higher than that of the generic NF-B site. Importantly, in the context of an infectious virus, the subtype-specific Sp1III motif demonstrates a profound loss of function in association with the generic NF-B motif. An additional substitution of the Sp1III motif fully restores viral replication suggesting that the subtype C specific Sp1III has evolved to function with the variant, but not generic, NF-B motif. A change of only two base pairs in the central NF-B motif completely suppresses viral transcription from the provirus and converts the promoter into heterochromatin refractory to TNF-aalpha; induction. The present work represents the first demonstration of functional incompatibility between an otherwise functional NF-B motif and a unique Sp1 site in the context of HIV-1 promoter. Our work provides important leads as per the evolution of HIV-1 subtype C viral promoter with relevance for gene expression regulation and viral latency.

    Importance: Subtype-specific genetic variations provide a powerful tool to examine how these variations offer a replication advantage to specific viral subtypes if any. Only in subtype C of HIV-1, two genetically distinct transcription factor binding sites are positioned at the most critical location of the viral promoter. Since a single promoter regulates viral gene expression, the promoter variations can play a critical role in determining the replication fitness of the viral strains. Our work for the first time provides a scientific explanation for the presence of a unique NF-B binding motif in subtype C, a major HIV-1 genetic family responsible for half of the global HIV-1 infections. The results offer compelling evidence that subtype C viral promoter is not only stronger but also is endowed with a qualitative-gain-of-function advantage. The genetically variant NF-B, and the Sp1III motifs may be responsive to specific cell signal-pathways differentially, and these mechanisms must be examined.

  • Identification and validation of small molecules that enhance recombinant Adeno-associated virus transduction following high throughput screen [PublishAheadOfPrint]

  • While the recent success of AAV-mediated gene therapy in clinical trials is promising, challenges still face the widespread applicability of recombinant AAV(rAAV). A major goal is to enhance the transduction efficiency of vectors in order to achieve therapeutic levels of gene expression at a vector dose that is below the immunological response threshold. In an attempt to identify novel compounds that enhance rAAV transduction, we performed two high-throughput screens comprising 2396 compounds. We identified 13 compounds that were capable of enhancing transduction, 12 of which demonstrated vector-specific effects and one that could also enhance vector-independent transgene expression. Many of these compounds shared similar properties and could be categorized into five groups: Epipodophyllotoxins (group 1), inducers of DNA damage (group 2), effectors of epigenetic modification (group 3), anthracyclines (group 4), and proteasome inhibitors (group 5). We optimized dosing for the identified compounds in several immortalized human cell lines as well as normal diploid cells. We found that the group 1 epipodophyllotoxins (teniposide and etoposide) consistently produced the greatest transduction enhancement. We also explored transduction enhancement among single-stranded, self-complementary, and fragment vectors and found that the compounds could impact fragmented rAAV2 transduction to an even greater extent than single-stranded vectors. In vivo analysis of rAAV2 and all of the clinically-relevant compounds revealed that, consistent with our in vitro results, teniposide exhibited the greatest level of transduction enhancement. Finally, we explored the capability of teniposide to enhance transduction of fragment vectors in vivo using an AAV8 capsid, which is known to exhibit robust liver tropism. Consistent with our in vitro results, teniposide co-administration greatly enhanced fragmented rAAV8 transduction at 48h and 8 days. This study provides a foundation based on rAAV small molecule screen methodology which is ideally studied for more diverse libraries of compounds that can be tested for potentiating rAAV transduction.

    IMPORTANCE This study seeks to enhance the ability of adeno-associated viral vectors for therapeutic gene delivery applicable to the treatment of diverse diseases. To do this, a comprehensive panel of FDA-approved drugs was tested in human cells and in animal models to determine if they increased adeno-associated virus gene delivery. The results demonstrate that particular groups of drugs enhance adeno-associated virus gene delivery by unknown mechanisms. In particular, the enhancement of gene delivery was approximately 50-100 times better using teniposide, which is also used as chemotherapy for cancer. Collectively, these results highlight the potential for FDA-approved drug enhancement of adeno-associated virus gene therapy which could result in a safer and effective treatments for diverse acquired or genetic diseases.

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