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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]

  • Influenza Virus Reservoirs and Intermediate Hosts: Dogs, Horses, and New Possibilities for Influenza Virus Exposure of Humans [Gem]

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

  • Severe Acute Respiratory Syndrome-Associated Coronavirus Vaccines Formulated with Delta Inulin Adjuvants Provide Enhanced Protection while Ameliorating Lung Eosinophilic Immunopathology [Vaccines and Antiviral Agents]

  • Although the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) epidemic was controlled by nonvaccine measures, coronaviruses remain a major threat to human health. The design of optimal coronavirus vaccines therefore remains a priority. Such vaccines present major challenges: coronavirus immunity often wanes rapidly, individuals needing to be protected include the elderly, and vaccines may exacerbate rather than prevent coronavirus lung immunopathology. To address these issues, we compared in a murine model a range of recombinant spike protein or inactivated whole-virus vaccine candidates alone or adjuvanted with either alum, CpG, or Advax, a new delta inulin-based polysaccharide adjuvant. While all vaccines protected against lethal infection, addition of adjuvant significantly increased serum neutralizing-antibody titers and reduced lung virus titers on day 3 postchallenge. Whereas unadjuvanted or alum-formulated vaccines were associated with significantly increased lung eosinophilic immunopathology on day 6 postchallenge, this was not seen in mice immunized with vaccines formulated with delta inulin adjuvant. Protection against eosinophilic immunopathology by vaccines containing delta inulin adjuvants correlated better with enhanced T-cell gamma interferon (IFN-) recall responses rather than reduced interleukin-4 (IL-4) responses, suggesting that immunopathology predominantly reflects an inadequate vaccine-induced Th1 response. This study highlights the critical importance for development of effective and safe coronavirus vaccines of selection of adjuvants based on the ability to induce durable IFN- responses.

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

  • The Genome of the Nucleopolyhedrosis-Causing Virus from Tipula oleracea Sheds New Light on the Nudiviridae Family [Genetic Diversity and Evolution]

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

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

  • Reverse Genetics System for Severe Fever with Thrombocytopenia Syndrome Virus [Genome Replication and Regulation of Viral Gene Expression]

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

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

  • Identification and Characterization of Nuclear and Nucleolar Localization Signals in the Adeno-Associated Virus Serotype 2 Assembly-Activating Protein [Virus-Cell Interactions]

  • Assembly-activating protein (AAP) of adeno-associated virus serotype 2 (AAV2) is a nucleolar-localizing protein that plays a critical role in transporting the viral capsid VP3 protein to the nucleolus for assembly. Here, we identify and characterize AAV2 AAP (AAP2) nuclear (NLS) and nucleolar (NoLS) localization signals near the carboxy-terminal region of AAP2 (amino acid positions 144 to 184) (AAP2144nndash;184). This region contains five basic-amino-acid-rich (BR) clusters, KSKRSRR (AAP2BR1), RRR (AAP2BR2), RFR (AAP2BR3), RSTSSR (AAP2BR4), and RRIK (AAP2BR5), from the amino terminus to the carboxy terminus. We created 30 AAP2BR mutants by arginine/lysine-to-alanine mutagenesis or deletion of AAP2BRs and 8 and 1 green fluorescent protein (GFP)-AAP2BR and bbeta;-galactosidasenndash;AAP2BR fusion proteins, respectively, and analyzed their intracellular localization in HeLa cells by immunofluorescence microscopy. The results showed that AAP2144nndash;184 has redundant multipartite NLSs and that any combinations of 4 AAP2BRs, but not 3 or less, can constitute a functional NLS-NoLS; AAP2BR1 and AAP2BR2 play the most influential role for nuclear localization, but either one of the two AAP2BRs is dispensable if all 4 of the other AAP2BRs are present, resulting in 3 different, overlapping NLS motifs; and the NoLS is shared redundantly among the five AAP2BRs and functions in a context-dependent manner. AAP2BR mutations not only resulted in aberrant intracellular localization, but also attenuated AAP2 protein expression to various degrees, and both of these abnormalities have a significant negative impact on capsid production. Thus, this study reveals the organization of the intermingling NLSs and NoLSs in AAP2 and provides insights into their functional roles in capsid assembly.

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

  • Human Cytomegalovirus Exploits Interferon-Induced Transmembrane Proteins To Facilitate Morphogenesis of the Virion Assembly Compartment [Structure and Assembly]

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

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

  • Analysis of the Functional Interchange between the IE1 and pp71 Proteins of Human Cytomegalovirus and ICP0 of Herpes Simplex Virus 1 [Genome Replication and Regulation of Viral Gene Expression]

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

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

  • Discovery of a Novel Coronavirus, China Rattus Coronavirus HKU24, from Norway Rats Supports the Murine Origin of Betacoronavirus 1 and Has Implications for the Ancestor of Betacoronavirus Lineage A [Genetic Diversity and Evolution]

  • We discovered a novel Betacoronavirus lineage A coronavirus, China Rattus coronavirus (ChRCoV) HKU24, from Norway rats in China. ChRCoV HKU24 occupied a deep branch at the root of members of Betacoronavirus 1, being distinct from murine coronavirus and human coronavirus HKU1. Its unique putative cleavage sites between nonstructural proteins 1 and 2 and in the spike (S) protein and low sequence identities to other lineage A betacoronaviruses (bbeta;CoVs) in conserved replicase domains support ChRCoV HKU24 as a separate species. ChRCoV HKU24 possessed genome features that resemble those of both Betacoronavirus 1 and murine coronavirus, being closer to Betacoronavirus 1 in most predicted proteins but closer to murine coronavirus by G+C content, the presence of a single nonstructural protein (NS4), and an absent transcription regulatory sequence for the envelope (E) protein. Its N-terminal domain (NTD) demonstrated higher sequence identity to the bovine coronavirus (BCoV) NTD than to the mouse hepatitis virus (MHV) NTD, with 3 of 4 critical sugar-binding residues in BCoV and 2 of 14 contact residues at the MHV NTD/murine CEACAM1a interface being conserved. Molecular clock analysis dated the time of the most recent common ancestor of ChRCoV HKU24, Betacoronavirus 1, and rabbit coronavirus HKU14 to about the year 1400. Cross-reactivities between other lineage A and B bbeta;CoVs and ChRCoV HKU24 nucleocapsid but not spike polypeptide were demonstrated. Using the spike polypeptide-based Western blot assay, we showed that only Norway rats and two oriental house rats from Guangzhou, China, were infected by ChRCoV HKU24. Other rats, including Norway rats from Hong Kong, possessed antibodies only against N protein and not against the spike polypeptide, suggesting infection by bbeta;CoVs different from ChRCoV HKU24. ChRCoV HKU24 may represent the murine origin of Betacoronavirus 1, and rodents are likely an important reservoir for ancestors of lineage A bbeta;CoVs.

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

  • Transcriptome Analysis of Kaposi's Sarcoma-Associated Herpesvirus during De Novo Primary Infection of Human B and Endothelial Cells [Genome Replication and Regulation of Viral Gene Expression]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) infects many target cells (e.g., endothelial, epithelial, and B cells, keratinocytes, and monocytes) to establish lifelong latent infections. Viral latent-protein expression is critical in inducing and maintaining KSHV latency. Infected cells are programmed to retain the incoming viral genomes during primary infection. Immediately after infection, KSHV transcribes many lytic genes that modulate various cellular pathways to establish successful infection. Analysis of the virion particle showed that the virions contain viral mRNAs, microRNAs, and other noncoding RNAs that are transduced into the target cells during infection, but their biological functions are largely unknown. We performed a comprehensive analysis of the KSHV virion packaged transcripts and the profiles of viral genes transcribed after de novo infections of various cell types (human peripheral blood mononuclear cells [PBMCs], CD14+ monocytes, and telomerase-immortalized vascular endothelial [TIVE] cells), from viral entry until latency establishment. A next-generation sequence analysis of the total transcriptome showed that several viral RNAs (polyadenylated nuclear RNA, open reading frame 58 [ORF58], ORF59, T0.7, and ORF17) were abundantly present in the KSHV virions and effectively transduced into the target cells. Analysis of the transcription profiles of each viral gene showed specific expression patterns in different cell lines, with the majority of the genes, other than latent genes, silencing after 24 h postinfection. We differentiated the actively transcribing genes from the virion-transduced transcripts using a nascent RNA capture approach (Click-iT chemistry), which identified transcription of a number of viral genes during primary infection. Treating the infected cells with phosphonoacetic acid (PAA) to block the activity of viral DNA polymerase confirmed the involvement of lytic DNA replication during primary infection. To further understand the role of DNA replication during primary infection, we performed de novo PBMC infections with a recombinant ORF59-deleted KSHV virus, which showed significantly reduced numbers of viral copies in the latently infected cells. In summary, the transduced KSHV RNAs as well as the actively transcribed genes control critical processes of early infection to establish KSHV latency.

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

  • The Herpes Simplex Virus 1 UL51 Protein Interacts with the UL7 Protein and Plays a Role in Its Recruitment into the Virion [Structure and Assembly]

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

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

  • Serine Phosphorylation of the Hepatitis C Virus NS5A Protein Controls the Establishment of Replication Complexes [Virus-Cell Interactions]

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

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

  • Heterosubtypic Antibodies to Influenza A Virus Have Limited Activity against Cell-Bound Virus but Are Not Impaired by Strain-Specific Serum Antibodies [Vaccines and Antiviral Agents]

  • The majority of influenza virus-specific antibodies elicited by vaccination or natural infection are effective only against the eliciting or closely related viruses. Rare stem-specific heterosubtypic monoclonal antibodies (hMAbs) can neutralize multiple strains and subtypes by preventing hemagglutinin (HA)-mediated fusion of the viral membrane with the endosomal membrane. The epitopes recognized by these hMAbs are therefore considered promising targets for the development of pan-influenza virus vaccines. Here, we report the isolation of a novel human HA stem-reactive monoclonal antibody, hMAb 1.12, with exceptionally broad neutralizing activity encompassing viruses from 15 distinct HA subtypes. Using MAb 1.12 and two other monoclonal antibodies, we demonstrate that neutralization by hMAbs is virtually irreversible but becomes severely impaired following virus attachment to cells. In contrast, no interference by human anti-influenza virus serum antibodies was found, indicating that apically binding antibodies do not impair access to the membrane-proximal heterosubtypic epitopes. Our findings therefore encourage development of new vaccine concepts aiming at the induction of stem-specific heterosubtypic antibodies, as we provide support for their effectiveness in individuals previously exposed to influenza virus.

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

  • Mutations Conferring a Noncytotoxic Phenotype on Chikungunya Virus Replicons Compromise Enzymatic Properties of Nonstructural Protein 2 [Genome Replication and Regulation of Viral Gene Expression]

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

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

  • Differential Effects of the G118R, H51Y, and E138K Resistance Substitutions in Different Subtypes of HIV Integrase [Vaccines and Antiviral Agents]

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

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

  • Analysis of Geminivirus AL2 and L2 Proteins Reveals a Novel AL2 Silencing Suppressor Activity [Virus-Cell Interactions]

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

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

  • Heat Shock Protein 70 Regulates Degradation of the Mumps Virus Phosphoprotein via the Ubiquitin-Proteasome Pathway [Virus-Cell Interactions]

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

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

  • Efficient Virus Assembly, but Not Infectivity, Determines the Magnitude of Hepatitis C Virus-Induced Interferon Alpha Responses of Plasmacytoid Dendritic Cells [Virus-Cell Interactions]

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

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

  • Impact of the Adenoviral E4 Orf3 Protein on the Activity and Posttranslational Modification of p53 [Virus-Cell Interactions]

  • Our previous studies have established that the p53 populations that accumulate in normal human cells exposed to etoposide or infected by an E1B 55-kDa protein-null mutant of human adenovirus type 5 carry a large number of posttranslational modifications at numerous residues (C. J. DeHart, J. S. Chahal, S. J. Flint, and D. H. Perlman, Mol Cell Proteomics 13:1nndash;17, 2014, http://dx.doi.org/10.1074/mcp.M113.030254). In the absence of this E1B protein, the p53 transcriptional program is not induced, and it has been reported that the viral E4 Orf3 protein inactivates p53 (C. Soria, F. E. Estermann, K. C. Espantman, and C. C. O'Shea, Nature 466:1076nndash;1081, 2010, http://dx.doi.org/10.1038/nature09307). As the latter protein disrupts nuclear Pml bodies, sites at which p53 is modified, we used mass spectrometry to catalogue the posttranscriptional modifications of the p53 population that accumulates when neither the E1B 55-kDa nor the E4 Orf3 protein is made in infected cells. Eighty-five residues carrying 163 modifications were identified. The overall patterns of posttranslational modification of this population and p53 present in cells infected by an E1B 55-kDa-null mutant were similar. The efficiencies with which the two forms of p53 bound to a consensus DNA recognition sequence could not be distinguished and were lower than that of transcriptionally active p53. The absence of the E4 Orf3 protein increased expression of several p53-responsive genes when the E1B protein was also absent from infected cells. However, expression of these genes did not attain the levels observed when p53 was activated in response to etoposide treatment and remained lower than those measured in mock-infected cells.

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

  • Synthetic Toll-Like Receptor 4 (TLR4) and TLR7 Ligands as Influenza Virus Vaccine Adjuvants Induce Rapid, Sustained, and Broadly Protective Responses [Vaccines and Antiviral Agents]

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

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

  • Conserved Motifs in a Tombusvirus Polymerase Modulate Genome Replication, Subgenomic Transcription, and Amplification of Defective Interfering RNAs [Genome Replication and Regulation of Viral Gene Expression]

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

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

  • Vpu Is the Main Determinant for Tetraspanin Downregulation in HIV-1-Infected Cells [Virus-Cell Interactions]

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

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

  • Stability of Structured Kaposi's Sarcoma-Associated Herpesvirus ORF57 Protein Is Regulated by Protein Phosphorylation and Homodimerization [Virus-Cell Interactions]

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

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

  • The Interface between Hepatitis B Virus Capsid Proteins Affects Self-Assembly, Pregenomic RNA Packaging, and Reverse Transcription [Structure and Assembly]

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

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

  • Contribution of MxB Oligomerization to HIV-1 Capsid Binding and Restriction [Virus-Cell Interactions]

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

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

  • Postchallenge Administration of Brincidofovir Protects Healthy and Immune-Deficient Mice Reconstituted with Limited Numbers of T Cells from Lethal Challenge with IHD-J-Luc Vaccinia Virus [Vaccines and Antiviral Agents]

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

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

  • High Preexisting Serological Antibody Levels Correlate with Diversification of the Influenza Vaccine Response [Vaccines and Antiviral Agents]

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

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

  • The Aberrant Gene-End Transcription Signal of the Matrix M Gene of Human Parainfluenza Virus Type 3 Downregulates Fusion F Protein Expression and the F-Specific Antibody Response In Vivo [Pathogenesis and Immunity]

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

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

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

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

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

  • Constitutive but Not Inducible Attenuation of Transforming Growth Factor {beta} Signaling Increases Natural Killer Cell Responses without Directly Affecting Dendritic Cells Early after Persistent Viral Infection [Cellular Response to Infection]

  • Rapid innate responses to viral encounters are crucial to shaping the outcome of infection, from viral clearance to persistence. Transforming growth factor bbeta; (TGF-bbeta;) is a potent immune suppressor that is upregulated early upon viral infection and maintained during chronic infections in both mice and humans. However, the role of TGF-bbeta; signaling in regulating individual cell types in vivo is still unclear. Using infections with two different persistent viruses, murine cytomegalovirus (MCMV) and lymphocytic choriomeningitis virus (LCMV; Cl13), in their natural rodent host, we observed that TGF-bbeta; signaling on dendritic cells (DCs) did not dampen DC maturation or cytokine production in the early stages of chronic infection with either virus in vivo. In contrast, TGF-bbeta; signaling prior to (but not during) chronic viral infection directly restricted the natural killer (NK) cell number and effector function. This restriction likely compromised both the early control of and host survival upon MCMV infection but not the long-term control of LCMV infection. These data highlight the context and timing of TGF-bbeta; signaling on different innate cells that contribute to the early host response, which ultimately influences the outcome of chronic viral infection in vivo.

    IMPORTANCE In vivo host responses to pathogens are complex processes involving the cooperation of many different immune cells migrating to specific tissues over time, but these events cannot be replicated in vitro. Viruses causing chronic infections are able to subvert this immune response and represent a human health burden. Here we used two well-characterized viruses that are able to persist in their natural mouse host to dissect the role of the suppressive molecule TGF-bbeta; in dampening host responses to infection in vivo. This report presents information that allows an increased understanding of long-studied TGF-bbeta; signaling by examining its direct effect on different immune cells that are activated very early after in vivo viral infection and may aid with the development of new antiviral therapeutic strategies.

  • Hepatitis C Virus-Mediated Enhancement of MicroRNA miR-373 Impairs the JAK/STAT Signaling Pathway [Virus-Cell Interactions]

  • Hepatitis C virus (HCV) is a serious global health problem and establishes chronic infection in a significant number of infected humans worldwide. Interferon (IFN) and IFN-stimulated genes (ISGs) are amplified during HCV infection but fail to eliminate virus from the liver in a large number of infected patients, and the mechanism is not fully understood. MicroRNAs (miRNAs) have been implicated in the control of many biological processes, including IFN signaling. To gain more insights into the role of cellular miRNAs in possible countermeasures of HCV for suppression of the host antiviral response, a miRNA array was performed by using primary human hepatocytes infected with in vitro cell culture-grown HCV. A group of miRNAs were modulated in HCV-infected primary human hepatocytes. We focused on miR-373, as this miRNA was significantly upregulated in HCV-infected primary human hepatocytes. Here, we analyzed the function of miR-373 in the context of HCV infection. HCV infection upregulates miR-373 expression in hepatocytes and HCV-infected liver biopsy specimens. Furthermore, we discovered that miR-373 directly targets Janus kinase 1 (JAK1) and IFN-regulating factor 9 (IRF9), important factors in the IFN signaling pathway. The upregulation of miR-373 by HCV also inhibited STAT1 phosphorylation, which is involved in ISG factor 3 (ISGF3) complex formation and ISG expression. The knockdown of miR-373 in hepatocytes enhanced JAK1 and IRF9 expression and reduced HCV RNA replication. Taken together, our results demonstrated that miR-373 is upregulated during HCV infection and negatively regulated the type I IFN signaling pathway by suppressing JAK1 and IRF9. Our results offer a potential therapeutic approach for antiviral intervention.

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

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

  • Uracil DNA glycosylases (UNG) are highly conserved proteins that preserve DNA fidelity by catalyzing the removal of mutagenic uracils. All herpesviruses encode a viral UNG (vUNG), and yet the role of the vUNG in a pathogenic course of gammaherpesvirus infection is not known. First, we demonstrated that the vUNG of murine gammaherpesvirus 68 (MHV68) retains the enzymatic function of host UNG in an in vitro class switch recombination assay. Next, we generated a recombinant MHV68 with a stop codon in ORF46/UNG (UNG) that led to loss of UNG activity in infected cells and a replication defect in primary fibroblasts. Acute replication of MHV68UNG in the lungs of infected mice was reduced 100-fold and was accompanied by a substantial delay in the establishment of splenic latency. Latency was largely, yet not fully, restored by an increase in virus inoculum or by altering the route of infection. MHV68 reactivation from latent splenocytes was not altered in the absence of the vUNG. A survey of host UNG activity in cells and tissues targeted by MHV68 indicated that the lung tissue has a lower level of enzymatic UNG activity than the spleen. Taken together, these results indicate that the vUNG plays a critical role in the replication of MHV68 in tissues with limited host UNG activity and this vUNG-dependent expansion, in turn, influences the kinetics of latency establishment in distal reservoirs.

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

  • A Native-Like SOSIP.664 Trimer Based on an HIV-1 Subtype B env Gene [Vaccines and Antiviral Agents]

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

    IMPORTANCE The cleaved, trimeric envelope protein complex is the only neutralizing antibody target on the HIV-1 surface. Many vaccine strategies are based on inducing neutralizing antibodies. For HIV-1, one approach involves using recombinant, soluble protein mimics of the native trimer. At present, the only reliable way to make native-like, soluble trimers in practical amounts is via the introduction of specific sequence changes that confer stability on the cleaved form of Env. The resulting proteins are known as SOSIP.664 gp140 trimers, and the current paradigm is based on the BG505 subtype A env gene. Here, we describe the production and characterization of a SOSIP.664 protein derived from a subtype B gene (B41), together with a simple, one-step method to purify native-like trimers by affinity chromatography with a trimer-specific bNAb, PGT145. The resulting trimers will be useful for structural and immunogenicity experiments aimed at devising ways to make an effective HIV-1 vaccine.

  • Sp1 Sites in the Noncoding Control Region of BK Polyomavirus Are Key Regulators of Bidirectional Viral Early and Late Gene Expression [Genome Replication and Regulation of Viral Gene Expression]

  • In kidney transplant patients with BK polyomavirus (BKPyV) nephropathy, viral variants arise bearing rearranged noncoding control regions (rr-NCCRs) that increase viral early gene expression, replicative fitness, and cytopathology. rr-NCCRs result from various deletions and duplications of archetype NCCR (ww-NCCR) sequences, which alter transcription factor binding sites (TFBS). However, the role of specific TFBS is unclear. We inactivated 28 TFBS in the archetype NCCR by selective point mutations and examined viral gene expression in bidirectional reporter constructs. Compared to the archetype, group 1 mutations increased viral early gene expression similar to rr-NCCR and resulted from inactivating one Sp1 or one Ets1 TFBS near the late transcription start site (TSS). Group 2 mutations conferred intermediate early gene activation and affected NF1, YY1, and p53 sites between early and late TSS. Group 3 mutations decreased early and late gene expression and included two other Sp1 sites near the early TSS. Recombinant viruses bearing group 1 NCCRs showed increased replication in human renal epithelial cells similar to clinical rr-NCCR variants. Group 2 and 3 viruses showed intermediate or no replication, respectively. A literature search revealed unnoticed group 1 mutations in BKPyV nephropathy, hemorrhagic cystitis, and disseminated disease.

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

  • Impact of Natural IgM Concentration on Gene Therapy with Adenovirus Type 5 Vectors [Gene Delivery]

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

  • Inhibition of H3K27me3-Specific Histone Demethylases JMJD3 and UTX Blocks Reactivation of Herpes Simplex Virus 1 in Trigeminal Ganglion Neurons [Genome Replication and Regulation of Viral Gene Expression]

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

  • Development of a Mouse-Adapted Live Attenuated Influenza Virus That Permits In Vivo Analysis of Enhancements to the Safety of Live Attenuated Influenza Virus Vaccine [Vaccines and Antiviral Agents]

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

  • Parainfluenza Virus 5 Expressing the G Protein of Rabies Virus Protects Mice after Rabies Virus Infection [Vaccines and Antiviral Agents]

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

  • Overlapping Local and Long-Range RNA-RNA Interactions Modulate Dengue Virus Genome Cyclization and Replication [Genome Replication and Regulation of Viral Gene Expression]

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

  • On the Stability of Parainfluenza Virus 5 F Proteins [Structure and Assembly]

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

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    JVI Accepts: Articles Published Ahead of Print

  • Cellular Transformation of Mouse Embryo Fibroblasts in the Absence of Activator E2Fs [PublishAheadOfPrint]

  • The E2F family of transcription factors, broadly divided into activator and repressor E2Fs, regulates cell cycle genes. Current models indicate that activator E2Fs are necessary for cell cycle progression and tumorigenesis, and are also required to mediate transformation induced by DNA tumor viruses. E2Fs are negatively regulated by the RB family of tumor suppressor proteins, and viral-encoded oncogenes disrupt the RB-E2F repressor complexes. This results in the release of activator E2Fs and induction of E2F-dependent genes. In agreement, expression of large T antigens (TAg) encoded by polyomaviruses in mammalian cells results in increased transcriptional levels of E2F target genes. In addition, tumorigenesis induced by transgenic expression of SV40 TAg in choroid plexus or intestinal villi requires at least one activator E2F. In contrast, we show that SV40 TAg-induced transformation in mouse embryonic fibroblasts is independent of activator E2Fs. This work, coupled with recent studies showing that proliferation in stem and progenitor cells is independent of activator E2Fs, suggest the presence of parallel pathways governing cell proliferation and tumorigenesis.

    Importance The RB-E2F pathway is altered in many cancers and also targeted by DNA tumor viruses. Viral oncoprotein action on RBs results in the release of activator E2Fs and up-regulation of E2F target genes, thus activator E2Fs are considered essential for normal and tumorigenic cell proliferation. However, we have observed that SV40 large T antigen can induce cell proliferation and transformation in the absence of activator E2Fs. Our results also suggest that TAg action on pRBs regulates both E2F dependent and independent pathways that govern proliferation. Thus, specific cell proliferation pathways affected by RB alterations in cancer may be a factor in tumor behavior and response to therapy.

  • Infection History Determines the Differentiation State of Human CD8+ T-cells [PublishAheadOfPrint]

  • After resolution of the acute phase of infection, otherwise quiescent antigen-experienced CD8+ T-cells confer rapid protection upon re-infection with viral pathogens, or in case of persistent viruses, help to maintain control of the infection. Depending on the type of virus, antigen-specific CD8+ T-cells have distinct traits, ranging from typical memory cell properties in the case of rapidly cleared viruses, to immediate effector functions for persistent viruses. We here show that both the differentiation stage defined by the expression of cell surface markers, such as CD45RA, CCR7, CD28 and CD27 and distinct expression levels of T-bet and eomesodermin (Eomes) predict the functional profile of antigen-experienced CD8+ T cells. Furthermore, virus-specific CD8+ T cells targeting different respiratory syncytial virus-, influenza A virus-, Epstein-Barr virus-, human cytomegalovirus- and HIV-1-specific epitopes, adopt distinct T-bet and Eomes expression patterns that appear to be installed early during the primary response. Importantly, the associations between surface phenotype, T-bet/Eomes expression levels and expression of markers that predict CD8+ T-cell function, change according to viral infection history, particularly against the background of human immunodeficiency virus-1, and to lesser extent, also of human cytomegalovirus and/or Epstein-Barr virus infection. Thus, functionality of human antigen-experienced CD8+ T cells follows at least two dimensions, one outlined by the surface phenotype and another by the T-bet/Eomes expression level, which is determined by previous or persistent viral challenges.

    Importance Functional human CD8+ T-cell subsets have been defined using surface markers like CD45RA, CCR7, CD28 and CD27. However, the induction of function-defining traits, like granzyme B expression, is controlled by transcription factors like T-bet and Eomes. Here we describe how T-bet and Eomes levels distinctly relate to the expression of molecules predictive for CD8+ T-cell function in a surface phenotype-independent manner. Importantly, we found that central-memory- and effector-memory CD8+ T-cell subsets differentially express T-bet and Eomes and molecules predictive for function according to viral infection history, particularly so in the context of HIV-1 infection and to lesser extent also of latent EBV and/or hCMV nndash;infected, otherwise healthy adults. Finally, we show that the distinct phenotypes and T-bet/Eomes levels of different virus-specific CD8+ T-cell populations are imprinted early during the acute phase of primary infection in vivo. These findings broaden our understanding of CD8+ T-cell differentiation.

  • Anti-APOBEC3G activity of HIV-1 Vif protein is attenuated in elite controllers [PublishAheadOfPrint]

  • HIV-1-infected individuals who control viremia to below the limit of detection without antiviral therapy have been termed elite controllers (EC). Functional attenuation of some HIV-1 proteins has been reported in EC. The HIV-1 accessory protein Vif enhances viral infectivity through APOBEC3G degradation; however, little is known regarding Vif function in EC. Here, the anti-APOBEC3G activity of clonal, plasma HIV RNA-derived Vif sequences from 46 EC, 46 non-controllers (NC) and 44 individuals with acute infection (AI) were compared. VSV-G-pseudotyped viruses were generated by co-transfecting 293T cells with expression plasmids encoding patient-derived Vif, human APOBEC3G, VSV-G and a vif/env-deficient luciferase-reporter HIV-1 proviral DNA clone. Viral stocks were used to infect 293T cells, and Vif anti-APOBEC3G activity was quantified in terms of luciferase signal. On average, the anti-APOBEC3G activities of EC-derived Vif sequences [median log10 RLU 4.54 (IQR 4.30 - 4.66)] were significantly lower than those derived from NC [4.75 (4.60 - 4.92), pllt;0.0001] and AI [4.74 (4.62 - 4.94), pllt;0.0001]. Reduced Vif activities were not associated with particular HLA class I alleles expressed by the host. Vif functional motifs were highly conserved in all patient groups. No single viral polymorphism could explain the reduced anti-APOBEC3G activity of EC-derived Vif, suggesting that various combinations of minor polymorphisms may underlie these effects. These results further support relative attenuation of viral protein function in EC-derived HIV sequences.

    Importance HIV-1 Elite Controllers (EC) are rare individuals who are able to control plasma viremia to undetectable levels without antiretroviral therapy. Understanding the pathogenesis and mechanisms underpinning this rare phenotype may provide important insights for HIV vaccine design. The EC phenotype is associated with beneficial host immunogenetic factors (such as HLA-B*57) as well as attenuated viral protein functions (e.g. Gag, Pol and Nef). In this study, we demonstrate that HIV-1 Vif sequences isolated from EC display relative impairments in their ability to counteract the host restriction factor APOBEC3G compared to Vif sequences from normal progressors and acutely-infected individuals. This result extends the growing body of evidence demonstrating attenuated HIV-1 protein function in EC, and in particular supports the relevance of viral factors in contributing to this rare HIV-1 phenotype.

  • A novel endogenous betaretrovirus in the common vampire bat (Desmodus rotundus) suggests multiple independent infection and cross-species transmission events [PublishAheadOfPrint]

  • The Desmodus rotundus endogenous betaretrovirus (DrERV) is fixed in the vampire bat D. rotundus population and in other phyllostomid bats, but is not present in all species from this family. DrERV is not phylogenetically related to Old World bat betaretroviruses, but to betaretroviruses from rodents and New World primates suggesting recent cross-species transmission. A recent integration age estimation of the provirus in some taxa indicates that an exogenous counterpart might have been in circulation.

  • Human papillomavirus type 16 oncoprotein expression is controlled by the cellular splicing factor SRSF2 (SC35). [PublishAheadOfPrint]

  • High risk human papillomaviruses (HR-HPV) cause anogenital cancers, including cervical cancer, and head and neck cancers. Human papillomavirus type 16 (HPV16) is the most prevalent HR-HPV. HPV oncogenesis is driven by two viral oncoproteins, E6 and E7, which are expressed through alternative splicing of a polycistronic RNA to yield four major splice isoforms (E6 full length, E6*I, E6*II, E6*X). Multiple RNA production from a single gene is controlled by SR splicing factors (SRSFs) and HPV16 infection induces overexpression of a subset of these, SRSFs1, 2 and 3. In this study we examined whether these proteins could control HPV16 oncoprotein expression. siRNA depletion experiments revealed that SRSF1 did not affect oncoprotein RNA levels. While SRSF3 knockdown caused some reduction in E6E7 expression depletion of SRSF2 resulted in a significant loss of E6E7 RNAs resulting in reduced levels of the E6-regulated p53 proteins and E7 oncoprotein itself. SRSF2 contributed to the tumour phenotype of HPV16-positive cervical cancer cells as its depletion resulted in decreased cell proliferation, reduced colony formation and increased apoptosis. SRSF2 did not affect transcription from the P97 promoter that controls viral oncoprotein expression. Rather, RNA decay experiments showed that SRSF2 is required to maintain stability of E6E7 mRNAs. These data show that SRSF2 is a key regulator of HPV16 oncoprotein expression and cervical tumour maintenance.

    Importance Expression of the HPV 16 oncoproteins E7 and E6 drives HPV-associated tumour formation. Although increased transcription may yield increased levels of E6E7 mRNAs, it is known that the RNAs can have increased stability upon integration into the host genome. SR splicing factors (SRSFs) control splicing but can also control other events in the RNA life cycle including RNA stability. Previously, we demonstrated increased levels of SRSFs 1-3 during cervical tumour progression. Now we show that SRSF2 is required for expression of E6E7 mRNAs in cervical tumour, but not nontumour cells, and may act by inhibiting their decay. SRSF2 depletion in W12 tumour cells resulted in increased apoptosis, decreased proliferation and decreased colony formation suggesting that SRSF2 has oncogenic functions in cervical tumour progression. SRSF function can be targeted by known drugs that inhibit SRSF phosphorylation suggesting a possible new avenue in abrogating HPV oncoprotein activity.

  • Activation of NF{kappa}B by human papillomavirus 16 E1 limits E1-dependent viral replication through degradation of E1. [PublishAheadOfPrint]

  • NFB is a family of transcription factors that regulate gene expression involved in many processes such as the inflammatory response and cancer progression. Little is known about associations of NFB with the HPV life cycle. We have developed a tissue culture system to conditionally induce E1-dependent replication of HPV16 genome in human cervical keratinocytes, and found that expression of HPV16 E1, a viral helicase, results in reduction of IBaalpha; and subsequent activation of NFB in a manner dependent on helicase activity. Exogenous expression of a degradation-resistant mutant of IBaalpha;, which inhibits the activation of NFB, enhanced E1-dependent replication of the viral genome. Wortmannin, a broad inhibitor of phosphoinositide 3-kinases (PI3Ks), and to a lesser extent, VE-822, an ATR kinase inhibitor, but not KU55933, an ATM kinase inhibitor, suppressed the activation of NFB and augmented E1-dependent replication of HPV16 genome. Interestingly, the enhancement of E1-dependent replication of the viral genome was associated with increased stability of E1 in the presence of Wortmannin as well as the IBaalpha; mutant. Collectively, we propose that expression of E1 induces NFB activation at least in part through the ATR-dependent DNA damage response and NFB in turn limits E1-dependent replication of HPV16 through degradation of E1, so that E1 and NFB may constitute a negative feedback loop.

    Importance A major risk factor in HPV-associated cancers is persistent infection with high-risk HPVs. To eradicate viruses from infected tissue, it is important to understand molecular mechanisms underlying the establishment and maintenance of persistent infection. In this study, we obtained evidence that HPV16 E1, a viral DNA helicase essential for amplification of the viral genomes, induces NFB activation and that this limits E1-dependent genome replication of HPV16. These results suggest that NFB mediates a negative feedback loop to regulate HPV replication and this feedback loop could be associated with control of the viral copy numbers. We could thus show for the first time that NFB activity is involved in the establishment and maintenance of persistent HPV infection.

  • Cellular STAT3 functions via PCBP2 to restrain EBV lytic activation in B lymphocytes [PublishAheadOfPrint]

  • A major hurdle to killing EBV-infected tumor cells using oncolytic therapy is the presence of a substantial fraction of EBV-infected cells that does not support the lytic phase of EBV despite exposure to lytic-cycle promoting agents. To determine the mechanism(s) underlying this refractory state, we developed a strategy to separate lytic from refractory EBV+ cells. By examining the cellular transcriptome in separated cells, we previously discovered that high levels of host STAT3 (signal transducer and activator of transcription 3) curtail susceptibility of latently-infected cells to lytic-cycle activation signals. The goals of the present study were two-fold: 1) determine the mechanism of STAT3-mediated resistance to lytic-activation, and 2) exploit our findings to enhance susceptibility to lytic-activation. We therefore analyzed our microarray dataset, cellular proteomes of separated lytic and refractory cells, and a publically available STAT3 ChIP-Seq dataset to identify cellular PCBP2 [poly(C) binding protein 2], an RNA-binding protein, as a transcriptional target of STAT3 in refractory cells. Using Burkitt lymphoma cells and EBV+ cell lines from patients with hypomorphic STAT3 mutations, we demonstrate that single cells expressing high levels of PCBP2 are refractory to spontaneous and induced EBV lytic activation, STAT3 functions via cellular PCBP2 to regulate lytic susceptibility, and suppression of PCBP2 levels is sufficient to increase the number of EBV lytic cells. We expect these findings and the genome-wide resources that they provide, to accelerate our understanding of a long-standing mystery in EBV biology and guide efforts to improve oncolytic therapy for EBV-associated cancers.

    Importance Most humans are infected with Epstein-Barr virus (EBV), a cancer-causing virus. While EBV generally persists silently in B lymphocytes, periodic lytic (re-)activation of latent virus is central to its life cycle and to most EBV-related diseases. However, a substantial fraction of EBV-infected B cells and tumor cells in a population is refractory to lytic activation. This resistance to lytic activation directly and profoundly impacts viral persistence and effectiveness of oncolytic therapy for EBV+ cancers. To identify the mechanisms that underlie susceptibility to EBV-lytic activation, we used host gene- and protein-expression profiling of separated-lytic and -refractory cells. We find that STAT3, a transcription factor overactive in many cancers, regulates PCBP2, a protein important in RNA biogenesis, to regulate susceptibility to lytic-cycle activation signals. These findings advance our understanding of EBV-persistence and provide important leads on devising methods to improve viral oncolytic therapies.

  • A role for 3-O sulfated heparan sulfate in promoting human cytomegalovirus infection in human iris cell [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) has emerged as a clinically opportunistic pathogen which targets multiple types of ocular cells and tissues including the iris region of the uveal tract during anterior uveitits. In this report we used primary cultures of human iris stroma (HIS) cells derived from human eye donors to investigate HCMV entry. The following lines of evidence suggested the role of 3-O sulfated heparan sulfate (3-OS HS) during HCMV mediated entry and cell-to-cell fusion in HIS cells. First, 3-OST-3 expression in HIS cell promoted HCMV internalization, while pre-treatment of HIS cells with heparinase enzyme or with anti-3-OS HS (G2) peptide significantly reduced the HCMV mediated plaque/foci formation. Second, co-culture of the HCMV infected HIS cells with CHO-K1 cells expressing 3-OS HS significantly enhanced cell fusion. Finally, a similar trend of enhanced fusion was observed with HCMV glycoproteins (gB, gO, gH-gL)-expressing cells co-cultured with 3-OS HS cells. Taken together, these results highlight the role of 3-OS HS during HCMV plaque and cell-to-cell fusion and identify a novel target for future therapeutic interventions.

  • Tegument protein ORF33 of a gammaherpesvirus is associated with intranuclear capsids at an early stage of tegumentation process [PublishAheadOfPrint]

  • Herpesvirus nascent capsids, after assembly in the nucleus, must acquire a variety of tegument proteins during maturation. However, little is known about the identity of the tegument proteins that are associated with capsids in the nucleus or the molecular mechanisms involved in nuclear egress of capsids into cytoplasm, especially for the two human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), due to lack of efficient lytic replication systems. Murine gammaherpesvirus 68 (MHV-68) is genetically related to human gammaherpesviruses and serves as an excellent model to study de novo lytic replication of gammaherpesviruses. We have previously shown that open reading frame 33 (ORF33) of MHV-68 is a tegument protein of mature virions and is essential for virion assembly and egress. However, it remained unclear how ORF33 is incorporated into virions. In this study, we first showed that the endogenous ORF33 protein co-localizes with capsid proteins at discrete areas in the nucleus during viral infection. Co-sedimentation analysis as well as immunoprecipitation assay demonstrated that ORF33 is associated with both nuclear and cytoplasmic capsids. Immunogold labeling experiment using an anti-ORF33 monoclonal antibody revealed that ORF33-rich areas in the nucleus are surrounded by immature capsids. Moreover, ORF33 is associated with nucleocapsids prior to primary envelopment as well as with mature virions in the cytoplasm. Finally, we showed that ORF33 interacts with two capsid proteins, suggesting that nucleocapsids may interact with ORF33 in a direct manner. In summary, we identified ORF33 as a tegument protein that is associated with intranuclear capsids prior to primary envelopment, likely through interacting with capsid proteins in a direct manner.

    Importance Morphogenesis is an essential step in virus propagation that leads to the generation of progeny virions. For herpesviruses, this is a complicated process that starts in the nucleus. Although capsid assembly and genome packaging is relatively well understood, how capsids acquire tegument (the layer between capsid and envelope in a herpesvirion) and whether the initial tegumentation process takes place in the nucleus remain unclear. We previously showed that ORF33 of MHV-68 is a tegument protein and functions both in the nuclear egress of capsids and final virion maturation in the cytoplasm. In the present study, we showed that ORF33 is associated with intranuclear capsids prior to primary envelopment and identified novel interactions between ORF33 and two capsid proteins. Our work provides new insights into the association between tegument proteins and nucleocapsids an early stage of virion maturation process for herpesviruses.

  • Oropouche virus infection and pathogenesis is restricted by MAVS, IRF-3, IRF-7, and type I IFN signaling pathways in non-myeloid cells [PublishAheadOfPrint]

  • Oropouche virus (OROV) is a member of the Orthobunyavirus genus in the Bunyaviridae family and a prominent cause of insect-transmitted viral disease in Central and South America. Despite its clinical relevance, little is known about OROV pathogenesis. To define the host defense pathways that control OROV infection and disease, we evaluated OROV pathogenesis and immune responses in primary cells and mice that were deficient in the RIG-I-like receptor signaling pathway (MDA5, RIG-I, or MAVS), downstream regulatory transcription factors (IRF-3 or IRF-7), IFN-bbeta;, or the receptor for type I IFN signaling (IFNAR). OROV replicated to higher levels in primary fibroblasts and dendritic cells lacking MAVS signaling, the transcription factors IRF-3 and IRF-7, or IFNAR. In mice, deletion of IFNAR, MAVS, or IRF-3 and IRF-7 resulted in uncontrolled OROV replication, hypercytokinemia, extensive liver damage, and death whereas wild-type (WT) congenic animals failed to develop disease. Unexpectedly, mice with a selective deletion of IFNAR on myeloid cells (CD11c Cre+ Ifnarf/f or LysM Cre+ Ifnarf/f) did not sustain enhanced disease with OROV or La Crosse virus, a closely related encephalitic orthobunyavirus. In bone marrow chimera studies, recipient irradiated Ifnar-/- mice reconstituted with WT hematopoietic cells sustained high levels of OROV replication and liver damage, whereas WT mice reconstituted with Ifnar-/- bone marrow were resistant to disease. Collectively, these results establish a dominant protective role for MAVS, IRF-3 and IRF-7, and IFNAR in restricting OROV virus infection and tissue injury, and suggest that IFN signaling in non-myeloid cells contributes to the host defense against orthobunyaviruses.

    IMPORTANCE Oropouche virus (OROV) is an emerging arthropod-transmitted orthobunyavirus that causes episodic outbreaks of a debilitating febrile illness in humans in countries of South and Central America. The continued expansion of the range and number of its arthropod vectors increases the likelihood that OROV will spread into new regions. At present, the pathogenesis of OROV in humans or other vertebrate animals remains poorly understood. To define cellular mechanisms of control of OROV infection, we performed infection studies in a series of primary cells and mice that were deficient in key innate immune genes involved in pathogen recognition and control. Our results establish that a MAVS-dependent type I IFN signaling pathway has a dominant role in restricting OROV infection and pathogenesis in vivo.

  • Prominent amphibian (Xenopus laevis) tadpole type III interferon response to the Frog Virus 3 ranavirus [PublishAheadOfPrint]

  • Ranaviruses (Iridoviridae) are posing an increasing threat to amphibian populations, with anuran tadpoles being particularly susceptible to these viral infections. Moreover, amphibians are the most basal phylogenetic class of vertebrates known to possess both type I and type III interferon-mediated immunity. Moreover, little is known regarding the respective roles of these mediators during amphibian antiviral defenses. Accordingly, we transcriptionally and functionally compared the amphibian Xenopus laevis type I (IFN) and III (IFN) IFNs in the context of infections by the ranavirus Frog Virus 3 (FV3). X. laevis IFN and IFN displayed distinct tissue expression profiles. In contrast to our previous findings that X. laevis tadpoles exhibit delayed and modest type I IFN responses to FV3 infections compared to adults, here we report that tadpoles mount timely and robust type III IFN gene responses. Recombinant forms of these cytokines (rXlIFN, rXlIFN) elicited antiviral gene expression in the kidney-derived A6 cell line as well as in tadpole leukocytes and tissues. However, in comparison to rXlIFN, rXlIFN was less effective in preventing FV3 replication in A6 cells and tadpoles, and inferior at promoting tadpole survival. Intriguingly, FV3 impaired the A6 cell and tadpole kidney type III IFN receptor gene expression. Furthermore, compared to rXlIFN, rXlIFN conferred equal or greater protection of A6 cultures against recombinant viruses deficient for the putative immune evasion genes, vCARD or a truncated vIF2-aalpha;. Thus, in contrast to previous beliefs, tadpoles possess intact antiviral defenses reliant on type III IFNs, which are overcome by FV3 pathogens.

    IMPORTANCE Anuran tadpoles, including those of Xenopus laevis are particularly susceptible to infection by ranavirus such as FV3. We investigated the respective roles of X. laevis type I and type III interferons (IFN and IFN, respectively) during FV3 infections. Notably, tadpoles mounted timely and more robust IFN gene expression responses to FV3 than adults, contrasting with the poorer tadpole type I IFN responses. However, a recombinant X. laevis (rXl) IFN conferred less protection to tadpoles and the A6 cell line than rXlIFN, which may be explained by the FV3 impairment of IFN receptor gene expression. The importance of IFN in tadpole anti-FV3 defenses is underlined by the critical involvement of two putative immune-evasion genes in FV3 resistance to IFN and IFN-mediated responses. These findings challenge the view that tadpoles have defective antiviral immunity and rather suggest that their antiviral responses are predominated by IFN responses, which are overcome by FV3.

  • Functional analysis of the short isoform of orf virus protein OV20.0 [PublishAheadOfPrint]

  • Orf virus (ORFV) OV20.0L is an ortholog of vaccinia virus (VACV) gene E3L. The function of VACV E3 protein as a virulence factor is well studied, but OV20.0 has received less attention. Here we show that like VACV E3L, OV20.0L encodes two proteins, a full-length protein and a shorter form (sh20). The shorter sh20 is an N-terminal truncated OV20.0 isoform generated when a downstream AUG codon is used for initiating translation. These isoforms differed in cellular localisation, with full-length OV20.0 and sh20 found throughout the cell and predominantly in the cytoplasm, respectively. None-the-less, both OV20.0 isoforms were able to bind dsRNA-activated protein kinase (PKR) and dsRNA. Moreover, both isoforms strongly inhibited PKR activation as shown by decreased phosphorylation of the translation initiation factor eIF2aalpha; subunit and protection of Sindbis virus infection against the activity of interferon (IFN). In spite of this apparent conservation of function in vitro, a recombinant ORFV that could only express the sh20 isoform was attenuated in a mouse model.

    Importance The OV20.0 protein of Orf virus (ORFV) has two isoforms and contributes to the virulence, but the roles of the two forms are not known. This study shows that the shorter isoform (sh20) arises due to use of a downstream initiation codon and is amino-terminally truncated. The sh20 form also differs in expression kinetics and cellular localisation compared with full length OV20.0. Similar to the full length isoform, sh20 is able to bind dsRNA and PKR, inactivate PKR and thus act as an antagonist of the interferon response in vitro. In vivo however, wild type OV20.0 could not be replaced with sh20 alone without a loss of virulence suggesting that the functions of the isoforms are not simply redundant.

  • Broad-spectrum inhibitors against 3C-like proteases of feline coronaviruses and feline caliciviruses [PublishAheadOfPrint]

  • Feline infectious peritonitis and virulent, systemic-calicivirus infection are caused by certain types of feline coronaviruses (FCoV) and feline caliciviruses (FCV), respectively, and are important infectious diseases with high fatality rates in members of the Felidae family. While FCoV and FCV belong to two distinct virus families of Coronaviridae and Caliciviridae, respectively, they share dependence on viral 3C-like protease (3CLpro) for their replication. Since 3CLpro is functionally and structurally conserved among these viruses and essential for viral replication, 3CLpro is considered a potential target for antiviral drug design with broad-spectrum activities against these distinct and highly important viral infections. However, small molecule 3CLpro inhibitors for FCoV and FCV have not been previously identified. In this study, derivatives of peptidyl compounds targeting 3CLpro were synthesized and evaluated against FCoV and FCV. Structures of compounds that show potent dual antiviral activities with a wide margin of safety were identified and discussed. Furthermore, the in vivo efficacy of 3CLpro inhibitors was evaluated using a mouse model of coronavirus infection. Intraperitoneal administration of two 3CLpro inhibitors in mice infected with murine hepatitis virus-A59, a hepatotrophic coronavirus, resulted in significant reduction in virus titers and pathological lesions in the liver compared to controls. These results suggest that the series of 3CLpro inhibitors described here may have a potential to be further developed as therapeutic agents for these important viruses in domestic and wild cats. This study provides important insights into the structure and function relationships in 3CLpro for the antiviral drug design with broader antiviral activities.

    Importance Feline infectious peritonitis virus (FIPV) is the leading cause of death in young cats and virulent, systemic calicivirus (vs-FCV) causes a highly fatal disease in cats for which no preventive or therapeutic measure is available. These distinct viruses that belong to different virus families encode structurally and functionally conserved 3C-like protease (3CLpro) which is a potential target for broad-spectrum antiviral drug development. However no studies have previously reported a structural platform for antiviral drug design for these viruses or the efficacy of 3CLpro inhibitors against coronavirus infection in experimental animals. In this study, we explored the structure-activity relationships of the derivatives of 3CLpro inhibitors and identified inhibitors with potent dual activities against these viruses. In addition, the efficacy of the 3CLpro inhibitors was demonstrated in mice infected with a murine coronavirus. Overall, our study provides the first insight into a structural platform for anti-FIPV and FCV drug development.

  • Viral DNA replication-dependent DNA damage response activation during BK polyomavirus infection [PublishAheadOfPrint]

  • BK polyomavirus (BKPyV) reactivation is associated with severe human disease in kidney and bone marrow transplant patients. The interplay between viral and host factors that regulates the productive infection process remains poorly understood. We have previously reported that the cellular DNA damage response (DDR) is activated upon lytic BKPyV infection and that activation is required for optimal viral replication in primary kidney epithelial cells. In this report, we set out to determine what viral components are responsible for activating the two major phosphatidylinositol 3-kinase-like kinases (PI3KKs) involved in the DDR: ataxia telangiectasia mutated (ATM), and ATM and Rad3-related (ATR) kinase. Using a combination of UV treatment, lentivirus transduction, and mutant virus infection experiments, our results demonstrate that neither the input virus nor the expression of large T antigen (TAg) alone is sufficient to trigger the activation of ATM or ATR in our primary culture model. Instead, our data suggest that the activation of both ATM- and ATR-mediated DDR pathways is linked to viral DNA replication. Intriguingly, a TAg mutant virus that is unable to activate the DDR causes substantial host DNA damage. Our study provides insight into how DDRs are activated by polyomaviruses in primary cells with intact cell cycle checkpoints and how the activation might be linked to the maintenance of host genome stability.

    Importance Polyomaviruses are opportunistic pathogens that are associated with several human diseases under immunosuppressed conditions. BK polyomavirus (BKPyV) affects mostly kidney and bone marrow transplant patients. The detailed replication mechanism of these viruses remains to be determined. We have previously reported that BKPyV activates the host DNA damage response (DDR), a response normally used by the host cell to combat genotoxic stress, to aid its own replication. In this study we identified that the trigger for DDR activation is viral replication. Furthermore, we show that the virus is able to cause host DNA damage in the absence of viral replication and DDR activation. These results suggest an intricate relationship between viral replication, DDR activation, and host genome instability.

  • Host Proteome Correlates of Vaccine-Mediated Enhanced Disease in a Mouse Model of Respiratory Syncytial Virus Infection [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants. Despite over 50 years of research, to date no safe and efficacious RSV vaccine has been licensed. Many experimental vaccination strategies failed to induce balanced T-helper (Th) responses and were associated with adverse effects such as hypersensitivity and immunopathology upon challenge. In this study we explored the well-established recombinant vaccinia virus (rVV) RSV F/G vaccination-challenge mouse model to study phenotypically distinct vaccine mediated host immune responses at the proteome level. In this model, rVV-G priming and not rVV-F priming results in the induction of Th2 skewed host responses upon RSV challenge. Mass spectrometry-based spectral count comparisons enabled us to identify seven host proteins for which expression in lung tissue is associated with an aberrant Th2 skewed response characterized by the influx of eosinophils and neutrophils. These proteins are involved in processes related to the influx of eosinophils directly (Epx), chemotaxis and extravasion processes (Chil3) as well as eosinophil and neutrophil homing signals to the lung (Itgam). In addition, the increased protein levels of Arg1 and Chil3 point at a functional and regulatory role for alternatively activated macrophages and type 2 innate lymphoid cells in Th2 cytokine driven RSV vaccine-mediated enhanced disease.

    Importance RSV alone is responsible for 80% of acute bronchiolitis cases in infants worldwide and causes substantial mortality in developing countries. Clinical trials with formalin-inactivated RSV vaccine preparations in the 1960s failed to induce protection upon natural RSV infection, and even predisposed for enhanced disease. Despite the clinical need, to date no safe and efficacious RSV vaccine has been licensed. Since RSV vaccines have a tendency to prime for unbalanced responses associated with an exuberant influx of inflammatory cells and enhanced disease, detailed characterization of primed host responses has become a crucial element in RSV vaccine research. We investigated the lung proteome of mice challenged with RSV upon priming with vaccine preparations known to induce phenotypically distinct host responses. Seven host proteins have been identified which expression levels associated with vaccine mediated enhanced disease. The identified protein biomarkers support the development, as well as detailed evaluation of next generation RSV vaccines.

  • A basic cluster in the N-terminus of yellow fever virus NS2A contributes to infectious particle production [PublishAheadOfPrint]

  • The flavivirus NS2A protein is involved in the assembly of infectious particles. To further understand its role in this process, a charged-to-alanine scanning analysis was performed on NS2A encoded by an infectious cDNA clone of yellow fever virus (YFV). Fifteen mutants containing single, double or triple charged-to-alanine changes were tested. Five of them did not produce infectious particles, whereat efficient RNA replication was detectable for two of the five NS2A mutants (R22A-K23A-R24A and R99A-E100A-R101A). Prolonged cultivation of transfected cells resulted in the recovery of pseudorevertants. Besides suppressor mutants in NS2A, a compensating second site mutation in NS3 (D343G) arose for the NS2A R22A-K23A-R24A mutant. We found this NS3 mutation previously to be suppressive for the NS2Aaalpha; cleavage site mutant Q189S also deficient in virion assembly. Here, the subsequently suggested interaction between NS2A and NS3 was proven by co-immunoprecipitation analyses. Using selectively permeabilized cells, we could demonstrate that the regions encompassing R22A-K23A-R24A and Q189S in NS2A are localized to the cytoplasm where NS3 is also known to reside. However, the defect in particle production observed for the NS2A R22A-K23A-R24A and Q189S mutants was not due to a defect in physical interaction between NS2A and NS3 as the NS2A mutations did not interrupt NS3 interaction. In fact, a region just upstream of R22-K23-R24 was mapped to be critical for NS2A-NS3 interaction. Taken together, these data support a complex interplay between YFV NS2A and NS3 in virion assembly and identify a basic cluster in the NS2A N-terminus to be critical in this process.

    IMPORTANCE Despite an available vaccine, yellow fever remains endemic in tropical areas of South America and Africa. To control the disease, antiviral drugs are required and understanding the determinants of virion assembly is central to their development. Here, we identified a basic cluster of amino acids in the N-terminus of YFV NS2A, which inhibited virion assembly upon mutation. The defect was rescued by a spontaneously occurring mutation in NS3. Our study proofs an interaction between NS2A and NS3, which, remarkably, was maintained for the NS2A mutant in the presence and absence of the NS3 mutation. This suggests a role for other viral and/or cellular proteins in virion assembly. Residues important for YFV virion production reported here only partially coincided with those reported for other flaviviruses suggesting the determinants for particle production are virus-specific. Reconstruction of a YFV encoding tagged NS2A paves the way to identify further NS2A interaction partners.

  • Peptides designed to spatially depict the Epstein-Barr virus major virion glycoprotein gp350 neutralization epitope elicit antibodies that block virus neutralizing antibody 72A1 interaction with the native gp350 molecule [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) is the etiologic agent of infectious mononucleosis and the root cause of B-cell lymphoproliferative disease in individuals with a weakened immune system, as well as a principal cofactor in nasopharyngeal carcinoma, various lymphomas and other cancers. The EBV major virion surface glycoprotein (gp)350 is viewed as the best vaccine candidate to prevent infectious mononucleosis in healthy EBV-naive persons and EBV-related cancers in at-risk individuals. Previous epitope mapping of gp350 reveals only one dominant neutralizing epitope, which has been shown to be the target of the monoclonal antibody 72A1. Computer modeling of 72A1 antibody interaction with the gp350 amino terminus was used to identify gp350 amino acids that could form strong ionic, electrostatic or hydrogen bonds with the 72A1 antibody. Peptide DDRTTLQLAQNPVYIPETYPYIKWDN (designated as peptide 2) and peptide GSAKPGNGSYFASVKTEMLGNEID (designated as peptide 3) were designed to spatially represent the gp350 amino acids predicted to interact with the 72A1 antibody paratope. Peptide 2 bound to the 72A1 antibody and blocked 72A1 antibody recognition of the native gp350 molecule. Peptide 2 and peptide 3 were recognized by human IgG and shown to elicit murine antibodies that could target gp350 and block its recognition by the 72A1 antibody. This work provides a structural mapping of the interaction between the EBV neutralizing antibody 72A1 and the major virion surface protein gp350. Gp350 mimetic peptides that spatially depict the EBV neutralizing epitope would be useful as a vaccine to focus the immune system exclusively to this important virus epitope.

    Importance The production of virus-neutralizing antibodies targeting the Epstein-Barr virus (EBV) major surface glycoprotein gp350 is important for the prevention of infectious mononucleosis and EBV-related cancers. The data presented here provide the first in silico map of gp350 interaction with a virus blocking monoclonal antibody. Immunization of gp350 peptides identified by in silico mapping generated antibodies that cross-react with the EBV gp350 molecule and block recognition of the gp350 molecule by a virus neutralizing antibody. Through its ability to focus the immune system exclusively on the gp350 sequence important for viral entry, these peptides may form the basis of an EBV vaccine candidate. This strategy would side-step the production of other irrelevant gp350 antibodies that divert the immune system from generating a protective antiviral response or that impede access to the virus blocking epitope by protective antibodies.

  • ORF57 overcomes the detrimental sequence bias of Kaposi sarcoma herpesvirus lytic genes [PublishAheadOfPrint]

  • Kaposi's sarcoma herpesvirus (KSHV) encodes ORF57, which enhances the expression of intronless KSHV genes on multiple post-transcriptional levels. Yet, it remains elusive how ORF57 recognizes viral RNAs. Here, we demonstrate that ORF57 also increases the expression of the multiple intron-containing K15 gene. The nucleotide bias of the K15 cDNA revealed an unusual high A/T content. Thus, we optimized the K15 cDNA by raising the frequency of G/C nucleotides yielding an ORF57-independent version. To further prove the importance of the sequence bias of ORF57-dependent RNAs, we grouped KSHV mRNAs according to their A/T content and found a correlation between A/T-richness and ORF57-dependency. More importantly, latent genes, which have to be expressed in the absence of ORF57, have a low A/T content and are indeed ORF57-independent. The nucleotide composition of K15 resembles that of HIV gag, which cannot be expressed unless RNA export is facilitated by the HIV Rev protein. Interestingly, ORF57 can partially rescue HIV Gag expression. Thus, the KSHV target RNAs of ORF57 and HIV gag RNA may share certain motifs based on the nucleotide bias. A bioinformatic comparison between wild type and sequence-optimized K15 revealed a higher density for hnRNP-binding motifs in the former. We speculate that binding of particular hnRNPs to KSHV lytic transcripts is the prerequisite for ORF57 to enhance their expression.

    Importance The mostly intron-less genes of KSHV are only expressed in presence of the viral regulator protein ORF57, but how ORF57 recognizes viral RNAs remains elusive. We focused on the multiple intron-containing KSHV gene K15 and revealed that its expression is also increased by ORF57. Moreover, sequences in the K15 cDNA mediate this enhancement. The quest for a target sequence or a response element for ORF57 in the lytic genes was not successful. Instead we found the nucleotide bias to be the critical determinant of ORF57 dependency. Based on the fact that ORF57 has only a weak affinity for nucleic acids, we speculate that a cellular RNA-binding protein provides the sequence preference for ORF57. This study provides evidence that herpesviral RNA regulator proteins use the sequence bias of lytic genes and the resulting composition of the viral mRNP to distinguish between viral and cellular mRNAs.

  • Transfection of Sclerotinia sclerotiorum with in vitro transcripts of a naturally occurring interspecific recombinant of Sclerotinia sclerotiorum hypovirus 2 significantly reduces virulence of the fungus [PublishAheadOfPrint]

  • A recombinant strain of Sclerotinia sclerotiorum hypovirus 2 (SsHV2) was identified from a North American Sclerotinia sclerotiorum isolate (#328) from lettuce (Lactuca sativa L.) by high-throughput sequencing of total RNA. The 5rrsquo; and 3rrsquo; terminal regions of the genome were determined by rapid amplification of cDNA ends. The assembled nucleotide sequence was up to 92% identical to two recently reported SsHV2 strains, but contained a deletion near its 5rrsquo; terminus of more than 1.2 kb relative to the other SsHV2 strains and an insertion of 524 nt that was distantly related to Valsa ceratosperma hypovirus 1. This suggests that the new isolate is a heterologous recombinant of SsHV2 with a yet uncharacterized hypovirus. We named the new strain Sclerotinia sclerotiorum hypovirus 2 Lactuca (SsHV2L) and deposited the sequence in Genbank with accession number KF898354. Sclerotinia sclerotiorum isolate #328 was coinfected with a strain of Sclerotinia sclerotiorum endornavirus 1 and debilitated compared to cultures of the same isolate that had been cured of virus infection by cycloheximide treatment and hyphal tipping. To determine whether SsHV2L alone could induce hypovirulence in S. sclerotiorum, a full-length cDNA of the 14,538-nt viral genome was cloned. Transcripts corresponding to the viral RNA were synthesized in vitro and transfected into a virus-free isolate of S. sclerotiorum, DK3. Isolate DK3 transfected with SsHV2L was hypovirulent on soybean and lettuce and exhibited delayed maturation of sclerotia relative to virus-free DK3, completing Koch's postulates for the association of hypovirulence with SsHV2L.

    IMPORTANCE A cosmopolitan fungus, S. sclerotiorum infects more than 400 plant species and causes a plant disease known as white mold that produces significant yield losses in major crops annually. Mycoviruses have been used successfully to reduce losses caused by fungal plant pathogens, but definitive relationships between hypovirus infections and hypovirulence in S. sclerotiorum was lacking. By establishing a cause-and-effect relationship between SsHV2L infection and the reduction in host virulence, we showed direct evidence that hypoviruses have the potential to reduce the severity of white mold disease. In addition to intraspecific recombination, this study showed that recent interspecific recombination is an important factor shaping viral genomes. The construction of an infectious clone of SsHV2L allows future exploration of the interactions between SsHV2L and S. sclerotiorum, a widespread fungal pathogen of plants.

  • A survey of the interactome of KSHV ORF45 revealed its binding to viral ORF33 and cellular USP7 resulting in stabilization of ORF33 that is required for production of progeny viruses [PublishAheadOfPrint]

  • ORF45 of Kaposi's sarcomanndash;associated herpesvirus (KSHV) is a gamma herpesvirus-specific, immediate-early, and tegument protein. Our previous studies have revealed its crucial roles in both early and late stages of KSHV infection. In this study, we surveyed the interactome of ORF45 using a panel of monoclonal antibodies. In addition to the previously identified extracellular regulated kinase (ERK) and p90 ribosomal S6 kinase (RSK) proteins, we found several other co-purified proteins, including prominent ones of ~38 kDa and ~130 kDa. Mass spectrometry revealed that the 38 kDa protein is viral ORF33 and the 130 kDa protein is cellular USP7 (ubiquitin-specific protease 7). We mapped the ORF33-binding domain to the highly conserved carboxyl terminal 19-aa of ORF45, and the USP7-binding domain to the reported consensus motif in the central region of ORF45. Using immunofluorescence staining, we observed colocalization of ORF45 with ORF33 or USP7, in both transfected conditions and KSHV-infected cells. Moreover, we noticed an ORF45-dependent relocalization of a portion of ORF33/USP7 from the nucleus to the cytoplasm. We found that ORF45 caused an increase in ORF33 protein accumulation which was abolished if either the ORF33- or USP7-binding domain in ORF45 was deleted. Furthermore, deletion of the conserved carboxyl terminus of ORF45 in the KSHV genome drastically reduced the level of ORF33 protein in KSHV-infected cells and abolished production of progeny virions. Collectively, our results not only reveal new components of the ORF45-interactome, but also demonstrate that the interactions among these proteins are crucial for KSHV lytic replication.

    IMPORTANCE STATEMENT Kaposi's Sarcoma-associated Herpesvirus (KSHV) is the causative agent of several human cancers. KSHV ORF45 is a multifunctional protein that is required for KSHV lytic replication, but the exact mechanisms by which ORF45 performs its critical functions are unclear. Our previous studies revealed that all ORF45 protein in cells exists in high molecular weight complexes. We therefore sought to characterize the interactome of ORF45 to provide insights into its roles during lytic replication. Using a panel of monoclonal antibodies, we surveyed the ORF45 interactome in KSHV-infected cells. We identified two new binding partners of ORF45: the viral protein ORF33 and cellular ubiquitin-specific protease 7 (USP7). We further demonstrate that the interaction between ORF45 and ORF33 is crucial for the efficient production of KSHV viral particles, suggesting that the targeted interference of this interaction may represent a novel strategy to inhibit KSHV lytic replication.

  • Oncogenic HTLV-1 Tax Suppression of Primary Innate Immune Signaling Pathways [PublishAheadOfPrint]

  • Human T-cell Lymphotropic virus I (HTLVI) is an oncogenic retrovirus considered to be the etiological agent of adult T-cell leukemia (ATL). The viral transactivator Tax is regarded as the oncoprotein responsible for contributing towards the transformation process. Here, we demonstrate that Tax potently inhibits the activity of DEx(D/H) box helicases RIG-I and MDA5 as well as Toll-dependent TRIF which function as cellular sensors or mediators of viral RNA and facilitate innate immune responses including the production of type I IFN. Tax manifested this function by binding to the RHIM domains of TRIF and RIP1 to disrupt IRF7 activity, a critical type I IFN transcription factor. This data provides further mechanistic insight into HTLV-I-mediated subversion of cellular host defense responses, which may help explain HTLV1 related pathogenesis and oncogenesis.

    Importance It is predicted that up to 15% of all human cancers may involve virus infection. For example, Human T-cell Lymphotropic virus I (HTLVI) has been reported to infect up to 25 million people worldwide and is the causative agent of adult T-cell leukemia (ATL). We show here that HTLV1 may be able to successfully infect the T cells and remain latent due to the viral encoded product Tax inhibiting a key host defense pathway. Understanding the mechanisms by which Tax subverts the immune system may lead to the development of therapeutic to help treat HTLV1-mediated disease.

  • Expression of the novel HCV core+1/ARF protein in the context of JFH1-based replicons [PublishAheadOfPrint]

  • Hepatitis C virus contains a second open reading frame within the core gene, designated as core+1/ARF. Here we demonstrate for the first time expression of core+1/ARF protein in the context of a bicistronic JFH1-based replicon and report the production of two isoforms, core+1/L (long) and core+1/S (short), with different kinetics.

  • Murine coronavirus ubiquitin-like domain is important for papain-like protease stability and viral pathogenesis [PublishAheadOfPrint]

  • Ubiquitin-like domains (Ubls) are now recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in Severe Acute Respiratory Syndrome CoV, Middle East Respiratory Syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, viral replication and pathogenesis. Using deletion and substitution approaches, we identified sites within the Ubl domain, residues 785-787 of nonstructural protein 3, which negatively affect protease activity, and valine residues 785 and 787, which negatively affect deubiquitinating activity. Using reverse genetics, we engineered Ubl-mutant viruses and found that AM2 (V787S) and AM3 (V785S) viruses replicate efficiently at 37ddeg;C, but generate smaller plaques than WT virus, and AM2 is defective for replication at higher temperatures. To evaluate the effect of the mutation on protease activity, we purified WT and Ubl-mutant PLP2 and found that the proteases exhibit similar specific activities at 25ddeg;C. However, the thermal stability of the Ubl-mutant PLP2 was significantly reduced at 30ddeg;C thereby reducing the total enzymatic activity. To determine if the destabilizing mutation affects viral pathogenesis, we infected C57BL/6 mice with WT or AM2 and found that the mutant virus is highly attenuated, yet replicates sufficiently to elicit protective immunity. These studies revealed that modulating the Ubl domain adjacent to the PLP reduces protease stability and viral pathogenesis, revealing a novel approach to coronavirus attenuation.

    Importance Introducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered the viral protease activity or affected viral replication or pathogenesis. Our studies using purified wild-type and Ubl-mutant proteases revealed that mutations in the viral Ubl domain destabilize and inactivate the adjacent viral protease. Furthermore, we show that a CoV encoding the mutant Ubl domain is unable to replicate at high temperature or cause lethal disease in mice. Our results identify the coronavirus Ubl domain as a novel modulator of viral protease stability and reveal manipulating the Ubl domain as a new approach for attenuating coronavirus replication and pathogenesis.

  • Evidence supporting a role for TopBP1 and Brd4 in the initiation but not continuation of human papillomavirus 16 E1/E2 mediated DNA replication [PublishAheadOfPrint]

  • To replicate the double stranded human papillomavirus 16 (HPV16) DNA genome viral proteins E1 and E2 associate with the viral origin of replication, E2 can also regulate transcription from adjacent promoters. E2 interacts with host proteins in order to regulate both transcription and replication; TopBP1 and Brd4 are cellular proteins that interact with HPV16 E2. Previous work with E2 mutants demonstrated Brd4 requirement for the transactivation properties of E2 while TopBP1 is required for DNA replication induced by E2 from the viral origin of replication in association with E1. More recent studies have also implicated Brd4 in the regulation of DNA replication by E2 and E1. Here we demonstrate that both TopBP1 and Brd4 are present at the viral origin of replication and that interaction with E2 is required for optimal initiation of DNA replication. Both cellular proteins are present in E1-E2 containing nuclear foci and the viral origin of replication is required for the efficient formation of these foci. shRNA against either TopBP1 or Brd4 destroys the E1-E2 nuclear bodies but has no effect on E1-E2 mediated levels of DNA replication. An E2 mutation in the context of the complete HPV16 genome that compromises Brd4 interaction fails to efficiently establish episomes in primary human keratinocytes. Overall the results suggest that interactions between TopBP1 and E2 and Brd4 and E2 are required to correctly initiate DNA replication but are not required for continuing DNA replication which may be mediated by alternative processes such as rolling circle amplification and/or homologous recombination.

    Importance Human papillomavirus 16 is causative in many human cancers including cervical and head and neck and is responsible for the annual deaths of hundreds of thousands of people worldwide. The current vaccine will save lives in future generations but anti-virals targeting HPV16 are required for the alleviation of disease burden on the current, and future, generations. Targeting viral DNA replication that is mediated by two viral proteins E1 and E2 in association with cellular proteins such as TopBP1 and Brd4 would have therapeutic benefits. This report suggests a role for these cellular proteins in the initiation of viral DNA replication by HPV16 E1-E2 but not for continuing replication. This is important if viral replication is to be effectively targeted: we need to understand the viral and cellular proteins required at each phase of viral DNA replication so that it can be effectively disrupted.

  • Adenovirus replaces mitotic checkpoint controls [PublishAheadOfPrint]

  • Infection with adenovirus triggers the cellular DNA damage response, elements of which include cell death and cell cycle arrest. Early adenoviral proteins including the E1B-55K and E4orf3 proteins inhibit signaling in response to DNA damage. A fraction of cells infected with an adenovirus mutant unable to express the E1B-55K and E4orf3 genes appeared to arrest in a mitotic-like state. Cells infected early in G1 of the cell cycle were predisposed to arrest in this state at late times of infection. This arrested state, which displays hallmarks of mitotic catastrophe, was prevented by expression of either the E1B-55K or the E4orf3 genes. However, E1B-55K-mutant virus-infected cells became trapped in a mitotic-like state in the presence of the microtubule poison colcemid, suggesting that the two viral proteins restrict entry into mitosis or facilitate exit from mitosis in order to prevent infected cells from arresting in mitosis. The E1B-55K protein appeared to prevent inappropriate entry into mitosis through its interaction with the cellular tumor suppressor protein p53. The E4orf3 protein facilitated exit from mitosis by possibly mislocalizing and functionally inactivating cyclin B1. When expressed in non-infected cells, E4orf3 overcame the mitotic arrest caused by the degradation-resistant R42A cyclin B1 variant.

    IMPORTANCE Cells that are infected with adenovirus type 5 early in G1 of the cell cycle are predisposed to arrest in a mitotic-like state in a p53-dependent manner. The adenoviral E1B-55K protein prevents entry into mitosis. This newly described activity for the E1B-55K protein appears to depend on the interaction between the E1B-55K protein and the tumor suppressor p53. The adenoviral E4orf3 protein facilitates exit from mitosis, possibly by altering the intracellular distribution of cyclin B1. By preventing entry into mitosis and by promoting exit from mitosis, these adenoviral proteins act to prevent the infected cell from arresting in a mitotic-like state.

  • The Retinoblastoma Tumor Suppressor Promotes Efficient Human Cytomegalovirus Lytic Replication [PublishAheadOfPrint]

  • The retinoblastoma (Rb) tumor suppressor controls cell cycle, DNA damage, apoptotic, and metabolic pathways. DNA tumor virus oncoproteins reduce Rb function by either inducing Rb degradation or physically disrupting complexes between Rb and its myriad binding proteins. Human cytomegalovirus (HCMV), a betaherpesvirus being investigated for potential roles in human cancers, encodes multiple lytic phase proteins that inactivate Rb in distinct ways, leading to the hypothesis that reduced Rb levels and/or activity would benefit HCMV lytic infection. Paradoxically, we found that Rb knockdown prior to infection, whether transient or constitutive, impaired HCMV lytic infection at multiple stages, notably viral DNA replication, late protein expression, and infectious virion production. The existence of differentially modified forms of Rb, the temporally and functionally distinct means by which HCMV proteins interact with Rb, and the necessity of Rb for efficient HCMV lytic replication combine to highlight the complex relationship between the virus and this critical tumor suppressor.

    Importance Initial work examining viral protein modulation of cell cycle progression and oncogenic transformation revealed these proteins inactivated the function of cellular tumor suppressor proteins. However, subsequent work, including experiments described here using human cytomegalovirus, demonstrate a more nuanced interaction that includes the necessity of cellular tumor suppressors for efficient viral replication. Understanding the positive impacts that cellular tumor suppressors have on viral infections may reveal new activities of these well studied yet incompletely understood proteins. The basis for oncolytic viral therapy is the selective replication of viruses in transformed cells in which tumor suppressor function may be compromised. Understanding how tumor suppressors support viral infections may allow for the generation of modified oncolytic viruses with greater selective tumor cell replication and killing.

  • Maturation of human herpesvirus-6A glycoprotein O requires coexpression of gH and gL [PublishAheadOfPrint]

  • Glycoprotein O (gO) is conserved among beta-herpesviruses, but little is known about the maturation process of gO in human herpesvirus-6 (HHV-6). We found that HHV-6 gO maturation was accompanied with cleavage of its carboxyl-terminus and required co-expression of gH and gL, which promoted the export of gO out of the endoplasmic reticulum (ER). Finally, we also found that gO was not required for HHV-6A growth in T cell.

  • HSV-1 upregulates p35, alters CDK-5 localization, and stimulates CDK-5 kinase activity during acute infection in neurons [PublishAheadOfPrint]

  • The cyclin-dependent kinase (CDK) -5 activating protein, p35, is important for acute HSV-1 replication in mice. This report shows that HSV-1 increases p35 levels, changes the primary localization of CDK-5 from the nucleus to the cytoplasm, and enhances CDK-5 activity during lytic or acute infection. Infected neurons also stained positive for the DNA damage response (DDR) marker, H2AX. We propose that CDK-5 is activated by the DDR to protect infected neurons from apoptosis.

  • CCCTC-Binding Factor Recruitment to the Early Region of the Human Papillomavirus Type 18 Genome Regulates Viral Oncogene Expression [PublishAheadOfPrint]

  • Host cell differentiation-dependent regulation of human papillomavirus (HPV) gene expression is required for productive infection. The host cell CCCTC-binding factor (CTCF) functions in genome-wide chromatin organization and gene regulation. We have identified a conserved CTCF binding site in the E2 open reading frame of high-risk HPV types. Using organotypic raft cultures of primary human keratinocytes containing high-risk HPV18 genomes, we show that CTCF recruitment to this conserved site regulates viral gene expression in differentiating epithelia. Mutation of the CTCF binding site increases the expression of the viral oncoproteins E6 and E7, and promotes host cell proliferation. Loss of CTCF binding results in a reduction of a specific alternatively spliced transcript expressed from the early gene region concomitant with an increase in the abundance of unspliced early transcripts. We conclude that high-risk HPV types have evolved to recruit CTCF to the early gene region to control the balance and complexity of splicing events that regulate viral oncoprotein expression.

    Importance The establishment and maintenance of human papillomavirus (HPV) infection in undifferentiated basal cells of the squamous epithelia requires activation of a subset of viral genes, termed early genes. Differentiation of infected cells initiates expression of the late viral transcripts allowing completion of the virus life cycle. This tightly controlled balance of differentiation-dependent viral gene expression allows the virus to stimulate cellular proliferation to support viral genome replication with minimal activation of the host immune response, thus promoting virus productivity. Alternative splicing of viral mRNAs further increases the complexity of viral gene expression. In this study, we show that the essential host cell protein CCCTC-binding factor (CTCF), which functions in genome-wide chromatin organization and gene regulation, is recruited to the HPV genome and plays an essential role in the regulation of early viral gene expression and transcript processing. These data highlight a novel virus-host interaction important for HPV pathogenicity.

  • The laminin receptor is an attachment cellular receptor for classical swine fever virus [PublishAheadOfPrint]

  • Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious, economically important viral disease in many countries. The Erns and E2 envelope glycoproteins are responsible for the binding to and entry of CSFV into the host cell. To date, only one cellular receptor, heparan sulfate (HS), has been identified as being involved in CSFV attachment. HS is also present on the surface of various cells that are non-permissive to CSFV. Hence, there must be other receptor(s) unidentified to date. In this study, we used a set of small interfering RNAs (siRNAs) against a number of porcine cell membrane protein genes to screen cellular proteins involved in the CSFV infection. This approach resulted in the identification of several proteins, of which the laminin receptor (LamR) has been demonstrated to be a cellular receptor for several viruses. Confocal analysis showed that LamR was co-located with CSFV virions on the membrane and co-immunoprecipitation assay indicated that LamR interacted with the CSFV Erns protein. In inhibition assays, anti-LamR antibodies, soluble laminin or LamR protein significantly inhibited the CSFV infection in a dose-dependent manner. Transduction of PK-15 cells with the recombinant lentivirus expressing LamR yielded higher viral titers. Moreover, attachment assay demonstrated that LamR functioned during virus attachment. We also demonstrated that LamR acted as an alternative attachment receptor, especially in SK6 cells. These results indicate that LamR is an attachment cellular receptor for CSFV.

    IMPORTANCE Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an economically important viral disease affecting the pig industry in many countries. To date, only heparan sulfate (HS) has been identified as an attachment receptor for CSFV. Here, we identified the laminin receptor (LamR) as another attachment receptor by RNA interference screening, using small interfering RNAs (siRNAs) against a number of porcine membrane protein genes. We demonstrated the involvement of LamR in the virus attachment together with HS, and we elucidated the relationship between LamR and HS. LamR also serves as an attachment receptor for many viral pathogens, including dengue virus, a fatal human flavivirus. The study will help to enhance our understanding of the life cycle and the development of antiviral strategies for flaviviruses.

  • Inflammatory monocytes recruited to the liver within 24 hours after virus-induced inflammation, resemble Kupffer cells, but are functionally distinct [PublishAheadOfPrint]

  • Due to a scarcity of immunocompetent animal models for viral hepatitis, little is known about the early innate immune responses in the liver. In various hepatotoxic models both pro- and anti-inflammatory activities of recruited monocytes have been described. In this study, we compared the effect of liver inflammation induced by the TLR4 ligand LPS with that of a persistent virus, lymphocytic choriomeningitis virus (LCMV) Clone 13, on early innate intrahepatic immune responses in mice. LCMV infection induces a remarkable influx of inflammatory monocytes in the liver within 24 hours, accompanied by increased transcript levels of several pro-inflammatory cytokines and chemokines in whole liver. Importantly, while a single LPS injection results in similar recruitment of inflammatory monocytes to the liver, the functional properties of the infiltrating cells are dramatically different in response to LPS versus LCMV infection. In fact, intrahepatic inflammatory monocytes are skewed towards a secretory phenotype with impaired phagocytosis in LCMV-induced liver inflammation, but exhibit increased endocytic capacity after LPS challenge. In contrast, F4/80high-Kupffer cells retain their steady-state endocytic functions upon LCMV infection. Strikingly, the gene expression levels of inflammatory monocytes dramatically change upon LCMV exposure and resemble those of Kupffer cells. Since inflammatory monocytes outnumber Kupffer cells 24 hours after LCMV infection, it is highly likely that inflammatory monocytes contribute to the intrahepatic inflammatory response during the early phase of infection. Our findings are instrumental in understanding the early immunological events during virus-induced liver disease and point towards inflammatory monocytes as potential target cells for future treatment options in viral hepatitis.

    Importance Insights in how the immune system deals with HBV and HCV are scarce due to the lack of adequate animal model systems. This knowledge is however crucial to develop new antiviral strategies aimed at eradicating these chronic infections. We model virus-host interactions during the initial phase of liver inflammation 24 hours after inoculating mice with LCMV. We show that infected Kupffer cells are rapidly outnumbered by infiltrating inflammatory monocytes, which secrete pro-inflammatory cytokines but are less phagocytic. Nevertheless, these recruited inflammatory monocytes start to resemble Kupffer cells on a transcript level. Specificity of these cellular changes for viral induced liver inflammation, is corroborated by demonstrating opposite functions of monocytes after LPS challenge. Overall this demonstrates the enormous functional and genetic plasticity of infiltrating monocytes and identifies them as an important target cell for future treatment regimens.

  • Dissecting the roles of the 5' exoribonucleases Xrn1 and Xrn2 in restricting hepatitis C virus replication [PublishAheadOfPrint]

  • The replication of hepatitis C virus (HCV) is uniquely dependent on a host microRNA, miR-122. Previous studies using genotype 1a H77S.3 virus demonstrated that miR-122 acts in part by protecting the RNA genome from 5rrsquo; decay mediated by the cytoplasmic 5rrsquo; exoribonuclease, Xrn1. However, this finding has been challenged by a recent report suggesting that a predominantly nuclear exoribonuclease, Xrn2, mediates degradation of genotype 2a JFH1 RNA. Here, we dissect the roles of these two 5rrsquo; exoribonucleases in restricting replication of different HCV strains and mediating decay of HCV RNA. siRNA depletion experiments indicated that Xrn1 restricts replication of all HCV strains tested: JFH1, H77S.3, H77D (a robustly replicating genotype 1a variant), and HJ3-5, a genotype 1a/2a chimeric virus. In contrast, the antiviral effects of Xrn2 were limited to JFH1 and H77D virus. Moreover, such effects were not apparent in cells infected with a JFH1 luciferase reporter virus. Whereas Xrn1 depletion significantly slowed decay of JFH1 and HJ3-5 RNAs, Xrn2 depletion marginally enhanced JFH1 RNA half-life and had no effect on HJ3-5 RNA decay. The positive effects of Xrn1 depletion on JFH1 replication were largely redundant and non-additive with those of exogenous miR-122 supplementation, whereas Xrn2 depletion acted additively and thus independently of miR-122. We conclude that Xrn1 is the dominant 5rrsquo; exoribonuclease mediating decay of HCV RNA and against which miR-122 provides protection. The restriction of JFH1 and H77D replication by Xrn2 is likely indirect in nature and possibly linked to cytopathic effects of these robustly replicating viruses.

    Importance HCV is a common cause of liver disease both within and outside the United States. Its replication is dependent upon a small, liver-specific noncoding RNA, miR-122. Although this requirement has been exploited for the development of an anti-miR-122 antagomir as a host-targeting antiviral, molecular mechanisms underpinning the host factor activity of miR-122 remain incompletely defined. Conflicting reports suggest miR-122 protects the viral RNA against decay mediated by distinct cellular 5rrsquo; exoribonucleases, Xrn1 or Xrn2. Here, we compare the roles of these two exoribonucleases in HCV-infected cells, and confirm that Xrn1, not Xrn2, is primarily responsible for decay of RNA in cells infected with multiple virus strains. Our results clarify previously published research and add to current understanding of the host factor requirement for miR-122.

  • Deep sequencing of virus derived small interfering RNAs and RNA from viral particles shows highly similar mutational landscape of a plant virus population [PublishAheadOfPrint]

  • RNA viruses exist within a host as a population of mutant sequences, often referred to as quasispecies. Within a host, sequences of RNA viruses constitute several distinct, but interconnected pools, such as RNA packed in viral particles, double stranded RNA and virus derived small interfering RNAs. We aimed to test if the same representation of within-host viral population structure could be obtained by sequencing different viral sequence pools. Using ultra deep Illumina sequencing, the diversity of two coexisting Potato virus Y sequence pools present within a plant was investigated: RNA isolated form viral particles and virus derived small interfering RNAs (the derivates of a plant RNA silencing mechanism). The mutational landscape of the within-host virus population was highly similar between both pools, with no notable hotspots across the viral genome. Notably, all the single nucleotide polymorphisms with frequency higher than 1.6% were found in both pools. Some unique SNPs with very low frequencies were found in each of the pools, more of them occurring in sRNA pool, possibly arising through genetic drift in localized virus populations within a plant and the errors introduced during the amplification of silencing signal. Sequencing of viral particle pool enhanced the efficiency of consensus viral genome sequence reconstruction. Non-homologous recombinations were commonly detected in viral particle pool, with a hotspot in 3ˋ untranslated and coat protein region of the genome. We stress that they present an important, but often overlooked aspect of virus population diversity.

    Significance This study is the most comprehensive whole-genome characterization of a within-plant virus population to date and the first study comparing diversity of different pools of viral sequences within a host. We show that both virus derived small RNAs and RNA from viral particles could be used for diversity assessment of within-plant virus population, since they show highly congruent portrayal of the virus mutational landscape within a plant. The study is an important baseline for future studies of virus population dynamics, for example during the adaptation to a new host. The comparison of the two virus sequence enrichment techniques, sequencing of virus derived small interfering RNA and RNA from purified viral particles, shows the strength of the latter for the detection of recombinant viral genomes and reconstruction of complete consensus viral genome sequence.

  • HIV-1-induced impairment of dendritic cell cross-talk with {gamma}{delta} T lymphocytes [PublishAheadOfPrint]

  • The interplay between dendritic cells (DC) and T lymphocytes represents a network of paracrine and cell contact interactions important for an integrated immune response to pathogens. HIV-1 infection dramatically affects the number and functions of both cell populations, and DC/ T cell cross-talk may represent a target of virus-induced immune escape. We investigated whether HIV-exposed DC could deliver aberrant signals to interacting T cells. Here we report that the interaction of human T lymphocytes with HIV-1-exposed autologous monocyte-derived DC, but not direct exposure to the virus, impairs lymphocyte expansion and IFN- production in response to phosphoantigens. This effect is independent of virus strain and occurs in the 55% of donors analyzed. The donor-dependent variation observed relies on the responsiveness of DC to HIV-1 and is strictly related to the capacity of the virus to suppress the maturation-induced expression of IL-12. In fact, T cell response to phosphoantigens is almost completely recovered when this cytokine is exogenously added to the DC/lymphocyte co-cultures. Interestingly, we show that T lymphocytes are recruited by HIV-1-exposed DC through a CCR5-mediated mechanism, and exert a CCL4-mediated control on virus dissemination within DC and susceptible CD4+ T lymphocytes.

    These results demonstrate an association between HIV-induced DC dysfunction and alterations of T cell responses. The aberrant cross-talk between these two cell populations may contribute to the pathogenesis of HIV infection by further reducing the strength of antiviral immune response.

    IMPORTANCE This study provides new evidence on the mechanisms exploited by HIV-1 to evade the host immune response. We report for the first time that HIV-1 impairs the cross-talk between DC and T lymphocytes, by reducing the capacity of DC to promote functional T cell activation. Interestingly, the virus does not per se interfere with T cell activation thus highlighting the key role of early DC-HIV-1 interaction in this phenomenon. Furthermore, the results achieved unravel the novel role of T cells in controlling HIV-1 dissemination within the DC population as well as virus transfer to susceptible CD4+ T lymphocytes. The interactions of DC with innate lymphocytes represent a major control mechanism for an integrated immune response to infection. Understanding how HIV-1 harnesses these pathways may provide important insights on the pathogenesis of disease and offer new opportunities for therapeutic interventions.

  • Monoclonal Antibodies against Extracellular Domains of Claudin-1 Block Hepatitis C Virus Infection in A Mouse Model [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) entry into host cells is a complex process requiring multiple host factors, including claudin-1 (CLDN1). Safe and effective therapeutic entry inhibitors need to be developed. We isolated a human hepatic Huh7.5.1-derived cell mutant nonpermissive to HCV, and comparative microarray analysis showed that the mutant was CLDN1 defective. Four hybridomas were obtained, which produced monoclonal antibodies (mAbs) that interacted with the parental Huh7.5.1 cell but not with the CLDN1-defective mutant. All mAbs produced by these hybridomas specifically bound to human CLDN1 with very high affinity and prevented HCV infection of Huh7.5.1 cells in a dose-dependent manner, without apparent cytotoxicity. Two selected mAbs also inhibited HCV infection of human liver-chimeric mice without significant adverse effects. CLDN1 may be a potential target to prevent HCV infection in vivo. Anti-CLDN1 mAbs may hence be promising candidates as novel anti-HCV agents.

    IMPORTANCE Safe and effective therapeutic entry inhibitors against Hepatitis C virus (HCV) are very useful for combination therapies with other anti-HCV drugs such as direct-acting antivirals. In this study, we first showed the effective strategy to develop functional monoclonal antibodies (mAbs) against extracellular domains of a multimembrane-spanning target protein, claudin-1 (CLDN1), using parental cells expressing the intact target membrane protein and the target-defective cells. The established mAbs against CLDN1, which had very high affinity with intact CLDN1, efficiently inhibited in vitro and in vivo HCV infection. These anti-CLDN1 mAbs are promising leads for novel entry inhibitors against HCV.

  • Ebola virus disease in mice transplanted with human hematopoietic stem cells [PublishAheadOfPrint]

  • The development of treatments for Ebola virus disease (EVD) has been hampered by the lack of small animal models that mimick human disease. Here we show that mice transplanted with human hematopoetic stem cells reproduce typical features of EVD. Infection with Ebola virus was associated with viremia, cell damage, liver steatosis, signs of hemorrhage, and high lethality. Our study provides a small animal model with human components for the development of EVD therapies.

  • Generation of mutant Uukuniemi viruses lacking the nonstructural protein NSs by reverse genetics indicates that NSs is a weak interferon antagonist. [PublishAheadOfPrint]

  • Uukuniemi virus (UUKV) is a tick-borne member of the Phlebovirus genus (family Bunyaviridae) and has been widely used as a safe laboratory model to study aspects of bunyavirus replication. Recently, a number of new tick-borne phleboviruses have been discovered, some of which, like severe fever with thrombocytopenia syndrome virus and Heartland virus, are highly pathogenic in man. UUKV could now serve as a useful comparator to understand the molecular basis for the different pathogenicities of these related viruses. We established a reverse genetics system to recover UUKV entirely from cDNA clones. We generated two recombinant viruses, one in which the nonstructural protein NSs open reading frame was deleted from the S segment and one in which the NSs gene was replaced with GFP, allowing convenient visualization of viral infection. We show that the UUKV NSs protein acts as a weak interferon antagonist in human cells, but it is unable to completely counteract the interferon response, which could serve as an explanation for its inability to cause disease in man.

    Importance Uukuniemi virus (UUKV) is a tick-borne phlebovirus that is apathogenic for man and has been used as a convenient model to investigate aspects of phlebovirus replication. Recently new tick-borne phleboviruses have emerged, such as severe fever with thrombocytopenia syndrome virus in China and Heartland virus in the US, that are highly pathogenic, and UUKV will now serve as a comparison to aid understanding of the molecular basis for the virulence of these new viruses. To help such investigations, we have developed a reverse genetics system for UUKV that permits manipulation of the viral genome. We generated viruses lacking the nonstructural protein NSs and show that UUKV NSs is a weak interferon antagonist. In addition, we created a virus that expresses GFP and thus allows convenient monitoring of virus replication. These new tools represent a significant advance in the study of tick-borne phleboviruses.

  • Dimerization of Matrix protein is required for budding of Respiratory Syncytial Virus [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) infects epithelial cells of the respiratory tract and is a major cause of bronchiolitis and pneumonia in children and the elderly. The virus assembles and buds through the plasma membrane, forming elongated membrane filaments, but details of how this happens remain obscure. Oligomerization of the matrix protein (M) is a key step in the process of assembly and infectious virus production. In addition, it was suggested to affect fusion protein conformation in the mature virus, the major current target for RSV antivirals. The structure and assembly of M are thus key parameters in the RSV antiviral development strategy. The structure of RSV M was previously published as a monomer. Other paramyxovirus M proteins have been shown to dimerize and biochemical data suggest that RSV M also dimerizes. Here, using Size Exclusion Chromatography nndash; Multi Angle Light Scattering, we show that the protein is dimeric in solution. We also crystallized M in two crystal forms and show that it assembles into equivalent dimers in both lattices. Dimerization interface mutants destabilize the M dimer in vitro. To assess the biological relevance of dimerization, we used confocal imaging to show that dimerization interface mutants of M fail to assemble into viral filaments on the plasma membrane. Additionally, M mutants that fail to form filaments prevent budding and release of virus-like particles. Importantly, we show that M is biologically active as a dimer and that the switch from M dimers to higher oligomers triggers viral filament assembly and virus production.

    Importance Human respiratory syncytial virus (RSV) is the most frequent cause of infantile bronchiolitis and pneumonia. The enormous burden of RSV makes it a major unmet target for a vaccine and anti-viral drug therapy. Oligomerization of the matrix protein is a key step in the process of assembly and production of infectious virus, but the molecular mechanism of RSV assembly is still poorly understood. Here we show that RSV matrix forms dimers in solution and in crystals; the dimer is essential for formation of higher order oligomers. Destabilizing the dimer interface resulted in loss of RSV filament formation and lack of budding of virus-like particles. Importantly, our findings can potentially lead to new structure-based RSV inhibitors targeting the assembly process.

  • Cell Surface Nucleolin Facilitates Enterovirus 71 Binding and Infection [PublishAheadOfPrint]

  • Because the pathogenesis of enterovirus 71 (EV71) remains mostly ambiguous, identifying the factors that mediate viral binding and entry to host cells is indispensable to ultimately uncover the mechanisms that underlie virus infection and pathogenesis. Despite the identification of several receptors/attachment molecules for EV71, the binding, entry, and infection mechanisms of EV71 remain unclear. Herein, we employed glycoproteomic approaches to identify human nucleolin as a novel binding receptor for EV71. Glycoproteins purified by lectin chromatography from the membrane extraction of human cells were treated with sialidase followed by immunoprecipitation with EV71 particles. Among the sixteen proteins identified by tandem mass spectrometry analysis, cell-surface nucleolin attracted our attention. We found that EV71 interacted directly with nucleolin via the VP1 capsid protein, and an anti-nucleolin antibody reduced the binding of EV71 to human cells. In addition, the knockdown of cell surface nucleolin decreased EV71 binding, infection, and production in human cells. Furthermore, the expression of human nucleolin on the cell surface of a mouse cell line increased EV71 binding and conferred EV71 infection and production in the cells. These results strongly indicate that human nucleolin can mediate EV71 binding to and infection of cells. Our findings also demonstrate that the use of glycoproteomic approaches is a reliable methodology to discover novel receptors for pathogens.

    IMPORTANCE Outbreaks of EV71 have been reported in Asia Pacific countries and caused thousands of deaths in young children during the last two decades. The discovery of new EV71-interacting molecules to understand the infection mechanism has become an emergent issue. Hence, this study uses glycoproteomic approaches to comprehensively investigate the EV71-interacting glycoproteins. Several EV71-interacting glycoproteins are identified, and the role of cell surface nucleolin in mediating the attachment and entry of EV71 is characterized and validated. Our findings not only indicate a novel target for uncovering EV71 infection mechanism and anti-EV71 drug discovery, but also provides a new strategy for virus receptor identification.

  • HPV Carcinogenesis - An Identity Crisis in the Retinoblastoma Tumor Suppressor Pathway [PublishAheadOfPrint]

  • Viruses are obligate intracellular parasites and need to reprogram host cells to establish long-term persistent infection and/or to produce viral progeny. Cellular changes initiated by the virus trigger cellular defense responses to cripple viral replication, and viruses have evolved countermeasures to neutralize them. Established models have suggested that human papillomaviruses target the Retinoblastoma (RB1) and TP53 tumor suppressor networks to usurp cellular replication, which drives carcinogenesis. More recent studies, however, suggest that modulating the activity of the Polycomb family of transcriptional repressors and the resulting changes in epigenetic regulation are proximal steps in the rewiring of cellular signaling circuits. Consequently, RB1 inactivation evolved to tolerate the resulting cellular alterations. Therefore, epigenetic reprograming results in cellular addictions to pathways for survival. Inhibition of such a pathway could cause "synthetic lethality" in adapted cells while not markedly affecting normal cells and could prove to be an effective therapeutic approach.

  • Structure and receptor binding preferences of recombinant hemagglutinins from avian and human H6 and H10 influenza A virus subtypes [PublishAheadOfPrint]

  • During 2013, three new avian influenza A virus subtypes, A(H7N9), A(H6N1) and A(H10N8), resulted in human infections. While the A(H7N9) virus resulted in a significant epidemic in China across 19 provinces and municipalities, both A(H6N1) and A(H10N8) viruses resulted in only a few human infections. This study focuses on the major surface glycoprotein hemagglutinin from both of these novel human viruses. The detailed structural and glycan microarray analyses presented here highlight that both A(H6N1) and A(H10N8) virus hemagglutinins retain a strong avian receptor binding preference, and thus currently pose a low risk for sustained human infections.

    Importance Human infections with zoonotic influenza virus subtypes continue to be a great public health concern. We report detailed structural analysis and glycan microarray data for recombinant hemagglutinins from A(H6N1) and A(H10N8) viruses, isolated from human infections in 2013, and compare them with hemagglutinins of avian origin. This is the first structural report of an H6 HA, and our results should further the understanding of these viruses, and provide useful information to aid continuous surveillance of these zoonotic influenza viruses.

  • The acetyltransferase Tip60 is a critical regulator of the differentiation-dependent amplification of human papillomaviruses [PublishAheadOfPrint]

  • The life cycle of human papillomaviruses (HPVs) is dependent upon differentiation of the infected host epithelial cell as well as activation of the ataxia-telangiectasia mutated (ATM) DNA repair pathway that in normal cells acts to repair double strand DNA breaks. In normal cells following DNA damage, the acetyltransferase Tip60 must acetylate ATM proteins prior to their full activation by autophosphorylation. E6 proteins have been shown to induce the degradation of Tip60 suggesting that Tip60 action may not be required for activation of the ATM pathway in HPV positive cells. We investigated what role, if any, Tip60 played in regulating the differentiation-dependent HPV life cycle. Our study indicates that Tip60 levels and activity are increased in cells that stably maintain complete HPV genomes as episomes, while low levels are seen in cells that express only HPV E6 and E7 proteins. Knockdown of Tip60 with shRNAs in cells that maintain HPV episomes blocked ATM induction and differentiation-dependent genome amplification demonstrating the critical role of Tip60 in the viral life cycle. The JAK/STAT transcription factor, STAT-5, has been previously shown to regulate the phosphorylation of ATM. Our studies demonstrate that STAT-5 regulates Tip60 activation and this occurs in part by targeting the glycogen synthase kinase, GSK3bbeta;. Inhibition of either STAT-5, Tip60 or GSK3bbeta; blocks differentiation-dependent genome amplification. Taken together, our findings identify Tip60 as an important regulator of HPV genome amplification whose activity during the viral life cycle is controlled by STAT-5 and the kinase, GSK3bbeta;.

    Importance Human papillomaviruses (HPVs) are the etiological agents of cervical and other anogenital cancers. HPVs regulate their differentiation-dependent life cycle by activation of DNA damage pathways. This study demonstrates that HPVs regulate the ATM DNA damage pathway through the action of the acetyltransferase, Tip60. Furthermore, the innate immune regulator, STAT-5 and the kinase GSK3bbeta; mediate the activation of Tip60 in HPV positive cells. This study identifies critical regulators of the HPV life cycle.

  • A mutant Tat protein inhibits HIV-1 reverse transcription by targeting the reverse transcription complex [PublishAheadOfPrint]

  • Previously we reported that a mutant of Tat referred to as Nullbasic inhibits HIV-1 reverse transcription although the mechanism of action is unknown. Here we show that Nullbasic is a reverse transcriptase (RT) binding protein that targets the reverse transcription complex rather than directly inhibiting RT activity. An interaction between Nullbasic and RT was observed by co-immunoprecipitation, pull-down assays, and a direct interaction was measured by biolayer interferometry (BLI) assay. Mixtures of recombinant 6x-His-RT and Nullbasic-FLAG-V5-6xHis at molar ratios of up to 1:20,000 did not inhibit RT activity in standard homopolymer primer template assays. An analysis of virus made by cells that co-expressed Nullbasic showed that Nullbasic co-purified with virus particles indicating it was a virion protein. In addition, analysis of reverse transcription complexes (RTCs) isolated from cells infected with wild type or Nullbasic-treated HIV-1 showed that Nullbasic reduced levels of viral DNA in RTC fractions. In addition, a shift in the distribution of viral DNA and CAp24 to less dense non-RTC fractions was observed, indicating that RTC activity from Nullbasic treated virus was impaired. Further analysis showed that viral cores isolated from Nullbasic treated HIV undergo increased disassembly in vitro compared to untreated HIV-1. To our knowledge this is the first description of an antiviral protein that inhibits reverse transcription by targeting the RTC and affecting core stability.

    IMPORTANCE HIV-1 infection is treated by using combinations of anti-retroviral drugs that target independent steps of virus replication. A newly described antiviral protein called Nullbasic can also inhibit a combination of different steps of virus replication (transcription, reverse transcription, and Rev mediated viral mRNA transport) although the precise mechanism of action is unknown. This study shows that Nullbasic can inhibit reverse transcription by binding to the viral enzyme called reverse transcriptase, which results in accelerated uncoating of the viral core and instability of the viral apparatus called the reverse transcription complex (RTC). This unique antiviral activity may inform development of other RTC inhibitors as well as providing a unique investigative tool for dissecting the RTC cellular composition.

  • Contrasting Effects of W781V and W780V Mutations in Helix N of Herpes Simplex Virus-1 and Human Cytomegalovirus DNA Polymerases on Antiviral Drug Susceptibility [PublishAheadOfPrint]

  • DNA polymerases of Herpesviridae and bacteriophage RB69 belong to aalpha;-like DNA polymerase family. In spite of similarities in structure and function, the RB69 enzyme is relatively resistant to foscarnet requiring the mutation V478W in helix N to promote the closed conformation of the enzyme to make it susceptible to the antiviral. Here, we generated recombinant herpes simplex virus-1 (HSV-1) and human cytomegalovirus (HCMV) harboring the revertant in UL30 (W781V) and UL54 (W780V) DNA polymerases, respectively, to further investigate the impact of this tryptophan on antiviral drug susceptibility and viral replicative capacity. The mutation W781V in HSV-1 induced resistance to foscarnet, acyclovir and ganciclovir (3-, 14- and 3-fold increase in EC50 values, respectively). The recombinant HCMV harboring mutation W780V was slightly resistant to foscarnet (1.9-fold increase) and susceptible to ganciclovir. Recombinant HSV-1 and HCMV mutants had altered viral replication kinetics. The apparent inhibition constant value of foscarnet against mutant UL30 and UL54 DNA polymerases were 45- and 4.9-fold higher than that of their wild-type counterparts, respectively. Structural evaluation of the tryptophan position in UL54 DNA polymerase suggests that the bulkier phenylalanine (fingers domain) and isoleucine (N-terminal domain) could induce a greater tendency towards the closed conformation than for UL30 and explains the modest effect of W780V mutation on foscarnet susceptibility. Our results further suggest a role of the tryptophan in helix N to confer susceptibility of HCMV, and especially HSV-1, to foscarnet and the possible contribution of other residues localized at the interface between fingers and N-terminal domains.

    Importance DNA polymerases of Herpesviridae and bacteriophage RB69 belong to aalpha;-like DNA polymerase family. However, the RB69 DNA polymerase is relatively resistant to the broad-spectrum antiviral agent foscarnet. The mutation V478W in helix N of the fingers domain caused the enzyme to adopt a closed conformation and to become susceptible to the antiviral. We generated recombinant herpes simplex virus-1 (HSV-1) and human cytomegalovirus (HCMV) harboring the revertant in UL30 (W781V) and UL54 (W780V) DNA polymerases, respectively, to further investigate the impact of this tryptophan on antiviral drug susceptibility. The mutation W781V in HSV-1 induced resistance to foscarnet whereas the W780V in HCMV slightly decreased drug susceptibility. This study suggests that the different susceptibility profile of HSV-1 and HCMV mutants to foscarnet could be related to subtle conformational changes resulting from the interaction between residues specific to each enzyme that are located at the interface between the fingers and the N-terminal domains.

  • The MET gene is a common integration target in Avian Leukosis Virus subgroup J induced chicken hemangiomas [PublishAheadOfPrint]

  • Avian leukosis virus, subgroup J (ALV-J), is a simple retrovirus that can cause hemangiomas and myeloid tumors in chickens and is currently a major economic problem in Asia. Here we characterize ALV-J strain PDRC-59831, a newly studied US isolate of ALV-J. Five-day-old chicken embryos were infected with this virus, and the chickens developed myeloid leukosis and hemangiomas within two months after hatching. To investigate the mechanism of pathogenesis, we employed high-throughput sequencing to analyze proviral integration sites in these tumors. We found expanded clones with integrations in the MET gene in two of the five hemangiomas studied. This integration locus was not seen in earlier work characterizing ALV-J-induced myeloid leukosis. MET is a known proto-oncogene that acts through a diverse set of signaling pathways and is involved in many neoplasms. We show that tumors harboring MET integrations exhibit strong overexpression of the MET mRNA.

    Importance These data suggest that ALV-J induces oncogenesis by insertional mutagenesis, and integrations in the MET oncogene can drive overexpression of MET and contribute to the development of hemangiomas.

  • Regulation and Function of Phosphorylation on VP8, the Major Tegument Protein of Bovine Herpesvirus-1 [PublishAheadOfPrint]

  • The major tegument protein of bovine herpesvirus-1 (BoHV-1), VP8, is essential for viral replication in cattle. VP8 is phosphorylated in vitro by casein kinase 2 (CK2) and BoHV-1 unique short protein 3 (US3). In this study VP8 was found to be phosphorylated in both transfected and infected cells, but was detected as a non-phosphorylated form in mature virions. This suggests that phosphorylation of VP8 is strictly controlled during different stages of the viral lifecycle. The regulation and function of VP8 phosphorylation by US3 and CK2 was further analyzed. An in vitro kinase assay, site-directed mutagenesis and liquid chromatographynndash;mass spectrometry were used to identify the active sites for US3 and CK2. The two kinases phosphorylate VP8 at different sites, resulting in distinct phosphopeptide patterns. S16 is a primary phosphoreceptor for US3 and it subsequently triggers phosphorylation at S32. CK2 has multiple active sites, among which T107 appears to be the preferred residue. Additionally, CK2 consensus motifs in the N-terminus of VP8 are essential for the phosphorylation. Based on these results, a non-phosphorylated VP8 mutant was constructed, and used for further studies. In transfected cells phosphorylation was not required for nuclear localization of VP8. Phosphorylated VP8 appeared to recruit promyelocytic leukemia (PML) protein, and to remodel the distribution of PML in the nucleus; however, PML protein did not show an association with non-phosphorylated VP8. This suggests that VP8 plays a role in resisting PML-related host antiviral defenses by re-distributing PML protein, and that this function depends on the phosphorylation of VP8.

    Importance The progression of VP8 phosphorylation over time and its function in BoHV-1 replication have not been characterized. This study demonstrates that activation of S16 initiates further phosphorylation at S32 by US3. Additionally, VP8 is phosphorylated by CK2 at several residues, with T107 having the highest level of phosphorylation. Evidence for a difference in phosphorylation status of VP8 in host cells and mature virus is presented for the first time. The phosphorylation was found to be a critical modification, which enables VP8 to attract and to redistribute PML protein in the nucleus. This might promote viral replication through interference with PML-mediated antiviral defense. This study provides new insights into the regulation of VP8 phosphorylation and suggests a novel, phosphorylation-dependent function for VP8 in the lifecycle of BoHV-1, which is important in view of the fact that VP8 is essential for viral replication in vivo.

  • Characterization of HSV-2 primary miRNA transcript regulation [PublishAheadOfPrint]

  • In order to understand factors that may influence LAT transcription and the LAT-associated phenotypes, we studied expression of the HSV-2 latency-associated transcript (LAT)-associated microRNAs. We mapped transcription initiation sites of all three primary miRNA transcripts, and identified ICP4-binding sequences at the transcription initiation sites of both HSV-2 LAT (pri-miRNA for miR-I and miR-II, which target ICP34.5, and miR-III, which targets ICP0) and L/ST (a pri-miRNA for miR-I and miR-II), but not at that of the primary miR-H6 (for which the target is unknown). We confirmed activity of the putative HSV-2 L/ST promoter and found that ICP4 trans-activates the L/ST promoter when the ICP4-binding site at its transcription initiation site is mutated, suggesting that ICP4 may play a dual role in regulating transcription of L/ST and consequently, of miR-I and miR-II. LAT exon 1 (containing LAT enhancer sequences) together with the LAT promoter region comprise a bidirectional promoter required for the expression of both LAT-encoded miRNAs and miR-H6 in latently infected mice ganglia. The ability of ICP4 to suppress ICP34.5-targeting miRNAs and to activate lytic viral genes suggests that ICP4 could play a key role in the switch between latency and reactivation.

    Importance The HSV-2 LAT and viral miRNAs expressed in the LAT region are the most abundant viral transcripts during HSV latency. The balance between the expression of LAT and LAT-associated miRNAs and the expression of lytic viral transcripts from the opposite strand appears to influence whether individual HSV-infected neurons will be latently or productively infected. The outcome of neuronal infection may thus depend on regulation of gene expression of the corresponding primary miRNAs. In the present study, we characterize promoter sequences responsible for miRNA expression, including identification of the primary miRNA 5rrsquo; ends and evaluation of ICP4 response. These findings provide further insight into the virus' strategy to tightly control expression of lytic cycle genes (especially the neurovirulence factor, ICP34.5) and suggest a mechanism (via ICP4) for the transition from latency to reactivated productive infection.

  • A(H7N9) virus results in early induction of proinflammatory cytokine responses in both human lung epithelial and endothelial cells and shows increased human adaption compared with avian H5N1 virus. [PublishAheadOfPrint]

  • Similar to H5N1 viruses, A(H7N9) influenza viruses have been associated with severe respiratory disease and fatal outcomes in humans. While high viral load, hypercytokinemia, and pulmonary endothelial cell involvement are known to be hallmarks of H5N1 virus infection, the pathogenic mechanism of the A(H7N9) virus in humans is largely unknown. Here, we assessed the ability of A(H7N9) virus to infect, replicate, and elicit innate immune responses in both human bronchial epithelial cells and pulmonary microvascular endothelial cells, compared with seasonal H3N2, avian H7N9, and H5N1 viruses. In epithelial cells, A(H7N9) virus replicated efficiently, but did not elicit robust induction of cytokines like that observed for H5N1 virus. In pulmonary endothelial cells, A(H7N9) virus efficiently initiated infection, however, no released infectious virus was detected. The magnitude of induction of host cytokine responses was comparable between A(H7N9) and H5N1 virus infection. Additionally, we utilized differentiated human primary bronchial/tracheal epithelial cells to investigate cellular tropism using transmission electron microscopy and the impact of temperature on virus replication. Interestingly, A(H7N9) virus budded from the surface of both ciliated and mucin-secretory cells. Furthermore, A(H7N9) virus replicated to a significantly higher titer at 37ddeg;C than at 33ddeg;C, with improved replication capacity at 33ddeg;C compared to H5N1 virus. These findings suggest that a high viral load from lung epithelial cells, coupled with induction of host responses in endothelial cells may contribute to the severe pulmonary disease observed following H7N9 virus infection. Improved adaption of A(H7N9) virus to human upper airway poses an important threat to public health.

    IMPORTANCE A(H7N9) influenza viruses have caused over 400 documented human infections with a 30% fatality rate since early 2013. However, these novel viruses lack many molecular determinants previously identified with mammalian pathogenicity, necessitating a closer examination of how these viruses elicit host responses which could be detrimental. This study provides greater insight into the interaction of this virus with host lung epithelial cells and endothelial cells, which results in high viral load, epithelial cell death, and elevated immune response in lung, revealing the mechanism of pathogenesis and disease development among A(H7N9)-infected patients. In particular, we characterized the involvement of pulmonary endothelial cells, a cell type in the human lung accessible to influenza virus following damage of the epithelial monolayer, and its potential role in the development of severe pneumonia caused by A(H7N9) infection in humans.

  • BAFFR deficiency results in limited CD169+ macrophage function during viral infection [PublishAheadOfPrint]

  • The B cell activating factor of the TNF family (BAFF) is critical for B-cell development and humoral immunity in mice and humans. While the role of BAFF in B cells has been widely described, its role in innate immunity remains unknown. Using BAFFR deficient mice we characterized BAFFR related innate and adaptive immune functions following infection with vesicular stomatitis virus (VSV) and lymphocytic choriomeningitis virus (LCMV). We identified a critical role for BAFFR signalling in the generation and maintenance of the CD169+ macrophage compartment. Consequently, Baffrnndash;/nndash; mice exhibited limited induction of innate type I interferon production after viral infection. Lack of BAFFR signalling reduced virus amplification and presentation following viral infection, resulting in highly reduced anti-viral adaptive immune responses. As a consequence, BAFFR deficient mice showed exacerbated and fatal disease after viral infection. Mechanistically, transient lack of B cells in Baffrnndash;/nndash; animals resulted in limited lymphotoxin expression, which was critical for maintenance of CD169+ cells. In conclusion, BAFFR signalling affects both innate and adaptive immune activation during viral infections.

    Importance Viruses cause acute and chronic infections in humans resulting in millions of deaths every year. Innate immunity is critical for the outcome of a viral infection. Innate type I interferon production can limit viral replication while adaptive immune priming by innate immune cells induces pathogen specific immunity with long term protection. Here we show that BAFFR deficiency not only perturbed B cells, but also resulted in limited CD169+ macrophages. These macrophages are critical in amplifying viral particles to trigger type I interferon production and initiate adaptive immune priming. Consequently, BAFFR deficiency resulted in reduced enforced viral replication, limited type I interferon production, and reduced adaptive immunity when compared to BAFFR competent controls. As a result, BAFFR deficient mice were predisposed to fatal viral infections. Thus, BAFFR expression is critical for innate immune activation and anti-viral immunity.

  • A novel rhabdovirus isolated from the straw-coloured fruit bat Eidolon helvum with signs of antibodies in swine and humans [PublishAheadOfPrint]

  • Bats have been implicated as reservoirs of emerging viruses. Bat species forming large social groups and roosting in proximity to human communities are of particular interest. Here we sampled a colony of ca. 350,000 individuals of the straw-coloured fruit bat Eidolon helvum in Kumasi, the second largest city of Ghana. A novel rhabdovirus (Kumasi rhabdovirus, KRV) was isolated in E. helvum cell cultures and passaged to Vero cells as well as interferon-competent human and primate cells (A549 and MA104). Genome composition was typical for a rhabdovirus. KRV was detected in 5.1% of 487 animals, showing association with the spleen but not the brain. Antibody prevalence was 11.5% by immunofluorescence and 6.4% by plaque-reduction virus neutralization test. Detection throughout 3 sampling years was pronounced in both annual wet seasons, of which only one overlaps the post parturition season. Juvenile bats showed increased viral prevalence. No evidence of infection was obtained in 1240 female mosquitos (6 different genera) trapped in proximity to the colony to investigate potential vector association. Antibodies were found in 28.9% (5.4% by PRNT) of 107 swine sera, but not in similarly large collections of sheep-, goat-, or cattle sera. Antibody detection rate in human subjects with occupational exposure to the bat colony was 11% (5/45 persons), which was significantly higher than in unexposed adults (0.8%; 1/118; ChiSquare, pllt;0.001). KRV is a novel bat-associated rhabdovirus potentially transmitted to humans and swine. Disease associations should be investigated.

    Importance Bats are thought to carry a huge number of as-yet undiscovered viruses that may pose epidemic threats to humans and livestock. Here we describe a novel dimarhabdovirus which we isolated from a large colony of the straw coloured fruit bat Eidolon helvum in Ghana. As these animals are exposed to humans and several livestock species, we looked for antibodies indicating infection in humans, cattle, swine, sheep, and goats. Signs of infection were found in swine and humans, with increased antibody findings in humans who are occupationally exposed to the bat colony. Our data suggest it is worth-while to look for diseases caused by the novel virus in humans and livestock.

  • Competitive Fitness of Influenza B Viruses with Neuraminidase Inhibitor-Resistant Substitutions in a Co-infection Model of the Human Airway Epithelium [PublishAheadOfPrint]

  • Influenza A and B viruses are regarded almost equally as human pathogens with significant disease burden. Neuraminidase (NA) inhibitors (NAIs) are the only class of drugs available to treat influenza A and B virus infections, so development of NAI-resistant viruses with superior fitness is a public health concern. Fitness of NAI-resistant influenza B viruses is not widely studied. Here we examined the replicative capacity and relative fitness in normal human bronchial epithelial (NHBE) cells of recombinant (rg) influenza B/Yamanashi/166/1998 viruses containing a single amino acid substitution in NA that is associated with NAI-resistance. Replication in NHBE cells of viruses with reduced inhibition by oseltamivir (rg-E119A, rg-D198E, rg-I222T, rg-H274Y, rg-N294S, and rg-R371K, N2 numbering) or zanamivir (rg-E119A and rg-R371K) failed to be inhibited by the presence of the respective NAI. In a fluorescence-based assay, detection of rg-E119A was easily masked by the presence of NAI-susceptible virus. We co-infected NHBE cells with NAI-susceptible and -resistant viruses and used next-generation deep-sequencing to reveal the order of relative fitness nndash;compared to that of wild-type (WT) virus: rg-H274Y ggt; rg-WT ggt; rg-I222T ggt; rg-N294S ggt; rg-D198E ggt; rg-E119A ggt;ggt; rg-R371K. Based on the lack of attenuated replication of rg-E119A in NHBE cells in the presence of oseltamivir or zanamivir, and the fitness advantage of rg-H274Y over rg-WT, we emphasize the importance of these substitutions in the NA glycoprotein. Human infections with influenza B viruses carrying E119A or H274Y substitutions could limit therapeutic options for those infected; emergence of such viruses should be closely monitored.

    IMPORTANCE Influenza B viruses are important human respiratory pathogens contributing to a significant portion of seasonal influenza infections worldwide. The development of resistance to a single class of available antivirals, the neuraminidase (NA) inhibitors (NAIs), is a public health concern. Amino acid substitutions in the NA glycoprotein of influenza B virus can not only confer antiviral resistance but also alter viral fitness. Here we use normal human bronchial epithelial (NHBE) cells, a model of the human upper respiratory tract, to examine replicative capacities and fitness of NAI-resistant influenza B viruses. We show that virus with an E119A NA substitution can replicate efficiently in NHBE cells in the presence of oseltamivir or zanamivir and that virus with the H274Y NA substitution has a relative fitness greater than that of the wild-type NAI-susceptible virus. This study is the first to use NHBE cells to determine the fitness of NAI-resistant influenza B viruses.

  • The glycosylation status of PrPC is a key factor in determining TSE transmission between species. [PublishAheadOfPrint]

  • The risk of transmission of Transmissible Spongiform Encephalopathies (TSE) between different species has been notoriously unpredictable because the mechanisms of transmission are not fully understood. A transmission barrier between species often prevents infection of a new host with a TSE agent. Nonetheless, some TSE agents are able to cross this barrier and infect new species with devastating consequences. The host PrPC misfolds during disease pathogenesis and has a major role in controlling the transmission of agents between species, but sequence compatibility between host and agent PrPC does not fully explain host susceptibility. PrPC is post-translationally modified by the addition of glycan moieties which have an important role in the infectious process. Here we show in vivo that glycosylation of the host PrPC has a significant impact on the transmission of TSE between different host species. We infected mice carrying different glycosylated forms of PrPC with two human agents (sCJDMM2 and vCJD) and one hamster strain (263K). The absence of glycosylation at both or the first PrPC glycosylation site in the host results in almost complete resistance to disease. Absence of the second site of N-glycan has a dramatic effect on the barrier to transmission between host species facilitating the transmission of sCJDMM2 to a host normally resistant to this agent. These results highlight glycosylation of PrPC as a key factor in determining the transmission efficiency of TSEs between different species.

    Importance The risks of transmission of Transmissible Spongiform Encephalopathies (TSE) between different species are difficult to predict due to a lack of knowledge over the mechanisms of disease transmission; some strains of TSE are able to cross a species barrier, whilst others do not. The host protein, PrPC, plays a major role in disease transmission. PrPC undergoes post-translational glycosylation and the addition of these glycans may play a role in disease transmission. We infected mice that express different forms of glycosylated PrPC with three different TSE agents. We demonstrate that changing the glycosylation status of the host can have profound effects on disease transmission, changing host susceptibility and incubation times. Our results show that PrPC glycosylation is a key factor in determining risks of TSE transmission between species.

  • Hepatitis C virus attenuates mitochondrial lipid {beta}-oxidation by down-regulating mitochondrial trifunctional protein expression [PublishAheadOfPrint]

  • The course of HCV infection and disease progression involves alterations in lipid metabolism, leading to symptoms such as hypocholesterolemia and steatosis. Steatosis can be induced by multiple mechanisms including increases in lipid biosynthesis and uptake, impaired lipoprotein secretion and/or attenuation of lipid bbeta;-oxidation. However, little is known about the effects of HCV on lipid bbeta;-oxidation. A previous proteomics study revealed that HCV interacted with both aalpha; and bbeta;-subunits of the mitochondrial trifunctional protein (MTP), an enzyme complex which catalyses the last 3 steps of mitochondrial lipid bbeta;-oxidation for cellular energy production. Here we show that in HCV infected Huh7.5 cells lipid bbeta;-oxidation was significantly attenuated. Consistent with this, MTP protein and mRNA levels were suppressed by HCV infection. A loss of function study showed that MTP depletion rendered cells less responsive to IFNaalpha; treatment by impairing IFN-stimulated gene expression. These aspects of host/virus interaction explain how HCV alters host energy homeostasis and may also contribute to the establishment of persistent infection in the liver.

    Importance HCV infection triggers metabolic alterations, which lead to significant disease outcomes such as fatty liver (steatosis). This study revealed that HCV impairs mitochondrial lipid bbeta;-oxidation, which results in low lipid combustion. On the other hand, the HCV-induced defects in metabolic status played an important role in the control of the type I interferon system. Under the conditions of impaired lipid bbeta;-oxidation, the host cells were less responsive to the ability of exogenously added interferon aalpha; (IFNaalpha;) to suppress HCV replication. This suggests that interference with lipid bbeta;-oxidation could assist the virus in the establishment of a long-term, persistent infection. Further understanding of this aspect of virus/host interaction may leads to improvements in current standard therapy.

  • A triple arginine motif in the amino-terminal domain and oligomerization are required for HIV-1 inhibition by human MX2 [PublishAheadOfPrint]

  • We have employed molecular genetic approaches to understand the domain organization of the HIV-1 resistance factor, myxovirus resistance 2 (MX2). First, we describe an essential triple arginine motif in the amino-terminal domain. Second, we demonstrate that this 91 residue domain mediates anti-viral activity when appended to heterologous proteins, and provide genetic evidence that protein oligomerization is required for MX2 function. These insights will facilitate future work aiming to elucidate MX2's mechanism of action.

  • Anti-V3/glycan and anti-MPER neutralizing antibodies, but not anti-V2/glycan-site antibodies are strongly associated with higher anti-HIV-1 neutralization breadth and potency [PublishAheadOfPrint]

  • The Membrane Proximal External Region (MPER), the V2/glycan site (initially defined by PG9 and PG16 antibodies), and the V3/glycans, (initially defined by PGT121-128 antibodies) are targets of broadly-neutralizing antibodies and potential targets for anti-HIV-1 antibody-based vaccines. Recent evidence shows that antibodies with moderate neutralization breadth are frequently attainable, with 50% of sera from chronically-infected individuals neutralizing gge;50% of a large, diverse set of viruses. Nonetheless, there is little systematic information addressing which specificities are preferentially targeted among such commonly found, moderately broad neutralizing sera. We explored associations between neutralization breadth and potency and presence of neutralizing antibodies targeting MPER, V2/glycan site and V3/glycans in sera from 177 antiretroviral-naive HIV-1-infected (ggt;1yr) individuals. Recognition of both MPER and V3/glycans was associated with increased breadth and potency. MPER-recognizing sera neutralized 4.62 more panel viruses than MPER-negative sera (95% prediction interval (PI) 4.41, 5.20), and V3/glycan-recognizing sera neutralized 3.24 more panel viruses than V3/glycan-negative sera (95%PI 3.15, 3.52). In contrast, V2/glycan site-recognizing sera neutralized only 0.38 more panel viruses (95%PI 0.20, 0.45) than V2/glycan site-negative sera and no association between V2/glycan site recognition and breadth or potency was observed. Despite autoreactivity of many neutralizing antibodies recognizing MPER and V3/glycans, antibodies to these sites are major contributors to neutralization breadth and potency in this cohort. It may therefore be appropriate to focus on developing immunogens based upon the MPER and V3/glycans.

    Importance Previous candidate HIV vaccines have failed to either induce wide-coverage neutralizing antibodies or substantially protect vaccinees. Therefore, current efforts focus on novel approaches never before successfully used in vaccine design, including modeling epitopes. Candidate immunogen models identified by broadly neutralizing antibodies include the membrane proximal eternal region (MPER), V3/glycans and the V2/glycan site. Autoreactivity and polyreactivity of anti-MPER and anti-V3/glycan antibodies are thought to pose both direct and indirect barriers to achieving neutralization breadth. We found that antibodies to the MPER and the V3/glycans contribute substantially to neutralization breadth/potency. In contrast, antibodies to the V2/glycan-site were not associated with neutralization breadth/potency. This suggests that the autoreactivity effect is not critical and that the MPER and the V3/glycans should remain high priority vaccine candidates. The V2/glycan site result is surprising because broadly neutralizing antibodies to this site have been repeatedly observed. Vaccine design priorities should shift towards the MPER and V3/glycans.

  • A Mutant H3N2 Influenza Virus Uses an Alternative Activation Mechanism in TMPRSS2 Knockout Mice by Loss of an Oligosaccharide in the Hemagglutinin Stalk Region [PublishAheadOfPrint]

  • The host protease TMPRSS2 plays an essential role for proteolytic activation of influenza A virus (IAV) HA protein possessing a monobasic cleavage site. However, after passages in TMPRSS2 knockout mice, an H3N2 subtype IAV became to undergo cleavage activation of HA showing high virulence for the mice by loss of an oligosaccharide at position 8 in the HA stalk region. Thus, the H3N2 IAV acquired the cleavability by an alternative activation mechanism/protease(s) for HA.

  • Detergent-Resistant Membrane Association of NS2 and E2 during Hepatitis C Virus Replication [PublishAheadOfPrint]

  • Previously, we have demonstrated that the efficiency of HCV E2-p7 processing regulates p7-dependent NS2 localization to putative virus assembly sites near lipid droplets (LD). In this study, we have employed subcellular fractionations and membrane flotation assays to demonstrate that NS2 associates with detergent-resistant membranes (DRM) in a p7-dependent manner. However, p7 likely plays an indirect role in this process, since only the background level of p7 was detectable in the DRM fractions. Our data also suggest that the p7-NS2 precursor is not involved in NS2 recruitment to the DRM, despite its apparent targeting to this location. Deletion of NS2 specifically inhibited E2 localization to the DRM, indicating that NS2 regulates this process. Treatment of cells with the methyl-bbeta;-cyclodextrin (Mbbeta;CD) significantly reduced the DRM association of Core, NS2 and E2 and reduced infectious HCV production. Since disruption of the DRM localization of NS2 and E2, either due to p7- and NS2-defects, respectively, or by Mbbeta;CD treatment, inhibited infectious HCV production, these proteins' associations with the DRM likely play an important role during HCV assembly. Interestingly, we detected the HCV replication-dependent accumulation of ApoE in the DRM fractions. Considering that ApoE was shown to be a major determinant for infectious HCV particle production at the post-envelopment step and that HCV Core protein strongly associates with the DRM, recruitment of E2 and ApoE to the DRM may allow the efficient coordination of Core particle envelopment and post-envelopment events at the DRM to generate infectious HCV production.

    Importance The biochemical nature of HCV assembly sites is currently unknown. In this study, we investigated the correlation between NS2 and E2 localization to the detergent-resistant membranes (DRM) and HCV particle assembly. We determined that although NS2's DRM localization is dependent on p7, p7 was not targeted to these membranes. We then showed that NS2 regulates E2 localization to the DRM, consistent with its role in recruiting E2 to the virus assembly sites. We also showed that short-term treatment with the cholesterol-extracting agent methyl-bbeta;-cyclodextrin (Mbbeta;CD) not only disrupted the DRM localization of Core, NS2, and E2 but also specifically inhibited intracellular virus assembly without affecting HCV RNA replication. Thus, our data support the role of the DRM as a platform for particle assembly process.

  • An anti-H5N1 influenza virus FcDART antibody is an highly efficacious therapeutic and prophylactic against H5N1 influenza virus infection. [PublishAheadOfPrint]

  • Highly pathogenic H5N1 avian influenza viruses are associated with severe disease in humans and continue to be a pandemic threat. Whilst vaccines are available, other approaches are required for patients that typically respond poorly to vaccination, such as the elderly and the immunocompromised. To produce a therapeutic that is highly efficacious at low doses and is broadly specific against antigenically-drifted H5N1 influenza viruses, we developed two neutralizing monoclonal antibodies and combined them into a single bispecific-Fc fusion protein (FcDART). In mice, a single 2.5mg/kg therapeutic or prophylactic dose of either monoclonal antibody provided 100% protection against challenge with A/Vietnam/1203/04 (H5N1) or the antigenically-drifted strain A/Whooper swan/Mongolia/244/05 (H5N1). In ferrets, a single 1mg/kg prophylactic dose provided 100% protection against A/Vietnam/1203/04 challenge. FcDART was also effective, as a single 2.5mg/kg therapeutic or prophylactic dose in mice provided 100% protection against A/Vietnam/1203/04 challenge. Antibodies bound to conformational epitopes in antigenic sites on the globular head of the hemagglutinin protein, based on antibody escape mutations. Whilst it was possible to generate escape mutants in vitro, they were neutralized in vivo by the antibodies, as mice infected with escape mutants were 100% protected after only a single therapeutic dose of the antibody used to generate the escape mutant in vitro. In summary, we have combined the antigen specificities of two highly efficacious anti-H5N1 influenza virus antibodies into a bispecific FcDART, which represents a strategy to produce broadly neutralizing antibodies that are effective against antigenically diverse influenza viruses.

    Importance: Highly pathogenic H5N1 avian influenza viruses are associated with severe disease in humans and are a pandemic threat. A vaccine is available but other approaches are required for patients that typically respond poorly to vaccination, such as the elderly and the immunocompromised. The variability of the virus means that such an approach must be broad spectrum. To achieve this, we developed two antibodies that neutralize H5N1 influenza viruses. These antibodies provided complete protection in mice against a spectrum of H5N1 influenza viruses at a single low dose. We then combined the two antibodies into a single molecule, FcDART, which combined the broad spectrum activity and protective efficacy of both antibodies. This treatment strategy represents a novel and effective therapeutic or prophylactic against highly pathogenic H5N1 avian influenza viruses.

  • Domestic pigs are susceptible to infection with influenza B viruses [PublishAheadOfPrint]

  • Influenza B virus (IBV) causes seasonal epidemics in humans. Although IBV has been isolated from seals, humans are considered to be the primary host and reservoir of this important pathogen. It is unclear whether other animal species can support the replication of IBV and serve as a reservoir. Swine are naturally infected with both influenza A and C viruses. To determine the susceptibility of pigs to IBV infection, we conducted a serological survey for U.S. Midwest domestic swine herds from 2010 to 2012. Results of this study showed that antibodies to IBVs were detected in 38.5% (20/52) of sampled farms and 7.3% (41/560) of tested swine serum samples were positive for IBV antibodies. Furthermore, swine herds infected with porcine reproductive and respiratory syndrome virus (PRRSV) showed a higher prevalence of IBV antibodies in our 2014 survey. In addition, IBV was detected in 3 nasal swabs collected from PRRSV seropositive pigs by real-time RT-PCR and sequencing. Finally, an experimental infection in pigs, via intranasal and intratracheal routes, was performed using one representative virus from each of the two genetically and antigenically distinct lineages of IBVs: B/Brisbane/60/2008 (Victoria lineage) and B/Yamagata/16/1988 (Yamagata lineage). Pigs developed influenza-like symptoms, lung lesions, and seroconverted post virus inoculation. Pigs infected with B/Brisbane/60/2008 virus successfully transmitted the virus to sentinel animals. Taken together, our data demonstrate that pigs are susceptible to IBV infection and therefore warrant further surveillance and investigation of swine as a potential host for human IBV.

    Importance Influenza B virus (IBV) is an important human pathogen, but its ability to infect other species, for example pigs, are not well understood. We showed serological evidence that antibodies to two genetically and antigenically distinct lineages of IBVs were present among domestic pigs, especially in swine herds previously infected with porcine reproductive and respiratory syndrome virus (PRRSV), an immunosuppressive virus. IBV was detected in 3 nasal swabs from PRRSV seropositive pigs by real-time RT-PCR and sequencing. Moreover, both lineages of IBV were able to infect pigs under experimental conditions with transmissibility of influenza B/Victoria lineage virus among pigs observed. Our results demonstrate that pigs are susceptible to IBV infections, indicating that IBV may be a potential swine pathogen and swine may serve as a natural reservoir of IBVs. In addition, pigs may serve as a model to study the mechanisms of transmission and pathogenesis of IBVs.

  • Triatoma virus recombinant VP4 protein induces membrane permeability through dynamic pores [PublishAheadOfPrint]

  • In naked viruses, membrane breaching is a key step that must be performed for genome transfer into the target cells. Despite its importance, the mechanisms behind this process remain poorly understood. The small protein VP4, codified in the genome of most Picornavirales order viruses, has been shown to be involved in membrane alterations. Here we have analyzed the permeabilization activity of the natively non-myristoylated VP4 protein from Triatoma virus (TrV), a virus belonging to the Dicistroviridae family within the Picornavirales order. The VP4 protein was produced as a C-terminal Maltose Binding Protein (MBP) fusion to achieve its successful expression. This recombinant VP4 protein is able to produce membrane permeabilization in model membranes in a membrane composition-dependent manner. The induced permeability was also influenced by the pH, being greater at higher pH values. We demonstrate that the permeabilization activity elicited by the protein occurs through discrete pores that are inserted on the membrane. Sizing experiments using fluorescent dextrans, cryo-electron microscopy imaging and other additional techniques showed that recombinant VP4 forms heterogeneous proteo-lipidic pores rather than common proteinaceous channels. These results suggest that VP4 protein may be involved in the membrane alterations required for genome transfer or cell entry steps during dicistrovirus infection.

    IMPORTANCE During viral infection, viruses need to overcome the membrane barrier in order to enter the cell and replicate their genome. In non-enveloped viruses membrane fusion is not possible and hence, other mechanisms are implemented. Among other proteins, like the capsid forming proteins and the proteins required for viral replication, several Picornaviral order viruses contains a small protein called VP4 that has been shown to be involved in membrane alterations. Here we show that Triatoma virus VP4 protein is able to produce membrane permeabilization in model membranes by the formation of heterogeneous dynamic pores. These pores formed by VP4 may be involved in the genome transfer or cell entry steps during viral infection.

  • Chimeric Rhinoviruses obtained Via Genetic Engineering or Artificially- Induced Recombination are Only Viable if the Polyprotein Coding Sequence Derives from the Same Species [PublishAheadOfPrint]

  • Recombination is a widespread phenomenon that ensures both the stability and variation of RNA viruses. This phenomenon occurs with different frequencies within species of the Enterovirus genus. Intraspecies recombination is frequently described among non-rhinovirus enteroviruses, but appears sporadic in rhinoviruses. Interspecies recombination is even rarer for rhinoviruses and mostly related to ancient events, which contributed to the speciation of these viruses. We reported that artificially engineered 5rrsquo; UTR interspecies rhinovirus/rhinovirus or rhinovirus/non-rhinovirus enterovirus recombinants are fully viable.

    Using a similar approach, we demonstrated in this study that exchanges of the P1-2A polyprotein region between members of the same rhinovirus species, but not between members of different species, give rise to competent chimeras. To further assess the rhinovirus intra- and interspecies recombination potential, we used artificially-induced recombination by co-transfection of 5rrsquo; end deleted and 3rrsquo; end deleted and replication-deficient genomes. In this system, intraspecies recombination also resulted in viable viruses with high frequency, whereas no interspecies rhinovirus recombinants could be recovered. Mapping intraspecies recombination sites within the polyprotein highlighted recombinant hotspots in non-structural genes and at gene boundaries. Notably, all recombinants occurring at gene junctions presented in frame sequence duplications, whereas most intragenic recombinants were homologous. Taken together, our results suggest that only intraspecies recombination gives rise to viable rhinovirus chimeras in the polyprotein coding region and that recombination hotspots map to non-structural genes with in-frame duplications at gene boundaries. These data provide new insight regarding the mechanism and limitations of rhinovirus recombination.

    Importance Recombination represents a means to ensure both the stability and the variation of RNA viruses. While intraspecies recombination is frequently described among non-rhinovirus enteroviruses, it seems to occur more rarely in rhinoviruses. Interspecies recombination is even rarer in this virus group and is mostly related to ancient events, which contributed to its speciation. We used engineered chimeric genomes and artificially-induced RNA recombination to study experimentally the recombination potential of rhinoviruses and analyze recombination sites. Our results suggest that only intraspecies recombination gives rise to viable chimeras in the polyprotein coding region. Furthermore, characterization of intraspecies chimeras provides new insight into putative recombination hotspots within the polyprotein. In summary, we applied two powerful and complementary experimental approaches to improve current knowledge on rhinovirus recombination.

  • Insect Inhibitor of Apoptosis IAPs are Negatively Regulated by Signal-Induced N-Terminal Degrons Absent within Viral IAPs [PublishAheadOfPrint]

  • Inhibitor of apoptosis (IAP) proteins are key regulators of the innate antiviral response by virtue of their capacity to respond to signals affecting cell survival. In insects, wherein the host IAP provides a primary restriction to apoptosis, diverse viruses trigger rapid IAP depletion that initiates caspase-mediated apoptosis, thereby limiting virus multiplication. We report here that the N-terminal leader of two insect IAPs, Spodoptera frugiperda SfIAP and Drosophila melanogaster DIAP1, contain distinct instability motifs that regulate IAP turnover and apoptotic consequences. Functioning as a protein degron, the cellular IAP leader dramatically shortened the lifespan of a long-lived viral IAP (Op-IAP3) when fused to its N terminus. The SfIAP degron contains mitogen-activated kinase (MAPK)-like regulatory sites, responsible for MAPK inhibitor-sensitive phosphorylation of SfIAP. Hyperphosphorylation correlated with increased SfIAP turnover independently of the E3 ubiquitin-ligase activity of the SfIAP RING, which also regulated IAP stability. Together, our findings suggest that the SfIAP phospho-degron responds rapidly to a signal-activated kinase cascade, which regulates SfIAP levels and thus apoptosis. The N-terminal leader of dipteran DIAP1 also conferred virus-induced IAP depletion by a caspase-independent mechanism. DIAP1 instability mapped to previously unrecognized motifs, not found in lepidopteran IAPs. Thus, the leaders of cellular IAPs from diverse insects carry unique signal-responsive degrons that control IAP turnover. Rapid response pathways that trigger IAP degradation and initiate apoptosis independent of canonical prodeath gene (Reaper-Grim-Hid) expression may provide important innate immune advantages. Furthermore, the elimination of these response motifs within viral IAPs, including those of baculoviruses, explains their unusual stability and their potent anti-apoptotic activity.

    IMPORTANCE Apoptosis is an effective means by which a host controls virus infection. In insects, inhibitor of apoptosis (IAP) proteins act as regulatory sentinels by responding to cellular signals that determine the fate of infected cells. We discovered that lepidopteran (moth and butterfly) IAPs, which are degraded upon baculovirus infection, are controlled by a conserved phosphorylation-sensitive degron within the IAP N-terminal leader. The degron likely responds to virus-induced kinase-specific signals for degradation through SKP1/Cullin/F-box complex-mediated ubiquitination. Such signal-induced destruction of cellular IAPs is distinct from degradation caused by well-known IAP antagonists, which act to expel IAP-bound caspases. The major implication of this study is that insects have multiple signal-responsive mechanisms by which the sentinel IAPs are actively degraded to initiate host apoptosis. Such diversity of pathways likely provides insects with rapid and efficient strategies for pathogen control. Furthermore, the absence of analogous degrons in virus-encoded IAPs explains their relative stability and anti-apoptotic potency.

  • Fighting fire with fire: Endogenous retrovirus envelopes as restriction factors [PublishAheadOfPrint]

  • A considerable portion of vertebrate genomes are made up of endogenous retroviruses (ERVs). While aberrant or uncontrolled ERV expression has been perceived as a potential cause of disease, there is mounting evidence that some ERVs have become integral components of normal host development and physiology. Here we revisit the longstanding concept that some of the gene products encoded by ERVs and other endogenous viral elements may offer to the host protection against viral infection. Notably proteins produced from envelope (env) genes have been shown to act as restriction factors against related exogenous retroviruses in chicken, sheep, mice, and cats. Based on the proposed mode of restriction and the domain architecture of known antiretroviral env, we argue that many more env-derived restriction factors await discovery in vertebrate genomes, including the human genome.

  • Reduced Virion-Associated {sigma}1 Fibers on Oncolytic Reovirus Variants Promotes Adaptation towards Tumorigenic Cells [PublishAheadOfPrint]

  • Serotype 3 reovirus (T3wt) is non-pathogenic in humans but preferentially infects and kills cancer cells in culture and demonstrates promising anti-tumor activity in vivo. Using forward genetics, we previously isolated two variants of reovirus, T3v1 and T3v2, with increased infectivity towards a panel of cancer cell lines and improved in vivo oncolysis in a murine melanoma model relative to T3wt. Our current study explored how mutations in T3v1 and T3v2 promote infectivity. Reovirions contain trimers of 1, the reovirus cell attachment protein, at icosahedral capsid vertices. Quantitative western blot analysis showed that purified T3v1 and T3v2 virions had approximately 2- and 4- fold lower levels of 1 fiber than T3wt. Importantly, using RNA interference to reduce 1 levels during T3wt production, we were able to generate wild-type reovirus with reduced 1 per virion. As 1 levels reduced, virion infectivity increased by 2-5 fold per equivalent bound virions, demonstrating a causal relationship between virion 1 levels and infectivity of incoming virions. During infection of tumorigenic L929 cells, T3wt, T3v1 and T3v2 uncoated outer capsid proteins 3 and mmu;1C at similar rates. However, having started with fewer 1 molecules, complete loss of 1 was achieved sooner for T3v1 and T3v2. Distinct from intracellular uncoating, chymotrypsin digestion as a mimic of natural enteric infection resulted in more-rapid 3 and mmu;1C removal, unique disassembly intermediates and rapid loss of infectivity for T3v1 and T3v2 compared to T3wt. Optimal infectivity towards natural versus therapeutic niches may therefore require distinct reovirus structures and 1 levels.

    Importance Wild-type reovirus is currently in clinical trials as a potential cancer therapy. Our molecular studies on variants of reovirus with enhanced oncolytic activity in vitro and in vivo, now shows that distinct reovirus structures promote adaptation towards cancer cells and away from conditions that mimic natural routes of infection. Specifically, we found that reovirus particles with fewer molecules of cell-attachment protein 1 became more infectious towards transformed cells. Reduced 1 conferred a benefit to incoming particles only, resulting in earlier depletion of 1 and higher probability of establishing productive infection. Conversely, reovirus variants with fewer 1 showed reduced stability, infectivity, and distinct disassembly when exposed to conditions that mimic natural intestinal proteolysis. These findings support a model where the mode of infection dictates the precise optimum of reovirus structure, and provides molecular rationale for considering alternative reovirus structures during oncolytic therapy.

  • Fine mapping and characterization of the L polymerase-binding domain of the respiratory syncytial virus phosphoprotein [PublishAheadOfPrint]

  • The minimum requirement for an active RNA-dependent RNA polymerase of respiratory syncytial virus is a complex made of two viral proteins, the polymerase large protein L and the phosphoprotein P. Here, we have investigated the domain on P responsible for this critical P-L interaction. Using recombinant proteins and serial deletions, a L binding site was mapped in the C-terminal region of P, just upstream from the N-RNA binding site. The role of this molecular recognition element of about 30 amino acid residues in L-P interaction and RNA polymerase activity was evaluated in cellula using an RSV minigenome system and site-directed mutagenesis. The results highlighted the critical role of hydrophobic residues located in this region.

    Importance. Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine or good antivirals are available against RSV, it is essential to better understand how the viral machinery functions in order to develop new antiviral strategies. Like all negative strand RNA viruses, RSV codes for its own machinery to replicate and transcribe its genome. The core of this machinery is composed of two proteins, the phosphoprotein P and the large protein L. Here, using recombinant proteins, we have mapped and characterized the P domain responsible for this L-P interaction and formation of an active L-P complex. These findings extend our understanding of the mechanism of action of RSV RNA polymerase and allow us to define a new target for the development of drugs against RSV.

  • Stress granule components G3BP1 and G3BP2 play a proviral role early in chikungunya virus replication [PublishAheadOfPrint]

  • Stress granules (SGs) are protein-mRNA aggregates that are formed in response to environmental stresses, resulting in translational inhibition. SGs are generally believed to play an antiviral role and are manipulated by many viruses, including various alphaviruses. GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1) is a key component and commonly used marker of SGs. Its homolog G3BP2 is a less extensively studied SG component. Here, we demonstrate that chikungunya virus (CHIKV) infection induces cytoplasmic G3BP1- and G3BP2-containing granules that differ from bona fide SGs in terms of morphology, composition, and behavior. For several Old World alphaviruses it has been shown that nonstructural protein 3 (nsP3) interacts with G3BPs, presumably to inhibit SG formation, and we have confirmed this interaction in CHIKV-infected cells. Surprisingly, CHIKV also relied on G3BPs for efficient replication, as simultaneous depletion of G3BP1 and G3BP2 reduced viral RNA levels, CHIKV protein expression, and viral progeny titers. The G3BPs colocalized with CHIKV nsP2 and nsP3 in cytoplasmic foci, but no colocalization with nsP1, nsP4, or dsRNA was observed. Furthermore, G3BPs could not be detected in a cellular fraction enriched for CHIKV replication/transcription complexes, suggesting they are not directly involved in CHIKV RNA synthesis. Depletion of G3BPs did not affect viral entry, translation of incoming genomes, or nonstructural polyprotein processing, but resulted in severely reduced levels of negative-stranded (and consequently also positive-stranded) RNA. This suggests a role for the G3BPs in the switch from translation to genome amplification, although the exact mechanism by which they act remains to be explored.

    IMPORTANCE Chikungunya virus (CHIKV) causes a severe polyarthritis that has affected millions of people since its re-emergence in 2004. The lack of approved vaccines or therapeutic options, and the ongoing explosive outbreak in the Caribbean underline the importance to better understand CHIKV replication. Stress granules (SGs) are cytoplasmic protein-mRNA aggregates formed in response to various stresses, including viral infection. The RNA-binding proteins G3BP1 and G3BP2 are essential SG components. SG formation and the resulting translational inhibition are generally considered an antiviral response, and many viruses manipulate or block this process. Late in infection, we and others have observed CHIKV nonstructural protein 3 in cytoplasmic G3BP1- and G3BP2-containing granules. These virally induced foci differed from true SGs and did not appear to represent replication complexes. Surprisingly, we found that G3BP1 and G3BP2 were also needed for efficient CHIKV replication, likely by facilitating the switch from translation to genome amplification early in infection.

  • African Green Monkey TRIM5{alpha} Restriction in SIV-Specific Rhesus Macaque Effector CD4 T Cells Enhances Their Survival and Antiviral Function [PublishAheadOfPrint]

  • Expression of xenogeneic TRIM5aalpha; proteins can restrict infection in various retrovirus/host cell pairings. Previously, we have shown that African green monkey TRIM5aalpha; (AgmTRIM5aalpha;) potently restricts both HIV-1 and SIVmac239 replication in a transformed human T-cell line. To assess AgmTRIM5aalpha; restriction in primary cells, we transduced AgmTRIM5aalpha; into primary rhesus macaque CD4 T cells and infected them with SIVmac239. Experiments with T-cell clones revealed that AgmTRIM5aalpha; could reproducibly restrict SIVmac239 replication and that this restriction synergizes with an intrinsic resistance to infection present in some CD4 T-cell clones. AgmTRIM5aalpha; transduction of virus-specific CD4 T-cell clones increased and prolonged their ability to suppress SIV spread in CD4 target cells. This increased antiviral function was strongly linked to decreased viral replication in the AgmTRIM5aalpha;-expressing effectors, consistent with restriction preventing virus-induced cytopathogenicity thereby disabling effector function. Taken together, our data show that AgmTRIM5aalpha; restriction, though not absolute, reduces SIV replication in primary rhesus CD4 T cells which, in turn, increases their antiviral function. These results support prior in vivo data indicating that the contribution of virus-specific CD4 T-cell effectors to viral control is limited due to infection.

    IMPORTANCE The potential of effector CD4 T cells to immunologically modulate SIV/HIV infection is likely limited by their susceptibility to infection and subsequent inactivation or elimination. Here, we show that AgmTRIM5aalpha; expression inhibits SIV spread in primary effector CD4 T cells in vitro. Importantly, protection of effector CD4 T cells by AgmTRIM5aalpha; markedly enhanced their antiviral function by delaying SIV infection, thereby extending their viability despite the presence of virus. Our in vitro data support prior in vivo HIV-1 studies suggesting that the antiviral CD4 effector response is impaired due to infection and subsequent cytopathogenicity. The ability of AgmTRIM5aalpha; expression to restrict SIV infection in primary rhesus effector CD4 T cells now opens an opportunity to use the SIV/rhesus macaque model to further elucidate the potential and scope of anti-AIDS virus effector CD4 T-cell function.

  • A Nucleotide Exchange Factor Promotes ER-to-cytosol Membrane Penetration of the Non-enveloped Virus SV40 [PublishAheadOfPrint]

  • The non-enveloped simian polyomavirus (PyV) SV40 hijacks the endoplasmic reticulum (ER) quality control machinery to penetrate the ER membrane and reach the cytosol, a critical infection step. During entry, SV40 traffics to the ER where host-induced conformational changes render the virus hydrophobic. The hydrophobic virus binds and integrates into the ER lipid bilayer to initiate membrane penetration. However, prior to membrane transport, the hydrophobic SV40 recruits the ER-resident Hsp70 BiP which holds the virus in a transport-competent state until it is ready to cross the ER membrane. Here we probed how BiP disengages from SV40 to enable the virus to penetrate the ER membrane. We find that the nucleotide exchange factor (NEF) Grp170 induces nucleotide exchange of BiP and releases SV40 from BiP. Importantly, this reaction promotes SV40 ER-to-cytosol transport and infection. The human BK PyV also relies on Grp170 for successful infection. Interestingly, SV40 mobilizes a pool of Grp170 into discrete puncta in the ER called foci. These foci, postulated to represent the ER membrane penetration site, harbor ER components including BiP known to facilitate viral ER-to-cytosol transport. Our results thus identify a nucleotide exchange activity essential for catalyzing the most proximal event before ER membrane penetration of PyVs.

    Importance PyVs are known to cause debilitating human diseases. During entry, this virus family including the monkey SV40 and human BK PyV hijacks ER protein quality control machinery to breach the ER membrane and access the cytosol, a decisive infection step. In this study, we pinpoint an ER-resident factor that executes a crucial role in promoting ER-to-cytosol membrane penetration of PyVs. Identifying a host factor that facilitates entry of the PyV family thus provides additional therapeutic targets to combat PyV-induced diseases.

  • CD40L-Adjuvanted DNA/MVA SIV Vaccine Enhances Protection Against Neutralization Resistant Mucosal SIV Infection [PublishAheadOfPrint]

  • Here, we show that a CD40L-adjuvanted DNA/MVA SIV vaccine enhances protection against a pathogenic neutralization-resistant mucosal SIV infection, improves long-term viral control and prevents AIDS. Analyses of serum IgG antibodies to linear peptides of SIV Env revealed a strong response to V2 with targeting of fewer epitopes in gp41-ID and V1 regions as a correlate for enhanced protection. Higher expansion of anti-viral CD8 T cells in the gut correlated with long-term viral control.

  • HIV-1 Dolutegravir-Resistance Substitution R263K Cannot Co-Exist in Combination with Many Classical Integrase Inhibitor Resistance Substitutions [PublishAheadOfPrint]

  • The new integrase strand transfer inhibitor (INSTI) dolutegravir (DTG) displays limited cross-resistance with older drugs of this class and selects for the R263K substitution in treatment-experienced patients. We performed tissue culture selections with DTG using viruses resistant to older INSTIs, infectivity and resistance assays, and showed that the presence of the E92Q or N155H substitutions was compatible with the emergence of R263K whereas the G140S/Q148R, E92Q/N155H, G140S, Y143R and Q148R substitutions were not.

  • Dynamics of the Cytotoxic T Cell Response to a Model of Acute Viral Infection [PublishAheadOfPrint]

  • A detailed characterization of the dynamics and breadth of the immune response to an acute viral infection, as well as the determinants of recruitment to immunological memory, can greatly contribute to our basic understanding of the mechanics of the human immune system, and can ultimately guide the design of effective vaccines. In addition to neutralizing antibodies, T cells have been shown to be critical for the effective resolution of acute viral infections. We report the first in-depth analysis of the dynamics of the CD8+ T cell repertoire at the level of individual T cell clonal lineages upon vaccination of human volunteers with a single dose of YF-17D. This live attenuated yellow fever vaccine yields sterile, long-term immunity and has been previously used as a model to understand the immune response to a controlled acute viral infection. We identified and enumerated unique CD8+ T cell clones specifically induced by this vaccine through a combined experimental and statistical approach that includes high throughput sequencing of the CDR3 variable region of the T cell receptor bbeta; chain and an algorithm that detects significantly expanded T cell clones. This allowed us to establish that: (a) on average ~2,000 CD8+ T cell clones were induced by YF-17D, (b) 5-6% of the responding clones were recruited to long-term memory three months post-vaccination, (c) the most highly-expanded effector clones were preferentially recruited to the memory compartment, and (d) a fraction of the YF-17D-induced clones can be identified from peripheral blood lymphocytes solely by measuring clonal expansion.

    IMPORTANCE The exhaustive investigation of pathogen-induced effector T cells is essential to accurately quantify the dynamics of the human immune response. The yellow fever vaccine (YFV) has been broadly used as a model to understand how a controlled, self-resolving acute viral infection induces an effective and long-term protective immune response. Here, we extend this previous work by reporting the identity of activated effector T cell clones that expand in response to the YFV two weeks post-vaccination (as defined by their unique T-cell receptor gene sequence), and by tracking clones that enter the memory compartment 6 weeks later. This is the first study to use high-throughput sequencing of immune cells to characterize the breadth of the anti-viral effector cell response, and to determine the contribution of unique virus-induced clones to the long-lived memory T cell repertoire. Thus, it establishes a benchmark against which future vaccines can be compared to predict their efficacy.

  • Expression of human decay-accelerating factor on intestinal epithelium of transgenic mice does not facilitate infection by the enteral route [PublishAheadOfPrint]

  • In vitro, infection of polarized human intestinal epithelial cells by Coxsackievirus B3 (CVB3) depends on virus interaction with decay accelerating factor (DAF), a receptor expressed on the apical cell surface. Although mice are highly susceptible to CVB3 infection when virus is delivered by intraperitoneal injection, infection by the enteral route is very inefficient. Murine DAF, unlike human DAF, does not bind virus, and we hypothesized that the absence of an accessible receptor on the intestinal surface is an important barrier to infection by the oral route. We generated transgenic mice that express human DAF specifically on intestinal epithelium, and measured their susceptibility to infection by a DAF-binding CVB3 isolate. Human DAF permitted CVB3 to bind to the intestinal surface ex vivo, and to infect polarized monolayers of small-intestinal epithelial cells derived from DAF-transgenic mice. However, expression of human DAF did not facilitate infection by the enteral route, either in immunocompetent animals or in animals deficient in the interferon alpha/beta receptor. These results indicate that the absence of an apical receptor on intestinal epithelium is not the major barrier to infection of mice by the oral route.

    IMPORTANCE CVB3 infection of human intestinal epithelial cells depends on DAF at the apical cell surface, and expression of human DAF on murine intestinal epithelial cells permits their infection in vitro. However, expression of human DAF on the intestinal surface of transgenic mice did not facilitate infection by the oral route. Although the role of intestinal DAF in human infection has not been directly examined, these results suggest that DAF is not the critical factor in mice.

  • Glycosylation of Mouse DPP4 Plays a Role in Inhibiting Middle East Respiratory Syndrome Coronavirus Infection [PublishAheadOfPrint]

  • Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes dipeptidyl peptidase 4 (DPP4) as an entry receptor. Mouse DPP4 (mDPP4) does not support MERS-CoV entry; however, changes at positions 288 and 330 can confer permissivity. Position 330 changes the charge and glycosylation state of mDPP4. We show that glycosylation is a major factor impacting DPP4 receptor function. These results provide insight into DPP4 species-specific differences impacting MERS-CoV host range and may inform MERS-CoV mouse model development.

  • Analysis by live imaging of effects of the adenovirus E4orf4 protein on passage through mitosis of H1299 tumor cells [PublishAheadOfPrint]

  • The adenovirus E4orf4 protein expressed at high levels kills cancer cells but not normal human primary cells. Previous studies suggested that disruption of processes that regulate mitosis may underlie E4orf4 toxicity. Here we have used live imaging to show that E4orf4 induces a slowed defective transit through mitosis, exhibiting a delay or often failure in cytokinesis that may account for an accumulation of G1 tetraploids in the population of dying E4orf4-expressing cells.

  • Role of type I interferon signaling in human metapneumovirus pathogenesis and control of viral replication. [PublishAheadOfPrint]

  • Type I interferon receptor (IFNAR) signaling regulates the expression of proteins that are crucial contributors to immune responses. Paramyxoviruses including human metapneumovirus (HMPV) have evolved mechanisms to inhibit IFNAR signaling, but the specific contribution of IFNAR signaling to control of HMPV replication, pathogenesis, and adaptive immunity is unknown. We used IFNAR-deficient (IFNAR-/-) mice to assess the effect of IFNAR signaling on HMPV replication and CD8+ T cell response. HMPV-infected IFNAR-/- mice had a higher peak of early viral replication, but cleared virus with similar kinetics to wild-type (WT) mice. However, IFNAR-/- mice infected with HMPV displayed less airway dysfunction and lung inflammation. CD8+ T cells in IFNAR-/- mice post-HMPV expressed similar levels of the inhibitory receptor programmed death-1 (PD-1) as WT. However, despite lower expression of the inhibitory ligand PD-L1, HMPV-specific CD8+ T cells in IFNAR-/- mice were more functionally impaired than WT and upregulated the inhibitory receptor TIM-3. Analysis of the antigen presenting cell subsets in the lungs revealed that the expansion of PD-L1lo dendritic cells (DCs), but not PD-L1high alveolar macrophages, was dependent on IFNAR signaling. Collectively, our results indicate a role for IFNAR signaling in the early control of HMPV replication, disease progression, and the development of an optimal adaptive immune response. Moreover, our findings suggest an IFNAR-independent mechanism of lung CD8+ T cell impairment.

    Importance Human metapneumovirus (HMPV) is a leading cause of acute respiratory illness. CD8+ T cells are critical for clearing viral infection, yet recent evidence shows that HMPV and other respiratory viruses induce CD8+ T cell impairment via PD-1:PD-L1 signaling. We sought to understand the role of type I interferon (IFN) in the innate and adaptive immune response to HMPV using a mouse model lacking IFN signaling. Although HMPV titers were higher in the absence of type I IFN, virus was nonetheless cleared and mice were less ill, indicating that type I IFN is not required to resolve HMPV infection but contributes to pathogenesis. Further, despite lower levels of the inhibitory ligand PD-L1 in mice lacking type I IFN, CD8+ T cells were more impaired in these mice than in WT mice. Our data suggest that specific antigen-presenting cell subsets and the inhibitory receptor TIM-3 may contribute to CD8+ T cell impairment.

  • ATP1A1-mediated Src signaling inhibits coronavirus entry into host cells [PublishAheadOfPrint]

  • Besides by transporting ions the multi-subunit Na+,K+-ATPase also functions by relaying cardiotonic steroid-binding induced signals into cells. In this study we analyzed the role of Na+,K+-ATPase and in particular of its ATP1A1 aalpha;-subunit during coronavirus (CoV) infection. As controls, the vesicular stomatitis virus (VSV) and influenza A virus (IAV) were taken along. Using gene silencing, the ATP1A1 protein was shown to be critical for infection of cells with murine hepatitis virus (MHV), feline infectious peritonitis virus (FIPV) and VSV, but not with IAV. Lack of ATP1A1 did not affect virus binding to host cells, but resulted inhibited entry of MHV and VSV. Consistently, nanomolar concentrations of the cardiotonic steroids ouabain or bufalin, which are known not to affect the transport function of Na+,K+-ATPase, inhibited infection of cells with MHV, FIPV, MERS-CoV, and VSV, but not IAV, when the compounds were present during virus inoculation. Cardiotonic steroids were shown to inhibit entry of MHV at an early stage, resulting in accumulation of virions close to the cell surface and as a consequence in reduced fusion. In agreement with an early block in infection, the inhibition of VSV by CTSs could be bypassed by low-pH shock. Viral RNA replication was not affected when these compounds were added after virus entry. The anti-viral effect of ouabain could be relieved by the addition of different Src kinase inhibitors, indicating that Src signaling mediated via ATP1A1 plays a crucial role in the inhibition of CoV and VSV infections.

    Importance Coronaviruses (CoVs) are important pathogens of animals and humans as demonstrated by the recent emergence of new human CoVs of zoonotic origin. Antiviral drugs targeting CoV infections are lacking. In the present study we show that the ATP1A1 subunit of Na+,K+-ATPase, an ion transporter and signaling transducer, supports CoV infection. Targeting ATP1A1 either by gene silencing or by low concentrations of the ATP1A1-binding cardiotonic steroids ouabain and bufalin, resulted in inhibition of infection with murine, feline and MERS-CoVs at an early entry stage. Infection with the control virus VSV was also inhibited. Src signaling mediated by ATP1A1 was shown to play a crucial role in the inhibition of virus entry by ouabain and bufalin. These results suggest that targeting the Na+,K+-ATPase using cardiotonic steroids, several of which are FDA-approved compounds, may be an attractive therapeutic approach against CoV and VSV infections.

  • X-ray structure of the pestivirus NS3 helicase and its conformation in solution [PublishAheadOfPrint]

  • Pestiviruses form a genus in the Flaviviridae family of small enveloped viruses with a positive-sense single-stranded RNA genome. Viral replication in this family requires the activity of a superfamily 2 RNA helicase contained in the C-terminal domain of the non-structural protein 3 (NS3). NS3 features two conserved RecA-like domains (D1 and D2) with ATPase activity, plus a third domain (D3) that is important for unwinding nucleic acid duplexes. We report here the X-ray structure of the pestivirus NS3 helicase domain (pNS3h) at 2.5 AAring; resolution. The structure deviates significantly from that of NS3 of other genera in the Flaviviridae family in D3, which contains two important insertions that result in a narrower nucleic acid binding groove. We also show that mutations in pNS3h that rescue viruses with deleted core protein map to D3, suggesting that this domain may be involved in interactions that facilitate particle assembly. Finally, structural comparisons of the enzyme in different crystalline environments, together with small angle X-ray scattering studies in solution, show that D2 is mobile with respect to the rest of the enzyme, oscillating between closed and open conformations. Binding of a non-hydrolyzable ATP analog locks pNS3h in a conformation that is more compact than the closest apo-form in our crystals. Together, our results provide new insight and bring up new questions about pNS3h function during pestivirus replication.

    Importance Although pestivirus infections impose an important toll on the livestock industry worldwide, little information is available about the non-structural proteins essential for viral replication, such as the NS3 helicase. We provide here a comparative structural and functional analysis of pNS3h with respect to its orthologs in other viruses of the same family, the flaviviruses and the hepatitis C virus. Our studies reveal differences in the nucleic acid binding groove that could have implications for understanding the unwinding specificity of pNS3h, which is only active on RNA duplexes. We also show that pNS3h has a highly dynamic behavior - a characteristic probably shared with NS3 helicases from all Flaviviridae members - that could be targeted for drug design by using recent algorithms to specifically block molecular motion. Compounds that lock the enzyme in a single conformation, or limit its dynamic range of conformations are indeed likely to block its helicase function.

  • Inhibition of Hepatitis B Virus Gene Expression and Replication by Hepatocyte Nuclear Factor 6 [PublishAheadOfPrint]

  • Hepatitis B virus (HBV), a small enveloped DNA virus, chronically infects more than 350 million people worldwide and causes liver diseases from hepatitis to cirrhosis and liver cancer. Here, we report that hepatocyte nuclear factor 6 (HNF6), a liver-enriched transcription factor, can inhibit HBV gene expression and DNA replication. Overexpression of HNF6 inhibited, while knockdown of HNF6 expression enhanced HBV gene expression and replication in hepatoma cells. Mechanistically, SP2 promoter was inhibited by HNF6, which partly accounts for the inhibition on S mRNA. Detailed analysis showed that a cis element on HBV genome (nt 3009-3019) was responsible for the inhibition of SP2 promoter by HNF6. Moreover, further analysis showed that HNF6 reduced viral pregenomic RNA (pgRNA) posttranscriptionally via accelerating the degradation of HBV pgRNA independent of La protein. Furthermore, by using truncated mutation experiments, we demonstrated that the N-terminal region of HNF6 was responsible for its inhibitory effects. Importantly, introduction of an HNF6 expression construct with HBV genome into the mouse liver using hydrodynamic injection resulted in a significant reduction in viral gene expression and DNA replication. Overall, our data demonstrated that HNF6 is a novel host factor that can restrict HBV replication via both transcriptional and posttranscriptional mechanisms.

    Importance HBV is a major human pathogen whose replication is regulated by host factors. Liver enriched transcription factors are critical for many liver functions including metabolism, development and cell proliferation, and some of them have been shown to regulate HBV gene expression or replication in different manners. In this paper, we showed that HNF6 could inhibit the gene expression and DNA replication of HBV via both transcriptional and posttranscriptional mechanisms. As HNF6 is differentially expressed in men and women, the current results may suggest a role of HNF6 in the gender dimorphism of HBV infection.

  • p53-derived Host Restriction of HIV-1 Replication by PKR-mediated Tat Phosphorylation and Inactivation [PublishAheadOfPrint]

  • Tumor suppressor p53 has been suggested to be a host restriction factor against HIV-1 replication, but the detailed molecular mechanism has remained elusive for decades. Here, we demonstrate that p53-mediated HIV-1 suppression is attributed to dsRNA-dependent protein kinase (PKR)-mediated HIV-1 trans-activator (Tat) phosphorylation and inactivation. p53-silencing significantly enhanced HIV-1 replication in infected cells. Ectopic expression of p53 suppressed Tat activity, which was rescued by PKR-silencing. In addition, ectopic expression of PKR abolished Tat activity in p53-/- and eIF2aCA cells. Finally, we found that HIV-1 infection activates p53, followed by the induction and activation of PKR. PKR directly interacted with HIV-1 Tat and phosphorylates the first exon of Tat exclusively at five Ser/Thr residues (T23, T40, S46, S62 and S68), which inhibits Tat-mediated provirus transcription in three critical steps: i) phosphorylation near the ARM inhibits Tat translocation into the nucleus, ii) accumulation of Tat phosphorylation abolishes Tat-TAR binding, and iii) Tat phosphorylation at T23 and/or T40 obliterates the Tat-cyclin T1 interaction. These five Ser/Thr sites on Tat were highly conserved in HIV-1 strains prevalent in Europe and the United. Taken together, our findings indicate that p53-derived host restriction of HIV-1 replication is likely attributed, at least in parts, to a non-canonical p53/PKR/Tat phosphorylation and inactivation pathway in HIV-1 infection and AIDS pathogenesis.

    IMPORTANCE HIV-1-mediated disease progression to AIDS lasts for years to decades after primary infection. Host restriction and associated viral latency have been studied for several decades. p53 has been suggested as an important host restriction factor against HIV-1 replication. However the detailed molecular mechanism is still unclear. In the present study, we found that the p53-mediated HIV-1 restriction is attributed to a p53/PKR/Tat-inactivation pathway. HIV-1 infection activated p53, which subsequently induced PKR expression and activation. PKR directly phosphorylated Tat exclusively at five specific Ser/Thr residues, which were accompanied by significant suppression of HIV-1 replication. Accumulation of Tat phosphorylation at these sites inhibited Tat function by blocking Tat nuclear localization, Tat binding to TAR, and Tat-cyclin T1 interaction. Our findings provide a better understanding of the p53-derived host restriction mechanism against HIV-1 replication in AIDS pathogenesis and may contribute to further research focusing on the investigation of potential therapeutic targets for HIV-1.

  • Rice stripe Tenuivirus nonstructural protein 3 hijacks the 26S proteasome of the small brown planthopper, via direct interaction with regulatory-particle non-ATPase subunit 3 [PublishAheadOfPrint]

  • The ubiquitin/26S proteasome system plays a vital role in regulating host defenses against pathogens. Previous studies have highlighted different roles for the ubiquitin/26S proteasome in defense during virus infection in both mammals and plants. But their role in the vectors that transmit those viruses is still unclear. In this study, we determined that the 26S proteasome present in the small brown planthopper (SBPH, Laodelphgax striatellus), and has similar components to that in plants and mammals. There was an increase in the accumulation of Rice stripe virus (RSV) in the transmitting vector SBPH after disrupting the 26S proteasome, indicating that the SBPH 26S proteasome play a role in defense against RSV infection by regulating RSV accumulation. Yeast two-hybrid analysis determined that a subunit of the 26S proteasome named RPN3 could interact with RSV NS3. Transient over-expression of RPN3 had no effect on the RNA silencing suppressor activity of RSV NS3. However, NS3 could inhibit the ability of SBPH rpn3 to complement an rpn3 mutant in yeast. Our findings also indicate that the direct interaction between RPN3 and NS3 was responsible for inhibiting the complementation ability of RPN3. In vivo, we found an accumulation of ubiquitinated protein in SBPH tissues where RSV titer was high, and silencing of rpn3 results in malfunction of the SBPH proteasome-mediated proteolysis. Consequently, viruliferous SBPH in which RPN3 was repressed transmitted the virus more effectively as a result of higher accumulation of RSV. Our results suggest that the RSV NS3 protein is able to hijack the 26S proteasome in SBPH, via a direct interaction with the RPN3 subunit to attenuate the host defense response.

    IMPORTANCE: This study shows, for the first time, that the 26S proteasome components present in the small brown planthopper, and play a role in defense against its vectored plant virus (RSV). In turn, RSV can encode a protein to subvert the SBPH 26S proteasome via direct interaction with the 26S proteasome subunit RPN3. Our results imply that the molecular arms race observed in plant hosts can be extended to the insect vector that transmits those viruses.

  • The sequence of pathogenic events in cynomolgus macaques infected with aerosolized monkeypox virus. [PublishAheadOfPrint]

  • To evaluate new vaccines when human efficacy studies are not possible, the FDA's "Animal Rule" requires well characterised models of infection. Thus in the present study the early pathogenic events of monkeypox infection in nonhuman primates, a surrogate for variola virus infection, were characterised. Cynomolgus macaques were exposed to aerosolized monkeypox virus (105 PFU). Clinical observations, viral load, immune responses and pathological changes were examined on days 2, 4, 6, 8, 10 and 12 postchallenge. Viral DNA (vDNA) was detected in the lungs 2 days post-challenge and viral antigen, by immunostaining, in the epithelium of bronchi, bronchioles and alveolar walls. Lesions comprised rare foci of dysplastic and sloughed cells in respiratory bronchioles. By day 4, vDNA was detected in the pharynx, tonsil and spleen and monkeypox antigen was detected in lung, hilar and submandibular lymph nodes, spleen, and colon. Lung lesions comprised focal epithelial necrosis and inflammation. Body temperature peaked on day 6, pox lesions appeared on the skin and lesions, with positive immunostaining, were present in lung, tonsil, spleen, lymph nodes and colon. By day 8, vDNA was present in 9/13 tissues. Blood concentrations of IL-1ra, IL-6 and IFN-gamma increased markedly. By day 10, circulating IgG antibody concentrations increased and on day 12 animals showed early signs of recovery. These results define early events occurring in an inhalational, macaque, monkeypox infection model, supporting its use as a surrogate model for human smallpox.

    Importance Bioterrorism poses a major threat to public health as the delibrate release of infectious agents such smallpox or a related virus, monkeypox, would have catastrophic consequences. The development and testing of new medical countermeasures, e.g. vaccines, is thus a priority however tests for efficacy cannot be performed in an endemic/naiiuml;ve population, as this is neither ethical nor feasible. To overcome this the US Food and Drug Administration may grant marketing approval of a new product based upon the "Animal Rule" where interventions are tested for efficacy in well-characterised animal models. Monkeypox virus infection of non-human primates (NHPs) presents a potential surrogate disease model for smallpox. Previously, the later stages of monkeypox infection were defined but the early course of infection remains unstudied. Here the early pathogenic events of inhalational monkeypox infection in NHPs were characterised and the results support the use of this surrogate model for testing human smallpox interventions.

  • Isolation, genetic characterization and seroprevalence of Adana virus a novel phlebovirus belonging to the Salehabad virus complex in Turkey [PublishAheadOfPrint]

  • A new phlebovirus, Adana virus, was isolated from a pool of Phlebotomus sp. (Diptera; Psychodidae) in the province of Adana, Mediterranean region of Turkey. Genetic analysis based on complete coding genomic sequences indicated that Adana virus belongs to the Salehabad virus species of the genus Phlebovirus in the family Bunyaviridae. Adana virus is the third virus of the Salehabad virus species for which the complete sequence has been determined. To understand the epidemiology of Adana virus, a seroprevalence study using microneutralization assay was performed to detect the presence of specific antibodies in human and domestic animal sera collected in Adana as well as Mersin province, located 147km west of Adana. The results demonstrate that (i) the virus is present in both provinces, (ii) high seroprevalence rates in goats, sheep and dogs support intensive exposure to Adana virus in the region, which have not been previously reported for any virus included in the Salehabad serocomplex (iii) low seroprevalence rates in humans suggest that Adana virus is not likely to constitute an important public health problem in exposed human populations but this deserves further studies.

    IMPORTANCE Until recently, in the genus Phlebovirus, the Salehabad virus species consisted of two viruses: Salehabad virus isolated from sandflies in Iran, and Arbia virus isolated from sandflies in Italy. Here we present the isolation and complete genome characterization of the Adana virus which we propose to be included in the Salehabad virus species. To our knowledge, this is the first report of the isolation and complete genome characterization, from sandflies in Turkey, of a Salehabad-related phlebovirus with supporting seropositivity in the Mediterranean, Aegean, and Central Anatolian region where phleboviruses have been circulating and causing outbreaks. Salehabad species viruses have generally been considered to be a group of viruses with little medical or veterinary interest. This view deserves to be revisited according to our results which indicate a high animal infection rate of Adana virus and recent evidence of human infection with Adria virus in Greece.

  • Scanning Mutagenesis Studies Reveal Potential Intramolecular Interaction within the C-terminal Half of Dengue Virus NS2A Involved in Viral RNA Replication and Virus Assembly/Secretion [PublishAheadOfPrint]

  • The NS2A protein of Dengue virus (DENV) has eight predicted transmembrane segments (pTMS1-8). NS2A has been shown to participate in RNA replication, virion assembly, and the host antiviral response. However, the role of the amino acid residues within the pTMS regions of NS2A during the virus life cycle is poorly understood. Here, we explore the function of DENV NS2A by introducing a series of double- or triple-alanine substitutions into the C-terminal half (pTMS4-8) of NS2A in the context of a DENV infectious clone or subgenomic replicon. Fourteen (eight within pTMS8) out of thirty-five NS2A mutants displayed a lethal phenotype due to impairment of RNA replication by replicon assay. Three NS2A mutants within pTMS7, CM20, 25, and 27, displayed similar phenotypes, low virus yields (ggt;100-fold reduction), wild-type-like replicon activity, and low infectious virus-like particle yields by transient trans-packaging experiments, suggesting a defect in virus assembly/secretion. The sequencing of revertant viruses derived from CM20, 25, and 27 mutant viruses revealed a consensus reversion mutation, leucine (L)-to-phenylalanine (F), at codon 181 within pTMS7. The introduction of an L181F mutation into a full-length NS2A mutant, i.e., the CM20, 25, and 27 constructs, completely restored wild-type infectivity. Notably, L181F also substantially rescued the other severely RNA replication-defective mutants within pTMS4, 6, and 8, i.e., CM2, 3, 13, 31, and 32. In conclusion, the results revealed the essential roles of pTMS4-8 of NS2A in RNA replication and/or virus assembly/secretion. The intramolecular interaction between pTMS7 with pTMS4, 6, or 8 of the NS2A protein was also implicated.

    Importance: The reported characterization of the C-terminal half of dengue virus NS2A is the first comprehensive mutagenesis study to investigate the function of flavivirus NS2A involved in the steps of the virus life cycle. In particular, detailed mapping of the amino acid residues within the predicted transmembrane segments (pTMSs) of NS2A involved in RNA replication and/or virus assembly/secretion was performed. A revertant genetics study also revealed that L181F within pTMS7 is a consensus reversion mutation that rescues both RNA replication- and virus assembly/secretion-defective mutants within the other three pTMSs of NS2A. Collectively, these findings elucidate the role played by NS2A during the virus life cycle, possibly through the intricate intramolecular interaction between pTMS7 and other pTMSs within the NS2A protein.

  • MiR-24-3p Promotes Porcine Reproductive and Respiratory Syndrome Virus Replication through Suppression of Heme Oxygenase-1 Expression [PublishAheadOfPrint]

  • Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important viruses affecting the swine industry worldwide. Our previous research showed that PRRSV down-regulates the expression of heme oxygenase-1 (HO-1), a pivotal cytoprotective enzyme, post-infection and overexpression of HO-1 inhibits PRRSV replication. MicroRNAs regulate gene expression at the posttranscriptional level and have recently been demonstrated to play vital roles in pathogen-host interactions. The present study sought to determine whether microRNAs modulate HO-1 expression, and by doing so, regulate PRRSV replication. Using bioinformatic prediction and experimental verification, we demonstrate that HO-1 expression is regulated by miR-24-3p. A direct interaction between miR-24-3p and HO-1 mRNA was confirmed using a number of approaches. Overexpression of miR-24-3p significantly decreased HO-1 mRNA and protein levels. PRRSV infection induced miR-24-3p expression to facilitate viral replication. The suppressive effect of HO-1 induction by protoporphyrin IX cobalt chloride (CoPP, a classical inducer of HO-1 expression) on PRRSV replication in MARC-145 cells and primary porcine alveolar macrophages could also be reversed by overexpression of miR-24-3p. Collectively, these results suggested that miR-24-3p promotes PRRSV replication through suppression of HO-1 expression, which not only provides new insights into virus-host interactions during PRRSV infection, but also suggests potential new antiviral strategies against PRRSV infection.

    IMPORTANCE microRNAs (miRNAs) play vital roles in viral infections by regulating the expression of viral or host gene at posttranscriptional level. Heme oxygenase-1 (HO-1), a pivotal cytoprotective enzyme, has antiviral activity for a number of viruses such as Ebola virus, hepatitis C virus and human immunodeficiency virus, including our focus, PRRSV, which causes great economic losses each year in the swine industry worldwide. Here we show that PRRSV infection induces host miRNA miR-24-3p expression, miR-24-3p regulates HO-1 expression through both mRNA degradation and translation repression. Suppression of HO-1 expression by miR-24-3p facilitates PRRSV replication. This work lends credibility to the hypothesis that an arterivirus can manipulate cellular miRNAs to enhance virus replication by regulating antiviral responses following viral infection.. Therefore, our findings provide new insights into the pathogenesis of PRRSV.

  • AAK1 and GAK Regulate Hepatitis C Virus Entry and Are Potential Drug Targets [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) enters its target cell via clathrin-mediated endocytosis. AP2-associated protein kinase-1 (AAK1) and cyclin G-associated kinase (GAK) are host kinases that regulate clathrin adaptor proteins (APs)-mediated trafficking in the endocytic and secretory pathways. We previously reported that AAK1 and GAK regulate HCV assembly by stimulating binding of the mmu; subunit of AP-2, AP2M1, to HCV core. We also discovered that AAK1 and GAK inhibitors, including approved anticancer drugs, sunitinib and erlotinib, could block HCV assembly. Here, we hypothesized that AAK1 and GAK regulate HCV entry independently of their effect on HCV assembly. Indeed, silencing AAK1 and GAK expression inhibited entry of pseudoparticles and cell culture grown HCV and internalization of Dil-labeled HCV particles with no effect on HCV attachment or RNA replication. AAK1 or GAK depletion impaired epidermal growth factor (EGF)-mediated enhanced HCV entry and endocytosis of EGF receptor (EGFR), a HCV entry co-factor and erlotinib's cancer target. Moreover, either RNA interference-mediated depletion of AP2M1 or NUMB, the substrates of AAK1 and/or GAK, or overexpression of their phosphorylation-site mutants inhibited HCV entry. Last, in addition to their effect on assembly, sunitinib and erlotinib inhibited HCV entry at a postbinding step, their combination was synergistic, and their antiviral effect was reversed by either AAK1 or GAK overexpression. Together, these results validate AAK1 and GAK as critical regulators of HCV entry that function in part by activating EGFR, AP2M1, and NUMB, and as the molecular targets underlying the antiviral effect of sunitinib and erlotinib (in addition to EGFR), respectively.

    Importance Understanding the host pathways hijacked by HCV is critical for developing host-centered anti-HCV approaches. Entry represents a potential target for antiviral strategies; however, no FDA-approved HCV entry inhibitors are currently available. We reported that two host kinases, AAK1 and GAK, regulate HCV assembly. Here, we provide evidence that AAK1 and GAK regulate HCV entry independently of their role in HCV assembly and define the mechanisms underlying AAK1- and GAK-mediated HCV entry. By regulating temporally distinct steps in the HCV lifecycle, AAK1 and GAK represent "master regulators" of HCV infection and potential targets for antiviral strategies. Indeed, approved anticancer drugs that potently inhibit AAK1 or GAK, inhibit HCV entry in addition to assembly. These results contribute to understanding the mechanisms of HCV entry and reveal attractive host targets for antiviral strategies as well as approved candidate inhibitors of these targets, with potential implications to other viruses that hijack clathrin-mediated pathways.

  • Influenza HA stem region mutations that stabilize or destabilize the structure of multiple HA subtypes [PublishAheadOfPrint]

  • Influenza A viruses enter host cells through endosomes, where acidification induces irreversible conformational changes of the viral hemagglutinin (HA) that drive the membrane fusion process. The pre-fusion conformation of the HA is metastable, and the pH of fusion can vary significantly amongst HA strains and subtypes. Furthermore, an accumulating body of evidence implicates HA stability properties as partial determinants of influenza host range, transmission phenotype, and pathogenic potential. Though previous studies have identified HA mutations that can affect HA stability, these have been limited to a small selection of HA strains and subtypes. Here we report a mutational analysis of HA stability utilizing a panel of expressed HAs representing a broad range of HA subtypes and strains, including avian representatives across the phylogenetic spectrum and several human strains. We focused on two highly conserved residues in the HA stem region, HA2 position 58 at the membrane distal tip of the short helix of the hairpin loop structure, and HA2 position 112, located in the long helix in proximity to the fusion peptide. We demonstrate that a K58I mutation confers an acid-stable phenotype for nearly all HAs examined, whereas a D112G mutation consistently leads to elevated fusion pH. The results enhance our understanding of HA stability across multiple subtypes and provide an additional tool for risk assessment for circulating strains that may have other hallmarks of human adaptation. Furthermore, the K58I mutants in particular, may be of interest for potential use in the development of vaccines with improved stability profiles.

    Importance The influenza A hemagglutinin glycoprotein (HA) mediates the receptor binding and membrane fusion functions that are essential for virus entry into host cells. While receptor binding has long been recognized for its role in host species specificity and transmission, membrane fusion and associated properties of HA stability have only recently been appreciated as potential determinants. Here we show that mutations can be introduced at highly conserved positions to stabilize or destabilize the HA structure of multiple HA subtypes, expanding our knowledge base for this important phenotype. The practical implications of these findings extend to the field of vaccine design, as the HA mutations characterized here could potentially be utilized across a broad spectrum of influenza subtypes to improve the stability of vaccine strains or components.

  • Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein [PublishAheadOfPrint]

  • It is hypothesized that targeting stable cellular factors involved in viral replication instead of viral-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. Here we investigated the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for small cellular GTPases Arf. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of BFA-resistance phenotype in both HeLa and Vero cells. Moreover, BFA-resistant phenotype of poliovirus mutants is also cell type-dependent in different cells of human origin and results in fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus rational approach to development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses.

    Importance Compared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. Yet viruses are highly adaptable and can easily generate mutants resistant to practically any compounds targeting viral proteins. An alternative approach is to target stable cellular factors recruited for the virus-specific functions. In this study we analyzed the factors permitting and restricting the establishment of resistance of poliovirus, a small (+)RNA virus, to BFA, a drug targeting a cellular component of the viral replication complex. We found that emergence and replication potential of resistant mutants is cell-type dependent and that BFA resistance reduces the virus fitness. Our data provide a rational approach to development of antiviral therapeutics targeting host factors.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

  • Zombies in TCGA [PublishAheadOfPrint]

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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