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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:

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  • Current Issue of Journal of Virology

    JVI Current Issue

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  • Role of Viral Hemorrhagic Septicemia Virus Matrix (M) Protein in Suppressing Host Transcription [Virus-Cell Interactions]

  • Viral hemorrhagic septicemia virus (VHSV) is a pathogenic fish rhabdovirus found in discrete locales throughout the Northern Hemisphere. VHSV infection of fish cells leads to upregulation of the host's virus detection response, but the virus quickly suppresses interferon (IFN) production and antiviral gene expression. By systematically screening each of the six VHSV structural and nonstructural genes, we identified matrix protein (M) as the virus' most potent antihost protein. Only M of VHSV genotype IV sublineage b (VHSV-IVb) suppressed mitochondrial antiviral signaling protein (MAVS) and type I IFN-induced gene expression in a dose-dependent manner. M also suppressed the constitutively active simian virus 40 (SV40) promoter and globally decreased cellular RNA levels. Chromatin immunoprecipitation (ChIP) studies illustrated that M inhibited RNA polymerase II (RNAP II) recruitment to gene promoters and decreased RNAP II C-terminal domain (CTD) Ser2 phosphorylation during VHSV infection. However, transcription directed by RNAP I to III was suppressed by M. To identify regions of functional importance, M proteins from a variety of VHSV strains were tested in cell-based transcriptional inhibition assays. M of a particular VHSV-Ia strain, F1, was significantly less potent than IVb M at inhibiting SV40/luciferase (Luc) expression yet differed by just 4 amino acids. Mutation of D62 to alanine alone, or in combination with an E181-to-alanine mutation (D62A E181A), dramatically reduced the ability of IVb M to suppress host transcription. Introducing either M D62A or D62A E181A mutations into VHSV-IVb via reverse genetics resulted in viruses that replicated efficiently but exhibited less cytotoxicity and reduced antitranscriptional activities, implicating M as a primary regulator of cytopathicity and host transcriptional suppression.

    IMPORTANCE Viruses must suppress host antiviral responses to replicate and spread between hosts. In these studies, we identified the matrix protein of the deadly fish novirhabdovirus VHSV as a critical mediator of host suppression during infection. Our studies indicated that M alone could block cellular gene expression at very low expression levels. We identified several subtle mutations in M that were less potent at suppressing host transcription. When these mutations were engineered back into recombinant viruses, the resulting viruses replicated well but elicited less toxicity in infected cells and activated host innate immune responses more robustly. These data demonstrated that VHSV M plays an important role in mediating both virus-induced cell toxicity and viral replication. Our data suggest that its roles in these two processes can be separated to design effective attenuated viruses for vaccine candidates.

  • Integrity of the Linker of Nucleoskeleton and Cytoskeleton Is Required for Efficient Herpesvirus Nuclear Egress [Virus-Cell Interactions]

  • Herpesvirus capsids assemble in the nucleus, while final virion maturation proceeds in the cytoplasm. This requires that newly formed nucleocapsids cross the nuclear envelope (NE), which occurs by budding at the inner nuclear membrane (INM), release of the primary enveloped virion into the perinuclear space (PNS), and subsequent rapid fusion with the outer nuclear membrane (ONM). During this process, the NE remains intact, even at late stages of infection. In addition, the spacing between the INM and ONM is maintained, as is that between the primary virion envelope and nuclear membranes. The linker of nucleoskeleton and cytoskeleton (LINC) complex consists of INM proteins with a luminal SUN (Sad1/UNC-84 homology) domain connected to ONM proteins with a KASH (Klarsicht, ANC-1, SYNE homology) domain and is thought to be responsible for spacing the nuclear membranes. To investigate the role of the LINC complex during herpesvirus infection, we generated cell lines constitutively expressing dominant negative (dn) forms of SUN1 and SUN2. Ultrastructural analyses revealed a significant expansion of the PNS and the contiguous intracytoplasmic lumen, most likely representing endoplasmic reticulum (ER), especially in cells expressing dn-SUN2. After infection, primary virions accumulated in these expanded luminal regions, also very distant from the nucleus. The importance of the LINC complex was also confirmed by reduced progeny virus titers in cells expressing dn-SUN2. These data show that the intact LINC complex is required for efficient nuclear egress of herpesviruses, likely acting to promote fusion of primary enveloped virions with the ONM.

    IMPORTANCE While the viral factors for primary envelopment of nucleocapsids at the inner nuclear membrane are known to the point of high-resolution structures, the roles of cellular components and regulators remain enigmatic. Furthermore, the machinery responsible for fusion with the outer nuclear membrane is unsolved. We show here that dominant negative SUN2 interferes with efficient herpesvirus nuclear egress, apparently by interfering with fusion between the primary virion envelope and outer nuclear membrane. This identifies a new cellular component important for viral egress and implicates LINC complex integrity in nonconventional nuclear membrane trafficking.

  • Cross-Linking of a CD4-Mimetic Miniprotein with HIV-1 Env gp140 Alters Kinetics and Specificities of Antibody Responses against HIV-1 Env in Macaques [Vaccines and Antiviral Agents]

  • Evaluation of the epitope specificities, locations (systemic or mucosal), and effector functions of antibodies elicited by novel HIV-1 immunogens engineered to improve exposure of specific epitopes is critical for HIV-1 vaccine development. Utilizing an array of humoral assays, we evaluated the magnitudes, epitope specificities, avidities, and functions of systemic and mucosal immune responses elicited by a vaccine regimen containing Env cross-linked to a CD4-mimetic miniprotein (gp140-M64U1) in rhesus macaques. Cross-linking of gp140 Env to M64U1 resulted in earlier increases of both the magnitude and avidity of the IgG binding response than those with Env protein alone. Notably, IgG binding responses at an early time point correlated with antibody-dependent cellular cytotoxicity (ADCC) function at the peak immunity time point, which was higher for the cross-linked Env group than for the Env group. In addition, the cross-linked Env group developed higher IgG responses against a linear epitope in the gp120 C1 region of the HIV-1 envelope glycoprotein. These data demonstrate that structural modification of the HIV-1 envelope immunogen by cross-linking of gp140 with the CD4-mimetic M64U1 elicited an earlier increase of binding antibody responses and altered the specificity of the IgG responses, correlating with the rise of subsequent antibody-mediated antiviral functions.

    IMPORTANCE The development of an efficacious HIV-1 vaccine remains a global priority to prevent new cases of HIV-1 infection. Of the six HIV-1 efficacy trials to date, only one has demonstrated partial efficacy, and immune correlate analysis of that trial revealed a role for binding antibodies and antibody Fc-mediated effector functions. New HIV-1 envelope immunogens are being engineered to selectively expose the most vulnerable and conserved sites on the HIV-1 envelope, with the goal of eliciting antiviral antibodies. Evaluation of the humoral responses elicited by these novel immunogen designs in nonhuman primates is critical for understanding how to improve upon immunogen design to inform further testing in human clinical trials. Our results demonstrate that structural modifications of Env that aim to mimic the CD4-bound conformation can result in earlier antibody elicitation, altered epitope specificity, and increased antiviral function postimmunization.

  • Monoclonal Antibodies, Derived from Humans Vaccinated with the RV144 HIV Vaccine Containing the HVEM Binding Domain of Herpes Simplex Virus (HSV) Glycoprotein D, Neutralize HSV Infection, Mediate Antibody-Dependent Cellular Cytotoxicity, and Protect Mice from Ocular Challenge with HSV-1 [Vaccines and Antiviral Agents]

  • The RV144 HIV vaccine trial included a recombinant HIV glycoprotein 120 (gp120) construct fused to a small portion of herpes simplex virus 1 (HSV-1) glycoprotein D (gD) so that the first 40 amino acids of gp120 were replaced by the signal sequence and the first 27 amino acids of the mature form of gD. This region of gD contains most of the binding site for HVEM, an HSV receptor important for virus infection of epithelial cells and lymphocytes. RV144 induced antibodies to HIV that were partially protective against infection, as well as antibodies to HSV. We derived monoclonal antibodies (MAbs) from peripheral blood B cells of recipients of the RV144 HIV vaccine and showed that these antibodies neutralized HSV-1 infection in cells expressing HVEM, but not the other major virus receptor, nectin-1. The MAbs mediated antibody-dependent cellular cytotoxicity (ADCC), and mice that received the MAbs and were then challenged by corneal inoculation with HSV-1 had reduced eye disease, shedding, and latent infection. To our knowledge, this is the first description of MAbs derived from human recipients of a vaccine that specifically target the HVEM binding site of gD. In summary, we found that monoclonal antibodies derived from humans vaccinated with the HVEM binding domain of HSV-1 gD (i) neutralized HSV-1 infection in a cell receptor-specific manner, (ii) mediated ADCC, and (iii) reduced ocular disease in virus-infected mice.

    IMPORTANCE Herpes simplex virus 1 (HSV-1) causes cold sores and neonatal herpes and is a leading cause of blindness. Despite many trials, no HSV vaccine has been approved. Nectin-1 and HVEM are the two major cellular receptors for HSV. These receptors are expressed at different levels in various tissues, and the role of each receptor in HSV pathogenesis is not well understood. We derived human monoclonal antibodies from persons who received the HIV RV144 vaccine that contained the HVEM binding domain of HSV-1 gD fused to HIV gp120. These antibodies were able to specifically neutralize HSV-1 infection in vitro via HVEM. Furthermore, we showed for the first time that HVEM-specific HSV-1 neutralizing antibodies protect mice from HSV-1 eye disease, indicating the critical role of HVEM in HSV-1 ocular infection.

  • Effects of Inner Nuclear Membrane Proteins SUN1/UNC-84A and SUN2/UNC-84B on the Early Steps of HIV-1 Infection [Virus-Cell Interactions]

  • Human immunodeficiency virus type 1 (HIV-1) infection of dividing and nondividing cells involves regulatory interactions with the nuclear pore complex (NPC), followed by translocation to the nucleus and preferential integration into genomic areas in proximity to the inner nuclear membrane (INM). To identify host proteins that may contribute to these processes, we performed an overexpression screen of known membrane-associated NE proteins. We found that the integral transmembrane proteins SUN1/UNC84A and SUN2/UNC84B are potent or modest inhibitors of HIV-1 infection, respectively, and that suppression corresponds to defects in the accumulation of viral cDNA in the nucleus. While laboratory strains (HIV-1NL4.3 and HIV-1IIIB) are sensitive to SUN1-mediated inhibition, the transmitted founder viruses RHPA and ZM247 are largely resistant. Using chimeric viruses, we identified the HIV-1 capsid (CA) protein as a major determinant of sensitivity to SUN1, and in vitro-assembled capsid-nucleocapsid (CANC) nanotubes captured SUN1 and SUN2 from cell lysates. Finally, we generated SUN1nndash;/nndash; and SUN2nndash;/nndash; cells by using CRISPR/Cas9 and found that the loss of SUN1 had no effect on HIV-1 infectivity, whereas the loss of SUN2 had a modest suppressive effect. Taken together, these observations suggest that SUN1 and SUN2 may function redundantly to modulate postentry, nuclear-associated steps of HIV-1 infection.

    IMPORTANCE HIV-1 causes more than 1 million deaths per year. The life cycle of HIV-1 has been studied extensively, yet important steps that occur between viral capsid release into the cytoplasm and the expression of viral genes remain elusive. We propose here that the INM components SUN1 and SUN2, two members of the linker of nucleoskeleton and cytoskeleton (LINC) complex, may interact with incoming HIV-1 replication complexes and affect key steps of infection. While overexpression of these proteins reduces HIV-1 infection, disruption of the individual SUN2 and SUN1 genes leads to a mild reduction or no effect on infectivity, respectively. We speculate that SUN1/SUN2 may function redundantly in early HIV-1 infection steps and therefore influence HIV-1 replication and pathogenesis.

  • Effects of the NEDD8-Activating Enzyme Inhibitor MLN4924 on Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus [Genome Replication and Regulation of Viral Gene Expression]

  • The switch of Kaposi's sarcoma-associated herpesvirus (KSHV) from latency to lytic replication is a key event for viral dissemination and pathogenesis. MLN4924, a novel neddylation inhibitor, reportedly causes the onset of KSHV reactivation but impairs later phases of the viral lytic program in infected cells. Thus far, the molecular mechanism involved in the modulation of the KSHV lytic cycle by MLN4924 is not yet fully understood. Here, we confirmed that treatment of different KSHV-infected primary effusion lymphoma (PEL) cell lines with MLN4924 substantially induces viral lytic protein expression. Due to the key role of the virally encoded ORF50 protein in the latent-to-lytic switch, we investigated its transcriptional regulation by MLN4924. We found that MLN4924 activates the ORF50 promoter (ORF50p) in KSHV-positive cells (but not in KSHV-negative cells), and the RBP-J-binding elements within the promoter are critically required for MLN4924 responsiveness. In KSHV-negative cells, reactivation of the ORF50 promoter by MLN4924 requires the presence of the latency-associated nuclear antigen (LANA). Under such a condition, LANA acts as a repressor to block the ORF50p activity, whereas MLN4924 treatment relieves LANA-mediated repression. Importantly, we showed that LANA is a neddylated protein and can be deneddylated by MLN4924. On the other hand, we revealed that MLN4924 exhibits concentration-dependent biphasic effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)- or sodium butyrate (SB)-induced viral reactivation in PEL cell lines. In other words, low concentrations of MLN4924 promote activation of TPA- or SB-mediated viral reactivation, whereas high concentrations of MLN4924, conversely, inhibit the progression of TPA- or SB-mediated viral lytic program.

    IMPORTANCE MLN4924 is a neddylation (NEDD8 modification) inhibitor, which currently acts as an anti-cancer drug in clinical trials. Although MLN4924 has been reported to trigger KSHV reactivation, many aspects regarding the action of MLN4924 in regulating the KSHV lytic cycle are not fully understood. Since the KSHV ORF50 protein is the key regulator of viral lytic reactivation, we focus on its transcriptional regulation by MLN4924. We here show that activation of the ORF50 gene by MLN4924 involves the relief of LANA-mediated transcriptional repression. Importantly, we find that LANA is a neddylated protein. To our knowledge, this is the first report showing that neddylation occurs in viral proteins. Additionally, we provide evidence that different concentrations of MLN4924 have opposite effects on TPA-mediated or SB-mediated KSHV lytic cycle activation. Therefore, in clinical application, we propose that MLN4924 needs to be used with caution in combination therapy to treat KSHV-positive subjects.

  • Permissivity of Dipeptidyl Peptidase 4 Orthologs to Middle East Respiratory Syndrome Coronavirus Is Governed by Glycosylation and Other Complex Determinants [Virus-Cell Interactions]

  • Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes dipeptidyl peptidase 4 (DPP4) as an entry receptor. While bat, camel, and human DPP4 support MERS-CoV infection, several DPP4 orthologs, including mouse, ferret, hamster, and guinea pig DPP4, do not. Previous work revealed that glycosylation of mouse DPP4 plays a role in blocking MERS-CoV infection. Here, we tested whether glycosylation also acts as a determinant of permissivity for ferret, hamster, and guinea pig DPP4. We found that, while glycosylation plays an important role in these orthologs, additional sequence and structural determinants impact their ability to act as functional receptors for MERS-CoV. These results provide insight into DPP4 species-specific differences impacting MERS-CoV host range and better inform our understanding of virus-receptor interactions associated with disease emergence and host susceptibility.

    IMPORTANCE MERS-CoV is a recently emerged zoonotic virus that is still circulating in the human population with an ~35% mortality rate. With no available vaccines or therapeutics, the study of MERS-CoV pathogenesis is crucial for its control and prevention. However, in vivo studies are limited because MERS-CoV cannot infect wild-type mice due to incompatibilities between the virus spike and the mouse host cell receptor, mouse DPP4 (mDPP4). Specifically, mDPP4 has a nonconserved glycosylation site that acts as a barrier to MERS-CoV infection. Thus, one mouse model strategy has been to modify the mouse genome to remove this glycosylation site. Here, we investigated whether glycosylation acts as a barrier to infection for other nonpermissive small-animal species, namely, ferret, guinea pig, and hamster. Understanding the virus-receptor interactions for these DPP4 orthologs will help in the development of additional animal models while also revealing species-specific differences impacting MERS-CoV host range.

  • Genetic Stability of Parainfluenza Virus 5-Vectored Human Respiratory Syncytial Virus Vaccine Candidates after In Vitro and In Vivo Passage [Vaccines and Antiviral Agents]

  • Human respiratory syncytial virus (RSV) is the leading etiologic agent of lower respiratory tract infections in children, but no licensed vaccine exists. Previously, we developed two parainfluenza virus 5 (PIV5)-based RSV vaccine candidates that protect mice against RSV challenge. PIV5 was engineered to express either the RSV fusion protein (F) or the RSV major attachment glycoprotein (G) between the hemagglutinin-neuraminidase (HN) and RNA-dependent RNA polymerase (L) genes of the PIV5 genome [PIV5-RSV-F (HN-L) and PIV5-RSV-G (HN-L), respectively]. To investigate the stability of the vaccine candidates in vitro, they were passaged in Vero cells at high and low multiplicities of infection (MOIs) for 11 generations and the genome sequences, growth kinetics, and protein expression of the resulting viruses were compared with those of the parent viruses. Sporadic mutations were detected in the consensus sequences of the viruses after high-MOI passages, and mutation rates increased under low-MOI-passage conditions. None of the mutations abolished antigen expression. Increased numbers of mutations correlated with increased growth rates in vitro, indicating that the viruses evolved through the course of serial passages. We also examined the in vivo stability of the vaccine candidates after a single passage in African green monkeys. No mutations were detected in the consensus sequences of viruses collected from the bronchoalveolar lavage (BAL) fluid of the animals. In vivo, mutations in RSV G and PIV5 L were found in individual isolates of PIV5-RSV-G (HN-L), but plaque isolates of PIV5-RSV-F (HN-L) had no mutations. To improve upon the PIV5-RSV-F (HN-L) candidate, additional vaccine candidates were generated in which the gene for RSV F was inserted into earlier positions in the PIV5 genome. These insertions did not negatively impact the sequence stability of the vaccine candidates. The results suggest that the RSV F and G gene insertions are stable in the PIV5 genome. However, the function of the foreign gene insertion may need to be considered when designing PIV5-based vaccines.

    IMPORTANCE The genetic stability of live viral vaccines is important for safety and efficacy. PIV5 is a promising live viral vector and has been used to develop vaccines. In this work, we examined the genetic stability of a PIV5-based RSV vaccine in vitro and in vivo. We found that insertions of foreign genes, such as the RSV F and G genes, were stably maintained in the PIV5 genome and there was no mutation that abolished the expression of RSV F or G. Interestingly, the function of the inserted gene may have an impact on PIV5 genome stability.

  • Parainfluenza Virus 5 Expressing Wild-Type or Prefusion Respiratory Syncytial Virus (RSV) Fusion Protein Protects Mice and Cotton Rats from RSV Challenge [Vaccines and Antiviral Agents]

  • Human respiratory syncytial virus (RSV) is the leading cause of pediatric bronchiolitis and hospitalizations. RSV can also cause severe complications in elderly and immunocompromised individuals. There is no licensed vaccine. We previously generated a parainfluenza virus 5 (PIV5)-vectored vaccine candidate expressing the RSV fusion protein (F) that was immunogenic and protective in mice. In this work, our goal was to improve the original vaccine candidate by modifying the PIV5 vector or by modifying the RSV F antigen. We previously demonstrated that insertion of a foreign gene at the PIV5 small hydrophobic (SH)nndash;hemagglutinin-neuraminidase (HN) junction or deletion of PIV5 SH increased vaccine efficacy. Additionally, other groups have demonstrated that antibodies against the prefusion conformation of RSV F have more potent neutralizing activity than antibodies against the postfusion conformation. Therefore, to improve on our previously developed vaccine candidate, we inserted RSV F at the PIV5 SH-HN gene junction or used RSV F to replace PIV5 SH. We also engineered PIV5 to express a prefusion-stabilized F mutant. The candidates were tested in BALB/c mice via the intranasal route and induced both humoral and cell-mediated immunity. They also protected against RSV infection in the mouse lung. When they were administered intranasally or subcutaneously in cotton rats, the candidates were highly immunogenic and reduced RSV loads in both the upper and lower respiratory tracts. PIV5-RSV F was equally protective when administered intranasally or subcutaneously. In all cases, the prefusion F mutant did not induce higher neutralizing antibody titers than wild-type F. These results show that antibodies against both pre- and postfusion F are important for neutralizing RSV and should be considered when designing a vectored RSV vaccine. The findings also that indicate PIV5-RSV F may be administered subcutaneously, which is the preferred route for vaccinating infants, who may develop nasal congestion as a result of intranasal vaccination.

    IMPORTANCE Despite decades of research, human respiratory syncytial virus (RSV) is still a major health concern for which there is no vaccine. A parainfluenza virus 5-vectored vaccine expressing the native RSV fusion protein (F) has previously been shown to confer robust immunity against RSV infection in mice, cotton rats, and nonhuman primates. To improve our previous vaccine candidate, we developed four new candidates that incorporate modifications to the PIV5 backbone, replace native RSV F with a prefusion-stabilized RSV F mutant, or combine both RSV F and PIV5 backbone modifications. In this work, we characterized the new vaccine candidates and tested their efficacies in both murine and cotton rat models of RSV infection. Most importantly, we found that PIV5-based RSV vaccine candidates were efficacious in preventing lower respiratory tract infection as well as in reducing the nasal viral load when administered via the subcutaneous route.

  • The Translesion Polymerase Pol {eta} Is Required for Efficient Epstein-Barr Virus Infectivity and Is Regulated by the Viral Deubiquitinating Enzyme BPLF1 [Virus-Cell Interactions]

  • Epstein-Barr virus (EBV) infection and lytic replication are known to induce a cellular DNA damage response. We previously showed that the virally encoded BPLF1 protein interacts with and regulates several members of the translesion synthesis (TLS) pathway, a DNA damage tolerance pathway, and that these cellular factors enhance viral infectivity. BPLF1 is a late lytic cycle gene, but the protein is also packaged in the viral tegument, indicating that BPLF1 may function both early and late during infection. The BPLF1 protein expresses deubiquitinating activity that is strictly conserved across the Herpesviridae; mutation of the active site cysteine results in a loss of enzymatic activity. Infection with an EBV BPLF1 knockout virus results in decreased EBV infectivity. Polymerase eta (Pol ), a specialized DNA repair polymerase, functions in TLS and allows for DNA replication complexes to bypass lesions in DNA. Here we report that BPLF1 interacts with Pol and that Pol protein levels are increased in the presence of functional BPLF1. BPLF1 promotes a nuclear relocalization of Pol molecules which are focus-like in appearance, consistent with the localization observed when Pol is recruited to sites of DNA damage. Knockdown of Pol resulted in decreased production of infectious virus, and further, Pol was found to bind to EBV DNA, suggesting that it may allow for bypass of damaged viral DNA during its replication. The results suggest a mechanism by which EBV recruits cellular repair factors, such as Pol , to sites of viral DNA damage via BPLF1, thereby allowing for efficient viral DNA replication.

    IMPORTANCE Epstein-Barr virus is the causative agent of infectious mononucleosis and infects approximately 90% of the world's population. It causes lymphomas in individuals with acquired and innate immune disorders and is strongly associated with Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large B-cell lymphomas, nasopharyngeal carcinoma (NPC), and lymphomas that develop in organ transplant recipients. Cellular DNA damage is a major determinant in the establishment of oncogenic processes and is well studied, but there are few studies of endogenous repair of viral DNA. This work evaluates how EBV's BPLF1 protein and its conserved deubiquitinating activity regulate the cellular DNA repair enzyme polymerase eta and recruit it to potential sites of viral damage and replication, resulting in enhanced production of infectious virus. These findings help to establish how EBV enlists and manipulates cellular DNA repair factors during the viral lytic cycle, contributing to efficient infectious virion production.

  • ISG15 Modulates Type I Interferon Signaling and the Antiviral Response during Hepatitis E Virus Replication [Cellular Response to Infection]

  • Hepatitis E virus (HEV), a single-stranded positive-sense RNA virus, generally causes self-limiting acute viral hepatitis, although chronic HEV infection has recently become a significant clinical problem in immunocompromised individuals, especially in solid-organ transplant recipients. Innate immunity, via the type I interferon (IFN) response, plays an important role during the initial stages of a viral infection. IFN-stimulated gene 15 (ISG15), an IFN-induced ubiquitin-like protein, is known to have an immunomodulatory role and can have a direct antiviral effect on a wide spectrum of virus families. In the present study, we investigated the antiviral effect as well as the potential immunomodulatory role of ISG15 during HEV replication. The results revealed that HEV induced high levels of ISG15 production both in vitro (Huh7-S10-3 liver cells) and in vivo (liver tissues from HEV-infected pigs); however, ISG15 is not required for virus replication. We also demonstrated that ISG15 silencing potentiates enhanced type I IFN-mediated signaling, resulting in an increase in the type I IFN-mediated antiviral effect during HEV replication. This observed enhanced type I IFN signaling correlated with an increase in IFN-stimulated gene expression levels during HEV replication. Furthermore, we showed that PKR and OAS1 played important roles in the ISG15-mediated type I IFN sensitivity of HEV. Taken together, the results from this study suggest that ISG15 plays an important immunomodulatory role and regulates HEV sensitivity to exogenous type I IFN.

    IMPORTANCE Hepatitis E virus (HEV) infection typically causes self-limiting acute viral hepatitis. However, chronic HEV infection has recently become a significant clinical problem in immunocompromised patients. Pegylated interferon (IFN) has been used to treat chronic HEV infection in solid-organ transplant patients with some success. However, the mechanism behind the type I IFN-mediated antiviral effect against HEV remains unclear. This report demonstrates that ISG15 induced by HEV replication in Huh7-S10-3 human liver cells plays an immunomodulatory role by negatively regulating type I IFN signaling and, thus, HEV sensitivity to type I IFN. Our results also show that PKR and OAS1 play important roles in the ISG15-mediated type I IFN sensitivity of HEV.

  • Species Specificity of Vaccinia Virus Complement Control Protein for the Bovine Classical Pathway Is Governed Primarily by Direct Interaction of Its Acidic Residues with Factor I [Pathogenesis and Immunity]

  • Poxviruses display species tropismmmdash;variola virus is a human-specific virus, while vaccinia virus causes repeated outbreaks in dairy cattle. Consistent with this, variola virus complement regulator SPICE (smallpox inhibitor of complement enzymes) exhibits selectivity in inhibiting the human alternative complement pathway and vaccinia virus complement regulator VCP (vaccinia virus complement control protein) displays selectivity in inhibiting the bovine alternative complement pathway. In the present study, we examined the species specificity of VCP and SPICE for the classical pathway (CP). We observed that VCP is ~43-fold superior to SPICE in inhibiting bovine CP. Further, functional assays revealed that increased inhibitory activity of VCP for bovine CP is solely due to its enhanced cofactor activity, with no effect on decay of bovine CP C3-convertase. To probe the structural basis of this specificity, we utilized single- and multi-amino-acid substitution mutants wherein 1 or more of the 11 variant VCP residues were substituted in the SPICE template. Examination of these mutants for their ability to inhibit bovine CP revealed that E108, E120, and E144 are primarily responsible for imparting the specificity and contribute to the enhanced cofactor activity of VCP. Binding and functional assays suggested that these residues interact with bovine factor I but not with bovine C4(H2O) (a moiety conformationally similar to C4b). Mapping of these residues onto the modeled structure of bovine C4b-VCP-bovine factor I supported the mutagenesis data. Taken together, our data help explain why the vaccine strain of vaccinia virus was able to gain a foothold in domesticated animals.

    IMPORTANCE Vaccinia virus was used for smallpox vaccination. The vaccine-derived virus is now circulating and causing outbreaks in dairy cattle in India and Brazil. However, the reason for this tropism is unknown. It is well recognized that the virus is susceptible to neutralization by the complement classical pathway (CP). Because the virus encodes a soluble complement regulator, VCP, we examined whether this protein displays selectivity in targeting bovine CP. Our data show that it does exhibit selectivity in inhibiting the bovine CP and that this is primarily determined by its amino acids E108, E120, and E144, which interact with bovine serine protease factor I to inactivate bovine C4bmmdash;one of the two subunits of CP C3-convertase. Of note, the variola complement regulator SPICE contains positively charged residues at these positions. Thus, these variant residues in VCP help enhance its potency against the bovine CP and thereby the fitness of the virus in cattle.

  • Propagation of Astrovirus VA1, a Neurotropic Human Astrovirus, in Cell Culture [Virus-Cell Interactions]

  • Astrovirus VA1/HMO-C (VA1; mamastrovirus 9) is a recently discovered astrovirus genotype that is divergent from the classic human astroviruses (mamastrovirus 1). The gastrointestinal tract is presumed to be the primary site of infection and pathogenicity for astroviruses. However, VA1 has been independently detected in brain tissue of five cases of human encephalitis. Studies of the pathogenicity of VA1 are currently impossible because there are no reported cell culture systems or in vivo models that support VA1 infection. Here, we describe successful propagation of VA1 in multiple human cell lines. The initial inoculum, a filtered clinical stool sample from the index gastroenteritis case cluster that led to the discovery of VA1, was first passaged in Vero cells. Serial blind passage in Caco-2 cells yielded increasing copies of VA1 RNA, and multistep growth curves demonstrated a ggt;100-fold increase in VA1 RNA 72 h after inoculation. The full-length genomic and subgenomic RNA strands were detected by Northern blotting, and crystalline lattices of viral particles of ~26-nm diameter were observed by electron microscopy in infected Caco-2 cells. Unlike other human astrovirus cell culture systems, which require addition of exogenous trypsin for continued propagation, VA1 could be propagated equally well with or without the addition of trypsin. Furthermore, VA1 was sensitive to the type I interferon (IFN-I) response, as VA1 RNA levels were reduced by pretreatment of Caco-2 cells with IFN-bbeta;1a. The ability to propagate VA1 in cell culture will facilitate studies of the neurotropism and neuropathogenesis of VA1.

    IMPORTANCE Astroviruses are an emerging cause of central nervous system infections in mammals, and astrovirus VA1/HMO-C is the most prevalent astrovirus in cases of human encephalitis. This virus has not been previously propagated, preventing elucidation of the biology of this virus. We describe the first cell culture system for VA1, a key step necessary for the study of its ability to cause disease.

  • Pseudorabies Virus US3-Induced Tunneling Nanotubes Contain Stabilized Microtubules, Interact with Neighboring Cells via Cadherins, and Allow Intercellular Molecular Communication [Virus-Cell Interactions]

  • Tunneling nanotubes (TNTs) are long bridge-like structures that connect eukaryotic cells and mediate intercellular communication. We found earlier that the conserved alphaherpesvirus US3 protein kinase induces long cell projections that contact distant cells and promote intercellular virus spread. In this report, we show that the US3-induced cell projections constitute TNTs. In addition, we report that US3-induced TNTs mediate intercellular transport of information (e.g., green fluorescent protein [GFP]) in the absence of other viral proteins. US3-induced TNTs are remarkably stable compared to most TNTs described in the literature. In line with this, US3-induced TNTs were found to contain stabilized (acetylated and detyrosinated) microtubules. Transmission electron microscopy showed that virus particles are individually transported in membrane-bound vesicles in US3-induced TNTs and are released along the TNT and at the contact area between a TNT and the adjacent cell. Contact between US3-induced TNTs and acceptor cells is very stable, which correlated with a marked enrichment in adherens junction components beta-catenin and E-cadherin at the contact area. These data provide new structural insights into US3-induced TNTs and how they may contribute to intercellular communication and alphaherpesvirus spread.

    IMPORTANCE Tunneling nanotubes (TNT) represent an important and yet still poorly understood mode of long-distance intercellular communication. We and others reported earlier that the conserved alphaherpesvirus US3 protein kinase induces long cellular protrusions in infected and transfected cells. Here, we show that US3-induced cell projections constitute TNTs, based on structural properties and transport of biomolecules. In addition, we report on different particular characteristics of US3-induced TNTs that help to explain their remarkable stability compared to physiological TNTs. In addition, transmission electron microscopy assays indicate that, in infected cells, virions travel in the US3-induced TNTs in membranous transport vesicles and leave the TNT via exocytosis. These data generate new fundamental insights into the biology of (US3-induced) TNTs and into how they may contribute to intercellular virus spread and communication.

  • rgs-CaM Detects and Counteracts Viral RNA Silencing Suppressors in Plant Immune Priming [Pathogenesis and Immunity]

  • Primary infection of a plant with a pathogen that causes high accumulation of salicylic acid in the plant typically via a hypersensitive response confers enhanced resistance against secondary infection with a broad spectrum of pathogens, including viruses. This phenomenon is called systemic acquired resistance (SAR), which is a plant priming for adaption to repeated biotic stress. However, the molecular mechanisms of SAR-mediated enhanced inhibition, especially of virus infection, remain unclear. Here, we show that SAR against cucumber mosaic virus (CMV) in tobacco plants (Nicotiana tabacum) involves a calmodulin-like protein, rgs-CaM. We previously reported the antiviral function of rgs-CaM, which binds to and directs degradation of viral RNA silencing suppressors (RSSs), including CMV 2b, via autophagy. We found that rgs-CaM-mediated immunity is ineffective against CMV infection in normally growing tobacco plants but is activated as a result of SAR induction via salicylic acid signaling. We then analyzed the effect of overexpression of rgs-CaM on salicylic acid signaling. Overexpressed and ectopically expressed rgs-CaM induced defense reactions, including cell death, generation of reactive oxygen species, and salicylic acid signaling. Further analysis using a combination of the salicylic acid analogue benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) and the Ca2+ ionophore A23187 revealed that rgs-CaM functions as an immune receptor that induces salicylic acid signaling by simultaneously perceiving both viral RSS and Ca2+ influx as infection cues, implying its autoactivation. Thus, secondary infection of SAR-induced tobacco plants with CMV seems to be effectively inhibited through 2b recognition and degradation by rgs-CaM, leading to reinforcement of antiviral RNA silencing and other salicylic acid-mediated antiviral responses.

    IMPORTANCE Even without an acquired immune system like that in vertebrates, plants show enhanced whole-plant resistance against secondary infection with pathogens; this so-called systemic acquired resistance (SAR) has been known for more than half a century and continues to be extensively studied. SAR-induced plants strongly and rapidly express a number of antibiotics and pathogenesis-related proteins targeted against secondary infection, which can account for enhanced resistance against bacterial and fungal pathogens but are not thought to control viral infection. This study showed that enhanced resistance against cucumber mosaic virus is caused by a tobacco calmodulin-like protein, rgs-CaM, which detects and counteracts the major viral virulence factor (RNA silencing suppressor) after SAR induction. rgs-CaM-mediated SAR illustrates the growth versus defense trade-off in plants, as it targets the major virulence factor only under specific biotic stress conditions, thus avoiding the cost of constitutive activation while reducing the damage from virus infection.

  • Enterovirus 3A Facilitates Viral Replication by Promoting Phosphatidylinositol 4-Kinase III{beta}-ACBD3 Interaction [Virus-Cell Interactions]

  • Like other enteroviruses, enterovirus 71 (EV71) relies on phosphatidylinositol 4-kinase IIIbbeta; (PI4KB) for genome RNA replication. However, how PI4KB is recruited to the genome replication sites of EV71 remains elusive. Recently, we reported that a host factor, ACBD3, is needed for EV71 replication by interacting with viral 3A protein. Here, we show that ACBD3 is required for the recruitment of PI4KB to RNA replication sites. Overexpression of viral 3A or EV71 infection stimulates the interaction of PI4KB and ACBD3. Consistently, EV71 infection induces the production of phosphatidylinositol-4-phosphate (PI4P). Furthermore, PI4KB, ACBD3, and 3A are all localized to the viral-RNA replication sites. Accordingly, PI4KB or ACBD3 depletion by small interfering RNA (siRNA) leads to a reduction in PI4P production after EV71 infection. I44A or H54Y substitution in 3A interrupts the stimulation of PI4KB and ACBD3. Further analysis suggests that stimulation of ACBD3-PI4KB interaction is also important for the replication of enterovirus 68 but disadvantageous to human rhinovirus 16. These results reveal a mechanism of enterovirus replication that involves a selective strategy for recruitment of PI4KB to the RNA replication sites.

    IMPORTANCE Enterovirus 71, like other human enteroviruses, replicates its genome within host cells, where viral proteins efficiently utilize cellular machineries. While multiple factors are involved, it is largely unclear how viral replication is controlled. We show that the 3A protein of enterovirus 71 recruits an enzyme, phosphatidylinositol 4-kinase IIIbbeta;, by interacting with ACBD3, which alters cellular membranes through the production of a lipid, PI4P. Consequently, the viral and host proteins form a large complex that is necessary for RNA synthesis at replication sites. Notably, PI4KB-ACBD3 interaction also differentially mediates the replication of enterovirus 68 and rhinovirus 16. These results provide new insight into the molecular network of enterovirus replication.

  • Pace of Coreceptor Tropism Switch in HIV-1-Infected Individuals after Recent Infection [Genetic Diversity and Evolution]

  • HIV-1 entry into target cells influences several aspects of HIV-1 pathogenesis, including viral tropism, HIV-1 transmission and disease progression, and response to entry inhibitors. The evolution from CCR5- to CXCR4-using strains in a given human host is still unpredictable. Here we analyzed timing and predictors for coreceptor evolution among recently HIV-1-infected individuals. Proviral DNA was longitudinally evaluated in 66 individuals using Geno2pheno[coreceptor]. Demographics, viral load, CD4+ and CD8+ T cell counts, CCR532 polymorphisms, GB virus C (GBV-C) coinfection, and HLA profiles were also evaluated. Ultradeep sequencing was performed on initial samples from 11 selected individuals. A tropism switch from CCR5- to CXCR4-using strains was identified in 9/49 (18.4%) individuals. Only a low baseline false-positive rate (FPR) was found to be a significant tropism switch predictor. No minor CXCR4-using variants were identified in initial samples of 4 of 5 R5/non-R5 switchers. Logistic regression analysis showed that patients with an FPR of ggt;40.6% at baseline presented a stable FPR over time whereas lower FPRs tend to progressively decay, leading to emergence of CXCR4-using strains, with a mean evolution time of 27.29 months (range, 8.90 to 64.62). An FPR threshold above 40.6% determined by logistic regression analysis may make it unnecessary to further determine tropism for prediction of disease progression related to emergence of X4 strains or use of CCR5 antagonists. The detection of variants with intermediate FPRs and progressive FPR decay over time not only strengthens the power of Geno2pheno in predicting HIV tropism but also indirectly confirms a continuous evolution from earlier R5 variants toward CXCR4-using strains.

    IMPORTANCE The introduction of CCR5 antagonists in the antiretroviral arsenal has sparked interest in coreceptors utilized by HIV-1. Despite concentrated efforts, viral and human host features predicting tropism switch are still poorly understood. Limited longitudinal data are available to assess the influence that these factors have on predicting tropism switch and disease progression. The present study describes longitudinal tropism evolution in a group of recently HIV-infected individuals to determine the prevalence and potential correlates of tropism switch. We demonstrated here that a low baseline FPR determined by the Geno2pheno[coreceptor] algorithm can predict tropism evolution from CCR5 to CXCR4 coreceptor use.

  • Selective Expression of CCR10 and CXCR3 by Circulating Human Herpes Simplex Virus-Specific CD8 T Cells [Pathogenesis and Immunity]

  • Herpes simplex virus (HSV) infection is restricted to epithelial cells and neurons and is controlled by CD8 T cells. These cells both traffic to epithelial sites of recurrent lytic infection and to ganglia and persist at the dermal-epidermal junction for up to 12 weeks after lesion resolution. We previously showed that cutaneous lymphocyte-associated antigen (CLA), a functional E-selectin ligand (ESL), is selectively expressed on circulating HSV-2-specific CD8 T cells. CLA/ESL mediates adhesion of T cells to inflamed vascular endothelium. Later stages in T-cell homing involve chemokines (Ch) and lymphocyte chemokine receptors (ChR) for vascular wall arrest and diapedesis. Several candidate ChR have been implicated in skin homing. We measured cell surface ChR on HSV-specific human peripheral blood CD8 T cells and extended our studies to HSV-1. We observed preferential cell surface expression of CCR10 and CXCR3 by HSV-specific CD8 T cells compared to CD8 T cells specific for control viruses, Epstein-Barr virus (EBV) and cytomegalovirus (CMV), and compared to bulk memory CD8 T cells. CXCR3 ligand mRNA levels were selectively increased in skin biopsy specimens from persons with recurrent HSV-2, while the mRNA levels of the CCR10 ligand CCL27 were equivalent in lesion and control skin. Our data are consistent with a model in which CCL27 drives baseline recruitment of HSV-specific CD8 T cells expressing CCR10, while interferon-responsive CXCR3 ligands recruit additional cells in response to virus-driven inflammation.

    IMPORTANCE HSV-2 causes very localized recurrent infections in the skin and genital mucosa. Virus-specific CD8 T cells home to the site of recurrent infection and participate in viral clearance. The exit of T cells from the blood involves the use of chemokine receptors on the T-cell surface and chemokines that are present in infected tissue. In this study, circulating HSV-2-specific CD8 T cells were identified using specific fluorescent tetramer reagents, and their expression of several candidate skin-homing-associated chemokine receptors was measured using flow cytometry. We found that two chemokine receptors, CXCR3 and CCR10, are upregulated on HSV-specific CD8 T cells in blood. The chemokines corresponding to these receptors are also expressed in infected tissues. Vaccine strategies to prime CD8 T cells to home to HSV lesions should elicit these chemokine receptors if possible to increase the homing of vaccine-primed cells to sites of infection.

  • Increased, Durable B-Cell and ADCC Responses Associated with T-Helper Cell Responses to HIV-1 Envelope in Macaques Vaccinated with gp140 Occluded at the CD4 Receptor Binding Site [Vaccines and Antiviral Agents]

  • Strategies are needed to improve the immunogenicity of HIV-1 envelope (Env) antigens (Ag) for more long-lived, efficacious HIV-1 vaccine-induced B-cell responses. HIV-1 Env gp140 (native or uncleaved molecules) or gp120 monomeric proteins elicit relatively poor B-cell responses which are short-lived. We hypothesized that Env engagement of the CD4 receptor on T-helper cells results in anergic effects on T-cell recruitment and consequently a lack of strong, robust, and durable B-memory responses. To test this hypothesis, we occluded the CD4 binding site (CD4bs) of gp140 by stable cross-linking with a 3-kDa CD4 miniprotein mimetic, serving to block ligation of gp140 on CD4+ T cells while preserving CD4-inducible (CDi) neutralizing epitopes targeted by antibody-dependent cellular cytotoxicity (ADCC) effector responses. Importantly, immunization of rhesus macaques consistently gave superior B-cell (P llt; 0.001) response kinetics and superior ADCC (P llt; 0.014) in a group receiving the CD4bs-occluded vaccine compared to those of animals immunized with gp140. Of the cytokines examined, Ag-specific interleukin-4 (IL-4) T-helper enzyme-linked immunosorbent spot (ELISpot) assays of the CD4bs-occluded group increased earlier (P = 0.025) during the inductive phase. Importantly, CD4bs-occluded gp140 antigen induced superior B-cell and ADCC responses, and the elevated B-cell responses proved to be remarkably durable, lasting more than 60 weeks postimmunization.

    IMPORTANCE Attempts to develop HIV vaccines capable of inducing potent and durable B-cell responses have been unsuccessful until now. Antigen-specific B-cell development and affinity maturation occurs in germinal centers in lymphoid follicles through a critical interaction between B cells and T follicular helper cells. The HIV envelope binds the CD4 receptor on T cells as soluble shed antigen or as antigen-antibody complexes, causing impairment in the activation of these specialized CD4-positive T cells. We proposed that CD4-binding impairment is partly responsible for the relatively poor B-cell responses to HIV envelope-based vaccines. To test this hypothesis, we blocked the CD4 binding site of the envelope antigen and compared it to currently used unblocked envelope protein. We found superior and durable B-cell responses in macaques vaccinated with an occluded CD4 binding site on the HIV envelope antigen, demonstrating a potentially important new direction in future design of new HIV vaccines.

  • Cholesterol 25-Hydroxylase Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication through Enzyme Activity-Dependent and -Independent Mechanisms [Virus-Cell Interactions]

  • Cholesterol 25-hydroxylase (CH25H) has recently been identified as a host restriction factor that exerts antiviral effects by catalyzing the production of 25-hydroxycholesterol (25HC). CH25H can be rapidly induced upon infection with some viruses. Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, has ranked among the most important swine pathogens since it was discovered in the late 1980s. In this study, we found that PRRSV infection significantly downregulated the expression of CH25H in cells by a so-far unknown mechanism, suggesting that CH25H exerts antiviral activity against PRRSV. Indeed, overexpression of CH25H inhibited PRRSV replication, whereas knockdown of CH25H by short interfering RNA (siRNA) promoted PRRSV infection. The anti-PRRSV effect of 25HC operates via inhibition of viral penetration. Interestingly, a CH25H mutant (CH25H-M) lacking hydroxylase activity still inhibited PRRSV infection. Screening using a yeast two-hybrid system followed by coimmunoprecipitation and immunofluorescence colocalization analyses confirmed that both CH25H and CH25H-M interact with the nonstructural protein 1 alpha (nsp1aalpha;) of PRRSV. Unexpectedly, the expression of nsp1aalpha; decreased following coexpression with CH25H or CH25H-M. Detailed analyses demonstrated that CH25H/CH25H-M could degrade nsp1aalpha; through the ubiquitin-proteasome pathway and that site K169 in the nsp1aalpha; protein is the key site of ubiquitination. Taken together, our findings demonstrate that CH25H restricts PRRSV replication by targeting viral penetration as well as degrading nsp1aalpha;, revealing a novel antiviral mechanism used by CH25H.

    IMPORTANCE PRRSV has been a continuous threat to the global swine industry, and current vaccines are insufficient to provide sustainable control. CH25H has been found to exert a broad antiviral effect; thus, it is an attractive target for the development of anti-PRRSV drugs. Here, we demonstrate that CH25H is an interferon-stimulated gene that is highly expressed in porcine alveolar macrophages. CH25H exerts its anti-PRRSV effect not only via the production of 25HC to inhibit viral penetration but also by degrading viral protein through the ubiquitin-proteasome pathway, suggesting that CH25H is a candidate for the development of antiviral therapeutics. However, PRRSV infection appears to actively decrease CH25H expression to promote viral replication, highlighting the complex game between PRRSV and its host.

  • Whole-Genome Sequencing and Variant Analysis of Human Papillomavirus 16 Infections [Genetic Diversity and Evolution]

  • Human papillomavirus (HPV) is a strongly conserved DNA virus, high-risk types of which can cause cervical cancer in persistent infections. The most common type found in HPV-attributable cancer is HPV16, which can be subdivided into four lineages (A to D) with different carcinogenic properties. Studies have shown HPV16 sequence diversity in different geographical areas, but only limited information is available regarding HPV16 diversity within a population, especially at the whole-genome level. We analyzed HPV16 major variant diversity and conservation in persistent infections and performed a single nucleotide polymorphism (SNP) comparison between persistent and clearing infections. Materials were obtained in the Netherlands from a cohort study with longitudinal follow-up for up to 3 years. Our analysis shows a remarkably large variant diversity in the population. Whole-genome sequences were obtained for 57 persistent and 59 clearing HPV16 infections, resulting in 109 unique variants. Interestingly, persistent infections were completely conserved through time. One reinfection event was identified where the initial and follow-up samples clustered differently. Non-A1/A2 variants seemed to clear preferentially (P = 0.02). Our analysis shows that population-wide HPV16 sequence diversity is very large. In persistent infections, the HPV16 sequence was fully conserved. Sequencing can identify HPV16 reinfections, although occurrence is rare. SNP comparison identified no strongly acting effect of the viral genome affecting HPV16 infection clearance or persistence in up to 3 years of follow-up. These findings suggest the progression of an early HPV16 infection could be host related.

    IMPORTANCE Human papillomavirus 16 (HPV16) is the predominant type found in cervical cancer. Progression of initial infection to cervical cancer has been linked to sequence properties; however, knowledge of variants circulating in European populations, especially with longitudinal follow-up, is limited. By sequencing a number of infections with known follow-up for up to 3 years, we gained initial insights into the genetic diversity of HPV16 and the effects of the viral genome on the persistence of infections. A SNP comparison between sequences obtained from clearing and persistent infections did not identify strongly acting DNA variations responsible for these infection outcomes. In addition, we identified an HPV16 reinfection event where sequencing of initial and follow-up samples showed different HPV16 variants. Based on conventional genotyping, this infection would incorrectly be considered a persistent HPV16 infection. In the context of vaccine efficacy and monitoring studies, such infections could potentially cause reduced reported efficacy or efficiency.

  • Bam35 Tectivirus Intraviral Interaction Map Unveils New Function and Localization of Phage ORFan Proteins [Genetic Diversity and Evolution]

  • The family Tectiviridae comprises a group of tailless, icosahedral, membrane-containing bacteriophages that can be divided into two groups by their hosts, either Gram-negative or Gram-positive bacteria. While the first group is composed of PRD1 and nearly identical well-characterized lytic viruses, the second one includes more variable temperate phages, like GIL16 or Bam35, whose hosts are Bacillus cereus and related Gram-positive bacteria. In the genome of Bam35, nearly half of the 32 annotated open reading frames (ORFs) have no homologs in databases (ORFans), being putative proteins of unknown function, which hinders the understanding of their biology. With the aim of increasing knowledge about the viral proteome, we carried out a comprehensive yeast two-hybrid analysis of all the putative proteins encoded by the Bam35 genome. The resulting protein interactome comprised 76 unique interactions among 24 proteins, of which 12 have an unknown function. These results suggest that the P17 protein is the minor capsid protein of Bam35 and P24 is the penton protein, with the latter finding also being supported by iterative threading protein modeling. Moreover, the inner membrane transglycosylase protein P26 could have an additional structural role. We also detected interactions involving nonstructural proteins, such as the DNA-binding protein P1 and the genome terminal protein (P4), which was confirmed by coimmunoprecipitation of recombinant proteins. Altogether, our results provide a functional view of the Bam35 viral proteome, with a focus on the composition and organization of the viral particle.

    IMPORTANCE Tailless viruses of the family Tectiviridae can infect commensal and pathogenic Gram-positive and Gram-negative bacteria. Moreover, they have been proposed to be at the evolutionary origin of several groups of large eukaryotic DNA viruses and self-replicating plasmids. However, due to their ancient origin and complex diversity, many tectiviral proteins are ORFans of unknown function. Comprehensive protein-protein interaction (PPI) analysis of viral proteins can eventually disclose biological mechanisms and thus provide new insights into protein function unattainable by studying proteins one by one. Here we comprehensively describe intraviral PPIs among tectivirus Bam35 proteins determined using multivector yeast two-hybrid screening, and these PPIs were further supported by the results of coimmunoprecipitation assays and protein structural models. This approach allowed us to propose new functions for known proteins and hypothesize about the biological role of the localization of some viral ORFan proteins within the viral particle that will be helpful for understanding the biology of tectiviruses infecting Gram-positive bacteria.

  • Evaluation of the Biological Properties and Cross-Reactive Antibody Response to H10 Influenza Viruses in Ferrets [Vaccines and Antiviral Agents]

  • The recent outbreak of avian origin H10N7 influenza among seals in northern Europe and two fatal human infections with an avian H10N8 virus in China have demonstrated that H10 viruses can spread between mammals and cause severe disease in humans. To gain insight into the potential for H10 viruses to cross the species barrier and to identify a candidate vaccine strain, we evaluated the in vitro and in vivo properties and antibody response in ferrets to 20 diverse H10 viruses. H10 virus infection of ferrets caused variable weight loss, and all 20 viruses replicated throughout the respiratory tract; however, replication in the lungs was highly variable. In glycan-binding assays, the H10 viruses preferentially bound "avian-like" aalpha;2,3-linked sialic acids. Importantly, several isolates also displayed strong binding to long-chain "human-like" aalpha;2,6-linked sialic acids and exhibited comparable or elevated neuraminidase activity relative to human H1N1, H2N2, and H3N2 viruses. In hemagglutination inhibition assays, 12 antisera cross-reacted with gge;14 of 20 H10 viruses, and 7 viruses induced neutralizing activity against gge;15 of the 20 viruses. By combining data on weight loss, viral replication, and the cross-reactive antibody response, we identified A/mallard/Portugal/79906/2009 (H10N7) as a suitable virus for vaccine development. Collectively, our findings suggest that H10 viruses may continue to sporadically infect humans and other mammals, underscoring the importance of developing an H10 vaccine for pandemic preparedness.

    IMPORTANCE Avian origin H10 influenza viruses sporadically infect humans and other mammals; however, little is known about viruses of this subtype. Thus, we characterized the biological properties of 20 H10 viruses in vitro and in ferrets. Infection caused mild to moderate weight loss (5 to 15%), with robust viral replication in the nasal tissues and variable replication in the lung. H10 viruses preferentially bind "avian-like" sialic acids, although several isolates also displayed binding to "human-like" sialic acid receptors. This is consistent with the ability of H10 viruses to cross the species barrier and warrants selection of an H10 vaccine strain. By evaluating the cross-reactive antibody response to the H10 viruses and combining this analysis with viral replication and weight loss findings, we identified A/mallard/Portugal/79906/2009 (H10N7) as a suitable H10 vaccine strain.

  • Experimental Transmission of the Chronic Wasting Disease Agent to Swine after Oral or Intracranial Inoculation [Prions]

  • Chronic wasting disease (CWD) is a naturally occurring, fatal neurodegenerative disease of cervids. The potential for swine to serve as hosts for the agent of CWD is unknown. The purpose of this study was to investigate the susceptibility of swine to the CWD agent following experimental oral or intracranial inoculation. Crossbred piglets were assigned to three groups, intracranially inoculated (n = 20), orally inoculated (n = 19), and noninoculated (n = 9). At approximately the age at which commercial pigs reach market weight, half of the pigs in each group were culled ("market weight" groups). The remaining pigs ("aged" groups) were allowed to incubate for up to 73 months postinoculation (mpi). Tissues collected at necropsy were examined for disease-associated prion protein (PrPSc) by Western blotting (WB), antigen capture enzyme immunoassay (EIA), immunohistochemistry (IHC), and in vitro real-time quaking-induced conversion (RT-QuIC). Brain samples from selected pigs were also bioassayed in mice expressing porcine prion protein. Four intracranially inoculated aged pigs and one orally inoculated aged pig were positive by EIA, IHC, and/or WB. By RT-QuIC, PrPSc was detected in lymphoid and/or brain tissue from one or more pigs in each inoculated group. The bioassay was positive in four out of five pigs assayed. This study demonstrates that pigs can support low-level amplification of CWD prions, although the species barrier to CWD infection is relatively high. However, detection of infectivity in orally inoculated pigs with a mouse bioassay raises the possibility that naturally exposed pigs could act as a reservoir of CWD infectivity.

    IMPORTANCE We challenged domestic swine with the chronic wasting disease agent by inoculation directly into the brain (intracranially) or by oral gavage (orally). Disease-associated prion protein (PrPSc) was detected in brain and lymphoid tissues from intracranially and orally inoculated pigs as early as 8 months of age (6 months postinoculation). Only one pig developed clinical neurologic signs suggestive of prion disease. The amount of PrPSc in the brains and lymphoid tissues of positive pigs was small, especially in orally inoculated pigs. Regardless, positive results obtained with orally inoculated pigs suggest that it may be possible for swine to serve as a reservoir for prion disease under natural conditions.

  • Binding of Herpes Simplex Virus 1 UL20 to GODZ (DHHC3) Affects Its Palmitoylation and Is Essential for Infectivity and Proper Targeting and Localization of UL20 and Glycoprotein K [Virus-Cell Interactions]

  • Herpes simplex virus 1 (HSV-1) UL20 plays a crucial role in the envelopment of the cytoplasmic virion and its egress. It is a nonglycosylated envelope protein that is regulated as a 1 gene. Two-hybrid and pulldown assays demonstrated that UL20, but no other HSV-1 gene-encoded proteins, binds specifically to GODZ (also known as DHHC3), a cellular Golgi apparatus-specific Asp-His-His-Cys (DHHC) zinc finger protein. A catalytically inactive dominant-negative GODZ construct significantly reduced HSV-1 replication in vitro and affected the localization of UL20 and glycoprotein K (gK) and their interactions but not glycoprotein C (gC). GODZ is involved in palmitoylation, and we found that UL20 is palmitoylated by GODZ using a GODZ dominant-negative plasmid. Blocking of palmitoylation using 2-bromopalmitate (2-BP) affected the virus titer and the interaction of UL20 and gK but did not affect the levels of these proteins. In conclusion, we have shown that binding of UL20 to GODZ in the Golgi apparatus regulates trafficking of UL20 and its subsequent effects on gK localization and virus replication. We also have demonstrated that GODZ-mediated UL20 palmitoylation is critical for UL20 membrane targeting and thus gK cell surface expression, providing new mechanistic insights into how UL20 palmitoylation regulates HSV-1 infectivity.

    IMPORTANCE HSV-1 UL20 is a nonglycosylated essential envelope protein that is highly conserved among herpesviruses. In this study, we show that (i) HSV-1 UL20 binds to GODZ (also known as DHHC3), a Golgi apparatus-specific Asp-His-His-Cys (DHHC) zinc finger protein; (ii) a GODZ dominant-negative mutant and an inhibitor of palmitoylation reduced HSV-1 titers and altered the localization of UL20 and glycoprotein K; and (iii) UL20 is palmitoylated by GODZ, and this UL20 palmitoylation is required for HSV-1 infectivity. Thus, blocking of the interaction of UL20 with GODZ, using a GODZ dominant-negative mutant or possibly GODZ shRNA, should be considered a potential alternative therapy in not only HSV-1 but also other conditions in which GODZ processing is an integral component of pathogenesis.

  • A Unique T-Cell Receptor Amino Acid Sequence Selected by Human T-Cell Lymphotropic Virus Type 1 Tax301-309-Specific Cytotoxic T Cells in HLA-A24:02-Positive Asymptomatic Carriers and Adult T-Cell Leukemia/Lymphoma Patients [Cellular Response to Infection]

  • We previously reported that the T-cell receptor (TCR) repertoire of human T-cell lymphotropic virus type 1 (HTLV-1) Tax301-309-specific CD8+ cytotoxic T cells (Tax301-309-CTLs) was highly restricted and a particular amino acid sequence motif, the PDR motif, was conserved among HLA-A*24:02-positive (HLA-A*24:02+) adult T-cell leukemia/lymphoma (ATL) patients who had undergone allogeneic hematopoietic cell transplantation (allo-HSCT). Furthermore, we found that donor-derived PDR+ CTLs selectively expanded in ATL long-term HSCT survivors with strong CTL activity against HTLV-1. On the other hand, the TCR repertoires in Tax301-309-CTLs of asymptomatic HTLV-1 carriers (ACs) remain unclear. In this study, we directly identified the DNA sequence of complementarity-determining region 3 (CDR3) of the TCR-bbeta; chain of Tax301-309-CTLs at the single-cell level and compared not only the TCR repertoires but also the frequencies and phenotypes of Tax301-309-CTLs between ACs and ATL patients. We did not observe any essential difference in the frequencies of Tax301-309-CTLs between ACs and ATL patients. In the single-cell TCR repertoire analysis of Tax301-309-CTLs, 1,458 Tax301-309-CTLs and 140 clones were identified in this cohort. Tax301-309-CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and PDR, the unique motif in TCR-bbeta; CDR3, was exclusively observed in all ACs and ATL patients. However, there was no correlation between PDR+ CTL frequencies and HTLV-1 proviral load (PVL). In conclusion, we have identified, for the first time, a unique amino acid sequence, PDR, as a public TCR-CDR3 motif against Tax in HLA-A*24:02+ HTLV-1-infected individuals. Further investigations are warranted to elucidate the role of the PDR+ CTL response in the progression from carrier state to ATL.

    IMPORTANCE ATL is an aggressive T-cell malignancy caused by HTLV-1 infection. The HTLV-1 regulatory protein Tax aggressively promotes the proliferation of HTLV-1-infected lymphocytes and is also a major target antigen for CD8+ CTLs. In our previous evaluation of Tax301-309-CTLs, we found that a unique amino acid sequence motif, PDR, in CDR3 of the TCR-bbeta; chain of Tax301-309-CTLs was conserved among ATL patients after allo-HSCT. Furthermore, the PDR+ Tax301-309-CTL clones selectively expanded and showed strong cytotoxic activities against HTLV-1. On the other hand, it remains unclear how Tax301-309-CTL repertoire exists in ACs. In this study, we comprehensively compared Tax-specific TCR repertoires at the single-cell level between ACs and ATL patients. Tax301-309-CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and PDR, the unique motif in TCR-bbeta; CDR3, was conserved in all ACs and ATL patients, regardless of clinical subtype in HTLV-1 infection.

  • Panels of HIV-1 Subtype C Env Reference Strains for Standardized Neutralization Assessments [Vaccines and Antiviral Agents]

  • In the search for effective immunologic interventions to prevent and treat HIV-1 infection, standardized reference reagents are a cost-effective way to maintain robustness and reproducibility among immunological assays. To support planned and ongoing studies where clade C predominates, here we describe three virus panels, chosen from 200 well-characterized clade C envelope (Env)-pseudotyped viruses from early infection. All 200 Envs were expressed as a single round of replication pseudoviruses and were tested to quantify neutralization titers by 16 broadly neutralizing antibodies (bnAbs) and sera from 30 subjects with chronic clade C infections. We selected large panels of 50 and 100 Envs either to characterize cross-reactive breadth for sera identified as having potent neutralization activity based on initial screening or to evaluate neutralization magnitude-breadth distributions of newly isolated antibodies. We identified these panels by downselection after hierarchical clustering of bnAb neutralization titers. The resulting panels represent the diversity of neutralization profiles throughout the range of virus sensitivities identified in the original panel of 200 viruses. A small 12-Env panel was chosen to screen sera from vaccine trials or natural-infection studies for neutralization responses. We considered panels selected by previously described methods but favored a computationally informed method that enabled selection of viruses representing diverse neutralization sensitivity patterns, given that we do not a priori know what the neutralization-response profile of vaccine sera will be relative to that of sera from infected individuals. The resulting 12-Env panel complements existing panels. Use of standardized panels enables direct comparisons of data from different trials and study sites testing HIV-1 clade C-specific products.

    IMPORTANCE HIV-1 group M includes nine clades and many recombinants. Clade C is the most common lineage, responsible for roughly half of current HIV-1 infections, and is a focus for vaccine design and testing. Standard reference reagents, particularly virus panels to study neutralization by antibodies, are crucial for developing cost-effective and yet rigorous and reproducible assays against diverse examples of this variable virus. We developed clade C-specific panels for use as standardized reagents to monitor complex polyclonal sera for neutralization activity and to characterize the potency and breadth of cross-reactive neutralization by monoclonal antibodies, whether engineered or isolated from infected individuals. We chose from 200 southern African, clade C envelope-pseudotyped viruses with neutralization titers against 16 broadly neutralizing antibodies and 30 sera from chronic clade C infections. We selected panels to represent the diversity of bnAb neutralization profiles and Env neutralization sensitivities. Use of standard virus panels can facilitate comparison of results across studies and sites.

  • Murine Gammaherpesvirus 68 Expressing Kaposi Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen (LANA) Reveals both Functional Conservation and Divergence in LANA Homologs [Pathogenesis and Immunity]

  • Latency-associated nuclear antigen (LANA) is a multifunctional protein encoded by members of the Rhadinovirus genus of gammaherpesviruses. Studies using murine gammaherpesvirus 68 (MHV68) demonstrated that LANA is important for acute replication, latency establishment, and reactivation in vivo. Despite structural similarities in their DNA-binding domains (DBDs), LANA homologs from Kaposi sarcoma-associated herpesvirus (KSHV) and MHV68 exhibit considerable sequence divergence. We sought to determine if KSHV and MHV68 LANA homologs are functionally interchangeable. We generated an MHV68 virus that encodes KSHV LANA (kLANA) in place of MHV68 LANA (mLANA) and evaluated the virus's capacity to replicate, establish and maintain latency, and reactivate. kLANA knock-in (KLKI) MHV68 was replication competent in vitro and in vivo but exhibited slower growth kinetics and lower titers than wild-type (WT) MHV68. Following inoculation of mice, KLKI MHV68 established and maintained latency in splenocytes and peritoneal cells but did not reactivate efficiently ex vivo. kLANA repressed the MHV68 promoter for ORF50, the gene that encodes the major lytic transactivator protein RTA, while mLANA did not, suggesting a likely mechanism for the KLKI MHV68 phenotypes. Bypassing this repression by providing MHV68 RTA in trans rescued KLKI MHV68 replication in tissue culture and enabled detection of KLKI MHV68 reactivation ex vivo. These data demonstrate that kLANA and mLANA are functionally interchangeable for establishment and maintenance of latency and suggest that repression of lytic replication by kLANA, as previously shown with KSHV, is a kLANA-specific function that is transferable to MHV68.

    IMPORTANCE Kaposi sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) are members of the Rhadinovirus genus of gammaherpesviruses. These viruses establish lifelong infections that place their respective human and murine hosts at risk for cancer. Latency-associated nuclear antigen (LANA) is a conserved Rhadinovirus protein that is necessary for long-term chronic infection by these viruses. To better understand the conserved functions performed by LANA homologs, we generated a recombinant MHV68 virus that encodes the KSHV LANA protein in place of the MHV68 LANA homolog. We determined that the KSHV LANA protein is capable of supporting MHV68 latency in a mouse model of chronic infection but also functions to repress viral replication. This work describes an in vivo model system for defining evolutionarily conserved and divergent functions of LANA homologs in Rhadinovirus infection and disease.

  • Identification of Vaccinia Virus Replisome and Transcriptome Proteins by Isolation of Proteins on Nascent DNA Coupled with Mass Spectrometry [Genome Replication and Regulation of Viral Gene Expression]

  • Poxviruses replicate within the cytoplasm and encode proteins for DNA and mRNA synthesis. To investigate poxvirus replication and transcription from a new perspective, we incorporated 5-ethynyl-2'-deoxyuridine (EdU) into nascent DNA in cells infected with vaccinia virus (VACV). The EdU-labeled DNA was conjugated to fluor- or biotin-azide and visualized by confocal, superresolution, and transmission electron microscopy. Nuclear labeling decreased dramatically after infection, accompanied by intense labeling of cytoplasmic foci. The nascent DNA colocalized with the VACV single-stranded DNA binding protein I3 in multiple puncta throughout the interior of factories, which were surrounded by endoplasmic reticulum. Complexes containing EdU-biotin-labeled DNA cross-linked to proteins were captured on streptavidin beads. After elution and proteolysis, the peptides were analyzed by mass spectrometry to identify proteins associated with nascent DNA. The known viral replication proteins, a telomere binding protein, and a protein kinase were associated with nascent DNA, as were the DNA-dependent RNA polymerase and intermediate- and late-stage transcription initiation and elongation factors, plus the capping and methylating enzymes. These results suggested that the replicating pool of DNA is transcribed and that few if any additional viral proteins directly engaged in replication and transcription remain to be discovered. Among the host proteins identified by mass spectrometry, topoisomerases IIaalpha; and IIbbeta; and PCNA were noteworthy. The association of the topoisomerases with nascent DNA was dependent on expression of the viral DNA ligase, in accord with previous proteomic studies. Further investigations are needed to determine possible roles for PCNA and other host proteins detected.

    IMPORTANCE Poxviruses, unlike many well-characterized animal DNA viruses, replicate entirely within the cytoplasm of animal cells, raising questions regarding the relative roles of viral and host proteins. We adapted newly developed procedures for click chemistry and iPOND (Isolation of proteins on nascent DNA) to investigate vaccinia virus (VACV), the prototype poxvirus. Nuclear DNA synthesis ceased almost immediately following VACV infection, followed swiftly by the synthesis of viral DNA within discrete cytoplasmic foci. All viral proteins known from genetic and proteomic studies to be required for poxvirus DNA replication were identified in the complexes containing nascent DNA. The additional detection of the viral DNA-dependent RNA polymerase and intermediate and late transcription factors provided evidence for a temporal coupling of replication and transcription. Further studies are needed to assess the potential roles of host proteins, including topoisomerases IIaalpha; and IIbbeta; and PCNA, which were found associated with nascent DNA.

  • Stability of Cucumber Necrosis Virus at the Quasi-6-Fold Axis Affects Zoospore Transmission [Structure and Assembly]

  • Cucumber necrosis virus (CNV) is a member of the genus Tombusvirus and has a monopartite positive-sense RNA genome. CNV is transmitted in nature via zoospores of the fungus Olpidium bornovanus. As with other members of the Tombusvirus genus, the CNV capsid swells when exposed to alkaline pH and EDTA. We previously demonstrated that a P73G mutation blocks the virus from zoospore transmission while not significantly affecting replication in plants (K. Kakani, R. Reade, and D. Rochon, J Mol Biol 338:507nndash;517, 2004, https://doi.org/10.1016/j.jmb.2004.03.008). P73 lies immediately adjacent to a putative zinc binding site (M. Li et al., J Virol 87:12166nndash;12175, 2013, https://doi.org/10.1128/JVI.01965-13) that is formed by three icosahedrally related His residues in the N termini of the C subunit at the quasi-6-fold axes. To better understand how this buried residue might affect vector transmission, we determined the cryo-electron microscopy structure of wild-type CNV in the native and swollen state and of the transmission-defective mutant, P73G, under native conditions. With the wild-type CNV, the swollen structure demonstrated the expected expansion of the capsid. However, the zinc binding region at the quasi-6-fold at the bbeta;-annulus axes remained intact. By comparison, the zinc binding region of the P73G mutant, even under native conditions, was markedly disordered, suggesting that the bbeta;-annulus had been disrupted and that this could destabilize the capsid. This was confirmed with pH and urea denaturation experiments in conjunction with electron microscopy analysis. We suggest that the P73G mutation affects the zinc binding and/or the bbeta;-annulus, making it more fragile under neutral/basic pH conditions. This, in turn, may affect zoospore transmission.

    IMPORTANCE Cucumber necrosis virus (CNV), a member of the genus Tombusvirus, is transmitted in nature via zoospores of the fungus Olpidium bornovanus. While a number of plant viruses are transmitted via insect vectors, little is known at the molecular level as to how the viruses are recognized and transmitted. As with many spherical plant viruses, the CNV capsid swells when exposed to alkaline pH and EDTA. We previously demonstrated that a P73G mutation that lies inside the capsid immediately adjacent to a putative zinc binding site (Li et al., J Virol 87:12166nndash;12175, 2013, https://doi.org/10.1128/JVI.01965-13) blocks the virus from zoospore transmission while not significantly affecting replication in plants (K. Kakani, R. Reade, and D. Rochon, J Mol Biol 338:507nndash;517, 2004, https://doi.org/10.1016/j.jmb.2004.03.008). Here, we show that the P73G mutant is less stable than the wild type, and this appears to be correlated with destabilization of the bbeta;-annulus at the icosahedral 3-fold axes. Therefore, the bbeta;-annulus appears not to be essential for particle assembly but is necessary for interactions with the transmission vector.

  • Insufficient Innate Immunity Contributes to the Susceptibility of the Castaneous Mouse to Orthopoxvirus Infection [Pathogenesis and Immunity]

  • The castaneous (CAST) mouse, a wild-derived inbred strain, is highly susceptible to orthopoxvirus infection by intranasal and systemic routes. The 50% lethal intraperitoneal dose of vaccinia virus (VACV) was 3 PFU for CAST mice, whereas BALB/c mice survived 106 PFU. At all times and in all organs analyzed, virus titers were higher in CAST than in BALB/c mice. In individual CAST mice, luciferase-expressing VACV was seen to replicate rapidly leading to death, whereas virus levels increased for a few days and then declined in BALB/c mice. Increases in gamma interferon (IFN-) and tumor necrosis factor alpha (TNF-aalpha;) were delayed and low in CAST mice compared to BALB/c mice following VACV infection or poly(I-C) inoculation, consistent with differences in innate immune responses. In addition, naive CAST mice had considerably lower numbers of NK and T cells than BALB/c mice. The percentage of IFN--producing CD4+ and CD8+ T cells increased following infection of CAST mice only after considerable virus spread, and the absolute cell numbers remained low. Administration of exogenous IFN- or -aalpha; to CAST mice before or during the first days of infection suppressed virus replication and prolonged survival, allowing the mice to make adaptive CD4+ and CD8+ T cell responses that were necessary to clear the virus after cessation of interferon treatment. Thus, insufficient innate cytokine and cellular immune responses contribute to the unique susceptibility of CAST mice to VACV, whereas the adaptive immune response can be protective only if virus replication is suppressed during the first several days of infection.

    IMPORTANCE Most inbred mouse strains are relatively resistant to orthopoxviruses. The castaneous (CAST) mouse is a notable exception, exhibiting extreme vulnerability to monkeypox virus, cowpox virus, and vaccinia virus and thus providing a unique model for studying pathogenicity, immunity, vaccines, and antiviral drugs. To fully utilize the CAST mouse for such purposes, it is necessary to understand the basis for virus susceptibility. We showed that naive CAST mice make low IFN- and TNF-aalpha; responses and have low levels of NK cells and CD4+ and CD8+ T cells compared to a resistant classical inbred mouse strain. Attenuating virus replication with one or more doses of exogenous IFN-aalpha; or - before or during the first few days of infection enabled the development of adaptive cellular immunity and clearance of virus. Further genetic studies may reveal the basis for the low innate immunity.

  • NKp46 Recognizes the Sigma1 Protein of Reovirus: Implications for Reovirus-Based Cancer Therapy [Pathogenesis and Immunity]

  • The recent approval of oncolytic virus for therapy of melanoma patients has increased the need for precise evaluation of the mechanisms by which oncolytic viruses affect tumor growth. Here we show that the human NK cell-activating receptor NKp46 and the orthologous mouse protein NCR1 recognize the reovirus sigma1 protein in a sialic-acid-dependent manner. We identify sites of NKp46/NCR1 binding to sigma1 and show that sigma1 binding by NKp46/NCR1 leads to NK cell activation in vitro. Finally, we demonstrate that NCR1 activation is essential for reovirus-based therapy in vivo. Collectively, we have identified sigma1 as a novel ligand for NKp46/NCR1 and demonstrated that NKp46/NCR1 is needed both for clearance of reovirus infection and for reovirus-based tumor therapy.

    IMPORTANCE Reovirus infects much of the population during childhood, causing mild disease, and hence is considered to be efficiently controlled by the immune system. Reovirus also specifically infects tumor cells, leading to tumor death, and is currently being tested in human clinical trials for cancer therapy. The mechanisms by which our immune system controls reovirus infection and tumor killing are not well understood. We report here that natural killer (NK) cells recognize a viral protein named sigma1 through the NK cell-activating receptor NKp46. Using several mouse tumor models, we demonstrate the importance of NK cells in protection from reovirus infection and in reovirus killing of tumors in vivo. Collectively, we identify a new ligand for the NKp46 receptor and provide evidence for the importance of NKp46 in the control of reovirus infections and in reovirus-based cancer therapy.

  • Structural and Functional Basis for an EBNA1 Hexameric Ring in Epstein-Barr Virus Episome Maintenance [Genome Replication and Regulation of Viral Gene Expression]

  • Epstein-Barr virus (EBV) establishes a stable latent infection that can persist for the life of the host. EBNA1 is required for the replication, maintenance, and segregation of the latent episome, but the structural features of EBNA1 that confer each of these functions are not completely understood. Here, we have solved the X-ray crystal structure of an EBNA1 DNA-binding domain (DBD) and discovered a novel hexameric ring oligomeric form. The oligomeric interface pivoted around residue T585 as a joint that links and stabilizes higher-order EBNA1 complexes. Substitution mutations around the interface destabilized higher-order complex formation and altered the cooperative DNA-binding properties of EBNA1. Mutations had both positive and negative effects on EBNA1-dependent DNA replication and episome maintenance with OriP. We found that one naturally occurring polymorphism in the oligomer interface (T585P) had greater cooperative DNA binding in vitro, minor defects in DNA replication, and pronounced defects in episome maintenance. The T585P mutant was compromised for binding to OriP in vivo as well as for assembling the origin recognition complex subunit 2 (ORC2) and trimethylated histone 3 lysine 4 (H3K4me3) at OriP. The T585P mutant was also compromised for forming stable subnuclear foci in living cells. These findings reveal a novel oligomeric structure of EBNA1 with an interface subject to naturally occurring polymorphisms that modulate EBNA1 functional properties. We propose that EBNA1 dimers can assemble into higher-order oligomeric structures important for diverse functions of EBNA1.

    IMPORTANCE Epstein-Barr virus is a human gammaherpesvirus that is causally associated with various cancers. Carcinogenic properties are linked to the ability of the virus to persist in the latent form for the lifetime of the host. EBNA1 is a sequence-specific DNA-binding protein that is consistently expressed in EBV tumors and is the only viral protein required to maintain the viral episome during latency. The structural and biochemical mechanisms by which EBNA1 allows the long-term persistence of the EBV genome are currently unclear. Here, we have solved the crystal structure of an EBNA1 hexameric ring and characterized key residues in the interface required for higher-order complex formation and long-term plasmid maintenance.

  • Synthetic Toll-Like Receptor 4 (TLR4) and TLR7 Ligands Work Additively via MyD88 To Induce Protective Antiviral Immunity in Mice [Vaccines and Antiviral Agents]

  • We previously demonstrated that the combination of synthetic small-molecule 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 murine challenge models. Combining the TLR4 and TLR7 ligands balances Th1 and Th2-type immune responses for long-lived cellular and neutralizing humoral immunity against the viral hemagglutinin. Here, we demonstrate that the protective response induced in mice by this combined adjuvant is dependent upon TLR4 and TLR7 signaling via myeloid differentiation primary response gene 88 (MyD88), indicating that the adjuvants function in vivo via their known receptors, with negligible off-target effects, to induce protective immunity. The combined adjuvant acts via MyD88 in both bone marrow-derived and non-bone marrow-derived radioresistant cells to induce hemagglutinin-specific antibodies and protect mice against influenza virus challenge. The protective efficacy generated by immunization with this adjuvant and recombinant hemagglutinin antigen is transferable with serum from immunized mice to recipient mice in a homologous, but not a heterologous, H1N1 viral challenge model. Depletion of CD4+ cells after an established humoral response in immunized mice does not impair protection from a homologous challenge; however, it does significantly impair recovery from a heterologous challenge virus, highlighting an important role for vaccine-induced CD4+ cells in cross-protective vaccine efficacy. The combination of the two TLR agonists allows for significant dose reductions of each component to achieve a level of protection equivalent to that afforded by either single agent at its full dose.

    IMPORTANCE Development of novel adjuvants is needed to enhance immunogenicity to provide better protection from seasonal influenza virus infection and improve pandemic preparedness. We show here that several dose combinations of synthetic TLR4 and TLR7 ligands are potent adjuvants for recombinant influenza virus hemagglutinin antigen induction of humoral and cellular immunity against viral challenges. The components of the combined adjuvant work additively to enable both antigen and adjuvant dose sparing while retaining efficacy. Understanding an adjuvant's mechanism of action is a critical component for preclinical safety evaluation, and we demonstrate here that a combined TLR4 and TLR7 adjuvant signals via the appropriate receptors and the MyD88 adaptor protein. This novel adjuvant combination contributes to a more broadly protective vaccine while demonstrating an attractive safety profile.

  • B Cell-Specific Expression of Ataxia-Telangiectasia Mutated Protein Kinase Promotes Chronic Gammaherpesvirus Infection [Pathogenesis and Immunity]

  • Manipulation of host cellular pathways is a strategy employed by gammaherpesviruses, including mouse gammaherpesvirus 68 (MHV68), in order to negotiate a chronic infection. Ataxia-telangiectasia mutated (ATM) plays a unique yet incompletely understood role in gammaherpesvirus infection, as it has both proviral and antiviral effects. Chronic gammaherpesvirus infection is poorly controlled in a host with global ATM insufficiency, whether the host is a mouse or a human. In contrast, ATM facilitates replication, reactivation, and latency establishment of several gammaherpesviruses in vitro, suggesting that ATM is proviral in the context of infected cell cultures. The proviral role of ATM is also evident in vivo, as myeloid-specific ATM expression facilitates MHV68 reactivation during the establishment of viral latency. In order to better understand the complex relationship between host ATM and gammaherpesvirus infection, we depleted ATM specifically in B cells, a cell type critical for chronic gammaherpesvirus infection. B cell-specific ATM deficiency attenuated the establishment of viral latency due to compromised differentiation of ATM-deficient B cells. Further, we found that during long-term infection, peritoneal B-1b, but not related B-1a, B cells display the highest frequency of gammaherpesvirus infection. While ATM expression did not affect gammaherpesvirus tropism for B-1 B cells, B cell-specific ATM expression was necessary to support viral reactivation from peritoneal cells during long-term infection. Thus, our study reveals a role of ATM as a host factor that promotes chronic gammaherpesvirus infection of B cells.

    IMPORTANCE Gammaherpesviruses infect a majority of the human population and are associated with cancer, including B cell lymphomas. ATM is a unique host kinase that has both proviral and antiviral roles in the context of gammaherpesvirus infection. Further, there is insufficient understanding of the interplay of these roles in vivo during chronic infection. In this study, we show that ATM expression by splenic B cells is required for efficient establishment of gammaherpesvirus latency. We also show that ATM expression by peritoneal B cells is required to facilitate viral reactivation during long-term infection. Thus, our study defines a proviral role of B cell-specific ATM expression during chronic gammaherpesvirus infection.

  • Rab5 and Rab11 Are Required for Clathrin-Dependent Endocytosis of Japanese Encephalitis Virus in BHK-21 Cells [Virus-Cell Interactions]

  • During infection Japanese encephalitis virus (JEV) generally enters host cells via receptor-mediated clathrin-dependent endocytosis. The trafficking of JEV within endosomes is controlled by Rab GTPases, but which Rab proteins are involved in JEV entry into BHK-21 cells is unknown. In this study, entry and postinternalization of JEV were analyzed using biochemical inhibitors, RNA interference, and dominant negative (DN) mutants. Our data demonstrate that JEV entry into BHK-21 cells depends on clathrin, dynamin, and cholesterol but not on caveolae or macropinocytosis. The effect on JEV infection of dominant negative (DN) mutants of four Rab proteins that regulate endosomal trafficking was examined. Expression of DN Rab5 and DN Rab11, but not DN Rab7 and DN Rab9, significantly inhibited JEV replication. These results were further tested by silencing Rab5 or Rab11 expression before viral infection. Confocal microscopy showed that virus particles colocalized with Rab5 or Rab11 within 15 min after virus entry, suggesting that after internalization JEV moves to early and recycling endosomes before the release of the viral genome. Our findings demonstrate the roles of Rab5 and Rab11 on JEV infection of BHK-21 cells through the endocytic pathway, providing new insights into the life cycle of flaviviruses.

    IMPORTANCE Although Japanese encephalitis virus (JEV) utilizes different endocytic pathways depending on the cell type being infected, the detailed mechanism of its entry into BHK-21 cells is unknown. Understanding the process of JEV endocytosis and postinternalization will advance our knowledge of JEV infection and pathogenesis as well as provide potential novel drug targets for antiviral intervention. With this objective, we used systematic approaches to dissect this process. The results show that entry of JEV into BHK-21 cells requires a low-pH environment and that the process occurs through dynamin-, actin-, and cholesterol-dependent clathrin-mediated endocytosis that requires Rab5 and Rab11. Our work provides a detailed picture of the entry of JEV into BHK-21 cells and the cellular events that follow.

  • UV Radiation Activates Toll-Like Receptor 9 Expression in Primary Human Keratinocytes, an Event Inhibited by Human Papillomavirus 38 E6 and E7 Oncoproteins [Virus-Cell Interactions]

  • Several lines of evidence indicate that cutaneous human papillomavirus (HPV) types belonging to the beta genus of the HPV phylogenetic tree synergize with UV radiation in the development of skin cancer. Accordingly, the E6 and E7 oncoproteins from some beta HPV types are able to deregulate pathways related to immune response and cellular transformation. Toll-like receptor 9 (TLR9), in addition to playing a role in innate immunity, has been shown to be involved in the cellular stress response. Using primary human keratinocytes as experimental models, we have shown that UV irradiation (and other cellular stresses) activates TLR9 expression. This event is closely linked to p53 activation. Silencing the expression of p53 or deleting its encoding gene affected the activation of TLR9 expression after UV irradiation. Using various strategies, we have also shown that the transcription factors p53 and c-Jun are recruited onto a specific region of the TLR9 promoter after UV irradiation. Importantly, the E6 and E7 oncoproteins from beta HPV38, by inducing the accumulation of the p53 antagonist Np73aalpha;, prevent the UV-mediated recruitment of these transcription factors onto the TLR9 promoter, with subsequent impairment of TLR9 gene expression. This study provides new insight into the mechanism that mediates TLR9 upregulation in response to cellular stresses. In addition, we show that HPV38 E6 and E7 are able to interfere with this mechanism, providing another explanation for the possible cooperation of beta HPV types with UV radiation in skin carcinogenesis.

    IMPORTANCE Beta HPV types have been suggested to act as cofactors in UV-induced skin carcinogenesis by altering several cellular mechanisms activated by UV radiation. We show that the expression of TLR9, a sensor of damage-associated molecular patterns produced during cellular stress, is activated by UV radiation in primary human keratinocytes (PHKs). Two transcription factors known to be activated by UV radiation, p53 and c-Jun, play key roles in UV-activated TLR9 expression. The E6 and E7 oncoproteins from beta HPV38 strongly inhibit UV-activated TLR9 expression by preventing the recruitment of p53 and c-Jun to the TLR9 promoter. Our findings provide additional support for the role that beta HPV types play in skin carcinogenesis by preventing activation of specific pathways upon exposure of PHKs to UV radiation.

  • Classic Spotlight, 2004 and 2005: Articles of Significant Interest Selected from the Journal of Virology Archives by the Editors [Editorial]

  • Intracellular Colocalization of Influenza Viral RNA and Rab11A Is Dependent upon Microtubule Filaments [Virus-Cell Interactions]

  • Influenza A virus (IAV) consists of eight viral RNA (vRNA) segments that are replicated in the host cell nucleus and transported to the plasma membrane for packaging into progeny virions. We have previously proposed a model where subcomplexes of vRNA are exported from the nucleus and assembled en route to the plasma membrane. However, the role of host cytoskeletal proteins in the cytoplasmic assembly of IAV vRNA segments remains unknown. Previous studies have suggested that IAV vRNA segments are transported via Rab11A-containing recycling endosomes (RE) and use both microtubules (MT) and actin. Rab11A RE transport primarily along MT; therefore, investigation of the role of MT in vRNA assembly is warranted. We explored the role of MT in vRNA assembly and replication by using multiple IAV strains in various cell types, including primary human airway epithelial cells. We observed that Rab11A localization was altered in the presence of MT-depolymerizing drugs, but growth of IAV in all of the cell types tested was unchanged. Fluorescent in situ hybridization was performed to determine the role of MT in the assembly of multiple vRNA segments. Unexpectedly, we found that vRNA-vRNA association in cytoplasmic foci was independent of MT. Given the disparity of localization between Rab11A and vRNA segments in the absence of intact MT filaments, we analyzed the three-dimensional spatial relationship between Rab11A and vRNA in the cytoplasm of infected cells. We found that Rab11A and vRNA colocalization is dependent upon dynamic MT filaments. Taken together, our data suggest that cytoplasmic transport of influenza vRNA may include a Rab11A RE-independent mechanism.

    IMPORTANCE IAV infections cause a large public health burden through seasonal epidemics and sporadic pandemics. Pandemic IAVs emerge through reassortment of vRNA in animal or human hosts. Elucidation of the mechanism of intracellular dynamics of IAV assembly is necessary to understand reassortment. Our results describing the role of MT in vRNA transport and assembly expand upon previous studies characterizing vRNA assembly. This study is the first to assess the role of MT in influenza virus replication in human bronchial airway epithelial cells. In addition, we present novel data on the role of MT in facilitating the association between distinct vRNA segments. Interestingly, our results suggest that progressive assembly of vRNA segments may be cell type dependent and that vRNA may be transported through the cytoplasm without Rab11A RE in the absence of intact MT. These results enhance our understanding of vRNA assembly and the role of cytoskeletal proteins in that process.

  • Human Immunodeficiency Virus and Simian Immunodeficiency Virus Maintain High Levels of Infectivity in the Complete Absence of Mucin-Type O-Glycosylation [Structure and Assembly]

  • A highly conserved threonine near the C terminus of gp120 of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) was investigated for its contributions to envelope protein function and virion infectivity. When this highly conserved Thr residue was substituted with anything other than serine (the other amino acid that can accept O-glycosylation), the resulting virus was noninfectious. We found that this Thr was critical for the association of gp120 with the virion and that amino acid substitution increased the amount of dissociated gp120 in the cell culture supernatant. When HIV virions were generated in cells overexpressing polypeptide N-acetylgalactosaminyltransferase 1 (GalNAcT1), viral infectivity was increased 2.5-fold compared to that of virus produced in wild-type HEK293T cells; infectivity was increased 8-fold when the Thr499Ser mutant was used. These infectivity enhancements were not observed when GalNAcT3 was used. Using HEK293T knockout cell lines totally devoid of the ability to perform O-linked glycosylation, we demonstrated production of normal levels of virions and normal levels of infectivity in the complete absence of O-linked carbohydrate. Our data indicate that O-glycosylation is not necessary for the natural replication cycle of HIV and SIV. Nonetheless, it remains theoretically possible that the repertoire of GalNAc transferase isoforms in natural target cells for HIV and SIV in vivo could result in O-glycosylation of the threonine residue in question and that this could boost the infectivity of virions beyond the levels seen in the absence of such O-glycosylation.

    IMPORTANCE Approximately 50% of the mass of the gp120 envelope glycoprotein of both HIV and SIV is N-linked carbohydrate. One of the contributions of this N-linked carbohydrate is to shield conserved peptide sequences from recognition by humoral immunity. This N-linked glycosylation is one of the reasons that primary isolates of HIV and SIV are so heavily resistant to antibody-mediated neutralization. Much less studied is any potential contribution from O-linked glycosylation. The literature on this topic to date is somewhat confusing and ambiguous. Our studies described in this report demonstrate unambiguously that O-linked glycosylation is not necessary for the natural replication cycle of HIV and SIV. However, the door is not totally closed because of the diversity of numerous GalNAc transferase enzymes that initiate O-linked carbohydrate attachment and the theoretical possibility that natural target cells for HIV and SIV in vivo could potentially complete such O-linked carbohydrate attachment to further increase infectivity.

  • Articles of Significant Interest Selected from This Issue by the Editors [Spotlight]

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    JVI Accepts: Articles Published Ahead of Print

  • Vaccination with Recombinant Parainfluenza Virus 5 Expressing Neuraminidase Protects against Homologous and Heterologous Influenza Virus Challenge [PublishAheadOfPrint]

  • Seasonal human influenza virus continues to cause morbidity and mortality annually, and highly pathogenic avian influenza (HPAI) along with other emerging influenzas continue to pose pandemic threats. Vaccination is considered the most effective measure for controlling influenza; however, current strategies rely on a precise vaccine match with currently circulating virus strains for efficacy, requiring constant surveillance and regular development of matched vaccines. Current vaccines focus on eliciting specific antibody responses against the hemagglutinin (HA) surface glycoprotein; however, the diversity of HAs across species and antigenic drift of circulating strains enables evasion of virus-inhibiting antibody responses, resulting in vaccine failure. The neuraminidase (NA) surface glycoprotein, while diverse, has a conserved enzymatic site and presents an appealing target for priming broadly effective antibody responses. Here we show that vaccination with parainfluenza virus 5 (PIV5), a promising live viral vector expressing the NA from avian (H5N1) or pandemic (H1N1) influenza virus elicited NA-specific antibody and T cell responses, which conferred protection against a homologous and heterologous influenza virus challenge. Vaccination with the PIV5-N1 NA provided cross-protection against challenge with a heterosubtypic (H3N2) virus. Experiments using antibody transfer indicate that antibodies to NA have an important role in the protection. The findings indicate that PIV5 expressing NA may be effective as a broadly protective vaccine against seasonal influenza and emerging pandemic threats.

    IMPORTANCE Seasonal influenza viruses cause considerable morbidity and mortality annually while emerging viruses pose potential pandemic threats. Currently licensed influenza vaccines rely on the antigenic match of the hemagglutinin (HA) for vaccine strain selection and most vaccines rely on HA inhibition titer to determine efficacy, despite growing awareness of the contribution of neuraminidase (NA) as contributing to influenza vaccine efficacy. Although NA is immunologically subdominant to the HA and clinical studies have shown variable NA responses to vaccination, in this study we show that vaccination with parainfluenza virus 5 recombinant vaccine candidate expressing NA (PIV5-NA) from a pandemic influenza (pdmH1N1) virus or highly pathogenic avian influenza (H5N1) virus elicits robust, cross-reactive protection from influenza virus infection in two animal models. New vaccination strategies incorporating NA, including PIV5-NA could improve seasonal influenza vaccine efficacy and provide protection against emerging influenza viruses.

  • Conformational Changes in the 5' End of the HIV-1 Genome Dependent on the Debranching Enzyme DBR1 During Early Stages of Infection [PublishAheadOfPrint]

  • Previous studies in our laboratory showed that the RNA debranching enzyme (DBR1) is not required for early steps in HIV cDNA formation, but is necessary for synthesis of intermediate and late cDNA products. To further characterize this effect, we evaluated the topology of the 5' end of the HIV-1 RNA genome during early infection with and without inhibition of DBR1 synthesis. Cells were transfected with DBR1 shRNA followed 48 hours later by infection with an HIV-1 derived vector containing an RNase H deficient reverse transcriptase. RNA was isolated at several times post-infection and treated with various RNA modifying enzymes prior to rapid amplification of 5' ends (5' RACE) for HIV-1 RNA and quantitative RT-PCR. In infected cells, DBR1 knockdown inhibited detection of free HIV-1 RNA 5' ends at all time points. The difference in detection of free HIV-1 RNA 5' ends in infected DBR1 knockdown vs. control cells was eliminated by in vitro incubation of infected cell RNAs with yeast or human DBR1 enzyme prior to 5' RACE and qRT-PCR. This was dependent on the 2' -5' phosphatase activity of DBR1, since it did not occur when we used the catalytically inactive DBR1 N85A mutant. Finally, HIV-1 RNA from infected DBR1 knockdown cells was resistant to RNase R that degrades linear RNAs but not RNAs in circular or lariat-like conformations. These results provide evidence for formation of a lariat-like structure involving the 5' end of HIV-1 RNA during an early step in infection, and the involvement of DBR1 in resolving it.

    IMPORTANCE Our findings support a new view of the early steps in HIV genome replication. We show that the HIV genomic RNA is rapidly de-capped and forms a lariat-like structure after entering a cell. The lariat-like structure is subsequently resolved by the cellular enzyme, DBR1, leaving a 5' phosphate. This pathway is similar to the formation and resolution of pre-mRNA intron lariats and therefore suggests that similar mechanisms may be used by HIV. Our work therefore opens a new area of investigation in HIV replication and may ultimately uncover new targets for inhibiting HIV replication and for preventing the development of AIDS.

  • Vertical transmission of hepatitis C virus: variable transmission bottleneck and evidence of mid-gestation in utero infection [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) can be transmitted from mother to child during pregnancy and childbirth. However, the timing and precise biologic mechanisms that are involved in this process are incompletely understood, as are the determinants that influence transmission of particular HCV variants. Here we report results of a longitudinal assessment of HCV quasispecies diversity and composition in 5 cases of vertical HCV transmission, including 3 women coinfected with HIV-1. The population structure of HCV variant spectra based on E2 envelope gene sequences (nucleotide positions 1491-1787), including hypervariable regions 1 and 2, was characterized using next-generation sequencing and median joining network analysis. Compatible with a loose transmission bottleneck, larger numbers of shared HCV variants were observed in presence of maternal coinfection. Coalescent Bayesian Markov chain Monte Carlo simulations revealed median times of transmission between 24.9 weeks and 36.1 weeks of gestation, with some confidence intervals ranging into the 1st trimester, hence considerably earlier than previously thought. Using recombinant autologous HCV pseudoparticles, differences were uncovered in HCV-specific antibody responses between coinfected mothers and mothers infected with HCV alone, in whom generalized absence of neutralization was observed. Finally, shifts in HCV quasispecies composition were seen in children around 1 year of age, compatible with the disappearance of passively transferred maternal immunoglobulins and/or the development of HCV-specific humoral immunity. Taken together, these results provide insights into the timing, dynamics, and biologic mechanisms involved in vertical HCV transmission and inform preventative strategies.

    IMPORTANCE Although it is well established that hepatitis C virus (HCV) can be transmitted from mother to child, the manner and the moment at which transmission operates have been the subject of conjecture. By carrying out a detailed examination of viral sequences, we showed that transmission could take place comparatively early in pregnancy. In addition, we showed that when the mother also carried human immunodeficiency virus type 1 (HIV-1), many more HCV variants were shared between her and child, suggesting that the mechanism and/or the route of transmission of HCV differed in presence of coinfection with HIV-1. These results could explain why cesarean section is ineffective in preventing vertical HCV transmission and guide the development of interventions to avert pediatric HCV infection.

  • Protective Humoral Immunity in the CNS Requires Peripheral CD19-Dependent Germinal Center Formation Following Coronavirus Encephalomyelitis [PublishAheadOfPrint]

  • B cell subsets with phenotypes characteristic of naïve, non-isotype-switched, memory (Bmem), and antibody-secreting cells (ASC) accumulate in various models of central nervous system (CNS) inflammation, including viral encephalomyelitis. During neurotropic coronavirus JHMV infection infiltration of protective ASC occurs after T cell mediated viral control, and is preceded by accumulation of non-isotype switched IgD+ and IgM+ B cells. However, the contribution of peripheral activation events in cervical lymph nodes (CLN) in driving humoral immune responses in the infected CNS is poorly defined. CD19, a signaling component of the B cell receptor complex, is one of multiple regulators driving B cell differentiation and germinal center (GC) formation by lowering the threshold of antigen-driven activation. JHMV infected CD19-/- mice were thus used to determine how CD19 affects CNS recruitment of B cell subsets. Early polyclonal ASC expansion, GC formation, and virus-specific ASC were all significantly impaired in CLN of CD19-/- mice compared to wild type (wt) mice, consistent with lower and unsustained virus-specific serum Ab. ASC were also significantly reduced in the CNS resulting in increased infectious virus during persistence. Nevertheless, CD19 deficiency did not affect early CNS IgD+ B cell accumulation. The results support that CD19 independent factors drive early B cell mobilization and recruitment to the infected CNS, while delayed accumulation of virus-specific, isotype switched ASC requires CD19 dependent GC formation in CLN. CD19 is thus essential for both sustained serum Ab as well as protective local Ab within the CNS following JHMV encephalomyelitis.

    IMPORTANCE CD19 activation is known to promote GC formation and sustain serum Ab responses following antigen immunization and viral infections. However, the contribution of CD19 in the context of CNS infections has not been evaluated. This study demonstrates that antiviral protective ASC in the CNS are dependent on CD19 activation and peripheral GC formation, while accumulation of early-recruited IgD+ B cells is CD19-independent. This indicates that IgD+ B cells commonly found early in the CNS do not give rise to local ASC differentiation and that only antigen-primed, peripheral GC-derived ASC infiltrate the CNS, thereby limiting potentially harmful non-specific Ab secretion. Expanding our understanding of activation signals driving CNS migration of distinct B cell subsets during neuroinflammatory insults is critical for preventing and managing acute encephalitic infections, as well as preempting reactivation of persistent viruses during immune suppressive therapies targeting B cells in multiple sclerosis (MS), such as Rituximab and Ocrelizumab.

  • Zika Virus Encoding Non-Glycosylated Envelope Protein is Attenuated and Defective in Neuroinvasion [PublishAheadOfPrint]

  • Zika virus (ZIKV), a mosquito-transmitted flavivirus, responsible for sporadic outbreaks of mild and febrile illness in Africa and Asia, re-emerged in the last decade causing serious human diseases including microcephaly, congenital malformations, and Guillain-Barreeacute; syndrome. Although genomic and phylogenetic analyses suggest that genetic evolution may have led to enhanced virulence of ZIKV, experimental evidence supporting the role of specific genetic changes in virulence is currently outstanding. One sequence motif, VNDT, containing an N-linked glycosylation site in the envelope (E) protein, is polymorphic, being absent in many of the African isolates while present in all isolates from the recent outbreaks. In the present study, we interrogated the role of this sequence motif and glycosylation of the E protein in pathogenicity of ZIKV. We first constructed a stable full-length cDNA clone of ZIKV in a novel linear vector from which infectious virus was recovered. The recombinant ZIKV generated from the infectious clone, which contains the VNDT motif, is highly pathogenic and causes lethality in a mouse model. In contrast, recombinant viruses from which the VNDT motif is deleted or from which N-linked glycosylation site is mutated by single amino acid substitution, are highly attenuated and non-lethal. The mutant viruses replicate poorly in the brain of infected mice when inoculated subcutaneously but replicate well following intracranial inoculation. Our findings provide the first evidence that N-linked glycosylation of the E protein is an important determinant of ZIKV virulence and neuroinvasion.

    IMPORTANCE Recent emergence of Zika virus (ZIKV) in the Americas has caused major worldwide public health concern. The virus appears to have gained significant pathogenicity, causing serious human diseases including microcephaly and Guillain-Barreeacute; syndrome. The factors responsible for the emergence of pathogenic ZIKV are not understood at this time, although genetic changes have been shown to facilitate virus transmission. All isolates from the recent outbreaks contain an N-linked glycosylation site within the viral envelope (E) protein whereas many isolates of the African lineage virus lack this site. To elucidate the functional significance of glycosylation in ZIKV pathogenicity, recombinant ZIKVs from infectious clones with or without the glycan on the E protein were generated. ZIKVs lacking the glycan were highly attenuated for their ability to cause mortality in mouse model and were severely compromised for neuroinvasion. Our studies suggest glycosylation of the E protein as an important factor contributing to ZIKV pathogenicity.

  • A DNA vaccine that targets hemagglutinin to antigen presenting cells protects mice against H7 influenza [PublishAheadOfPrint]

  • Zoonotic influenza H7 viral infections have a case fatality rate of about 40%. Currently no or limited human to human spread has occurred, but we may be facing a severe pandemic threat if the virus acquires the ability to transmit between humans. Novel vaccines that can be rapidly produced for global distribution are urgently needed, and DNA vaccines may be the only type of vaccine that allows for the speed necessary to quench an emerging pandemic. Here, we have constructed DNA vaccines encoding the hemagglutinin (HA) from influenza A/chicken/Italy/13474/99 (H7N1). In order to increase the efficacy of DNA vaccination, HA was targeted to either major histocompatibility complex (MHC) class II molecules or chemokine receptors 1, 3 and 5 (CCR1/3/5) that are expressed on antigen presenting cells (APC). A single DNA vaccination with APC-targeted HA significantly increased antibody levels in sera, as compared to non-targeted control vaccines. The antibodies were confirmed neutralizing in an H7 pseudotype-based neutralization assay. Furthermore, the APC-targeted vaccines increased the levels of antigen-specific cytotoxic T-cells, and a single DNA vaccination could confer protection against a lethal challenge with influenza A/turkey/Italy/3889/1999 (H7N1) in mice. In conclusion, we have developed a vaccine that rapidly could contribute protection against a pandemic threat from avian influenza.

    IMPORTANCE: Highly pathogenic avian influenza H7 constitute a pandemic threat that can cause severe illness and death in infected individuals. Vaccination is the main method of prophylaxis against influenza, but current vaccine strategies fall short in a pandemic situation due to a prolonged production time and insufficient production capabilities. In contrast, a DNA vaccine can be rapidly produced and deployed to prevent the potential escalation of a highly pathogenic influenza pandemic. We here demonstrate that a single DNA delivery of hemagglutinin from an H7 influenza could mediate full protection against a lethal challenge with H7N1 influenza in mice. Vaccine efficacy was contingent on targeting of the secreted vaccine protein to antigen presenting cells.

  • p21 restricts HIV-1 in monocyte-derived dendritic cells through the reduction of dNTP biosynthesis and regulation of SAMHD1 antiviral activity [PublishAheadOfPrint]

  • HIV-1 infection of non-cycling cells, such as dendritic cells (DCs), is impaired due to limited availability of dNTPs, which are needed for HIV-1 reverse transcription. The levels of dNTPs are tightly regulated during cell cycle and depend on the balance between dNTPs biosynthesis and degradation. SAMHD1 potently blocks HIV-1 replication in DCs, although the underlying mechanism is still unclear. SAMHD1 has been reported to be able to degrade dNTPs and viral nucleic acids, which may both hamper HIV-1 reverse transcription. The relative contribution of these activities may differ in cycling and non-cycling cells. Here we show that inhibition of HIV-1 replication in monocyte-derived DCs (MDDCs) is associated to an increased expression of p21cip1/waf, a cell cycle regulator that is involved in the differentiation and maturation of DCs. Induction of p21 in MDDCs decreases the pool of dNTPs and increases the antiviral active isoform of SAMHD1. Although both processes are complementary in inhibiting HIV-1 replication, the antiviral activity of SAMHD1 in our primary cell model appears to be, at least partially, independent of its dNTPase activity. The reduction in the pool of dNTPs in MDDCs appears rather mostly due to a p21-mediated suppression of several enzymes involved in dNTP synthesis (i.e., RNR2, TYMS, and TK-1). These results are important to better understand the interplay between HIV-1 and DCs and may inform the design of new therapeutic approaches to decrease viral dissemination and improve immune responses against HIV-1.

    IMPORTANCE Dendritic cells (DCs) play a key role in the induction of immune responses against HIV. However, HIV has evolved ways to exploit these cells, facilitating immune evasion and virus dissemination. We have found that the expression of p21, a cyclin-dependent kinase inhibitor involved in cell cycle regulation and monocyte differentiation and maturation, can potentially contribute to the inhibition of HIV-1 replication in monocyte-derived DCs through multiple mechanisms. p21 decreased the size of the intracellular dNTP pool. In parallel, p21 prevented SAMHD1 phosphorylation and promoted SAMHD1 dNTPase-independent antiviral activity. Induction of p21 thus resulted in conditions that allowed the effective inhibition of HIV-1 replication through complementary mechanisms. Overall, p21 appears to be a key regulator of HIV infection in myeloid cells.

  • Susceptibility of Human Endogenous Retrovirus Type-K to Reverse Transcriptase Inhibitors [PublishAheadOfPrint]

  • Human Endogenous Retroviruses make up 8% of the human genome. The HERV-K HML-2 (HK2) family contains proviruses that are the most recent entrants into the human germline and are transcriptionally active. In HIV-1 infection and cancer, HK2 genes produce retroviral particles that appear to be infectious, yet the replication capacity of these viruses and potential pathogenicity has been difficult to ascertain. In this report, we screened the efficacy of commercially available reverse transcriptase inhibitors (RTIs) at inhibiting the enzymatic activity of the HK2 RT and HK2 genomic replication. Interestingly, only one provirus, K103, was found to encode a functional RT among those examined. Several nucleoside analogue RTIs (NRTIs) blocked K103 RT activity and consistently inhibited the replication of HK2 genomes. The NRTIs zidovudine (AZT), stavudine (d4T), didanosine (ddI) and lamivudine (3TC), and the nucleotide RTI inhibitor tenofovir (TDF), show efficacy in blocking K103 RT. HIV-1 specific non-nucleoside RTIs (NNRTIs), protease inhibitors (PIs), and integrase inhibitors (IIs) did not affect HK2, except for the NNRTI etravirine (ETV). The inhibition of HK2 infectivity by NRTIs appears to take place at either the reverse transcription step of the viral genome prior to HK2 viral particle formation and/or in the infected cells. Inhibition of HK2 by these drugs will be useful in suppressing HK2 infectivity if these viruses prove to be pathogenic in cancer, neurological disorders, or other diseases associated with HK2. The present studies also elucidate a key aspect of the life cycle of HK2, specifically addressing how they do, and/or did, replicate.

    IMPORTANCE Endogenous retroviruses are relics of ancestral virus infections in the human genome. The most recent of these infections was caused by HK2. While HK2 often remains silent in the genome, this group of viruses is activated in HIV-1-infected and cancer cells. Recent evidence suggests that these viruses might be infectious, and the potential exists for HK2 to contribute to disease. We show that HK2, and specifically the enzyme that mediates virus replication, can be inhibited by a panel of drugs that are commercially available. We show that several drugs block HK2 with different efficacies. The inhibition of HK2 replication by antiretroviral drugs appears to occur in the virus itself as well as after infection of cells. Therefore, these drugs might prove to be an effective treatment by suppressing HK2 infectivity in diseases where these viruses have been implicated, such as cancer and neurological syndromes.

  • Natural reassortants between potentially zoonotic avian influenza viruses H5N1 and H9N2 from Egypt display distinct pathogenic phenotypes in experimentally infected chickens and ferrets [PublishAheadOfPrint]

  • Co-circulation of zoonotic highly pathogenic avian influenza virus (HPAIV) H5N1 and AIV of subtype H9N2 among poultry in Egypt for at least six years should render this country a hypothetical hotspot for the emergence of reassortant, phenotypically altered viruses, yet no reassortants have been detected in Egypt. The present investigations proved that reassortants between Egyptian H5N1 clade 2.2.1.2 and H9N2 of the G1-B lineage can be generated by co-amplification in embryonated chicken eggs. Reassortants were restricted to the H5N1 subtype and had acquired between two and all six of the internal segments of the H9N2 virus. Five selected plaque-purified reassortant clones expressed a broad phenotypic spectrum both in vitro and in vivo. Two groups of reassortants were characterized to have retarded growth characteristics in vitro compared to the parental H5N1 virus. One clone provoked reduced mortality in inoculated chickens, although characteristics of an HP phenotype were retained. Enhanced zoonotic properties were not predicted for any of these clones, which was confirmed by ferret inoculation experiments: Neither the parental H5N1 virus nor two selected clones induced severe clinical symptoms or were contact-transmitted to sentinel ferrets. While the emergence of reassortants of Egyptian HPAI H5N1 viruses with internal gene segments of co-circulating H9N2 viruses is possible in principle, spread of such viruses is expected to be governed by their fitness to outcompete the parental viruses in the field. Eventual spread of attenuated phenotypes, however, would impact syndrome surveillance in poultry farms negatively and might foster enzootic virus circulation.

    IMPORTANCE Despite almost six years of continuous co-circulation of highly pathogenic avian influenza virus H5N1 and avian influenza virus H9N2 in poultry in Egypt, no reassortants between the two subtypes were reported. Here, the principal compatibility of the two subtypes is shown by forcing reassortment of co-passaged H5N1 und H9N2 in embryonated chicken eggs. Resulting reassortant viruses displayed a wide range of pathogenicity including attenuated phenotypes in chickens, but did not show enhanced zoonotic propensities in the ferret model.

  • Comparative transcriptomics highlights the role of the AP1 transcription factor in the host response to Ebolavirus [PublishAheadOfPrint]

  • Ebolavirus and Marburgvirus comprise two genera of negative-sense single-stranded RNA viruses that cause severe hemorrhagic fevers in humans. Despite considerable research efforts, the molecular events following Ebola virus (EBOV) infection are poorly understood. With the view of identifying host factors that underpin EBOV pathogenesis, we compared the transcriptomes of EBOV-infected human, pig and bat kidney cells using an RNAseq approach. Despite a significant difference in viral transcription/replication between the cell lines, all cells responded to EBOV infection through a robust induction of extracellular growth factors. Furthermore, a significant up-regulation of AP1 transcription factor complex members FOS and JUN was observed in permissive cell lines. Functional studies focusing on human cells showed that EBOV infection induces protein expression, phosphorylation and nuclear accumulation of JUN, and to a lesser degree, FOS. Using a luciferase-based reporter, we showed that EBOV infection induces AP1 transactivation activity at 48 and 72 hours post infection within human cells. Finally, we show that JUN knockdown decreases the expression of EBOV-induced host gene expression. Taken together, our study highlights the role of AP1 in promoting the host gene expression profile that defines EBOV pathogenesis.

    Non-technical summary Many questions remain about the molecular events that underpin filovirus pathophysiology. The rational design of new intervention strategies, such as post-exposure therapeutics, will be significantly enhanced through an in-depth understanding of these molecular events. We believe new insights into the molecular pathogenesis of EBOV may be possible by examining the transcriptomic response of taxonomically diverse cell lines (derived from human, pig and bat). We first identified the responsive pathways using an RNAseq-based transcriptomics approach. Further functional and computational analysis mmdash; focusing on human cells mmdash; highlighted an important role for the AP1 transcription factor in mediating the transcriptional response to EBOV infection. Our study sheds new light on how host transcription factors respond and promote the transcriptional landscape that follows viral infection.

  • IKK{gamma} mimetic peptides block the resistance to apoptosis associated with KSHV infection. [PublishAheadOfPrint]

  • Primary effusion lymphoma (PEL) is a lymphogenic disorder associated with KSHV infection. Key to the survival and proliferation of PEL is the canonical NF-kB pathway that becomes constitutively activated following overexpression of the viral oncoprotein ks-vFLIP. This arises from its capacity to form a complex with the modulatory subunit of the IKK kinase, IKK (or NEMO) resulting in the overproduction of proteins that promote cellular survival and prevent apoptosis; both of which are important drivers of tumourigenesis. Using a combination of cell based and biophysical assays together with structural techniques, we show that the observed resistance to cell death is largely independent of autophagy or major death receptor signalling pathways and demonstrate that direct targeting of the ks-vFLIP-IKK interaction both in cells and in vitro can be achieved using IKK mimetic peptides. Our results further reveal that these peptides not only induce cell killing, but potently sensitise PEL to the pro-apoptotic agents tumour necrosis factor alpha and etoposide and are the first to confirm ks-vFLIP as a tractable target for the treatment of PEL and related disorders.

    IMPORTANCE KSHV vFLIP (ks-vFLIP) has been shown to have a crucial role in cellular transformation where it is vital for the survival and proliferation of primary effusion lymphoma (PEL), an aggressive malignancy associated with infection that is resistant to the majority of chemotherapeutic drugs. It operates via subversion of the canonical NF-B pathway that requires a physical interaction between ks-vFLIP and the IKK kinase modulatory subunit IKK. Whilst this interaction has been directly linked to protection against apoptosis, it is unclear whether the suppression of other cell death pathways implicated in ks-vFLIP pathogenesis are additional contributors. We demonstrate that the interaction between ks-vFLIP and IKK is pivotal in conferring resistance to apoptosis. Additionally, we show that the ks-vFLIP-IKK complex can be disrupted using peptides leading to direct killing and the sensitisation of PEL cells to pro-apoptotic agents. Our studies thus provide a framework for future therapeutic interventions.

  • Insights into the impact of CD8+ immune modulation on HIV evolutionary dynamics in distinct anatomical compartments using SIV-infected macaque models of AIDS progression [PublishAheadOfPrint]

  • A thorough understanding of the role of HIV intra-host evolution in AIDS pathogenesis has been limited by the need for longitudinally sampled viral sequences from the vast target space within the host, which are often difficult to obtain from human subjects. CD8+ lymphocyte-depleted macaques infected with simian immunodeficiency virus (SIV) provide an increasingly utilized model of pathogenesis due to similar clinical manifestations as HIV-1 infection and AIDS progression, as well as characteristic rapid disease onset. Comparison of this model with SIV-infected non-CD8+ lymphocyte-depleted macaques also provides a unique opportunity to investigate the role of CD8+ cells in viral evolution and population dynamics throughout the duration of infection. Using several different phylogenetic methods, we analyzed viral gp120 sequences obtained from extensive longitudinal sampling of multiple tissues and enriched leukocyte populations from SIVmac251-infected macaques, with or without CD8+ lymphocyte depletion. SIV evolutionary and selection patterns in non-CD8+ lymphocyte-depleted animals were characterized by sequential population turnover and continual viral adaptation, a scenario readily comparable to intra-host evolutionary patterns during human HIV infection in the absence of antiretroviral therapy. Alternatively, animals that were CD8+ lymphocyte depleted exhibited greater variation in population dynamics among tissues and cell populations over the course of infection. Our findings highlight the major role for CD8+ lymphocytes in prolonging disease progression through continual control of SIV sub-populations from varying anatomical compartments and the potential for greater independent viral evolutionary behavior among these compartments in response to immune modulation.

    IMPORTANCE Although developments in combined antiretroviral therapy (cART) strategies have successfully prolonged AIDS onset in HIV-1-infected individuals, a functional cure has yet to be found. Improvement of drug interventions for a virus that is able to infect a wide range of tissues and cell types requires a thorough understanding of viral adaptation and infection dynamics within this target milieu. Although difficult to accomplish in the human host, longitudinal sampling of multiple anatomical locations is readily accessible in the SIV-infected macaque models of neuroAIDS. The significance of our research is in identifying the impact of immune modulation, through differing immune selective pressure, on viral evolutionary behavior in a multitude of anatomical compartments. The results provide evidence encouraging the development of a more sophisticated model that considers a network of individual viral subpopulations within the host with differing infection and transmission dynamics, which is necessary for more effective treatment strategies.

  • Structurally guided removal of deISGylase biochemical activity from papain-Like protease originating from the Middle East Respiratory Syndrome Virus [PublishAheadOfPrint]

  • Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging human pathogen that is the causative agent for Middle East respiratory syndrome (MERS). With MERS outbreaks resulting in over 35% fatalities and now spread to 27 countries, MERS-CoV poses a significant on-going threat to global human health. As part of its viral genome, MERS-CoV encodes for a papain-like protease (PLpro) that has been observed to act as a deubiquitinase and deISGylase to antagonize IFN-I immune pathways. This activity is in addition to its viral polypeptide cleavage function. Although the overall impact of MERS-CoV PLpro function is observed to be essential, difficulty has been encountered in delineating the importance of its separate functions, particularly its deISGylase activity. As a result, the interface of MERS-CoV and human interferon stimulated gene product 15 (hISG15) was probed with isothermal calorimetry suggesting the C-terminal domain of hISG15 to be principally responsible for interactions. Subsequently, the structure of MERS-CoV PLpro was solved to 2.4 AAring; in complex with the C-terminal domain of hISG15. Utilizing this structural information, mutants were generated that lacked appreciable deISGylase activity but retained wild-type deubiquitinase and peptide cleavage activities. Hence, this provides a new platform for understanding viral deISGylase activity within MERS-CoV and other CoVs.

    IMPORTANCE Coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV), encode a papain-like protease (PLpro) that possesses the ability to antagonize interferon immune pathways through the removal of ubiquitin and interferon-stimulated gene product 15 (ISG15) from target proteins. The lack of CoV proteases with attenuated deISGylase activity has been a key obstacle in delineating the impact between deubiquitinase and deISGylase activities on viral host evasion and pathogenesis. Here, biophysical techniques revealed that MERS-CoV PLpro chiefly engages human ISG15 occurs through its C-terminal domain. The first structure of MERS-CoV PLpro in complex with this domain exposed the interface between these two entities. Employing these structural insights, mutations were employed to selectively remove deISGylase activity with no appreciable impact to its other deubiquitinase and peptide cleavage biochemical properties. Excitingly, this study introduces a new tool to probe pathogenesis of MERS-CoV and related viruses through the removal of viral deISGylase activity.

  • A selective bottleneck shapes the evolutionary mutant spectra of enterovirus A71 during viral dissemination in humans [PublishAheadOfPrint]

  • RNA viruses accumulate mutations to rapidly adapt to environmental changes. Enterovirus A71 (EV-A71) causes various clinical manifestations with occasional severe neurological complications. However, the mechanism by which EV-A71 evolves within the human body is unclear. Utilizing deep sequencing and haplotype analyses of viruses from various tissues of an autopsy patient, we sought to define the evolutionary pathway by which enterovirus A71 evolves fitness for invading the central nervous system in humans. Broad mutant spectra with divergent mutations were observed at the initial infection sites in the respiratory and digestive systems. After viral invasion, we identified a haplotype switch and dominant haplotype, with glycine at VP1 residue 31 in viral particles disseminated into the integumentary and central nervous systems. In vitro viral-growth and fitness analyses indicated that VP1-31G conferred growth and a fitness advantage in human neuronal cells, whereas VP1-31D showed enhanced replication in human colorectal cells. A higher proportion of VP1-31G was also found among fatal cases, suggesting that it may facilitate central nervous system infection in humans. Our data provide the first glimpse of EV-A71 quasispecies from oral tissues to central nervous system within humans, showing broad implications for the surveillance and pathogenesis of this re-emerging viral pathogen.

    IMPORTANCE EV-A71 continues to be a worldwide burden to public health. Although EV-A71 is the major etiological agent of hand-foot-and-mouth disease, it can also cause neurological pulmonary edema, encephalitis, and even death, especially in children. Understanding selection processes enabling dissemination and accurately estimating EV-A71 diversity during invasion in humans are critical for applications in viral pathogenesis and vaccine studies. Here, we define a selection bottleneck appearing in respiratory and digestive tissues. Glycine substitution at VP1 residue 31 helps viruses break through the bottleneck and invade the central nervous system. This substitution is also advantageous for replication in neuronal cells in vitro. Considering that fatal cases contain enhanced glycine substitution at VP1-31, we suggest that the increased prevalence of VP1-31G may alter viral tropism and aid central nervous system invasion. Our findings provide new insights into a dynamic mutant spectral switch active during acute viral infection with emerging viral pathogens.

  • Pathogenic Correlates of the Simian Immunodeficiency Virus (SIV)-Associated B Cell Dysfunction [PublishAheadOfPrint]

  • We compared and contrasted pathogenic (pigtailed macaques-PTMs) and nonpathogenic (African green monkeys-AGMs) SIVsab infections to assess the significance of the B-cell dysfunction observed in SIV/HIV infection. We report that the loss of B cells is specifically associated with the pathogenic SIV infection, while in the nonpathogenic natural hosts B cells rapidly increase in both LNs and intestine. SIV-associated B-cell dysfunction associated to the pathogenic SIV infection is characterized by loss of naïve B cells; loss of resting memory B cells due to their redistribution to the gut; increases of the activated B cells and circulating tissue-like memory B cells and expansion of the B regulatory cells. While circulating B cells are virtually restored to preinfection levels during the chronic pathogenic SIV infection, restoration is mainly due to an expansion of the "exhausted", virus-specific B cells, i.e., activated memory cells and tissue-like memory B cells. Despite of the B-cell dysfunction, SIV-specific Ab production was higher in the PTMs than in AGMs, with the caveat that rapid disease progression in PTMs was strongly associated with lack of anti-SIV Ab. Neutralization titers, the avidity and maturation of immune responses did not differ between pathogenic and nonpathogenic infections, with the exception of the conformational epitope recognition, which evolved from low to high conformations in the nonpathogenic host. The patterns of humoral immune responses in the natural host are therefore more similar to those observed in HIV-infected subjects, suggesting that natural hosts may be more appropriate for modeling the immunization strategies aimed at preventing HIV disease progression. The numerous differences between the pathogenic and nonpathogenic infections with regard to dynamics of the memory B-cell subsets point to their role in the pathogenesis of HIV/SIV infections, and suggest that monitoring B cells may be a reliable approach for assessing disease progression.

    IMPORTANCE We report here that the HIV/SIV-associated B cell dysfunction (defined by loss of total and memory B cells, increased Breg counts and B cell activation and apoptosis) is specifically associated to pathogenic SIV infection and absent during the course of nonpathogenic SIV infection in natural NHP hosts. Alterations of the B cell population are not correlated with production of neutralizing antibodies, which is similar in both species. Rapid progressive infections associate a severe impairment in SIV-specific antibody production. While we did not find major differences in avidity and maturation between the pathogenic and nonpathogenic SIV infection, we identified a major difference in conformational epitope recognition, with the nonpathogenic infection being characterized by an evolution from low to high conformations. B cell dysfunction should be considered in designing immunization strategies aimed at preventing HIV disease progression.

  • Host noncoding retrotransposons induced by DNA viruses: a SINE of infection? [PublishAheadOfPrint]

  • Our genomes are dominated by repetitive elements. The majority of these elements derive from retrotransposons, which expand throughout the genome through a process of reverse transcription and integration. Short interspersed nuclear elements, or SINEs, are an abundant class of retrotransposons that are transcribed by RNA polymerase III, thus generating exclusively noncoding RNA (ncRNA) that must hijack the machinery required for their transposition. SINE loci are generally transcriptionally repressed in somatic cells, but can be robustly induced upon infection with multiple DNA viruses. Recent research has focused on the gene expression and signaling events that are modulated by SINE ncRNAs, particularly during gammaherpesvirus infection. Here, we review the biology of these SINE ncRNAs, explore how DNA virus infection may lead to their induction, and describe how novel gene regulatory and immune-related functions of these ncRNAs may impact the viral lifecycle.

  • Molecular evolution of herpes simplex virus 2 complete genomes: Comparison between primary and recurrent infections [PublishAheadOfPrint]

  • Herpes simplex virus (HSV)-1 and -2 are large, double-stranded DNA viruses that cause lifelong persistent infections characterized by periods of quiescence and recurrent disease. How HSV evolves within an infected individual experiencing multiple episodes of reactivation over time is not known. We determined genome sequences of viruses isolated from two subjects in the Herpevac Trial for Women who experienced primary HSV-2 genital disease and compared them with sequences of viruses isolated from the subsequent fifth or sixth episode of recurrent disease in the same individuals. Each of the HSV-2 genome sequences was initially obtained using next-generation sequencing and completed with Sanger sequencing. Polymorphisms over the entire genomes were mapped and amino acid variants resulting from non-synonymous changes were analyzed based on the secondary and tertiary structure of a previously crystallized protein. A phylogenetic reconstruction was used to assess relationships among the four HSV-2 samples, other North American sequences, and reference sequences. Little genetic drift was detected in viruses shed by the same subjects following repeated reactivation events, suggesting strong selective pressure on the viral genome to maintain sequence fidelity during reactivations from its latent state within an individual host. Our results also demonstrate that some primary HSV-2 isolates from North America more closely resemble the HG52 laboratory strain from Scotland than the low-passage clinical isolate SD90e from South Africa or laboratory strain 333. Thus, one of the sequences reported herein would make a logical choice as a reference strain for inclusion in future studies of North American HSV-2 isolates.

    IMPORTANCE The extent to which the HSV-2 genome evolves during multiple episodes of reactivation from its latent state within an infected individual is not known. We used next generation sequencing techniques to determine whole genome sequences of four viral samples from two subjects in the Herpevac Trial. The sequence of each subject's well-documented primary infection was compared with the sequence of the isolate from their fifth or sixth episode of recurrent disease. Only nineteen genetic polymorphisms unique to the primary or recurrent isolate were identified, 10 in Subject A and 9 in Subject B. These observations indicate remarkable genetic conservation between primary and recurrent episodes of HSV-2 infection, and imply strong selection pressures exist to maintain fidelity of the viral genome during repeated reactivations from its latent state. The genome conservation observed also has implications for the potential success of a therapeutic vaccine.

  • Caenorhabditis elegans as an Emerging Model for Virus-Host Interactions [PublishAheadOfPrint]

  • Since 1999, Caenorhabditis elegans has been extensively used to study microbe-host interactions due to its simple culture, genetic tractability, and susceptibility to numerous bacterial and fungal pathogens. In contrast, virus studies have been hampered by a lack of convenient virus infection models in nematodes. The recent discovery of a natural viral pathogen of C. elegans and development of diverse artificial infection models are providing new opportunities to explore virus-host interplay in this powerful model organism.

  • Exploring the Human-Nipah virus protein-protein interactions [PublishAheadOfPrint]

  • Nipah virus is an emerging, highly pathogenic, zoonotic virus of the paramyxoviridiae family. Human transmission occurs by close contact with infected animals, the consumption of contaminated food, or, occasionally, via other infected individuals. Currently, we lack therapeutic or prophylactic treatments for Nipah virus. To develop these agents we must now improve our understanding of the host-virus interactions that underpin a productive infection. This aim led us to perform the present work, in which we identified (101) human-Nipah virus protein-protein interactions (PPIs), most of which (88) are novel. This dataset provides a comprehensive view of the host complexes that are manipulated by viral proteins. Host targets include the PRP19 complex and the miRNA processing machinery. Furthermore, we explored the biologic consequences of the interaction with the PRP19 complex and found that the Nipah virus W protein is capable of altering p53 control and gene expression. We anticipate that these data will help in guiding the development of novel interventional strategies to counter this emerging viral threat.

    IMPORTANCE Nipah virus is recently discovered virus that infects a wide range of mammals, including humans. Since its discovery there have been yearly outbreaks and in some of them the mortality rate has reach 100% of the confirmed cases. However, the study of Nipah virus has been largely neglected and currently we lack treatments for this infection. To develop these agents we must now improve our understanding of the host-virus interactions that underpin a productive infection. In the present work we identified 101 human-Nipah virus protein-protein interactions using an affinity purification approach coupled with mass spectrometry. Additionally, we explored the cellular consequences of some of these interactions. Globally, this dataset offers a comprehensive and detailed view of the host machinery's contribution to the Nipah virus's life cycle. Furthermore, our data present a large number of putative drug targets that could be exploited for the treatment of this infection.

  • Vaccinia virus encodes a novel inhibitor of apoptosis that associates with the apoptosome. [PublishAheadOfPrint]

  • Apoptosis is an important anti-viral host defense mechanism. Here we report the identification of a novel apoptosis inhibitor encoded by the vaccinia virus (VACV) M1L gene. M1L is absent in the attenuated MVA strain of VACV, a strain that stimulates apoptosis in several types of immune cells. M1 expression increased the viability of MVA-infected THP-1 and Jurkat cells and reduced several biochemical hallmarks of apoptosis such as PARP-1 and procaspase-3 cleavage. Furthermore, ectopic M1L expression decreased staurosporine-induced (intrinsic) apoptosis in HeLa cells. We then identified the molecular basis for M1 inhibitory function. M1 allowed mitochondrial depolarization but blocked procaspase-9 processing, suggesting that M1 targeted the apoptosome. In support of this model, we found that M1 promoted survival in yeast over-expressing human Apaf-1 and procaspase-9, critical components of the apoptosome, or only over-expressing conformationally active caspase-9. In mammalian cells, M1 co-immunoprecipitated with Apaf-1-procaspase-9 complexes. The current model is that M1 associates with and allows the formation of the apoptosome, but prevents apoptotic functions of the apoptosome. The M1 protein features 14 predicted ankyrin (ANK) repeat domains, and M1 is the first ANK-containing protein reported to use this inhibitory strategy. Since ANK-containing proteins are encoded by many large DNA viruses and found in all domains of life, studies of M1 may lead to a better understanding of the roles of ANK proteins in virus-host interactions.

    IMPORTANCE Apoptosis selectively eliminates dangerous cells such as virus-infected cells. Poxviruses express apoptosis antagonists to neutralize this anti-viral host defense. The vaccinia virus (VACV) M1 ankyrin (ANK) protein, a protein with no previously ascribed function, inhibits apoptosis. M1 interacts with the apoptosome and prevents procaspase-9 processing as well as downstream procaspase-3 cleavage in several cell types and under multiple conditions. M1 is the first poxviral protein reported to associate with and prevent the function of the apoptosome, giving a more detailed picture of the threats VACV encounters during infection. Dysregulation of apoptosis is associated with several human diseases. One potential treatment of apoptosis-related diseases is through the use of designed ANK repeat proteins (DARPins), similar to M1, as caspase inhibitors. Thus, the study of the novel anti-apoptosis effects of M1 via apoptosome association will be helpful for understanding how to control apoptosis using either natural or synthetic molecules.

  • Differential Inhibitory Receptor Expression on T Cells Delineates Functional Capacities in Chronic Viral Infection [PublishAheadOfPrint]

  • Inhibitory receptors have been extensively described for their importance in regulating immune responses in chronic infections and cancers. Blocking the function of inhibitory receptors such as PD-1, CTLA-4, 2B4, Tim-3, and LAG-3 have shown promise for augmenting CD8 T cell activity and boosting pathogen-specific immunity. However, the prevalence of inhibitory receptors on CD4 T cells and their relative influence on CD4 T cell functionality in chronic HIV infection remains poorly described. We therefore determined and compared inhibitory receptor expression patterns of 2B4, CTLA-4, LAG-3, PD-1, and Tim-3 on virus-specific CD4 and CD8 T cells in relation to their functional T cell profile. In chronic HIV infection, inhibitory receptor distribution differed markedly between cytokine-producing T cell subsets with IFN-- and TNF-aalpha;-producing cells displaying the highest and lowest prevalence of inhibitory receptors, respectively. Blockade of inhibitory receptors differentially impacted cytokine production by cells in response to SEB stimulation. CTLA-4 blockade increased IFN- and CD40L production, while PD-1 blockade strongly augmented IFN-, IL-2, and TNF-aalpha; production. In a Friend retrovirus infection model, CTLA-4 blockade in particular was able to improve control of viral replication. Together these results show that inhibitory receptor distribution on HIV-specific CD4 T cells varies markedly with respect to the functional subset of CD4 T cell being analyzed. Furthermore, the differential effects of receptor blockade suggest novel methods of immune response modulation, which could be important in the context of HIV vaccination or therapeutic strategies.

    IMPORTANCE Inhibitory receptors are important to limit damage by the immune system during acute infections. In chronic infections however, their expression limits immune system responsiveness. Studies have shown that blocking inhibitory receptors augments CD8 T cell functionality in HIV infection, but their influence on CD4 T cells remains unclear. We assessed the expression of inhibitory receptors on HIV-specific CD4 T cells and their relationship with T cell functionality. We uncovered differences in inhibitory receptor expression depending on the CD4 T cell function. We also found differences in functionality of CD4 T cells following blocking different inhibitory receptors, and confirmed our results in a Friend Virus retroviral model infection in mice. Our results show that inhibitory receptor expression on CD4 T cells is linked to CD4 T cell functionality, and could be sculpted by blockade of specific inhibitory receptors. These data reveal exciting possibilities for the development of novel treatments and immunotherapeutics.

  • Calcitonin gene-related peptide induces HIV-1 proteasomal degradation in mucosal Langerhans cells [PublishAheadOfPrint]

  • The neuro-immune dialogue between peripheral neurons and Langerhans cells (LCs) within mucosal epithelia protects against incoming pathogens. LCs rapidly internalize human immunodeficiency virus type 1 (HIV-1) upon its sexual transmission and then trans-infect CD4+ T-cells. We recently found that the neuropeptide calcitonin gene-related peptide (CGRP), secreted mucosally from peripheral neurons, inhibits LCs-mediated HIV-1 trans-infection. Herein, we investigated the mechanism of CGRP-induced inhibition, focusing on HIV-1 degradation in LCs and its interplay with trans-infection. We first show that HIV-1 degradation occurs in endo-lysosomes in untreated LCs, and functionally blocking such degradation with lysosomotropic agents results in increased trans-infection. We demonstrate that CGRP acts via its cognate receptor and at a viral post-entry step, to induce faster HIV-1 degradation, but without affecting the kinetics of endo-lysosomal degradation. Unexpectedly, we reveal that CGRP shifts HIV-1 degradation from endo-lysosomes towards the proteasome, providing the first evidence for functional HIV-1 proteasomal degradation in LCs. Such efficient proteasomal degradation significantly inhibits the first phase of trans-infection, and proteasomal, but not endo-lysosomal, inhibitors abrogate CGRP-induced inhibition. Together, our results establish that CGRP controls HIV-1 degradation mode in LCs. The presence of endogenous CGRP within innervated mucosal tissues, especially during the sexual response to which CGRP contributes, suggests that HIV-1 proteasomal degradation predominates in-vivo. Hence, proteasomal, rather than endo-lysosomal, HIV-1 degradation in LCs should be enhanced clinically to effectively restrict HIV-1 trans-infection.

    IMPORTANCE During sexual transmission, HIV-1 is internalized and degraded in LCs, the resident antigen-presenting cells in mucosal epithelia. Yet, during trans-infection, infectious virions escaping degradation are transferred to CD4+ T-cells, the principal HIV-1 targets. We previously found that the neuro-immune dialogue between LCs and peripheral neurons, innervating mucosal epithelia, significantly inhibits trans-infection via the action of the secreted neuropeptide CGRP on LCs. Herein, we investigated whether CGRP-induced inhibition of trans-infection is linked to CGRP-controlled HIV-1 degradation in LCs. We show that in untreated LCs, HIV-1 is functionally degraded in endo-lysosomes. In sharp contrast, we reveal that in CGRP-treated LCs, HIV-1 is diverted towards and degraded via another cytosolic protein degradative pathway, namely the proteasome. These results establish that CGRP regulates HIV-1 degradation in LCs. As CGRP contributes to the sexual response and present within mucosal epithelia, HIV-1 proteasomal degradation in LCs might predominate in-vivo and should be enhanced clinically.

  • Three conserved regions in baculovirus sulfhydryl oxidase, P33, are critical for enzymatic activity and function [PublishAheadOfPrint]

  • Baculoviruses encode a conserved sulfhydryl oxidase P33, which is necessary for budded virus (BV) production and multi-nucleocapsid occlusion-derived virus (ODV) formation. Here, the structural and functional relationship of P33 was revealed by X-ray crystallography, site-directed mutagenesis, and functional analysis. Based on crystallographic characterization and structural analysis, a series of P33 mutants within three conserved regions, i.e. the active site, dimer interface, and the R127-E183 salt bridge, were constructed. In vitro experiments showed that mutations within the active site and the dimer interface severely impaired the sulfhydryl oxidase activity of P33, while the mutations in the salt bridge had a relatively minor influence. Recombinant viruses containing mutated P33 were constructed and assayed in vivo. Except for the active site mutant AXXA, all other mutants produced infectious BVs, although certain mutants had a decreased BV production. The active site mutant H114A, the dimer interface mutant H227D, and the salt bridge mutant R127A-E183A were further analyzed by electron microscopy and bioassays. The occlusion bodies (OBs) of mutants H114A and R127A-E183A had a ragged surface and contained mostly ODVs with single nucleocapsid. The OBs of all three mutants contained lower numbers of ODVs and had a significantly reduced oral infectivity in comparison to control virus. Crystallographic analyses further revealed that all three regions may coordinate with one another to achieve optimal function of P33. Taken together, our data revealed that all the three conserved regions are involved in P33 activity and are crucial for virus morphogenesis and per oral infectivity.

    IMPORTANCE Sulfhydryl oxidase catalyzes disulfide bond formation of substrate proteins. P33, a baculovirus-encoded sulfhydryl oxidase, is different from other cellular and viral sulfhydryl oxidases, bearing unique features in tertiary and quaternary structure organizations. In this study, we found that three conserved regions, i.e. the active site, dimer interface, and the R127-E183 salt bridge, play important roles in the enzymatic activity and function of P33. Previous observations showed that deletion of p33 results in a total loss of budded virus (BV) production and in morphological changes in occlusion derived virus (ODV). Our study revealed that certain P33 mutants lead to occlusion bodies (OBs) with a ragged surface, decreased embedded ODVs and reduced oral infectivity. Interestingly, some P33 mutants with impaired ODV/OB still retained BV productivity, indicating the impact on BV and on ODV/OB are two distinctly different functions of P33, which are likely to be performed via different substrate proteins.

  • The Role of Herpes Simplex Virus Type 1 {gamma}34.5 in the Regulation of IRF3 Signaling. [PublishAheadOfPrint]

  • During viral infection, pattern recognition receptors (PRRs) and their associated adaptors recruit TANK-binding kinase 1 (TBK1) to activate interferon regulatory factor 3 (IRF3), resulting in production of type I interferons (IFNs). ICP0 and ICP34.5 are amongst the proteins encoded by herpes simplex virus 1 (HSV-1) that modulate type I IFN signaling. We constructed a recombinant virus (XX) which lacks amino acids 87 - 106, a portion of the previously described TBK1-binding domain of the 34.5 gene (D. Verpooten, Y. Ma, S. Hou, Z. Yan, and B. He, J Biol Chem 284 (2):1097-105, 2009, doi:10.1074/JBC.M805905200). These 20 residues are outside of the 34.5 beclin1-binding domain (BBD) that interacts with Beclin1 and regulates autophagy. Unexpectedly, XX showed no deficit in replication in vivo in a variety of tissues and showed comparable virulence to wild-type and marker-rescued viruses following intracerebral infection. XX was fully capable of mediating the dephosphorylation of eIF2aalpha;, and this virus was capable of controlling the phosphorylation of IRF3. In contrast, a null mutant in 34.5 failed to control IRF3 phosphorylation due to an inability of this mutant to sustain expression of ICP0. Our data show that while 34.5 regulates IRF3 phosphorylation, the TBK1-binding domain itself has no impact on IRF3 phosphorylation, or on replication and pathogenesis in mice.

    IMPORTANCE Interferons (IFNs) are potent activators of a variety of host responses that serve to control virus infections. The Herpesviridae have evolved countermeasures to IFN responses. Herpes simplex virus 1 (HSV-1) encodes the multifunctional neurovirulence protein, ICP34.5. In this study, we investigated the biological relevance of the interaction between ICP34.5 and TANK-binding kinase 1 (TBK1), an activator of IFN responses. Here, we establish that although ICP34.5 binds TBK1 under certain conditions through a TBK1-binding domain (TBD), there was no direct impact of the TBD on viral replication or virulence in mice. Furthermore, we showed that activation of IRF3, a substrate of TBK1, was independent of the TBD. Instead, we provided evidence that the ability of ICP34.5 to control IRF3 activation is through its ability to reverse translational shutoff, and sustain the expression of other IFN inhibitors encoded by the virus. This work provides new insights into the immunomodulatory functions of ICP34.5.

  • The dose of retroviral infection determines the induction of anti-viral NK cell responses [PublishAheadOfPrint]

  • Natural killer (NK) cells are part of the innate immune system and recognize virus-infected cells as well as tumor cells. Conflicting data about the beneficial or even detrimental role of NK cells in different infectious diseases were described. While the type of pathogen strongly influences NK cell functionality, less is known about how the infection dose influences the quality of a NK cell response against retroviruses. Here we used the well-established Friend retrovirus (FV) mouse model to investigate the impact of virus dose on the induction of anti-viral NK cell functions. High-dose virus inoculation increased initial virus replication compared to medium- or low-dose viral challenge and significantly improved NK cell activation. Anti-viral NK cell activity, including in vivo cytotoxicity towards infected target cells, was also enhanced by high-dose virus infection. NK cell activation following high-dose viral challenge was likely mediated by activated dendritic cells (DCs) and macrophages and the NK cell-stimulating cytokines, IL-15 and IL-18. Neutralization of these cytokines decreased NK cell functions and increased viral loads whereas IL-15 and IL-18 therapy improved the NK cell activity. Here we demonstrate that virus dose positively correlates with anti-viral NK cell activity and function, which is at least partly driven by IL-15 and IL-18. Our results suggest that NK cell activity may be therapeutically enhanced by administering IL-15 and IL-18 in virus infections that inadequately activate NK cells.

    IMPORTANCE In infections with retroviruses, like HIV and FV infection of mice, NK cells clearly mediate anti-viral activities, but they are usually not sufficient to prevent severe pathology. Here, we show that the initial infection dose impacts the induction of an anti-viral NK cell response during an acute retroviral infection, which was not investigated so far. High-dose infection resulted in a strong NK cell functionality, whereas no anti-viral activities were detected after low- or medium-dose infection. Interestingly, DCs and macrophages were highly activated after high-dose FV challenge, which corresponded with increased levels of NK cell-stimulating cytokines, IL-15 and IL-18. IL-15 and IL-18 neutralization decreased NK cell functions whereas IL-15 and IL-18 therapy improved the NK cell activity. Here, we show the importance of cytokines for NK cell activation in retroviral infections and may suggest that immunotherapy combining the well-tolerable cytokines IL-15 and IL-18 might be an interesting approach for anti-retroviral treatment.

  • Vector order determines protection against pathogenic simian immunodeficiency virus infection in a triple component vaccine by balancing CD4+ and CD8+ T-cell responses [PublishAheadOfPrint]

  • An effective AIDS vaccine should elicit strong humoral and cellular immune responses while maintaining low levels of CD4+ T cell activation to avoid the generation of target cells for viral infection. The present study investigated two prime-boost regimens, both starting vaccination with single cycle immunodeficiency virus, followed by two mucosal boosts either with recombinant adenovirus (rAd) or fowlpox virus (rFWPV) expressing SIVmac239 or SIVmac251 gag/pol and env genes, respectively. Finally, vectors were switched and systemically administered to the reciprocal group of animals. Only mucosal rFWPV immunizations followed by systemic rAd boost significantly protected animals against a repeated low-dose, intrarectal challenge with pathogenic SIVmac251 resulting in a vaccine efficacy (i.e. risk reduction per exposure) of 68%. Delayed viral acquisition was associated with higher levels of activated CD8+ T cells and Gag-specific IFN- secreting CD8+ cells, low virus-specific CD4+ T cell responses and low Env antibody titers. In contrast, the systemic rFWPV boost induced strong virus-specific CD4+ T cell activity. rAd and rFWPV also induced differential patterns of the innate immune responses, thereby possibly shaping the specific immunity. Plasma CXCL10 levels after final immunization correlated directly with virus-specific CD4+ T cell responses and inversely with the number of exposures to infection. Also, the percentage of activated CD69+ CD8+ T cells correlated with the number of exposures to infection. Differential stimulation of the immune response likely provided the basis for the diverging levels of protection afforded by this vaccine regimen.

    IMPORTANCE A failed phase II AIDS vaccine trial led to the hypothesis that CD4+ T cell activation can abrogate any potentially protective effects delivered by vaccination or promote acquisition of the virus because CD4+ T helper cells, required for an effective immune response, also represent the target cells for viral infection. We compared two vaccination protocols that elicited similar levels of Gag-specific immune responses in rhesus macaques. Only the animal group that had a low level of virus-specific CD4+ T cells in combination with high levels of activated CD8+ T cells was significantly protected from infection. Notably, protection was achieved despite the lack of appreciable Env-antibody titers. Moreover, we show that both, the vector and the route of immunization affected the level of CD4+ T cell responses. Thus, mucosal immunization with FWPV-based vaccines should be considered as potent prime in prime-boost vaccination protocols.

  • Mutations in the fusion protein of measles virus that confer resistance to the membrane fusion inhibitors carbobenzoxy-D-Phe-L-Phe-Gly and AS-48 [PublishAheadOfPrint]

  • The inhibitors Z-d-Phe-l-Phe Gly (fusion inhibitor peptide, FIP) and AS-48 have similar efficacy in blocking membrane fusion and syncytia formation mediated by measles virus. Other homologues such as Z-d-Phe are less effective, but may act through the same mechanism. In an attempt to map the site of action of these inhibitors, we generated mutant viruses which were resistant to the inhibitory effects of Z-d-Phe-l-Phe Gly. These 10 mutations were localized to the heptad repeat region (HRB) of the fusion protein and no changes were observed in the viral hemagglutinin, which is the receptor attachment protein. Mutations were validated in a luciferase based membrane fusion assay, using transfected fusion and hemagglutinin expression plasmids, or with syncytia based assays in Vero, Vero-SLAM, and Vero-Nectin 4 cell lines. The changes I452T, D458N, D458G/V459A, N462K, N462H, G464E, and I483R conferred resistance to both FIP and AS-48, without compromising membrane fusion. The inhibitors did not block hemagglutinin protein mediated binding to the target cell. Edmonston vaccine/laboratory and IC323 wild type strains were equally affected by the inhibitors. Escape mutations were mapped upon a 3D structure modeled from the published crystal structure of parainfluenzavirus 5 fusion protein. The most effective mutations were situated in a region located near the base of the globular head and its junction with the alpha helical stalk of the prefusion protein. We hypothesize that the fusion inhibitors could interfere with the structural changes that occur between the prefusion and postfusion conformations of the fusion protein.

    IMPORTANCE Due to lapses in vaccination worldwide that have caused localized outbreaks, measles virus (MeV) has regained importance as a pathogen. Antiviral agents against measles virus are not commercially available, but could be useful in conjunction with MeV eradication vaccine programs, and as a safeguard in oncolytic viral therapy. Three decades ago, the small hydrophobic peptide, Z-d-Phe-l-Phe Gly (FIP), was shown to block MeV infections and syncytia formation in monkey kidney cell lines. The exact mechanism of its action has yet to be determined but it does appear to have similar properties to another chemical inhibitor, AS-48, which appears to interfere with the conformational change in the viral F protein that is required to elicit membrane fusion. Escape mutations were used to map the site of action for FIP. Knowledge from these studies could help design new inhibitors against morbilliviruses and provide additional knowledge concerning the mechanism of virus-mediated membrane fusion.

  • Epstein-Barr Virus BKRF4 Gene Product Is Required for Efficient Progeny Production [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV), a member of human gammaherpesvirus, infects mainly B cells. EBV has two alternative life cycles, latent and lytic, and is reactivated occasionally from the latent stage to the lytic cycle. To combat EBV-associated disorders, understanding the molecular mechanisms of the EBV lytic replication cycle is also important. Here, we focused on an EBV lytic gene, BKRF4. Using our anti-BKRF4 antibody, we revealed that the BKRF4 gene product is expressed during the lytic cycle with late kinetics. To characterize the role of BKRF4, we constructed BKRF4-knockout mutants using the bacterial artificial chromosome (BAC) and CRISPR/Cas9 systems. While disruption of the BKRF4 gene had almost no effect on viral protein expression and DNA synthesis, it significantly decreased progeny virion levels in HEK293 and Akata cells. Furthermore, we show that BKRF4 is involved not only in production of progeny virions but also in increasing the infectivity of the virus particles. Immunoprecipitation assays revealed that BKRF4 interacted with a virion protein, BGLF2. We showed that the C-terminal region of BKRF4 was critical for this interaction and for efficient progeny production. Immunofluorescence analysis revealed that BKRF4 partially colocalized with BGLF2 in the nucleus and perinuclear region. Finally, we showed that BKRF4 is a phosphorylated, possible tegument protein and that the EBV protein kinase BGLF4 may be important for this phosphorylation. Taken together, our data suggest that BKRF4 is involved in the production of infectious virions.

    IMPORTANCE While the latent genes of EBV have been studied extensively, the lytic genes are less well characterized. This study focused on one such lytic gene, BKRF4, which is conserved only among gammaherpesviruses (ORF45 of Kaposi's sarcoma-associated herpesvirus or murine herpesvirus-68). After preparing the BKRF4 knockout virus using B95-8 EBV-BAC, we demonstrated that the BKRF4 gene was involved in infectious progeny particle production. Importantly, we successfully generated a BKRF4 knockout virus of Akata using CRISPR/Cas9 technology, confirming the phenotype in this separate strain. We further showed that BKRF4 interacted with another virion protein, BGLF2, and demonstrated the importance of this interaction in infectious virion production. These results shed light on the elusive process of EBV progeny maturation in the lytic cycle. Notably, this study describes a successful example of the generation and characterization of an EBV construct with a disrupted lytic gene using CRISPR/Cas9 technology.

  • Type 1 interferon signalling to dendritic cells limits Murid Herpesvirus-4 spread from the olfactory epithelium [PublishAheadOfPrint]

  • Murid Herpesvirus-4 (MuHV-4) is a B cell-tropic gamma-herpesvirus that can be studied in vivo. Despite viral evasion, type 1 interferons (IFN-I) limit its spread. After MuHV-4 inoculation into footpads, IFN-I protect lymph node subcapsular sinus macrophages (SSM) against productive infection; after peritoneal inoculation, they protect splenic marginal zone macrophages; and they limit MuHV-4 replication in the lungs. While invasive infections can test specific aspects of host colonization, it is important also to understand natural infection. MuHV-4 taken up spontaneously by alert mice enters them via olfactory neurons. We determined how IFN-I act in this context. Blocking IFN-I signalling did not increase neuronal infection, but allowed its spread to the adjacent respiratory epithelium. In lymph nodes a complete IFN-I signalling block increased MuHV-4 lytic infection in SSM and increased the number of dendritic cells (DC) expressing viral GFP independently of lytic infection. A CD11c+ cell-directed signalling block increased only DC infection. However this was sufficient to increase down-stream infection, consistent with DC providing the main viral route to B cells. The capacity of IFN-I to limit DC infection indicated that viral IFN-I evasion was only partly effective. Therefore DC are a possible target for IFN-I-based interventions to reduce host colonization.

    IMPORTANCE Human gamma-herpesviruses infect B cells and cause B cell cancers. Interventions to block virus binding to B cells have not stopped their infection. Therefore we must identify other control points that are relevant to natural infection. Human infections are difficult to analyse. However gamma-herpesviruses colonize all mammals. A related gamma-herpesvirus of mice reaches B cells not directly but via infected dendritic cells. We show that type 1 interferons, an important general anti-viral defence, limit gamma-herpesvirus B cell infection by acting on dendritic cells. Therefore dendritic cell infection is a potential point of interferon-based therapeutic intervention.

  • Heterogeneity of the Epstein-Barr virus major internal repeat reveals evolutionary mechanisms of EBV and a functional defect in the prototype EBV strain B95-8. [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) is a ubiquitous pathogen of humans that can cause several types of lymphoma and carcinoma. Like other herpesviruses, EBV has diversified both through co-evolution with its host, and genetic exchange between virus strains. Sequence analysis of the EBV genome is unusually challenging, because of the large number and length of repeat regions within the virus. Here we describe the sequence assembly and analysis of the large internal repeat of EBV (IR1 or BamW repeats) from over 70 strains.

    Diversity of the latency protein EBNA-LP resides predominantly within the exons downstream of IR1. The integrity of the putative BWRF1 ORF is retained in over 80% of strains, and deletions truncating IR1 always spare BWRF1. Conserved regions include the IR1 latency promoter (Wp), and one zone upstream of and two within BWRF1.

    IR1 is heterogeneous in 70% of strains, and this heterogeneity arises from sequence exchange between strains as well as spontaneous mutation, with inter-strain recombination more common in tumour-derived viruses. This genetic exchange often incorporates regions of llt;1kb, and allelic gene conversion changes the frequency of small regions within the repeat, but not close to the flanks. These observations suggest that IR1 mmdash; and by extension EBV mmdash; diversifies through both recombination and breakpoint repair, while concerted evolution of IR1 is driven by gene conversion of small regions. Finally, the prototype EBV strain B95-8 contains four non-consensus variants within a single IR1 repeat unit, including a STOP codon in EBNA-LP. Repairing IR1 improves EBNA-LP levels and the quality of transformation by the B95-8 BAC.

    IMPORTANCE Epstein-Barr virus (EBV) infects the majority of the world population, but only causes illness in a small minority. Nevertheless, over 1% of cancers worldwide are attributable to EBV. Recent sequencing projects investigating virus diversity, to see if different strains have different disease impacts, have excluded regions of repeating sequence, as they are more technically challenging. Here we analyse the sequence of the largest repeat in EBV (IR1). We first characterised the variations in protein sequences encoded across IR1. In studying variations within the repeat of each strain, we identified a mutation in the main laboratory strain of EBV that impairs virus function, and suggest that tumour-associated viruses may be more likely to contain DNA mixed from two strains. Patterns of this mixing suggest that sequences can spread between strains (and also within the repeat) by copying sequence from another strain (or repeat unit) to repair DNA damage.

  • A distinct mechanism for the formation of the ribonucleoprotein complex of the Tomato spotted wilt virus [PublishAheadOfPrint]

  • The tomato spotted wilt virus (TSWV) belongs to the Tospovirus genus of the Bunyaviridae family and represents the sole plant-infecting group within bunyavirus. TSWV encodes a nucleocapsid protein (N) which encapsidates the RNA genome to form a ribonucleoprotein complex (RNP). In addition, the N has multiple roles during the infection of plant cells. Here, we report the crystal structure of the full-length TSWV N. The N features a body domain consisting of an N- and C-lobe. These lobes clamp a positively charged groove which may constitute the RNA binding site. Furthermore, the body domains are flanked by N- and C-terminal arms which mediate homotypic interactions to the neighboring subunits, resulting in a ring-shaped N trimer. Interestingly, the C-terminus of one protomer forms an additional interaction with the protomer of an adjacent trimer in the crystal, which may constitute a higher-order oligomerization contact. In this way, this study provides insights into the structure and trimeric assembly of TSWV N which help to explain previous functional findings, but also suggests distinct N interactions within a higher-order RNP.

    Importance TSWV is one of the most devastating plant pathogens that cause severe diseases in numerous agronomic and ornamental crops worldwide. TSWV is also the prototypic member of the Tospovirus genus, which is the sole group of plant-infecting viruses in bunyavirus family. This study determined the structure of full-length TSWV N in an oligomeric state. The structural observations explain previously identified biological properties of TSWV N. Most importantly, the additional homotypic interaction between the C-terminus of one protomer with another protomer indicates there is a distinct mechanism of RNP formation in the bunyavirus family, thereby enhancing current knowledge of --ssRNA virus-encoded N. TSWV N is the last remaining representative N with an unknown structure in the bunyavirus family. Combined with previous studies, the structure of TSWV N helps to build a complete picture of the bunyavirus-encoded N family and reveals a close evolutionary relationship between orthobunyavirus, phlebovirus, hantavirus and tospovirus.

  • Discrete dynamical modeling of influenza infection suggests age-dependent differences in immunity [PublishAheadOfPrint]

  • Immunosenescence, an age-related decline in immune function, is a major contributor to morbidity and mortality in the elderly. Older hosts exhibit delayed onset of immunity and prolonged inflammation after an infection, leading to excess damage and greater likelihood of death. Our study applies a rule-based model to infer which components of immune response are most changed in an aged host. Two groups of BALB/c mice (age 12-16 wks and 72-76 wks) were infected at 2 inocula: a survivable 50 PFU dose and a lethal 500 PFU dose. Data were measured at 10 points over 19 days in the sublethal case and 6 points over 7 days in the lethal case, after which all mice had died. Data varies primarily in the onset of immunity, particularly the inflammatory response, which leads to a 2-day delay in clearance of the virus from the older host in the sublethal cohort. We developed a Boolean model to describe the interactions between virus and 21 immune components including cells, chemokines, and cytokines from innate and adaptive immunity. The model identifies distinct sets of rules for each age group using Boolean operators to describe the complex series of interactions that activate and deactivate immune components. Our model accurately simulates the immune response for both ages and both inocula included in the data (95% accurate for younger mice, 94% accurate for older mice), and shows distinct rule choices for the innate immunity arm of the model between younger and aging mice in response to influenza A virus.

    Importance Influenza virus infection causes high morbidity and mortality every year, especially in the elderly. The elderly tend to have delayed onset of many immune responses, as well as prolonged inflammatory responses, leading to an overall weakened response to infection. Many of the details of immune mechanisms that change with age are currently not well understood. We present a rule-based model of the intra-host immune response to influenza viral infection. The model is fit to experimental data for young and older mice infected with influenza. We generated distinct sets of rules for each age group to capture the temporal differences seen in the immune responses of these mice. These rules describe a network of interactions leading to either clearance of the virus or death of the host, depending on the initial dosage of virus. Our models clearly demonstrate the differences in these two age groups, particularly in the innate immune responses.

  • Characterization of a replicating mammalian orthoreovirus with tetracysteine tagged {mu}NS for live cell visualization of viral factories [PublishAheadOfPrint]

  • Within infected host cells, mammalian orthoreovirus (MRV) forms viral factories (VFs) which are sites of viral transcription, translation, assembly, and replication. MRV non-structural protein, mmu;NS, comprises the structural matrix of VFs and is involved in recruiting other viral proteins to VF structures. Previous attempts have been made to visualize VF dynamics in live cells but due to current limitations in recovery of replicating reoviruses carrying large fluorescent protein tags, researchers have been unable to directly assess VF dynamics from virus-produced mmu;NS. We set out to develop a method to overcome this obstacle by utilizing the 6 amino acid (CCPGCC) tetracysteine (TC)-tag and FlAsH-EDT2 reagent. The TC-tag was introduced into eight sites throughout mmu;NS, and the capacity of the TC-mmu;NS fusion proteins to form virus factory-like (VFL) structures and colocalize with virus proteins was characterized. Insertion of the TC-tag interfered with recombinant virus rescue in six of the eight mutants, likely as a result of loss of VF formation or important virus protein interactions. However, two recombinant (r)TC-mmu;NS viruses were rescued and VF formation, colocalization with associating virus proteins, and characterization of virus replication were subsequently examined. Furthermore the rTC-mmu;NS viruses were utilized to infect cells and examine VF dynamics using live cell microscopy. These experiments demonstrate active VF movement with fusion events as well as transient interactions between individual VFs, and demonstrate the importance of microtubule stability for VF fusion during MRV infection. This work provides important groundwork for future in depth studies of VF dynamics and host cell interactions.

    IMPORTANCE MRV has historically been used as a model to study the double-stranded RNA (dsRNA) Reoviridae family, which infect and cause disease in humans, animals, and plants. During infection, MRV forms VFs that play a critical role in virus infection, but remain to be fully characterized. To study VFs, researchers have focused on visualizing the non-structural protein mmu;NS which forms the VF matrix. This work provides the first evidence of recovery of replicating reoviruses in which VFs can be labeled in live cells via introduction of a TC-tag into the mmu;NS open reading frame. Characterization of each recombinant reovirus sheds light on mmu;NS interactions with viral proteins. Moreover, utilizing the TC labeling FlAsH-EDT2 biarsenical reagent to visualize VFs, evidence is provided of dynamic VF movement and interactions at least partially dependent on intact microtubules.

  • Filling gaps about mimivirus entry, uncoating and morphogenesis [PublishAheadOfPrint]

  • Since the discovery of mimivirus, its unusual structural and genomic features have raised great interest in the study of its biology; however, many aspects concerning its replication cycle remain uncertain. In this study, extensive analyses of electron microscope images, as well as biological assays, shed light on unclear points concerning the mimivirus replication cycle. Here, we demonstrate that cytochalasin treatment, a phagocytosis inhibitor, impacts negatively on the incorporation of mimivirus particles by Acanthamoeba castellanii, causing a negative effect on viral growth in amoebae monolayers. The treatment of amoebas with bafilomicin impacts significantly on mimivirus uncoating and replication. In conjunction with microscopic analyses, these data suggest that mimiviruses indeed depend on phagocytosis for entry into amoebas, and particle uncoating (and stargate opening) appears to be dependent on phagosome acidification. In depth analyses of particle morphogenesis suggests that the mimivirus capsids are assembled from growing lamellar structures. Despite proposals of previous studies that genome acquisition occurs before the acquisition of fibrils, our results clearly demonstrate that the genome and fibrils can be acquired simultaneously. Our data suggest the existence of a specific area surrounding the core of the viral factory, where particles acquire the surface fibrils. Furthermore, we reinforce that defective particles can be formed even in the absence of virophages. Our work provides new information about unexplored steps in the life cycle of mimiviruses.

    IMPORTANCE Investigating the viral life cycle is essential to a better understanding of viruses' biology. The combination of biological assays and microscope images allows a clear view of the biological features of viruses. Since the discovery of mimivirus, many studies have been conducted to characterize its replication cycle, but many gaps remain to be filled. In this study, we conducted a new examination of the replication cycle of mimiviruses, providing new evidence concerning some stages of the cycle which were previously unclear, mainly entry, uncoating and morphogenesis. Furthermore, we demonstrated that atypical virion morphologies can occur even in the absence of virophages. Our results, along with previous data, lead us to present an ultimate model for the mimivirus replication cycle.

  • Novel Stable Ebola Virus Minigenome Replicon Reveals Remarkable Stability of the Viral Genome [PublishAheadOfPrint]

  • Ebola virus (EBOV) causes severe hemorrhagic fever in humans and other primates with a high case fatality rate. No approved drug or vaccine of EBOV is available, which necessitates better understanding of the virus life cycle. Studies on EBOV have been hampered because experimentations involving live virus are restricted to biosafety level 4 (BSL-4) laboratories. EBOV minigenome system has provided researchers with the opportunity to study EBOV under BSL-2 conditions. Here, we developed a novel EBOV minigenome replicon which, to our knowledge, is the first EBOV cell culture system that can stably replicate and transcribe EBOV minigenome. The minigenomic RNA harboring a Gaussia Luciferase and hygromycin-resistant marker can replicate for months in a helper cell stably expressing viral NP, VP35, VP30 and L proteins. Quantification of vRNA, cRNA and mRNA levels of EBOV minigenome demonstrated that the stable EBOV replicon had much more active minigenome replication than previously developed transient transfection-based EBOV minigenome systems which recapitulate viral primary transcription more than genome replication. Interestingly, minigenome replication in the stable EBOV replicon cells was insensitive to interferon treatment or RNA interference. Moreover, RNase digestion of the replicon cell lysates revealed the remarkably stable nature of EBOV minigenomic vRNA ribonucleoprotein complex, which may help understand EBOV persistence in convalescent patients.

    IMPORTANCE The scope and severity of the recent Ebola outbreak in the Western Africa justified more comprehensive investigation of the causative Risk Group 4 agent Ebola virus (EBOV). Study of EBOV replication and antiviral development can be facilitated by developing a cell culture system that allows for experimentations in the biosafety level 2 conditions. Here, we developed a novel stable EBOV minigenome replicon which, to our knowledge, is the first EBOV cell culture system that can stably replicate and transcribe EBOV minigenome. Compared to previously developed transient transfection-based EBOV minigenome systems, the replicon system had more active genome replication, providing a convenient surrogate system to study EBOV replication. Furthermore, self-replicating minigenomic vRNA in the replicon cells displayed strong stability in response to interferon treatment, RNA silencing and RNase digestion, which may provide an explanation for the persistence of EBOV in survivors.

  • Cooperation of the Ebola proteins VP40 and GP1,2 with BST2 to activate NF-{kappa}{beta} independently of virus-like particle trapping [PublishAheadOfPrint]

  • BST2 is a host protein with dual functions in response to viral infections: it traps newly assembled enveloped virions at the plasma membrane of infected cells, and it induces NF-bbeta; activity, especially in the context of retroviral assembly. In this study, we examined whether Ebola virus proteins affect BST2-mediated induction of NF-bbeta;. We found that the Ebola matrix protein, VP40, and envelope glycoprotein, GP, each cooperate with BST2 to induce NF-bbeta; activity, with maximal activity when all three proteins are expressed. Unlike the Human Immunodeficiency Virus type 1 Vpu protein, which antagonizes both virion-entrapment and the activation of NF-bbeta; by BST2, Ebola GP does not inhibit NF-bbeta; signaling even while it antagonizes the entrapment of virus-like particles. GP from Reston Ebola virus, a non-pathogenic species in humans, showed a similar phenotype to GP from Zaire Ebola, a highly pathogenic species, in terms of both the activation of NF-bbeta; and the antagonism of virion-entrapment. Although Ebola VP40 and GP both activate NF-bbeta; independently of BST2, VP40 is the more potent activator. Activation of NF-bbeta; by the Ebola proteins either alone or together with BST2 requires the canonical NF-bbeta; signaling pathway. Mechanistically, the maximal NF-bbeta; activation by GP, VP40, and BST2 together requires the ectodomain cysteines needed for BST2-dimerization, the putative BST2 tetramerization residue L70, and Y6 of a potential hemi-ITAM motif in BST2's cytoplasmic domain. BST2 with a GPI anchor signal-deletion, which is not expressed at the plasma membrane and is unable to entrap virions, activated NF-bbeta; in concert with the Ebola proteins at least as effectively as wild type BST2. Signaling by the GPI-anchor mutant also depended on Y6 of BST2. Overall, our data show that activation of NF-bbeta; by BST2 is independent of virion-entrapment in the case of Ebola virus. Nonetheless, BST2 may induce or amplify proinflammatory signaling during Ebola virus infection, potentially contributing to the dysregulated cytokine response that is a hallmark of Ebola virus disease.

    IMPORTANCE Understanding how the host responds to viral infections informs the development of therapeutics and vaccines. We asked how proinflammatory signaling by the host protein BST2/tetherin, which is mediated by the transcription factor NF-bbeta;, responds to Ebola proteins. Although the Ebola virus envelope glycoprotein (GP1,2) antagonizes the trapping of newly formed virions at the plasma membrane by BST2, we found that it does not inhibit BST2's ability to induce NF-bbeta; activity. This distinguishes GP1,2 from the HIV-1 protein Vpu, the prototype BST2-antagonist, which inhibits both virion-entrapment and the induction of NF-bbeta; activity. Ebola GP1,2, the Ebola matrix protein VP40, and BST2 are at least additive with respect to the induction of NF-bbeta; activity. The effects of these proteins converge on an intracellular signaling pathway that depends on a protein-modification termed neddylation. Better mechanistic understanding of these phenomena could provide targets for therapies that modulate the inflammatory response during Ebola virus disease.

  • Mechanical barriers restrict invasion of herpes simplex virus 1 into human oral mucosa [PublishAheadOfPrint]

  • Oral mucosa is one of the main target tissues of the human pathogen herpes simplex virus 1 (HSV-1). How the virus overcomes the protective epithelial barriers and penetrates into the tissue to reach its receptors and initiate infection is still unclear. Here, we established an ex vivo infection assay with human oral mucosa which allows viral entry studies in a natural target tissue. The focus was on the susceptibility of keratinocytes in the epithelium, and the characterization of cellular receptors that mediate viral entry. Upon ex vivo infection of gingiva or vestibular mucosa, we observed that intact human mucosa samples were protected from viral invasion. In contrast, the basal layer of the oral epithelium was efficiently invaded once the connective tissue and the basement membrane were removed. Later during infection, HSV-1 spread from basal keratinocytes to upper layers demonstrating the susceptibility of the stratified squamous epithelium to HSV-1. The analysis of potential receptors revealed nectin-1 on most mucosal keratinocytes, whereas HVEM was found only on a subpopulation of cells suggesting that nectin-1 acts as primary receptor for HSV-1 in human oral mucosa. To mimic the supposed entry route of HSV-1 via microlesions in vivo, we mechanically wounded the mucosa prior to infection. While we observed a limited number of infected keratinocytes in some wounded mucosa samples, other samples showed no infected cells. Thus, we conclude that mechanical wounding of mucosa is insufficient for the virus to efficiently overcome epithelial barriers and to make entry-mediating receptors accessible.

    IMPORTANCE To invade the target tissue of its human host during primary infection, herpes simplex virus (HSV) must overcome the epithelial barriers of mucosa, skin or cornea. For most viruses the mechanisms underlying the invasion into the target tissues of their host organism are still open. Here, we established an ex vivo infection model of human oral mucosa to explore how HSV can enter its target tissue. Our results demonstrate that intact mucosa samples and even compromised tissue allow only very limited access of HSV to keratinocytes. Detailed understanding of barrier functions is an essential precondition to unravel how HSV bypasses the barriers and approaches its receptors in tissue, and why it is beneficial for the virus to use a cell-cell adhesion molecule, such as nectin-1, as receptor.

  • IRF9 prevents CD8+ T cell exhaustion in an extrinsic manner during acute LCMV infection [PublishAheadOfPrint]

  • Effective CD8+ T cell responses play an important role in determining the course of a viral infection. Overwhelming antigen exposure can result in suboptimal CD8+ T cell responses, leading to chronic infection. This altered CD8+ T cell differentiation state, termed exhaustion, is characterized by reduced effector function, upregulation of inhibitory receptors and altered expression of transcription factors. Prevention of overwhelming antigen exposure which limits CD8+ T cell exhaustion is of significant interest for controlling chronic infection. The transcription factor interferon regulatory factor 9 (IRF9) is a component of type I interferon (IFN-I) signaling downstream of the IFN-I receptor (IFNAR). Using acute infection of mice with lymphocytic choriomeningitis virus (LCMV)-Armstrong, we show for the first time that IRF9 limited early LCMV replication by regulating expression of interferon-stimulated genes and IFN-I, and by controlling levels of IRF7, a transcription factor essential for IFN-I production. Infection of IRF9- or IFNAR-deficient mice led to a loss of early restriction of viral replication and impaired anti-viral responses in dendritic cells, resulting in CD8+ T cell exhaustion and chronic infection. Differences in the antiviral activity of IRF9- and IFNAR-deficient mice and dendritic cells provided further evidence for IRF9-independent IFN-I signaling. Thus, our findings illustrate a CD8+ T cell extrinsic function for IRF9, as a downstream signaling factor of IFNAR, in preventing overwhelming antigen exposure resulting in CD8+ T cell exhaustion and ultimately in chronic infection.

    IMPORTANCE During early viral infection, overwhelming antigen exposure can cause functional exhaustion of CD8+ T cells and lead to chronic infection. Here we show that the transcription factor interferon regulatory factor (IRF) 9 plays a decisive role in preventing CD8+ T cell exhaustion. Using acute infection of mice with the LCMV-Armstrong strain, we found that IRF9 limited early LCMV replication by regulating expression of interferon-stimulated genes and Irf7, a transcription factor crucial for of type I interferon (IFN-I) production, as well as by controlling the levels IFN-I. Infection of IRF9-deficient mice led to chronic infection that was accompanied by CD8+ T cell exhaustion due to T cell extrinsic defects. Our findings illustrate an essential role for IRF9 as a mediator downstream of IFNAR in preventing overwhelming antigen exposure causing CD8+ T cell exhaustion and leading to chronic viral infection.

  • Lysine 242 within helix 10 of the pseudorabies virus nuclear egress complex pUL31 component is critical for primary envelopment of nucleocapsids [PublishAheadOfPrint]

  • Newly assembled herpesvirus nucleocapsids are translocated from the nucleus to the cytosol by a vesicle-mediated process engaging the nuclear membranes. This transport is governed by the conserved nuclear egress complex (NEC), consisting of the alphaherpesviral pUL34 and pUL31 homologs. The NEC is not only required for efficient nuclear egress but also sufficient for vesicle formation from the inner nuclear membrane (INM) as well as from synthetic lipid bilayers. The recently solved crystal structures for the NECs from different herpesviruses revealed molecular details of this membrane deformation and scission machinery uncovering the interfaces involved in complex and coat formation. However, the interaction domain with the nucleocapsid remained undefined. Since the NEC assembles a curved hexagonal coat on the nucleoplasmic side of the INM consisting of tightly interwoven pUL31/pUL34 heterodimers arranged in hexamers, only the membrane-distal end of the NEC formed by pUL31 residues appears accessible for interaction with the nucleocapsid cargo. To identify the amino acids involved in capsid incorporation we mutated the corresponding regions in the alphaherpesvirus pseudorabies virus (PrV). Site-specifically mutated pUL31 were tested for localization, interaction with pUL34 and complementation of PrV-UL31. Here, we identify a conserved lysine residue at amino acid position 242 in PrV pUL31 located in the alpha-helical domain H10 exposed on the membrane-distal end of the NEC as a key residue for nucleocapsid incorporation into the nascent primary particle.

    IMPORTANCE Vesicular transport through the nuclear envelope is a focus of research but still not well understood. Herpesviruses pioneered this mechanism for translocation of the newly assembled nucleocapsid from the nucleus into the cytosol via vesicles derived from the inner nuclear membrane which fuse in a well-tuned process with the outer nuclear membrane to release their content. The structure of the viral nuclear membrane budding and scission machinery has been solved recently providing in-depth molecular details. However, how cargo is incorporated remained unclear. Here, we identified a conserved lysine residue in the membrane-distal portion of the nuclear egress complex required for capsid uptake into inner nuclear membrane-derived vesicles.

  • The effect of polymorphisms at codon 146 of the goat PRNP gene on susceptibility to challenge with classical scrapie by different routes [PublishAheadOfPrint]

  • This report presents the results of experimental challenges of goats with scrapie by both the intracerebral and oral routes, exploring the effects of polymorphisms at codon 146 of the goat PRNP gene on resistance to disease. The results of these studies illustrate that while goats of all genotypes can be infected by intracerebral (ic) challenge, the survival distribution of the animals homozygous for asparagine at codon 146 was significantly shorter than for all other genotypes (chi2 =10.8. P=0.001). In contrast, only those animals homozygous for asparagine at codon 146 (NN) succumb to oral challenge. The results also indicate that any cases in non-NN animals can be detected by the current confirmatory test (immunohistochemistry), although successful detection with the ELISA rapid test was more variable, and dependent on the polymorphism. Together with previous studies of goats exposed to infection in the field, these data support the previously published observations that polymorphism at this codon has a profound effect on susceptibility to disease. It is concluded that only animals homozygous for asparagine at codon 146 succumb to scrapie under natural conditions.

    IMPORTANCE In goats, like in sheep, there are PRNP polymorphisms which are associated with susceptibility/resistance to scrapie. However, in contrast to sheep, they are more numerous and may be restricted to certain breeds or geographical regions. Therefore, eradication programs must be specifically designed depending on the identification of suitable polymorphisms. Initial analysis of surveillance data suggested that such a polymorphism in Cypriot goats may lie in codon 146. In this study, we demonstrate experimentally that NN animals are highly susceptible after ic inoculation. Presence of D or S prolonged incubation periods significantly and only in NN animals prions were detected in peripheral tissues. In oral challenges, prions were detected only in NN animals and presence of D or S at this position conferred resistance to the disease. This study provides an experimental transmission model for assessing genetic susceptibility of goats to scrapie.

  • How germinal centers evolve broadly neutralizing antibodies: the breadth of the follicular helper T cell response. [PublishAheadOfPrint]

  • Many HIV-1 infected patients evolve broadly neutralizing antibodies (bnAbs). This evolutionary process typically takes several years, and is poorly understood as selection taking place in germinal centers occurs on the basis of antibody affinity. B cells with the highest affinity receptors tend to acquire the most antigen from the FDC network, and present the highest density of cognate peptides to follicular helper T cells (Tfh), which provide survival signals to the B cell. BnAbs are therefore only expected to evolve when the B cell lineage evolving breadth is consistently capturing and presenting more peptides to Tfh cells than other lineages of more specific B cells. Here we develop mathematical models of Tfh in germinal centers to explicitly define the mechanisms of selection in this complex evolutionary process.

    Our results suggest that broadly reactive B cells presenting a high density of pMHC are readily outcompeted by B cells responding to lineages of HIV-1 that transiently dominate the within host viral population. Conversely, if broadly reactive B cells acquire a large variety of several HIV-1 proteins from the FDC network and present a high diversity of several pMHC, they be rescued by a large fraction of the Tfh repertoire in the germinal center. Under such circumstances the evolution of bnAbs is much more consistent. Increasing the magnitude of the Tfh response, or the breadth of the Tfh repertoire, both markedly facilitate the evolution of bnAbs. Because both can be increased by vaccination with several HIV-1 proteins, this calls for experiments testing.

    Importance Many HIV-infected patients slowly evolve antibodies that can neutralize a large variety of viruses. Such "broadly neutralizing antibodies" (bnAbs) could in the future become therapeutic agents. BnAbs appear very late and patients are typically not protected by them. At the moment we fail to understand why this takes so long, and how the immune system selects for broadly neutralizing capacity. Typically antibodies are selected based on affinity and not on breadth. We develop mathematical models to study two different mechanisms by which the immune system can select for broadly neutralizing capacity. One of these is based upon the repertoire of different follicular helper T cells (Tfh) in germinal centers. We suggest that broadly reactive B cells may interact with a larger fraction of this repertoire, and demonstrate that this would select for bnAbs. Intriguingly, this suggest that broadening the Tfh repertoire by vaccination may speed up the evolution of bnAbs.

  • Human Herpesvirus 8 Interleukin-6 Interacts with Calnexin Cycle Components and Promotes Protein Folding [PublishAheadOfPrint]

  • Viral interleukin-6 (vIL-6) encoded by human herpesvirus 8 (HHV-8) is believed to contribute via mitogenic, survival, and angiogenic activities to HHV-8-associated Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease through autocrine or paracrine mechanisms during latency or productive replication. There is direct evidence that vIL-6 promotes latently infected PEL cell viability and proliferation and also viral productive replication in PEL and endothelial cells. These activities are mediated largely through endoplasmic reticulum (ER)-localized vIL-6, which can induce signal transduction via the gp130 signaling receptor, activating mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription (STAT) signaling, and interactions of vIL-6 with the ER membrane protein vitamin K epoxide reductase complex subunit 1 variant 2 (VKORC1v2). The latter functional axis involves suppression of pro-apoptotic lysosomal protein cathepsin D by promotion of the ER-associated degradation of ER-transiting, pre-proteolytically processed pro-cathepsin D. Other interactions of VKORC1v2 and activities of vIL-6 via the receptor have not been reported. Here, we show that both vIL-6 and VKORC1v2 interact with calnexin cycle proteins UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1), catalyzing monoglucosylation of N-glycans, and oppositely-acting glucosidase II (GlucII) and that vIL-6 can promote protein folding. This activity was found to require VKORC1v2 and UGGT1, to involve vIL-6 associations with VKORC1v2, UGGT1 and GlucII, and to operate in the context of productively infected cells. These findings document new VKORC1v2-associated interactions and activities of vIL-6, revealing novel mechanisms of vIL-6 function within the ER compartment.

    IMPORTANCE HHV-8 vIL-6 pro-survival (latent) and pro-replication functions are mediated from the ER compartment through both gp130 receptor-mediated signal transduction and interaction of vIL-6 with the ER membrane protein VKORC1v2. This report identifies interactions of vIL-6 and VKORC1v2 with calnexin cycle enzymes GlucII and UGGT1, which are involved in glycan processing and nascent protein folding. The presented data show that vIL-6 and VKORC1v2 can co-complex with GlucII and UGGT1, that vIL-6 promotes protein folding, and that VKORC1v2, UGGT1, and vIL-6 interactions with GlucII and UGGT1 are important for the pro-folding activity of vIL-6, which can be detected in the context of infected cells. This newly identified ER activity of vIL-6 involving VKORC1v2 may promote viral latency (in PEL cells) and productive replication by limiting the damaging effects of unfolded protein response signaling in addition to enhancing viral protein folding. This is the first report of such a function for a cytokine.

  • MetSV, a novel archaeal lytic virus targeting Methanosarcina strains [PublishAheadOfPrint]

  • A novel archaeal lytic virus targeting species of the genus Methanosarcina was isolated using Methanosarcina mazei strain Gö1 as host. Due to its spherical morphology the virus was designated Methanosarcina spherical virus (MetSV). Molecular analysis demonstrated that MetSV contains double stranded linear DNA with a genome size of 10,567 bp containing 22 open reading frames (ORFs) all oriented in the same direction. Functions were predicted for some of these ORFs, i. e. like DNA polymerase, ATPase, DNA-binding protein, as well as envelope (structural) protein. MetSV-derived spacers in CRISPR loci were detected in several published Methanosarcina draft genomes using bioinformatic tools, revealing the potential PAM motif (TTA/T). Transcription and expression of several predicted viral ORFs were validated by RT-PCR, PAGE analysis and LC-MS based proteomics. Analysis of core lipids by APCI mass spectrometry showed that MetSV and M. mazei both contain archaeol and glycerol dialkyl glycerol tetraether without cyclopentane moiety (GDGT-0). The MetSV host range is limited to Methanosarcina strains growing as single cells (M. mazei, M. bakeri and M. soligelidi). In contrast, strains growing as sarcina-like aggregates were apparently protected from infection. Heterogeneity related to morphology phases in M. mazei cultures allowed acquisition of resistance to MetSV after challenge by growing as sarcina-like aggregates. CRISPR/Cas mediated resistance was excluded since neither of the two CRISPR arrays showed MetSV-derived spacer acquisition. Based on these findings, we propose that changing the morphology from single cells to sarcina-like aggregates upon rearrangement of the envelope structure prevents infection and subsequent lysis by MetSV.

    IMPORTANCE Methanoarchaea are among the most abundant organisms on the planet since they are present in high numbers in major anaerobic environments. They convert various carbon sources e.g. acetate, methylamines or methanol to methane and carbon dioxide, thus they have a significant impact on the emission of major greenhouse gases. Today very little is known about viruses specifically infecting methanoarchaea, which most probably impact the abundance of methanoarchaea in microbial consortia. Here we characterize the first identified Methanosarcina infecting virus (MetSV) and show a mechanism for acquiring resistance against MetSV. Based on our results we propose that growth as sarcina-like aggregates prevents infection and subsequent lysis. These findings allow new insights into virus-host relationship in methanogenic community structures, their dynamics and their phase heterogeneity. Moreover, the availability of a specific virus provides new possibilities to deepen our knowledge on defence mechanisms of potential hosts and offers tools for genetic manipulation.

  • Localisation of double-strand break repair proteins to viral replication compartments following lytic reactivation of KSHV [PublishAheadOfPrint]

  • Double-strand breaks (DSBs) in DNA are recognised by the Ku70/80 heterodimer and the MRE11-RAD50-NBS1 (MRN) complex and result in activation of the DNA-PK and ATM kinases that play key roles in regulating the cellular DNA damage response (DDR). DNA tumour viruses such as Kaposi's sarcoma-associated herpesvirus (KSHV) are known to interact extensively with the DDR during the course of their replicative cycles. Here we show that during lytic amplification of KSHV DNA, the Ku70/80 heterodimer and the MRN complex consistently co-localise with viral genomes in replication compartments (RCs) whereas other DSB repair proteins form foci outside of RCs. Depletion of MRE11 and abrogation of its exonuclease activity negatively impacts on viral replication, while in contrast, knockdown of Ku80 and inhibition of the DNA-PK enzyme, which are involved in non-homologous end joining repair (NHEJ), enhances amplification of viral DNA. Although the recruitment of DSB sensing proteins to KSHV RCs is a consistent occurrence across multiple cell types, activation of the ATM-CHK2 pathway during viral replication is a cell line-specific event indicating that recognition of viral DNA by the DDR does not necessarily result in activation of downstream signalling pathways. We have also observed that newly replicated viral DNA is not associated with cellular histones. Since the presence and modification of these DNA packaging proteins provides a scaffold for docking of multiple DNA repair factors, the absence of histone deposition may allow the virus to evade localisation of DSB repair proteins that would otherwise have a detrimental effect on viral replication.

    Importance Tumour viruses are known to interact with machinery responsible for detection and repair of double-strand breaks (DSBs) in DNA, although detail concerning how Kaposi's sarcoma-associated herpesvirus (KSHV) modulates these cellular pathways during its lytic replication phase was previously lacking. By undertaking a comprehensive assessment of the localisation of DSB repair proteins during KSHV replication, we have determined that a DNA damage response (DDR) is directed to viral genomes but is distinct from the response to cellular DNA damage. We also demonstrate that although recruitment of the MRE11-RAD50-NBS1 (MRN) DSB sensing complex to viral genomes and activation of the ATM kinase can promote KSHV replication, proteins involved in non-homologous end joining (NHEJ) repair restrict amplification of viral DNA. Overall this study extends our understanding of the viral-host interactions that occur during lytic replication of KSHV and provides a deeper insight into the how the DDR is manipulated during viral infection.

  • Foot-and-mouth disease virus 3C protease mutant L127P: Implications for FMD vaccine development [PublishAheadOfPrint]

  • The foot-and-mouth disease virus (FMDV) afflicts livestock in more than 80 countries limiting food production and global trade. Production of foot-and-mouth disease (FMD) vaccines requires cytosolic expression of the FMDV 3C protease to cleave the P1 polyprotein into mature capsid proteins, but the FMDV 3C protease is toxic to host cells. To identify less toxic isoforms of the FMDV 3C protease, we screened 3C mutants for increased transgene output over wild-type 3C using a Gaussia luciferase reporter system. The novel point-mutation 3C(L127P) increased yields of recombinant FMDV subunit proteins in mammalian and bacterial cells expressing P1-3C transgenes and retained the ability to process P1 polyproteins from multiple FMDV serotypes. The 3C(L127P) mutant produced crystalline-arrays of FMDV virus-like particles in mammalian and bacterial cells, potentially providing a practical method of rapid, inexpensive FMD vaccine production in bacteria.

    Importance: The mutant FMDV 3C protease L127P significantly increased yields of recombinant FMDV subunit antigens and produced virus-like particles in mammalian and bacterial cells. The L127P mutation provides a novel advancement for economical FMD vaccine production.

  • Potent Plasmablast-Derived Antibodies Elicited by the NIH Dengue Vaccine [PublishAheadOfPrint]

  • Exposure to dengue virus (DENV) is thought to elicit lifelong immunity, mediated by DENV-neutralizing antibodies (nAbs). However, Abs generated by primary infections confer serotype-specific protection, and immunity against other serotypes only develops after subsequent infections. Accordingly, the induction of these nAb responses acquired after serial DENV infections has been a long sought-after goal for vaccination. Nonetheless, it is still unclear if tetravalent vaccines can elicit or recall nAbs. Here, we have characterized the responses from a volunteer who had been previously exposed to DENV and was immunized with the live attenuated tetravalent vaccine Butantan-DV, developed by the NIH and Butantan. Eleven days after vaccination, we observed a ~70-fold expansion of the plasmablast population. We generated 21 monoclonal (m)Abs from singly-sorted plasmablasts. These mAbs were the result of clonal expansions and had significant levels of somatic hypermutation (SHM). Nineteen mAbs (90.5%) neutralized at least one DENV serotype at concentrations of 1 mmu;g/ml or less; six of the 21 mAbs neutralized three or more serotypes. Despite the tetravalent composition of the vaccine, we observed a neutralization bias in the induced repertoire: DENV3 was targeted by 18 of the 19 neutralizing (n)mAbs. Furthermore, the P3D05 nmAb neutralized DENV3 with extraordinary potency (Neut50 0.03 mmu;g/ml). Thus, the Butantan-DV vaccine engendered a mature, antigen-selected B cell repertoire. Our results suggest that pre-existing responses elicited by a previous DENV3 infection were recalled by immunization.

    IMPORTANCE The dengue epidemic presents a global public health challenge that causes widespread economic burden and remains largely unchecked by existing control strategies. Successful control of the dengue epidemic will require effective prophylactic and therapeutic interventions. Several vaccine clinical efficacy trials are approaching completion, and the chances that one or more live attenuated tetravalent vaccines (LATVs) will be introduced worldwide is higher than ever. While it is widely accepted that dengue virus (DENV)-neutralizing antibody (nAb) titers are associated with protection, the Ab repertoire induced by LATVs remain uncharacterized. Here, we describe the isolation of potent (Neut50 llt; 0.1 mmu;g/ml) nAbs from a DENV-seropositive volunteer immunized with the tetravalent vaccine Butantan-DV, which is currently in Phase III trials.

  • High-throughput Protein Engineering Improves the Antigenicity and Stability of Soluble HIV-1 Envelope Glycoprotein SOSIP Trimers [PublishAheadOfPrint]

  • Soluble envelope glycoprotein (Env) trimers (SOSIP.664 gp140) are attractive HIV-1 vaccine candidates, with structures that mimic the native membrane-bound Env spike (gp160). Since engineering trimers can be limited by the difficulty of rationally predicting beneficial mutations, here we used a more comprehensive mutagenesis approach with the goal of identifying trimer variants with improved antigenic and stability properties. We created 341 cysteine pairs at predicted points of stabilization throughout gp140, 149 proline residue substitutions at every residue of the gp41 ectodomain, and 362 space-filling residue substitutions at every hydrophobic and aromatic residue in gp140. The parental protein target, the clade B strain B41 SOSIP.664 gp140, does not bind the broadly neutralizing antibody PGT151, so was used here to identify improved variants that also provide insight into the structural basis for Env antigenicity. Each of the 852 mutants was expressed in human cells and screened for antigenicity using four different monoclonal antibodies (mAbs), including PGT151. We identified 29 trimer variants with antigenic improvements derived from each of the three mutagenesis strategies. We selected four variants (Q203F, T538F, I548F, and M629P) for more comprehensive biochemical, structural, and antigenicity analyses. The T538F substitution had the most beneficial effect overall, including restoration of the PGT151 epitope. The improved B41 SOSIP.664 trimer variants identified here may be useful for vaccine and structural studies.

    IMPORTANCE Soluble Env trimers have become attractive HIV-1 vaccine candidates, but the prototype designs are capable of further improvement through protein engineering. Using a high-throughput screening technology (shotgun mutagenesis) to create and evaluate 852 variants, we were able to identify sequence changes that were beneficial to the antigenicity and stability of soluble trimers based on the clade B B41 env gene. The strategies described here may be useful for identifying a wider range of antigenically and structurally improved soluble trimers based on multiple genotypes for use in programs intended to create a broadly protective HIV-1 vaccine.

  • The characterization of the quasi-enveloped hepatitis E virus particles released by the cellular exosomal pathway [PublishAheadOfPrint]

  • Our previous studies demonstrated that membrane-associated hepatitis E virus (HEV) particlesmmdash;now considered "quasi-enveloped particles"mmdash;are present in the multivesicular body with intraluminal vesicles (exosomes) in infected cells, and that the release of HEV virions is related to the exosomal pathway. In this study, we characterized exosomes purified from culture supernatant of HEV-infected PLC/PRF/5 cells. Purified CD63-, CD9-, or CD81-positive exosomes derived from the culture supernatant of HEV-infected cells that had been cultivated in serum-free medium were found to contain HEV RNA and the viral capsid (ORF2) and ORF3 proteins, as determined by a RT-PCR and Western blotting, respectively. Furthermore, immunoelectron microscopy, with or without prior detergent and protease treatment, revealed the presence of virus-like particles in the exosome fraction. These particles were 39.6 pplusmn; 1.0 nm in diameter, and were covered with a lipid membrane. After treatment with detergent and protease, the diameter of these virus-like particles was 26.9 pplusmn; 0.9 nm, and the treated particles became accessible with an anti-HEV ORF2 monoclonal antibody (mAb). The HEV particles in the exosome fraction were capable of infecting native PLC/PRF/5 cells, but were not neutralized by an anti-HEV ORF2 mAb, which can efficiently neutralize the non-enveloped HEV particles in cell culture. These results indicate that the membrane-wrapped HEV particles released by the exosomal pathway are co-purified with the exosomes in the exosome fraction, and suggest that the capsids of HEV particles are individually covered by lipid membranes resembling those of exosomes, similarly to enveloped viruses.

    IMPORTANCE Hepatitis E, caused by HEV, is an important infectious disease that is spreading worldwide. HEV infection can cause acute or fulminant hepatitis and can become chronic in immunocompromised hosts, including patients after organ transplantation. The HEV particles present in feces and bile are non-enveloped, while those in circulating blood and culture supernatant are covered with a cellular membrane, similar to enveloped viruses. Furthermore, these membrane-associated and -unassociated HEV particles can be propagated in cultured cells. The significance of our research is that the capsids of HEV particles are individually covered by a lipid membrane that resembles the membrane of exosomes, similar to enveloped viruses, and are released from infected cells via the exosomal pathway. These data will help elucidate the entry mechanisms and receptors for HEV infection in the future. This is the first report to characterize the detailed morphological features of membrane-associated HEV particles.

  • Zika virus escapes NK cell detection by upregulating MHC class I molecules [PublishAheadOfPrint]

  • NK cells are innate lymphocytes that participate in many immune processes encompassing cancer, bacterial and fungal infection, autoimmunity and even pregnancy and are specialize in anti-viral defense. NK cells express inhibitory and activating receptors and kill their targets when activating signals overpower the inhibitory ones. The NK cell inhibitory receptors compose of a uniquely diverse array of proteins named Killer-cell immunoglobulin-like receptors (KIRs) the CD94 family and the leukocyte immunoglobulin like receptor family (LIR). The NK inhibitory receptors recognize mostly MHC class I proteins. Zika virus has recently emerged as a major threat due to its association with birth defects and its pandemic potential. How Zika virus interacts with the immune system, especially with NK cells is unclear. Here we show that Zika virus infection is barely sensed by NK cells, as little or no increase in the expression of activating NK cell ligands was observed. In contrast, we demonstrate that Zika virus infection leads to the upregulation of MHC class I proteins and consequently to inhibition of NK cell killing. Mechanistically, we show that the upregulation of MHC class I occurs via the RIGI-IRF3 pathway, and is mediated via IFNbbeta;. Potentially, countering MHC class I upregulation during Zika virus infection could be used as a prophylactic treatment against Zika virus.

    IMPORTANCE NK cells are innate lymphocytes, which recognize and eliminate various pathogens, and are mostly known for their role in controlling viral infections. NK cells express inhibitory and activating receptors and kill or spare their targets based on integration of inhibitory and activating signals. Zika virus has recently emerged as a major threat to humans due to its pandemic potential and association with birth defects. The role of NK cells in Zika virus infection is largely unknown. Here, we demonstrate that Zika virus infection is almost undetected by NK cells, as expression of activating ligands for NK cells are not induced following Zika infection. We identified a mechanism whereby Zika virus sensing via the RIGI IRF3 pathway resulted in an IFNbbeta; mediated upregulation of MHC-I molecules, and inhibition of NK cell activity. Countering MHC class I upregulation and boosting NK cell activity may be employed as a prophylactic measure to combat Zika virus infection.

  • Effect of the phosphorylation of the CM2 protein on influenza C virus replication [PublishAheadOfPrint]

  • CM2 is the second membrane protein of the influenza C virus and has been demonstrated to play a role in the uncoating and genome packaging processes in influenza C virus replication. Although the effects of N-linked glycosylation, disulfide-linked oligomerization, and palmitoylation of CM2 on virus replication have been analyzed, the effect of the phosphorylation of CM2 on virus replication remains to be determined. In this study, phosphorylation site(s) at residues 78 and/or 103 of CM2 were substituted with alanine residue(s) and the effects of the loss of phosphorylation on influenza C virus replication were analyzed. No significant differences were observed in the packaging of the reporter gene between influenza C virus-like particles (VLPs) produced from 293T cells expressing wild-type CM2 and those from the cells expressing the CM2 mutants lacking the phosphorylation site(s). Reporter gene expression in HMV-II cells infected with VLPs containing the CM2 mutants was inhibited in comparison with that in cells infected with wild-type VLPs. The virus production of the recombinant influenza C virus possessing CM2 mutants containing a serine-to-alanine substitution at residue 78 was significantly lower than that of wild-type recombinant influenza C virus. Furthermore, the virus growth of the recombinant viruses possessing CM2 with a serine-to-aspartic acid substitution at position 78, to mimic constitutive phosphorylation, was virtually identical to that of the wild-type virus. These results suggest that phosphorylation of CM2 plays a role in efficient virus replication, probably through the addition of a negative charge to the Ser78 phosphorylation site.

    IMPORTANCE It is well-known that many host and viral proteins are post-translationally modified by phosphorylation, which plays a role in the functions of these proteins. In influenza A and B viruses, phosphorylation of viral proteins, NP, M1, NS1 and NEP, which are not integrated into the membranes, affects the functions of these proteins, thereby affecting virus replication. However, it was reported that phosphorylation of influenza A virus M2 ion channel protein, which is integrated into the membrane, has no effect on virus replication in vitro or in vivo. We previously demonstrated that influenza C virus CM2 ion channel protein is modified by N-glycosylation, oligomerization, palmitoylation, and phosphorylation, and have analyzed the effects of these modifications, except phosphorylation, on virus replication. This is the first report demonstrating that phosphorylation of the influenza C virus CM2 ion channel protein, unlike the influenza A virus M2 protein, plays a role in virus replication.

  • HCMV particles treated with specific antibodies induce intrinsic and adaptive but not innate immune responses [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) persistently infects 40% to 100% of the human population worldwide. Experimental and clinical evidence indicates that humoral immunity to HCMV plays an important role in restricting virus dissemination and protecting the infected host from disease. Specific immunoglobulin preparations from pooled plasma of adults selected for high titers of HCMV antibodies, have been used for prevention of CMV disease in transplant recipients and pregnant women. Even though incubation of HCMV particles with these preparations leads to neutralization of the viral infectivity, it is still unclear whether the antibody-treated HCMV particles (here defined as HCMV-Ab) enter into the cells and modulate anti-viral immune responses. Here we demonstrate that HCMV-Ab did enter macrophages. HCMV-Ab did not initiate the expression of immediate early antigens (IEAs) in macrophages, but they induced an antiviral state and rendered the cells less susceptible to HCMV infection upon challenge. Resistance against HCMV infection seemed to be due to the activation of intrinsic restriction factors and was independent of interferons. In contrast to actively infected cells, autologous NK cells did not degranulate against HCMV-Ab treated macrophages suggesting that these cells may not become eliminated by innate effector cells. Interestingly, HCMV-Ab treated macrophages stimulated the proliferation of autologous adaptive CD4+ and CD8+ T cells. Our findings not only expand the current knowledge on virus-antibody immunity, but may also be relevant for future vaccination strategies.

    IMPORTANCE Human cytomegalovirus (HCMV), a common herpesvirus, establishes benign but persistent infections in immunocompetent hosts. However, in subjects with an immature or dysfunctional immune system, HCMV is a major cause of morbidity and mortality. Passive immunization has been used in different clinical settings with variable clinical results. Intravenous hyperimmune globulin preparations (IVIg) are obtained from pooled adult human plasma selected for high anti-CMV antibody titers. While HCMV neutralization can be shown in vitro using different systems, data are lacking regarding the cross influence of IVIg administration on the cellular immune responses. The aim of this study was to evaluate the effects of IVIg on distinct components of the immune response against HCMV, including antigen presentation by macrophages, degranulation of innate Natural Killer cells, and proliferation of adaptive CD4+ and CD8+ T cells.

  • Zika virus infects human Sertoli cells and modulates the integrity of the in vitro blood-testis barrier model [PublishAheadOfPrint]

  • Confirmed reports of ZIKV in human seminal fluid for months after the clearance of viremia suggest the ability of ZIKV to establish persistent infection in the seminiferous tubules, an immune privileged site in the testis protected by the blood-testis barrier, also called the Sertoli cell barrier (SCB). However, cellular targets of ZIKV in human testis and mechanisms by which the virus enters seminiferous tubules remain unclear. We demonstrate that primary human SCs were highly susceptible to ZIKV as compared to the closely related dengue virus and induced expression of IFN-aalpha;, key cytokines and cell-adhesion molecules (VCAM-1 and ICAM-1). Further, using an in vitro SCB model, we show ZIKV was released on the adluminal side of the SCB model with higher efficiency than the blood-brain barrier. ZIKV-infected SCs exhibited enhanced adhesion of leukocytes that correlated with decrease in the SCB integrity. ZIKV infection did not affect the expression of tight and adherens junction proteins such as ZO-1, claudin and JAM-A, however exposure of SCs with inflammatory mediators derived from ZIKV-infected macrophages led to the degradation of ZO-1 protein that correlated with increased SCB permeability. Taken together, our data suggest that infection of SCs may be one of the crucial steps by which ZIKV gains access to the site of spermatozoa development and identify SCs as a therapeutic target to clear testicular infections. The SCB model opens up opportunities to assess interactions of SCs with other testicular cells and test the ability of anti-ZIKV drugs to cross the barrier.

    IMPORTANCE Recent outbreaks of ZIKV, a neglected mosquito-borne flavivirus, have identified sexual transmission as a new route of disease spread, not reported for other flaviviruses. To be able to sexually transmit for months after clearance of the viremia, ZIKV must establish infection in the seminiferous tubules, a site for spermatozoa development. However, little is known about the cell types that support ZIKV infection in the human testis. Currently there are no models to study mechanisms of virus persistence in the seminiferous tubules. We provide evidence that ZIKV infection of human Sertoli cells, important component of the seminiferous tubules, is robust and induce strong antiviral response. The use in vitro Sertoli cell barrier to describe how ZIKV or inflammatory mediators derived from ZIKV-infected macrophages compromise the barrier integrity will enable studies to explore interaction of other testicular cells with Sertoli cells and test novel antivirals for clearing testicular ZIKV infection.

  • Minocycline has anti-inflammatory effects and reduces cytotoxicity in an ex vivo spinal cord slice culture model of West Nile virus infection [PublishAheadOfPrint]

  • West Nile virus (WNV) is a neurotropic flavivirus that can cause significant neurological disease. Mouse models of WNV infection demonstrate that a pro-inflammatory environment is induced within the central nervous system (CNS) after WNV infection leading to entry of activated peripheral immune cells. We utilized ex vivo spinal cord slice cultures (SCSC) to demonstrate that anti-inflammatory mechanisms may also play a role in WNV-induced pathology and/or recovery. Microglia are a type of macrophage that function as resident CNS immune cells. Similar to mouse models, infection of SCSC with WNV induces the up-regulation of pro-inflammatory genes and proteins that are associated with microglial activation, including the microglia activation marker Iba1 and CC-motif chemokines CCL2, CCL3, and CCL5. This suggests that microglia assume a pro-inflammatory phenotype in response to WNV infection similar to the pro-inflammatory (M1) activation that can be displayed by other macrophages. We now show that the WNV-induced expression of these and other pro-inflammatory genes was significantly decreased in the presence of minocycline, which has anti-neuroinflammatory properties, including the ability to inhibit pro-inflammatory microglial responses. Minocycline also caused a significant increase in the expression of anti-inflammatory genes associated with alternative anti-inflammatory (M2) macrophage activation, including IL-4, IL-13, and FIZZ1. Minocycline-dependent alterations to M1/M2 gene expression were associated with a significant increase in survival of neurons, microglia, and astrocytes in WNV-infected slices and markedly decreased levels of iNOS. These results demonstrate that an anti-inflammatory environment induced by minocycline reduces viral cytotoxicity during WNV infection in ex vivo CNS tissue.

    IMPORTANCE West Nile virus (WNV) causes substantial morbidity and mortality, with no specific therapeutic treatments available. Antiviral inflammatory responses are a crucial component of WNV pathology, and understanding how they are regulated is important for tailoring effective treatments. Pro-inflammatory responses during WNV infection have been extensively studied, but anti-inflammatory responses (and their potential protective and reparative capabilities) following WNV infection have not been investigated. Minocycline induced the expression of genes associated with the anti-inflammatory (M2) activation of CNS macrophages (microglia) in WNV-infected SCSC whilst inhibiting the expression of genes associated with pro-inflammatory (M1) macrophage activation, and was protective for multiple CNS cell types indicating its potential use as a therapeutic reagent. This ex vivo culture system can uniquely address the ability of CNS parenchymal cells (neurons, astrocytes, microglia) to respond to minocycline and modulate the inflammatory environment and cytotoxicity in response to WNV infection without peripheral immune cell involvement.

  • HLA-B*14:02-RESTRICTED ENV-SPECIFIC CD8+ T-CELL ACTIVITY HAS HIGHLY POTENT ANTIVIRAL EFFICACY ASSOCIATED WITH IMMUNE CONTROL OF HIV INFECTION [PublishAheadOfPrint]

  • Immune control of human immunodeficiency virus type 1 (HIV) infection is typically associated with effective Gag-specific CD8+ T-cell responses. We here focus on HLA-B*14, that protects against HIV disease progression, but the immunodominant HLA-B*14-restricted anti-HIV response is Env-specific (ERYLKDQQL, llsquo;HLA-B*14-EL9rrsquo;). A subdominant HLA-B*14-restricted response targets Gag (DRYFKTLRA, llsquo;HLA-B*14-DA9rrsquo;). Using HLA-B*14/peptide-saporin conjugated tetramers, we show that HLA-B*14-EL9 is substantially more potent at inhibiting viral replication than HLA-B*14-DA9. HLA-B*14-EL9 also has significantly higher functional avidity (pllt;0.0001) and drives stronger selection pressure on the virus than HLA-B*14-DA9. However, these differences were HLA-B*14 subtype-specific, applying only to HLA-B*14:02 and not HLA-B*14:01. Furthermore, the HLA-B*14-associated protection against HIV disease progression is significantly greater for HLA-B*14:02 than for HLA-B*14:01, consistent with the superior antiviral efficacy of the HLA-B*14-EL9 response. Thus, although Gag-specific CD8+ T-cell responses may usually have greater anti-HIV efficacy, factors independent of protein specificity, including functional avidity of individual responses, are also critically important to immune control of HIV.

    IMPORTANCE In HIV infection, although CTL play a potentially critical role in eradication of viral reservoirs, the features that constitute an effective response remain poorly defined. We focus on HLA-B*14, unique among HLA associated with control of HIV in that the dominant CTL response is Env-specific, not Gag. We demonstrate that Env-specific HLA-B*14-restricted activity is substantially more efficacious than the subdominant HLA-B*14-restricted Gag response. Env immunodominance over Gag, and strong Env-mediated selection pressure on HIV, are only observed in subjects expressing HLA-B*14:02, and not HLA-B*14:01. This reflects increased functional avidity of Env response over Gag, substantially more marked for HLA-B*14:02. Finally, we show that HLA-B*14:02 is significantly more strongly associated with viraemic control than HLA-B*14:01. These findings indicate that, although Gag-specific CTL may usually have greater anti-HIV efficacy than Env responses, factors independent of protein specificity, including functional avidity, may carry greater weight in mediating effective control of HIV.

  • Population diversity and collective interactions during influenza virus replication and evolution [PublishAheadOfPrint]

  • Influenza A virus (IAV) continues to pose an enormous and unpredictable global public health threat, largely due to the continual evolution of escape from pre-existing immunity and the potential for zoonotic emergence. Understanding how the unique genetic make-up and structure of IAV populations influences their transmission and evolution is essential for developing more effective vaccines, therapeutics, and surveillance capabilities. Owing to their mutation-prone replicase and unique genome organization, IAV populations exhibit enormous amounts of diversity both in terms of sequence and functional gene content. Here, I review what is currently known about the genetic and genomic diversity present within IAV populations, and how this diversity may shape the replicative and evolutionary dynamics of these viruses.

  • Epstein-Barr Virus LMP1 Mediated Oncogenicity [PublishAheadOfPrint]

  • Epstein-Barr virus Latent Membrane Protein 1 (LMP1) is expressed in multiple human malignancies, including nasopharynegeal carcinoma, Hodgkin and immunosuppression-associated lymphomas. LMP1 mimics CD40 signaling to activate multiple growth and survival pathways, in particular NF-B. LMP1 has critical roles in EBV-driven B-cell transformation, and its expression causes fatal lymphoproliferative disease in immunosuppressed mice. Here, we review recent developments in studies of LMP1 signaling, LMP1-induced host dependency factors, mouse models of LMP1 lymphomagenesis and anti-LMP1 immunotherapy approaches.

  • Interferon regulatory factor-1 (IRF-1) protects against chikungunya virus induced immunopathology by restricting infection in muscle cells [PublishAheadOfPrint]

  • The innate immune system protects cells against viral pathogens in part through the autocrine and paracrine actions of interferons (IFN)-aalpha;/bbeta; (type I), - (type II), and - (type III). The transcription factor interferon regulatory factor (IRF)-1 has a demonstrated role in shaping innate and adaptive antiviral immunity by inducing the expression of IFN stimulated genes (ISGs) and mediating signals downstream of IFN-. Although ectopic expression experiments have suggested an inhibitory function of IRF-1 against infection of alphaviruses in cell culture, its role in vivo remains unknown. Here, we infected Irf1-/- mice with two distantly related arthritogenic alphaviruses, chikungunya (CHIKV) and Ross River (RRV), and assessed the early antiviral functions of IRF-1 prior to induction of adaptive B and T cell responses. IRF-1 expression limited CHIKV-induced foot swelling in joint-associated tissues and prevented dissemination of CHIKV and RRV at early time points. Virological and histological analysis revealed greater infection of muscle tissues in Irf1-/- compared to wild-type mice. The antiviral actions of IRF-1 appeared independent of the induction of type I IFN or effects of type II and III IFNs but were associated with altered local pro-inflammatory cytokine and chemokine responses and differential infiltration of myeloid cell subsets. Collectively, our in vivo experiments suggest that IRF-1 restricts CHIKV and RRV infection in stromal cells, especially muscle cells, and this controls local inflammation and joint-associated swelling.

    IMPORTANCE Interferon regulatory factor (IRF)-1 is a transcription factor that regulates the expression of a broad range of antiviral host defense genes. In this study, using Irf1-/- mice, we investigated the role of IRF-1 in modulating pathogenesis of two related arthritogenic alphaviruses, chikungunya and Ross River viruses. Our studies show that IRF-1 controlled alphavirus replication and swelling in joint-associated tissues within days of infection. Detailed histopathological and virological analyses revealed that IRF-1 preferentially restricted CHIKV infection in cells of non-hematopoietic lineage, including muscle cells. The antiviral actions of IRF-1 resulted in decreased local inflammatory responses in joint associated tissues, which prevented immunopathology.

  • HPV-16, HPV-18 and HPV-31 E6 override the normal phospho-regulation of E6AP enzymatic activity. [PublishAheadOfPrint]

  • The Human Papillomavirus (HPV) E6 oncoproteins recruit the cellular ubiquitin ligase, E6AP/UBE3A, to target cellular substrates for proteasome-mediated degradation, and one consequence of this activity is the E6 stimulation of E6AP auto-ubiquitination and degradation. Recent studies identified an autism-linked mutation within E6AP at T485, which was identified as a PKA phospho-acceptor site and which could directly regulate E6AP ubiquitin ligase activity. In this study we have analysed how T485-mediated regulation of E6AP might affect E6 targeting of some of its known substrates. We show that, modulation of T485 has no effect on the ability of E6 to direct either p53 or Dlg for degradation. Furthermore, T485 regulation has no effect on HPV-16 or HPV-31 E6-induced auto-degradation of E6AP, but does affect HPV-18 E6-induced auto-degradation of E6AP. In cells derived from cervical cancers, we find low levels of both phosphorylated and non-phosphorylated E6AP in the nucleus. However, ablation of E6 results in a dramatic accumulation of phospho-E6AP in the cytoplasm, whereas non-phosphorylated E6AP accumulates primarily in the nucleus. Interestingly, E6AP phosphorylation at T485 confers association with 14-3-3 proteins, and this interaction seems to be important, in part, for the ability of E6 to recruit phospho-E6AP into the nucleus. These results demonstrate that HPV E6 overrides the normal phospho-regulation of E6AP, both in terms of its enzymatic activity and its sub-cellular distribution.

    Importance Recent reports demonstrate the importance of phospho-regulation of E6AP for its normal enzymatic activity. Here we show that HPV E6 is capable of overriding this regulation and can promote degradation of p53 and Dlg regardless of the phosphorylation status of E6AP. Furthermore, E6 interaction with E6AP also significantly alters how E6AP is subject to auto-degradation, and suggests that this is not a simple stimulation of an already-existing activity, but rather a re-direction of E6AP activity towards itself. Furthermore, E6 mediated regulation of the subcellular distribution of phospho-E6AP appears to be dependent, in part, upon the 14-3-3 family of proteins.

  • Expression and subcellular localization of the KSHV K15P protein during latency and lytic reactivation in primary effusion lymphoma cells. [PublishAheadOfPrint]

  • The K15P membrane protein of Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with multiple cellular signaling pathways, and is thought to play key roles in KSHV-associated endothelial cell angiogenesis, regulation of B-cell receptor (BCR) signaling and the survival, activation and proliferation of BCR-negative primary effusion lymphoma (PEL) cells. Although full-length K15P is ~45kDa in size, numerous lower molecular weight forms of the protein exist as a result of differential splicing and poorly characterized post-translational processing. K15P has been reported to localize to numerous subcellular organelles in heterologous expression studies, but there is limited data concerning the sorting of K15P in KSHV-infected cells. The relationship between the various molecular weight forms of K15P, their subcellular distribution and how these may differ in latent and lytic KSHV infections is poorly understood. Here we report that a cDNA encoding a full-length ~45kDa K15P reporter protein is expressed as a ~23/34kDa species that colocalizes with the trans Golgi network marker TGN46 in KSHV-infected PEL cells. Following lytic reactivation by sodium butyrate the levels of the ~23-24kDa protein diminish and full-length ~45kDa K15P accumulates. This is accompanied by apparent fragmentation of the TGN and redistribution of K15P to a dispersed peripheral location. Similar results were seen when lytic reactivation was stimulated by the KSHV replication and transcription activator (RTA), and during spontaneous reactivation. We speculate that expression of differing molecular weight forms of K15P, in distinct cellular locations, reflects the alternative demands placed upon the protein in the latent and lytic phases.

    IMPORTANCE The K15P protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is thought to play key roles in disease, including KSHV-associated angiogenesis and the survival and growth of primary effusion lymphoma cells (PELs). The protein exists in multiple molecular weight forms and its intracellular trafficking is poorly understood. Here we demonstrate that the molecular weight form of a reporter K15P molecule, and its intracellular distribution, change when KSHV switches from its latent (quiescent) phase to the lytic, infectious state. We speculate that expression of differing molecular weight forms of K15P in distinct cellular locations reflects the alternative demands placed upon the protein in the viral latent and lytic stages.

  • PrP P102L and nearby lysine mutations promote spontaneous in vitro formation of transmissible prions [PublishAheadOfPrint]

  • Accumulation of fibrillar protein aggregates is a hallmark of many diseases. While numerous proteins form fibrils by prion-like seeded polymerization in vitro, only some are transmissible and pathogenic in vivo. To probe the structural features that confer transmissibility to prion protein (PrP) fibrils, we have analyzed synthetic PrP amyloids with or without the human prion disease-associated P102L mutation. The formation of infectious prions from PrP molecules in vitro has required cofactors and/or unphysiological denaturing conditions. Here, we demonstrate that, under physiologically compatible conditions without cofactors, the P102L mutation in recombinant hamster PrP promoted prion formation when seeded by minute amounts of scrapie prions in vitro. Surprisingly, combination of the P102L mutation with charge-neutralizing substitutions of four nearby lysines promoted spontaneous prion formation. When inoculated into hamsters, both of these types of synthetic prions initiated substantial accumulation of prion seeding activity and protease-resistant PrP without TSE clinical signs or notable glial activation. Our evidence suggests PrP's centrally located proline and lysine residues act as conformational switches in the in vitro formation of transmissible PrP amyloids.

    IMPORTANCE Many diseases involve the damaging accumulation of specific misfolded proteins in thread-like aggregates. These threads (fibrils) are capable of growing on the ends by seeding the refolding and incorporation of the normal form of the given protein. In many cases such aggregates can be infectious and propagate like prions when transmitted from one individual host to another. Some transmitted aggregates can cause fatal disease, as with human iatrogenic prion diseases, while other aggregates appear to be relatively innocuous. The factors that distinguish infectious and pathogenic protein aggregates from more innocuous ones are poorly understood. Here we have compared the combined effects of prion seeding and mutations of prion protein (PrP) on the structure and transmission properties of synthetic PrP aggregates. Our results highlight the influence of specific sequence features in the normally unstructured region of PrP that influence the infectious and neuropathogenic properties of PrP-derived aggregates.

  • Inherited chromosomally integrated human herpesvirus 6 genomes are ancient, intact and potentially able to reactivate from telomeres [PublishAheadOfPrint]

  • The genomes of human herpesviruses 6A and 6B (HHV-6A and HHV-6B) have the capacity to integrate into telomeres, the essential capping structures of chromosomes that play roles in cancer and ageing. About 1% of people worldwide are carriers of chromosomally integrated HHV-6 (ciHHV-6), which is inherited as a genetic trait. Understanding the consequences of integration for the evolution of the viral genome, for the telomere and for the risk of disease associated with carrier status is hampered by a lack of knowledge about ciHHV-6 genomes. Here, we report an analysis of 28 ciHHV-6 genomes and show that they are significantly divergent from the few modern non-integrated HHV-6 strains for which complete sequences are currently available. In addition ciHHV-6B genomes in Europeans are more closely related to each other than to ciHHV-6B genomes from China and Pakistan, suggesting regional variation of the trait. Remarkably, at least one group of European ciHHV-6B carriers has inherited the same ciHHV-6B genome, integrated in the same telomere allele, from a common ancestor estimated to have existed 24,500 pplusmn;10,600 years ago. Despite the antiquity of some, and possibly most, germline HHV-6 integrations, the majority of ciHHV-6B (95%) and ciHHV-6A (72%) genomes contain a full set of intact viral genes and therefore appear to have the capacity for viral gene expression and full reactivation.

    IMPORTANCE Inheritance of HHV-6A or HHV-6B integrated into a telomere occurs at a low frequency in most populations studied to date but its characteristics are poorly understood. However, stratification of ciHHV-6 carriers in modern populations due to common ancestry is an important consideration for genome-wide association studies that aim to identify disease risks for these people. Here we present full sequence analysis of 28 ciHHV-6 genomes and show that ciHHV-6B in many carriers with European ancestry most likely originated from ancient integration events in a small number of ancestors. We propose that ancient ancestral origins for ciHHV-6A and ciHHV-6B are also likely in other populations. Moreover, despite their antiquity, all of the ciHHV-6 genomes appear to retain the capacity to express viral genes, and most are predicted to be capable of full viral reactivation. These discoveries represent potentially important considerations in immune-compromised patients, in particular in organ transplantation and in stem cell therapy.

  • Nipah and Hendra Virus Nucleoproteins Inhibit Nuclear Accumulation of STAT1 and STAT2 by Interfering with Their Complex Formation [PublishAheadOfPrint]

  • Henipaviruses, such as Nipah (NiV) and Hendra (HeV) viruses, are highly pathogenic zoonotic agents within the Paramyxoviridae family. The phosphoprotein (P) gene products of the paramyxoviruses have been well characterized in their interferon (IFN) antagonist activity and their contribution to viral pathogenicity. In this study, we demonstrated that the nucleoprotein (N) of henipaviruses also prevents the host IFN signaling pathway. Reporter assays demonstrated that NiV and HeV N proteins (NiV-N and HeV-N, respectively) suppressed both type-I and type-II IFN responses dose-dependently, and the inhibitory effect was mediated by their core-domains. Additionally, NiV-N prevented the nuclear transport of STAT1 and STAT2. However, NiV-N did not associate with Impaalpha;5, Impbbeta;1, or Ran, which are members of the nuclear transport system for STATs. Although P protein is known as a binding partner of N protein and actively retains N protein in the cytoplasm, the IFN antagonist activity of N protein was not abolished by the co-expression of P protein. This suggests that the IFN inhibition by N protein occurs in the cytoplasm. Furthermore, we demonstrated that the complex formation of STATs was hampered in the N protein-expressing cells. As a result, STAT nuclear accumulation was reduced, causing a subsequent downregulation of ISGs (interferon-stimulated genes) due to low promoter occupancy by STAT complexes. This novel route for preventing host IFN responses by Henipavirus N proteins provides new insight for understanding the pathogenesis of these viruses.

    IMPORTANCE Paramyxoviruses are well known for suppressing interferon (IFN)-mediated innate immunity with their phosphoprotein (P) gene products, and the henipaviruses also possess P, V, W, and C proteins for evading host antiviral responses. There are numerous studies providing evidence for the relationship between viral pathogenicity and antagonistic activities against IFN responses by P gene products. Meanwhile, little attention has been paid to the influence of nucleoprotein (N) on host innate immune responses. In this study, we demonstrated that both NiV and HeV N proteins have an antagonistic activity against the JAK/STAT signaling pathway by preventing the nucleocytoplasmic trafficking of STAT1 and STAT2. This inhibitory effect was due to an impairment of their ability to form complexes. These results provide new insight into the involvement of N protein in viral pathogenicity via its IFN antagonism.

  • Attenuated human parainfluenza virus type 1 (HPIV1) expressing the respiratory syncytial virus (RSV) fusion F glycoprotein from an added gene: effects of pre-fusion stabilization and packaging of RSV F [PublishAheadOfPrint]

  • Human respiratory syncytial virus (RSV) is the most prevalent worldwide cause of severe respiratory tract infection in infants and young children. Human parainfluenza virus type 1 (HPIV1) also causes severe pediatric respiratory illness, especially croup. Both viruses lack vaccines. Here, we describe the preclinical development of a bivalent RSV/HPIV1 vaccine based on a recombinant HPIV1 vector, attenuated by a stabilized mutation, that expresses RSV F protein modified for increased stability in the pre-fusion (pre-F) conformation by previously-described disulfide bond (DS) and hydrophobic cavity-filling (Cav1) mutations. RSV F was expressed from the first or second gene position as the full-length protein or as a chimeric protein with its transmembrane (TM) and cytoplasmic tail (CT) domains substituted with those of HPIV1 F in an effort to direct packaging in the vector particles. All constructs were recovered by reverse genetics. The TMCT versions of RSV F were packaged in the rHPIV1 particles much more efficiently than their full-length counterparts. In hamsters, the presence of the RSV F gene, and in particular the TMCT versions, was attenuating and resulted in reduced immunogenicity. However, the vector expressing full-length RSV F from the pre-N position was immunogenic for RSV and HPIV1. It conferred complement-independent high-quality RSV-neutralizing antibodies at titers similar to those of wild type RSV and provided protection against RSV challenge. The vectors exhibited stable RSV F expression in vitro and in vivo. In conclusion, an attenuated rHPIV1 vector expressing a pre-F-stabilized form of RSV F demonstrated promising immunogenicity and should be further developed as an intranasal pediatric vaccine.

    Importance. RSV and HPIV1 are major viral causes of acute pediatric respiratory illness for which no vaccines or suitable antiviral drugs are available. The RSV F glycoprotein is the major RSV neutralization antigen. We used a rHPIV1 vector, bearing a stabilized attenuating mutation, to express the RSV F glycoprotein bearing amino acid substitutions that increase its stability in the pre-F form, the most immunogenic form that elicits highly functional virus-neutralizing antibodies. RSV F was expressed from the pre-N or N-P gene position of the rHPIV1 vector as a full-length protein or as a chimeric form with its TMCT domain derived from HPIV1 F. TMCT modification greatly increased packaging of RSV F into the vector particles but also increased vector attenuation in vivo, resulting in reduced immunogenicity. In contrast, full-length RSV F expressed from the pre-N position was immunogenic, eliciting complement-independent RSV-neutralizing antibodies and providing protection against RSV challenge.

  • Murine Olfactory Bulb Interneurons Survive Infection with a Neurotropic Coronavirus [PublishAheadOfPrint]

  • Viral infection of the central nervous system is complicated by the mostly irreplaceable nature of neurons, as the loss of neurons has the potential to result in permanent damage to brain function. However, whether neurons or other cells in the CNS sometimes survive infection and the effects of infection on neuronal function are largely unknown. To address this question, we used the rJHM strain (rJ) of mouse hepatitis virus, (MHV), a neurotropic coronavirus, which causes acute encephalitis in susceptible strains of mice. To determine whether neurons or other CNS cells survive acute infection with this virulent virus, we developed a recombinant JHMV that expresses Cre recombinase (rJ-Cre) and infected mice that universally expressed a silent (floxed) version of tdTomato. Infection of these mice with rJ-Cre resulted in expression of tdTomato in host cells. The results showed that some cells were able to survive the infection, as demonstrated by continued tdTomato expression after virus antigen could no longer be detected. Most notably, interneurons in the olfactory bulb, which are known to be inhibitory, represented a large fraction of the surviving cells. In conclusion, our results indicated that some neurons are resistant to virus-mediated cell death and provide a framework for studying the effects of prior coronavirus infection on neuron function.

    Importance. We developed a novel recombinant virus that allows for the study of cells that survive an infection by a central nervous system-specific strain of murine coronavirus. Using this virus, we identified neurons and to a lesser extent, non-neuronal cells in the brain that were infected during the acute phase of the infection and survived for approximately two weeks until the mice succumbed to the infection. We focused on neurons and glial cells within the olfactory bulb because the virus enters the brain at this site. Our results show that interneurons of the olfactory bulb were the primary cell type able to survive infection. Further, these results indicate that this system will be useful for functional and gene expression studies of cells in the brain that survive acute infection.

  • The uncoupling of protease trans-cleavage and helicase activities in the pestivirus NS3 [PublishAheadOfPrint]

  • The nonstructural protein NS3 from the Flaviviridae family is a multi-functional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the Classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at 2.35 AAring; resolution. The structure reveals a previously unidentified ~2200-AAring;2 intra-molecular protease-helicase interface comprising three clusters of interactions, representing a "closed" global conformation related to the NS3-NS4A cis-cleavage event. Although this conformation is incompatible with protease trans-cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities in vitro and virus production in vivo. Collectively, our work reveals important features of protease-helicase coordination in pestivirus NS3, and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein.

    IMPORTANCE Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the Flaviviridae family viruses are quite unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease cis-cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through different conformational states.

  • ZIC2 is Essential for Maintenance of Latency and is a Target of an Immediate-Early Protein during KSHV Lytic Reactivation [PublishAheadOfPrint]

  • Bivalent histone modifications are defined as repressive and activating epigenetic marks that simultaneously decorate the same genomic region. The H3K27me3 mark silences gene expression while H3K4me3 mark prevents the region from becoming permanently silenced and prepares the domain for activation when needed. Specific regions of Kaposi's sarcoma-associated herpesvirus (KSHV) latent episomes are "poised" to be activated by the KSHV replication and transcription activator (K-Rta). How KSHV episomes are prepared such that they maintain latent infection and switch to lytic replication by K-Rta remains unclear. K-Rta transactivation activity requires a protein degradation function, thus we hypothesized that identification of cellular substrates of K-Rta may provide insight into the maintenance of KSHV latent infection and the switch to lytic replication.

    Here we show that a zinc finger protein, ZIC2, a key regulator for central nervous system development, is a substrate of K-Rta, and is responsible for maintaining latency. K-Rta directly interacted with ZIC2 and functioned as an E3 ligase to ubiquitinate ZIC2. ZIC2 localized at immediate-early and early gene cluster regions of the KSHV genome, contributed to tethering Polycomb Repressive Complex 2 though physical interaction thus maintaining H3K27me3 marks at the K-Rta promoter. Accordingly, depletion of ZIC2 shifted balance of bivalent histone modifications towards more active forms and induced KSHV reactivation in naturally infected cells. We suggest that ZIC2 turnover by K-Rta is a strategy employed by KSHV to favor the transition from latency to lytic replication.

    IMPORTANCE Post-translational histone modifications regulate the accessibility of transcriptional factors to DNA; thus, they have profound effects on gene expression (e.g., viral reactivation). KSHV episomes are known to possess bivalent chromatin domains. How such KSHV chromatin domains are maintained to be reactivatable by K-Rta remains unclear. We found that ZIC2, an essential transcriptional factor for stem cell pluripotency, plays a role in maintaining KSHV latent infection in naturally infected cells. We found that ZIC2 degradation by K-Rta shifts bivalent histone marks to a more active configuration, leading to KSHV reactivation. ZIC2 interacts and maintains Polycomb Repressor Complex 2 at the K-Rta promoter. Our findings uncover (i) a mechanism utilized by KSHV to maintain latent infection, (ii) a latency-lytic cycle switch operated by K-Rta, and (iii) a molecular mechanism of ZIC2 mediated local histone modification.

  • HIV-1 resistance to dolutegravir is affected by cellular histone acetyltransferase activity [PublishAheadOfPrint]

  • Integrase strand transfer inhibitors are the newest class of antiretrovirals to have been approved for the treatment of HIV. Canonical resistance to these competitive inhibitors develops through substitutions in the integrase active site that disrupt drug-protein interactions. However resistance against the newest integrase inhibitor dolutegravir (DTG) is associated with a R263K substitution at the C-terminus of integrase that causes resistance through an unknown mechanism. The integrase C-terminal domain is involved in many processes over the course of infection and is post-translationally modified via acetylation of three lysine residues, that are important for enzyme activity, integrase multimerization, and protein-protein interactions. Here, we report that regulation of the acetylation of integrase is integral to the replication of HIV in the presence of DTG and that R263K specifically disrupts this regulation, likely due to enhancement of interactions with the histone deacetylase I complex, as suggested by co-immunoprecipitation. Although no detectable differences were observed in levels of cell-free acetylation of wild-type (WT) and mutated R263K enzymes, the inhibition of cellular histone acetyltransferase enzymes sensitized NL4.3WT viruses to DTG while NL4.3R263K was almost completely unaffected. When levels of endogenous acetylation were manipulated in virus-producing cells, inhibitors of acetylation enhanced the replication of NL4.3R263K whereas inhibition of deacetylation greatly diminished the replication of NL4.3WT. Taken together, these results point to a pivotal role of acetylation in the resistance mechanism of HIV to some second-generation integrase strand transfer inhibitors such as DTG.

    IMPORTANCE This is, to our knowledge, the first report of the influence of post-translational modifications on HIV drug resistance. Both viral replication and resistance to second-generation integrase strand transfer inhibitors of both WT and INSTI-resistant HIV were differentially affected by acetylation, likely as a result of altered interactions between integrase and the cellular deacetylation machinery. Many "shock and kill" strategies to eradicate HIV manipulate endogenous levels of acetylation in order to reactivate latent HIV. However, our results suggest that some drug resistant viruses may differentially respond to such stimulation, which may complicate the attainment of this goal. Our future work will further illuminate the mechanisms involved.

  • HIV-1 Cross-reactive Primary Virus Neutralizing Antibody Response Elicited by Immunization in Non-human Primate [PublishAheadOfPrint]

  • Elicitation of broadly neutralizing antibody (bNAb) responses is a major goal for the development of an HIV-1 vaccine. Current HIV-1 envelope glycoprotein (Env) vaccine candidates predominantly elicit tier 1 and/or autologous tier 2 virus neutralizing antibody (NAb) responses, as well as weak and/or sporadic cross-reactive tier 2 virus NAb responses with unknown specificity. To delineate the specificity of vaccine-elicited cross-reactive tier 2 virus NAb responses, we performed single memory B cell sorting from the peripheral blood of a rhesus macaque immunized with YU2gp140-F trimers in adjuvant, using JR-FL SOSIP.664, a native Env trimer mimetic as sorting probe to isolate monoclonal Abs (MAbs). We found striking genetic and functional convergence of the SOSIP-sorted Ig repertoire, with pre-dominant VH4 or VH5 gene family usage and Env V3-specificity. Of these vaccine-elicited V3-specific mAbs, nearly 20% (6/33) displayed cross-reactive tier 2 virus neutralization, which recapitulated the serum neutralization capacity. Substantial similarities in binding specificity, neutralization breadth and potency, and sequence/structural homology were observed between selected macaque cross-reactive V3 NAbs elicited by vaccination and prototypic V3 NAbs derived from natural infections in humans, highlighting the convergence of this subset of primate V3-specific B cell repertories. Our study demonstrated that cross-reactive primary virus neutralizing B cell lineages could be elicited by vaccination as detected using a standardized panel of tier 2 viruses. Whether these lineages could be expanded to acquire increased breadth and potency of neutralization merits further investigation.

    IMPORTANCE Elicitation of antibody responses capable of neutralizing diverse HIV-1 primary virus isolates (designated as broadly neutralizing antibodies, bNAbs) remains a high priority for the vaccine field. bNAb responses were so far only observed in response to natural infection and only in a subset of individuals. To achieve this goal, an improved understanding of vaccine-elicited responses including at the monoclonal Ab level is essential. Here, we isolated and characterized a panel of vaccine-elicited cross-reactive neutralizing mAbs targeting Env V3 loop that moderately neutralized several primary viruses and recapitulated the serum neutralizing antibody response. Striking similarities between the cross-reactive V3 NAbs elicited by vaccination in macaques and natural infections in humans illustrate commonalities between the vaccine- and infection-induced responses to V3 and support the feasibility of exploring the V3 epitope as HIV-1 vaccine target in non-human primates.

  • CD8+ T Cell Immune Response in Immunocompetent Mice during Zika Virus Infection [PublishAheadOfPrint]

  • Zika virus (ZIKV) infection cause neurologic complications, including Guillain-Barreeacute; syndrome (GBS) in adults and central nerve system (CNS) abnormalities in fetuses. We investigated the immune response, especially CD8+ T cell response in C57BL/6 (B6) wild type (WT) mouse during ZIKV infection. We found that robust CD8+ T cell response was elicited, MHC-I restricted CD8+ T cell epitopes were identified and a tetramer that recognized ZIKV specific CD8+ T cells was developed. Finally, virus specific memory CD8+ T cells were generated in these mice. The CD8+ T cells from these infected mice were functional, as evidenced by that adoptive transfer of ZIKV specific CD8+ T cells could protect CNS against ZIKV infection and cross protective to Dengue virus (DENV) infection. Thus, our findings provided comprehensive insight into the immune responses against ZIKV and demonstrated that WT mouse could be a natural and easy access model for evaluating immune response to ZIKV infection.

    IMPORTANCE ZIKV infection cause severe clinical consequences, including Guillain-Barreeacute; syndrome (GBS) in adults, microcephaly, congenital malformations in fetuses and newborn infants, therefore, study of the immune response especially adaptive immune response to ZIKV infection is important to understand the disease caused by ZIKV infection. The importance of our finding is that we characterized the CD8+ T cell immune response to ZIKV in a comprehensive manner, and identified ZIKV epitopes. Using the identified immunodominant epitope, we developed a tetramer that could recognize ZIKV specific CD8+ T cells in vivo, which simplified detection and evaluation of ZIKV specific immune responses. In addition, the finding that a tetramer positive memory CD8+ T cell responses were generated and that CD8+ T cells could traffic to ZIKV infected brain greatly enhanced our understanding of ZIKV infection and will provide important insight for ZIKV vaccines design.

  • Amino acid substitution K186E in the canine influenza virus H3N8 NS1 protein restores its ability to inhibit host gene expression [PublishAheadOfPrint]

  • Canine influenza viruses (CIVs) are the causative agents of canine influenza, a contagious respiratory disease in dogs, and include the equine-origin H3N8 and the avian-origin H3N2. Influenza A virus (IAV) non-structural protein 1 (NS1) is a virulence factor essential for counteracting the innate immune response. Here, we evaluated the ability of H3N8 CIV NS1 to inhibit host innate immune responses. We found that H3N8 CIV NS1 was able to efficiently counteract interferon (IFN) responses but was unable to block general gene expression in human or canine cells. Such ability was restored by a single amino acid substitution in position 186 (K186E) that resulted in NS1 binding to the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), a cellular protein involved in pre-mRNA processing. We also examined the frequency distribution of K186 and E186 among H3N8 CIVs and equine influenza viruses (EIVs), the ancestors of H3N8 CIV, and experimentally determined the impact of amino acid 186 in the ability of different CIV and EIV NS1s to inhibit general gene expression. In all cases, the presence of E186 was responsible for the control of host gene expression. Contrastingly, the NS1 protein of H3N2 CIV harbors E186 and blocks general gene expression in canine cells. Altogether, our results confirm previous studies on the strain-dependent ability of NS1 to block general gene expression. Moreover, the observed polymorphism on amino acid 186 between H3N8 and H3N2 CIVs might be the result of adaptive changes acquired during long-term circulation of avian-origin IAVs in mammals.

    IMPORTANCE Canine influenza is a respiratory disease of dogs caused by two CIV subtypes, the H3N8 and H3N2 viruses of equine and avian origin, respectively. Influenza NS1 is the main viral factor responsible for the control of host innate immune responses and changes in NS1 can play an important role in host adaptation. Here we assessed the ability of H3N8 CIV NS1 to inhibit host innate immune responses and gene expression. The H3N8 CIV NS1 did not block host gene expression but this activity was restored by a single amino acid substitution (K186E), which was responsible for NS1 binding to the host factor CPSF30. In contrast, the H3N2 CIV NS1, that contains E186, blocks general gene expression. Our results suggest that the ability to block host gene expression is not required for influenza replication in mammals but might be important in the long-term adaptation of avian-origin influenza viruses to mammals.

  • Cysteines and N-glycosylation Sites Conserved Among all Alphaherpesviruses Regulate Membrane Fusion in Herpes Simplex Virus Type 1 Infection [PublishAheadOfPrint]

  • Neurotropism is a defining characteristic of alphaherpesvirus pathogenicity. Glycoprotein K (gK) is a conserved virion glycoprotein of all alphaherpesviruses that is not found in other herpesvirus subfamilies. The extracellular amino terminus of gK has been shown to be important in the ability of the prototypic alphaherpesvirus HSV-1 to enter neurons via axonal termini. Herein, we determined the role of the two conserved N-linked glycosylation (N48 and N58) sites of gK in virus-induced cell fusion and replication. We found that N-linked glycosylation is important to the regulation of HSV-1-induced membrane fusion, since mutating N58 to alanine caused extensive virus-induced cell fusion. Due to the known contributions of N-linked glycosylation to protein processing and correct disulfide bond formation, we investigated whether the conserved extracellular cysteine residues within the amino terminus of gK contributed the regulation of HSV-1-induced membrane fusion. We found that mutation of C37 and C114 residues led to a gK-null phenotype characterized by very small plaque formation and drastic reduction in infectious virus production, while mutation of C82 and C243 caused extensive virus-induced cell fusion. Comparison of N-linked glycosylation and cysteine mutant replication kinetics identified disparate effects on infectious virion egress from infected cells. Specifically, cysteine mutations caused defects in the accumulation of infectious virus in both the cellular and supernatant fractions, while glycosylation site mutants did not adversely affect virion egress from infected cells. These results demonstrate a critical role for the N glycosylation sites and cysteines for the structure and function of the amino terminus of gK.

    IMPORTANCE We have previously identified important entry and neurotropic determinants in the amino terminus of HSV-1 glycoprotein K (gK). Alphaherpesvirus-mediated membrane fusion is a complex and highly regulated process that is not clearly understood. gK and UL20, which are highly conserved across all alphaherpesviruses, play important roles in the regulation of HSV-1 fusion in the context of infection. A greater understanding of mechanisms governing alphaherpesvirus membrane fusion is expected to inform the rational design of therapeutic and prevention strategies to combat herpesviral infection and pathogenesis. This work adds to the growing reports regarding the importance of gK to alphaherpesvirus pathogenesis and details important structural features of gK that are involved in gK-mediated regulation of virus-induced membrane fusion.

  • Asymptomatic primary infection with Epstein-Barr virus: observations on young adult cases [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) is typically acquired asymptomatically in childhood. By contrast, infection later in life often leads to infectious mononucleosis (IM), a febrile illness characterised by anti-EBV IgM antibody-positivity, high loads of circulating latently-infected B cells, and a marked lymphocytosis caused by hyper-expansion of EBV-specific CD8+ T cells plus milder expansion of CD56dim NKG2A+ KIRnndash; NK cells. How the two situations compare is unclear due to the paucity of studies on clinically-silent infection. Here we describe five prospectively-studied asymptomatic infections identified in a sero-epidemiological survey of University entrants. In each case the key blood sample had high cell-associated viral loads without marked IM-like CD8 lymphocytosis or NK cell disturbance. Two of the highest viral load cases showed a coincident expansion of activated EBV-specific CD8+ T cells but overall CD8+ T cell numbers were either unaffected or only mildly increased. Two slightly lower load cases, which serology suggests may have been caught earlier in the course of infection, also showed no T or NK cell expansion at the time. Interestingly, in another higher load case where T and NK cell responses were undetectable in the primary infection bleed, EBV-specific T cell responses did not appear until several months later, by which time virus loads in the blood had already fallen. Thus some asymptomatic primary infections have very high circulating viral loads and a cell-mediated immune response that is qualitatively similar to IM but of lower magnitude. However, others may be quite different and ultimately could reveal novel mechanisms of host control.

    IMPORTANCE Epstein-Barr virus (EBV) is transmitted orally, replicates in the throat and then invades the B lymphocyte pool through a growth-transforming latent infection. While primary infection in childhood is usually asymptomatic, delayed infection is associated with infectious mononucleosis (IM), a febrile illness with high circulating viral loads and an exaggerated virus-induced immune response involving both CD8+ T cells and natural-killer (NK) cells. Here we show that in five cases of asymptomatic infection, virus loads in the blood are as high as in acute IM whereas cell-mediated responses, even where they resemble IM in timing and quality, are never as exaggerated. We infer that IM symptoms arise as a consequence not of the virus infection per se, but of the hyper-activated immune response. Interestingly, there were idiosyncratic differences among asymptomatic cases in the relationship between viral load and response kinetics, emphasising how much there is still to learn about primary EBV infection.

  • Structural transitions of the conserved and metastable hantaviral glycoprotein envelope [PublishAheadOfPrint]

  • Hantaviruses are zoonotic pathogens with a near-global distribution that can cause severe hemorrhagic fever and pulmonary syndrome. The outer membrane of the hantavirus envelope displays a lattice of two glycoproteins, Gn and Gc, which orchestrate host cell recognition and entry. Here, we describe the crystal structure of the Gn glycoprotein ectodomain from the Asiatic Hantaan virus (HTNV), the most prevalent pathogenic hantavirus. Structural overlay analysis reveals that the HTNV Gn fold is highly similar to the Gn of Puumala virus (PUUV), a genetically and geographically distinct and less pathogenic hantavirus found predominantly in North-Eastern Europe, confirming that the hantaviral Gn fold is architecturally conserved across hantavirus clades. Interestingly, HTNV Gn crystallized at acidic pH, in a compact tetrameric configuration distinct from the organization at neutral pH. Analysis of the Gn, both in solution and in the context of the virion, confirms the pH-sensitive oligomeric nature of the glycoprotein, indicating that the hantaviral Gn undergoes structural transitions during host cell entry. These data allow us to present a structural model for how acidification during endocytic uptake of the virus triggers the dissociation of the metastable Gn-Gc lattice to enable insertion of the Gc-resident hydrophobic fusion loops into the host cell membrane. Together, these data reveal the dynamic plasticity of the structurally conserved hantaviral surface.

    IMPORTANCE Although the outbreaks of Korean hemorrhagic fever were first recognized during the Korean War (1950-53), it was not until 1978 that they were known to be caused by Hantaan virus (HTNV), the most prevalent pathogenic hantavirus. Here, we describe the crystal structure of HTNV envelope glycoprotein Gn, an integral component of the Gn-Gc glycoprotein spike complex responsible for host cell entry. HTNV Gn is structurally conserved with the Gn of a genetically and geographically distal hantavirus, Puumala virus, indicating that the observed aalpha;/bbeta; fold is well-preserved across the Hantaviridae family. Combination of our crystal structure with solution state analysis of recombinant protein and electron cryo-microscopy of acidified hantavirus allows us to propose a model for endosome-induced reorganization of the hantaviral glycoprotein lattice. This provides a molecular-level rationale for the exposure of the hydrophobic fusion loops on the Gc, a process required for fusion of viral and cellular membranes.

  • Baculovirus molecular evolution via gene turnover and recurrent positive selection of key genes [PublishAheadOfPrint]

  • Hosts and viruses are locked in an evolutionary arms race. Hosts are constantly evolving to suppress virulence and replication, while viruses, reliant on host machinery for survival and reproduction, develop counter-strategies to escape this immune defense. Viruses must also adapt to novel conditions when establishing themselves in a host species. Both processes provide strong selection for viral adaptation. Understanding adaptive evolution in insect viruses can help us better understand adaptive evolution in general, and is important due to their use as biocontrol agents, and for protecting ecologically or economically important species from outbreaks. Here we examine the molecular evolution of baculoviruses and nudiviruses, a group of insect-infecting viruses with key roles in biocontrol. We look for signatures of selection between baculoviruses genomes infecting a range of species and within a population of baculoviruses. Both analyses find only a few strong signatures of positive selection, primarily in replication aamp; transcription associated genes, and several structural proteins. In both analyses, we detect a conserved complex of genes, including helicase, showing consistently high levels of adaptive evolution, suggesting they may be key in the antagonistic co-evolution, to escape host suppression. These genes are integral to the baculoviruses life cycle and be good focal genes for developing baculoviruses as effective biocontrol agents, or for targeting baculoviruses infecting ecologically relevant species. Recombination and complex genomes make evolution in these double stranded DNA viruses more efficient than in smaller RNA viruses with error prone replication, seen via signatures of selection in specific genes within a population of baculoviruses.

    Importance Most viral evolutionary studies focus on RNA viruses. While these viruses cause many human and animal diseases, it leaves us with a lesser understanding of how DNA viruses adapt to hosts and how the host responds to these pathogens. In this paper, we focus on the evolution of baculovirus, a group of insect-infecting DNA viruses, many of which have been used in biocontrol. We find that most the genome is under purifying selection, with only a few key genes evolving adaptively. Our results provide a glimpse into how DNA viruses differ from RNA viruses in their evolutionary dynamics and identifies genes key to DNA virus adaptation, improving our understanding of how this group of pathogens evolves.

  • HIV fusion in Dendritic cells mainly occurs at the surface and is limited by low CD4 levels. [PublishAheadOfPrint]

  • HIV-1 poorly infects monocyte-derived dendritic cells (MDDCs). This is in a large part due to SAMHD1, which restricts viral reverse transcription. Pseudotyping HIV-1 with VSV-G strongly enhances infection, suggesting that earlier steps of viral replication, including fusion, are also inefficient in MDDCs. The site of HIV-1 fusion remains controversial and may depend on the cell type, with reports indicating that it occurs at the plasma membrane or contrarily, in an endocytic compartment. Here, we examined the pathways of HIV-1 entry in MDDCs. Using a combination of temperature shift and fusion inhibitors, we show that HIV-1 fusion mainly occurs at the cell surface. We then asked whether surface levels or intracellular localization of CD4 modulate HIV-1 entry. Increasing CD4 levels strongly enhanced fusion and infection with various HIV-1 isolates, including reference and transmitted/founder strains, but not with BaL, which uses low CD4 levels for entry. Overexpressing co-receptors did not facilitate viral infection. To further study the localization of fusion events, we generated CD4 mutants carrying heterologous cytoplasmic tails (LAMP1 or TLR7) to redirect the molecule to intracellular compartments. The intracellular CD4 mutants did not facilitate HIV-1 fusion and replication in MDDCs. Fusion of an HIV-2 isolate with MDDCs was also enhanced by increasing surface CD4 levels. Our results demonstrate that MDDCs are inefficiently infected by various HIV-1 and HIV-2 strains in part because of low CD4 levels. In these cells, viral fusion occurs mainly at the surface, and probably not after internalization.

    IMPORTANCE Dendritic Cells (DCs) are professional antigen-presenting cells inducing innate and adaptive immune responses. DCs express the HIV receptor CD4 and are potential target cells for HIV. There is debate about the sensitivity of DCs to productive HIV-1 and HIV-2 infection. The fusion step of the viral replication cycle is inefficient in DCs, and the underlying mechanisms are poorly characterized. We show that increasing levels of CD4 at the plasma membrane allows more HIV fusion and productive infection in DCs. We further demonstrate that HIV fusion occurs mainly at the cell surface and not in an intracellular compartment. Our results help understand why DCs are poorly sensitive to HIV infection.

  • HIV-1 exploits dynamic multi-aminoacyl-tRNA synthetase complex to enhance viral replication [PublishAheadOfPrint]

  • A hallmark of retroviruses such as human immunodeficiency virus type 1 (HIV-1) is reverse transcription of genomic RNA to DNA, a process that is primed by cellular tRNAs. HIV-1 recruits human tRNALys3 to serve as the reverse transcription primer via an interaction between lysyl-tRNA synthetase (LysRS) and the HIV-1 Gag polyprotein. LysRS is normally sequestered in a multi-aminoacyl-tRNA synthetase complex (MSC). Previous studies demonstrated that components of the MSC can be mobilized in response to certain cellular stimuli, but how LysRS is redirected from the MSC to viral particles for packaging is unknown. Here, we show that upon HIV-1 infection, a free pool of non-MSC associated LysRS is observed and partially relocalized to the nucleus. Heat inactivation of HIV-1 blocks nuclear localization of LysRS but treatment with a reverse transcriptase inhibitor does not, suggesting that the trigger for relocalization occurs prior to reverse transcription. A reduction in HIV-1 infection is observed upon treatment with an inhibitor to mitogen-activated protein kinase that prevents phosphorylation of LysRS on Ser207, release of LysRS from the MSC and nuclear localization. A phosphomimetic mutant of LysRS (S207D), that lacked the capability to aminoacylate tRNALys3, localized to the nucleus, rescued HIV-1 infectivity, and was packaged into virions. In contrast, a phosphoablative mutant (S207A) remained cytosolic and maintained full aminoacylation activity, but failed to rescue infectivity and was not packaged. These findings suggest that HIV-1 takes advantage of the dynamic nature of the MSC to redirect and co-opt cellular translation factors to enhance viral replication.

    IMPORTANCE Human tRNALys3, the primer for reverse transcription, and LysRS are essential host factors packaged into HIV-1 virions. Previous studies found that tRNALys3 packaging depends on interactions between LysRS and HIV-1 Gag; however, many details regarding the mechanism of tRNALys3 and LysRS packaging remain unknown. LysRS is normally sequestered in a high molecular weight multi-aminoacyl-tRNA synthetase complex (MSC), restricting the pool of free LysRS:tRNALys. Mounting evidence suggests that LysRS is released under a variety of stimuli to perform alternative functions within the cell. Here, we show that HIV-1 infection results in a free pool of LysRS that is re-localized to the nucleus of target cells. Blocking this pathway in HIV-1 producing cells resulted in less infectious progeny virions. Understanding the mechanism by which LysRS is recruited into the viral assembly pathway can be exploited for the development of specific and effective therapeutics targeting this non-translational function.

  • Proteomic and functional analyses of the virion transmembrane proteome of cyprinid herpesvirus 3 [PublishAheadOfPrint]

  • Virion transmembrane proteins (VTPs) mediate key functions in the herpesvirus infectious cycle. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses. The present study was devoted to CyHV-3 VTPs. Using mass spectrometry approaches, we identified 16 VTPs of the CyHV-3 FL strain. Mutagenesis experiments demonstrated that eight of these proteins are essential for viral growth in vitro (ORF32, ORF59, ORF81, ORF83, ORF99, ORF106, ORF115, and ORF131), and eight are non-essential (ORF25, ORF64, ORF65, ORF108, ORF132, ORF136, ORF148, and ORF149). Among the non-essential proteins, deletion of ORF25, ORF132, ORF136, ORF148, or ORF149 affects viral replication in vitro, and deletion of ORF25, ORF64, ORF108, ORF132, or ORF149 impacts plaque size. Lack of ORF148 or ORF25 causes attenuation in vivo to a minor or major extent, respectively. The safety and efficacy of a virus lacking ORF25 were compared to those of a previously described vaccine candidate deleted for ORF56 and ORF57 (56-57). Using quantitative PCR, we demonstrated that the ORF25 deleted virus infects fish through skin infection and then spreads to internal organs as reported previously for the wild-type parental virus and the 56-57 virus. However, compared to the parental wild-type virus, the replication of the ORF25 deleted virus was reduced in intensity and duration to levels similar to those observed for the 56-57 virus. Vaccination of fish with a virus lacking ORF25 was safe but had low efficacy at the doses tested. This characterization of the virion transmembrane proteome of CyHV-3 provides a firm basis for further research on alloherpesvirus VTPs.

    IMPORTANCE Virion transmembrane proteins play key roles in the biology of herpesviruses. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses and the causative agent of major economic losses in common and koi carp worldwide. In this study of the virion transmembrane proteome of CyHV-3, the major findings were: (i) the FL strain encodes 16 virion transmembrane proteins; (ii) eight of these proteins are essential for viral growth in vitro; (iii) seven of the non-essential proteins affect viral growth in vitro, and two affect virulence in vivo; and (iv) a mutant lacking ORF25 is highly attenuated but induces moderate immune protection. This study represents a major breakthrough in understanding the biology of CyHV-3 and will contribute to the development of prophylactic methods. It also provides a firm basis for the further research on alloherpesvirus virion transmembrane proteins.

  • Mutation of the Putative Immunosuppressive Domain of the Retroviral Envelope Glycoprotein Compromises Infectivity [PublishAheadOfPrint]

  • The envelope glycoprotein of diverse endogenous and exogenous retroviruses is considered inherently immunosuppressive. Extensive work mapped the immunosuppressive activity to a highly conserved domain, termed the immunosuppressive domain (ISD), in the transmembrane (TM) subunit of the envelope glycoprotein and identified two naturally polymorphic key residues that afford immunosuppressive activity to distinct envelope glycoproteins. Concurrent mutation of these two key residues (E14R and A20F) in the envelope glycoprotein of Friend murine leukemia virus (F-MLV) ISD has been reported to abolish its immunosuppressive activity, without affecting its fusogenicity, and to weaken the ability of the virus to replicate specifically in immunocompetent hosts. Here, we show that mutation of these key residues did in fact result in substantial loss of F-MLV infectivity, independent of host immunity, challenging associations between the two. Notably, loss of infectivity incurred by the E14R and A20F double ISD mutant F-MLV was conditional on expression of the ecotropic envelope receptor mCAT1 (cationic amino acid transporter -1) in the virus-producing cell. Indeed the mutant F-MLV retained infectivity when produced by human cells, naturally lacking mCAT1 expression, but not by murine cells. Furthermore, mCAT1 overexpression in human cells impaired the infectivity of both double mutant and wild-type F-MLV, suggesting a finely-tuned relationship between levels of mCAT1 in the producer cell and infectivity of the virions produced. An adverse effect on this relationship, rather than disruption of the putative ISD, is therefore more likely to explain the loss of F-MLV infectivity incurred by mutations in the key E14 and A20 ISD residues.

    IMPORTANCE Retroviruses can interact with their hosts in ways that, although not entirely understood, can greatly influence their pathogenic potential. One such example is a putative immunosuppressive activity, mapped to a conserved domain of the retroviral envelope glycoprotein of several exogenous, as well as endogenous retroviruses. In this study, mutations naturally found in some envelope glycoproteins lacking immunosuppressive activity were shown to affect retrovirus infectivity, only if the host cell that produces the retrovirus also expresses the cellular entry receptor. These findings shed light on a novel role for this conserved domain in providing the necessary stability to the envelope glycoprotein, in order to withstand interaction with the cellular receptor during virus formation. This function of the domain is critical for further elucidation of the mechanism of immunosuppression mediated by the retroviral envelope glycoprotein.

  • Structure-Guided Functional Annotation of the Influenza A Virus NS1 Protein Reveals Dynamic Evolution of the p85{beta}-Binding Site During Circulation in Humans [PublishAheadOfPrint]

  • Rational characterization of virulence and host-adaptive markers in the multifunctional influenza A virus NS1 protein is hindered by a lack of comprehensive knowledge about NS1-host protein-protein interfaces. Here, we surveyed the impact of amino-acid variation in NS1 at its structurally-defined binding site for host p85bbeta;, a regulator of phosphoinositide-3 kinase (PI3K) signaling. Structure-guided alanine-scanning of all viral residues at this interface defined 10 positions contributing to the interaction, with residues 89, 95, 98, 133, 145 and 162 being most important. Bioinformatic study of ggt;24,000 publicly-available NS1 sequences derived from viruses infecting different hosts highlighted several prevalent amino-acid variants at the p85bbeta; interface that either enhanced (I95) or weakened (N135, T145, L161, Y161, S164) p85bbeta;-binding. Interestingly, analysis of viruses circulating in humans since the 1918 pandemic revealed temporal acquisition of functionally relevant variants at this interface. I95 (enhanced p85bbeta;-binding) quickly became prevalent in the 1940s, and experimentally conferred a fitness advantage to a recombinant 1930s-based H1N1 virus in human lung epithelial cells. Surprisingly, both H1N1 and H3N2 viruses recently acquired T145 or N135, respectively, which diminished p85bbeta;-binding, but apparently not overall fitness in the human population. Evolutionary analyses revealed co-variation of the NS1-p85bbeta; binding phenotype in humans with functional changes at multiple residues in other viral proteins, suggesting unexplored compensatory or synergistic interplay between phenotypes in vivo. Overall, our data provide a resource to understand consequences of the NS1-p85bbeta; binding spectrum of different influenza viruses, and highlight dynamic evolution of this property in viruses circulating in humans.

    IMPORTANCE In humans, influenza A viruses are responsible for causing seasonal epidemics and occasional pandemics. These viruses also circulate and evolve in other animal species, creating a reservoir from which novel viruses with distinct properties can emerge. The viral non-structural protein, NS1, is an important host-range determinant and virulence factor that exhibits strain-specific interactions with several host factors, although few have been characterized extensively. Here, we comprehensively surveyed the impact of natural and unnatural NS1 variation on the binding of NS1 to host p85bbeta;, a subunit of phosphoinositide-3 kinase that regulates intracellular metabolism and contributes to virus replication and virulence. We define the p85bbeta;-binding site on NS1 and provide a predictive resource to assess this NS1 ability in viruses from different hosts. Strikingly, we uncover a spectrum of p85bbeta;-binding by different NS1s, and reveal that viruses evolving in humans have undergone dynamic changes in this NS1 function over the last century.

  • BA71{Delta}CD2: A new recombinant live attenuated African swine fever virus with cross-protective capabilities [PublishAheadOfPrint]

  • African swine fever is a highly contagious viral disease of mandatory declaration to the World Organization for Animal Health (OIE). Lack of available vaccines make its control difficult and thus ASFV represents a major threat to the swine industry. Inactivated vaccines do not confer solid protection against African swine fever virus (ASFV). Conversely, live attenuated viruses (LAV), either naturally isolated or obtained by genetic manipulation, have demonstrated reliable protection against homologous ASFV strains, albeit little or no protection has been demonstrated against heterologous viruses. Safety concerns are a major issue for the use of ASFV attenuated vaccine candidates, and has hampered their implementation in the field so far. While trying to develop safer and efficient ASFV vaccines, we found and demonstrate here that the deletion of the viral CD2v (EP402R) gene, highly attenuated the virulent BA71 strain in vivo. Inoculation of pigs with the deletion mutant virus BA71CD2 conferred protection not only against the lethal challenge with the parental BA71 but also against the heterologous E75 (both genotype I strains). The protection induced was dose-dependent and the cross-protection observed in vivo correlated with the ability of BA71CD2 to induce specific CD8+ T-cells capable of recognizing both BA71 and E75 viruses in vitro. Interestingly, 100% of the pigs immunized with BA71CD2 also survived lethal challenge with Georgia 2007/1, the genotype II strain of ASFV currently circulating in Continental Europe. These results open new avenues to design ASFV cross-protective vaccines, essential to fight ASFV in endemic areas where multiple viruses are circulating.

    Importance African swine fever virus (ASFV) remains endemic in most countries of Sub-Saharan Africa, today representing a major threat for the development of their swine industry. The uncontrolled presence of ASFV has favored its periodic exportation to other countries, the last event being Georgia in 2007. Since then, ASFV has spread towards neighboring countries reaching the European Union's East border in 2014. Lack of available vaccines against ASFV make its control difficult and so far, only live attenuated viruses have demonstrated solid protection against homologous experimental challenges, but have failed at inducing solid cross-protective immunity against heterologous viruses. Here we describe a new LAV candidate with unique cross-protective abilities: BA71CD2. Thus, inoculation of BA71CD2 protected pigs not only against the experimental challenge with BA71, the virulent parental strain, but also against heterologous viruses, including Georgia 2007/1, the genotype II strain of ASFV currently circulating in East Europe.

  • Screening of FDA-Approved Drugs for Inhibitors against Japanese Encephalitis Virus Infection [PublishAheadOfPrint]

  • Japanese encephalitis virus (JEV), an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. There is no approved drug available for JEV-specific treatment, and the vaccines are not effective against all clinical JEV isolates. Herein, a high-throughput screening was performed against JEV from an FDA-approved drug library. Five hit drugs were identified that inhibited JEV infection with a selective index ggt; 10. Antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors were utilized that confirmed calcium was essential for JEV infection, most likely during viral replication. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the non-structural NS4B protein while relatively conserved in flavivirus, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca2+ channel (VGCC) inhibitor, without apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing viral load in brain, while abrogating histopathological changes associated with JEV infection. This study provided five anti-flavivirus candidates and identified cytoplasmic calcium as a novel antiviral target for treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flavivirus.

    IMPORTANCE Currently there is no approved therapy to treat Japanese Encephalitis Virus infection. Repurposing of the approved drugs will accelerate the development of the therapeutic stratagem. In this study, we screened an FDA-drugs library and identified five hit drugs, especially calcium inhibitors, exerting anti-flavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a JEV-infected mouse model and the viral target was identified by generating adaptive mutant.

  • Escape of tick-borne flavivirus from 2 -C-methylated nucleoside antivirals is mediated by a single conservative mutation in NS5 that has a dramatic effect on viral fitness [PublishAheadOfPrint]

  • Tick-borne encephalitis virus (TBEV) causes a severe and potentially fatal neuroinfection in humans. Despite its high medical relevance, no specific antiviral therapy is currently available. Here we demonstrate that treatment with a nucleoside analog, 7-deaza-2rrsquo; -C-methyladenosine (7-deaza-2rrsquo; -CMA), substantially improved disease outcome, increased survival, and reduced signs of neuroinfection and viral titers in the brains of mice infected with a lethal dose of TBEV. To investigate the mechanism of action of 7-deaza-2rrsquo; -CMA, two drug-resistant TBEV clones were generated and characterized. The two clones shared a signature amino acid substitution, S603T, in the viral NS5 RNA-dependent RNA polymerase (RdRp) domain. This mutation conferred resistance to various 2rrsquo; -C-methylated nucleoside derivatives, but no cross-resistance was seen to other nucleoside analogs, such as 4rrsquo; -C-azidocytidine and 2rrsquo; -deoxy-2rrsquo; -beta-hydroxy-4rrsquo; -azidocytidine (RO-9187). All-atom molecular dynamics simulations revealed that the S603T RdRp mutant repels a water molecule that coordinates the position of a metal ion cofactor as 2rrsquo; -C-methylated nucleoside analogs approach the active site. To investigate its phenotype, the S603T mutation was introduced into a recombinant TBEV (Oshima-IC) generated from an infectious cDNA clone and into a TBEV replicon that expresses a reporter luciferase gene (Oshima-REP-luc2A). The mutants were replication-impaired, showing reduced growth and small plaque size in mammalian cell culture and reduced levels of neuroinvasiveness and neurovirulence in rodent models. These results indicate that TBEV resistance to 2rrsquo; -C-methylated nucleoside inhibitors is conferred by a single conservative mutation that causes a subtle atomic effect within the active site of viral NS5 RdRp and is associated with strong attenuation of the virus.

    Importance This study found that the nucleoside analog 7-deaza-2rrsquo; -C-methyladenosine (7-deaza-2rrsquo; -CMA) has high antiviral activity against tick-borne encephalitis virus (TBEV), a pathogen that causes severe human neuroinfections in large areas of Europe and Asia and for which there is currently no specific therapy. Treating mice infected with a lethal dose of TBEV with 7-deaza-2rrsquo; -CMA resulted in significantly higher survival rates, reduced the severity of neurological signs of the disease. Thus, this compound shows promise for further development as an anti-TBEV drug. It is important to generate drug-resistant mutants to understand how the drug works and to develop guidelines for patient treatment. We generated TBEV mutants that were resistant not only to 7-deaza-2rrsquo; -CMA but also to a broad range of other 2rrsquo; -C-methylated antiviral medications. Our findings suggest that combination therapy could be used to improve treatment and reduce the emergence of drug-resistant viruses during nucleoside analog therapy for TBEV infection.

  • Extracellular Matrix Proteins Mediate HIV-1 gp120 Interactions with {alpha}4{beta}7 [PublishAheadOfPrint]

  • Gut-homing aalpha;4bbeta;7high CD4+ T lymphocytes have been shown to be preferentially targeted by Human Immunodeficiency Virus-1 (HIV), and are implicated in HIV pathogenesis. Previous studies demonstrated that HIV envelope protein gp120 binds and signals through aalpha;4bbeta;7, and that this likely contributes to the infection of aalpha;4bbeta;7high T cells and promotes cell-to-cell virus transmission. Structures within the second variable loop (V2) of gp120, including the tripeptide motif LDV/I, are thought to mediate gp120-aalpha;4bbeta;7 binding. However, lack of aalpha;4bbeta;7 binding has been reported in gp120 proteins containing LDV/I, and the precise determinants of gp120-aalpha;4bbeta;7 binding are not fully defined. In this work, we report the novel finding that fibronectins mediate indirect gp120-aalpha;4bbeta;7 interactions. We show that Chinese Hamster Ovary (CHO) cells used to express recombinant gp120 produced fibronectins and other extracellular matrix proteins that co-purified with gp120. CHO fibronectins were able to mediate the binding of a diverse panel of gp120 proteins to aalpha;4bbeta;7 in an in vitro cell binding assay. The V2 loop was not required for fibronectin-mediated binding of gp120 to aalpha;4bbeta;7, nor did V2-specific antibodies block this interaction. Removal of fibronectin through anion exchange chromatography abrogated V2-independent gp120-aalpha;4bbeta;7 binding. Additionally, we showed a recombinant human fibronectin fragment mediated gp120-aalpha;4bbeta;7 interactions in a similar manner to CHO fibronectin. These findings provide an explanation for the apparent contradictory observations regarding the gp120-aalpha;4bbeta;7 interaction and offer new insights into the potential role of fibronectin and other extracellular matrix proteins in HIV-1 biology.

    IMPORTANCE Immune tissues within the gut are severely damaged by HIV, and this plays an important role in the development of AIDS. Integrin aalpha;4bbeta;7 plays a major role in the trafficking of lymphocytes, including CD4+ T cells, into gut lymphoid tissues. Previous reports indicate that some HIV gp120 envelope proteins bind to and signal through aalpha;4bbeta;7, which may help explain the preferential infection of gut CD4+ T cells. In this study, we demonstrate that extracellular matrix proteins can mediate interactions between gp120 and aalpha;4bbeta;7. This suggests that the extracellular matrix may be an important mediator of HIV interaction with aalpha;4bbeta;7nndash;expressing cells. These findings provide new insight into the nature of HIV-aalpha;4bbeta;7 interactions, and how these interactions may represent targets for therapeutic intervention.

  • FDG Uptake in Lymphoid Tissue serves as a Predictor of Disease Outcome in the Nonhuman Primate Model of Monkeypox Infection [PublishAheadOfPrint]

  • Real-time bioimaging of infectious disease processes may aid countermeasure development and lead to an improved understanding of pathogenesis. However, few studies have identified biomarkers for monitoring infections using in vivo imaging. Previously, we demonstrated that positron emission tomography/computed tomography (PET/CT) imaging with [18F]-fluorodeoxyglucose (FDG) can monitor monkeypox disease progression in vivo in nonhuman primates (NHPs). In this study, we investigated [18F]-FDG-PET/CT imaging of immune processes in lymphoid tissues to identify patterns of inflammation in the monkepox NHP model and to determine the value of [18F]-FDG-PET/CT as a biomarker for disease and treatment outcomes. Quantitative analysis of [18F]-FDG-PET/CT images revealed differences between moribund and surviving animals at two sites vital to the immune response to viral infections, bone marrow and lymph nodes (LN). Moribund NHPs demonstrated increased [18F]-FDG uptake in bone marrow 4 days post-infection when compared to surviving NHPs. In surviving, treated NHPs, increase in LN volume correlated to [18F]-FDG uptake and peaked 10 days post-infection while minimal lymphadenopathy and higher glycolytic activity were observed in moribund NHPs early in infection. Imaging data were supported by standard virology, pathology, and immunology findings. Even with the limited number of subjects, imaging was able to differentiate the difference between disease outcomes warranting additional studies to demonstrate whether [18F]-FDG-PET/CT can identify other subtler effects. Visualizing altered metabolic activity at sites involved in the immune response by [18F]-FDG-PET/CT imaging is a powerful tool for identifying key disease-specific time points and locations that are most relevant for pathogenesis and treatment.

    IMPORTANCE Positron emission tomography and computed tomography (PET/CT) imaging is a universal tool in oncology and neuroscience. The application of this technology to the infectious diseases is far less developed. We used PET/CT imaging with 18F-labeled deoxyglucose ([18F]-FDG) in monkeys after monkeypox virus exposure to monitor the immune response in lymphoid tissues. In lymph nodes of surviving monkeys, changes in [18F]-FDG uptake positively correlated with enlargement of the lymph nodes and peaked on day 10 post-infection. In contrast, the bone marrow and lymph nodes of non-survivors showed increased [18F]-FDG uptake by day 4 post-infection with minimal lymph node enlargement indicating that elevated cell metabolic activity early after infection is predictive of disease outcome. [18F]-FDG-PET/CT imaging can provide real-time snapshots of metabolic activity changes in response to viral infections and identify key time points and locations most relevant for monitoring the development of pathogenesis and for potential treatment to be effective.

  • A Single-Amino-Acid Substitution at Position 225 in HA Alters the Transmissibility of Eurasian Avian-like H1N1 Swine Influenza Virus in Guinea Pigs [PublishAheadOfPrint]

  • Efficient transmission from human to human is the prerequisite for an influenza virus to cause a pandemic; however, the molecular determinants of influenza virus transmission are still largely unknown. In this study, we explored the molecular basis for transmission of Eurasian avian-like H1N1 (EAH1N1) swine influenza viruses by comparing two viruses that are genetically similar but differ in their transmissibility in guinea pigs: the A/swine/Guangxi/18/2011 virus (GX/18) is highly transmissible by respiratory droplet in guinea pigs, whereas the A/swine/Heilongjiang/27/2012 virus (HLJ/27) does not transmit in this animal model. We used reverse genetics to generate a series of reassortants and mutants in the GX/18 background and tested their transmissibility in guinea pigs. We found that a single amino acid substitution of glycine (G) for glutamic acid (E) at position 225 (E225G) in the HA1 protein completely abolished the respiratory droplet transmission of GX/18, whereas the substitution of E for G at the same position (G225E) in HA1 enabled HLJ/27 to transmit in guinea pigs. We investigated the underlying mechanism and found that viruses bearing 225E in HA1 replicated more rapidly than viruses bearing 225G due to differences in assembly and budding efficiency. Our study indicates that the amino acid 225E in HA1 plays a key role in EAH1N1 swine influenza virus transmission and provides important information for evaluating the pandemic potential of field influenza strains.

    IMPORTANCE Efficient transmission among humans is a prerequisite for a novel influenza virus to cause a human pandemic. Transmissibility of influenza viruses is a polygenic trait, and understanding the genetic determinants for transmissibility will provide useful insights for evaluating the pandemic potential of influenza viruses in the field. Several amino acids in the hemagglutinin (HA) protein of influenza viruses have been shown to be important for transmissibility, usually by increasing virus affinity for human-type receptors. In this study, we explored the genetic basis of the transmissibility difference between two Eurasian avian-like H1N1 (EAH1N1) swine influenza viruses in guinea pigs, and found that the amino acid glutamic acid at position 225 in the HA1 protein plays a critical role in the transmission of EAH1N1 virus by increasing the efficiency of viral assembly and budding.

  • Zinc salts block Hepatitis E virus replication by inhibiting the activity of viral RNA-dependent RNA polymerase [PublishAheadOfPrint]

  • Hepatitis E virus (HEV) causes an acute, self limiting hepatitis in normal individuals and leads to chronic disease in immunocompromised individuals. HEV infection in pregnant women results in a more severe outcome, with the mortality rate going upto 30%. Though the virus usually causes sporadic infection, epidemics have been reported in developing and resource starved countries. No specific anti-viral exists against HEV. A combination of interferon and ribavirin therapy has been used to control the disease with some success. Zinc is an essential micronutrient that plays crucial roles in multiple cellular processes. Zinc salts are known to be effective in reducing infections caused by few viruses. Here, we investigated the effect of zinc salts on HEV replication. In human hepatoma cell (Huh7) culture model, zinc salts inhibited the replication of g-1 (genotype-1), g-3 HEV replicons and g-1 HEV infectious genomic RNA in a dose dependent manner. Analysis of a replication defective mutant of g-1 HEV genomic RNA under similar conditions ruled out the possibility of zinc salts acting on replication independent processes. An ORF4-Huh7 cell line based infection model of g-1 HEV further confirmed the above observations. Zinc salts did not show any effect on the entry of g-1 HEV into the host cell. Furthermore, our data reveals that zinc salts directly inhibit the activity of viral RNA-dependent RNA polymerase (RdRp), leading to inhibition of viral replication. Taken together, these studies unravel the ability of zinc salts in inhibiting HEV replication, suggesting their possible therapeutic value in controlling HEV infection.

    Importance Hepatitis E virus (HEV) is a public health concern in resource starved countries due to frequent outbreaks. It is also emerging to be a health concern in developed countries owing to its ability to cause acute and chronic infection in organ transplant and immunocompromised individuals. Although antivirals such as Ribavirin has been used to treat HEV cases, there are known side effects and limitations of such therapy. Our discovery of the ability of zinc salts to block HEV replication by virtue of their ability to inhibit the activity of viral RdRp is important because these findings pave the way to test the efficacy of zinc supplementation therapy in HEV infected patients. Since zinc supplementation therapy is known to be safe in normal individuals and high dose zinc is used in the treatment of Wilson's disease, it may be possible to control HEV associated health problems following a similar treatment regimen.

  • Contributions of individual domains to function of the HIV-1 Rev response element [PublishAheadOfPrint]

  • The HIV-1 Rev response element (RRE) is a 351-base element in unspliced and partially spliced viral RNA; binding of the RRE by the viral Rev protein induces nuclear export of RRE-containing RNAs, as required for virus replication. It contains one long, imperfect double helix (domain I), one branched domain (domain II) containing a high-affinity Rev-binding site, and two or three additional domains. We previously reported that the RRE assumes an "A" shape in solution and suggested that the location of the Rev binding sites in domains I and II, opposite each other on the two legs of the A, is optimal for Rev binding and explains Rev's specificity for RRE-containing RNAs. Using SAXS and a quantitative functional assay, we have now analyzed a panel of RRE mutants. All the results support the essential role of the A shape for RRE function. Moreover, they suggest that the distal portion of domain I and the three crowning domains all contribute to the maintenance of the A shape. Domains I and II are necessary and sufficient for substantial RRE function, provided they are joined by a flexible linker that allows the two domains to face each other.

    IMPORTANCE Retroviral replication requires that some of the viral RNAs transcribed in the cell nucleus be exported to the cytoplasm without being spliced. To achieve this, HIV-1 encodes a protein, Rev, which binds to a complex, highly structured element within viral RNA, the Rev Response Element (RRE), and escorts RRE-containing RNAs from the nucleus. We previously reported that the RRE is "A"-shaped and suggested that this architecture, with the 2 legs opposite one another, can explain the specificity of Rev for the RRE. We have analyzed the functional contributions of individual RRE domains, and now report that several domains contribute, with some redundancy, to maintenance of the overall RRE shape. The data strongly support the hypothesis that the opposed placement of the 2 legs is essential for RRE function.

  • Zoonotic Risk, Pathogenesis, and Transmission of Avian-Origin H3N2 Canine Influenza Virus [PublishAheadOfPrint]

  • Two subtypes of influenza A virus (IAV), avian-origin canine influenza virus H3N2 (CIV-H3N2) and equine-origin CIV-H3N8, are enzootic in the canine population. Dogs have demonstrated seroconversion to diverse IAVs and naturally occurring reassortants of CIV-H3N2 and the 2009 H1N1 pandemic virus (pdmH1N1) have been isolated. We conducted a thorough phenotypic evaluation of CIV-H3N2 in order to assess its threat to human health. Using ferret-generated antisera we determined that CIV-H3N2 is antigenically distinct from contemporary human H3N2 IAVs, suggesting there may be minimal herd immunity in humans. We assessed the public health risk of CIV-H3N2xpdmH1N1 reassortants by characterizing in vitro genetic compatibility and in vivo pathogenicity and transmissibility. Using a luciferase minigenome assay, we quantified the polymerase activity of all possible 16 ribonucleoprotein (RNP) complexes (PB2, PB1, PA, NP) between CIV-H3N2 and pdmH1N1 identifying some combinations that were more active than either parental virus complex. Using reverse genetics, and fixing the CIV-H3N2 HA, we found that 51 of the 127 possible reassortant viruses were viable and able to be rescued. Nineteen of these reassortant viruses had high in vitro growth phenotypes and 13 of these replicated in mice lungs. A single reassortant with the NP and HA gene segments from CIV-H3N2 was selected for characterization in ferrets. The reassortant efficiently transmitted by contact but not airborne routes and was pathogenic in ferrets. Our results suggest that CIV-H3N2 reassortants may pose a moderate risk to public health and that the canine host should be monitored for emerging IAVs.

    Importance IAV pandemics are caused by the introduction of novel viruses that are capable of efficient and sustained human transmission into a human population with limited herd immunity. The dog, as a potential "mixing vessel" for avian and mammalian IAVs, represents a human health concern due to their susceptibility to infection, large global population and close physical contact with humans. Our results suggest that humans are likely to have limited preexisting immunity to CIV-H3N2 and that CIV-H3N2xpdmH1N1 reassortants have moderate genetic compatibility and are transmissible by direct contact in ferrets. Our study contributes to the increasing evidence that IAV surveillance in the canine population is an important component of pandemic preparedness.

  • Characterization of cis-acting RNA elements of Zika virus by using a self-splicing ribozyme-dependent infectious clone [PublishAheadOfPrint]

  • Zika virus (ZIKV) has caused significant outbreaks and epidemics in the Americas recently, raising global concern due to its ability to cause microcephaly and other neurological complications. A stable and efficient infectious clone of ZIKV is urgently needed. However, the instability and toxicity of flavivirus cDNA clones in E. coli hosts has hindered the development of ZIKV infectious clones. Herein, using a novel self-splicing ribozyme-based strategy, we have generated a stable infectious cDNA clone of a contemporary ZIKV strain imported from Venezuela to China in 2016. The constructed clone contained a modified version of the group II self-splicing intron P.li.LSUI2 near the junction between the E and NS1 genes, which were removed from the RNA transcripts by an easy-to-establish in vitro splicing reaction. Transfection of the spliced RNAs into BHK-21 cells led to the production of infectious progeny virus that resembled the parental virus. Finally, potential cis-acting RNA elements in ZIKV genomic RNA were identified based on this novel reverse genetics system, and the critical role of 5rrsquo; -SLA promoter and 5rrsquo; -3rrsquo; cyclization sequences (CS) were characterized by a combination of different assays. Our results provide another stable and reliable reverse genetics system for ZIKV that will help study ZIKV infection and pathogenesis, and the novel self-splicing intron-based strategy could be further expanded for the construction of infectious clones from other emerging and re-emerging flaviviruses.

    IMPORTANCE The ongoing Zika virus (ZIKV) outbreaks have drawn global concern due to the unexpected causal link to fetus microcephaly and other severe neurological complications. The infectious cDNA clones of ZIKV are critical for the research community to study the virus, understand the disease, and inform vaccine design and antiviral screening. A panel of existing technologies have been utilized to develop ZIKV infectious clones. Here, we successfully generated a stable infectious clone of a 2016 ZIKV strain using a novel self-splicing ribozyme-based technology that abolished the potential toxicity of ZIKV cDNA clones to the E. coli host. Moreover, two crucial cis-acting replication elements (5rrsquo; -SLA and 5rrsquo; -CS) of ZIKV were first identified using this novel reverse genetics system. This novel self-splicing ribozyme-based reverse genetics platform will be widely utilized in future ZIKV studies and provide insight for the development of infectious clones of other emerging viruses.

  • Mammalian Orthoreovirus Factories Modulate Stress Granule Protein Localization by Interaction with G3BP1 [PublishAheadOfPrint]

  • Mammalian orthoreovirus (MRV) infection induces phosphorylation of translation initiation factor eIF2aalpha; which promotes formation of discrete cytoplasmic inclusions, termed stress granules (SGs). SGs are emerging as a component of the innate immune response to virus infection, and modulation of SG assembly is a common mechanism employed by viruses to counter this antiviral response. We previously showed that MRV infection induces SGs early, then interferes with SG formation as infection proceeds. In this work, we found that SG associated proteins localized to the periphery of virus-encoded cytoplasmic structures, termed virus factories (VFs), where viral transcription, translation, and replication occur. The localization of SG proteins to VFs was dependent on polysome dissociation and occurred via association of SG effector protein, G3BP1, with MRV non-structural protein NS, which localizes to VFs via association with VF nucleating protein, mmu;NS. Deletion analysis of the NS RNA binding domain and G3BP1 RNA (RRM) and ribosomal (RGG) binding domains showed that NS association and VF localization phenotypes of G3BP1 are not occurring solely through RNA or ribosomal binding, but require both RRM and RGG domains of G3BP1 for maximal VFL localization and NS association. Co-expression of NS and mmu;NS resulted in disruption of normal SG puncta, and in cells lacking G3BP1, MRV replication was enhanced in a manner correlating with strain-dependent induction of host translation shutoff. These results suggest that NS association with and relocalization of G3BP1 to the VF periphery plays a role in SG disruption to facilitate MRV replication in the host translational shutoff environment.

    IMPORTANCE SGs and SG effector proteins have emerged as important, yet poorly understood, players in the host's innate immune response to virus infection. MRV infection induces SGs early during infection that are dispersed and/or prevented from forming during late stages of infection despite continued activation of the eIF2aalpha; signaling pathway. Cellular and viral components involved in disruption of SGs during late stages of MRV infection remain to be elucidated. This work provides evidence that MRV disruption of SGs may be facilitated by association of MRV non-structural protein NS with major SG effector protein G3BP1 and subsequent localization of G3BP1 and other SG associated proteins around the periphery of virus encoded factories, interrupting the normal formation of SGs. Our findings also reveal the importance of G3BP1 as an inhibitor of MRV replication during infection for the first time.

  • Ancient Evolution and Dispersion of Human Papillomavirus Type 58 Variants [PublishAheadOfPrint]

  • Human papillomavirus type 58 is found in 10-18% of cervical cancers in East Asia but rather uncommon elsewhere. The distribution and oncogenic potential of HPV58 variants appear to be heterogeneous since the E7 T20I/G63S variant is more prevalent in East Asia and confers 7-9 fold higher risk for cervical precancer and cancer. However, the underlying genomic mechanisms that explain the geographic and carcinogenic diversity of HPV58 variants are still poorly understood. In this study, we used a combination of phylogenetic analyses and bioinformatics to investigate the deep evolutionary history of HPV58 complete genome variants. The initial splitting of HPV58 variants was estimated to occur 478,600 (95% HPD 391,000 nndash; 569,600) years ago. This divergence time is well among the era of the speciation between Homo sapiens and Neanderthal/Denisova, and around three times longer than the modern Homo sapiens divergence times. The expansion of present-day variants in Eurasia could be the consequence of viral transmission from Neanderthal/Denisova to non-African modern human populations through gene flow. A whole genome sequence signature analysis identified 3 amino acid changes, 16 synonymous nucleotide changes and a 12-bp insertion strongly associated with the E7 T20I/G63S variant that represents A3 sublineage and carries higher carcinogenetic potential. Compared with the capsid proteins, the oncogenes E7 and E6 had increased substitution rates indicative of higher selection pressure. These data provide a comprehensive evolutionary history and genomic basis of HPV58 variants to assist further investigation on carcinogenic association and development of diagnostic and therapeutic strategies.

    Importance Papillomaviruses (PVs) are an ancient and heterogeneous group of double stranded DNA viruses preferentially infecting the cutaneous and mucocutaneous epithelium of vertebrates. Persistent infection by specific oncogenic human papillomavirus (HPV) types including HPV58 has been established as a necessary cause of cervix cancer. In this work, we reveal the complex evolutionary history of HPV58 variants that well explains the heterogeneity of oncogenic potential and geographic predilection. Our data suggests that HPV58 variants may have co-evolved with archaic hominins and dispersed across the planet through intrahost gene flow. Certain genes and codons of HPV58 variants representing higher carcinogenic potential and/or under positive selection may have important implications of viral host specificity, pathogenesis and disease prevention.

  • Persistent TGEV infection enhances ETEC K88 adhesion by promoting epithelial-mesenchymal transition in intestinal epithelial cells [PublishAheadOfPrint]

  • Transmissible gastroenteritis virus (TGEV) is a coronavirus, characterized by diarrhea, high morbidity, and the mortality is 100% in piglets less than 2 weeks old. Pigs infected with TGEV are often suffer secondary infection with other pathogens, which aggravates the severity of diarrhea, but the mechanisms remain unknown. Here, we hypothesized that persistent TGEV infection stimulates the epithelialnndash;mesenchymal transition (EMT), thereby generating cells that more easily adhere to enterotoxigenic Escherichia coli (ETEC). Intestinal epithelial cells are the primary targets of TGEV and ETEC infection. We found that TGEV can persistently infect porcine intestinal columnar epithelial cells (IPEC-J2), and cause EMT, consistent with multiple changes in key cell characteristics. Infected cells display fibroblast-like shapes, exhibit increases in mesenchymal markers with a corresponding loss of epithelial markers, have enhanced expression of IL-1bbeta;, IL-6, IL-8, TGF-bbeta;, and TNF-aalpha; mRNAs, and demonstrate increases in migratory and invasive behaviors. Additional experiments showed that activation of the PI3K/Akt and ERK signaling pathways via TGF-bbeta; are critical for the TGEV-mediated EMT process. Cellular uptake is also modified in cells that have undergone EMT. TGEV-infected cells have higher levels of integrin aalpha;5 and fibronectin and exhibit enhanced adhesion of ETEC K88. Reversal of EMT reduces ETEC K88 adhesion and inhibits the expression of integrin aalpha;5 and fibronectin. Overall, these results suggest that TGEV infection induces EMT in IPEC-J2 cells, increasing the adhesion of ETEC K88 in the intestine and facilitating dual infection.

    Importance Transmissible gastroenteritis virus (TGEV) causes pig diarrhea and is often followed by secondary infection with other pathogens. In this study, we showed that persistent TGEV infection induces EMT in porcine intestinal columnar epithelial cells (IPEC-J2) and enhances adhesion of secondary pathogen ETEC K88. Additional experiments suggest that integrin aalpha;5 and fibronectin play an important role in TGEV-enhanced ETEC K88 adhesion. Reversal of EMT reduces the expression of integrin aalpha;5 and fibronectin and also reduces ETEC K88 adhesion. We conclude that TGEV infection triggers EMT and facilitates dual infection. Our results provide new insights into secondary infection, and suggest that anti-EMT targeted therapy may have implications for prevention and treatment of secondary infection.

  • HEPATITIS C VIRUS CORE PROTEIN MODULATES ENDOGLIN (CD105) SIGNALING PATHWAY FOR LIVER PATHOGENESIS [PublishAheadOfPrint]

  • Endoglin is part of the TGF-bbeta; receptor complex and has a crucial role in fibrogenesis and angiogenesis. It is also an important protein for tumor growth, survival, and cancer cell metastasis. In our previous study, we have shown that hepatitis C virus (HCV) infection induces epithelial-mesenchymal transition state (EMT) and cancer stem-like cell (CSC) properties in human hepatocytes. Our array data suggested that endoglin (CD105) mRNA is significantly upregulated in HCV associated CSCs. In this study, we have observed increased endoglin expression on the cell surface of HCV core expressing hepatocellular carcinoma (HepG2) cell line or immortalized human hepatocytes (IHH) and activation of its downstream signaling molecules. The status of phospho-SMAD1/5 and the expression of inhibitor of DNA binding protein 1 (ID1) were upregulated in HCV infected cells or viral core gene transfected cells. Additionally, we observed upregulation of endoglin/ID1 mRNA expression in chronic HCV patient liver biopsy samples. CSC generation by HCV core protein was dependent on the endoglin signaling pathway using activin receptor-like kinase (ALK)-1 Fc blocking peptide and endoglin siRNA. Further, follow-up from in vitro analysis suggested that anti-apoptosis Bcl2 protein, proliferation related Cyclin D1 protein, and CSC associated Hes1, Notch1, Nanog and Sox2 proteins are enhanced during infection or ectopic expression of HCV core protein.

    IMPORTANCE Endoglin plays a crucial role in fibrogenesis and angiogenesis, and is an important protein for tumor growth, survival, and cancer cell metastasis. Endoglin enhances ALK1/SMAD1/5 signaling in different cell types, leading to increased proliferation and migration responses. We have observed endoglin expression on HCV core expressing cell surface of human hepatocyte origin and activation of phospho-SMAD1/5 and ID1 downstream signaling molecules. ID1 protein plays a role in CSC properties and we found this pathway is important for anti-apoptitic and cell proliferation signaling. Blocking of endoglin-Alk1-SMAD1/5 might be a good therapeutic candidate for liver cancer stem cells together with liver cirrhosis.

  • Retrovirus-based Virus-Like Particle Immunogenicity and its Modulation by Toll-Like Receptor Activation [PublishAheadOfPrint]

  • Retrovirus-derived virus-like particles (VLPs) are particularly interesting vaccine platforms as they trigger efficient humoral and cellular immune responses and can be used to display heterologous antigens. In this study, we characterized the intrinsic immunogenicity of VLPs and investigated their possible adjuvantization by incorporation of toll-like receptor (TLR) ligands. We designed a non-coding single-stranded RNA (ncRNA) that could be encapsidated by VLPs and induce TLR7/8-signaling. We found that VLPs efficiently induce in vitro dendritic cell activation, which can be improved by ncRNA encapsidation (ncRNAVLPs). Transcriptome studies of dendritic cells harvested from the spleen of immunized mice identified antigen presentation and immune activation as the main gene expression signatures induced by VLPs, while TLR signaling and Th1 signatures characterize ncRNAVLPs. In vivo and compared with standard VLPs, ncRNAVLPs promoted Th1 responses and improved CD8+ T cell proliferation in a MyD88-dependent manner. In an HIV vaccine mouse model, HIV-pseudotyped ncRNAVLPs elicited stronger antigen-specific cellular and humoral responses than VLPs. Altogether our findings provide molecular evidence for a strong vaccine potential of retrovirus-derived VLPs that can be further improved by harnessing TLR-mediated immune activation.

    IMPORTANCE We previously reported that DNA vaccines encoding antigens displayed in/on retroviral VLPs are more efficient than standard DNA vaccines at inducing cellular and humoral immune responses. We aimed to decipher the mechanisms and investigated the VLPs immunogenicity independently of the DNA vaccination. We show that VLPs have the ability to activate antigen-presenting cells directly, thus confirming their intrinsic immunostimulatory properties and their potential to be used as an antigenic platform. Notably, this immunogenicity can be further improved and/or oriented by the incorporation into VLPs of ncRNA, which provides further TLR-mediated activation and Th1-type CD4+ and CD8+ T cell response orientation. Our results highlight the versatility of retrovirus--derived VLP design and the value of using ncRNA as an intrinsic vaccine adjuvant.

  • The UL21 tegument protein of herpes simplex virus type-1 is differentially required for the syncytial phenotype [PublishAheadOfPrint]

  • The initial goal of this study was to re-examine the requirement of UL21 for herpes simplex virus 1 (HSV-1) replication. Previous studies suggested that UL21 is dispensable for replication in cell cultures, but a recent report on HSV-2 challenges those findings. As was done for the HSV-2 study, a UL21-null virus was made and propagated on complementing cells to discourage selection of compensating mutations. This HSV-1 mutant was able to replicate in non-complementing cells, even at a low MOI, though a reduction in titer was observed. Also, increased proportions of empty capsids were observed in the cytoplasm, suggesting a role for UL21 in preventing their exit from the nucleus. Surprisingly, passage of the null mutant resulted in the rapid outgrowth of syncytial variants. This was unexpected because UL21 has been shown to be required for the Syn phenotype. However, earlier experiments only made use of syncytial mutant A855V of glycoprotein B (gB), and the Syn phenotype can also be produced with substitutions in glycoprotein K (gK), UL20, and UL24. Sequencing of the syncytial variants revealed mutations in the gK locus, but UL21 was shown to be dispensable for UL20Syn and UL24Syn. To test whether UL21 is needed only for mutant A855V, additional gBSyn derivatives were examined in the context of the null virus, and all produced lytic rather than syncytial sites of infection. Thus, UL21 is only required for the gBSyn phenotype. This is the first example of a differential requirement for a viral protein across the four syn loci.

    IMPORTANCE UL21 is conserved among alphaherpesviruses, but its role is poorly understood. This study shows that HSV-1 can replicate without UL21, although virus titers were greatly reduced. The null virus had greater proportions of empty (DNA-less) capsids in the cytoplasm of infected cells, suggesting that UL21 may play a role in retaining them in the nucleus. This is consistent with reports showing UL21 to be capsid associated and localized to the nuclei of infected cells. UL21 also appears to be needed for viral membrane activities. It was found to be required for virus-mediated cell fusion, but only for those mutants that harbor syncytial mutations in gB (not variants of gK, UL20, or UL24). The machinery needed for syncytial formation is similar to that needed for direct spread of the virus through cell junctions, and these studies show that UL21 is required for cell-to-cell spread, even in the absence of syncytial mutations.

  • Pseudorabies Virus dUTPase UL50 Induces Lysosomal Degradation of Type I Interferon Receptor 1 and Antagonizes the Alpha Interferon Response [PublishAheadOfPrint]

  • Alphaherpesviruses that establish persistent infection partly rely on their ability to evade host anti-viral responses, notably, the type I interferon (IFN) response. However, the mechanisms employed by alphaherpesviruses to avoid this response are not well understood. Pseudorabies virus (PRV) is an economically important pathogen and a useful model system to study alphaherpesvirus biology. To identify PRV proteins that antagonize type I IFN signaling, we performed a screen using an IFN-stimulated response element reporter in the swine cell line CRL. Unexpectedly, we identified the dUTPase UL50 as a strong inhibitor. We confirmed that UL50 has the ability to inhibit type I IFN signaling by ectopic expression of UL50 in cells and deletion of UL50 in PRV. Mechanistically, UL50 impeded type I IFN-induced STAT1 phosphorylation, likely by accelerating lysosomal degradation of IFN receptor 1 (IFNAR1). In addition, this UL50 activity was independent of its dUTPase activity and required amino acids 225-253 in the C-terminal region. The UL50 encoded by herpes simplex virus type 1 (HSV-1) also possessed similar activity. Moreover, UL50-deleted PRV was more susceptible to IFN than UL50 proficient PRV. Our results suggest that, in addition to its dUTPase activity, the UL50 of alphaherpesvirus possesses the ability to suppress type I IFN signaling by promoting lysosomal degradation of IFNAR1, thereby contributing to immune evasion. This finding reveals UL50 as a potential anti-viral target.

    IMPORTANCE Alphaherpesviruses can establish lifelong infections and cause many diseases in humans and animals. Pseudorabies virus (PRV) is a swine alphaherpesvirus that threatens pig production. Using PRV as a model, we found that alphaherpesvirus could utilize its encoded dUTPase UL50 to induce IFNAR1 degradation and inhibit type I IFN signaling in an enzymatic activity independent manner. Our finding reveals a mechanism employed by alphaherpesvirus to evade the immune response and indicates that UL50 is an important viral protein in pathogenesis and a potential target for anti-viral drug development.

  • Crystal structure of the DNA-binding domain of human herpesvirus 6A immediate early protein 2 [PublishAheadOfPrint]

  • Immediate early proteins of human herpesvirus 6A (HHV-6A) are expressed at the outset of lytic infection, and thereby regulate viral gene expression. Immediate early protein 2 (IE2) of HHV-6A is a trans-activator that drives a variety of promoters. The C-terminal region of HHV-6A IE2 is shared among IE2 homologs in beta-herpesviruses, and is involved in dimerization, DNA binding, and transcription factor binding. In this study, the structure of the C-terminal domain (IE2-CTD) was determined by X-ray crystallography at a resolution of 2.5 AAring;. IE2-CTD forms a homodimer stabilized by a bbeta;-barrel core with two interchanging long loops. Unexpectedly, the core structure resembles the gamma-herpesvirus factors Epstein-bar virus EBNA1 and Kaposi sarcoma herpesvirus LANA, but the interchanging loops are longer in IE2-CTD and form helix-turn-helix (HTH)-like motifs at their tips. The HTH and surrounding aalpha;-helices form a structural feature specific to the IE2 group. The apparent DNA-binding site (based on structural similarity with EBNA1 and LANA) resides on the opposite side of the HTH-like motifs, surrounded by positive electrostatic potential. Mapping analysis of conserved residues on the three dimensional structure delineated a potential factor-binding site adjacent to the expected DNA-binding site. The predicted bi- or tri-partite functional sites indicate a role for IE2-CTD as an adapter connecting the promoter and transcriptional factors that drive gene expression.

    Importance Human herpesvirus 6A and 6B (HHV-6A and -6B) belong to beta-herpesvirus subfamily. Both viruses establish life-long latency after primary infection, and their reactivation poses significant risk to immunocompromised patients. Immediate early protein 2 (IE2) of HHV-6A and HHV-6B is a trans-activator that triggers viral replication, and contains a DNA-binding domain shared with other beta-herpesviruses such as human herpesvirus 7 and human cytomegalovirus. In this study, an atomic structure of the DNA-binding domain of HHV-6A IE2 was determined and analyzed, enabling a structure-based understanding of the functions of IE2, specifically DNA recognition and interaction with transcription factors. Unexpectedly, the dimeric core resembles the DNA-binding domain of transcription regulators from gamma-herpesviruses showing structural conservation as a DNA binding domain, but with its own unique structural features. These findings facilitate further characterization of this key viral trans-activator.

  • Highly pathogenic New World arenavirus infection activates the pattern recognition receptor PKR without attenuating virus replication in human cells [PublishAheadOfPrint]

  • The arenavirus family consists of several highly pathogenic viruses including the Old World (OW) arenavirus Lassa fever virus (LASV) and the New World (NW) Juniiacute;n virus (JUNV) and Machupo virus (MACV). Host response to infection by these pathogenic arenaviruses is distinct in many aspects. NW JUNV and MACV infections readily induce an IFN response in human cells, while OW LASV infection usually triggers an undetectable or weak IFN response. JUNV induces IFN response through RIG-I, suggesting that the host non-self RNA sensor readily detects JUNV viral RNAs during infection and activates IFN response. Double-stranded RNA activated Protein Kinase R (PKR) is another host non-self RNA sensor classically known for its vRNA recognition activity. Herein we report that infection with NW arenavirus JUNV and MACV, but not OW LASV, activated PKR, concomitant with elevated phosphorylation of translation initiation factor eIF2aalpha;. Host protein synthesis was substantially suppressed in MACV- and JUNV-infected cells, but was only marginally reduced in LASV-infected cells. Despite the antiviral activity known for PKR against many other viruses, the replication of JUNV and MACV was not impaired, but slightly augmented in wt cells compared to in PKR deficient cells, suggesting that PKR or PKR activation did not negatively affect JUNV and MACV infection. Additionally, we found an enhanced IFN response in JUNV or MACV-infected, PKR deficient cells, which was inversely correlated with virus replication.

    IMPORTANCE The detection of viral RNA by host non-self RNA sensors including RIG-I and MDA5 is critical to the initiation of innate immune response to RNA virus infection. Among pathogenic arenaviruses, the OW LASV usually does not elicit an interferon response. However, the NW arenaviruses JUNV and MACV readily trigger IFN response in a RIG-I dependent manner. Herein, we demonstrate for the first time that pathogenic NW arenaviruses JUNV and MACV, but not the OW arenavirus LASV, activated the dsRNA-dependent PKR, another host non-self RNA sensor during infection. Interestingly, the replication of JUNV and MACV was not restricted, but rather slightly augmented in the presence of PKR. Our data provide new evidences for distinct interplay between host non-self RNA sensors and pathogenic arenaviruses, which also provide insights into the pathogenesis of arenaviruses and may facilitate the design of vaccines and treatments against arenavirus-caused diseases.

  • Adenovirus core protein VII down-regulates the DNA damage response on the host genome [PublishAheadOfPrint]

  • Viral manipulation of cellular proteins allows viruses to suppress host defenses and generate infectious progeny. Due to the linear double-stranded DNA nature of the adenovirus genome, the cellular DNA damage response (DDR) is considered a barrier for successful infection. The adenovirus genome is packaged with protein VII, a viral-encoded histone-like core protein that is suggested to protect incoming viral genomes from detection by cellular DNA damage machinery. We showed that protein VII localizes to host chromatin during infection, leading us to hypothesize that protein VII may affect DNA damage responses on the cellular genome. Here, we show that protein VII at cellular chromatin results in a significant decrease in accumulation of phosphorylated H2AX (H2AX) following irradiation, indicating that protein VII inhibits DDR signaling. The oncoprotein SET was recently suggested to modulate the DDR by affecting access of repair proteins to chromatin. Since protein VII binds SET, we investigated a role for SET in DDR inhibition by protein VII. We show that knockdown of SET partially rescues the protein VII-induced decrease in H2AX accumulation on the host genome, suggesting that SET is required for inhibition. Finally, we show that knockdown of SET also allows ATM to localize to incoming viral genomes bound by protein VII during infection with a mutant lacking early region E4. Together, our data suggest that the protein VII-SET interaction contributes to DDR evasion by adenovirus. Our results provide an additional example of a strategy used by adenovirus to manipulate the host DDR and show how viruses can modify cellular processes through manipulation of host chromatin.

    IMPORTANCE The DNA damage response (DDR) is a cellular network crucial for maintaining genome integrity. DNA viruses replicating in the nucleus challenge the resident genome and must overcome cellular responses, including the DDR. Adenoviruses are prevalent human pathogens that can cause a multitude of diseases such as respiratory infections and conjunctivitis. Here we describe how a small adenovirus core protein that localizes to host chromatin during infection can globally down-regulate the DDR. Our study focuses on key players in the damage signaling pathway and highlights how viral manipulation of chromatin may influence access of DDR proteins to the host genome.

  • The Investigation of Promoter Sequences of Marseilleviruses Highlights a Remarkable Abundance of AAATATTT motif in Intergenic Regions [PublishAheadOfPrint]

  • Viruses display a wide range of genomic profiles and, consequently, a variety of gene expression strategies. Specific sequences associated with transcriptional processes have been described in viruses, and putative promoter motifs have been elucidated for some nucleocytoplasmic large DNA viruses (NCLDV). Among NCLDV, the Marseilleviridae is a well-recognized family because of its genomic mosaicism. The marseilleviruses have an ability to incorporate foreign genes, especially from sympatric organisms inhabiting Acanthamoeba, its main known host. Here we identified for the first time an eight-nucleotide A/T-rich promoter sequence (AAATATTT) associated with 55% of marseillevirus genes and is conserved in all marseilleviruses lineages, more than any giant virus described to date. We instigated our prediction about the promoter motif by biological assays and by evaluating how single mutations in this octamer can impact gene expression. The investigation of sequences that regulate the expression of genes relative to lateral transfer revealed that the promoter motifs do not appear to be incorporated by marseilleviruses from donor organisms. Indeed, analyses of the intergenic regions that regulate lateral gene transfer-related genes have revealed an independent origin of the marseilleviruses intergenic regions that does not match gene-donor organisms. About 50% of AAATATTT motifs spread throughout intergenic regions of the marseilleviruses are present as multiple copies. We believe that such multiple motifs may be associated both to increased expression of a given gene or may be related to incorporation of foreign genes to the mosaic genome of marseilleviruses.

    Importance The marseilleviruses draws attention because of the peculiar features of its genome, however little is known about its gene expression patterns or the factors that regulate those expression patterns. The limited published research on the expression patterns of the marseilleviruses and their unique genome has led us to study the promoter motif sequences in the intergenic regions of the marseilleviruses. This work is the first to analyze promoter sequences in the genomes of the marseilleviruses. We also suggest a strong capacity to acquire foreign genes and to express those genes mediated by multiple copies of the promoter motifs available in intergenic regions. These findings contribute to an understanding of genomic expansion and plasticity observed in these giant viruses.

  • Structure-Based Design of Hepatitis C Virus Vaccines that Elicit Neutralizing Antibody Responses to a Conserved Epitope [PublishAheadOfPrint]

  • Despite recent advances in therapeutic options, hepatitis C virus (HCV) remains a severe global disease burden, and a vaccine can substantially reduce its incidence. Due to its extremely high sequence variability, HCV can readily escape the immune response, thus an effective vaccine must target conserved, functionally important epitopes. Using the structure of a broadly neutralizing antibody in complex with a conserved linear epitope from the HCV E2 envelope glycoprotein (residues 412-423; epitope I), we performed structure-based design of immunogens to induce antibody responses to this epitope. This resulted in epitope-based immunogens based on a cyclic defensin protein, as well as a bivalent immunogen with two copies of the epitope on the E2 surface. We solved the x-ray structure of a cyclic immunogen in complex with the HCV1 antibody and confirmed preservation of the epitope conformation and the HCV1 interface. Mice vaccinated with our designed immunogens produced robust antibody responses to epitope I, and their serum could neutralize HCV. Notably, the cyclic designs induced greater epitope-specific responses and neutralization than the native peptide epitope. Beyond successfully designing several novel HCV immunogens, this study demonstrates the principle that neutralizing anti-HCV antibodies can be induced by epitope-based, engineered vaccines and provides the basis for further efforts in structure-based design of HCV vaccines.

    IMPORTANCE Hepatitis C virus is a leading cause of liver disease and liver cancer, with approximately 3% of the world's population infected. To combat this virus, an effective vaccine would have distinct advantages over current therapeutic options, yet experimental vaccines have not been successful to date, due in part to the virus's high sequence variability leading to immune escape. In this study we rationally designed several vaccine immunogens based on the structure of a conserved epitope that is the target of broadly neutralizing antibodies. In vivo results in mice indicated that these antigens elicited epitope-specific neutralizing antibodies, with varying degrees of potency and breadth. These promising results suggest that a rational design approach can be used to generate an effective vaccine for this virus.

  • Interferon-Inducible CD169/Siglec1 Attenuates Anti-HIV-1 Effects of IFN-{alpha} [PublishAheadOfPrint]

  • A hallmark of HIV-1 infection in vivo is chronic immune activation concomitant with type I interferon (IFN) production. Although type I IFN induces an antiviral state in many cell types, HIV-1 can replicate in vivo via mechanisms that have remained unclear. We have recently identified a type I IFN-inducible protein CD169 as the HIV-1 attachment factor on dendritic cells (DCs) that can mediate robust infection of CD4+ T cells in trans. Since CD169 expression on macrophages is also induced by type I IFN, we hypothesized that type I IFN-inducible CD169 could facilitate productive HIV-1 infection in myeloid cells in cis and CD4+ T cells in trans and thus offset antiviral effects of type I IFN. In support of this hypothesis, infection of HIV-1 or MLV-Env pseudotyped HIV-1 particles was enhanced in IFN-aalpha;-treated THP1 monocytoid cells, and this enhancement was primarily dependent on CD169-mediated enhancement at the virus entry step, an observation phenocopied in HIV-1 infections of IFN-aalpha;-treated primary monocyte-derived macrophages (MDMs). Furthermore, expression of CD169, a marker of type I IFN-induced immune activation in vivo, was enhanced in lymph nodes from RT-SHIV-infected pigtailed macaques, compared to uninfected macaques, and interestingly, there was extensive co-localization of p27gag and CD169, suggesting productive infection of CD169+ myeloid cells in vivo. While cell-free HIV-1 infection of IFN-aalpha;-treated CD4+ T cells was robustly decreased, initiation of infection in trans via co-culture with CD169+ IFN-aalpha;-treated DCs restored infection suggesting that HIV-1 exploits CD169 in cis and in trans to attenuate a type I IFN-induced antiviral state.

    Structured Abstract

    HIV-1 infection in human causes immune activation characterized by elevated levels of pro-inflammatory cytokines including type I interferons (IFN). Although type I IFN induces an antiviral state in many cell types in vitro, HIV-1 can replicate in vivo via mechanisms that have remained unclear. In this report, we test the hypothesis that CD169, a type I IFN-inducible HIV-1 attachment factor, offsets antiviral effects of type I IFN. Infection of HIV-1 was rescued in IFN-aalpha;-treated myeloid cells via upregulation of CD169 and subsequent increase in CD169-dependent virus entry. Furthermore, extensive co-localization of viral Gag and CD169 was observed in lymph nodes of infected pigtailed macaques, suggesting productive infection of CD169+ cells in vivo. Treatment of dendritic cell (DC) nndash; T cell co-cultures with IFN-aalpha;, upregulated CD169 expression on DCs and rescued HIV-1 infection of CD4+ T cells in trans suggesting that HIV-1 exploits CD169 to attenuate type I IFN-induced restrictions.

  • Sorafenib impedes Rift Valley fever virus egress by inhibiting Valosin-containing protein function in the cellular secretory pathway [PublishAheadOfPrint]

  • There is an urgent need for therapeutic development to combat Rift Valley fever virus (RVFV) infections, which causes devastating disease in both humans and animals. In an effort to repurpose drugs for RVFV treatment, our previous studies screened a library of FDA-approved drugs. The most promising candidate identified was the hepatocellular and renal cell carcinoma drug, sorafenib. Mechanism of action studies indicated sorafenib targeted a late stage in virus infection and caused a buildup of virions within cells. In addition, siRNA knockdown studies suggested that non-classical targets of sorafenib are important for the propagation of RVFV. Here we extend our previous findings to identify the mechanism by which sorafenib inhibits release of RVFV virions from the cell. Confocal microscopy imaging revealed that Gn colocalizes and accumulates within the endoplasmic reticulum (ER), and the transport of Gn from the Golgi to the host cell membrane is reduced. Transmission electron microscopy demonstrated that sorafenib caused virions to be present inside large vacuoles inside the cells. P97/Vasolin-containing protein (VCP), a protein involved in membrane remodeling in the secretory pathway and a known target of sorafenib, was found to be important for RVFV egress. Knockdown of VCP resulted in decreased RVFV replication, reduced Gn Golgi localization, and increased Gn ER accumulation. An intracellular accumulation of RVFV virions was also observed in VCP siRNA transfected cells. Collectively these data indicate that sorafenib causes a disruption in viral egress by targeting VCP and the secretory pathway, resulting in a buildup of virions within dilated ER vesicles.

    IMPORTANCE In humans, symptoms of RVFV infection mainly include a self-limiting febrile illness. However in some cases, infected individuals can also experience hemorrhagic fever, neurological disorders, liver failure and blindness, which could collectively be lethal. The ability of RVFV to expand geographically outside sub-Saharan Africa is of concern, particularly to the Americas, where native mosquito species are capable of virus transmission. Currently, there are no FDA-approved therapeutics to treat RVFV infection, and thus there is an urgent need to understand the mechanisms by which the virus hijacks the host cell machinery to replicate. The significance of our research is in identifying the cellular target of sorafenib that inhibits RVFV propagation, so that this information can be used as a tool for further development of therapeutics used to treat RVFV infection.

  • Pathobiology of clade 2.3.4.4 H5Nx high pathogenicity avian influenza virus infections in minor gallinaceous poultry supports early backyard flock introductions in Western U.S., 2014-2015 [PublishAheadOfPrint]

  • In 2014-2015, the U.S. experienced an unprecedented outbreak of Eurasian clade 2.3.4.4 H5 highly pathogenic avian influenza (HPAI) virus. Initial cases affected mainly wild birds and mixed backyard poultry species, while later outbreaks affected mostly commercial chickens and turkeys. The pathogenesis, transmission, and intra-host evolutionary dynamics of initial Eurasian H5N8 and reassortant H5N2 clade 2.3.4.4 HPAI viruses in the U.S. were investigated in minor gallinaceous poultry species (i.e. species for which the U.S. commercial industries are small): Japanese quail, Bobwhite quail, Pearl guinea fowl, Chukar partridges, and Ring-necked pheasants. Low mean bird infectious doses (llt;2 to 3.7 log10) support direct introduction and infection of these species as observed in mixed backyard poultry during the early outbreaks. Pathobiological features and systemic virus replication in all species tested were consistent with HPAI virus infection. Sustained virus shedding with transmission to contact-exposed birds, alongside long incubation periods, could enable unrecognized dissemination and adaptation to other gallinaceous such as chickens and turkeys. Genome sequencing of excreted viruses revealed numerous low-frequency polymorphisms and 20 consensus-level substitutions in all genes and species but especially in Japanese quail and Pearl guinea fowl and in internal proteins PB1 and PB2. This genomic flexibility after only one passage indicates that influenza viruses can continue to evolve in Galliformes, increasing their opportunity to adapt to other species. Our findings suggest that these gallinaceous poultry are permissive for infection and sustainable transmissibility with 2014 initial wild bird-adapted clade 2.3.4.4 virus, with potential acquisition of mutations leading to host range adaptation.

    IMPORTANCE The outbreak of clade 2.3.4.4 H5 highly pathogenic avian influenza (HPAI) virus that occurred in the U.S. in 2014-2015 represents the worst livestock disease event in the country, with unprecedented socioeconomic and commercial consequences. Epidemiological and molecular investigations can identify transmission pathways of the HPAI virus. However, understanding the pathogenesis, transmission, and intra-host evolutionary dynamics of new HPAI viruses in different avian species is paramount. The significance of our research is in examining the susceptibility of minor gallinaceous species to HPAI virus, as this poultry sector also suffers from HPAI epizootics, and identifying its biological potential as epidemiological link between the waterfowl reservoir and the commercial chicken and turkey populations, with the ultimate goal of refining surveillance in these populations to enhance early detection, management, and control in future HPAI outbreaks.

  • Palmitoylation contributes to membrane curvature in Influenza A virus assembly and hemagglutinin-mediated membrane fusion [PublishAheadOfPrint]

  • The highly conserved cytoplasmic tail of influenza virus glycoprotein hemagglutinin (HA) contains three cysteines, post-translationally modified by covalently bound fatty acids. While viral HA acylation is crucial in virus replication, its physico-chemical role is unknown. We used virus-like particles (VLP) to study the effect of acylation on morphology, protein incorporation, lipid composition, and membrane fusion. De-acylation interrupted HA-M1 interactions since de-acylated mutant HA fail to incorporate an M1 layer within spheroidal VLP, and filamentous particles incorporated increased numbers of neuraminidase (NA). While HA acylation did not influence VLP shape, lipid composition, or HA lateral spacing, acylation significantly affected envelope curvature. Compared to wild type HA, de-acylated HA is correlated with released particles with flatter envelope curvature in the absence of the matrix (M1) protein layer. The spontaneous curvature of palmitate was calculated by molecular dynamic simulations, and was found to be comparable to the curvature values derived from VLP size distributions. Cell-cell fusion assays show a strain-independent failure of fusion pore enlargement amongst H2 (A/Japan/305/57), H3 (A/Aichi/2/68), H3 (A/Udorn/72). In contradistinction, acylation made no difference in the low pH-dependent fusion of isolated VLPs to liposomes: fusion pores formed and expanded as demonstrated by the presence of complete fusion products observed using cryo-electron tomography (cryo-ET). We propose that the primary mechanism of action of acylation is to control membrane curvature and to modify HA's interaction with M1 protein, while the stunting of fusion by deacylated HA acting in isolation may be balanced by other viral proteins, which help lower the energetic barrier to pore expansion.

    IMPORTANCE Influenza A virus is an air-borne pathogen causing seasonal epidemics and occasional pandemics. Hemagglutinin (HA), a glycoprotein abundant on the virion surface is important both in influenza A virus assembly and entry. HA is modified by acylation whose removal abrogates viral replication. Here we used cryo-electron tomography to obtain three-dimensional images to elucidate a role for HA acylation in virus-like particle (VLP) assembly. Our data indicates that HA acylation contributes to the capability of HA to bend membranes and to HA's interaction with the M1 scaffold protein during virus assembly. Furthermore, our data on VLP, and by hypothesis virus, suggests that HA acylation, while not critical to fusion pore formation, contributes to pore expansion in a target-dependent fashion.

  • Potent In Vivo NK Cell-mediated Elimination of HIV-1-Infected Cells Mobilized by a gp120-bispecific and Hexavalent Broadly Neutralizing Fusion Protein [PublishAheadOfPrint]

  • Antibodies bound to HIV-1 envelope protein expressed by infected cells mobilize antibody dependent cellular cytotoxicity (ADCC) to eliminate the HIV-1-infected cells and thereby suppress HIV-1 infection and delay disease progression. Studies treating HIV-1-infected individuals with latency reactivation agents to reduce their latent HIV-1 reservoirs indicated that their HIV-1-specific immune responses were insufficient to effectively eliminate the reactivated latent HIV-1-infected T cells. Mobilization of ADCC may facilitate elimination of reactivated latent HIV-1-infected cells to deplete the HIV-1 reservoir and contribute to functional HIV-1 cure. The most effective antibodies for controlling and eradicating HIV-1 infection would likely have the dual capacities of potently neutralizing a broad range of HIV-1 isolates and effectively mobilizing HIV-1-specific ADCC to eliminate HIV-1-infected cells. For this purpose, we constructed LSEVh-LS-F, a broadly neutralizing, defucosylated hexavalent fusion protein specific for both the CD4 and co-receptor gp120-binding sites. LSEVh-LS-F potently inhibited in vivo HIV-1 and SHIV infection in humanized mouse and macaque models, respectively, including in vivo neutralization of HIV-1 strains resistant to the broadly neutralizing antibodies VRC01 and 3BNC117. We developed a novel humanized mouse model to evaluate in vivo human NK cell-mediated elimination of HIV-1-infected cells by ADCC and utilized it to demonstrate that LSEVh-LS-F rapidly mobilized NK cells to eliminate ggt;80% of HIV-1-infected cells in vivo one day after its administration. The capacity of LSEVh-LS-F to eliminate HIV-1-infected cells via ADCC combined with its broad neutralization activity supports its potential use as an immunotherapeutic agent to eliminate reactivated latent cells and deplete the HIV-1 reservoir.

    IMPORTANCE Mobilization of antibody dependent cellular cytotoxicity (ADCC) to eliminate reactivated latent HIV-1-infected cells is a strategy which may contribute to depleting the HIV-1 reservoir and achieving a functional HIV-1 cure. To more effectively mobilize ADCC, we designed and constructed LSEVh-LS-F, a broadly neutralizing, defucosylated hexavalent fusion protein specific for both the CD4 and co-receptor gp120-binding sites. LSEVh-LS-F potently inhibited in vivo HIV-1 and SHIV infection in humanized mouse and macaque models, respectively, including in vivo neutralization of an HIV-1 strain resistant to the broadly neutralizing antibodies VRC01 and 3BNC117. Using a novel humanized mouse model, we demonstrated that LSEVh-LS-F rapidly mobilized NK cells to eliminate ggt;80% of HIV-1-infected cells in vivo one day after its administration. The capacity of LSEVh-LS-F to eliminate HIV-1-infected cells via ADCC combined with its broad neutralization activity supports its potential use as an immunotherapeutic agent to eliminate reactivated latent cells and deplete the HIV-1 reservoir.

  • HIV DNA-Adenovirus Multiclade Envelope Vaccine Induces Gp41 Antibody Immunodominance in Rhesus Macaques [PublishAheadOfPrint]

  • Dominant antibody responses in vaccinees who received the multiclade (A, B and C) envelope (Env) DNA/rAd5 vaccine studied in the HIV-1 vaccine trials network (HVTN) efficacy trial 505 (HVTN 505), targeted Env gp41 and cross-reacted with microbial antigens. In this study, we asked if the DNA/rAd5 vaccine induced a similar antibody response in rhesus macaques (RMs) that are commonly used as an animal model for human HIV-1 infections and for testing candidate HIV-1 vaccines. We also asked if gp41 immunodominance could be avoided by immunization of neonatal RMs during the early stages of microbial colonization. We found that the DNA/rAd5 vaccine elicited a higher frequency of gp41-reactive memory B cells compared to gp120-memory B cells in adult and neonatal RMs. Analysis of the vaccine-induced Env-reactive B cell repertoire revealed that the majority of HIV-1 Env-reactive antibodies in both adult and neonatal RMs were targeted to gp41. Interestingly, a subset of gp41-reactive antibodies isolated from RMs cross-reacted with host antigens, including autologous intestinal microbiota. Thus, gp41-containing DNA/rAd5 vaccine induced dominant gp41-microbiota cross-reactive antibodies derived from blood memory B cells in RMs as observed in the HVTN 505 efficacy trial. These data demonstrated that RMs can be used to investigate the gp41 immunodominance in candidate HIV-1 vaccines. Moreover, colonization of neonatal RMs occurred within the first week of life, and immunization of neonatal RMs during this time also induced a dominant gp41-reactive antibody response.

    IMPORTANCE Our results are critical to current work in the HIV-1 vaccine field evaluating the phenomenon of gp41 immunodominance induced by HIV-1 Env gp140 in RMs and humans. Our data demonstrate that RMs are an appropriate animal model to study this phenomenon and to determine the immunogenicity in new HIV-1 Env trimer vaccine designs. The demonstration of gp41 immunodominance in memory B cells of both adult and neonatal RMs indicated that early vaccination could not overcome gp41 dominant responses.

  • Combinatorial effects of the glucocorticoid receptor and Krüppel-like transcription factor 15 on bovine herpesvirus 1 transcription and productive infection. [PublishAheadOfPrint]

  • Bovine herpesvirus 1 (BoHV-1), an important bovine pathogen, establishes life-long latency in sensory neurons. Latently infected calves consistently reactivate from latency following a single intravenous injection of the synthetic corticosteroid dexamethasone. The immediate early transcription unit 1 (IEtu1) promoter, which drives bICP0 and bICP4 expression, is stimulated by dexamethasone because it contains two glucocorticoid receptor (GR) response elements (GREs). Several Krüppel-like transcription factors (KLF), including KLF15, are induced during reactivation from latency and they stimulate certain viral promoters and productive infection. In this study, we demonstrate that the GR and KLF15 were frequently expressed in the same TG neuron during reactivation, cooperatively stimulated productive infection, and IEtu1 GREs in mouse neuroblastoma cells (Neuro-2A). We further hypothesized that additional regions in the BoHV-1 genome are trans-activated by the GR or stress-induced transcription factors. To test this hypothesis, BoHV-1 DNA fragments (less than 400 bp) containing potential GR and KLF binding sites were identified and examined for transcriptional activation by stress-induced transcription factors. Intergenic regions within the unique long 52 gene (UL-52; component of DNA primase/helicase complex), bICP4, IEtu2, and unique short region were stimulated by KLF15 and the GR. Chromatin immuno-precipitation studies revealed that the GR and KLF15 interacted with sequences within IEtu1 GREs and UL-52 fragment. Co-immunoprecipitation studies demonstrated KLF15 and the GR were associated with each other in transfected cells. Since the GR stimulates KLF15 expression, we suggest these two transcription factors form a feed-forward loop that stimulates viral gene expression and productive infection following stressful stimuli.

    IMPORTANCE Bovine herpesvirus 1 (BoHV-1) is an important viral pathogen that causes respiratory disease and suppresses immune responses in cattle: consequently, life-threatening bacterial pneumonia can occur. Following acute infection, BoHV-1 establishes life-long latency in sensory neurons. Reactivation from latency is initiated by the synthetic corticosteroid dexamethasone. Dexamethasone stimulates lytic cycle viral gene expression in sensory neurons of calves latently infected with BoHV-1, culminating in virus shedding and transmission. Two stress induced cellular transcription factors, Krüppel-like transcription factor 15 (KLF15) and the glucocorticoid receptor (GR), cooperate to stimulate productive infection and viral transcription. Additional studies demonstrated KLF15 and the GR form a stable complex and these stress-induced transcription factors bind to viral DNA sequences, which correlates with transcriptional activation. The ability of the GR and KLF15 to synergistically stimulate viral gene expression and productive infection may be critical for the ability of BoHV-1 to reactivate from latency following stressful stimuli.

  • The reovirus {mu}1 340-343 loop controls entry related conformational changes [PublishAheadOfPrint]

  • Reovirus particles are covered with 200 mmu;1/3 heterohexamers. Following attachment to cell surface receptors, reovirus is internalized by receptor-mediated endocytosis. Within the endosome, particles undergo a series of step-wise disassembly events. First, the 3 protector protein is degraded by cellular proteases to generate infectious subviral particles (ISVPs). Second, the mmu;1 protein rearranges into a protease sensitive conformation to generate ISVP*s and releases two virally encoded peptides, mmu;1N and . The released peptides promote delivery of the genome-containing core by perforating the endosomal membrane. Thus, to establish a productive infection, virions must be stable in the environment but flexible to disassemble in response to the appropriate cellular cue. The reovirus outer capsid is stabilized by mmu;1 intratrimer, intertrimer, and trimer-core interactions. As a consequence of ISVP-to-ISVP* conversion, neighboring mmu;1 trimers unwind and separate. Located within the mmu;1 jelly-roll bbeta; barrel domain, which is a known regulator of ISVP* formation, residues 340-343 form a loop and were proposed to facilitate viral entry. To test this idea, we generated recombinant reoviruses that encoded deletions within this loop (341 and 342). Both deletions destabilized the outer capsid. Notably, 342 impaired the viral lifecycle; however, replicative fitness was restored by an additional change (V403A) within the mmu;1 jelly-roll bbeta; barrel domain. In the 341 and 342 backgrounds, V403A also rescued defects in ISVP-to-ISVP* conversion. Together, these findings reveal a new region that regulates reovirus disassembly and how perturbing a metastable capsid can compromise replicative fitness.

    IMPORTANCE Capsids of nonenveloped viruses are composed of protein complexes that encapsulate, or form a shell around, nucleic acid. The protein-protein interactions that form this shell must be stable to protect the viral genome but also flexible to disassemble during cell entry. Thus, capsids adopt conformations that undergo rapid disassembly in response to a specific cellular cue. In this work, we identify a new region within the mammalian orthoreovirus outer capsid that regulates particle stability. Amino acid deletions that destabilize this region impair the viral replication cycle. Nonetheless, replicative fitness is restored by a compensatory mutation that restores particle stability. Together, this work demonstrates the critical balance between assembling virions that are stable and maintaining conformational flexibility. Any factor that perturbs this balance has the potential to block a productive infection.

  • Recombinant origins of pathogenic and nonpathogenic mouse gammaretroviruses of polytropic host range [PublishAheadOfPrint]

  • Ecotropic, xenotropic and polytropic mouse leukemia viruses (E-, X-, P-MLVs) exist in mice as infectious viruses and endogenous retroviruses (ERVs) inserted into mouse chromosomes. All 3 MLV subgroups are linked to leukemogenesis, which involves generation of recombinants with polytropic host range. Although P-MLVs are deemed to be the proximal agents of disease induction, few biologically characterized infectious P-MLVs have been sequenced for comparative analysis. We analyzed the complete genomes of 16 naturally occurring infectious P-MLVs, 12 of which were typed for pathogenic potential. We sought to identify ERV progenitors, recombinational hotspots, and segments that are always replaced, never replaced, or linked to pathogenesis or host range. Each P-MLV has an E-MLV backbone with P- or X-ERV replacements that together cover 100% of the recombinant genomes, with different substitution patterns for X- and P-ERVs. Two segments are always replaced, in envelope (Env): the N-terminus of the surface subunit, and the cytoplasmic tail R peptide. Viral gag gene replacements are influenced by host restriction genes Fv1 and Apobec3. Pathogenic potential maps to the env transmembrane subunit segment encoding the N-heptad repeat (HR1). Molecular dynamics simulations identified three novel interdomain salt bridges in the lymphomagenic virus HR1 that could affect structural stability, entry or sensitivity to host immune responses. The long terminal repeats of lymphomagenic P-MLVs are differentially altered by recombinations, duplications or mutations. This analysis of the naturally occurring, sometimes pathogenic P-MLV recombinants defines the limits and extent of intersubgroup recombination, and identifies specific sequence changes linked to pathogenesis and host interactions.

    Importance During virus-induced leukemogenesis, ecotropic mouse leukemia viruses (MLVs) recombine with nonecotropic endogenous retroviruses (ERVs) to produce polytropic MLVs (P-MLVs). Analysis of 16 P-MLV genomes identified two segments consistently replaced, one at the envelope N-terminus that alters receptor choice, and one in the R peptide at the envelope C-terminus which is removed during virus assembly. Genome-wide analysis shows that nonecotropic replacements in the progenitor ecotropic MLV genome are more extensive than previously appreciated, covering 100% of the genome; contributions from xenotropic and polytropic ERVs differentially alter the regions responsible for receptor determination or subject to APOBEC3 and Fv1 restriction. All pathogenic viruses had modifications in the regulatory elements in their long terminal repeats and differed in a helical segment of envelope involved in entry and targeted by the host immune system. Virus-induced leukemogenesis thus involves generation of complex recombinants, and specific replacements are linked to pathogenesis and host restrictions.

  • In vitro enhancement of RSV infection by maternal antibodies does not explain disease severity in infants [PublishAheadOfPrint]

  • Respiratory syncytial virus (RSV) is the leading cause of severe respiratory illness in infants. At this young age, infants typically depend on maternally transferred antibodies (matAbs) and their innate immune system for protection against infections. RSV-specific matAbs are thought to protect from severe illness, yet severe RSV disease occurs mainly below 6 months of age, when neutralizing matAb levels are present. To investigate this discrepancy, we asked if disease severity is related to antibody properties other than neutralization. Some antibody effector functions are mediated via their Fc binding region. However, it has been shown that this binding may lead to antibody-dependent enhancement (ADE) of infection or reduction of neutralization, both possibly leading to more disease. In this study, we first showed that high levels of ADE of RSV infection occur in monocytic THP-1 cells in the presence of RSV antibodies and that neutralization by these antibodies was reduced in Vero cells when they were transduced with Fc gamma receptors. We then demonstrated that antibodies from cotton rats with formalin-inactivated- (FI-) RSV-induced pulmonary pathology were capable of causing ADE. Human matAbs also caused ADE and were less neutralizing in vitro in cells that carry Fc receptors. However, these effects were unrelated to disease severity because they were seen both in uninfected controls and in infants hospitalized with different levels of RSV disease severity. We conclude that ADE and reduction of neutralization are unlikely to be involved in RSV disease in infants with neutralizing matAbs.

    IMPORTANCE It is unclear why severity of RSV disease peaks at the age when infants have neutralizing levels of maternal antibodies. Additionally, the exact reason for FI-RSV-induced enhanced disease, as seen in the 1960's vaccine trials, is still unclear. We hypothesized that antibodies present in either of these conditions could contribute to disease severity. Antibodies can have effects that may lead to more disease instead of protection. We investigated two of those effects: antibody-dependent enhancement of infection (ADE) and neutralization reduction. We show that ADE occurs in vitro with antibodies from FI-RSV-immunized RSV-infected cotton rats. Moreover, passively acquired maternal antibodies from infants had the capacity to induce ADE and reduction of neutralization. However, no clear association with disease severity was seen, ruling out that these properties explain disease in the presence of maternal antibodies. Our data contribute to a better understanding of the impact of antibodies on RSV disease in infants.

  • Development of Virus like Particle Vaccine and Reporter Assay for Zika Virus [PublishAheadOfPrint]

  • Recent worldwide outbreaks of Zika virus (ZIKV) infection and the lack of an approved vaccine raises serious concerns regarding preparedness to combat this emerging virus. We used a Virus Like Particle (VLP) based approach to develop a vaccine and a microneutralization assay against ZIKV. Synthetic C-prM-E (Capsid-preMembrane-Envelope) gene construct of ZIKV was used to generate Reporter Virus Particles (RVPs) that package GFP reporter expressing WNV replicon. The assay was adapted to 96 well format, similar to the Plaque Reduction Neutralization Test (PRNT) and showed high reproducibility with specific detection of ZIKV neutralizing antibodies. Furthermore, C-prM-E and prM-E VLPs were tested as vaccine candidates in mice and compared to DNA vaccination. While ZIKV prM-E construct alone was sufficient for generating VLPs; efficient VLP production from the C-prM-E construct could be achieved in the presence of WNV NS2B-3 protease that cleaves C from prM allowing virus release. Immunization studies in mice showed that VLPs generated higher neutralizing antibody titers than DNA vaccine with C-prM-E VLPs slightly better than prM-E VLPs. The superiority of C-prM-E VLPs suggests that inclusion of capsid may have benefits for ZIKV and other flaviviral VLP vaccines. To facilitate the VLP platform, we generated a stable cell line expressing high levels of ZIKV prM-E proteins that constitutively produce VLPs as well as a cell line expressing ZIKV C-prM-E proteins for RVP production. While several vaccine platforms have been proposed for ZIKV, this study describes a safe, effective and economical VLP based vaccine against ZIKV.

    IMPORTANCE To address the growing Zika virus epidemic we undertook this study with two objectives. Firstly, to develop a safe effective and economical vaccine for ZIKV and secondly, to develop a rapid versatile assay to detect anti-ZIKV immune response. We generated cell lines stably expressing the ZIKV prM-E that produce high amounts of VLPs in the supernatants and ZIKV C-prM-E cell line that produces reporter virus particles upon transfection with GFP replicon plasmid. The prM-E VLPs induced a strong neutralizing antibody response in mice that was better when the capsid was included. VLP based vaccines showed significantly better neutralizing antibody response when compared to their DNA counterparts. The RVP based microneutralization assay worked similar to the PRNT assay with a rapid GFP readout in a 96 well format. Our VLP based platform provides a source for ZIKV vaccine and diagnosis that can be rapidly adapted to current outbreaks.

  • Coordinate Regulation of TET2 and EBNA2 Control DNA Methylation State of Latent Epstein-Barr Virus [PublishAheadOfPrint]

  • Epstein-Barr Virus (EBV) latency and its associated carcinogenesis are regulated by dynamic changes in DNA methylation of both virus and host genomes. We show here that the Ten-Eleven Translocation 2 (TET2) gene, implicated in hydroxymethylation and active DNA demethylation, is a key regulator of EBV latency type DNA methylation patterning. EBV latency types are defined by DNA methylation patterns that restrict expression of viral latency genes. We show that TET2 mRNA and protein expression correlate with the highly demethylated EBV type III latency program permissive for expression of EBNA2, EBNA3s, and LMP transcripts. We show that shRNA depletion of TET2 results in a decrease in latency gene expression, but can also trigger a switch to lytic gene expression. TET2 depletion results in the loss of hydroxymethylated cytosine, and corresponding increase in cytosine methylation at key regulatory regions on the viral and host genomes. This also corresponded to a loss of RBP-j binding, and decreased histone H3K4 trimethylation at these sites. Furthermore, we show that the TET2 gene, itself, is regulated similar to the EBV genome. ChIP-Seq revealed that TET2 gene contains EBNA2-dependent RBP-j and EBF1 binding sites, and is subject to DNA methylation associated transcriptional silencing similar to EBV latency type III genomes. Finally, we provide evidence that TET2 colocalizes with EBNA2-EBF1-RBP-j binding sites, and can interact with EBNA2 by co-immunoprecipitation. Taken together, these findings indicate that TET2 gene transcripts are regulated similarly to EBV type III latency genes, and that TET2 protein is a cofactor of EBNA2 and co-regulator of the EBV type III latency program and DNA methylation state..

    IMPORTANCE Epstein-Barr Virus (EBV) latency and carcinogenesis involves the selective epigenetic modification of viral and cellular genes. Here, we show that TET2, a cellular tumor suppressor involved in active DNA demethylation, plays a central role in regulating DNA methylation state during EBV latency. TET2 is coordinately regulated and functionally interacts with the viral oncogene EBNA2. TET2 and EBNA2 function cooperatively to demethylate genes important for EBV-driven B cells growth transformation.

  • No Substantial Evidence for Sexual Transmission of Minority HIV Drug Resistance Mutations in Men Who Have Sex with Men [PublishAheadOfPrint]

  • During primary HIV-infection, the presence of minority drug resistant mutations (DRM) may be a consequence of sexual transmission, de novo mutations or technical errors. Baseline blood samples were collected from 24 HIV-infected antiretroviral-naive, genetically- and epidemiologically- linked source and recipient partners, shortly after the recipient's estimated date of infection. An additional 32 longitudinal samples were available from 11 recipients. Deep-sequencing of HIV reverse transcriptase (RT) was performed (Roche/454) and screened for nucleoside and non-nucleoside RT inhibitor DRM. The likelihood of sexual transmission and persistence of DRM was assessed using Bayesian-based statistical modeling. While majority DRM (ggt;20%) were consistently transmitted from source to recipient, the probability to detect a minority DRM in the recipient was not increased when the same minority DRM was detected in the source (Bayes Factor, BF=6.37). Longitudinal analyses revealed an exponential decay of DRM (BF=0.05) while genetic diversity increased. Our analysis revealed no substantial evidence for sexual transmission of minority DRM. The presence of minority DRM during early infection, followed by a rapid decay is consistent with the "mutation-selection balance" hypothesis, in which deleterious mutations are more efficiently purged later during HIV infection when the larger effective population size allows more efficient selection. Future studies using more recent sequencing technologies that are less prone to single-base errors should confirm these results applying a similar Bayesian framework in other clinical settings.

    IMPORTANCE The advent of sensitive sequencing platforms has led to an increased identification of minority drug resistance mutations (DRM), including among antiretroviral therapy-naïve HIV-infected individuals. While transmission of DRM may impact future therapy options for newly infected individuals, the clinical significance of the detection of minority DRMs remains controversial. In the present study, we applied deep sequencing techniques within a Bayesian hierarchical framework to a cohort of 24 transmission pairs to investigate whether minority DRMs detected shortly after transmission were the consequence of: (i) sexual transmission from the source, (ii) de novo emergence shortly after infection followed by viral selection and evolution or (iii) technical errors/limitations of deep sequencing methods. We found no clear evidence to support the sexual transmission of minority resistant variants and our results suggested that minor resistant variants may emerge de novo shortly after transmission when the small effective population size limits efficient purge by natural selection.

  • Nidovirus-Associated Proliferative Pneumonia in the Green Tree Python (Morelia viridis) [PublishAheadOfPrint]

  • In 2014 we observed a noticeable increase in sudden deaths of green tree pythons (Morelia viridis). Pathological examination revealed accumulation of mucoid material within airways and lung, associated with enlargement of the entire lung. We performed full necropsy and histological examination on 12 affected green tree pythons from 7 different breeders to characterise the pathogenesis of this "mucinous" pneumonia. By histology we could show a marked hyperplasia of the airway epithelium and of faveolar type II pneumocytes. Since routine microbiological tests failed to identify a causative agent, we studied lung samples of a few diseased snakes by next-generation sequencing (NGS). From the NGS data we could assemble a piece of RNA genome llt;85% identical to nidoviruses previously identified in ball pythons and Indian pythons. We then employed RT-PCR to demonstrate the presence of the novel nidovirus in all diseased snakes. To attempt virus isolation, we established primary cell cultures of Morelia viridis liver and brain, which we inoculated with lung homogenates of infected individuals. Ultrastructural examination of concentrated cell culture supernatants showed the presence of nidovirus particles, and subsequent NGS analysis yielded the full genome of the novel virus, Morelia viridis nidovirus (MVNV). We then generated an antibody against MVNV nucleoprotein, which we used alongside RNA in situ hybridisation to demonstrate viral antigen and RNA in the affected lungs. This suggests that in natural infection MVNV damages the respiratory tract epithelium which then results in epithelial hyperplasia, most likely as an exaggerated regenerative attempt in association with increased epithelial turnover.

    Importance Fairly recently novel nidoviruses associated with severe respiratory disease were identified in ball pythons and Indian pythons. Herein we report isolation and identification of a further nidovirus from green tree pythons (Morelia viridis) with fatal pneumonia. We thoroughly characterize the pathological changes in the infected individuals, and show that nidovirus infection is associated with marked epithelial proliferation in the respiratory tract. We speculate that this and the associated excess mucus production can lead to the animals' death, by inhibitingthe normal gas exchange in the lung. The virus was predominantly detected in the respiratory tract, which renders transmission via the respiratory route likely. Nidoviruses cause sudden outbreaks with high mortality in breeding collections, most affected snakes die without prior clinical signs. These findings, together with those of other groups, indicate that nidoviruses are a likely cause of severe pneumonia in pythons.

  • Reverse genetics system demonstrates that rotavirus non-structural protein NSP6 is not essential for viral replication in cell culture [PublishAheadOfPrint]

  • The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two non-structural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6-deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture although the NSP6 protein seemed to promote efficient virus growth.

    IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1-VP4, VP6, and VP7) and six non-structural proteins (NSP1-NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmid only-based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can be hardly addressed by conventional approaches.

  • An Epstein-Barr Virus microRNA Blocks IL-1 Signaling By Targeting the Interleukin-1 Receptor 1 [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) encodes ggt;44 viral microRNAs (miRNAs) that are differentially expressed throughout infection, can be detected in EBV-positive tumors, and manipulate several biological processes including cell proliferation, apoptosis, and immune responses. Here, we show that EBV BHRF1-2 miRNAs block NF-kB activation following treatment with pro-inflammatory cytokines, specifically interleukin-1 beta. Analysis of EBV PAR-CLIP miRNA targetome datasets combined with pathway analysis revealed multiple BHRF1-2 miRNA targets involved in interleukin signaling pathways. By further analyzing changes in cellular gene expression patterns, we identified the interleukin-1 receptor 1 (IL1R1) as a direct target of miR-BHRF1-2-5p. Targeting of the IL1R1 3rrsquo; UTR by EBV miR-BHRF1-2-5p was confirmed using 3rrsquo; UTR luciferase reporter assays and western blot assays. Manipulation of EBV BHRF1-2 miRNA activity in latently-infected B cells altered steady-state cytokine levels and disrupted IL-1bbeta; responsiveness. These studies demonstrate functionally relevant BHRF1-2 miRNA interactions during EBV infection, which is an important step in understanding their roles in pathogenesis.

    Importance IL-1 signaling plays an important role in inflammation and early activation of host innate immune responses following virus infection. Here, we demonstrate that a viral miRNA downregulates the IL-1 receptor 1 during EBV infection, which consequently alters the responsiveness of cells to IL-1 stimuli and changes the cytokine expression levels within infected cell populations. We postulate that this viral miRNA activity not only disrupts IL-1 autocrine and paracrine signaling loops that can alert effector cells to sites of infection, but also provides a survival advantage by dampening excessive inflammation that may be detrimental to the infected cell.

  • Tumor Suppressor p53 Stimulates the Expression of Epstein-Barr Virus Latent Membrane Protein 1 [PublishAheadOfPrint]

  • Epstein-Barr virus (EBV) is associated with multiple human malignancies. EBV latent membrane protein 1 (LMP1) is required for the efficient transformation of primary B lymphocytes in vitro and possibly in vivo. The tumor suppressor p53 plays a seminal role in cancer development. In some EBV-associated cancers, p53 tends to be wild-type and overly expressed, however, the effects of p53 on LMP1 expression is not clear. We find LMP1 expression is associated with p53 expression in EBV-transformed cells under physiological and DNA damaging conditions. DNA damage stimulates LMP1 expression, and p53 is required for the stimulation. Ectopic p53 stimulates endogenous LMP1 expression. Moreover, endogenous LMP1 blocks DNA damage-mediated apoptosis. To address the mechanism of p53-mediated LMP1 expression, we find that interferon regulatory factor 5 (IRF5), a direct target of p53, is associated with both p53 and LMP1. IRF5 binds to and activates LMP1 promoter reporter construct. Ectopic IRF5 increases the expression of LMP1, while knockdown of IRF5 leads to reduction of LMP1. Furthermore, LMP1 blocks IRF5-mediated apoptosis in EBV-infected cells. All of the data suggest that cellular p53 stimulates viral LMP1 expression, IRF5 may be one of the factors for p53-mediated LMP1 stimulation. LMP1 may subsequently block DNA damage- and IRF5-mediated apoptosis for the benefits of EBV. The mutual regulation between p53 and LMP1 may play an important role in EBV infection, latency, and its related cancers.

    IMPORTANCE The tumor suppressor p53 is a critical cellular protein in response to various stresses and dictates cells for various responses including apoptosis. This work suggests that an Epstein-Bar virus (EBV) principal viral oncogene is activated by cellular p53. The viral oncogene blocks p53-mediated adverse effects during viral infection and transformation. Therefore, the induction of the viral oncogene by p53 provides a means for the virus to cope with infection and DNA damage-mediated cellular stresses. This seems to be the first report that p53 activates a viral oncogene, therefore, the discovery would be interesting to a broad readership from oncology to virology.

  • Redefining Memory: Building the Case for Adaptive NK cells [PublishAheadOfPrint]

  • Classically, natural killer (NK) cells have been defined by nonspecific innate killing of virus-infected and tumor cells. However, burgeoning evidence suggests that the functional repertoire of NK cells is far more diverse than has been previously appreciated, thus raising the possibility that there could be unexpected functional specialization and even adaptive capabilities among NK cell subpopulations. Some of the first evidence that NK cells respond in an antigen-specific fashion came from experiments revealing that subpopulations of murine NK cells could respond to a specific MCMV protein, and that in the absence of T and B cells, murine NK cells also mediated adaptive immune responses to a secondary challenge with specific haptens. These data have been followed by demonstrations of NK cell memory to viruses and viral antigens in mice and primates (Figure 1). Herein, we will discuss different forms of NK cell antigen specificity, how these responses may be tuned to specific viral pathogens, and provide assessment of the current literature that may explain molecular mechanisms of the novel phenomenon of NK cell memory.

  • Reverse engineering field isolates of myxoma virus demonstrates that some gene disruptions or loss of function do not explain virulence changes observed in the field [PublishAheadOfPrint]

  • The coevolution of myxoma virus (MYXV) and wild European rabbits in Australia and Europe is a paradigm for the evolution of a pathogen in a new host species. Genomic analyses have identified the mutations that have characterized this evolutionary process, but defining causal mutations in the pathways from virulence to attenuation and back to virulence has not been possible. Using reverse genetics we examined the role of six selected mutations found in Australian field isolates of MYXV that fall in known or potential virulence genes. Several of these mutations occurred in genes previously identified as virulence genes in whole gene knockout studies. Strikingly, no single or double mutation among the mutations tested had an appreciable impact on virulence. This suggests either that virulence evolution was defined by amino acid changes other than those analyzed here, or that combinations of multiple mutations, possibly involving epistatic interactions or non-coding sequences, have been critical in the ongoing evolution of MYXV virulence. In sum, our results show that single-gene knockout studies in a progenitor virus can have little power to predict the impact of individual mutations seen in the field. The genetic determinants responsible for this canonical case of virulence evolution remain to be determined.

    IMPORTANCE The species jump of myxoma virus (MYXV) from the South American tapeti into the European rabbit populations of Australia and Europe is a canonical example of host-pathogen coevolution. Detailed molecular studies have identified multiple genes in MYXV that are critical for virulence, and genome sequencing has revealed the evolutionary history of MYXV in Australia and Europe. However, it has not been possible to categorically identify the key mutations responsible for attenuation or reversion to virulence during this evolutionary process. Here we use reverse genetics to examine the role of mutations in viruses from early and late in the Australian radiation of MYXV. Surprisingly, none of the candidate mutations we identified as likely having roles in attenuation proved to be important for virulence. This indicates that considerable caution is warranted when interpreting the possible role of individual mutations during virulence evolution.

  • Crystal structure of the N-terminal half of the traffic controller UL37 from Herpes Simplex virus Type 1. [PublishAheadOfPrint]

  • Inner tegument protein UL37 is conserved among all three subfamilies of herpesviruses. Studies of UL37 homologs from two alphaherpesviruses, Herpes Simplex virus Type 1 (HSV-1) and pseudorabies virus (PRV), have suggested that UL37 plays an essential albeit poorly defined role in intracellular capsid trafficking. At the same time, HSV and PRV homologs cannot be swapped, which suggests that in addition to a conserved function, UL37 homologs may also have divergent virus-specific functions. Accurate dissection of UL37 functions requires detailed maps in the form of atomic-resolution structures. Previously, we reported the crystal structure of the N-terminal half of UL37 (UL37N) from PRV. Here, we report the crystal structure of HSV-1 UL37N. Comparison of the two structures reveals that UL37 homologs differ in their overall shapes, distribution of surface charges, and the location of projecting loops. In contrast, the previously identified R2 surface region is structurally conserved. We propose that within the N-terminal half of UL37, functional conservation is centered within the R2 surface region whereas divergent structural elements pinpoint regions mediating virus-specific functions and may engage different binding partners. Together, the two structures can now serve as templates for a structure-guided exploration of both conserved and virus-specific functions of UL37.

    IMPORTANCE The ability to move efficiently within host cell cytoplasm is essential for replication in all viruses. It is especially important in the neuroinvasive alphaherpesviruses, such as the human Herpes Simplex Viruses 1 and 2 (HSV-1, 2) and veterinarian pseudorabies virus (PRV), that infect the peripheral nervous system and have to travel long distances along axons. Capsid movement in these viruses is controlled by capsid-associated tegument proteins, yet their specific roles have not yet been defined. Systematic exploration of the roles of tegument proteins in capsid trafficking requires detailed navigational charts in the form of their three-dimensional structures. Here, we determined the crystal structure of the N-terminal half of a conserved tegument protein UL37 from HSV-1. This structure, along with our previously reported structure of UL37 homolog from PRV, provides a much needed 3-dimensional template for the dissection of both conserved and virus-specific functions of UL37 in intracellular capsid trafficking.

  • Inactivated H7 influenza virus vaccines protect mice despite low levels of neutralizing antibodies [PublishAheadOfPrint]

  • Avian influenza viruses of the H7 hemagglutinin (HA) subtype present a significant public health threat, as evidenced by the ongoing outbreak of human A(H7N9) infections in China. When evaluated by hemagglutinin inhibition (HI) and micro-neutralization (MN) assays, H7 viruses and vaccines induce lower level of neutralizing antibodies (nAb) than do their seasonal counterparts, making it difficult to develop and evaluate pre-pandemic vaccines. We have previously shown that purified recombinant H7 hemagglutinin (HA) appear to be poorly immunogenic in that they induce low levels of HI and MN antibodies. Here, we immunized mice with whole inactivated reverse genetics reassortant (RG) viruses expressing HA and NA from 3 different H7 viruses [A/Shanghai/2/2013 (H7N9), A/Netherlands/219/2003 (H7N7) and A/New York/107/2003 (H7N2)], or with human A(H1N1)pdm09 [A/California/07/2009-like] or A(H3N2) [A/Perth16/2009] viruses. Mice produced equivalent titers of antibodies to all viruses as measured by ELISA. However, the antibody titers induced by H7 viruses were significantly lower when measured by HI and MN assays. Despite inducing very low levels of nAb, H7 vaccines conferred complete protection against homologous virus challenge in mice, and the serum antibodies directed against the HA head region were capable of mediating protection. The apparently low immunogenicity associated with H7 viruses and vaccines may be at least partly related to measuring antibody titers with the traditional HI and MN assays, which may not provide a true measure of protective immunity associated with H7 immunization. This study underscores the need for development of additional correlates of protection for pre-pandemic vaccines.

    IMPORTANCE H7 avian influenza viruses present a serious risk to human health. Preparedness efforts include development of pre-pandemic vaccines. For seasonal influenza viruses, protection is correlated with antibody titers measured by hemagglutination inhibition (HI) and virus microneutralization (MN) assays. Since H7 vaccines typically induce low HI and MN titers, they have been considered to be poorly immunogenic. We show that in mice H7 whole inactivated virus (WIV) vaccines were as immunogenic as seasonal WIVs, as they induced similar levels of overall serum antibodies. However, a larger fraction of the antibodies induced by H7 WIV was non-neutralizing in vitro. Nevertheless, the H7 WIV completely protected mice against homologous viral challenge, and antibodies directed against the HA-head were the major contributor toward immune protection. Vaccines against H7 avian influenza viruses may be more effective than HI and virus neutralization assays suggest, and such vaccines may need other methods for evaluation.

  • Relevance of assembly-activating protein for Adeno-associated virus vector production and capsid protein stability in mammalian and insect cells [PublishAheadOfPrint]

  • The discovery that Adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both, mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1-3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1-3, but neither triggered assembly of functional capsids, nor did it promote nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5 which exhibited AAP-independent, albeit low-level particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knock-out. We conclude that AAP is key for assembly of genuine capsids from at least ten different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids.

    IMPORTANCE Assembly of Adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein AAP whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus-like particles solely composed of the major capsid protein VP3, AAP's role and relevance for assembly of genuine AAV capsids has remained largely unclear. Thus, we established a trans-complementation assay permitting to assess AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding of AAV/AAP biology, and it concomitantly provides insights into the importance of AAP for AAV vector production.

  • Protective Efficacy of Broadly Neutralizing Antibodies with Incomplete Neutralization Activity Against SHIV in Rhesus Monkeys [PublishAheadOfPrint]

  • HIV broadly neutralizing antibodies (bnAbs) have been shown to occasionally display unusual virus neutralization profiles with non-sigmoidal slopes and plateaus at llt;100% neutralization against a variety of viruses. The significance of incomplete neutralization for the ability of bnAbs to mediate protective effects in vivo however is undetermined. In the current study, we selected two bnAbs, PGT121 and 3BNC117, as they incompletely neutralize the clade C SHIV stock (SHIV-327c) at 85% and 70%, respectively, and performed a protection study in rhesus macaques. Animals were i.v. administered 10 mg/kg and 2 mg/kg, of PGT121 or 3BNC117 before being rectally challenged with a single high dose of SHIV-327c. PGT121 protected 6 out of 7 monkeys, while 6 out of 7 3BNC117-pretreated animals became infected, although with significantly delayed plasma viremia compared to the control animals. These data suggest that complete neutralization is not imperative for bnAbs to prevent infection but that with increasing levels of incomplete neutralization the sterilizing activity diminishes.

    Importance: Multiple antibodies have been identified that potently neutralize a broad range of circulating HIV strains. However, not every virus/antibody combination results in complete neutralization of the input virus suggesting that a fraction of virus particles is resistant to antibody neutralization, despite high antibody concentrations. This observation of "incomplete neutralization" is associated with non-sigmoidal neutralization curves plateauing below 100% neutralization but the significance of this phenomenon for the ability of neutralizing antibodies to mediate protective effects in-vivo is undetermined. In this study, we show that the broadly neutralizing antibody PGT121 that only neutralized up to 85% of the SHIV-327c challenge stock in-vitro protected 6 out of 7 rhesus macaques against infection while the antibody 3BNC117 that neutralized up to 70% of SHIV-327c in-vitro did not prevent though significantly delayed establishment of infection, suggesting with increasing levels of incomplete neutralization the ability of a bnAb to mediate sterilizing protection diminishes.

  • Immunodominance of adenovirus-derived CD8+ T cell epitopes interferes with the induction of transgene-specific immunity in adenovirus-based immunization [PublishAheadOfPrint]

  • Adenovirus (Ad)-based immunization is a popular approach in vaccine development, and Ad-based vectors are renowned for their potential to induce strong CD8+ T cell responses to the encoded transgene. Surprisingly, we found before in the mouse Friend retrovirus (FV) model that Ad-based immunization did not induce CD8+ T cell responses to the FV Leader-Gag-derived immunodominant epitope GagL85-93. We show now that induction of GagL85-93-specific CD8+ T cells was highly effective when Leader-Gag was delivered by plasmid DNA immunization, implying a role for Ad-derived epitopes in mediating unresponsiveness. Immunizing with DNA constructs encoding strings of GagL85-93 and the two Ad-derived epitopes DBP418-426 and Hexon486-494, we confirmed that Ad epitopes prevent induction of GagL85-93-specific CD8+ T cells. Interestingly, while DBP418-426 did not interfere with GagL85-93-specific CD8+ T cell induction, the H-2Dd-restricted Hexon486-494 suppressed the CD8+ T cell response to the H-2Db-restricted GagL85-93 strongly in H-2b/d, but not in H-2b/b mice. This finding indicates that competition occurs at the level of responding CD8+ T cells, and we could indeed demonstrate that co-immunization with an IL2-encoding plasmid restored GagL85-93-specific CD8+ T cell responses to epitope strings in the presence of Hexon486-494. IL2 co-delivery did not restore GagL85-93 responsiveness in Ad-based immunization, however, likely due to the presence of further epitopes in the Ad vector. Our findings show that seemingly immunodominant transgene epitopes can be dominated by Ad-derived epitopes. These findings underline the importance of thorough characterization of vaccine vectors, and modifications of vectors or immunogens may be required to prevent impaired transgene-specific immune responses.

    Importance Ad-based vectors are widely used in experimental pre-clinical and clinical immunization studies against numerous infectious agents such as human immunodeficiency virus, Ebola virus, plasmodium falciparum, or mycobacterium tuberculosis. Pre-existing immunity to Ad-based vectors is widely recognized as a hindrance to the widespread use of Ad-based vectors for immunizations in humans, however, our data shows that immune response to Ad-derived T cell epitopes can also result in loss or impairment of transgene-specific immune responses in pre-naïve vaccinees due to immune competition. Our results highlight that seemingly immunodominant epitopes may be affected by dominance of vector-derived epitopes, and modifications of the vector design or the immunogens employed in immunization may lead to more effective vaccines.

  • High Risk Alpha Papillomavirus Oncogenes Impair the Homologous Recombination Pathway. [PublishAheadOfPrint]

  • Persistent high risk genus aalpha; human papillomavirus (HPV) infections cause nearly every cervical carcinoma and a subset of tumors in the oropharyngeal tract. During the decades required for HPV-associated tumorigenesis, the cellular genome becomes significantly destabilized. Our analysis of cervical tumors from 4 separate data sets found a significant upregulation of the homologous recombination (HR) pathway genes. The increased abundance of HR proteins can be replicated in primary cells by expression of the two HPV oncogenes (E6 and E7) required for HPV-associated transformation. HPV E6 and E7 also enhanced the ability of HR proteins to form repair foci, yet both E6 and E7 reduce the ability of the HR pathway to complete double strand break (DSB) repair by about 50%. The HPV oncogenes hinder HR by allowing the process to begin at points in the cell cycle when the lack of a sister chromatid to serve as a homologous template prevents completion of the repair. Further, HPV E6 attenuates repair by causing RAD51 to be mislocalized away from both transient and persistent DSBs, while HPV E7 is only capable of impairing RAD51 localization to transient lesions. Finally, we show that the inability to robustly repair DSBs causes some of these lesions to be more persistent, a phenotype that correlates with increased integration of episomal DNA. Together these data support our hypothesis that HPV oncogenes contribute to the genomic instability observed in HPV-associated malignancies by attenuating the repair of damaged DNA.

    IMPORTANCE: This work expands the understanding of HPV biology, establishing a direct role for both HPV E6 and E7 in the destabilization of the host genome by blocking the homologous repair of DSBs. To our knowledge, this is the first time that both viral oncogenes were shown to disrupt this DSB repair pathway. We show that HPV E6 and E7 allow HR to initiate at an inappropriate part of the cell cycle. The mislocalization of RAD51 away from DSBs in cells expressing HPV E6 and E7 hinders HR through a distinct mechanism. These observations have broad implications. The impairment of HR by HPV oncogenes may be targeted for treatment of HPV+ malignancies. Further, this attenuation of repair suggests HPV oncogenes may contribute to tumorigenesis by promoting the integration of the HPV genome, a common feature of HPV-transformed cells. Our data support this idea as HPV E6 stimulates the integration of episomes.

  • KINASE ACTIVITY OF FIBROBLAST GROWTH FACTOR RECEPTOR-3 REGULATES ACTIVITY OF THE PAPILLOMAVIRUS E2 PROTEIN [PublishAheadOfPrint]

  • The papillomavirus (PV) E2 protein is a DNA binding, protein interaction platform that recruits viral and host factors necessary for transcription and replication. We recently discovered phosphorylation of a tyrosine (Y102) in bovine PV E2. To identify the responsible factor, we tested several candidate tyrosine kinases that are highly expressed in keratinocytes for binding to BPV-1 E2. Fibroblast growth factor receptor 3 (FGFR3) co-immunoprecipitated with the BPV-1 E2 protein, as did HPV-31 E2, which also co-localized with FGFR3 within the nucleus. A constitutively active mutant form of FGFR3 decreased BPV-1 and HPV-31 transient replication, though this result also occurred in a BPV-1 E2 mutant lacking a previous phosphorylation site of interest (Y102). Furthermore, FGFR3 depletion in cell lines that maintain HPV-31 episomes increased viral copy number. These results suggest that FGFR3 kinase activity may regulate the PV reproductive program through phosphorylation of the E2 protein, although this is unlikely to occur through the Y102 residue of HPV E2.

    IMPORTANCE The papillomavirus (PV) is a double stranded DNA tumor virus infecting cervix, mouth, and throat tissues. The viral protein E2 is responsible for the replication of the virus. Understanding the mechanisms of the replicative life cycle of the virus may bring to light direct targets and treatments against viral infection. We recently found that the fibroblast growth factor receptor 3 (FGFR3) interacts with and mediates PV E2 function through phosphorylation of the E2 protein. Our study suggests that the function of the E2 protein may be regulated through a direct FGFR3 target during the maintenance stage of the PV life cycle.

  • Monoclonal antibodies against Zika virus: Therapeutics and their implications for vaccine design [PublishAheadOfPrint]

  • Zika virus (ZIKV) has caused global concern due to its association with neurological complications in newborns and adults. Although no approved vaccines or antivirals against ZIKV infection to date, hundreds of monoclonal antibodies (MAbs) have been developed in a short period. Here, we first present a complete picture of the ZIKV MAbs and then focus on the neutralizing mechanisms and immune hot spot uncovered through structural studies, which provide insight for therapeutics and vaccine design.

  • The asymmetric trimeric ring structure of the nucleocapsid protein of Tospovirus [PublishAheadOfPrint]

  • Tomato spotted wilt virus (TSWV), belonging to the genus Tospovirus of the family Bunyaviridae, causes significant economic damage in several vegetables and ornamental plants worldwide. Similar to that in all other negative-strand RNA viruses, the nucleocapsid (N) protein plays very important roles in their viral life cycle. N proteins protect genomic RNAs by encapsidation and form a viral ribonucleoprotein complex (vRNP) with some RNA-dependent RNA polymerases. Here we show the crystal structure of the N protein from TSWV. Protomers of TSWV N proteins consist of three parts: N arm, C arm, and core domain. Unlike N proteins of other negative-strand RNA viruses, the TSWV N protein forms an asymmetric trimeric ring. To form the trimeric ring, the N and C arms of the N protein interact with the core domains of two adjacent N proteins. By solving the crystal structures of the TSWV N protein with nucleic acids, we showed that an inner cleft of the asymmetric trimeric ring is an RNA-binding site. These characters are similar to those of N proteins of other viruses in the family Bunyaviridae. Based on these observations, we discuss possibilities of TSWV encapsidation model.

    IMPORTANCE Tospoviruses cause significant crop losses throughout the world. Particularly, TSWV has an extremely wide host range (ggt;1,000 plant species including dicots and monocots), and their worldwide losses estimated to be in excess of US$1 billion annually. Despite such an importance, any proteins of Tospoviruses have not been elucidated so far. Among TSWV-encoded proteins, N protein is required for assembling the viral genomic RNA into viral ribonucleoprotein (vRNP), and that is involved in various steps of their life cycle, such as RNA replication, virus particle formation, and cell-to-cell movement. This study revealed the structure of TSWV N protein with or without nucleic acids as the first virus of the genus Tospovirus, so it completed our view of N proteins of the family Bunyaviridae.

  • Isolation and Characterization of Metallosphaera turreted icosahedral virus (MTIV), a founding member of a new family of archaeal viruses [PublishAheadOfPrint]

  • Our understanding of archaeal virus diversity and structure is just beginning to emerge. Here we describe a new archaeal virus, tentatively named Metallosphaera turreted icosahedral virus (MTIV), that was isolated from an acidic hot spring in Yellowstone National Park, USA. Two strains of the virus were identified and found to replicate in an archaeal host species closely related to Metallosphaera yellowstonensis. Each strain encodes for a 9.8-9.9 kb, linear dsDNA genome with large inverted terminal repeats. Each genome encodes for 21 ORFs. Between the strains the ORFs display high homology, but they are quite distinct from other known viral genes. The 70-nm diameter virion is built upon on a T=28 icosahedral lattice. Both single particle cryo-electron microscopy and cryo-tomography reconstructions reveal an unusual structure that has 42 turret-like projections: 12 from each of the 5-fold axes and 30 hexameric units positioned on icosahedral 2-fold axes. Both the virion structural properties and genome content support MTIV as the founding member of a new family of archaeal viruses.

    Importance: Many archaeal viruses are quite different than viruses infecting bacteria and eukaryotes. Initial characterization of MTIV reveals a virus distinct from other known bacterial, eukaryotic, and archaeal viruses; this finding suggests that viruses infecting Archaea are still an understudied group of viruses. As the first known virus infecting the Metallosphaera, MTIV provides a new system for exploring archaeal virology by examining host-virus interactions and the unique features of MTIV structure-function relationships. These studies will likely expand our understanding of virus ecology and evolution.

  • Human herpesvirus 8 infects and replicates in Langerhans cells and interstitial dermal dendritic cells and impairs their function [PublishAheadOfPrint]

  • The predominant types of dendritic cells (DC) in the skin and mucosa are Langerhans cells (LC) and interstitial dermal DC (iDDC). LC and iDDC process cutaneous antigens and migrate out of the skin and mucosa to the draining lymph nodes to present antigens to T and B cells. Because of the strategic location of LC and iDDC and the ability of these cells to capture and process pathogens, we hypothesized that they could be infected with human herpesvirus 8 (HHV-8; Kaposi's sarcoma-associated herpesvirus) and have an important role in the development of Kaposi's sarcoma (KS). We have previously shown that HHV-8 enters monocyte-derived dendritic cells (MDDC) through DC-SIGN, resulting in a nonproductive infection. Here we show that LC and iDDC generated from pluripotent cord blood CD34+ cell precursors support productive infection with HHV-8. Anti-DC-SIGN mAb inhibited HHV-8 infection of iDDC as shown by low expression of viral proteins and DNA. In contrast, blocking of both langerin and the receptor protein-tyrosine kinase ephrin A2 was required to inhibit HHV-8 infection of LC. Infection with HHV-8 did not alter cell surface expression of langerin on LC, but down-regulated expression of DC-SIGN on iDDC, as we previously reported for MDDC. HHV-8-infected LC and iDDC had reduced ability to stimulate allogeneic CD4+ T cells in the mixed lymphocyte reaction. These results indicate that HHV-8 can target both LC and iDDC for productive infection via different receptors and alter their function, supporting their potential role in HHV-8 pathogenesis and KS.

    IMPORTANCE: Here we show that HHV-8, a DNA tumor virus that causes Kaposi's sarcoma, infects three types of dendritic cells: monocyte-derived dendritic cells, Langerhans cells and interstitial dermal dendritic cells. We show that different receptors are used by this virus to infect these cells. DC-SIGN is a major receptor for infection of both monocyte-derived dendritic cells and interstitial dermal dendritic cells, yet the virus only fully replicates in the latter. HHV-8 uses langerin and the ephrin A2 receptor to infect Langerhans cells, which support full HHV-8 lytic replication. This infection of Langerhans cells and interstitial dermal dendritic cells results in impaired ability to stimulate CD4+ helper T cell responses. Taken together, our data show that HHV-8 utilizes alternate receptors to differentially infect and replicate in these tissue-resident DC, and supports the hypothesis that these cells play an important role in HHV-8 infection and pathogenesis.

  • Positive and negative regulation of type I Interferons by the human T cell leukemia virus antisense protein HBZ [PublishAheadOfPrint]

  • The pathogenesis of human T-cell leukemia virus type 1 (HTLV-1) is strongly linked to the viral regulatory proteins Tax1 and HBZ, whose opposing functions contribute to the clinical outcome of infection. Type I interferons aalpha; and bbeta; (IFNaalpha; and IFNbbeta;) are key cytokines involved in innate immunity and IFNaalpha;, in combination with other antivirals, is extensively used in the treatment of HTLV-1 infection. The relationship between HTLV-1 and IFN signaling is unclear and to date the effect of HBZ on this pathway has not been examined. Here we report that HBZ significantly enhances IRF7 induced IFNaalpha; and ISRE promoter activities and IFNaalpha; production and can counteract the inhibitory effect of Tax1. In contrast to this, we show that HBZ and Tax1 cooperate to inhibit the induction of IFNbbeta; and ISRE promoters by IRF3 and IFNbbeta; production. In addition we reveal that HBZ enhances ISRE activation by IFNaalpha;. We further show that HBZ enhances IRF7 and suppresses IRF3 activation by TBK1 and IKK. We demonstrate that HBZ has no effect on virus induced nuclear accumulation of IRF3 suggesting that it may inhibit IRF3 activity at a transcriptional level. We show that HBZ physically interacts with IRF7 and IKK but not with IRF3 or TBK1. Overall our findings suggest that both HBZ and Tax1 are negative regulators of immediate-early IFNbbeta; innate immune responses, while HBZ but not Tax1 positively regulates the induction of IFNaalpha; and downstream IFNaalpha; signaling.

    IMPORTANCE Type I interferons are powerful antiviral cytokines and are used extensively in the treatment of HTLV-1 induced ATL. To date the relationship between HTLV-1 and the IFN pathway is poorly understood and studies so far have focused on Tax1. Our study is unique in that it examines the effect of HBZ, alone or in combination with Tax1 on type I IFN signaling. This is important as HBZ is frequently the only viral protein expressed in infected cells, particularly at later stages of infection. A better understanding of the how HBZ regulates IFN signaling may lead to the development of therapeutics that can modify such responses and improve the clinical outcome of infected individuals.

  • Zinc finger-containing cellular transcription corepressor ZBTB25 promotes influenza virus RNA transcription and is a target for zinc-ejector drugs [PublishAheadOfPrint]

  • Influenza A virus (IAV) replication relies on an intricate interaction between virus and host cells. How the cellular proteins are usurped for IAV replication remains largely obscure. The aim of this study was to search for novel and potential cellular factors that participate in IAV replication. ZBTB25, a transcription repressor of a variety of cellular genes, was identified by an RNAi genomic library screening. Depletion of ZBTB25 significantly reduced IAV production. Conversely, overexpression of ZBTB25 enhanced it. ZBTB25 interacted with viral RNA-dependent RNA polymerase (RdRp) proteins and modulated its transcription activity. Besides, ZBTB25 also functioned as a viral RNA-binding protein, binding preferentially to the U-rich sequence within 5rrsquo; UTR of vRNA. Both protein-protein and protein-RNA interaction involving ZBTB25 facilitated viral RNA transcription and replication. In addition, ZBTB25 suppressed interferon production, further enhancing viral replication. ZBTB25-associated functions required an intact Zinc-finger domain and post-translational SUMO-1 modification of ZBTB25. Furthermore, treatment with disulfiram (a zinc-ejector) of ZBTB25-overexpressing cells showed significantly reduced IAV production as a result of reduced RNA synthesis. Our findings indicate that IAV usurps ZBTB25 for IAV RNA synthesis and serves as a novel and potential therapeutic anti-viral target.

    IMPORTANCE IAV-induced seasonal influenza causes severe illness and death in high risk populations. However, IAV develops resistance to current antiviral drugs due to its high mutation rate. Therefore, development of drugs targeting cellular factors required for IAV replication is an attractive alternative for IAV therapy. Here, we discovered a cellular protein, ZBTB25, that enhances viral RdRp activity by binding to both viral RdRp and viral RNA to stimulate viral RNA synthesis. A unique feature of ZBTB25 in the regulation of viral replication is its dual transcription functions; namely, promoting viral RNA transcription through binding to the U-rich region of vRNA and suppressing cellular interferon production. ZBTB25 contains a zinc-finger domain that is required for RNA-inhibitory activity by chelating zinc ions. Disulfiram treatment disrupts the zinc-finger functions, effectively repressed IAV replication. Altogether, we demonstrated that ZBTB25 regulates IAV RNA transcription and replication, and serves as a promising antiviral target for IAV treatment.

  • Presence of a Shared 5'-Leader Sequence in Ancestral Human and Mammalian Retroviruses and its Transduction into Feline Leukemia Virus. [PublishAheadOfPrint]

  • Recombination events induce significant genetic changes, and this process can result in virus genetic diversity or in the generation of novel pathogenicity. We discovered a new recombinant feline leukemia virus (FeLV) gag gene harboring an unrelated insertion, termed the "X-region", which was derived from FcERV-gamma4, a feline endogenous gammaretrovirus. The identified FcERV-gamma4 proviruses have lost their coding capability, but some can express their viral RNA in feline tissues. Although the X-region-carrying recombinant FeLVs appeared to be replication-defective viruses, they were detected in 6.4% of tested FeLV-infected cats. All isolated recombinant FeLV clones commonly incorporated a middle part of FcERV-gamma4 5rrsquo; -leader region as an X-region. Surprisingly, a sequence corresponding to the portion contained in all X-regions is also present in at least 13 endogenous retroviruses (ERVs) observed in the cat, human, primate, and pig genomes. We termed this shared genetic feature the "commonly-shared sequence" (CS-sequence). Despite our phylogenetic analysis indicating that all CS-sequence-carrying ERVs are classified as gammaretroviruses, no obvious closeness was revealed among these ERVs. However, the Shannon entropy in the CS-sequence was lower than that in other parts of the provirus genome. Notably, the CS-sequence of human endogenous retrovirus T had 73.8% similarity with that of FcERV-gamma4, and specific signals were detected in the human genome by southern blot analysis using a probe for the FcERV-gamma4 CS-sequence. Our results provide an interesting evolutionary history for CS-sequence circulation among several distinct ancestral viruses and a novel recombined virus over a prolonged period.

    IMPORTANCE Recombination among ERVs or modern viral genomes causes a rapid evolution of retroviruses, and this phenomenon can result in the serious situation of viral disease re-emergence. We identified a novel recombinant FeLV gag gene that contains an unrelated sequence, termed the X-region. This region originated from 5'-leader of FcERV-gamma4, a replication-incompetent feline ERV. Surprisingly, a sequence corresponding to the X-region is also present in the 5'-portion of other ERVs, including human endogenous retroviruses. Scattered copies of the ERVs carrying the unique genetic feature, here named the "commonly-shared sequence" (CS-sequence), were found in each host genome, suggesting that ancestral viruses may have captured and maintained the CS-sequence. More recently, a novel recombinant FeLV hijacked the CS-sequence from inactivated FcERV-gamma4 as the X-region. Therefore, tracing the CS-sequences can provide unique models for not only the modern reservoir of new recombinant viruses but also for the genetic features shared among ancient retroviruses.

  • Molecular Function Analysis of Rabies Virus RNA Polymerase L Protein by Using an L Gene-Deficient Virus [PublishAheadOfPrint]

  • While the RNA-dependent RNA polymerase L protein of rabies virus (RABV), a member of the genus Lyssavirus of the family Rhabdoviridae, has potential to be a therapeutic target for rabies, the molecular functions of this protein have remained largely unknown. In this study, to obtain a novel experimental tool for molecular function analysis of the RABV L protein, we established by using a reverse genetics approach an L gene-deficient RABV (Nishi-L/Nluc), which infects, propagates and correspondingly produces NanoLuc luciferase in cultured neuroblastoma cells [20013240291] transfected to express the L protein. The trans-complementation with wild-type L protein, but not that with a functionally defective L protein mutant, efficiently supported luciferase production by Nishi-L/Nluc, confirming its potential for function analysis of the L protein. Based on the findings obtained from comprehensive genetic analyses of L genes from various RABV and other lyssavirus species, we examined the functional importance of a highly conserved L protein region at positions 1,914-1,933 by a trans-complementation assay with Nishi-L/Nluc and a series of L protein mutants. The results revealed that the amino acid sequence at positions 1,929-1,933 (NPYNE) is functionally important, being also supported by other findings that this sequence is critical for binding of the L protein with its essential cofactor P protein, thereby also for L protein's RNA polymerase activity. Our findings provide useful information for the development of an anti-RABV drug targeting the L-P protein interaction.

    IMPORTANCE To the best of our knowledge, this is the first report on the establishment of an L gene-deficient, reporter gene-expressing virus in all viral species of the order Mononegavirales, also highlighting its applicability to a trans-complementation assay, which is useful for molecular function analyses of their L proteins. Moreover, this study revealed for the first time that the NPYNE sequence at positions 1,929-1,933 in the RABV L protein is important for L protein's interaction with the P protein, being consistent with and extending the results of a previous study showing that the P protein-binding domain in the L protein is located at its C-terminal region at positions 1,562-2,127. This study indicates that the NPYNE sequence is a promising target for the development of an inhibitor of viral RNA synthesis, which has high potential as a therapeutic drug for rabies.

  • Ubiquitin Ligase WWP1 Interacts with Ebola Virus VP40 to Regulate Egress [PublishAheadOfPrint]

  • Ebola virus (EBOV) is a member of the Filoviridae family and the cause of hemorrhagic fever outbreaks. The EBOV VP40 (eVP40) matrix protein is the main driving force for virion assembly and budding. Indeed, expression of eVP40 alone in mammalian cells results in the formation and budding of virus-like particles (VLPs), which mimics the budding process and morphology of authentic, infectious EBOV. To complete the budding process, eVP40 utilizes its PPxY L-domain motif to recruit a specific subset of host proteins containing one or more modular WW-domains that then function to facilitate efficient production and release of eVP40 VLPs. In this report, we identified additional host WW-domain interactors by screening for potential interactions between mammalian proteins possessing one or more WW-domains and WT or PPxY-mutant peptides of eVP40. We identified HECT-family E3 ubiquitin ligase WWP1 and all four of its WW-domains as strong interactors with the PPxY motif of eVP40. The eVP40-WWP1 interaction was confirmed by both peptide pulldown and co-immunoprecipitation assays, which also demonstrated that modular WW-domain #1 of WWP1 was most critical for binding to eVP40. Importantly, the eVP40-WWP1 interaction was found to be biologically relevant for VLP budding since: 1) siRNA knockdown of endogenous WWP1 resulted in inhibition of eVP40 VLP egress, 2) co-expression of WWP1 and eVP40 resulted in ubiquitination of eVP40 and a subsequent increase in eVP40 VLP egress, and 3) an enzymatically inactive mutant of WWP1 (C890A) did not ubiquitinate eVP40, nor enhance eVP40 VLP egress. Lastly, our data show that ubiquitination of eVP40 by WWP1 enhances egress of VLPs and concomitantly decreases cellular levels of higher MW oligomers of eVP40. In sum, these findings contribute to our fundamental understanding of the functional interplay between host E3 ligases, ubiquitination, and regulation of EBOV VP40-mediated egress.

    Importance: Ebola virus (EBOV) is a high-priority, emerging human pathogen that can cause severe outbreaks of hemorrhagic fever with high mortality rates. As there are currently no approved vaccines or treatments for EBOV, a better understanding of the biology and functions of EBOV-host interactions that promote or inhibit viral budding is warranted. Here we describe a physical and functional interaction between EBOV VP40 (eVP40) and WWP1, a host E3 ubiquitin ligase that ubiquitinates VP40 and regulates VLP egress. This viral PPxY-host WW-domain mediated interaction represents a potential new target for host-oriented inhibitors of EBOV egress.

  • PGC1{alpha} TRANSCRIPTIONAL ADAPTOR FUNCTION GOVERNS HEPATITIS B VIRUS REPLICATION BY CONTROLLING HBcAg/p21 PROTEIN MEDIATED CAPSID FORMATION [PublishAheadOfPrint]

  • In the human hepatoma cell line, Huh7, co-expression of the coactivators, peroxisome proliferator-activated receptor gamma coactivator 1aalpha; (PGC1aalpha;), cAMP responsive element binding protein binding protein (CBP), steroid receptor coactivator 1 (SRC1) and protein arginine methyltransferase 1 (PRMT1) only modestly increase HBV biosynthesis. However, utilizing the human embryonic kidney cell line, HEK293T, it was possible to demonstrate that PGC1aalpha; alone can support viral biosynthesis independently of additional coactivator or transcription factor expression. In contrast, additional coactivators failed to support robust HBV replication in the absence of PGC1aalpha;. These observations indicate that PGC1aalpha; represents a novel adaptor molecule capable of recruiting the necessary transcriptional machinery to the HBV nucleocapsid promoter to modestly enhance viral pregenomic 3.5kb RNA synthesis. Although this change in transcription is associated with a similar modest change in hepatitis B virus core antigen polypeptide (HBcAg/p21) synthesis, it mediates a dramatic increase in viral capsid production and robust viral replication. Therefore it is apparent that the synthesis of cytoplasmic HBcAg/p21 above a critical threshold level is required for the efficient assembly of HBV replication competent viral capsids.

    IMPORTANCE Hepatitis B virus (HBV) is a major human pathogens and novel targets for the development of additional therapeutic agents are urgently needed. Here we demonstrate that the coactivator, peroxisome proliferator-activated receptor gamma coactivator 1aalpha; (PGC1aalpha;), serves as a unique adapter molecule for the recruitment of additional coactivator proteins which can finely regulate HBV transcription. The consequence of this precise regulation of viral RNA levels by PGC1aalpha; is a subtle increase in cytoplasmic HBcAg/p21 polypeptide translation which shifts the equilibrium from dimer formation dramatically in favor of viral capsid assembly. These finding suggest that both PGC1aalpha; and capsid assembly may represent attractive targets for the development of antiviral agents against chronic HBV infection.

  • Identification of emerging mac-tropic HIV-1 R5 variants in brain tissue of patients without severe neurological complications [PublishAheadOfPrint]

  • Untreated HIV-1+ individuals frequently suffer from HIV associated neurocognitive disorders (HAND), with about 30% of AIDS patients suffering severe HIV associated dementias (HAD). Antiretroviral therapy has greatly reduced the incidence of HAND and HAD. However, there is a continuing problem of milder neurocognitive impairments in treated HIV+ patients that may be increasing with long term therapy.

    Here, we investigated whether envelope (env) genes could be amplified from proviral DNA or RNA derived from brain tissue of 12 individuals with normal neurology or minor neurological conditions (N/MC individuals). The tropism and characteristics of the brain-derived Envs were then investigated and compared with Envs derived from immune tissue. We showed that (1) macrophage-tropic R5 Envs could be detected in brain tissue of 4/12 N/MC individuals, (2) macrophage-tropic Envs in brain tissue formed compartmentalized clusters distinct from non-macrophage-tropic envs recovered from spleen or brain, (3) there was evidence consistent with active viral expression by macrophage-tropic variants in brain tissue of some individuals and (4) Envs from immune tissue of the N/MC individuals were nearly all tightly non-mac-tropic contrasting with previous data for neuro-AIDS patients where immune tissue Envs mediated a range of macrophage infectivity from background levels to modest infection, with a small number of Envs from some patients mediating high macrophage infection.

    In summary, data presented shows that compartmentalized and active macrophage-tropic HIV-1 variants are present in brain tissue of individuals before neurological disease becomes overt or serious.

    IMPORTANCE The detection of highly compartmentalized macrophage-tropic R5 Envs in brain tissue of HIV patients without serious neurological disease is consistent with their emergence from a viral population already established there, perhaps from early disease. The detection of active macrophage-tropic virus expression and probably replication indicates that anti-retroviral drugs with optimal penetration through the blood brain barrier should be considered even in patients without neuro-disease. Finally, our data are consistent with the brain forming a sanctuary site for latent virus and low level viral replication in the absence of neuro-disease.

  • Mechanism of Interferon Stimulated Gene Induction in HIV-1 Infected Macrophages [PublishAheadOfPrint]

  • Viruses manipulate the complex interferon and interferon stimulated gene (ISG) system in different ways. We have previously shown that HIV inhibits type I and III interferons in its key target cells but directly stimulates a subset of ggt;20 ISGs in macrophages and dendritic cells many of which are antiviral. Here we examined the mechanism of induction of ISGs and showed this occurred in two phases. The first phase was transient (0-24hpi) induced mainly by extracellular vesicles, and one of its component proteins HSP90aalpha;, contained within the HIV inoculum. The second dominant and persistent phase (ggt;48hpi) was induced via newly transcribed HIV RNA and sensed via RIGI, as shown by the reduction in ISG expression after the knock down of the RIGI adaptor, MAVS, by siRNA and the inhibition of both the initiation and elongation of HIV transcription, by shRNA transcriptional silencing. We further defined the induction pathway, showing sequential HIV RNA stimulation via Tat, RIGI, MAVS, IRF1 and IRF7 also identified by siRNA knockdown. IRF1 also plays a key role in the first phase. We also showed that the ISGs, IFITs 1-3 inhibited HIV production, measured as extracellular infectious virus. All induced antiviral ISGs probably lead to restriction of HIV replication in macrophages, contributing to a persistent, non-cytopathic infection while the inhibition of interferon facilitates spread to adjacent cells. Both may influence the size of macrophage HIV reservoirs in vivo. Elucidating the mechanisms of ISG induction may help devise immunotherapeutic strategies to limit the size of these reservoirs.

    IMPORTANCE HIV, like other viruses, manipulates the antiviral interferon and interferon stimulated gene (ISG) system to facilitate its initial infection and establishment of viral reservoirs. HIV specifically inhibits all type l and lll interferons in its target cells, including, macrophages, dendritic cells and T cells. It also induces a subset of over 20 ISGs of differing composition in each cell target. This occurs in two temporal phases in macrophages. Extracellular vesicles contained within the inoculum induced the first and transient phase of ISGs. Newly transcribed HIV RNA induced the second and dominant ISG phase and here the full induction pathway is defined. Therefore, HIV nucleic acids, which are potent inducers of interferon and ISGs, are initially concealed and antiviral ISGs are not fully induced until replication is well established. Theses antiviral ISGs may contribute to the persistent infection in macrophages and to the establishment of viral reservoirs in vivo.

  • THE REPLICATION OF JCV DNA IN THE G144 OLIGODENDROCYTE CELL LINE IS DEPENDENT UPON AKT. [PublishAheadOfPrint]

  • Progressive Multifocal Leukoencephalopathy (PML) is an often-fatal demyelinating disease of the central nervous system. PML results when oligodendrocytes within immunocompromised individuals are infected with the human JC virus (JCV). We have identified an oligodendrocyte precursor cell line, termed G144, which supports robust levels of JCV DNA replication, a central part of the JCV life cycle. In addition, we have determined that JC virus readily infects G144 cells. Furthermore, we have determined that JCV DNA replication in G144 cells is stimulated by myristoylated (i.e., constitutively active) Akt and reduced by the Akt specific inhibitor MK2206. Thus, this oligodendrocyte based model system will be useful for a number of purposes, such as studies of JCV infection, establishing key pathways needed for the regulation of JCV DNA replication and identifying inhibitors of this process.

    Importance: The disease PML (Progressive Multifocal Leukoencephalopathy) is due to the infection of particular brain cells, termed oligodendrocytes, by the JC virus. Studies of PML have, however, been hampered by the lack of an immortalized human cell line derived from oligodendrocytes. Herein, we report that the G144 oligodendrocyte cell line supports both infection by JC virus and robust levels of JCV DNA replication. Moreover, we have established that the Akt pathway regulates JCV DNA replication and that JCV DNA replication can be inhibited by MK2206, a compound that is specific for Akt. These and related findings suggest that we have established a powerful oligodendrocyte-based model system for studies of JCV dependent PML.

  • Non-encapsidated 5 copy-back defective-interfering genomes produced by recombinant measles viruses are recognized by RIG-I and LGP2 but not MDA5 [PublishAheadOfPrint]

  • Attenuated Measles virus (MV) is one of the most effective and safe vaccine available, making it an attractive candidate vector to prevent other infectious diseases. Yet the great capacity of this vaccine still needs to be understood at the molecular level. MV vaccine strains have different type-I interferon-inducing abilities, that partially depend on the presence of 5rrsquo; copy-back defective interfering genomes (DI-RNAs). DI-RNAs are pathogen associated molecular patterns recognized by RIG-I-like receptors (RLRs: RIG-I, MDA5 and LGP2) that activate innate immune signalling and shape the adaptive immune response. In this study, we characterized the DI-RNAs produced by various modified recombinant MV (rMV), including vaccine candidates, as well as wild-type MV. All tested rMVs produced 5rrsquo; copy-back DI-RNAs that were different in length and nucleotide sequence but still respected the so-called "rule of six". We correlated the presence of DI-RNAs with a higher stimulation of the IFN-bbeta; pathway and compared their immunostimulatory potential. Importantly, we revealed that encapsidation of DI-RNA molecules within the MV nucleocapsid abolished their immunoactive properties. Furthermore, we identified specific interactions of DI-RNAs with both RIG-I and LGP2, but not MDA5. Our results suggest that DI-RNAs produced by rMV vaccine candidates may indeed strengthen their efficiency by triggering RLR signalling.

    IMPORTANCE Administered to hundreds of millions of children, live-attenuated measles virus (MV) vaccine is the safest and widely used human vaccine providing high protection with long-term memory. Additionally, recombinant MV carring heterologous antigens are promising vectors for new vaccines. The great capacity of this vaccine still needs to be elucidated at the molecular level. Here we documented that recombinant MVs produce defective interfering genomes that have high immunostimulatory properties via their binding to RIG-I and LGP2 proteins, both cytosolic non-self RNA sensors of innate immunity. Defective interfering genomes production during viral replication should be considered of great importance due to their immunostimulatory properties as intrinsic adjuvants produced by the vector that increase recognition by the innate immune system.

  • Human Metapneumovirus M2-2 Protein Acts as a Negative Regulator of Alpha Interferon Production by Plasmacytoid Dendritic Cells [PublishAheadOfPrint]

  • Human metapneumovirus (HMPV) has the ability to inhibit TLR7- and TLR9-dependent alpha interferon (IFN-aalpha;) production by plasmacytoid dendritic cells (pDCs). However the inhibition mechanism remains largely unknown. To identify viral proteins responsible for this inhibition, we performed a screening of HMPV open reading frames (ORFs) for their abilities to block TLR7/9-dependent signaling reconstituted in HEK293T cells by transfection with MyD88, TRAF6, IKKaalpha;, and IRF7. This screening demonstrated that the M2-2 protein was the most potent inhibitor of TLR7/9-dependent IFN-aalpha; induction. A recombinant HMPV, in which the M2-2 ORF is silenced, indeed induced greater IFN-aalpha; production by human pDCs than wild type HMPV did. Immunoprecipitation experiments showed direct physical association of the M2-2 protein with the inhibitory domain (ID) of IRF7. As a natural consequence of this, transfection of the IRF7 lacking the ID, a constitutively active mutant, resulted in activation of the IFN-aalpha; promoter even in the presence of M2-2. Bioluminescence resonance energy transfer assay and split Renilla luciferase complementation assay revealed that M2-2 inhibited MyD88/TRAF6/IKKaalpha;-induced homodimerization of IRF7. By contrast, expression of the M2-2 protein did not result in inhibition of IPS-1-induced homodimerization and resultant activation of IRF7. This indicates that inhibition of the MyD88/TRAF6/IKKaalpha;-induced IRF7 homodimerization does not result from steric effect of the M2-2 binding. Instead, it was found that M2-2 inhibited MyD88/TRAF6/IKKaalpha;-induced phosphorylation of IRF7 on Ser477. These results suggest that M2-2 blocks TLR7/9-dependent IFN-aalpha; induction by preventing IRF7 homodimerization possibly through affecting phosphorylation status of IRF7.

    IMPORTANCE The family Paramyxoviridae is classified into two subfamilies, the Paramyxovirinae and the Pneumovirinae. Members of the subfamily Paramyxovirinae have the ability to inhibit TLR7/9-dependent alpha IFN production, and the underlying inhibition mechanism has been intensively studied. In contrast, little is known about how members of the subfamily Pneumovirinae regulate alpha IFN production by pDCs. We identified the M2-2 protein of HMPV, a member of the subfamily Pneumovirinae, as a negative regulator of alpha IFN production by pDCs, and uncovered the underlying mechanism. This study in part explains why the M2-2 knockout recombinant HMPV is attenuated, and further suggests that M2-2 is a potential target for HMPV therapy.

  • HRAS, EGFR, MET, and RON are recurrently activated by provirus insertion in MAV-2 retrovirus-induced liver tumors [PublishAheadOfPrint]

  • Myeloblastosis-associated virus-2 (MAV-2) is a highly tumorigenic simple avian retrovirus. Chickens infected in ovo with MAV-2 develop tumors in kidneys, lungs, and liver with a short latency of less then 8 weeks. Here we report the results of molecular analyses of MAV-2-induced liver tumors that fall into three classes: hepatic hemangiosarcomas (HHS), intrahepatic cholangiocarcinomas (ICC), and hepatocellular carcinomas (HCC). Comprehensive inverse PCR-based screening of 92 chicken liver tumors revealed that in ca 86 % of these tumors, MAV-2 provirus had integrated into one of four gene loci: HRAS, EGFR, MET, and RON. Insertionally mutated genes correlated with tumor type nndash; HRAS was hit in HHSs, MET in ICCs, RON mostly in ICCs and EGFR mostly in HCC. The provirus insertions lead to the overexpression of the affected genes, and in the case of EGFR and RON also to the truncation of exons encoding the extracellular ligand-binding domain of these transmembrane receptors. The structure of truncated EGFR and RON closely mimic the structure of oncogenic variants of these genes frequently found in human tumors (EGFRvIII and sfRON).

    IMPORTANCE These data describe the mechanisms of oncogenesis incited in chickens by the MAV-2 retrovirus. They also show that molecular processes converting cellular regulatory genes to cancer genes may be remarkably similar in chickens and humans. We suggest that the MAV-2 retrovirus-based model can complement experiments performed using mouse models and provide data that could translate to human medicine.

  • Targeted Genome Sequencing Reveals Varicella-Zoster Virus Open Reading Frame 12 Deletion [PublishAheadOfPrint]

  • The neurotropic herpesvirus varicella zoster virus (VZV) establishes a life-long latent infection in humans following primary infection. The low abundance of VZV nucleic acids in human neurons has hindered an understanding of the mechanisms that regulate viral gene transcription during latency. To overcome this critical barrier, we optimized a targeted capture protocol to enrich VZV DNA and cDNA prior to whole-genome/transcriptome sequence analysis. Since the VZV genome is remarkably stable, it was surprising to detect that VZV32, a VZV laboratory strain with no discernable growth defect in tissue culture, contained a 2158-bp deletion in open reading frame (ORF) 12. Consequently, ORF 12 and 13 protein expression was abolished, and Akt phosphorylation was inhibited. The discovery of the ORF 12 deletion, revealed through targeted genome sequencing analysis, points to the need to authenticate the VZV genome when the virus is propagated in tissue culture.

    IMPORTANCE Viruses isolated from clinical samples often undergo genetic modifications when cultured in the laboratory. Historically, varicella zoster virus (VZV) is among the most genetically stable herpesviruses, a notion supported by more than 60 complete genome sequences from multiple isolates and following multiple in vitro passages. However, application of enrichment protocols to targeted genome sequencing revealed the unexpected deletion of a significant portion of VZV ORF 12 following propagation in cultured human fibroblast cells. While the enrichment protocol did not introduce bias in either the virus genome or transcriptome, the findings indicate the need for authentication of VZV by sequencing when the virus is propagated in tissue culture.

  • Roles of cellular NSF protein in entry and nuclear egress of budded virions of Autographa californica multiple nucleopolyhedrovirus [PublishAheadOfPrint]

  • In eukaryotic cells, the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) proteins comprise the minimal machinery that triggers fusion of transport vesicles with their target membranes. Comparative studies revealed that genes encoding the components of the SNARE system are highly conserved in yeast, insect, and human genomes. Upon infection of insect cells by the virus AcMNPV, the transcript levels of most SNARE genes were initially up-regulated. We found that overexpression of dominant-negative (DN) forms of NSF or knock-down of the expression of NSF, the key regulator of the SNARE system, significantly affected the infectious AcMNPV production. In cells expressing DN NSF, entering virions were trapped in the cytoplasm or transported to the nucleus with low efficiency. The presence of DN NSF also moderately reduced trafficking of the viral envelope glycoprotein GP64 to the plasma membrane but dramatically inhibited production of infectious BV. TEM analysis of infections in cells expressing DN NSF revealed that progeny nucleocapsids were retained in a perinuclear space surrounded by inner and outer nuclear membranes. Several baculovirus conserved (core) proteins (Ac76, Ac78, GP41, Ac93, and Ac103) that are important for infectious budded virion production, were found to associate with NSF, and NSF was detected within the assembled BV. Together, these data indicate that the cellular SNARE system is involved in AcMNPV infection and that NSF is required for efficient entry and nuclear egress of budded virions of AcMNPV.

    Importance Little is known regarding the complex interplay between cellular factors and baculoviruses during viral entry and egress. Here we examined the cellular SNARE system, which mediates the fusion of vesicles in healthy cells, and its relation to baculovirus infection. Using a dominant negative (DN) approach and an RNAi knockdown, we demonstrated that a general disruption of the SNARE machinery significantly inhibited the production of infectious budded virions (BV) of AcMNPV. The presence of a DN NSF protein resulted in low efficiency entry of BV and the retention of progeny nucleocapsids in the perinuclear space during egress. Combined with these effects, we also found that several conserved (core) baculovirus proteins closely associate with NSF, and these results suggest their involvement in the egress of BV. Our findings are the first to demonstrate that the SNARE system is required for efficient entry of BV and nuclear egress of progeny nucleocapsids of baculoviruses.

  • A domain of Herpes simplex virus pUL33 required to release monomeric viral genomes from cleaved concatameric DNA [PublishAheadOfPrint]

  • Monomeric herpesvirus DNA is cleaved from concatamers and inserted into preformed capsids through the actions of the viral terminase. The terminase of herpes simplex virus (HSV) is composed of three subunits encoded by UL15, UL28, and UL33. The UL33 encoded protein (pUL33) interacts with pUL28 but its precise role in the DNA cleavage and packaging reaction is unclear. To investigate the function of pUL33 we generated a panel of recombinant viruses with either deletions or substitutions in the most conserved regions of UL33 using a bacterial artificial chromosome system. Deletion of 11 amino acids (residues 50-60, or residues 110-120) precluded viral replication, whereas truncation of the last 10 amino acids from the pUL33 C-terminus did not affect viral replication nor pUL33 interaction with pUL28. Mutations that replaced codons 110-Lysine and 111-arginine with alanine codons failed to replicate, and the mutant pUL33 interacted with pUL28 less efficiently. Interestingly, genomic termini of the L and S components were detected readily in cells infected with these mutants, indicating that concatameric DNA was cleaved efficiently. However, release of monomeric genomes as assessed on pulsed field gel electrophoresis was greatly diminished, and DNA-containing capsids were not observed. These results suggest that pUL33 is necessary for one of the two viral DNA cleavage events required to release individual genomes from concatemeric viral DNA.

    Importance This paper shows a role for pUL33 in one of the two DNA cleavage events required to release monomeric genomes from concatameric viral DNA. This is the first time such a phenotype has been observed and is the first identification of a function of this protein relevant to DNA packaging other than its interaction with other terminase components.

  • MUTATIONS IN THE E2 GLYCOPROTEIN AND THE 3 UNTRANSLATED REGION ENHANCE CHIKUNGUNYA VIRUS VIRULENCE IN MICE [PublishAheadOfPrint]

  • Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes debilitating musculoskeletal pain and inflammation and can persist for months to years after acute infection. Although studies in humans and experimentally-infected animals suggest that CHIKV infection persists in musculoskeletal tissues, the mechanisms for this remain poorly understood. To evaluate this further, we isolated CHIKV from the serum of persistently infected Rag1-/- mice at day 28. When inoculated into naïve WT mice, this persistently circulating CHIKV strain displayed a capacity for earlier dissemination and greater pathogenicity compared with the parental virus. Sequence analysis revealed a nonsynonymous mutation in the E2 glycoprotein (E2 K200R) and a deletion within the 3rrsquo; untranslated region (3rrsquo; -UTR). Introduction of these changes into the parental virus conferred enhanced virulence in mice although a primary tropism for musculoskeletal tissues was maintained. The E2 K200R mutation was largely responsible for enhanced viral dissemination and pathogenicity, although these effects were augmented by the 3rrsquo; -UTR deletion. Finally, studies in Irf3/Irf7-/- and Ifnar1-/- mice suggest that the E2 K200R mutation enhances viral dissemination from the site of inoculation independently of IRF3, IRF7, and IFNAR1 mediated responses. As our findings reveal viral determinants of CHIKV dissemination and pathogenicity, their further study should help to elucidate host-virus interactions that determine acute and chronic CHIKV infection.

    IMPORTANCE CHIKV is a globally-spreading, mosquito-transmitted virus that causes debilitating acute and chronic musculoskeletal disease in humans. The viral genetic determinants that dictate acute and chronic disease severity are not understood. To improve our understanding of CHIKV pathogenesis, we evaluated a CHIKV strain isolated from the serum of chronically-infected immunocompromised mice. Sequence analysis of this persistent CHIKV strain identified two mutations, an amino acid change in the E2 viral attachment protein and a deletion within the 3rrsquo; -UTR of the viral genome. We identified roles for these mutations in enhancement of viral dissemination from the inoculation site and in disease severity. These data improve our understanding of the viral determinants of CHIKV pathogenesis and adaptive changes that occur during viral persistence.

  • HCV induces the localization of lipid rafts to autophagosomes for its RNA replication [PublishAheadOfPrint]

  • Autophagy plays important roles in maintaining cellular homeostasis. It uses double- or multiple- membrane vesicles termed autophagosomes to remove protein aggregates and damaged organelles from the cytoplasm for recycling. Hepatitis C virus (HCV) has been shown to induce autophagy to enhance its own replication. Here we describe a procedure that combines membrane flotation and affinity chromatography for the purification of autophagosomes from cells that harbor a HCV subgenomic RNA replicon. The purified autophagosomes had double- or multiple- membrane structures with a diameter ranging from 200 nm to 600 nm. The analysis of proteins associated with HCV-induced autophagosomes by proteomics led to the identification of HCV nonstructural proteins as well as proteins involved in membrane trafficking. Notably, caveolin-1, caveolin-2 and annexin A2, which are proteins associated with lipid rafts, were also identified. The association of lipid rafts with HCV-induced autophagosomes was confirmed by western-blot, immunofluorescence microscopy and immunoelectron microscopy. Their association with autophagosomes was also confirmed in HCV-infected cells. The association of lipid rafts with autophagosomes was specific to HCV, as it was not detected in autophagosomes induced by nutrient starvation. Further analysis indicated that the autophagosomes purified from HCV replicon cells could mediate HCV RNA replication in a lipid raft-dependent manner, as the depletion of cholesterol, a major component of lipid rafts, from autophagosomes abolished HCV RNA replication. Our studies thus demonstrated that HCV could specifically induce the association of lipid rafts with autophagosomes for its RNA replication.

    IMPORTANCE HCV can cause severe liver diseases including cirrhosis and hepatocellular carcinoma and is one of the most important human pathogens. Its infection can lead to the reorganization of membrane structures in its host cells, including the induction of autophagosomes. In this report, we developed a procedure to purify HCV-induced autophagosomes and demonstrated that HCV could induce the localization of lipid rafts to autophagosomes to mediate its RNA replication. This finding provided important information for further understanding the life cycle of HCV and its interaction with the host cells.

  • STLV-1 infection of Papio Anubis: tax Sequence Heterogeneity and T Cell Recognition [PublishAheadOfPrint]

  • Natural infection of baboons with simian T lymphotropic virus (STLV) is a potentially useful model system for study of vaccination against the human virus HTLV. Here we expand the number of available full-length STLV-1 baboon sequences from one to three and relate T cell responses that recognize the immunodominant Tax protein to the tax sequences present in two individual baboons. Continuously growing T cell lines were established from two baboons, 12141 and 12752. Next-generation sequencing (NGS) of complete STLV genome sequences from these lines revealed them to be closely related but distinct from each other and from the STLV-1 baboon sequence in the NCBI sequence database. Overlapping peptides corresponding to each unique Tax sequence and to the reference baboon Tax sequence were used to analyze recognition by T cells from each baboon using intracellular cytokine staining (ICS). Individual baboons expressed more IFN- and TNF-aalpha; in response to Tax peptides corresponding to their own sequence than with Tax peptides corresponding to the reference baboon sequence. Thus, our analyses revealed distinct but closely related STLV-1 genome sequences in two baboons, extremely low heterogeneity of STLV sequences within each baboon, no evidence for superinfection within each baboon, and a ready ability of T cells in each baboon to recognize circulating Tax sequences. While amino acid substitutions that result in escape from CD8+ T cell recognition were not observed, premature stop codons were observed only in tax sequences obtained from PBMC at 7% in 12141 and 56% in 12752.

    Importance: It has been estimated that approximately 100,000 people suffer serious morbidity and 10,000 people die each year from the consequences associated with human T lymphotropic virus (HTLV) infection. There are no antiviral drugs and no preventive vaccine. A preventative vaccine would significantly impact the global burden associated with HTLV infections. Here we provide fundamental information on the simian T lymphotropic virus (STLV) being naturally transmitted in a colony of captive baboons. The limited viral sequence heterogeneity in individual baboons, the identity of the viral gene product that is the major target of cellular immune responses, the persistence of viral amino acid sequences that are the major targets of cellular immune responses, and the emergence in vivo of truncated variants in the major target of cellular immune responses all parallel what is seen with HTLV infection of humans. These results justify the use of STLV-baboon model systems for vaccine development efforts.

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