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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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  • MicroRNA 373 Facilitates the Replication of Porcine Reproductive and Respiratory Syndrome Virus by Its Negative Regulation of Type I Interferon Induction [Pathogenesis and Immunity]

  • MicroRNAs (miRNAs) play an important role in the regulation of immune responses. Previous studies have indicated that dysregulating the miRNAs leads to the immunosuppression of porcine reproductive and respiratory syndrome virus (PRRSV). However, it is not clear how PRRSV regulates the expression of host miRNA, which may lead to immune escape or promote the replication of the virus. The present work suggests that PRRSV upregulated the expression of miR-373 through elevating the expression of specificity protein 1 (Sp1) in MARC-145 cells. Furthermore, this work demonstrated that miR-373 promoted the replication of PRRSV, since miR-373 was a novel negative miRNA for the production of beta interferon (IFN-bbeta;) by targeting nuclear factor IA (NFIA), NFIB, interleukin-1 receptor-associated kinase 1 (IRAK1), IRAK4, and interferon regulatory factor 1 (IRF1). We also found that both NFIA and NFIB were novel proteins for inducing the production of IFN-bbeta;, and both of them could inhibit the replication of PRRSV. In conclusion, PRRSV upregulated the expression of miR-373 by elevating the expression of Sp1 and hijacked the host miR-373 to promote the replication of PRRSV by negatively regulating the production of IFN-bbeta;.

    IMPORTANCE PRRSV causes one of the most economically devastating diseases of swine, and there is no effective method for controlling PRRSV. It is not clear how PRRSV inhibits the host's immune response and induces persistent infection. Previous studies have shown that PRRSV inhibited the production of type I IFN, and the treatment of type I IFN could efficiently inhibit the replication of PRRSV, so it will be helpful to design new methods of controlling PRRSV by understanding the molecular mechanism by which PRRSV modulated the production of IFN. The current work shows that miR-373, upregulated by PRRSV, promotes PRRSV replication, since miR-373 impaired the production of IFN-bbeta; by targeting NFIA, NFIB, IRAK1, IRAK4, and IRF1, and both NFIA and NFIB were antiviral proteins to PRRSV. In conclusion, this paper revealed a novel mechanism of PRRSV that impaired the production of type I IFN by upregulating miR-373 expression in MARC-145 cells.

  • Coat Protein Regulation by CK2, CPIP, HSP70, and CHIP Is Required for Potato Virus A Replication and Coat Protein Accumulation [Virus-Cell Interactions]

  • We demonstrate here that both coat protein (CP) phosphorylation by protein kinase CK2 and a chaperone system formed by two heat shock proteins, CP-interacting protein (CPIP) and heat shock protein 70 (HSP70), are essential for potato virus A (PVA; genus Potyvirus) replication and that all these host proteins have the capacity to contribute to the level of PVA CP accumulation. An E3 ubiquitin ligase called carboxyl terminus Hsc70-interacting protein (CHIP), which may participate in the CPIP-HSP70-mediated CP degradation, is also needed for robust PVA gene expression. Residue Thr243 within the CK2 consensus sequence of PVA CP was found to be essential for viral replication and to regulate CP protein stability. Substitution of Thr243 either with a phosphorylation-mimicking Asp (CPADA) or with a phosphorylation-deficient Ala (CPAAA) residue in CP expressed from viral RNA limited PVA gene expression to the level of nonreplicating PVA. We found that both the CPAAA mutant and CK2 silencing inhibited, whereas CPADA mutant and overexpression of CK2 increased, PVA translation. From our previous studies, we know that phosphorylation reduces the RNA binding capacity of PVA CP and an excess of CP fully blocks viral RNA translation. Together, these findings suggest that binding by nonphosphorylated PVA CP represses viral RNA translation, involving further CP phosphorylation and CPIP-HSP70 chaperone activities as prerequisites for PVA replication. We propose that this mechanism contributes to shifting potyvirus RNA from translation to replication.

    IMPORTANCE Host protein kinase CK2, two host chaperones, CPIP and HSP70, and viral coat protein (CP) phosphorylation at Thr243 are needed for potato virus A (PVA) replication. Our results show that nonphosphorylated CP blocks viral translation, likely via binding to viral RNA. We propose that this translational block is needed to allow time and space for the formation of potyviral replication complex around the 3' end of viral RNA. Progression into replication involves CP regulation by both CK2 phosphorylation and chaperones CPIP and HSP70.

  • Design and In Vivo Characterization of Immunoconjugates Targeting HIV gp160 [Vaccines and Antiviral Agents]

  • The envelope (Env) glycoprotein of HIV is expressed on the surface of productively infected cells and can be used as a target for cytotoxic immunoconjugates (ICs), in which cell-killing moieties, including toxins, drugs, or radionuclides, are chemically or genetically linked to monoclonal antibodies (MAbs) or other targeting ligands. Such ICs could be used to eliminate persistent reservoirs of HIV infection. We have found that MAbs which bind to the external loop of gp41, e.g., MAb 7B2, make highly effective ICs, particularly when used in combination with soluble CD4. We evaluated the toxicity, immunogenicity, and efficacy of the ICs targeted with 7B2 in mice and in simian-human immunodeficiency virus-infected macaques. In the macaques, we tested immunotoxins (ITs), consisting of protein toxins bound to the targeting agent. ITs were well tolerated and initially efficacious but were ultimately limited by their immunogenicity. In an effort to decrease immunogenicity, we tested different toxic moieties, including recombinant toxins, cytotoxic drugs, and tubulin inhibitors. ICs containing deglycosylated ricin A chain prepared from ricin toxin extracted from castor beans were the most effective in killing HIV-infected cells. Having identified immunogenicity as a major concern, we show that conjugation of IT to polyethylene glycol limits immunogenicity. These studies demonstrate that cytotoxic ICs can target virus-infected cells in vivo but also highlight potential problems to be addressed.

    IMPORTANCE It is not yet possible to cure HIV infection. Even after years of fully effective antiviral therapy, a persistent reservoir of virus-infected cells remains. Here we propose that a targeted conjugate consisting of an anti-HIV antibody bound to a toxic moiety could function to kill the HIV-infected cells that constitute this reservoir. We tested this approach in HIV-infected cells grown in the lab and in animal infections. Our studies demonstrated that these immunoconjugates are effective both in vitro and in test animals. In particular, ITs constructed with the deglycosylated A chain prepared from native ricin were the most effective in killing cells, but their utility was blunted because they provoked immune reactions that interfered with the therapeutic effects. We then demonstrated that coating of the ITs with polyethylene glycol minimized the immunogenicity, as has been demonstrated with other protein therapies.

  • Glycosylphosphatidylinositol-Anchored Anti-HIV scFv Efficiently Protects CD4 T Cells from HIV-1 Infection and Deletion in hu-PBL Mice [Vaccines and Antiviral Agents]

  • Despite success in viral inhibition and CD4 T cell recovery by highly active antiretroviral treatment (HAART), HIV-1 is still not curable due to the persistence of the HIV-1 reservoir during treatment. One patient with acute myeloid leukemia who received allogeneic hematopoietic stem cell transplantation from a homozygous CCR5 32 donor has had no detectable viremia for 9 years after HAART cessation. This case has inspired a field of HIV-1 cure research focusing on engineering HIV-1 resistance in permissive cells. Here, we employed a glycosylphosphatidylinositol (GPI)-scFv X5 approach to confer resistance of human primary CD4 T cells to HIV-1. We showed that primary CD4 T cells expressing GPI-scFv X5 were resistant to CCR5 (R5)-, CXCR4 (X4)-, and dual-tropic HIV-1 and had a survival advantage compared to control cells ex vivo. In a hu-PBL mouse study, GPI-scFv X5-transduced CD4 T cells were selected in peripheral blood and lymphoid tissues upon HIV-1 infection. Finally, GPI-scFv X5-transduced CD4 T cells, after being cotransfused with HIV-infected cells, showed significantly reduced viral loads and viral RNA copy numbers relative to CD4 cells in hu-PBL mice compared to mice with GPI-scFv AB65-transduced CD4 T cells. We conclude that GPI-scFv X5-modified CD4 T cells could potentially be used as a genetic intervention against both R5- and X4-tropic HIV-1 infections.

    IMPORTANCE Blocking of HIV-1 entry is one of most promising approaches for therapy. Genetic disruption of the HIV-1 coreceptor CCR5 by nucleases in T cells is under 2 clinical trials and leads to reduced viremia in patients. However, the emergence of viruses using the CXCR4 coreceptor is a concern for therapies applying single-coreceptor disruption. Here, we report that HIV-1-permissive CD4 T cells engineered with GPI-scFv X5 are resistant to R5-, X4-, or dual-tropic virus infection ex vivo. In a preclinical study using hu-PBL mice, we show that CD4 T cells were protected and that GPI-scFv X5-transduced cells were selected in HIV-1-infected animals. Moreover, we show that GPI-scFv X5-transduced CD4 T cells exerted a negative effect on virus replication in vivo. We conclude that GPI-scFv X5-modified CD4 T cells could potentially be used as a genetic intervention against both R5- and X4-tropic HIV-1 infections.

  • Pre-mRNA Processing Factor Prp18 Is a Stimulatory Factor of Influenza Virus RNA Synthesis and Possesses Nucleoprotein Chaperone Activity [Genome Replication and Regulation of Viral Gene Expression]

  • The genome of influenza virus (viral RNA [vRNA]) is associated with the nucleoprotein (NP) and viral RNA-dependent RNA polymerases and forms helical viral ribonucleoprotein (vRNP) complexes. The NP-vRNA complex is the biologically active template for RNA synthesis by the viral polymerase. Previously, we identified human pre-mRNA processing factor 18 (Prp18) as a stimulatory factor for viral RNA synthesis using a Saccharomyces cerevisiae replicon system and a single-gene deletion library of Saccharomyces cerevisiae (T. Naito, Y. Kiyasu, K. Sugiyama, A. Kimura, R. Nakano, A. Matsukage, and K. Nagata, Proc Natl Acad Sci USA, 104:18235nndash;18240, 2007, In infected Prp18 knockdown (KD) cells, the synthesis of vRNA, cRNA, and viral mRNAs was reduced. Prp18 was found to stimulate in vitro viral RNA synthesis through its interaction with NP. Analyses using in vitro RNA synthesis reactions revealed that Prp18 dissociates newly synthesized RNA from the template after the early elongation step to stimulate the elongation reaction. We found that Prp18 functions as a chaperone for NP to facilitate the formation of NP-RNA complexes. Based on these results, it is suggested that Prp18 accelerates influenza virus RNA synthesis as an NP chaperone for the processive elongation reaction.

    IMPORTANCE Templates for viral RNA synthesis of negative-stranded RNA viruses are not naked RNA but rather RNA encapsidated by viral nucleocapsid proteins forming vRNP complexes. However, viral basic proteins tend to aggregate under physiological ionic strength without chaperones. We identified the pre-mRNA processing factor Prp18 as a stimulatory factor for influenza virus RNA synthesis. We found that one of the targets of Prp18 is NP. Prp18 facilitates the elongation reaction of viral polymerases by preventing the deleterious annealing of newly synthesized RNA to the template. Prp18 functions as a chaperone for NP to stimulate the formation of NP-RNA complexes. Based on these results, we propose that Prp18 may be required to maintain the structural integrity of vRNP for processive template reading.

  • Cryo-electron Microscopy Structures of Expanded Poliovirus with VHHs Sample the Conformational Repertoire of the Expanded State [Virus-Cell Interactions]

  • By using cryo-electron microscopy, expanded 80S-like poliovirus virions (poliovirions) were visualized in complexes with four 80S-specific camelid VHHs (Nanobodies). In all four complexes, the VHHs bind to a site on the top surface of the capsid protein VP3, which is hidden in the native virus. Interestingly, although the four VHHs bind to the same site, the structures of the expanded virus differ in detail in each complex, suggesting that each of the Nanobodies has sampled a range of low-energy structures available to the expanded virion. By stabilizing unique structures of expanded virions, VHH binding permitted a more detailed view of the virus structure than was previously possible, leading to a better understanding of the expansion process that is a critical step in infection. It is now clear which polypeptide chains become disordered and which become rearranged. The higher resolution of these structures also revealed well-ordered conformations for the EF loop of VP2, the GH loop of VP3, and the N-terminal extensions of VP1 and VP2, which, in retrospect, were present in lower-resolution structures but not recognized. These structural observations help to explain preexisting mutational data and provide insights into several other stages of the poliovirus life cycle, including the mechanism of receptor-triggered virus expansion.

    IMPORTANCE When poliovirus infects a cell, it undergoes a change in its structure in order to pass RNA through its protein coat, but this altered state is short-lived and thus poorly understood. The structures of poliovirus bound to single-domain antibodies presented here capture the altered virus in what appear to be intermediate states. A careful analysis of these structures lets us better understand the molecular mechanism of infection and how these changes in the virus lead to productive-infection events.

  • Suppression of Adenovirus Replication by Cardiotonic Steroids [Vaccines and Antiviral Agents]

  • The dependence of adenovirus on the host pre-RNA splicing machinery for expression of its complete genome potentially makes it vulnerable to modulators of RNA splicing, such as digoxin and digitoxin. Both drugs reduced the yields of four human adenoviruses (HAdV-A31, -B35, and -C5 and a species D conjunctivitis isolate) by at least 2 to 3 logs by affecting one or more steps needed for genome replication. Immediate early E1A protein levels are unaffected by the drugs, but synthesis of the delayed protein E4orf6 and the major late capsid protein hexon is compromised. Quantitative reverse transcription-PCR (qRT-PCR) analyses revealed that both drugs altered E1A RNA splicing (favoring the production of 13S over 12S RNA) early in infection and partially blocked the transition from 12S and 13S to 9S RNA at late stages of virus replication. Expression of multiple late viral protein mRNAs was lost in the presence of either drug, consistent with the observed block in viral DNA replication. The antiviral effect was dependent on the continued presence of the drug and was rapidly reversible. RIDK34, a derivative of convallotoxin, although having more potent antiviral activity, did not show an improved selectivity index. All three drugs reduced metabolic activity to some degree without evidence of cell death. By blocking adenovirus replication at one or more steps beyond the onset of E1A expression and prior to genome replication, digoxin and digitoxin show potential as antiviral agents for treatment of serious adenovirus infections. Furthermore, understanding the mechanism(s) by which digoxin and digitoxin inhibit adenovirus replication will guide the development of novel antiviral therapies.

    IMPORTANCE Despite human adenoviruses being a common and, in some instances, life-threating pathogen in humans, there are few well-tolerated therapies. In this report, we demonstrate that two cardiotonic steroids already in use in humans, digoxin and digitoxin, are potent inhibitors of multiple adenovirus species. A synthetic derivative of the cardiotonic steroid convallotoxin was even more potent than digoxin and digitoxin when tested with HAdV-C5. These drugs alter the cascade of adenovirus gene expression, acting after initiation of early gene expression to block viral DNA replication and synthesis of viral structural proteins. These findings validate a novel approach to treating adenovirus infections through the modulation of host cell processes.

  • Activation of COX-2/PGE2 Promotes Sapovirus Replication via the Inhibition of Nitric Oxide Production [Virus-Cell Interactions]

  • Enteric caliciviruses in the genera Norovirus and Sapovirus are important pathogens that cause severe acute gastroenteritis in both humans and animals. Cyclooxygenases (COXs) and their final product, prostaglandin E2 (PGE2), are known to play important roles in the modulation of both the host response to infection and the replicative cycles of several viruses. However, the precise mechanism(s) by which the COX/PGE2 pathway regulates sapovirus replication remains largely unknown. In this study, infection with porcine sapovirus (PSaV) strain Cowden, the only cultivable virus within the genus Sapovirus, markedly increased COX-2 mRNA and protein levels at 24 and 36 h postinfection (hpi), with only a transient increase in COX-1 levels seen at 24 hpi. The treatment of cells with pharmacological inhibitors, such as nonsteroidal anti-inflammatory drugs or small interfering RNAs (siRNAs) against COX-1 and COX-2, significantly reduced PGE2 production, as well as PSaV replication. Expression of the viral proteins VPg and ProPol was associated with activation of the COX/PGE2 pathway. We observed that pharmacological inhibition of COX-2 dramatically increased NO production, causing a reduction in PSaV replication that could be restored by inhibition of nitric oxide synthase via the inhibitor N-nitro-l-methyl-arginine ester. This study identified a pivotal role for the COX/PGE2 pathway in the regulation of NO production during the sapovirus life cycle, providing new insights into the life cycle of this poorly characterized family of viruses. Our findings also reveal potential new targets for treatment of sapovirus infection.

    IMPORTANCE Sapoviruses are among the major etiological agents of acute gastroenteritis in both humans and animals, but little is known about sapovirus host factor requirements. Here, using only cultivable porcine sapovirus (PSaV) strain Cowden, we demonstrate that PSaV induced the vitalization of the cyclooxygenase (COX) and prostaglandin E2 (PGE2) pathway. Targeting of COX-1/2 using nonsteroidal anti-inflammatory drugs (NSAIDs) such as the COX-1/2 inhibitor indomethacin and the COX-2-specific inhibitors NS-398 and celecoxib or siRNAs targeting COXs, inhibited PSaV replication. Expression of the viral proteins VPg and ProPol was associated with activation of the COX/PGE2 pathway. We further demonstrate that the production of PGE2 provides a protective effect against the antiviral effector mechanism of nitric oxide. Our findings uncover a new mechanism by which PSaV manipulates the host cell to provide an environment suitable for efficient viral growth, which in turn can be a new target for treatment of sapovirus infection.

  • Hepatitis C Virus-Induced Rab32 Aggregation and Its Implications for Virion Assembly [Virus-Cell Interactions]

  • Hepatitis C virus (HCV) is highly dependent on cellular factors for viral propagation. Using high-throughput next-generation sequencing, we analyzed the host transcriptomic changes and identified 30 candidate genes which were upregulated in cell culture-grown HCV (HCVcc)-infected cells. Of these candidates, we selected Rab32 for further investigation. Rab32 is a small GTPase that regulates a variety of intracellular membrane-trafficking events in various cell types. In this study, we demonstrated that both mRNA and protein levels of Rab32 were increased in HCV-infected cells. Furthermore, we showed that HCV infection converted the predominantly expressed GTP-bound Rab32 to GDP-bound Rab32, contributing to the aggregation of Rab32 and thus making it less sensitive to cellular degradation machinery. In addition, GDP-bound Rab32 selectively interacted with HCV core protein and deposited core protein into the endoplasmic reticulum (ER)-associated Rab32-derived aggregated structures in the perinuclear region, which were likely to be viral assembly sites. Using RNA interference technology, we demonstrated that Rab32 was required for the assembly step but not for other stages of the HCV life cycle. Taken together, these data suggest that HCV may modulate Rab32 activity to facilitate virion assembly.

    IMPORTANCE Rab32, a member of the Ras superfamily of small GTPases, regulates various intracellular membrane-trafficking events in many cell types. In this study, we showed that HCV infection concomitantly increased Rab32 expression at the transcriptional level and altered the balance between GDP- and GTP-bound Rab32 toward production of Rab32-GDP. GDP-bound Rab32 selectively interacted with HCV core protein and enriched core in the ER-associated Rab32-derived aggregated structures that were probably necessary for viral assembly. Indeed, we showed that Rab32 was specifically required for the assembly of HCV. Collectively, our study identifies that Rab32 is a novel host factor essential for HCV particle assembly.

  • Ablation of Programmed -1 Ribosomal Frameshifting in Venezuelan Equine Encephalitis Virus Results in Attenuated Neuropathogenicity [Pathogenesis and Immunity]

  • The alphaviruses Venezuelan equine encephalitis virus (VEEV), eastern equine encephalitis virus (EEEV), and western equine encephalitis virus (WEEV) are arthropod-borne positive-strand RNA viruses that are capable of causing acute and fatal encephalitis in many mammals, including humans. VEEV was weaponized during the Cold War and is recognized as a select agent. Currently, there are no FDA-approved vaccines or therapeutics for these viruses. The spread of VEEV and other members of this family due to climate change-mediated vector range expansion underscores the need for research aimed at developing medical countermeasures. These viruses utilize programmed nndash;1 ribosomal frameshifting (nndash;1 PRF) to synthesize the viral trans-frame (TF) protein, which has previously been shown to be important for neuropathogenesis in the related Sindbis virus. Here, the alphavirus nndash;1 PRF signals were characterized, revealing novel nndash;1 PRF stimulatory structures. nndash;1 PRF attenuation mildly affected the kinetics of VEEV accumulation in cultured cells but strongly inhibited its pathogenesis in an aerosol infection mouse model. Importantly, the decreased viral titers in the brains of mice infected with the mutant virus suggest that the alphavirus TF protein is important for passage through the blood-brain barrier and/or for neuroinvasiveness. These findings suggest a novel approach to the development of safe and effective live attenuated vaccines directed against VEEV and perhaps other closely related nndash;1 PRF-utilizing viruses.

    IMPORTANCE Venezuelan equine encephalitis virus (VEEV) is a select agent that has been weaponized. This arthropod-borne positive-strand RNA virus causes acute and fatal encephalitis in many mammals, including humans. There is no vaccine or other approved therapeutic. VEEV and related alphaviruses utilize programmed nndash;1 ribosomal frameshifting (nndash;1 PRF) to synthesize the viral trans-frame (TF) protein, which is important for neuropathogenesis. nndash;1 PRF attenuation strongly inhibited VEEV pathogenesis in mice, and viral replication analyses suggest that the TF protein is critical for neurological disease. These findings suggest a new approach to the development of safe and effective live attenuated vaccines directed against VEEV and other related viruses.

  • Mapping of Functional Subdomains in the Terminal Protein Domain of Hepatitis B Virus Polymerase [Genome Replication and Regulation of Viral Gene Expression]

  • Hepatitis B virus (HBV) encodes a multifunction reverse transcriptase or polymerase (P), which is composed of several domains. The terminal protein (TP) domain is unique to HBV and related hepadnaviruses and is required for specifically binding to the viral pregenomic RNA (pgRNA). Subsequently, the TP domain is necessary for pgRNA packaging into viral nucleocapsids and the initiation of viral reverse transcription for conversion of the pgRNA to viral DNA. Uniquely, the HBV P protein initiates reverse transcription via a protein priming mechanism using the TP domain as a primer. No structural homologs or high-resolution structure exists for the TP domain. Secondary structure prediction identified three disordered loops in TP with highly conserved sequences. A meta-analysis of mutagenesis studies indicated these predicted loops are almost exclusively where functionally important residues are located. Newly constructed TP mutations revealed a priming loop in TP which plays a specific role in protein-primed DNA synthesis beyond simply harboring the site of priming. Substitutions of potential sites of phosphorylation surrounding the priming site demonstrated that these residues are involved in interactions critical for priming but are unlikely to be phosphorylated during viral replication. Furthermore, the first 13 and 66 TP residues were shown to be dispensable for protein priming and pgRNA binding, respectively. Combining current and previous mutagenesis work with sequence analysis has increased our understanding of TP structure and functions by mapping specific functions to distinct predicted secondary structures and will facilitate antiviral targeting of this unique domain.

    IMPORTANCE HBV is a major cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. One important feature of this virus is its polymerase, the enzyme used to create the DNA genome from a specific viral RNA by reverse transcription. One region of this polymerase, the TP domain, is required for association with the viral RNA and production of the DNA genome. Targeting the TP domain for antiviral development is difficult due to the lack of homology to other proteins and high-resolution structure. This study mapped the TP functions according to predicted secondary structure, where it folds into alpha helices or unstructured loops. Three predicted loops were found to be the most important regions functionally and the most conserved evolutionarily. Identification of these functional subdomains in TP will facilitate its targeting for antiviral development.

  • Inclusion Body Fusion of Human Parainfluenza Virus Type 3 Regulated by Acetylated {alpha}-Tubulin Enhances Viral Replication [Virus-Cell Interactions]

  • Viral inclusion bodies (IBs), or replication factories, are unique structures generated by viral proteins together with some cellular proteins as a platform for efficient viral replication, but little is known about the mechanism underlying IB formation and fusion. Our previous study demonstrated that the interaction between the nucleoprotein (N) and phosphoprotein (P) of human parainfluenza virus type 3 (HPIV3), an enveloped virus with great medical impact, can form IBs. In this study, we found that small IBs can fuse with each other to form large IBs that enhance viral replication. Furthermore, we found that acetylated aalpha;-tubulin interacts with the N-P complex and colocalizes with IBs of HPIV3 but does not interact with the N-P complex of human respiratory syncytial virus or vesicular stomatitis virus and does not colocalize with IBs of human respiratory syncytial virus. Most importantly, enhancement of aalpha;-tubulin acetylation using the pharmacological inhibitor trichostatin A (TSA), RNA interference (RNAi) knockdown of the deacetylase enzymes histone deacetylase 6 (HDAC6) and sirtuin 2 (SIRT2), or expression of aalpha;-tubulin acetyltransferase 1 (aalpha;-TAT1) resulted in the fusion of small IBs into large IBs and effective viral replication. In contrast, suppression of acetylation of aalpha;-tubulin by overexpressing HDAC6 and SIRT2 profoundly inhibited the fusion of small IBs and viral replication. Our findings offer previously unidentified mechanistic insights into the regulation of viral IB fusion by acetylated aalpha;-tubulin, which is critical for viral replication.

    IMPORTANCE Inclusion bodies (IBs) are unique structures generated by viral proteins and some cellular proteins as a platform for efficient viral replication. Human parainfluenza virus type 3 (HPIV3) is a nonsegmented single-stranded RNA virus that mainly causes lower respiratory tract disease in infants and young children. However, no vaccines or antiviral drugs for HPIV3 are available. Therefore, understanding virus-host interactions and developing new antiviral strategies are increasingly important. Acetylation on lysine (K) 40 of aalpha;-tubulin is an evolutionarily conserved modification and plays an important role in many cellular processes, but its role in viral IB dynamics has not been fully explored. To our knowledge, our findings are the first to show that acetylated aalpha;-tubulin enhances viral replication by regulating HPIV3 IB fusion.

  • Phylogenetic Diversity of Koala Retrovirus within a Wild Koala Population [Genetic Diversity and Evolution]

  • Koala populations are in serious decline across many areas of mainland Australia, with infectious disease a contributing factor. Koala retrovirus (KoRV) is a gammaretrovirus present in most wild koala populations and captive colonies. Five subtypes of KoRV (A to E) have been identified based on amino acid sequence divergence in a hypervariable region of the receptor binding domain of the envelope protein. However, analysis of viral genetic diversity has been conducted primarily on KoRV in captive koalas housed in zoos in Japan, the United States, and Germany. Wild koalas within Australia have not been comparably assessed. Here we report a detailed analysis of KoRV genetic diversity in samples collected from 18 wild koalas from southeast Queensland. By employing deep sequencing we identified 108 novel KoRV envelope sequences and determined their phylogenetic diversity. Genetic diversity in KoRV was abundant and fell into three major groups; two comprised the previously identified subtypes A and B, while the third contained the remaining hypervariable region subtypes (C, D, and E) as well as four hypervariable region subtypes that we newly define here (F, G, H, and I). In addition to the ubiquitous presence of KoRV-A, which may represent an exclusively endogenous variant, subtypes B, D, and F were found to be at high prevalence, while subtypes G, H, and I were present in a smaller number of animals.

    IMPORTANCE Koala retrovirus (KoRV) is thought to be a significant contributor to koala disease and population decline across mainland Australia. This study is the first to determine KoRV subtype prevalence among a wild koala population, and it significantly expands the total number of KoRV sequences available, providing a more precise picture of genetic diversity. This understanding of KoRV subtype prevalence and genetic diversity will be important for conservation efforts attempting to limit the spread of KoRV. Furthermore, KoRV is one of the only retroviruses shown to exist in both endogenous (transmitted vertically to offspring in the germ line DNA) and exogenous (horizontally transmitted between infected individuals) forms, a division of fundamental evolutionary importance.

  • Activation and Inactivation of Primary Human Immunodeficiency Virus Envelope Glycoprotein Trimers by CD4-Mimetic Compounds [Vaccines and Antiviral Agents]

  • Human immunodeficiency virus type 1 (HIV-1) entry into cells is mediated by the viral envelope glycoproteins (Env), a trimer of three gp120 exterior glycoproteins, and three gp41 transmembrane glycoproteins. The metastable Env is triggered to undergo entry-related conformational changes when gp120 binds sequentially to the receptors, CD4 and CCR5, on the target cell. Small-molecule CD4-mimetic compounds (CD4mc) bind gp120 and act as competitive inhibitors of gp120-CD4 engagement. Some CD4mc have been shown to trigger Env prematurely, initially activating Env function, followed by rapid and irreversible inactivation. Here, we study CD4mc with a wide range of anti-HIV-1 potencies and demonstrate that all tested CD4mc are capable of activating as well as inactivating Env function. Biphasic dose-response curves indicated that the occupancy of the protomers in the Env trimer governs viral activation versus inactivation. One CD4mc bound per Env trimer activated HIV-1 infection. Envs with two CD4mc bound were activated for infection of CD4-negative, CCR5-positive cells, but the infection of CD4-positive, CCR5-positive cells was inhibited. Virus was inactivated when all three Env protomers were occupied by the CD4mc, and gp120 shedding from the Env trimer was increased in the presence of some CD4mc. Env reactivity and the on rates of CD4mc binding to the Env trimer were found to be important determinants of the potency of activation and entry inhibition. Cross-sensitization of Env protomers that do not bind the CD4mc to neutralization by an anti-V3 antibody was not evident. These insights into the mechanism of antiviral activity of CD4mc should assist efforts to optimize their potency and utility.

    IMPORTANCE The trimeric envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) mediate virus entry into host cells. Binding to the host cell receptors, CD4 and CCR5, triggers changes in the conformation of the HIV-1 envelope glycoprotein trimer important for virus entry. Small-molecule CD4-mimetic compounds inhibit HIV-1 infection by multiple mechanisms: (i) direct blockade of the interaction between the gp120 exterior envelope glycoprotein and CD4; (ii) premature triggering of conformational changes in the envelope glycoproteins, leading to irreversible inactivation; and (iii) exposure of cryptic epitopes to antibodies, allowing virus neutralization. The consequences of the binding of the CD4-mimetic compound to the HIV-1 envelope glycoproteins depends upon how many of the three subunits of the trimer are bound and upon the propensity of the envelope glycoproteins to undergo conformational changes. Understanding the mechanistic factors that influence the activity of CD4-mimetic compounds can help to improve their potency and coverage of diverse HIV-1 strains.

  • Infectious Bursal Disease Virus Activates c-Src To Promote {alpha}4{beta}1 Integrin-Dependent Viral Entry by Modulating the Downstream Akt-RhoA GTPase-Actin Rearrangement Cascade [Virus-Cell Interactions]

  • While the entry of infectious bursal disease virus (IBDV) is initiated by the binding of the virus to the two major receptors integrin and HSP90, the signaling events after receptor binding and how they contribute to virus entry remain elusive. We show here that IBDV activates c-Src by inducing the phosphorylation of the Y416 residue in c-Src both in DF-1 chicken fibroblasts and in vivo in the bursa of Fabricius from specific-pathogen-free (SPF) chickens. Importantly, inactivated IBDV fails to stimulate c-Src Y416 phosphorylation, and a very virulent IBDV strain induces a much higher level of c-Src Y416 phosphorylation than does an attenuated strain. Inhibition of c-Src activation by an Src kinase inhibitor or expression of a c-Src dominant negative mutant results in a significant decrease in the internalization of IBDV but has little effect on virus adhesion. Furthermore, short hairpin RNA (shRNA) downregulation of integrin, either the aalpha;4 or bbeta;1 subunit, but not HSP90 remarkably attenuates IBDV-induced c-Src Y416 phosphorylation, resulting in a decrease in IBDV internalization but not virus adhesion. Moreover, interestingly, inhibition of either c-Src downstream of the phosphatidylinositol 3-kinase (PI3K)/Akt-RhoA signaling cascade or actin rearrangement leads to a significant decrease in IBDV internalization irrespective of the IBDV-induced high levels of c-Src phosphorylation. Cumulatively, our results suggest a novel feed-forward model whereby IBDV activates c-Src for benefiting its cell entry via an integrin-mediated pathway by the activation of downstream PI3K/Akt-RhoA signaling and cytoskeleton actin rearrangement.

    IMPORTANCE While IBDV-caused immunosuppression is highly related to viral invasion, the molecular basis of the cellular entry of IBDV remains elusive. In this study, we demonstrate that IBDV activates c-Src by inducing the phosphorylation of the Y416 residue in c-Src to promote virus internalization but not virus adhesion. The ability to induce the level of c-Src Y416 phosphorylation correlates with the pathogenicity of an IBDV strain. IBDV-induced c-Src Y416 activation is aalpha;4bbeta;1 integrin but not HSP90 dependent and involves the activation of the downstream PI3K/Akt-RhoA GTPase-actin rearrangement cascade. Thus, our findings provide new insights into the IBDV infection process and the potential for c-Src as a candidate target for the development of IBDV therapeutic drugs.

  • The Crystal Structure of the Fifth Scavenger Receptor Cysteine-Rich Domain of Porcine CD163 Reveals an Important Residue Involved in Porcine Reproductive and Respiratory Syndrome Virus Infection [Structure and Assembly]

  • Porcine reproductive and respiratory syndrome (PRRS) has become an economically critical factor in swine industry since its worldwide spread in the 1990s. Infection by its causative agent, PRRS virus (PRRSV), was proven to be mediated by an indispensable receptor, porcine CD163 (pCD163), and the fifth scavenger receptor cysteine-rich domain (SRCR5) is essential for virus infection. However, the structural details and specific residues of pCD163 SRCR5 involved in infection have not been defined yet. In this study, we prepared recombinant pCD163 SRCR5 in Drosophila melanogaster Schneider 2 (S2) cells and determined its crystal structure at a high resolution of 2.0 AAring;. This structure includes a markedly long loop region and shows a special electrostatic potential, and these are significantly different from those of other members of the scavenger receptor cysteine-rich superfamily (SRCR-SF). Subsequently, we carried out structure-based mutational studies to identify that the arginine residue at position 561 (Arg561) in the long loop region is important for PRRSV infection. Further, we showed Arg561 probably takes effect on the binding of pCD163 to PRRSV during virus invasion. Altogether the current work provides the first view of the CD163 SRCR domain, expands our knowledge of the invasion mechanism of PRRSV, and supports a molecular basis for prevention and control of the virus.

    IMPORTANCE PRRS has caused huge economic losses to pig farming. The syndrome is caused by PRRSV, and PRRSV infection has been shown to be mediated by host cell surface receptors. One of them, pCD163, is especially indispensable, and its SRCR5 domain has been further demonstrated to play a significant role in virus infection. However, its structural details and the residues involved in infection are unknown. In this study, we determined the crystal structure of pCD163 SRCR5 and then carried out site-directed mutational studies based on the crystal structure to elucidate which residue is important. Our work not only provides structural information on the CD163 SRCR domain for the first time but also indicates the molecular mechanism of PRRSV infection and lays a foundation for future applications in prevention and control of PRRS.

  • Porcine Reproductive and Respiratory Syndrome Virus nsp1{alpha} Inhibits NF-{kappa}B Activation by Targeting the Linear Ubiquitin Chain Assembly Complex [Virus-Cell Interactions]

  • Linear ubiquitination, a newly discovered posttranslational modification, is catalyzed by the linear ubiquitin chain assembly complex (LUBAC), which is composed of three subunits: one catalytic subunit HOIP and two accessory molecules, HOIL-1L and SHARPIN. Accumulating evidence suggests that linear ubiquitination plays a crucial role in innate immune signaling and especially in the activation of the NF-B pathway by conjugating linear polyubiquitin chains to NF-B essential modulator (NEMO, also called IKK), the regulatory subunit of the IKK complex. Porcine reproductive and respiratory syndrome virus (PRRSV), an Arterivirus that has devastated the swine industry worldwide, is an ideal model to study the host's disordered inflammatory responses after viral infection. Here, we found that LUBAC-induced NF-B and proinflammatory cytokine expression can be inhibited in the early phase of PRRSV infection. Screening the PRRSV-encoded proteins showed that nonstructural protein 1aalpha; (nsp1aalpha;) suppresses LUBAC-mediated NF-B activation and its CTE domain is required for the inhibition. Mechanistically, nsp1aalpha; binds to HOIP/HOIL-1L and impairs the interaction between HOIP and SHARPIN, thus reducing the LUBAC-dependent linear ubiquitination of NEMO. Moreover, PRRSV infection also blocks LUBAC complex formation and NEMO linear-ubiquitination, the important step for transducing NF-B signaling. This unexpected finding demonstrates a previously unrecognized role of PRRSV nsp1aalpha; in modulating LUBAC signaling and explains an additional mechanism of immune modulation by PRRSV.

    IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS) is one of the most important veterinary infectious diseases in countries with intensive swine industries. PRRS virus (PRRSV) infection usually suppresses proinflammatory cytokine expression in the early stage of infection, whereas it induces an inflammatory storm in the late stage. However, precisely how the virus is capable of doing so remains obscure. In this study, we found that by blocking the interaction of its catalytic subunit HOIP and accessory molecule SHARPIN, PRRSV can suppress NF-B signal transduction in the early stage of infection. Our findings not only reveal a novel mechanism evolved by PRRSV to regulate inflammatory responses but also highlight the important role of linear ubiquitination modification during virus infection.

  • Potential for Low-Pathogenic Avian H7 Influenza A Viruses To Replicate and Cause Disease in a Mammalian Model [Pathogenesis and Immunity]

  • H7 subtype influenza A viruses are widely distributed and have been responsible for human infections and numerous outbreaks in poultry with significant impact. Despite this, the disease-causing potential of the precursor low-pathogenic (LP) H7 viruses from the wild bird reservoir has not been investigated. Our objective was to assess the disease-causing potential of 30 LP H7 viruses isolated from wild avian species in the United States and Canada using the DBA/2J mouse model. Without prior mammalian adaptation, the majority of viruses, 27 (90%), caused mortality in mice. Of these, 17 (56.7%) caused 100% mortality and 24 were of pathogenicity similar to that of A/Anhui/1/2013 (H7N9), which is highly pathogenic in mice. Viruses of duck origin were more pathogenic than those of shorebird origin, as 13 of 18 (72.2%) duck origin viruses caused 100% mortality while 4 of 12 (33.3%) shorebird origin viruses caused 100% mortality, despite there being no difference in mean lung viral titers between the groups. Replication beyond the respiratory tract was also evident, particularly in the heart and brain. Of the 16 viruses studied for fecal shedding, 11 were detected in fecal samples. These viruses exhibited a strong preference for avian-type aalpha;2,3-linked sialic acids; however, binding to mammalian-type aalpha;2,6-linked sialic acids was also detected. These findings indicate that LP avian H7 influenza A viruses are able to infect and cause disease in mammals without prior adaptation and therefore pose a potential public health risk.

    IMPORTANCE Low-pathogenic (LP) avian H7 influenza A viruses are widely distributed in the avian reservoir and are the precursors of numerous outbreaks of highly pathogenic avian influenza viruses in commercial poultry farms. However, unlike highly pathogenic H7 viruses, the disease-causing potential of LP H7 viruses from the wild bird reservoir has not been investigated. To address this, we studied 30 LP avian H7 viruses isolated from wild avian species in the United States and Canada using the DBA/2J mouse model. Surprisingly, the majority of these viruses, 90%, caused mortality in mice without prior mammalian adaptation, and 56.7% caused 100% mortality. There was also evidence of spread beyond the respiratory tract and fecal shedding. Therefore, the disease-causing potential of LP avian H7 influenza A viruses in mammals may be underestimated, and these viruses therefore pose a potential public health risk.

  • Modeling Severe Fever with Thrombocytopenia Syndrome Virus Infection in Golden Syrian Hamsters: Importance of STAT2 in Preventing Disease and Effective Treatment with Favipiravir [Vaccines and Antiviral Agents]

  • Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease endemic in parts of Asia. The etiologic agent, SFTS virus (SFTSV; family Bunyaviridae, genus Phlebovirus) has caused significant morbidity and mortality in China, South Korea, and Japan, with key features of disease being intense fever, thrombocytopenia, and leukopenia. Case fatality rates are estimated to be in the 30% range, and no antivirals or vaccines are approved for use for treatment and prevention of SFTS. There is evidence that in human cells, SFTSV sequesters STAT proteins in replication complexes, thereby inhibiting type I interferon signaling. Here, we demonstrate that hamsters devoid of functional STAT2 are highly susceptible to as few as 10 PFU of SFTSV, with animals generally succumbing within 5 to 6 days after subcutaneous challenge. The disease included marked thrombocytopenia and inflammatory disease characteristic of the condition in humans. Infectious virus titers were present in the blood and most tissues 3 days after virus challenge, and severe inflammatory lesions were found in the spleen and liver samples of SFTSV-infected hamsters. We also show that SFTSV infection in STAT2 knockout (KO) hamsters is responsive to favipiravir treatment, which protected all animals from lethal disease and reduced serum and tissue viral loads by 3 to 6 orders of magnitude. Taken together, our results provide additional insights into the pathogenesis of SFTSV infection and support the use of the newly described STAT2 KO hamster model for evaluation of promising antiviral therapies.

    IMPORTANCE Severe fever with thrombocytopenia syndrome (SFTS) is an emerging viral disease for which there are currently no therapeutic options or available vaccines. The causative agent, SFTS virus (SFTSV), is present in China, South Korea, and Japan, and infections requiring medical attention result in death in as many as 30% of the cases. Here, we describe a novel model of SFTS in hamsters genetically engineered to be deficient in a protein that helps protect humans and animals against viral infections. These hamsters were found to be susceptible to SFTSV and share disease features associated with the disease in humans. Importantly, we also show that SFTSV infection in hamsters can be effectively treated with a broad-spectrum antiviral drug approved for use in Japan. Our findings suggest that the new SFTS model will be an excellent resource to better understand SFTSV infection and disease as well as a valuable tool for evaluating promising antiviral drugs.

  • Identification of Human Anti-HIV gp160 Monoclonal Antibodies That Make Effective Immunotoxins [Vaccines and Antiviral Agents]

  • The envelope (Env) glycoprotein of HIV is the only intact viral protein expressed on the surface of both virions and infected cells. Env is the target of neutralizing antibodies (Abs) and has been the subject of intense study in efforts to produce HIV vaccines. Therapeutic anti-Env Abs can also exert antiviral effects via Fc-mediated effector mechanisms or as cytotoxic immunoconjugates, such as immunotoxins (ITs). In the course of screening monoclonal antibodies (MAbs) for their ability to deliver cytotoxic agents to infected or Env-transfected cells, we noted disparities in their functional activities. Different MAbs showed diverse functions that did not correlate with each other. For example, MAbs against the external loop region of gp41 made the most effective ITs against infected cells but did not neutralize virus and bound only moderately to the same cells that they killed so effectively when they were used in ITs. There were also differences in IT-mediated killing among transfected and infected cell lines that were unrelated to the binding of the MAb to the target cells. Our studies of a well-characterized antigen demonstrate that MAbs against different epitopes have different functional activities and that the binding of one MAb can influence the interaction of other MAbs that bind elsewhere on the antigen. These results have implications for the use of MAbs and ITs to kill HIV-infected cells and eradicate persistent reservoirs of HIV infection.

    IMPORTANCE There is increased interest in using antibodies to treat and cure HIV infection. Antibodies can neutralize free virus and kill cells already carrying the virus. The virus envelope (Env) is the only HIV protein expressed on the surfaces of virions and infected cells. In this study, we examined a panel of human anti-Env antibodies for their ability to deliver cell-killing toxins to HIV-infected cells and to perform other antiviral functions. The ability of an antibody to make an effective immunotoxin could not be predicted from its other functional characteristics, such as its neutralizing activity. Anti-HIV immunotoxins could be used to eliminate virus reservoirs that persist despite effective antiretroviral therapy.

  • A Novel Rabies Vaccine Expressing CXCL13 Enhances Humoral Immunity by Recruiting both T Follicular Helper and Germinal Center B Cells [Vaccines and Antiviral Agents]

  • Rabies remains a public health threat in most parts of the world, and approximately 99% of the cases are transmitted by dogs. There is an urgent need to develop an efficacious and affordable vaccine to control canine-transmitted rabies in developing countries. Our previous studies demonstrate that overexpression of chemokines/cytokines such as CCL-3 (MIP-1aalpha;) and granulocyte-macrophage colony-stimulating factor (GM-CSF) can enhance the immunogenicity of rabies vaccines. In the present study, the chemokine CXCL13 was inserted into the genome of the recombinant rabies virus (rRABV) strain LBNSE, and the effect of the chemokine CXCL13 on the immunogenicity of RABV was investigated. It was found that LBNSE-CXCL13 recruited follicular helper T (Tfh) and germinal center (GC) B cells, promoted the formation of GCs, and increased the population of plasma cells in immunized mice. Further studies showed that mice immunized with LBNSE-CXCL13 produced more rabies virus-neutralizing antibodies (VNAs) and developed better protection than those immunized with the parent virus LBNSE or the GM-CSF-expressing RABV (LBNSE-GM-CSF). Collectively, these findings provide a better understanding of the role of CXCL13 expression in the immunogenicity of the RABV, which may help in designing more-efficacious rabies vaccines.

    IMPORTANCE Rabies is endemic in most parts of the world, and more effort is needed to develop affordable and effective vaccines to control or eliminate this disease. The chemokine CXCL13 recruits both Tfh and B cells, which is essential for the homing of Tfh cells and the development of B cell follicles. In this study, the effect of the overexpression of CXCL13 on the immunogenicity of the RABV was evaluated in a mouse model. We found that CXCL13 expression promoted humoral immunity by recruiting Tfh and GC B cells, facilitating the formation of GCs, and increasing the number of plasma cells. As expected, the overexpression of CXCL13 resulted in enhanced virus-neutralizing antibody (VNA) production and protection against a virulent RABV challenge. These findings provide a better understanding of the role of CXCL13 in RABV-induced immune responses, which will help in designing more efficacious rabies vaccines.

  • The Many Faces of the Flavivirus NS5 Protein in Antagonism of Type I Interferon Signaling [Minireviews]

  • The vector-borne flaviviruses cause severe disease in humans on every inhabited continent on earth. Their transmission by arthropods, particularly mosquitoes, facilitates large emergence events such as witnessed with Zika virus (ZIKV) or West Nile virus in the Americas. Every vector-borne flavivirus examined thus far that causes disease in humans, from dengue virus to ZIKV, antagonizes the host type I interferon (IFN-I) response by preventing JAK-STAT signaling, suggesting that suppression of this pathway is an important determinant of infection. The most direct and potent viral inhibitor of this pathway is the nonstructural protein NS5. However, the mechanisms utilized by NS5 from different flaviviruses are often quite different, sometimes despite close evolutionary relationships between viruses. The varied mechanisms of NS5 as an IFN-I antagonist are also surprising given that the evolution of NS5 is restrained by the requirement to maintain function of two enzymatic activities critical for virus replication, the methyltransferase and RNA-dependent RNA polymerase. This review discusses the different strategies used by flavivirus NS5 to evade the antiviral effects of IFN-I and how this information can be used to better model disease and develop antiviral countermeasures.

  • Adeno-associated Virus (AAV) Assembly-Activating Protein Is Not an Essential Requirement for Capsid Assembly of AAV Serotypes 4, 5, and 11 [Gene Delivery]

  • Adeno-associated virus (AAV) vectors have made great progress in their use for gene therapy; however, fundamental aspects of AAV's capsid assembly remain poorly characterized. In this regard, the discovery of assembly-activating protein (AAP) sheds new light on this crucial part of AAV biology and vector production. Previous studies have shown that AAP is essential for assembly; however, how its mechanistic roles in assembly might differ among AAV serotypes remains uncharacterized. Here, we show that biological properties of AAPs and capsid assembly processes are surprisingly distinct among AAV serotypes 1 to 12. In the study, we investigated subcellular localizations and assembly-promoting functions of AAP1 to -12 (i.e., AAPs derived from AAV1 to -12, respectively) and examined the AAP dependence of capsid assembly processes of these 12 serotypes using combinatorial approaches that involved immunofluorescence and transmission electron microscopy, barcode-Seq (i. e., a high-throughput quantitative method using DNA barcodes and a next-generation sequencing technology), and quantitative dot blot assays. This study revealed that AAP1 to -12 are all localized in the nucleus with serotype-specific differential patterns of nucleolar association; AAPs and assembled capsids do not necessarily colocalize; AAPs are promiscuous in promoting capsid assembly of other serotypes, with the exception of AAP4, -5, -11, and -12; assembled AAV5, -8, and -9 capsids are excluded from the nucleolus, in contrast to the nucleolar enrichment of assembled AAV2 capsids; and, surprisingly, AAV4, -5, and -11 capsids are not dependent on AAP for assembly. These observations highlight the serotype-dependent heterogeneity of the capsid assembly process and challenge current notions about the role of AAP and the nucleolus in capsid assembly.

    IMPORTANCE Assembly-activating protein (AAP) is a recently discovered adeno-associated virus (AAV) protein that promotes capsid assembly and provides new opportunities for research in assembly. Previous studies on AAV serotype 2 (AAV2) showed that assembly takes place in the nucleolus and is dependent on AAP and that capsids colocalize with AAP in the nucleolus during the assembly process. However, through the investigation of 12 different AAV serotypes (AAV1 to -12), we find that AAP is not an essential requirement for capsid assembly of AAV4, -5, and -11, and AAP, assembled capsids, and the nucleolus do not colocalize for all the serotypes. In addition, we find that there are both serotype-restricted and serotype-promiscuous AAPs in their assembly roles. These findings challenge widely held beliefs about the importance of the nucleolus and AAP in AAV assembly and show the heterogeneous nature of the assembly process within the AAV family.

  • Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions [Structure and Assembly]

  • Palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms that are identical in their N termini but unique in their C termini due to a nndash;1 ribosomal frameshift during translation. In this study, we used cysteine (Cys) mutants to test differential palmitoylation of the Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N termini of 6K and TF, the four additional Cys residues in TF's unique C terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation occurs primarily in the N terminus. In contrast, 6K is not palmitoylated, even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to the wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane, and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a high incidence of abnormal particle morphologies compared to the wild-type virus as determined by transmission electron microscopy. We propose a model where the C terminus of TF modulates the palmitoylation of TF at the N terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding.

    IMPORTANCE Alphaviruses are a reemerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo. Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions, and optimal levels of TF correlate positively with wild-type-like particle morphology. In this study, we show that the palmitoylation of TF regulates its localization to the plasma membrane, which is the site of alphavirus budding. Mutants in which TF is not palmitoylated display drastically reduced plasma membrane localization, which effectively prevents TF from participating in budding or being incorporated into virus particles. Investigation of the regulation of TF will aid current efforts in the alphavirus field searching for approaches to mitigate alphaviral disease in humans.

  • Identification of New World Quails Susceptible to Infection with Avian Leukosis Virus Subgroup J [Genetic Diversity and Evolution]

  • The J subgroup of avian leukosis virus (ALV-J) infects domestic chickens, jungle fowl, and turkeys. This virus enters the host cell through a receptor encoded by the tvj locus and identified as Na+/H+ exchanger 1. The resistance to avian leukosis virus subgroup J in a great majority of galliform species has been explained by deletions or substitutions of the critical tryptophan 38 in the first extracellular loop of Na+/H+ exchanger 1. Because there are concerns of transspecies virus transmission, we studied natural polymorphisms and susceptibility/resistance in wild galliforms and found the presence of tryptophan 38 in four species of New World quails. The embryo fibroblasts of New World quails are susceptible to infection with avian leukosis virus subgroup J, and the cloned Na+/H+ exchanger 1 confers susceptibility on the otherwise resistant host. New World quails are also susceptible to new avian leukosis virus subgroup J variants but resistant to subgroups A and B and weakly susceptible to subgroups C and D of avian sarcoma/leukosis virus due to obvious defects of the respective receptors. Our results suggest that the avian leukosis virus subgroup J could be transmitted to New World quails and establish a natural reservoir of circulating virus with a potential for further evolution.

    IMPORTANCE Since its spread in broiler chickens in China and Southeast Asia in 2000, ALV-J remains a major enzootic challenge for the poultry industry. Although the virus diversifies rapidly in the poultry, its spillover and circulation in wild bird species has been prevented by the resistance of most species to ALV-J. It is, nevertheless, important to understand the evolution of the virus and its potential host range in wild birds. Because resistance to avian retroviruses is due particularly to receptor incompatibility, we studied Na+/H+ exchanger 1, the receptor for ALV-J. In New World quails, we found a receptor compatible with virus entry, and we confirmed the susceptibilities of four New World quail species in vitro. We propose that a prospective molecular epidemiology study be conducted to identify species with the potential to become reservoirs for ALV-J.

  • Alternative Polyadenylation of Human Bocavirus at Its 3' End Is Regulated by Multiple Elements and Affects Capsid Expression [Genome Replication and Regulation of Viral Gene Expression]

  • Alternative processing of human bocavirus (HBoV) P5 promoter-transcribed RNA is critical for generating the structural and nonstructural protein-encoding mRNA transcripts. The regulatory mechanism by which HBoV RNA transcripts are polyadenylated at proximal [(pA)p] or distal [(pA)d] polyadenylation sites is still unclear. We constructed a recombinant HBoV infectious clone to study the alternative polyadenylation regulation of HBoV. Surprisingly, in addition to the reported distal polyadenylation site, (pA)d, a novel distal polyadenylation site, (pA)d2, which is located in the right-end hairpin (REH), was identified during infectious clone transfection or recombinant virus infection. (pA)d2 does not contain typical hexanucleotide polyadenylation signal, upstream elements (USE), or downstream elements (DSE) according to sequence analysis. Further study showed that HBoV nonstructural protein NS1, REH, and cis elements of (pA)d were necessary and sufficient for efficient polyadenylation at (pA)d2. The distance and sequences between (pA)d and (pA)d2 also played a key role in the regulation of polyadenylation at (pA)d2. Finally, we demonstrated that efficient polyadenylation at (pA)d2 resulted in increased HBoV capsid mRNA transcripts and protein translation. Thus, our study revealed that all the bocaviruses have distal poly(A) signals on the right-end palindromic terminus, and alternative polyadenylation at the HBoV 3' end regulates its capsid expression.

    IMPORTANCE The distal polyadenylation site, (pA)d, of HBoV is located about 400 nucleotides (nt) from the right-end palindromic terminus, which is different from those of bovine parvovirus (BPV) and canine minute virus (MVC) in the same genus whose distal polyadenylation is located in the right-end stem-loop structure. A novel polyadenylation site, (pA)d2, was identified in the right-end hairpin of HBoV during infectious clone transfection or recombinant virus infection. Sequence analysis showed that (pA)d2 does not contain typical polyadenylation signals, and the last 42 nt form a stem-loop structure which is almost identical to that of MVC. Further study showed that NS1, REH, and cis elements of (pA)d are required for efficient polyadenylation at (pA)d2. Polyadenylation at (pA)d2 enhances capsid expression. Our study demonstrates alternative polyadenylation at the 3' end of HBoV and suggests an additional mechanism by which capsid expression is regulated.

  • Kaposi's Sarcoma-Associated Herpesvirus MicroRNAs Target GADD45B To Protect Infected Cells from Cell Cycle Arrest and Apoptosis [Virus-Cell Interactions]

  • Kaposi's sarcoma is one of the most common malignancies in HIV-infected individuals. The responsible agent, Kaposi's sarcoma-associated herpesvirus (KSHV; HHV8), expresses multiple microRNAs (miRNAs), but the targets and functions of these miRNAs are not completely understood. After infection in primary endothelial cells with KSHV, growth arrest DNA damage-inducible gene 45 beta (GADD45B) is one of the most repressed genes using genomic expression profiling. GADD45B was also repressed in mRNA expression profiling experiments when KSHV miRNAs were introduced to uninfected cells. We hypothesized that KSHV miRNAs target human GADD45B to protect cells from consequences of DNA damage, which can be triggered by viral infection. Expression of GADD45B protein is induced by the p53 activator, Nutlin-3, and KSHV miRNA-K9 inhibits this induction. In addition, Nutlin-3 increased apoptosis and cell cycle arrest based on flow cytometry assays. KSHV miR-K9 protected primary endothelial cells from apoptosis and cell cycle arrest following Nutlin-3 treatment. Similar protective phenotypes were seen for targeting GADD45B with short interfering RNAs (siRNAs), as with miR-K9. KSHV miR-K9 also decreased the protein levels of cleaved caspase-3, cleaved caspase-7, and cleaved poly(ADP-ribose) polymerase (PARP). In B lymphocytes latently infected with KSHV, specific inhibitors of KSHV miR-K9 led to increased GADD45B expression and apoptosis, indicating that miR-K9 is important for reducing apoptosis in infected cells. Furthermore, ectopic expression of GADD45B in KSHV-infected cells promoted apoptosis. Together, these results identify a new miRNA target and demonstrate that KSHV miRNAs are important for protecting infected cells from DNA damage responses.

    IMPORTANCE Kaposi's sarcoma-associated herpesvirus is a leading cause of cancers in individuals with AIDS. Promoting survival of infected cells is essential for maintaining viral infections. A virus needs to combat various cellular defense mechanisms designed to eradicate the viral infection. One such response can include DNA damage response factors, which can promote an arrest in cell growth and trigger cell death. We used a new approach to search for human genes repressed by small nucleic acids (microRNAs) expressed by a gammaherpesvirus (KSHV), which identified a gene called GADD45B as a target of microRNAs. Repression of GADD45B, which is expressed in response to DNA damage, benefited survival of infected cells in response to a DNA damage response. This information could be used to design new treatments for herpesvirus infections.

  • Correction for Lund et al., HIV-1-Neutralizing IgA Detected in Genital Secretions of Highly HIV-1-Exposed Seronegative Women on Oral Preexposure Prophylaxis [Author Correction]

  • Porcine Reproductive and Respiratory Syndrome Virus Antagonizes JAK/STAT3 Signaling via nsp5, Which Induces STAT3 Degradation [Pathogenesis and Immunity]

  • Signal transducer and activator of transcription 3 (STAT3) is a pleiotropic signaling mediator of many cytokines, including interleukin-6 (IL-6) and IL-10. STAT3 is known to play critical roles in cell growth, proliferation, differentiation, immunity and inflammatory responses. The objective of this study was to determine the effect of porcine reproductive and respiratory syndrome virus (PRRSV) infection on the STAT3 signaling since PRRSV induces a weak protective immune response in host animals. We report here that PRRSV infection of MARC-145 cells and primary porcine pulmonary alveolar macrophages led to significant reduction of STAT3 protein level. Several strains of both PRRSV type 1 and type 2 led to a similar reduction of STAT3 protein level but had a minimal effect on its transcripts. The PRRSV-mediated STAT3 reduction was in a dose-dependent manner as the STAT3 level decreased, along with incremental amounts of PRRSV inocula. Further study showed that nonstructural protein 5 (nsp5) of PRRSV induced the STAT3 degradation by increasing its polyubiquitination level and shortening its half-life from 24 h to ~3.5 h. The C-terminal domain of nsp5 was shown to be required for the STAT3 degradation. Moreover, the STAT3 signaling in the cells transfected with nsp5 plasmid was significantly inhibited. These results indicate that PRRSV antagonizes the STAT3 signaling by accelerating STAT3 degradation via the ubiquitin-proteasomal pathway. This study provides insight into the PRRSV interference with the JAK/STAT3 signaling, leading to perturbation of the host innate and adaptive immune responses.

    IMPORTANCE The typical features of immune responses in PRRSV-infected pigs are delayed onset and low levels of virus neutralizing antibodies, as well as weak cell-mediated immunity. Lymphocyte development and differentiation rely on cytokines, many of which signal through the JAK/STAT signaling pathway to exert their biological effects. Here, we discovered that PRRSV antagonizes the JAK/STAT3 signaling by inducing degradation of STAT3, a master transcription activator involved in multiple cellular processes and the host immune responses. The nsp5 protein of PRRSV is responsible for the accelerated STAT3 degradation. The PRRSV-mediated antagonizing STAT3 could lead to suppression of a broad spectrum of cytokines and growth factors to allow virus replication and spread in host animals. This may be one of the reasons for the PRRSV interference with the innate immunity and its poor elicitation of protective immunity. This finding provides insight into PRRSV pathogenesis and its interference with the host immune responses.

  • Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export [Virus-Cell Interactions]

  • HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding.

    IMPORTANCE HIV-1 infects more than 34 million people worldwide causing ggt;1 million deaths per year. Infectious virion production is activated by the essential viral Rev protein that mediates nuclear export of intron-bearing late-stage viral mRNAs. Rev's shuttling into and out of the nucleus is regulated by the antagonistic activities of both a peptide-encoded N-terminal nuclear localization signal and C-terminal nuclear export signal (NES). How Rev and related viral proteins balance strong import and export activities in order to achieve optimal levels of viral gene expression is incompletely understood. We provide evidence that multimerization provides a mechanism by which Rev transiently masks its NES peptide, thereby biasing its trafficking to and retention within the nucleus. Targeted pharmacological disruption of Rev-Rev interactions should perturb multiple Rev activities, both Rev-RNA binding and Rev's trafficking to the nucleus in the first place.

  • The Norovirus NS3 Protein Is a Dynamic Lipid- and Microtubule-Associated Protein Involved in Viral RNA Replication [Virus-Cell Interactions]

  • Norovirus (NoV) infections are a significant health burden to society, yet the lack of reliable tissue culture systems has hampered the development of appropriate antiviral therapies. Here we show that the NoV NS3 protein, derived from murine NoV (MNV), is intimately associated with the MNV replication complex and the viral replication intermediate double-stranded RNA (dsRNA). We observed that when expressed individually, MNV NS3 and NS3 encoded by human Norwalk virus (NV) induced the formation of distinct vesicle-like structures that did not colocalize with any particular protein markers to cellular organelles but localized to cellular membranes, in particular those with a high cholesterol content. Both proteins also showed some degree of colocalization with the cytoskeleton marker bbeta;-tubulin. Although the distribution of MNV and NV NS3s were similar, NV NS3 displayed a higher level of colocalization with the Golgi apparatus and the endoplasmic reticulum (ER). However, we observed that although both proteins colocalized in membranes counterstained with filipin, an indicator of cholesterol content, MNV NS3 displayed a greater association with flotillin and stomatin, proteins known to associate with sphingolipid- and cholesterol-rich microdomains. Utilizing time-lapse epifluorescence microscopy, we observed that the membrane-derived vesicular structures induced by MNV NS3 were highly motile and dynamic in nature, and their movement was dependent on intact microtubules. These results begin to interrogate the functions of NoV proteins during virus replication and highlight the conserved properties of the NoV NS3 proteins among the seven Norovirus genogroups.

    IMPORTANCE Many mechanisms involved in the replication of norovirus still remain unclear, including the role for the NS3 protein, one of seven nonstructural viral proteins, which remains to be elucidated. This study reveals that murine norovirus (MNV) NS3 is intimately associated with the viral replication complex and dsRNA. We observed that the NS3 proteins of both MNV and Norwalk virus (NV) induce prominent vesicular structures and that this formation is dependent on microtubules and cellular cholesterol. Thus, this study contributes to our understanding of protein function within different Norovirus genogroups and expands a growing knowledge base on the interaction between positive-strand RNA [(+)RNA] viruses and cellular membranes that contribute to the biogenesis of virus-induced membrane organelles. This study contributes to our understanding of viral protein function and the ability of a viral protein to recruit specific cellular organelles and lipids that enable replication.

  • Zika Virus Efficiently Replicates in Human Retinal Epithelium and Disturbs Its Permeability [Letters to the Editor]

  • Inhibition of Human Cytomegalovirus pUL89 Terminase Subunit Blocks Virus Replication and Genome Cleavage [Vaccines and Antiviral Agents]

  • The human cytomegalovirus terminase complex cleaves concatemeric genomic DNA into unit lengths during genome packaging and particle assembly. This process is an attractive drug target because cleavage of concatemeric DNA is not required in mammalian cell DNA replication, indicating that drugs targeting the terminase complex could be safe and selective. One component of the human cytomegalovirus terminase complex, pUL89, provides the endonucleolytic activity for genome cleavage, and the domain responsible is reported to have an RNase H-like fold. We hypothesize that the pUL89 endonuclease activity is inhibited by known RNase H inhibitors. Using a novel enzyme-linked immunosorbent assay (ELISA) format as a screening assay, we found that a hydroxypyridonecarboxylic acid compound, previously reported to be an inhibitor of human immunodeficiency virus RNase H, inhibited pUL89 endonuclease activity at low-micromolar concentrations. Further characterization revealed that this pUL89 endonuclease inhibitor blocked human cytomegalovirus replication at a relatively late time point, similarly to other reported terminase complex inhibitors. Importantly, this inhibitor also prevented the cleavage of viral genomic DNA in infected cells. Taken together, these results substantiate our pharmacophore hypothesis and validate our ligand-based approach toward identifying novel inhibitors of pUL89 endonuclease.

    IMPORTANCE Human cytomegalovirus infection in individuals lacking a fully functioning immune system, such as newborns and transplant patients, can have severe and debilitating consequences. The U.S. Food and Drug Administration-approved anti-human cytomegalovirus drugs mainly target the viral polymerase, and resistance to these drugs has appeared. Therefore, anti-human cytomegalovirus drugs from novel targets are needed for use instead of, or in combination with, current polymerase inhibitors. pUL89 is a viral ATPase and endonuclease and is an attractive target for anti-human cytomegalovirus drug development. We identified and characterized an inhibitor of pUL89 endonuclease activity that also inhibits human cytomegalovirus replication in cell culture. pUL89 endonuclease, therefore, should be explored as a potential target for antiviral development against human cytomegalovirus.

  • Classic Spotlight, 1971 to 1973: Articles of Significant Interest Selected from the Journal of Virology Archives by the Editors [Editorial]

  • Mechanism of Enhanced HIV Restriction by Virion Coencapsidated Cytidine Deaminases APOBEC3F and APOBEC3G [Virus-Cell Interactions]

  • The APOBEC3 (A3) enzymes, A3G and A3F, are coordinately expressed in CD4+ T cells and can become coencapsidated into HIV-1 virions, primarily in the absence of the viral infectivity factor (Vif). A3F and A3G are deoxycytidine deaminases that inhibit HIV-1 replication by inducing guanine-to-adenine hypermutation through deamination of cytosine to form uracil in minus-strand DNA. The effect of the simultaneous presence of both A3G and A3F on HIV-1 restriction ability is not clear. Here, we used a single-cycle infectivity assay and biochemical analyses to determine if coencapsidated A3G and A3F differ in their restriction capacity from A3G or A3F alone. Proviral DNA sequencing demonstrated that compared to each A3 enzyme alone, A3G and A3F, when combined, had a coordinate effect on hypermutation. Using size exclusion chromatography, rotational anisotropy, and in vitro deamination assays, we demonstrate that A3F promotes A3G deamination activity by forming an A3F/G hetero-oligomer in the absence of RNA which is more efficient at deaminating cytosines. Further, A3F caused the accumulation of shorter reverse transcripts due to decreasing reverse transcriptase efficiency, which would leave single-stranded minus-strand DNA exposed for longer periods of time, enabling more deamination events to occur. Although A3G and A3F are known to function alongside each other, these data provide evidence for an A3F/G hetero-oligomeric A3 with unique properties compared to each individual counterpart.

    IMPORTANCE The APOBEC3 enzymes APOBEC3F and APOBEC3G act as a barrier to HIV-1 replication in the absence of the HIV-1 Vif protein. After APOBEC3 enzymes are encapsidated into virions, they deaminate cytosines in minus-strand DNA, which forms promutagenic uracils that induce transition mutations or proviral DNA degradation. Even in the presence of Vif, footprints of APOBEC3-catalyzed deaminations are found, demonstrating that APOBEC3s still have discernible activity against HIV-1 in infected individuals. We undertook a study to better understand the activity of coexpressed APOBEC3F and APOBEC3G. The data demonstrate that an APOBEC3F/APOBEC3G hetero-oligomer can form that has unique properties compared to each APOBEC3 alone. This hetero-oligomer has increased efficiency of virus hypermutation, raising the idea that we still may not fully realize the antiviral mechanisms of endogenous APOBEC3 enzymes. Hetero-oligomerization may be a mechanism to increase their antiviral activity in the presence of Vif.

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

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    JVI Accepts: Articles Published Ahead of Print

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

  • Surveillance of bat coronaviruses in Kenya identifies relatives of human coronaviruses NL63 and 229E and their recombination history [PublishAheadOfPrint]

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

    IMPORTANCE Understanding the driving forces of cross-species virus transmission is central to understanding the nature of disease emergence. Previous studies have demonstrated that bats are the ultimate reservoir hosts for a number of coronaviruses (CoVs) including ancestors of SARS-CoV, MERS-CoV, and HCoV-229E. However, the evolutionary pathways of bat CoVs remain elusive. We provide evidence for natural recombination between distantly-related African bat coronaviruses associated with Triaenops afer and Hipposideros sp. bats that resulted in a NL-63 like virus, an ancestor of the human pathogen HCoV-NL63. These results suggest that inter-species recombination may play an important role in CoV evolution and the emergence of novel CoVs with zoonotic potential.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

  • Herpes Simplex Virus Type 1 Ubiquitin-specific Protease UL36 abrogates NF-{kappa}B Activation in DNA Sensing Signal Pathway [PublishAheadOfPrint]

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

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

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

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

  • Association of human papillomavirus type 16 E2 with Rad50-interacting protein 1 enhances viral DNA replication [PublishAheadOfPrint]

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

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

  • The structural interface between HIV-1 Vif and human APOBEC3H [PublishAheadOfPrint]

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

    Taken together, this virus-host interface model explains previous reported data and will help to identify novel drug targets to combat HIV-1 infection.

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

  • Screening for novel small molecule inhibitors targeting the assembling of influenza virus polymerase complex by a BiLC-based reporter system [PublishAheadOfPrint]

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

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

  • Severe symptomatic primary HCMV infection despite effective innate and adaptive immune responses [PublishAheadOfPrint]

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

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

  • Binding of the methyl donor SAM to MERS-CoV 2 -O-methyltransferase nsp16 promotes the recruitment of the allosteric activator nsp10. [PublishAheadOfPrint]

  • The Middle East respiratory syndrome coronavirus (MERS-CoV) non-structural protein 16 (nsp16) is an S-adenosyl-L-methionine (SAM)-dependent 2rrsquo; -O-methyltransferase (MTase) that is thought to methylate the ribose 2rrsquo; -OH of the first transcribed nucleotide (N1) of viral RNA cap structures. This 2rrsquo; -O MTase activity is regulated by nsp10. The 2rrsquo; -O methylation prevents virus detection by cell innate immunity mechanisms and viral translation inhibition by the interferon-stimulated IFIT-1 protein. To unravel the regulation of nsp10/nsp16 2rrsquo; -O-MTase activity, we used purified MERS-CoV nsp16 and nsp10. First, we showed that nsp16 recruited N7-methylated capped RNA and SAM. The SAM binding promotes then the assembly of the enzymatically active nsp10/nsp16 complex that converted 7mGpppG (cap-0) into 7mGpppG2rrsquo; Om (cap-1) RNA by 2rrsquo; -OH methylation of N1 in a SAM-dependent manner. The subsequent release of SAH speeds up nsp10/nsp16 dissociation that stimulates the reaction turnover. Alanine mutagenesis and RNA binding assays allowed the identification of the nsp16 residues involved in RNA recognition forming the RNA binding groove (K46, K170, E203, D133, R38, Y47 and Y181) and the cap-0 binding site (Y30, Y132 and H174). Finally, we found that nsp10/nsp16 2rrsquo; -O-MTase activity is sensitive to known MTase inhibitors, such as sinefungin and cap analogues. This characterization of the MERS-CoV 2rrsquo; -O-MTase is a preliminary step towards the development of molecules to inhibit cap 2rrsquo; -O methylation and to restore the host antiviral response.

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

  • The Zika virus methyltransferase: structure and functions for drug design perspectives [PublishAheadOfPrint]

  • The Flavivirus Zika virus (ZIKV) is the causing agent of neurological disorders like microcephaly in newborns or Guillain-Barre syndrome. Its NS5 protein embeds a methyltransferase (MTase) domain involved in the formation of the viral mRNA cap. We investigated the structural and functional properties of the ZIKV MTase. We show that the ZIKV MTase can methylate RNA cap structures at the N7 position of the cap, and at the 2rrsquo; O position on the ribose of the first nucleotide, yielding a cap-1 structure. In addition, the ZIKV MTase methylates the ribose 2rrsquo; -O position of internal adenosines of RNA substrates. Crystal structure of the ZIKV MTase determined at 2.01 AAring; resolution reveals a crystallographic homodimer. One chain is bound to the methyl-donor (SAM) and shows a high structural similarity to the Dengue virus (DENV) MTase. The second chain lacks SAM and displays conformational changes in the aalpha;X aalpha;-helix contributing to the SAM and RNA binding. These conformational modifications reveal a possible molecular mechanism of the enzymatic turnover involving a conserved Ser/Arg motif. In the second chain, the SAM binding site accommodates a sulphate close to a glycerol that could serve as a basis for structure-based drug design. In addition, compounds known to inhibit the DENV MTase show similar inhibition potency on the ZIKV MTase. Altogether these results contribute to a better understanding of the ZIKV MTase, a central player in viral replication and host innate immune response, and lay the basis development of potential antiviral drugs.

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

  • Barriers to infection of human cells by feline leukemia virus: insights into resistance to zoonosis [PublishAheadOfPrint]

  • The human genome displays a rich fossil record of past gamma-retrovirus infections, yet no current epidemic is evident, despite environmental exposure to viruses that infect human cells in vitro. Feline leukemia viruses (FeLVs) rank high on this list, but domestic or workplace exposure has not been associated with detectable serological responses. Non-specific inactivation of gamma-retroviruses by serum factors appears insufficient to explain these observations. To investigate further we explored the susceptibility of primary and established human cell lines to FeLV-B, the most likely zoonotic variant. Fully permissive infection was common in cancer-derived cell lines, but was also a feature of non-transformed keratinocytes and lung fibroblasts. Cells of haematopoietic origin were less generally permissive and formed discrete groups on the basis of high or low intracellular protein expression and virion release. Potent repression was observed in primary human blood mononuclear cells and a subset of leukemia cell lines. However, the early steps of reverse transcription and integration appear to be unimpaired in non-permissive cells. FeLV-B was subject to G-ggt;A hypermutation with a predominant APOBEC3G signature in partially permissive cells but was not mutated in permissive cells or in non-permissive cells that block secondary viral spread. Distinct cellular barriers that protect primary human blood cells are likely to be important in protection against zoonotic infection with FeLV.

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

  • AcMNPV AC83 is a PIF protein required for ODV and BV nucleocapsid assembly as well as assembly of the PIF complex in ODV envelopes [PublishAheadOfPrint]

  • Baculovirus occlusion derived virus (ODV) initiate infection of lepidopteran larval hosts by binding to the midgut epithelia which is mediated by per os infectivity factors (PIFs). Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes seven PIF proteins of which PIF1-4 form a core complex in ODV envelopes to which PIF0 and PIF6 loosely associate. Deletion of any pif results in ODV unable to bind or enter midgut cells. AC83 also associates with the PIF complex and this study further analyzed its role in oral infectivity to determine if it is a PIF protein. It had been proposed that AC83 possesses a chitin binding domain that enables transit through the peritrophic matrix; however, no chitin binding activity has ever been demonstrated. AC83 has been reported to be found only in the ODV envelopes but in contrast, the Orgyia pseudotsugata MNPV AC83 homolog is associated with both ODV nucleocapsids and envelopes. In addition, unlike known pif genes, deletion of ac83 eliminates nucleocapsid formation. We propose a new model for AC83 function and show AC83 is associated with both ODV nucleocapsids and envelopes. We also further define the domain required for nucleocapsid assembly. The cysteine-rich region of AC83 is also shown not to be a chitin binding, but a zinc finger domain required for the recruitment or assembly of the PIF complex to ODV envelopes. As such, AC83 has all the properties of a PIF protein and should be considered PIF8. In addition, pif7 (ac110) is reported as the 38th baculovirus core gene.

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

  • The Autographa californica nucleopolyhedrovirus ac83 gene contains a cis-acting element that is essential for nucleocapsid assembly [PublishAheadOfPrint]

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

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

  • Mapping the Human Memory B Cell and Serum Neutralizing Antibody Responses to DENV4 Infection and Vaccination [PublishAheadOfPrint]

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

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

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

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

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

  • An optimized hepatitis C virus E2 glycoprotein core adopts a functional homodimer that efficiently blocks virus entry [PublishAheadOfPrint]

  • The hepatitis C virus (HCV) envelope glycoprotein E2 is the major target of broadly neutralizing antibodies in vivo and is the focus of efforts in the rational design of a universal B cell vaccine against HCV. The E2 glycoprotein exhibits a high degree of amino-acid variability which localizes to three discrete regions: hypervariable region 1 (HVR1), hypervariable region 2 (HVR2) and the intergenotypic variable region (igVR). All three variable regions contribute to immune evasion and/or isolate-specific structural variations, both important considerations for vaccine design. High resolution structural definition of the intact HCV envelope glycoprotein complex containing E1 and E2 remains to be elucidated whilst crystallographic structures of a recombinant E2 ectodomain failed to resolve HVR1, HVR2 and a major neutralization determinant adjacent to HVR1. To obtain further information on E2, we characterized the role of all three variable regions in E2 ectodomain folding and function in the context of a recombinant ectodomain fragment (rE2). We report that removal of the variable regions accelerates binding to the major host-cell receptor CD81 and that simultaneous deletion of HVR2 and the igVR is required to maintain wild-type CD81-binding characteristics. The removal of the variable regions also rescued the ability of rE2 to form a functional homodimer. We propose that the phenotype of the D123 dimer provides novel insights into the role of the variable motifs in the higher order assembly of the E2 ectodomain and may have implications for E1E2 structure on the virion surface.

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

  • Residues in the gp41 Ectodomain Regulate HIV-1 Envelope Glycoprotein Conformational Transitions Induced by gp120-directed Inhibitors [PublishAheadOfPrint]

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

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

  • Lineage A Betacoronavirus NS2 proteins and homologous Torovirus Berne pp1a-carboxyterminal domain are phosphodiesterases that antagonize activation of RNase L [PublishAheadOfPrint]

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

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

  • Genesis and dissemination of highly pathogenic H5N6 avian influenza viruses [PublishAheadOfPrint]

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

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

  • Feline immunodeficiency virus cross-species transmission: Implications for emergence of new lentiviral infections. [PublishAheadOfPrint]

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

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

  • A potential role for a {beta}-catenin coactivator (high mobility group AT-hook 1 protein) during the latency-reactivation cycle of bovine herpesvirus 1 [PublishAheadOfPrint]

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

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

  • Mildly acidic pH triggers an irreversible conformational change in the fusion domain of herpes simplex virus 1 glycoprotein B and inactivation of viral entry [PublishAheadOfPrint]

  • Herpes simplex virus (HSV) entry into a subset of cells requires endocytosis and endosomal low-pH. Pre-exposure of isolated virions to mildly acidic pH of 5 to 6 partially inactivates HSV infectivity in an irreversible manner. Acid inactivation is a hallmark of viruses that enter via low-pH pathways; this occurs by pre-triggering conformational changes essential for fusion. The target and mechanism(s) of low-pH inactivation of HSV are unclear. Here, low-pH treated HSV-1 was defective in fusion activity yet retained normal levels of attachment to cell surface heparan sulfate and binding to nectin-1 receptor. Low-pH triggered conformational changes in gB reported to date are reversible, despite irreversible low-pH inactivation. gB conformational changes and their reversibility were measured by antigenic analysis with a panel of monoclonal antibodies and by detecting changes in oligomeric conformation. Multiple sequential treatments of HSV-1 virions with pH 5 followed by neutral pH or 3 hr treatment at pH 5 caused an irreversible ggt; 2.5 log infectivity reduction. While changes in several gB antigenic sites were reversible, alteration of the H126 epitope was irreversible. gB oligomeric conformational change remained reversible under all conditions tested. Altogether, our results reveal that oligomeric alterations and fusion domain changes represent distinct conformational changes in gB, and the latter correlates with irreversible low-pH inactivation of HSV. We propose that conformational change in the gB fusion domain is important for activation of membrane fusion during viral entry, and that in the absence of a host target membrane this change results in irreversible inactivation of virions.

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

  • Restricted TET2 expression in germinal center type B cells promotes stringent Epstein-Barr virus latency [PublishAheadOfPrint]

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

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

  • NS1 protein amino acid changes D189N and V194I affect interferon responses, thermosensitivity and virulence of circulating H3N2 human influenza A viruses [PublishAheadOfPrint]

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

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

  • Ribosome Profiling Reveals Translational Upregulation of Cellular Oxidative Phosphorylation mRNAs During Vaccinia Virus-induced Host Shutoff [PublishAheadOfPrint]

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

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

  • Disruption of stress granule formation by the multifunctional cricket paralysis virus 1A protein [PublishAheadOfPrint]

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

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

  • Non-structural Protein 4 of Porcine Reproductive and Respiratory Syndrome Virus Modulates Cell Surface Swine Leukocyte Antigen Class I Expression by Down-regulating {beta}2-Microglobulin Transcription [PublishAheadOfPrint]

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

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

  • Shifting clade distribution, reassortment, and emergence of new subtypes of highly pathogenic avian influenza A(H5) viruses collected in Vietnamese poultry from 2012 to 2015 [PublishAheadOfPrint]

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

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

  • Adenovirus serotype 5 vaccination results in suboptimal CD4 T helper 1 responses in mice. [PublishAheadOfPrint]

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

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

  • CD63 regulates Epstein-Barr virus LMP1 exosomal packaging, enhancement of vesicle production, and non-canonical NF-{kappa}B signaling [PublishAheadOfPrint]

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

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

  • Within-host evolution of simian arteriviruses in crab-eating macaques [PublishAheadOfPrint]

  • Simian arteriviruses are a diverse clade of viruses infecting captive and wild nonhuman primates. We recently reported that Kibale red colobus virus 1 (KRCV-1) causes a mild and self-limiting disease in experimentally infected crab-eating macaques, while simian hemorrhagic fever virus (SHFV) causes lethal viral hemorrhagic fever. Here we characterize how these viruses evolved during replication in cell culture and in experimentally infected macaques. During passage in cell culture, 68 substitutions became fixed in the KRCV-1 genome that were localized in open reading frames (ORFs) likely associated with host cell entry and exit. However, we did not detect any strong signatures of selection during replication in macaques. We uncovered patterns of evolution that were distinct from those observed in surveys of wild red colobus monkeys, suggesting that these species may exert different adaptive challenges for KRCV-1. During SHFV infection, we detected signatures of selection on ORF 5a and on a small subset of sites in the genome. Overall, our data suggest that patterns of evolution differ markedly among simian arteriviruses, and among host species.

    IMPORTANCE Certain RNA viruses can cross species barriers and cause disease in new hosts. Simian arteriviruses are a diverse group of related viruses that infect captive and wild nonhuman primates, with associated disease severity ranging from apparently asymptomatic infections to severe, viral hemorrhagic fevers. We infected nonhuman primate cell cultures and then crab-eating macaques with either simian hemorrhagic fever virus (SHFV) or Kibale red colobus virus 1 (KRCV-1) and assessed within-host viral evolution. We found that KRCV-1 quickly acquired a large number of substitutions in its genome during replication in cell culture, but that evolution in macaques was limited. In contrast, we detected selection focused on SHFV ORFs 5a and 5, which encode putative membrane proteins. These patterns suggest that in addition to diverse pathogenic phenotypes, these viruses may also exhibit distinct patterns of within-host evolution both in vitro and in vivo.

  • Channel inactivating mutations and their revertant mutants in the envelope protein of the infectious bronchitis virus. [PublishAheadOfPrint]

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

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

  • Human CD4+ T cell responses to an attenuated tetravalent dengue vaccine parallel those induced by natural infection, in magnitude, HLA restriction and antigen specificity [PublishAheadOfPrint]

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

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

  • Human adenovirus type 37 uses {alpha}V{beta}1 and {alpha}3{beta}1 integrins for infection of human corneal cells [PublishAheadOfPrint]

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

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

  • Thymidine Kinase-Negative Herpes Simplex Virus 1 Can Efficiently Establish Persistent Infection in Neural Tissues of Nude Mice [PublishAheadOfPrint]

  • Herpes simplex virus 1 (HSV-1) establishes latency in neural tissues of immunocompetent mice, but persists in both peripheral and neural tissues of lymphocyte-deficient mice. Thymidine kinase (TK) is believed to be essential for HSV-1 to persist in neural tissues of immunocompromised mice, because the infectious virus of a mutant with defects in both TK and UL24 is detected only in peripheral tissues, but not in neural tissues, of severe combined immunodeficiency mice (T. Valyi-Nagy et al., Virology 199:484-490, 1994). Here we find infiltration of CD4 and CD8 T cells in peripheral and neural tissues of mice infected with a TK-negative mutant. We therefore investigated the significance of viral TK and host T cells for HSV-1 to persist in neural tissues using three genetically engineered mutants with defects only in TK or in both TK and UL24 and two strains of nude mice. Surprisingly, all three mutants establish persistent infection in up to 100% of brain stems and 93% of trigeminal ganglia of adult nude mice 28 days post-infection as measured by the recovery of infectious virus. Thus, in mouse neural tissues, host T cells block persistent HSV-1 infection, and viral TK is dispensable for virus to establish persistent infection. Furthermore, we found 30- to 200-fold more virus in neural tissues than in the eye and detected glycoprotein C, a true late viral antigen in brainstem neurons of nude mice persistently infected with the TK-negative mutant, suggesting that adult mouse neurons can support the replication of TK-negative HSV-1.

    IMPORTANCE Acyclovir is used to treat herpes simplex virus 1 (HSV-1)-infected, immunocompromised patients, but the treatment is hindered by the emergence of drug-resistant viruses, mostly with mutations in viral thymidine kinase (TK) that activates acyclovir. TK mutants are detected in brains of immunocompromised patients with persistent infection. However, the questions as to whether TK-negative (TKnndash;) HSV-1 can establish persistent infection in brains of immunocompromised hosts and whether neurons in vivo are permissive for TKnndash; HSV-1 remain elusive. Using three genetically engineered HSV-1 TKnndash; mutants and two strains of nude mice deficient in T cells, we found that all three HSV-1 TKnndash; mutants can efficiently establish persistent infection in the brain stem and trigeminal ganglion and detected glycoprotein C, a true late viral antigen in brainstem neurons. Our study provides evidence that TKnndash; HSV-1 can persist in neural tissues and replicate in brain neurons of the immunocompromised host.

  • On the analysis of intra-host and inter-host viral populations: human cytomegalovirus as a case study of pitfalls and expectations [PublishAheadOfPrint]

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

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

  • African swine fever virus gets undressed: new insights on the entry pathway [PublishAheadOfPrint]

  • African swine fever virus (ASFV) is a large multienveloped DNA virus composed of a genome-containing core successively wrapped by an inner lipid envelope, an icosahedral protein capsid and an outer lipid envelope. In keeping with this structural complexity, recent studies have revealed an intricate entry program. This Gem article highlights how ASFV uses two alternative pathways, macropinocytosis and clathrin-mediated endocytosis, to enter into the host macrophage and how the endocytosed particles undergo a stepwise, low pH-driven disassembly leading to inner envelope fusion and core delivery in the cytoplasm.

  • Zika virus infects early- and mid-gestation human maternal-decidual tissues, inducing distinct innate tissue responses in the maternal-fetal interface [PublishAheadOfPrint]

  • Zika virus (ZIKV) has emerged as a cause of congenital brain anomalies and a range of placental-related abnormalities, highlighting the need to unveil the modes of maternal-to-fetal transmission. The most likely route of vertical ZIKV transmission is via the placenta. The earliest events of ZIKV transmission in the maternal decidua, representing the maternal-uterine aspect of the chimeric placenta, have remained unexplored. Here we show that ZIKV replicates in first-trimester human maternal-decidual tissues grown ex-vivo as 3D organ cultures. An efficient viral spread in the decidual tissues was demonstrated by the rapid upsurge and continued increase of tissue-associated ZIKV load and titers of infectious cell-free virus progeny, released from the infected tissues. Notably, maternal-decidual tissues obtained at mid-gestation remained similarly susceptible to ZIKV, whereas fetal-derived chorionic-villi demonstrated reduced ZIKV replication with increasing gestational age. A genome-wide transcriptome analysis revealed that ZIKV substantially upregulated the decidual tissue innate immune responses. Further comparison of the innate tissue-response patterns following parallel infections with ZIKV and human cytomegalovirus (HCMV), revealed that unlike HCMV, ZIKV did not induce immune-cell activation or trafficking responses in the maternal-fetal interface, but rather upregulated placental apoptosis and cell-death molecular functions. The data identify the maternal-uterine aspect of the human placenta as a likely site of ZIKV transmission to the fetus, and further reveal distinct patterns of innate tissue responses to ZIKV. Our unique experimental model and findings could further serve to study the initial stages of congenital ZIKV transmission and pathogenesis and evaluate the effect of new therapeutic interventions.

    IMPORTANCE In view of the rapid spread of the current ZIKV epidemic and the severe manifestations of congenital ZIKV infection, it is crucial to learn the fundamental mechanisms of viral transmission from the mother to the fetus. Our studies of ZIKV infection in the authentic tissues of the human maternal-fetal interface unveil a route of transmission whereby virus originating from the mother could reach the fetal compartment via efficient replication within the maternal decidual aspect of the placenta, cohabited by maternal and fetal cells. The identified distinct placental-tissue innate immune responses and damage pathways could provide a mechanistic basis for some of the placental developmental abnormalities associated with ZIKV infection. The findings in the unique model of the human decidua should pave the way to future studies examining the interaction of ZIKV with decidual immune cells, and to evaluation of therapeutic interventions aimed at the earliest stages of transmission.

  • Infectious Bursal Disease Virus Subverts Autophagic Vacuoles to Promote Viral Maturation and Release [PublishAheadOfPrint]

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

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

  • In vivo imaging with bioluminescent enterovirus 71 allows for real-time visualization of tissue tropism and viral spread [PublishAheadOfPrint]

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

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

  • VP24-karyopherin alpha binding affinities differ between Ebolavirus species influencing interferon inhibition and VP24 stability [PublishAheadOfPrint]

  • Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV) and Reston ebolavirus (RESTV) belong to the same genus, Ebolavirus, but exhibit different virulence properties. VP24 protein, a structural protein present in all family members, blocks interferon (IFN) signaling and likely contributes to virulence. Inhibition of IFN signaling by EBOV VP24 (eVP24) involves its interaction with the NPI-1 subfamily of karyopherin alpha (KPNA) nuclear transporters. Here, we evaluated eVP24, BDBV VP24 (bVP24) and RESTV VP24 (rVP24) interactions with three NPI-1 subfamily KPNAs (KPNA1, KPNA5 and KPNA6). Using purified proteins we demonstrated that each VP24 binds to each of the three NPI-1 KPNAs. bVP24, however, exhibited approximately a 10-fold lower KPNA binding affinity as compared to either eVP24 or rVP24. Cell-based assays also indicate that bVP24 exhibits decreased KPNA interaction, decreased suppression of IFN induced gene expression and a shorter half-life in transfected cells, as compared to eVP24 or rVP24. Amino acid sequence alignments between bVP24 and eVP24 also identified residues within and surrounding the previously-defined eVP24-KPNA5 binding interface that decrease eVP24-KPNA affinity or bVP24-KPNA affinity. VP24 mutations that cause reduced KPNA binding affinity also decrease IFN-inhibition and shorten VP24 half-lives. These data identify novel functional differences in VP24-KPNA interaction and reveal a novel impact of the VP24-KPNA interaction on VP24 stability.

    IMPORTANCE The interaction of Ebola virus (EBOV) VP24 protein with host karyopherin alpha (KPNA) proteins blocks type I interferon (IFN) signaling, which is a central component of the host innate immune response to viral infection. Here, we quantitatively compared the interactions of VP24 proteins from EBOV and two members of the Ebolavirus genus, Bundibugyo virus (BDBV) and Reston virus (RESTV). The data reveal lower binding affinity of the BDBV VP24 (bVP24) for KPNAs and demonstrate that the interaction with KPNA modulates inhibition of IFN signaling and VP24 stability The effect of KPNA interaction on VP24 stability is a novel functional consequence of this virus-host interaction and the differences identified between viral species may contribute to differences in pathogenesis.

  • RPLP1 and RPLP2 are essential flavivirus host factors that promote early viral protein accumulation [PublishAheadOfPrint]

  • The Flavivirus genus contains several arthropod-borne viruses that pose global health threats, including dengue viruses (DENV), yellow fever virus (YFV) and Zika virus (ZIKV). In order to understand how these viruses replicate in human cells, we previously conducted genome-scale RNAi screens to identify candidate host factors. In these screens we identified ribosomal proteins RPLP1 and RPLP2 (RPLP1/2) to be among the most crucial putative host factors required for DENV and YFV infection. RPLP1/2 are phosphoproteins that bind the ribosome through interaction with another ribosomal protein, RPLP0, to form a structure termed the ribosomal stalk. RPLP1/2 were validated as essential host factors for DENV, YFV, and ZIKV infection in two human cell lines: A549 lung adenocarcinoma and HuH-7 hepatoma, and for productive DENV infection of Aedes aegypti mosquitoes. Depletion of RPLP1/2 caused moderate cell-line specific effects on global protein synthesis as determined by metabolic labeling: In A549 global translation was increased while in HuH-7 it was reduced, albeit both of these effects were modest. In contrast, RPLP1/2 knockdown strongly reduced early DENV protein accumulation, suggesting a requirement for RPLP1/2 in viral translation. Furthermore, knockdown of RPLP1/2 reduced levels of DENV structural proteins expressed from an exogenous transgene. We postulate that these ribosomal proteins are required for efficient translation elongation through the viral open reading frame. In summary, this work identifies RPLP1/2 as critical flaviviral host factors required for translation.

    IMPORTANCE Flaviviruses cause important diseases in humans. Examples of mosquito-transmitted flaviviruses include dengue, yellow fever and Zika viruses. Viruses require a plethora of cellular factors to infect cells, and the ribosome plays an essential role in all viral infections. The ribosome is a complex macromolecular machine composed of RNA and proteins that is responsible for protein synthesis. We identified two specific ribosomal proteins that are strictly required for flavivirus infection of human cells and mosquitoes: RPLP1 and RPLP2 (RPLP1/2). These proteins are part of a structure known as the ribosomal stalk and help orchestrate the elongation phase of translation. We show that flaviviruses are particularly dependent on the function of RPLP1/2. Our findings suggest that ribosome composition is an important factor for virus translation and may represent a regulatory layer for translation of specific cellular mRNAs.

  • mTOR promotes anti-viral humoral immunity by differentially regulating CD4 helper T cell and B cell responses [PublishAheadOfPrint]

  • mTOR has important roles in regulation of both innate and adaptive immunity but whether and how mTOR modulates humoral immune responses is yet to be fully understood. To address this issue, we examined the effects of rapamycin, a specific inhibitor of mTOR, on B cell and CD4 T cell responses during acute infection with lymphocytic choriomeningitis virus. Rapamycin treatment resulted in suppression of virus-specific B cell responses by inhibiting proliferation of germinal center (GC) B cells. In contrast, the number of memory CD4 T cells was increased in rapamycin treated mice. However, the drug treatment caused a striking bias of CD4 T cell differentiation into Th1 and substantially impaired formation of Tfh cells, which are essential for humoral immunity. Further experiments in which mTOR signaling was modulated by RNAi revealed that B cells were the primary target cells of rapamycin for the impaired humoral immunity and that reduced Tfh formation in rapamycin-treated mice were due to lower GC B cell responses that are essential for Tfh generation. Additionally, we found that rapamycin had minimal effects on B cell responses activated by LPS that stimulates B cells in antigen independent manner, suggesting that rapamycin specifically inhibits B cell responses induced by B cell receptor stimulation with antigen. Together, these findings demonstrate that mTOR signals play an essential role in antigen-specific humoral immune responses by differentially regulating B cell and CD4 T cell responses during acute viral infection, and rapamycin treatment alters interplay of immune cell subsets involved in anti-viral humoral immunity.

    IMPORTANCE mTOR is a serine/threonine kinase involved in a variety of cellular activities. Although its specific inhibitor, rapamycin, is currently used as an immunosuppressive drug in transplant patients, it has been reported that rapamycin can also stimulate pathogen specific cellular immunity in certain circumstances. However, whether and how mTOR regulates humoral immunity is not well understood. Here, we found that rapamycin treatment predominantly inhibited GC B cell responses during viral infection, and this led to biased helper CD4 T cell differentiation as well as impaired antibody responses. Theses findings suggest that inhibition of B cell responses by rapamycin could play an important role in regulation of allograft-specific antibody responses to prevent organ rejection in transplant recipients. Our results also show that consideration for antibody responses is required when rapamycin is used to stimulate vaccine-induced immunity.

  • IRAV (FLJ11286), an Interferon Stimulated Gene with Antiviral Activity Against Dengue Virus, Interacts with MOV10 [PublishAheadOfPrint]

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

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

  • Mechanism of PKR Inhibition by Human Cytomegalovirus pTRS1 [PublishAheadOfPrint]

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

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

  • HCMV requires EGFR signaling to enter and initiate the early steps in the establishment of latency in CD34+ human progenitor cells [PublishAheadOfPrint]

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

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

  • The effect of HIV-1 Env on SERINC5 antagonism [PublishAheadOfPrint]

  • SERINC5 is able to restrict HIV-1 infection by drastically impairing the infectivity of viral particles. Current studies have shown that HIV-1 Nef protein counters SERINC5 through down regulating SERINC5 from the cell surface and preventing virion incorporation of SERINC5. In addition, the Env proteins of some HIV-1 strains can also overcome SERINC5 inhibition. However, it is unclear how HIV-1 Env does so and why HIV-1 has two mechanisms to resist SERINC5 inhibition. Results of this study show that neither Env nor Nef prevents high-level ectopic SERINC5 from incorporation into HIV-1 particles, except that Env, but not Nef, is able to resist the inhibition of virion-associated SERINC5. Testing a panel of HIV-1 Env proteins from different subtypes reveals a high frequency of SERINC5-resistant Envs. Interestingly, the SERINC5-bearing viruses, although not inhibited by SERINC5 itself, become more sensitive to the CCR5 inhibitor maraviroc and some neutralizing antibodies compared to the SERINC5-free viruses, which suggests a possible influence of SERINC5 on Env function. We conclude that HIV-1 Env is able to overcome SERINC5 without preventing SERINC5 virion incorporation.

    IMPORTANCE HIV-1 Nef has been known to enhance the infectivity of HIV-1 viral particles and maintain high viral loads in patients. However, the underlying molecular mechanism has remained elusive until the recent discovery of the antiviral activity of SERINC5. SERINC5 profoundly inhibits HIV-1, but is antagonized by Nef that prevents the incorporation of SERINC5 into viral particles. Here, we show that HIV-1 Env, but not Nef, is able to resist high-level SERINC5 without excluding SERINC5 from incorporation into viral particles. However, the virion-associated SERINC5 renders HIV-1 more sensitive to some broadly neutralizing antibodies. It is possible that, under the pressure of some neutralizing antibodies in vivo, HIV-1 needs Nef to remove SERINC5 from viral particles despite that viral Env is able to resist virion-associated SERINC5.

  • A temperature sensitive live-attenuated canine influenza virus H3N8 vaccine [PublishAheadOfPrint]

  • Canine influenza is a respiratory disease of dogs caused by canine influenza virus (CIV). CIV subtypes responsible for influenza in dogs include H3N8, which originated from the transfer of H3N8 equine influenza virus to dogs; and the H3N2 CIV, which is an avian-origin virus that adapted to infect dogs. Influenza infections are most effectively prevented through vaccination to reduce transmission and future infection. Currently, only inactivated influenza vaccines (IIVs) are available for the prevention of CIV in dogs. However, the efficacy of IIVs is suboptimal and novel approaches are necessary for the prevention of disease caused by this canine respiratory pathogen. Using reverse genetics techniques, we have developed a live-attenuated CIV vaccine (LACIV) for the prevention of H3N8 CIV. The H3N8 LACIV replicates efficiently in canine cells at 33oC but is impaired at 37-39oC temperatures, and was attenuated when compared to wild-type H3N8 CIV, in vivo and ex vivo. The LACIV was able to induce protection against H3N8 CIV challenge with a single intranasal inoculation in mice. Immunogenicity and protection efficacy were better than that observed with a commercial CIV H3N8 IIV, but provided limited cross-reactive immunity and heterologous protection against H3N2 CIV. These results demonstrate the feasibility of implementing a LAIV approach for the prevention and control of H3N8 CIV in dogs and suggest the need for a new LAIV for the control of H3N2 CIV.

    IMPORTANCE Two influenza A virus subtypes has been reported in dogs in the last 16 years - the canine influenza virus (CIV) H3N8 and H3N2 of equine and avian origin, respectively. To date, only inactivated influenza vaccines (IIVs) are available to prevent CIV infections. Here we report the generation of a recombinant, temperature sensitive H3N8 CIV as a live-attenuated influenza vaccine (LAIV), which was attenuated in mice and dog tracheal, explants when compared to CIV H3N8 wild-type. A single dose of H3N8 LACIV showed immunogenicity and protection against a homologous challenge that was better than that conferred with an H3N8 IIV, demonstrating the feasibility of implementing a LAIV approach for the improved control of H3N8 CIV infections in dogs.

  • Histidine 375 Modulates CD4 Binding in HIV-1 CRF01_AE Envelope Glycoproteins [PublishAheadOfPrint]

  • The envelope glycoproteins (Env) from human immunodeficiency virus type 1 (HIV-1) mediate viral entry. The binding of the HIV-1 gp120 glycoprotein to CD4 triggers conformational changes in gp120 that allow high-affinity binding to its coreceptors. Contrary to all other Envs from the same phylogenetic group M, which possess a serine (S) at position 375, those from CRF01_AE strains possess a histidine (H) at this location. This residue is part of the Phe43 cavity where residue 43 of CD4 (a phenylalanine) engages with the gp120. Here we evaluated the functional consequences of replacing this residue in two CRF01_AE Envs (CM244 and 92TH023) by a serine. We observed that reversion of the 375 amino acid to a serine (H375S) resulted in a loss of functionality of both CRF01_AE Envs as measured by a dramatic loss in infectivity and their ability to mediate cell-to-cell fusion. While no effects on processing or trimer stability of these variants were observed, decreased functionality could be linked to a major defect in CD4 binding induced by the substitution of H375 by a serine. Importantly, mutation of residues 61 (Layer 1), 105, 108 (Layer 2) and 474 to 476 (Layer 3) of the CRF01_AE gp120 inner domain layers to the consensus residues present in group M restored CD4 binding and wild-type levels of infectivity and cell-to-cell fusion. These results suggest a functional co-evolution between the Phe43 cavity and the gp120 inner domain layers. Altogether, our observations describe the functional importance of 375H amino acid in CRF01_AE envelopes.

    IMPORTANCE A highly conserved serine located at position 375 in group M is replaced by an histidine in CRF01_AE Envs. Here we show that H375 is required for efficient CRF01_AE Env binding to CD4. Moreover, this work suggests that specific residues of the gp120 inner domain layers have co-evolved with H375 in order to maintain its ability to mediate viral entry.

  • High prevalence of infectious adeno-associated virus (AAV) in human peripheral blood mononuclear cells indicative of T lymphocytes as sites of AAV persistence [PublishAheadOfPrint]

  • Seroepidemiology shows that infections with adeno-associated virus (AAV) are widespread, but diverse AAV serotypes isolated from man or non-human primates have so far not been proven as causes of human disease. In view of the increasing success of AAV-derived vectors in human gene therapy, the definition of the in vivo sites of wild-type AAV persistence and clinical consequences of its reactivation are becoming increasingly urgent.

    Here, we identified the presumed cell-type for AAV persistence in the human host, by highly sensitive AAV PCRs developed for the full spectrum of human AAV serotypes. In genomic DNA samples from leukocytes of 243 healthy blood donors, 34% were found to be AAV-positive, predominantly AAV2 (77%), AAV5 (19%), and additional serotypes. Roughly 11% of blood donors had mixed AAV infections. AAV prevalence was dramatically increased in immunosuppressed patients, where 76% were AAV-positive. Of these, at least 45% displayed mixed infections. Follow up of single blood donors over two years allowed repeated detection of the initial and/or additional AAV serotypes suggestive of fluctuating, persistent infection. Leukocyte separation revealed that AAV resided in CD3+ T-lymphocytes, perceived as putative in vivo site of AAV persistence. Moreover, infectious AAV of various serotypes could be rescued and propagated from numerous samples. The high prevalence and broad spectrum of human AAVs in leukocytes closely follows AAV seroepidemiology. Immunosuppression obviously enhances AAV replication in parallel to activation of human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6), reminiscent of herpesvirus-induced AAV activation.

    IMPORTANCE Adeno-associated virus is viewed as apathogenic and replication-defective, requiring coinfection with adenovirus or herpesvirus for productive infection. In vivo persistence of a defective virus requires latency in specialized cell types to escape the host immune response until viral spread becomes possible. Reactivation from latency can be induced by diverse stimuli including infections, typically induced upon host immunosuppression. We show for the first time that infectious AAV is highly prevalent in human leukocytes, specifically T-lymphocytes, and that AAV is strongly amplified upon immunosuppression along with reactivation of latent human herpesviruses. In the absence of an animal model to study the AAV life cycle, our findings in the human host will advance the understanding of AAV latency, reactivation, and in vivo pathogenesis.

  • Amino Acid Mutations in the NS4A Region of Hepatitis C Virus Contribute to Viral Replication and Infectious Virus Production [PublishAheadOfPrint]

  • Hepatitis C Virus (HCV) strain JFH-1, which belongs to genotype 2a, replicates autonomously in cultured cells, whereas another genotype 2a strain, J6CF, does not. Previously, we found that replacing the NS3 helicase and NS5B-to-3rrsquo; X regions of J6CF with those of JFH-1 confers J6CF replication competence. In this study, we aimed to identify the minimum modifications to establish replication-competent J6CF within these genomic regions. We previously identified 4 mutations in the NS5B-to-3rrsquo; X region that confer J6CF replication competence instead of replacing of this region. Here, we induced cell culture-adaptive mutations in J6CF by the long-term culture of J6CF/JFH-1 chimeras composed of JFH-1 NS5B-to-3rrsquo; X or individual parts of this but not the NS3 helicase region. After two months of culture, efficient HCV replication and infectious virus production in chimeric RNA-transfected cells were observed, and several amino acid mutations in NS4A were identified in replicating HCV genomes. The introduction of NS4A mutations into the J6CF/JFH-1 chimeras enhanced viral replication and infectious virus production. Immunofluorescence microscopy demonstrated that some of these mutations altered the subcellular localization of co-expressed NS3 protein and affected the interaction between NS3 and NS4A. Finally, introducing the most effective NS4A mutation, A1680E, into J6CF contributed to its replication competence in cultured cells when introduced in conjunction with four previously identified adaptive mutations in NS5B-to-3rrsquo; X. In conclusion, we identified an adaptive mutation in NS4A that confers J6CF replication competence when introduced in conjunction with 4 mutations in NS5B-to-3rrsquo; X and established a replication-competent J6CF strain with minimum essential modifications in cultured cells.

    IMPORTANCE The HCV cell culture system using JFH-1 strain and HuH-7 cells can be used to assess the complete HCV life cycle in cultured cells. This cell culture system has been used to develop direct-acting antivirals against HCV, and the ability to use various HCV strains within this system is important for future studies. In this study, we aimed to establish a novel HCV cell culture system using another HCV genotype 2a strain, J6CF, which replicates in chimpanzees but not in cultured cells. We identified an effective cell culture-adaptive mutation in NS4A and established a replication-competent J6CF strain in cultured cells with minimum essential modifications. The described strategy can be used in establishing a novel HCV cell culture system, and the replication-competent J6CF clone composed of the minimum essential modifications for cell culture adaptation will be valuable as another representative of genotype 2a strains.

  • Promising Role of Toll-Like Receptor 8 Agonist in Concert with Prostratin for Activation of Silent HIV [PublishAheadOfPrint]

  • The persistence of latently HIV-infected cells in patients under combined anti-retroviral treatment (cART) remains the major hurdle for HIV eradication. Thus far, individual compounds have not been sufficiently potent to reactivate latent virus and guarantee its elimination in vivo. Thus, we hypothesized that transcriptional enhancers, in concert with compounds triggering the innate immune system, are more efficient in reversing latency by creating a Th1 supportive milieu that acts against latently HIV-infected cells at various levels. To test our hypothesis, we screened six compounds on a co-culture of latently infected cells (J-lat) and monocyte-derived dendritic cells (MDDCs). The PKC agonist, Prostratin, with a TLR8 agonist, resulted in greater reversion of HIV latency than any single compound. This combinatorial approach led to a drastic phenotypic and functional maturation of the MDDCs. TNF and cell-cell interactions were crucial for the greater reversion observed. Similarly, we found a greater potency of the combination of Prostratin/TLR8 agonist in reversing HIV latency when applying it to primary cells of HIV-infected patients. Thus, we demonstrated here the synergistic interplay between TLR8-matured MDDCs and compounds acting directly on latently HIV-infected cells, targeting different mechanisms of latency, by triggering various signaling pathways. Moreover, TLR8 triggering may reverse exhaustion of HIV-specific cytotoxic T lymphocytes that might be essential for killing or constraining the latently infected cells.

    IMPORTANCE Curing HIV is the Holy Grail. The so-called "shock and kill strategy" relies on drug-mediated reversion of HIV latency and the subsequent death of those cells under combined anti-retroviral treatment. So far, no compound achieves efficient reversal of latency nor eliminates this latent reservoir. The compounds may not target all of the latency mechanisms in all latently infected cells. Moreover, HIV-associated exhaustion of the immune system hinders the efficient elimination of the reactivated cells. In this study, we demonstrated synergistic latency reversion by combining agonists for protein kinase C and toll-like receptor 8 in a co-culture of latently infected cells with myeloid dendritic cells. The drug Prostratin stimulates directly the transcriptional machinery of latently infected cells and the TLR8 agonist acts indirectly by maturing dendritic cells. These findings highlight the importance of the immune system and its activation, in combination with direct acting compounds, to reverse latency.

  • Cryo-EM study of genome release of dicistrovirus Israeli acute bee paralysis virus [PublishAheadOfPrint]

  • Viruses from the family Dicistroviridae can cause substantial economic damage by infecting agriculturally important insects. Israeli acute bee paralysis virus (IAPV) causes honeybee colony collapse disorder in the United States. High-resolution molecular details of the genome delivery mechanism of dicistroviruses are unknown. Here we present a cryo-EM analysis of IAPV virions induced to release their genomes in vitro. We determined structures of full IAPV virions primed to release their genomes to a resolution of 3.3 AAring; and of empty capsids to a resolution of 3.9 AAring;. We show that IAPV does not form expanded A particles before the genome release, as is the case in the related enteroviruses from the family Picornaviridae. The structural changes observed in the empty IAPV particles include the detachment of the VP4 minor capsid proteins from the inner face of the capsid and partial loss of the structure of the N-terminal arms of the VP2 capsid proteins. Unlike in many picornaviruses, the empty particles of IAPV are not expanded, relative to the native virions, and do not contain pores in their capsids that might serve as channels for the genome release. Therefore, re-arrangement of a unique region of the capsid is probably required for IAPV genome release.

    IMPORTANCE Honeybee populations in Europe and North America are declining due to pressure from pathogens including viruses. Israeli acute bee paralysis virus from the family Dicistroviridae causes honeybee colony collapse disorder in the United States. The delivery of virus genomes into host cells is necessary for the initiation of infection. Here we present structural cryo-electron microscopy analysis of IAPV particles induced to release their genomes. We show that the genome release is not preceded by an expansion of IAPV virions, as is the case in the related picornaviruses that infect vertebrates. Furthermore, minor capsid proteins detach from the capsid upon the genome release. The genome leaves behind empty particles that have compact protein shells.

  • Herpes Simplex Virus 1 UL41 Protein Suppresses the IRE1/XBP1 Signal Pathway of the Unfolded Protein Response via its RNase activity [PublishAheadOfPrint]

  • During viral infection, accumulation of viral proteins can cause stress in the endoplasmic reticulum (ER) and trigger the unfolded protein response (UPR) to restore the ER homeostasis. Inositol-requiring enzyme 1 (IRE1)-dependent pathway is the most conserved one of the three UPR signal pathways. Upon activation, IRE1 splices out an intron from the unspliced inactive form X-box binding protein 1 (XBP1(u)) mRNA and produces a transcriptionally potent spliced form (XBP1(s)). Previous studies have reported that the IRE1/XBP1 pathway is inhibited upon herpes simplex virus 1 (HSV-1) infection, however, the underlying molecular mechanism is still illusive. Here we uncovered a role of HSV-1 protein UL41 in inhibiting the IRE1/XBP1 signal pathway. Ectopic expression of UL41 decreased the expression of XBP1 and blocked XBP1 splicing activation induced by the ER stress inducer of thapsigargin. Wild-type (WT), but not the UL41-null mutant HSV-1 (R2621), decreased XBP1 mRNA induced by thapsigargin. Nevertheless, infection with both WT-HSV-1 and R2621 without drug pretreatment could reduce the mRNA and protein levels of XBP1(s), and additional mechanisms might contribute to this inhibition of XBP1(s) during the R2621 infection. Taking these findings together, our results reveal XBP1 as a novel target of UL41 and provide insights into the mechanism by which HSV-1 modulates the IRE1/XBP1 pathway.

    IMPORTANCE During viral infection, viruses hijack the host translation apparatus to produce large amounts of viral proteins which leads to ER stress. To restore ER homeostasis, cells initiate the UPR to alleviate effects of ER stress. The IRE1/XBP1 pathway is the most conserved UPR branch and it would activate ER-associated protein degradation (ERAD) to reduce the ER load. The IRE1/XBP1 branch is repressed during HSV-1 infection, while little is known about the underlying molecular mechanism. Our results show for the first time that UL41 suppressed the IRE1/XBP1 signal pathway via reducing the accumulation of XBP1 mRNA and characterization of the underlying molecular mechanism provides new insight into the modulation of UPR by HSV-1.

  • Lassa virus cell entry reveals new aspects of virus-host cell interaction [PublishAheadOfPrint]

  • Viral entry represents the first step of every viral infection and is a determinant for the host-range and disease potential of a virus. Here we review the latest developments on cell entry of the highly pathogenic Old World arenavirus Lassa, providing novel insights into the complex host cell interaction of this important human pathogen. We will cover new discoveries on the molecular mechanisms of receptor recognition, endocytosis, and the use of late endosomal entry factors.

  • Adjuvanting an SIV vaccine with TLR ligands encapsulated in nanoparticles induces persistent antibody responses and enhanced protection in TRIM5{alpha} restrictive macaques [PublishAheadOfPrint]

  • Our previous work has shown that antigens adjuvanted with specific ligands for toll-like receptor 4 (TLR4) and TLR7/8 encapsulated in poly (lactic-co-glycolic acid) (PLGA) based nanoparticles (NP), induced robust and durable immune responses in mice and macaques. We investigated the efficacy of these NP adjuvants in inducing protective immunity against simian immunodeficiency virus (SIV). Rhesus macaques (RMs) were immunized with NP containing TLR4 and TLR7/8 agonists mixed with soluble recombinant SIVmac239 derived envelope (Env) gp140 and Gag p55 (Protein), or with virus like particles (VLP) containing SIVmac239 Env and Gag. NP adjuvanted vaccines induced robust innate responses, a greater magnitude and persistence of antigen specific antibody responses, and enhanced plasmablast responses, compared to Alum adjuvanted vaccines. NP adjuvanted vaccines induced antigen specific, long-lived plasma cells (LLPCs), which persisted in the bone marrow for several months after vaccination. NP adjuvanted vaccines induced immune responses that were associated with enhanced protection against repeated low dose, intra-vaginal challenges with heterologous SIVsmE660, in animals that carried TRIM5aalpha; restrictive alleles. Protection induced by immunization with Protein + NP correlated with the pre-challenge titers of Env-specific IgG antibodies in serum and vaginal secretions. However, no such correlate was apparent for immunization with VLP + NP or alum adjuvanted groups. Transcriptional profiling of PBMCs isolated within the first few hours to days after primary vaccination revealed that NP adjuvanted vaccines induced a molecular signature similar to the live attenuated yellow fever viral vaccine. This systems approach identified early blood transcriptional signatures that correlate with Env-specific antibody responses in vaginal secretions and protection against infection. These results demonstrate the adjuvanticity of NP adjuvant in inducing persistent and protective antibody responses against SIV in RM with implications for vaccine design against HIV.

    Importance: The RV144 HIV vaccine trial highlighted modest protective vaccine efficacy but more importantly a need to develop strategies to prolong protection by enhancing the durability of antibody response. We attempted to address this issue by investigating nanoparticle encapsulated TLR 4/7/8 ligands or the NP adjuvant combined with SIV immunogens as a vaccine in a non-human primate model and compared immune responses with alum, an adjuvant currently approved for use with human vaccines and used in the RV144 trial. Our findings indicate advantages of using the NP adjuvant over alum when combining with SIV specific Protein immunogen in promoting high magnitude and persistence of antibody responses in serum and vaginal secretions, which also correlated with ability to protect against an intra-vaginal challenge with SIV. This strategy can be further developed by combining with the next generation of HIV immunogens currently developed to enhance magnitude and breadth of neutralizing antibody responses.

  • Mice expressing minimally humanized CD81 and occludin genes support hepatitis C virus uptake in vivo [PublishAheadOfPrint]

  • Hepatitis C virus (HCV) causes chronic infections in at least 150 million individuals world-wide. HCV has a narrow host range and robustly infects only humans and chimpanzees. The underlying mechanisms for this narrow host range are incompletely understood. At the level of entry differences in the amino acid sequences between the human and mouse orthologues of two essential host factors, the tetraspannin CD81 and the tight junction protein occludin (OCLN) explain at least in part HCV's limited ability to enter mouse hepatocytes. We have previously shown that adenoviral or transgenic overexpression of human CD81 and OCLN facilitates HCV uptake into mouse hepatocytes in vitro and in vivo. In efforts to refine these models we constructed knock-in mice in which the second extracellular loops of CD81 and OCLN were replaced with the respective human sequences, which contain the determinants that are critical of HCV uptake. We demonstrate that the humanized CD81 and OCLN are expressed at physiologic levels in a tissue-appropriate fashion. Mice bearing the humanized alleles form normal tight junctions and do not exhibit any immunologic abnormalities, indicating that interactions with their physiologic ligands are intact. HCV entry factor knock-in mice enable HCV uptake with similar efficiency as mice expressing HCV entry factors transgenically or adenovirally, demonstrating the utility of this model for studying HCV infection in vivo.

    IMPORTANCE At least 150 million individuals are chronically infected with hepatitis C virus (HCV). Chronic hepatitis C can result in progressive liver disease and liver cancer. New antiviral treatments can cure HCV in the majority of patients but a vaccine remains elusive. To gain a better understanding of the processes culminating in liver failure and cancer and to prioritize more efficiently vaccine candidates, small animal models are needed. Here, we describe the characterization of a new mouse model in which the parts of two host factors that are essential for HCV uptake, CD81 and occludin (OCLN) which differ between mice and men were humanized. We demonstrate that such minimally humanized mice develop normally, express the modified genes at physiological levels and support HCV uptake. This model is of considerable utility for studying viral entry in the three dimensional context of the liver and to test approaches aimed at preventing HCV entry.

  • Reassortment between swine H3N2 and 2009 pandemic H1N1 in the United States resulted in influenza A viruses with diverse genetic constellations with variable virulence in pigs [PublishAheadOfPrint]

  • Repeated spillovers of the H1N1 pandemic virus (H1N1pdm09) from humans to pigs resulted in substantial evolution of influenza A viruses infecting swine, contributing to the genetic and antigenic diversity of influenza A virus (IAV) currently circulating in swine. The reassortment with endemic swine viruses and maintenance of some of the H1N1pdm09 internal genes resulted in the circulation of different genomic constellations in pigs. Here, we performed a whole genome phylogenetic analysis of 496 IAV circulating in swine from 2009 to 2016 in the U.S. We identified 44 different genotypes, with the most common genotype (32.33%) containing a clade IV-A HA gene, a 2002-lineage NA gene, an M-pdm09 gene, and remaining gene segments of triple reassortant internal gene (TRIG) origin. To understand how different genetic constellations may relate to viral fitness, we compared the pathogenesis and transmission in pigs of six representative genotypes. Although all six genotypes efficiently infected pigs, they resulted in different degrees of pathology and viral shedding. These results highlight the vast H3N2 genetic diversity circulating in U.S. swine after 2009. This diversity has important implications to the control of this disease by the swine industry, as well as a potential risk for public health if swine-adapted viruses with H1N1pdm09 genes have an increased risk to humans, as occurred in the 2011-12 and 2016 human variant H3N2v cases associated with exhibition swine.

    IMPORTANCE People continue to spread the 2009 H1N1 pandemic (H1N1pdm09) influenza A virus (IAV) to pigs, allowing H1N1pdm09 to reassort with endemic swine IAV. In this study, we determined the 8 gene combinations of swine H3N2 IAV detected from 2009-2016. We identified 44 different genotypes of H3N2, the majority of which contained at least one H1N1pdm09 gene segment. We compared six of the representative genotypes of H3N2 in pigs. All six genotypes efficiently infected pigs, but they resulted in different degrees of lung damage and viral shedding. These results highlight the vast genetic diversity of H3N2 circulating in U.S. swine after 2009 with important implications to the control of IAV for the swine industry. Because the H1N1pdm09 is also highly adapted to humans, these swine viruses may have a potential risk to public health if swine-adapted viruses with H1N1pdm09 genes also have an increased risk for human infection.

  • Influence of Plasma Cell Niche Factors on the Recruitment and Maintenance of IRF4hi Plasma Cells and Plasmablasts in Vaccinated, SIV-Infected Rhesus Macaques with Low and High Viremia [PublishAheadOfPrint]

  • In a recent study, we found protection following SIV exposure correlated with rectal plasma cell frequency in vaccinated female rhesus macaques. We sought to determine if these same macaques maintained high mucosal plasma cell frequencies post-infection and if this translated to reduced viremia. Although delayed SIV acquisition did not predict subsequent viral control, alterations existed in the distribution of plasma cells and plasmablasts between macaques that exhibited high or low viremia. Flow cytometric analysis of cells from rectal biopsies, bone marrow, and mesenteric lymph nodes of vaccinated infected, unvaccinated infected, and uninfected macaques identified two main IRF4hi subsets of interest: CD138+ plasma cells, and CD138- plasmablasts. In rectal tissue, plasma cell frequency positively correlated with plasma viremia and unvaccinated macaques had increased plasma cells and plasmablasts compared to vaccinated animals. Likewise, plasmablast frequency in the mesenteric lymph node correlated with viremia. However in bone marrow, plasmablast frequency negatively correlated with viremia. Accordingly, low viremic macaques had a higher frequency of both bone marrow IRF4hi subsets compared to animals with high viremia. Significant reciprocal relationships between rectal and bone marrow plasmablasts suggested efficient trafficking to the bone marrow as opposed to the rectal mucosa was linked to viral control. mRNA expression analysis of proteins involved in establishment of plasma cell niches in sorted bone marrow and rectal cell populations further supported this model and offered a window into differential mRNA expression patterns in these tissues.

    IMPORTANCE As key antibody producers, plasma cells and plasmablasts are critical components of vaccine-induced immunity to HIV-1 in humans and SIV in the macaque model; however few have attempted to examine the role of these cells in viral suppression post-infection. Our results suggest plasmablast trafficking to and retention in the bone marrow plays a previously unappreciated role in viral control and contrasts the potential contribution of mucosal plasma cells to mediate protection at sites of infection with that of bone marrow plasmablasts and plasma cells to viral control during chronic infection. Manipulation of niche factors influencing the distribution and maintenance of these critical antibody-secreting cells may serve as potential therapeutic targets to enhance anti-viral responses post-vaccination and post-infection.

  • Expression of the Antisense-to-Latency Transcript Long Noncoding RNA in Kaposi's Sarcoma-Associated Herpesvirus [PublishAheadOfPrint]

  • The regulation of latency is central to herpesvirus biology. Recent transcriptome-wide surveys have uncovered evidence for promiscuous transcription across the entirety of the Kaposi sarcoma-associated herpesvirus genome and postulated the existence of multiple viral long noncoding RNAs (lncRNAs). NextGen sequencing studies are highly dependent on the specific experimental approach and particular algorithms of analysis, and therefore benefit from independent confirmation of the results. The antisense to latency transcripts (ALT) lncRNA was discovered by genome-tiling microarray (Chandriani et al. J. Virol. 16: 7934-7942, 2010, doi: 10.1128/JVI.00645-10). To characterize ALT in detail, we physically isolated this lncRNA by a strand-specific hybrid-capture assay and then employed RNA-seq and novel RT-PCR assays to distinguish all RNA species in the KSHV latency region. These methods confirm that ALT initiates at 120,739/121,012 and encodes a single splice site, which is shared with the 3rrsquo; -coterminal K14-vGPCR/ORF74, terminating at 130,873 (GQ994935), resulting in a ~10,000-nt transcript. No shorter ALT isoforms were identified. This study also identified a novel intron within the LANA 5rrsquo; UTR using a splice acceptor at 127,888. In sum, ALT joins PAN/nut1/T1.1 as a bona fide lncRNA of KSHV with potentially important roles in viral gene regulation and pathogenesis.

    IMPORTANCE Increasing data support the importance of noncoding RNAs (ncRNAs), including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), which have been shown to exert critical regulatory functions without coding for recognizable proteins. Defining the sequences of these ncRNAs is essential for future studies aiming to functionally characterize a specific ncRNA. Most lncRNA studies are highly dependent on high-throughput sequencing and bioinformatic analyses, few studies follow up on the initial predictions, and analyses are at times discordant. This manuscript characterizes one key viral lncRNA, ALT, by physically isolating ALT and by a sequencing-independent assay. It provides for a simple assay to follow lncRNA expression in experimental and clinical samples. ALT is expressed antisense to the major viral latency transcripts encoding LANA as well as the viral microRNAs, and thus has the potential to regulate this key part of the viral life cycle.

  • Structural Insights into Reovirus {sigma}1 Interactions with Two Neutralizing Antibodies [PublishAheadOfPrint]

  • Reovirus attachment protein 1 engages glycan receptors and junctional adhesion molecule-A (JAM-A) and is thought to undergo a conformational change during the proteolytic disassembly of virions to infectious subvirion particles (ISVPs) that accompanies cell entry. The 1 protein is also the primary target of neutralizing antibodies. Here we present a structural and functional characterization of two neutralizing antibodies that target 1 of serotypes 1 (T1) and 3 (T3) reoviruses. Crystal structures reveal that each antibody engages its cognate 1 protein within the head domain via epitopes distinct from the JAM-A-binding site. Surface-plasmon resonance and cell-binding assays indicate that both antibodies likely interfere with JAM-A engagement by steric hindrance. To define the interplay between carbohydrate receptor and antibody binding, we conducted hemagglutination inhibition assays using virions and ISVPs. The glycan-binding site of T1 1 is located in the head domain and is partly occluded by the bound Fab in the crystal structure. The T1-specific antibody inhibited hemagglutination by virions and ISVPs, probably via direct interference with glycan engagement. In contrast to T1 1, the carbohydrate-binding site of T3 1 is located in the tail domain, distal to the antibody epitope. The T3-specific antibody inhibited hemagglutination by T3 virions but not ISVPs, indicating that the antibody- and glycan-binding sites in 1 are in closer spatial proximity on virions than on ISVPs. Our results provide direct evidence for a structural rearrangement of 1 during virion-to-ISVP conversion and contribute new information about mechanisms of antibody-mediated neutralization of reovirus.

    IMPORTANCE Virus attachment proteins mediate binding to host cell receptors, serve critical functions in cell and tissue tropism, and are often targeted by the neutralizing antibody response. The structural investigation of antibody-antigen complexes can provide valuable information for understanding the molecular basis of virus neutralization. Studies with enveloped viruses such as HIV and influenza virus have helped to define sites of vulnerability and guide vaccination strategies. By comparison, less is known about antibody binding to nonenveloped viruses. Here, we structurally investigated two neutralizing antibodies that bind the attachment protein 1 of reovirus. We have furthermore characterized the neutralization efficiency, the binding affinity for 1, and the effect of the antibodies on reovirus receptor engagement. Our analysis defines reovirus interactions with two neutralizing antibodies, allows us to propose a mechanism by which they block virus infection, and provides evidence for a conformational change in the 1 protein during viral cell entry.

  • Increasing type-1 poliovirus capsid stability by thermal selection [PublishAheadOfPrint]

  • Poliomyelitis is a highly infectious disease caused by poliovirus (PV). It can result in paralysis and may be fatal. Integrated global immunisation programmes using live-attenuated oral (OPV) and/or inactivated PV vaccines (IPV) have systematically reduced its spread and paved the way for eradication. Immunisation will continue post-eradication to ensure against reintroduction of the disease, but there are biosafety concerns for both OPV and IPV. These could be addressed by the production and use of virus-free virus-like particle (VLP) vaccines which mimic the llsquo;empty' capsids (ECs) normally produced in viral infection. Although ECs are antigenically indistinguishable from mature virus particles, they are less stable and readily convert to an alternative conformation unsuitable for vaccine purposes. Stabilised ECs, expressed recombinantly as VLPs, could be ideal candidate vaccines for a polio-free world. However, although genome-free PV ECs have been expressed as VLPs in a variety of systems, their inherent antigenic instability has proved a barrier to further development. In this study, we have selected thermally-stable ECs of type-1 PV (PV-1). The ECs are antigenically stable at temperatures above the conversion temperature of wild type (wt) virion. We have identified mutations on the capsid surface and internal networks that are responsible for the EC stability. With reference to the capsid structure, we speculate on the roles of these residues in capsid stability and postulate that such stabilised VLPs could be used as novel vaccines.

    Importance Poliomyelitis is a highly infectious disease caused by PV and is on the verge of eradication. There are biosafety concerns of reintroduction of the disease from current vaccines which require live virus for production. Recombinantly-expressed virus-like particles could address these inherent problems. However, the genome-free capsids (ECs) of wt PV are unstable and readily change antigenicity to a form not suitable as a vaccine. Here, we demonstrate that the ECs of type-1 PV can be stabilised by selecting heat-resistant viruses. Our data show that some capsid mutations stabilise the ECs and could be applied as candidates to synthesise stable virus-like particles (VLPs) as future genome-free poliovirus vaccines.

  • Emergence of a viral RNA polymerase variant during gene copy number amplification promotes rapid evolution of vaccinia virus [PublishAheadOfPrint]

  • Viruses are under relentless selective pressure from host immune defenses. To study how poxviruses adapt to innate immune detection pathways, we performed serial infections of vaccinia virus in primary human cells. Independent courses of experimental evolution with a recombinant strain lacking E3L revealed several high frequency point mutations in conserved poxvirus genes, suggesting important roles for essential poxvirus proteins in innate immune subversion. Two distinct mutations were identified in the viral RNA polymerase gene A24R, which seem to act through different mechanisms to increase virus replication. Specifically, a Leu18Phe substitution in A24R conferred fitness tradeoffs, including increased activation of the antiviral factor Protein kinase R (PKR). Intriguingly, this A24R variant underwent a drastic selective sweep during passaging, despite enhanced PKR activity. We show that the sweep of this variant can be accelerated by the presence of copy number variation (CNV) at the K3L locus, which with multiple copies strongly reduces PKR activation. Therefore, adaptive cases of CNV can also facilitate the accumulation of point mutations separate from the expanded locus. This study reveals how rapid bouts of gene copy number amplification during accrual of distant point mutations can potently facilitate poxvirus adaptation to host defenses.

    IMPORTANCE Viruses can quickly evolve to defeat host immune functions. For poxviruses, little is known about how multiple adaptive mutations emerge in populations at the same time. In this study, we uncovered a means of vaccinia virus adaptation involving the accumulation of distinct genetic variants within a single population. We identified adaptive point mutations in the viral RNA polymerase gene A24R, and surprisingly, found that one of these mutations activates the nucleic acid sensing factor PKR. We also found that gene copy number variation (CNV) can provide dual benefits to evolving virus populations, including evidence that CNV facilitates the accumulation of a point mutation distant from the expanded locus. Our data suggest that transient CNV can accelerate the fixation of mutations conferring modest benefits, or even fitness tradeoffs, and highlight how structural variation might aid poxvirus adaptation through both direct and indirect action.

  • TLR2 ligation enhances HIV-1 replication in activated CCR6+CD4+ T cells by increasing virus entry and establishing a more permissive environment to infection [PublishAheadOfPrint]

  • In this study, we investigated the effect of TLR2 ligation on the permissiveness of activated CD4+ T cells to HIV-1 infection by focusing our experiments on the relative susceptibility of cell subsets based on their expression of CCR6. Purified primary human CD4+ T cells were first subjected to a CD3/CD28 costimulation before treatment with the TLR2 agonist Pam3CSK4. Finally, cells were inoculated with R5-tropic HIV-1 particles that permit to study the effect of TLR2 triggering on virus production at both population and single-cell levels. We report here that HIV-1 replication is augmented in CD3/CD28-costimulated CCR6+CD4+ T cells upon engagement of the cell surface TLR2. Additional studies indicate that a higher virus entry and polymerization of the cortical actin are seen in this cell subset following TLR2 stimulation. A TLR2-mediated increase in the level of phosphorylated NF-B p65 subunit was also detected in CD3/CD28-costimulated CCR6+CD4+ T cells. We propose that, upon antigenic presentation, an engagement of TLR2 acts specifically on CCR6+CD4+ T cells by promoting virus entry in an intracellular milieu more favourable for productive HIV-1 infection.

    IMPORTANCE Following primary infection, HIV-1 induces an immunological and structural disruption of the gut mucosa, leading to bacterial translocation and release of microbial components in the bloodstream. These pathogen-derived constituents include several agonists of Toll-like receptors that may affect gut-homing CD4+ T cells, such as those expressing the chemokine receptor CCR6, which are highly permissive to HIV-1 infection. We demonstrate that TLR2 ligation in CD3/CD28-costimulated CCR6+CD4+ T cells leads to an enhanced virus production. Our results highlight the potential impact of bacterial translocation on the overall permissiveness of CCR6+CD4+ T cells to productive HIV-1 infection.

  • Inhibition of an aquatic rhabdovirus demonstrates promise of a broad-spectrum antiviral for use in aquaculture [PublishAheadOfPrint]

  • Many enveloped viruses cause devastating disease in aquaculture, resulting in significant economic impact. LJ001 is a broad-spectrum antiviral compound that inhibits enveloped viral infections by specifically targeting phospholipids in the lipid bilayer via production of singlet oxygen (1O2). This stabilizes positive curvature and decreases membrane fluidity, which inhibits viral-cell membrane fusion during viral entry. Based on previous mammalian studies and the requirement of light for activation of LJ001, we hypothesized that LJ001 may be useful as a preventative and/or therapeutic agent for enveloped viral infections in aquaculture. Here, we report that LJ001 was more stable with a prolonged inhibitory half-life at relevant aquaculture temperatures (15ddeg;C), as compared to mammalian studies at 37ddeg;C. When LJ001 was pre-incubated with our model virus, infectious hematopoietic necrosis virus (IHNV), infectivity was significantly inhibited in vitro (using the EPC fish cell line) and in vivo (using rainbow trout fry) in a dose-dependent and time-dependent manner. While horizontal transmission of IHNV in a static co-habitation challenge model was reduced by LJ001, transmission was not completely blocked at established antiviral doses. Therefore, LJ001 may be best suited as a therapeutic for aquaculture settings that include viral infections with lower viral shedding rates than IHNV, or where higher viral titers are required to initiate infection of naïve fish. Importantly, our data also suggests that LJ001-inactivated IHNV elicited an innate immune response in the rainbow trout host, making LJ001 potentially useful for future vaccination approaches.

    IMPORTANCE Viral diseases in aquaculture are challenging because there are few preventative measures and/or treatments. Broad-spectrum antivirals are highly sought after and studied because they target common components of viruses. In our studies, we used LJ001, a broad-spectrum antiviral compound that specifically inhibits enveloped viruses. We used the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV) as a model to study aquatic enveloped virus diseases and their inhibition. We demonstrated inhibition of IHNV by LJ001 both in cell culture as well as in live fish. Additionally, we showed that LJ001 inhibited transmission of IHNV from infected fish to healthy fish, which lays the groundwork for using LJ001 as a possible therapeutic for aquatic viruses. Our results also suggest that virus inactivated by LJ001 induces an immune response, showing potential for future preventative (e.g. vaccine) applications.

  • A protective role for IL-1 signaling during mouse adenovirus type 1-induced encephalitis [PublishAheadOfPrint]

  • IL-1bbeta;, an inflammatory cytokine and IL-1 receptor ligand, has diverse activities in the brain. We examined whether IL-1 signaling contributes to the encephalitis observed in mouse adenovirus type 1 (MAV-1) infection, using mice lacking the IL-1 receptor, Il1r1-/-. Il1r1-/- mice had lower survival, higher disruption of the blood-brain barrier (BBB), higher brain viral loads, and higher brain inflammatory cytokines and chemokines than control C57BL/6 mice. We also examined infections of mice defective in IL-1bbeta; production (Pycard-/-mice) and mice defective in trafficking of TLRs to the endosome (Unc93b1-/- mice). Pycard-/- and Unc93b1-/- mice had lower survival (similar to Il1r1-/- mice) than control mice, but unlike Il1r1-/- mice, did not have increased brain viral loads or BBB disruption. Based on brain cytokine levels, MAV-1-infected Unc93b1-/- mice had a very different inflammatory profile from infected Il1r1-/- and Pycard-/- mice. Histological examination demonstrated pathological findings consistent with encephalitis in control and knockout mice; however, intranuclear viral inclusions were seen only in Il1r1-/- mice. A time course of infection of control and Il1r1-/- mice evaluating the kinetics of viral replication and cytokine production revealed differences between the mouse strains primarily at 7 - 8 days after infection, when mice began succumbing to MAV-1 infection. In the absence of IL-1 signaling, we noted an increase in the transcription of type I interferon (IFN)-stimulated genes. Together these results indicate that IL-1 signaling is important during MAV-1 infection and suggest that in its absence, increased IFN-bbeta; signaling may result in increased neuroinflammation.

    IMPORTANCE The investigation of encephalitis pathogenesis produced by different viruses is needed to characterize virus and host-specific factors that contribute to disease. MAV-1 produces viral encephalitis in its natural host, providing a good model for studying factors involved in encephalitis development. We investigated the role of IL-1 signaling during MAV-1-induced encephalitis. Unexpectedly, the lack of IL-1 signaling increased the mortality and inflammation in mice infected with MAV-1. Also there was an increase in the transcription of type I IFN-stimulated genes that correlated with the observed increased mortality and inflammation. The findings highlight the complex nature of encephalitis and suggests that IL-1 has a protective effect for the development of MAV-1-induced encephalitis.

  • Accumulation of Pol mutations selected by HLA-B*52:01-C*12:02 protective haplotype-restricted CTLs causes low plasma viral load due to low viral fitness of mutant viruses [PublishAheadOfPrint]

  • HLA-B*52:01-C*12:02, which is the most abundant haplotype in Japan, has a protective effect on disease progression in HIV-1-infected Japanese individuals, whereas HLA-B*57 and -B*27 protective alleles are very rare in Japan. A previous study on HLA-associated polymorphisms demonstrated that the number of HLA-B*52:01-associated mutations at four Pol positions was inversely correlated with plasma viral load (pVL) in HLA-B*52:01-negative individuals, suggesting that the transmission of HIV-1 with these mutations could modulate the pVL in the population. However, it remains unknown whether these mutations were selected by HLA-B*52:01-restricted CTLs and also reduced viral fitness. In this study, we identified two HLA-B*52:01-restricted and one HLA-C*12:02-restricted novel CTL epitopes in Pol. Analysis using CTLs specific for these three epitopes demonstrated that these CTLs failed to recognize mutant epitopes or more weakly recognized cells infected with mutant viruses than wild-type virus, supporting the idea that these mutations were selected by the HLA-B*52:01- or HLA-C*12:02-restricted T cells. We further showed that these mutations reduced viral fitness, although the effect of each mutation was weak. The present study demonstrated that the accumulation of these Pol mutations selected by HLA-B*52:01- or HLA-C*12:02-restricted CTLs impaired viral replication capacity and, thus, reduced the pVL. The fitness cost imposed by the mutations partially accounted for the effect of the HLA-B*52:01-C*12:02 haplotype on clinical outcome, together with the effect of HLA-B*52:01-restricted CTLs on viral replication, which had been previously demonstrated.

    IMPORTANCE Numerous population-based studies identified HLA-associated HIV-1 mutations to predict HIV-1 escape mutations from cytotoxic T lymphocytes (CTLs). However, the majority of these HLA-associated mutations have not been identified as CTL escape mutations. Our previous population-based study showed that five HLA-B*52:01-associated mutations at four Pol positions were inversely correlated with the plasma viral load in HLA-B*52:01-negative Japanese individuals. In the present study, we demonstrated that these mutations were indeed selected by CTLs specific for novel B*52:01- and C*12:02-restricted epitopes and that the accumulation of these mutations reduced the viral fitness in vitro. This study elucidated the mechanism by which the accumulation of these CTL escape mutations contributed to the protective effect of the HLA-B*52:01-HLA-C*12:02 haplotype on disease progression in HIV-1-infected Japanese individuals.

  • The human cytomegalovirus IE1 protein antagonizes PML nuclear body mediated intrinsic immunity via the inhibition of PML de novo SUMOylation [PublishAheadOfPrint]

  • PML nuclear bodies (NBs) are accumulations of cellular proteins embedded in a scaffold-like structure built by SUMO-modified PML/TRIM19. PML and other NB proteins act as cellular restriction factors against human cytomegalovirus (HCMV), however, this intrinsic defense is counteracted by the immediate-early protein 1 (IE1) of HCMV. IE1 directly interacts with the PML coiled-coil domain via its globular core region and disrupts NB foci by inducing a loss of PML SUMOylation. Here, we demonstrate that IE1 acts via abrogating the de novo SUMOylation of PML. In order to overcome reversible SUMOylation dynamics, we made use of a cell-based assay that combines inducible IE1 expression with a SUMO mutant resistant to SUMO proteases. Interestingly, we observed that IE1 expression did not affect preSUMOylated PML, however, it clearly prevented de novo SUMO conjugation. Consistent results were obtained by in vitro SUMOylation assays demonstrating that IE1 alone is sufficient for this effect. Furthermore, IE1 acts in a selective manner since K160 was identified as the main target lysine. This is strengthened by the fact that IE1 also prevents As2O3-mediated hyperSUMOylation of K160 thereby blocking PML degradation. Since IE1 did not interfere with coiled-coil mediated PML dimerization we propose that IE1 either affects PML autoSUMOylation by directly abrogating PML E3 ligase function or by preventing the access to SUMO sites. Thus, our data suggest a novel mechanism how a viral protein counteracts a cellular restriction factor by selectively preventing the de novo SUMOylation at specific lysine residues without affecting global protein SUMOylation.

    IMPORTANCE The human cytomegalovirus IE1 protein acts as an important antagonist of a cellular restriction mechanism that is mediated by a subnuclear structure termed PML nuclear bodies. This function of IE1 is required for efficient viral replication and thus constitutes a potential target for antiviral strategies. In this paper, we further elucidate the molecular mechanism how IE1 antagonizes PML-NBs. We show that tight binding of IE1 to PML interferes with the de novo SUMOylation of a distinct lysine residue that is also the target of stress-mediated hyperSUMOylation of PML. This is of importance since it represents a novel mechanism used by a viral antagonist of intrinsic immunity. Furthermore, it highlights the possibility to develop small molecules that specifically abrogate this PML-antagonistic activity of IE1 and thus inhibit viral replication.

  • CXCR6-mediated SIVagmSab Entry into Sabaeus African Green Monkey Lymphocytes Implicates Widespread Use of Non-CCR5 Pathways in Natural Host Infections [PublishAheadOfPrint]

  • African green monkeys (AGM) and sooty mangabeys (SM) are well-studied natural hosts of SIV, which do not progress to AIDS when infected with their species-specific viruses. SIV natural hosts express very low levels of the canonical entry coreceptor CCR5, and recent studies have shown that CCR5 is dispensable for SIV infection of SM in vivo, and blocking CCR5 does not prevent ex vivo infection of PBMC from SM or vervet AGM. In both hosts, CXCR6 is an efficient entry pathway in vitro. Here we investigated use of species-matched CXCR6 and other alternative coreceptors by SIVagmSab, which infects sabaeus AGM. We cloned sabaeus CD4 and ten candidate coreceptors. Species-matched CXCR6, CCR5 and GPR15 mediated robust entry into transfected cells by pseudotypes carrying the SIVagmSab92018ivTF Env, with lower level entry through GPR1 and APJ. We cloned genetically divergent envs from plasma of two wild-infected sabaeus and found similar coreceptor use patterns. Titration experiments showed that CXCR6 and CCR5 were more efficient than other coreceptors when tested at limiting CD4/coreceptor levels. Finally, blocking CXCR6 with its ligand CXCL16 significantly inhibited SIVagmSab replication in sabaeus PBMC and had greater impact than the CCR5 blocker maraviroc, confirming CXCR6 use in primary lymphocyte infection. These data suggest a new paradigm for SIV infection of natural host species, whereby a shared outcome of virus/host coevolution is use of CXCR6 or other alternative coreceptors for entry, which may direct SIV toward CD4+ T cell subsets and anatomical sites that support viral replication without disrupting immune homeostasis and function.

    Importance Natural hosts of SIV do not progress to AIDS, in stark contrast to pathogenic HIV-1/human and SIVmac/macaque infections. Identifying how natural hosts avoid immunodeficiency can elucidate key mechanisms of pathogenesis. It is known that despite high viral loads, natural hosts have a low frequency of CD4+ cells expressing the SIV coreceptor CCR5. In this study, we demonstrate efficient use of the coreceptor CXCR6 by SIVagmSab to infect sabaeus African green monkey lymphocytes. In conjunction with studies of SIVsmm that infects sooty mangabeys and SIVagmVer that infects vervet monkeys, our data suggest a unifying model whereby in natural hosts, in which CCR5 expression is low, use of CXCR6 or other coreceptors to mediate infection may target SIV towards distinct cell populations that are able to support high viral replication without causing loss of CD4+ T cell homeostasis and lymphoid tissue damage that lead to AIDS in HIV-1 and SIVmac infection.

  • Genetic Variability of Myxoma Virus Genomes [PublishAheadOfPrint]

  • Myxomatosis is a recurrent problem on rabbit farms throughout Europe despite the success of vaccines. To identify gene variations of field and vaccine strains that may be responsible for changes in virulence, immunomodulation, and immunoprotection, the genomes of 6 Myxoma virus strains (MYXV) were sequenced: German field isolates Munich-1, FLI-H, 2604, 3207, vaccine strain MAV, and challenge strain ZA. The analyzed genomes ranged from 147.6 kb (strain MAV) to 161.8 kb (strain 3207) in size. All sequences were affected by several mutations, covering 24-93 open reading frames (ORFs) and resulted in amino acid (aa) substitutions, insertions, or deletions. Only strains Munich-1 and MAV revealed the deletion of 10 (M007L-M015L) and 11 (M007L-M008.1L, M149R-M008.1R) ORFs, respectively. Major differences were observed in the 27 immunomodulatory proteins encoded by MYXV. Compared to the reference strain Lausanne, strains FLI-H, 2604, 3207, and ZA showed the highest aa identity (ggt;98.4%). In strains Munich-1 and MAV the deletion of 5 and 10 ORFs, respectively, was observed, encoding immunomodulatory proteins with ankyrin repeats or members of the family of serine protease inhibitors. Furthermore, putative immunodominant surface proteins with homology to vaccinia virus (VACV) were investigated in the sequenced strains. Only strain MAV revealed aa substitutions and frameshift mutations above average. Finally, we performed recombination analysis and found signs of recombination in vaccine strain MAV. Phylogenetic analysis showed a close relationship of strain MAV and the MSW strain of Californian MYXV. However, in a challenge model strain MAV provided full protection against lethal challenges with strain ZA.

    Importance Myxoma virus (MYXV) is pathogenic for European rabbits and two North American species. Due to sophisticated strategies in immune evasion and oncolysis, MYXV is an important model virus for immunological and pathological research. In its natural hosts MYXV causes a benign infection, whereas in European rabbits it causes the lethal disease myxomatosis. Since introduction of MYXV into Australia and Europe for biological control of European rabbits in the 1950s, a co-evolution of host and pathogen has started, selecting for attenuated virus strains and increased resistance in rabbits. Evolution of viruses is a continuous process and influences the protective potential of vaccines. In our analyses, we sequenced 6 MYXV field, challenge, and vaccine strains. We focused on genes encoding proteins involved in virulence, host-range, immunomodulation, and envelope composition. Genes affected most by mutations play a role in immunomodulation. However, attenuation cannot be linked to individual mutations or gene disruptions.

  • Structural protein VP2 of African horse sickness virus is not essential for virus replication in vitro [PublishAheadOfPrint]

  • The Reoviridae family consists of non-enveloped multi-layered viruses with a double stranded RNA genome consisting of 9 to 12 genome segments. The Orbivirus genus of the Reoviridae family contains African horse sickness virus (AHSV), bluetongue virus and epizootic haemorrhagic disease virus causing notifiable diseases and are spread by biting Culicoides species. Here, we used reverse genetics for AHSV to study the role of outer capsid protein VP2 encoded by genome segment 2 (Seg-2). Expansion of a previously found deletion in Seg-2 indicates that structural protein VP2 of AHSV is not essential for virus replication in vitro. In addition, in-frame replacement of RNA sequences in Seg-2 for that of green fluorescence protein (GFP) resulted in AHSV expressing GFP which further confirmed that VP2 is not essential for virus replication. In contrast to virus replication without VP2 expression in mammalian cells, virus replication in insect cells was strongly reduced and virus release from insect cells was completely abolished. Further, the other outer capsid protein VP5 was not co-purified with virions for virus mutants without VP2 expression. AHSV without VP5 expression however could not be recovered, indicating that outer capsid protein VP5 is essential for virus replication in vitro. Our results demonstrate for the first time that a structural viral protein is not essential for orbivirus replication in vitro, which opens new possibilities for research on other members of the Reoviridae family.

    Importance Members of the Reoviridae family cause major health problems worldwide ranging from lethal diarrhoea by rotavirus in humans to economic losses in livestock production by different orbiviruses. The orbivirus genus contains many virus species of which bluetongue virus, epizootic haemorrhagic disease virus, and African horse sickness virus (AHSV) are causing notifiable diseases according to the World Organisation of Animal Health. Recently, it has been shown that non-structural proteins NS3/NS3a and NS4 are not essential for virus replication in vitro, whereas it is generally assumed that structural proteins VP1-7 of these non-enveloped architectural complex virus particles are essential. Here we demonstrate for the first time that structural protein VP2 of AHSV is not essential for virus replication in vitro. Our findings are very important for virologists working in the field of non-enveloped viruses, in particular reoviruses.

  • LUBAC modulates LMP1 activation of NF{kappa}B and IRF7 [PublishAheadOfPrint]

  • Recently, Linear Ubiquitin Assembly Complex (LUBAC)-mediated linear ubiquitination has come into focus due to its emerging role in activation of NFB in different biological contexts. However, the role of LUBAC in LMP1 signaling leading to NFB and IRF7 activation has not been investigated. We have shown here that RNF31, the key component of LUBAC complex, interacts with LMP1 and IRF7 in EBV-transformed cells, and that LUBAC stimualtes linear ubiquitination of NEMO and IRF7. Consequently, LUBAC is required for LMP1 signaling to full activation of NFB, but inhibits LMP1-stimulated IRF7 transcriptional activity. The protein levels of RNF31 and LMP1 correlate in EBV-transformed cells. Knockdown of RNF31 in EBV-transformed IB4 cells by RNA interference negatively regulates expression of the genes downstream of LMP1 signaling, and results in a decrease of cell proliferation. These lines of evidence indicate that LUBAC-mediated linear ubiquitination play crucial roles in regulating LMP1 signaling and functions.

    Importance We have shown here that LUBAC-mediated linear ubiquitination is required for LMP1 activation of NFB, but inhibits LMP1-mediated IRF7 activation. Our findings provide novel mechanisms underlying EBV-mediated oncogenesis, and may have broad impact on IRF7-mediated immune responses.

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