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Jean-Yves Sgro
Inst. for Mol.Virology
731B Bock Labs
1525 Linden Drive Madison, WI 53706

Table of Contents for this page:

  • Current Issue
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  • Current Issue of Journal of Virology

    JVI Current Issue

  • Editorial Board [Masthead]

  • Citrus Psorosis Virus Movement Protein Contains an Aspartic Protease Required for Autocleavage and the Formation of Tubule-Like Structures at Plasmodesmata [Virus-Cell Interactions]

  • Plant virus cell-to-cell movement is an essential step in viral infections. This process is facilitated by specific virus-encoded movement proteins (MPs), which manipulate the cell wall channels between neighboring cells known as plasmodesmata (PD). Citrus psorosis virus (CPsV) infection in sweet orange involves the formation of tubule-like structures within PD, suggesting that CPsV belongs to "tubule-forming" viruses that encode MPs able to assemble a hollow tubule extending between cells to allow virus movement. Consistent with this hypothesis, we show that the MP of CPsV (MPCPsV) indeed forms tubule-like structures at PD upon transient expression in Nicotiana benthamiana leaves. Tubule formation by MPCPsV depends on its cleavage capacity, mediated by a specific aspartic protease motif present in its primary sequence. A single amino acid mutation in this motif abolishes MPCPsV cleavage, alters the subcellular localization of the protein, and negatively affects its activity in facilitating virus movement. The amino-terminal 34-kDa cleavage product (34KCPsV), but not the 20-kDa fragment (20KCPsV), supports virus movement. Moreover, similar to tubule-forming MPs of other viruses, MPCPsV (and also the 34KCPsV cleavage product) can homooligomerize, interact with PD-located protein 1 (PDLP1), and assemble tubule-like structures at PD by a mechanism dependent on the secretory pathway. 20KCPsV retains the protease activity and is able to cleave a cleavage-deficient MPCPsV in trans. Altogether, these results demonstrate that CPsV movement depends on the autolytic cleavage of MPCPsV by an aspartic protease activity, which removes the 20KCPsV protease and thereby releases the 34KCPsV protein for PDLP1-dependent tubule formation at PD.

    IMPORTANCE Infection by citrus psorosis virus (CPsV) involves a self-cleaving aspartic protease activity within the viral movement protein (MP), which results in the production of two peptides, termed 34KCPsV and 20KCPsV, that carry the MP and viral protease activities, respectively. The underlying protease motif within the MP is also found in the MPs of other members of the Aspiviridae family, suggesting that protease-mediated protein processing represents a conserved mechanism of protein expression in this virus family. The results also demonstrate that CPsV and potentially other ophioviruses move by a tubule-guided mechanism. Although several viruses from different genera were shown to use this mechanism for cell-to-cell movement, our results also demonstrate that this mechanism is controlled by posttranslational protein cleavage. Moreover, given that tubule formation and virus movement could be inhibited by a mutation in the protease motif, targeting the protease activity for inactivation could represent an important approach for ophiovirus control.

  • Phosphorylated VP30 of Marburg Virus Is a Repressor of Transcription [Genome Replication and Regulation of Viral Gene Expression]

  • The filoviruses Marburg virus (MARV) and Ebola virus (EBOV) cause hemorrhagic fever in humans and nonhuman primates, with high case fatality rates. MARV VP30 is known to be phosphorylated and to interact with nucleoprotein (NP), but its role in regulation of viral transcription is disputed. Here, we analyzed phosphorylation of VP30 by mass spectrometry, which resulted in identification of multiple phosphorylated amino acids. Modeling the full-length three-dimensional structure of VP30 and mapping the identified phosphorylation sites showed that all sites lie in disordered regions, mostly in the N-terminal domain of the protein. Minigenome analysis of the identified phosphorylation sites demonstrated that phosphorylation of a cluster of amino acids at positions 46 through 53 inhibits transcription. To test the effect of VP30 phosphorylation on its interaction with other MARV proteins, coimmunoprecipitation analyses were performed. They demonstrated the involvement of VP30 phosphorylation in interaction with two other proteins of the MARV ribonucleoprotein complex, NP and VP35. To identify the role of protein phosphatase 1 (PP1) in the identified effects, a small molecule, 1E7-03, targeting a noncatalytic site of the enzyme that previously was shown to increase EBOV VP30 phosphorylation was used. Treatment of cells with 1E7-03 increased phosphorylation of VP30 at a cluster of phosphorylated amino acids from Ser-46 to Thr-53, reduced transcription of MARV minigenome, enhanced binding to NP and VP35, and dramatically reduced replication of infectious MARV particles. Thus, MARV VP30 phosphorylation can be targeted for development of future antivirals such as PP1-targeting compounds.

    IMPORTANCE The largest outbreak of MARV occurred in Angola in 2004 to 2005 and had a 90% case fatality rate. There are no approved treatments available for MARV. Development of antivirals as therapeutics requires a fundamental understanding of the viral life cycle. Because of the close similarity of MARV to another member of Filoviridae family, EBOV, it was assumed that the two viruses have similar mechanisms of regulation of transcription and replication. Here, characterization of the role of VP30 and its phosphorylation sites in transcription of the MARV genome demonstrated differences from those of EBOV. The identified phosphorylation sites appeared to inhibit transcription and appeared to be involved in interaction with both NP and VP35 ribonucleoproteins. A small molecule targeting PP1 inhibited transcription of the MARV genome, effectively suppressing replication of the viral particles. These data demonstrate the possibility developing antivirals based on compounds targeting PP1.

  • The 5' Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation [Genome Replication and Regulation of Viral Gene Expression]

  • The capsid mRNA transcripts of human bocavirus 1 (HBoV1) can be generated by alternative splicing from the mRNA precursor transcribed from the P5 promoter. However, the alternative translation regulation mechanism of capsid mRNA transcripts is largely unknown. Here we report that the polycistronic capsid mRNA transcripts encode VP1, VP2, and VP3 in vitro and in vivo. The 5' untranslated regions (UTRs) of capsid mRNA transcripts, which consist of exons, affected not only the abundance of mRNA but also the translation pattern of capsid proteins. Further study showed that exons 2 and 3 were critical for the abundance of mRNA, while exon 4 regulated capsid translation. Alternative translation of capsid mRNA involved a leaky scan mechanism. Mutating the upstream ATGs (uATGs) located in exon 4 resulted in more mRNA transcripts polyadenylated at the proximal polyadenylation [(pA)p] site, leading to increased capsid mRNA transcripts. Moreover, uATG mutations induced more VP1 expression, while VP3 expression was decreased, which resulted in less progeny virus production. Our data show that the 5' UTR of HBoV1 plays a critical role in the modulation of mRNA abundance, alternative RNA processing, alternative translation, and progeny virus production.

    IMPORTANCE Alternative translation of HBoV1 capsid mRNAs is vital for the viral life cycle, as capsid proteins perform essential functions in genome packaging, assembly, and antigenicity. The 5' untranslated regions (UTRs) of capsid mRNAs are generated by alternative splicing, and they contain different exons. Our study shows that the 5' UTR not only modulates mRNA abundance but also regulates capsid expression. Two upstream ATGs (uATGs) that were upstream of the capsid translation initiation site in the 5' UTR were found to affect viral capsid mRNA polyadenylation, alternative translation, and progeny virus production. The results reveal that uATGs play an important role in the viral life cycle and represent a new layer to regulate HBoV1 RNA processing, which could be a target for gene therapy.

  • Cottontail Rabbit Papillomavirus E1 and E2 Proteins Mutually Influence Their Subcellular Localizations [Genome Replication and Regulation of Viral Gene Expression]

  • The papillomavirus (PV) E2 protein is a nuclear, sequence-specific DNA-binding protein that regulates transcription and nuclear retention of viral genomes. E2 also interacts with the viral E1 protein to replicate the viral genome. E2 residue K111 is highly conserved among PV and has been implicated in contributing to nuclear transport, transcription, and replication. Cottontail rabbit (Sylvilagus floridanus) PV (CRPV or SfPV1) E2 K111R, A, or Q mutations are transcription deficient and localized to the cytoplasm, comparable to other PV types. The addition of a nuclear localization signal (NLS) resulted in nuclear E2 K111 mutant proteins but did not restore transcriptional activation, and this is most likely due to an impaired binding to the cellular Brd4 protein. Surprisingly, coexpression of E1 with E2 K111 mutations resulted in their nuclear localization and, for K111A and R mutations, the activation of an E1/E2-dependent reporter construct. Interestingly, the nuclear localization of E2 K111Q mutant protein was independent from the presence of the conserved bipartite NLS in E1 and the direct interaction between E1 and E2. On the other hand, the cytoplasmic E1 NLS mutation could be targeted to the nucleus by wild-type E2, and this was dependent upon an interaction between E1 and E2. In summary, our studies have uncovered that E1 and E2 control each other's subcellular localization: direct binding of E2 to E1 can direct E1 to the nucleus independently from the E1 NLS, and E1 can direct E2 to the nucleus without an intact NLS or direct binding to E2.

    IMPORTANCE Papillomaviruses encode the DNA-binding E1 and E2 proteins, which form a complex and are essential for genome replication. Both proteins are targeted to the nucleus via nuclear localization signals. Our studies have uncovered that cytoplasmic mutant E1 or E2 proteins can be localized to the nucleus when E1 or E2 is also present. An interaction between E1 and E2 is necessary to target cytoplasmic E1 mutant proteins to the nucleus, but cytoplasmic E2 mutant proteins can be targeted to the nucleus without a direct interaction, which points to a novel function of E1.

  • Semen Exosomes Promote Transcriptional Silencing of HIV-1 by Disrupting NF-{kappa}B/Sp1/Tat Circuitry [Cellular Response to Infection]

  • Exosomes play various roles in host responses to cancer and infective agents, and semen exosomes (SE) inhibit HIV-1 infection and transmission, although the mechanism(s) by which this occurs is unclear. Here, we show that SE block HIV-1 proviral transcription at multiple transcriptional checkpoints, including transcription factor recruitment to the long terminal repeat (LTR), transcription initiation, and elongation. Biochemical and functional studies show that SE inhibit HIV-1 LTR-driven viral gene expression and virus replication. Through partitioning of the HIV-1 RNA, we found that SE reduced the optimal expression of various viral RNA species. Chromatin immunoprecipitationnndash;real-time quantitative PCR (ChIPnndash;RT-qPCR) and electrophoretic mobility shift assay (EMSA) analysis of infected cells identified the human transcription factors NF-B and Sp1, as well as RNA polymerase (Pol) II and the viral protein transcriptional activator (Tat), as targets of SE. Of interest, SE inhibited HIV-1 LTR activation mediated by HIV-1 or Tat, but not by the mitogen phorbol myristate acetate (PMA) or tumor necrosis factor alpha (TNF-aalpha;). SE inhibited the DNA binding activities of NF-B and Sp1 and blocked the recruitment of these transcription factors and Pol II to the HIV-1 LTR promoter. Importantly, SE directly blocked NF-B, Sp1, and Pol II binding to the LTR and inhibited the interactions of Tat/NF-B and Tat/Sp1, suggesting that SE-mediated inhibition of the functional quadripartite complex NF-Bnndash;Sp1nndash;Pol IInndash;Tat may be a novel mechanism of proviral transcription repression. These data provide a novel molecular basis for SE-mediated inhibition of HIV-1 and identify Tat as a potential target of SE.

    IMPORTANCE HIV is most commonly transmitted sexually, and semen is the primary vector. Despite progress in studies of HIV pathogenesis and the success of combination antiretroviral therapy in controlling viral replication, current therapy cannot completely control sexual transmission. Thus, there is a need to identify effective methods of controlling HIV replication and transmission. Recently, it was shown that human semen contains exosomes that protect against HIV infection in vitro. In this study, we identified a mechanism by which semen exosomes inhibited HIV-1 RNA expression. We found that semen exosomes inhibit recruitment of transcription factors NF-B and Sp1, as well as RNA Pol II, to the promoter region in the 5' long terminal repeat (LTR) of HIV-1. The HIV-1 early protein transcriptional activator (Tat) was a target of semen exosomes, and semen exosomes inhibited the binding and recruitment of Tat to the HIV-1 LTR.

  • In Situ Structures of the Polymerase Complex and RNA Genome Show How Aquareovirus Transcription Machineries Respond to Uncoating [Structure and Assembly]

  • Reoviruses carry out genomic RNA transcription within intact viruses to synthesize plus-sense RNA strands, which are capped prior to their release as mRNA. The in situ structures of the transcriptional enzyme complex (TEC) containing the RNA-dependent RNA polymerase (RdRp) and NTPase are known for the single-layered reovirus cytoplasmic polyhedrosis virus (CPV), but not for multilayered reoviruses, such as aquareoviruses (ARV), which possess a primed stage that CPV lacks. Consequently, how the RNA genome and TEC respond to priming in reoviruses is unknown. Here, we determined the near-atomic-resolution asymmetric structure of ARV in the primed state by cryo-electron microscopy (cryo-EM), revealing the in situ structures of 11 TECs inside each capsid and their interactions with the 11 surrounding double-stranded RNA (dsRNA) genome segments and with the 120 enclosing capsid shell protein (CSP) VP3 subunits. The RdRp VP2 and the NTPase VP4 associate with each other and with capsid vertices; both bind RNA in multiple locations, including a novel C-terminal domain of VP4. Structural comparison between the primed and quiescent states showed translocation of the dsRNA end from the NTPase to the RdRp during priming. The RNA template channel was open in both states, suggesting that channel blocking is not a regulating mechanism between these states in ARV. Instead, the NTPase C-terminal domain appears to regulate RNA translocation between the quiescent and primed states. Taking the data together, dsRNA viruses appear to have adapted divergent mechanisms to regulate genome transcription while retaining similar mechanisms to coassemble their genome segments, TEC, and capsid proteins into infectious virions.

    IMPORTANCE Viruses in the family Reoviridae are characterized by the ability to endogenously synthesize nascent RNA within the virus. However, the mechanisms for assembling their RNA genomes with transcriptional enzymes into a multilayered virion and for priming such a virion for transcription are poorly understood. By cryo-EM and novel asymmetric reconstruction, we determined the atomic structure of the transcription complex inside aquareoviruses (ARV) that are primed for infection. The transcription complex is anchored by the N-terminal segments of enclosing capsid proteins and contains an NTPase and a polymerase. The NTPase has a newly discovered domain that translocates the 5' end of plus-sense RNA in segmented dsRNA genomes from the NTPase to polymerase VP2 when the virus changes from the inactive (quiescent) to the primed state. Conformation changes in capsid proteins and transcriptional complexes suggest a mechanism for relaying information from the outside to the inside of the virus during priming.

  • Surfactin Inhibits Membrane Fusion during Invasion of Epithelial Cells by Enveloped Viruses [Vaccines and Antiviral Agents]

  • Because membrane fusion is a crucial step in the process by which enveloped viruses invade host cells, membrane fusion inhibitors can be effective drugs against enveloped viruses. We found that surfactin from Bacillus subtilis can suppress the proliferation of porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) in epithelial cells at a relatively low concentration range (15 to 50 mmu;g/ml), without cytotoxicity or viral membrane disruption. Membrane fusion inhibition experiments demonstrate that surfactin treatment significantly reduces the rate at which the virus fuses to the cell membrane. Thermodynamic experiments show that the incorporation of small amounts of surfactin hinders the formation of negative curvature by lamellar-phase lipids, suggesting that surfactin acts a membrane fusion inhibitor. A fluorescent lipopeptide similar to surfactin was synthesized, and its ability to insert into the viral membrane was confirmed by spectroscopy. In vivo experiments have shown that oral administration of surfactin to piglets protects against PEDV infection. In conclusion, our study indicates that surfactin is a membrane fusion inhibitor with activity against enveloped viruses. As the first reported naturally occurring wedge lipid membrane fusion inhibitor, surfactin is likely to be a prototype for the development of a broad range of novel antiviral drugs.

    IMPORTANCE Membrane fusion inhibitors are a rapidly emerging class of antiviral drugs that inhibit the infection process of enveloped viruses. They can be classified, on the basis of the viral components targeted, as fusion protein targeting or membrane lipid targeting. Lipid-targeting membrane fusion inhibitors have a broader antiviral spectrum and are less likely to select for drug-resistant mutations. Here we show that surfactin is a membrane fusion inhibitor and has a strong antiviral effect. The insertion of surfactin into the viral envelope lipids reduces the probability of viral fusion. We also demonstrate that oral administration of surfactin protects piglets from PEDV infection. Surfactin is the first naturally occurring wedge lipid membrane fusion inhibitor that has been identified and may be effective against many viruses beyond the scope of this study. Understanding its mechanism of action provides a foundation for the development of novel antiviral agents.

  • Epstein-Barr Virus Type 2 Infects T Cells and Induces B Cell Lymphomagenesis in Humanized Mice [Virus-Cell Interactions]

  • Epstein-Barr virus (EBV) has been classified into two strains, EBV type 1 (EBV-1) and EBV type 2 (EBV-2) based on genetic variances and differences in transforming capacity. EBV-1 readily transforms B cells in culture while EBV-2 is poorly transforming. The differing abilities to immortalize B cells in vitro suggest that in vivo these viruses likely use alternative approaches to establish latency. Indeed, we recently reported that EBV-2 has a unique cell tropism for T cells, infecting T cells in culture and in healthy Kenyan infants, strongly suggesting that EBV-2 infection of T cells is a natural part of the EBV-2 life cycle. However, limitations of human studies hamper further investigation into how EBV-2 utilizes T cells. Therefore, BALB/c Rag2null IL2rnull SIRPaalpha; humanized mice were utilized to develop an EBV-2 in vivo model. Infection of humanized mice with EBV-2 led to infection of both T and B cells, unlike infection with EBV-1, in which only B cells were infected. Gene expression analysis demonstrated that EBV-2 established a latency III infection with evidence of ongoing viral reactivation in both B and T cells. Importantly, EBV-2-infected mice developed tumors resembling diffuse large B cell lymphoma (DLBCL). These lymphomas had morphological features comparable to those of EBV-1-induced DLBCLs, developed at similar rates with equivalent frequencies, and expressed a latency III gene profile. Thus, despite the impaired ability of EBV-2 to immortalize B cells in vitro, EBV-2 efficiently induces lymphomagenesis in humanized mice. Further research utilizing this model will enhance our understanding of EBV-2 biology, the consequence of EBV infection of T cells, and the capacity of EBV-2 to drive lymphomagenesis.

    IMPORTANCE EBV is a well-established B cell-tropic virus. However, we have recently shown that the EBV type 2 (EBV-2) strain also infects primary T cells in culture and in healthy Kenyan children. This finding suggests that EBV-2, unlike the well-studied EBV-1 strain, utilizes the T cell compartment to persist. As EBV is human specific, studies to understand the role of T cells in EBV-2 persistence require an in vivo model. Thus, we developed an EBV-2 humanized mouse model, utilizing immunodeficient mice engrafted with human cord blood CD34+ stem cells. Characterization of the EBV-2-infected humanized mice established that both T cells and B cells are infected by EBV-2 and that the majority of infected mice develop a B cell lymphoma resembling diffuse large B cell lymphoma. This new in vivo model can be utilized for studies to enhance our understanding of how EBV-2 infection of T cells contributes to persistence and lymphomagenesis.

  • Acute-Phase CD4+ T Cell Responses Targeting Invariant Viral Regions Are Associated with Control of Live Attenuated Simian Immunodeficiency Virus [Cellular Response to Infection]

  • We manipulated SIVmac239nef, a model of major histocompatibility complex (MHC)-independent viral control, to evaluate characteristics of effective cellular responses mounted by Mauritian cynomolgus macaques (MCMs) that express the M3 MHC haplotype, which has been associated with poor control of pathogenic simian immunodeficiency virus (SIV). We created SIVnef-8x to test the hypothesis that effective SIV-specific T cell responses targeting invariant viral regions can emerge in the absence of immunodominant CD8+ T cell responses targeting variable epitopes and that control is achievable in individuals lacking known "protective" MHC alleles. Full-proteome gamma interferon (IFN-) enzyme-linked immunospot (ELISPOT) assays identified six newly targeted immunogenic regions following SIVnef-8x infection of M3/M3 MCMs. We deep sequenced circulating virus and found that four of the six newly targeted regions rarely accumulated mutations. Six animals infected with SIVnef-8x had T cell responses that targeted at least one of the four invariant regions and had a lower set point viral load than two animals that did not have T cell responses that targeted any invariant regions. We found that MHC class II molecules restricted all four of the invariant peptide regions, while the two variable regions were restricted by MHC class I molecules. Therefore, in the absence of immunodominant CD8+ T cell responses that target variable regions during SIVmac239nef infection, individuals without protective MHC alleles developed predominantly CD4+ T cell responses specific for invariant regions that may improve control of virus replication. Our results provide some evidence that antiviral CD4+ T cells during acute SIV infection can contribute to effective viral control and should be considered in strategies to combat HIV infection.

    IMPORTANCE Studies defining effective cellular immune responses to human immunodeficiency virus (HIV) and SIV have largely focused on a rare population that express specific MHC class I alleles and control virus replication in the absence of antiretroviral treatment. This leaves in question whether similar effective immune responses can be achieved in the larger population. The majority of HIV-infected individuals mount CD8+ T cell responses that target variable viral regions that accumulate high-frequency escape mutations. Limiting T cell responses to these variable regions and targeting invariant viral regions, similar to observations in rare "elite controllers," may provide an ideal strategy for the development of effective T cell responses in individuals with diverse MHC genetics. Therefore, it is of paramount importance to determine whether T cell responses can be redirected toward invariant viral regions in individuals without protective MHC alleles and if these responses improve control of virus replication.

  • Selective 4-Thiouracil Labeling of RNA Transcripts within Latently Infected Cells after Infection with Human Cytomegalovirus Expressing Functional Uracil Phosphoribosyltransferase [Virus-Cell Interactions]

  • Infections with human cytomegalovirus (HCMV) are highly prevalent in the general population as the virus has evolved the capacity to undergo distinct replication strategies resulting in lytic, persistent, and latent infections. During the latent life cycle, HCMV resides in subsets of cells within the hematopoietic cell compartment, including hematopoietic progenitor cells (HPCs) and peripheral blood monocytes. Since only a small fraction of these cell types harbor viral genomes during natural latency, identification and analysis of distinct changes mediated by viral infection are difficult to assess. In order to characterize latent infections of HPCs, we used an approach that involves complementation of deficiencies within the human pyrimidine salvage pathway, thus allowing for conversion of labeled uracil into rUTP. Here, we report the development of a recombinant HCMV that complements the defective human pyrimidine salvage pathway, allowing incorporation of thiol containing UTP into all RNA species that are synthesized within an infected cell. This virus grows to wild-type kinetics and can establish a latent infection within two distinct culture models of HCMV latency. Using this recombinant HCMV, we report the specific labeling of transcripts only within infected cells. These transcripts reveal a transcriptional landscape during HCMV latency that is distinct from uninfected cells. The utility of this labeling system allows for the identification of distinct changes within host transcripts and will shed light on characterizing how HCMV establishes and maintains latency.

    IMPORTANCE HCMV is a significant pathogen that accounts for a substantial amount of complications within the immunosuppressed and immunocompromised. Of particular significance is the capacity of HCMV to reactivate within solid tissue and bone marrow transplant recipients. While it is known that HCMV latency resides within a fraction of HPCs and monocytes, the exact subset of cells that harbor latent viral genomes during natural infections remain uncharacterized. The capacity to identify changes within the host transcriptome during latent infections is critical for developing approaches that therapeutically or physically eliminate latent viral genome containing cells and will represent a major breakthrough for reducing complications due to HCMV reactivation posttransplant. In this report, we describe the generation and use of a recombinant HCMV that allows specific and distinct labeling of RNA species that are produced within virally infected cells. This is a critical first step in identifying how HCMV affects the host cell during latency and more importantly, allows one to characterize cells that harbor latent HCMV.

  • Intrahepatic Cross-Presentation and Hepatocellular Antigen Presentation Play Distinct Roles in the Induction of Hepatitis B Virus-Specific CD8+ T Cell Responses [Pathogenesis and Immunity]

  • CD8+ T cells are the key cellular effectors mediating the clearance of hepatitis B virus (HBV) infections. However, early immunological events surrounding the priming of HBV-specific CD8+ T cell responses remain poorly understood. This study examined the importance of priming location and the relative contribution of endogenous antigen presentation by hepatocytes versus cross-presentation by bone marrow-derived cells to the induction of functional HBV-specific CD8+ T cell responses using the animal models of acute and chronic HBV infection. Functional HBV-specific CD8+ T cell responses could be induced to intrahepatically expressed HBV even when T cell homing to the lymphoid tissues was severely suppressed, suggesting that functional priming could occur in the liver. The expansion of HBV-specific CD8+ T cells was significantly reduced in the mice whose major histocompatibility complex (MHC) class I expression was mostly restricted to nonhematopoietic cells, suggesting the importance of cross-presentation by hematopoietic cells in the induction of HBV-specific CD8+ T cells. Strikingly, the expansion and cytolytic differentiation of HBV-specific CD8+ T cells were reduced even more severely in the mice whose MHC class I expression was restricted to hematopoietic cells. Collectively, these results indicate that cross-presentation is required but relatively inefficient in terms of inducing the cytolytic differentiation of HBV-specific CD8+ T cells by itself. Instead, the expansion and functional differentiation of HBV-specific CD8+ T cells are primarily dependent on hepatocellular antigen presentation.

    IMPORTANCE Hepatitis B virus (HBV) causes acute and chronic hepatitis. Approximately 260 million people are chronically infected with HBV and under an increased risk of developing cirrhosis and hepatocellular carcinoma. Host immune responses, particularly HBV-specific CD8+ T cell responses, largely determine the outcome of HBV infection. It is widely accepted that antigen inexperienced CD8+ T cells should be initially activated by professional antigen-presenting cells (pAPCs) in lymphoid tissues to differentiate into effector CD8+ T cells. However, this notion has not been tested for HBV-specific CD8+ T cells. In this study, we show that HBV-specific CD8+ T cell responses can be induced in the liver. Surprisingly, antigen presentation by hepatocytes is more important than cross-presentation by hematopoietic cells for the induction of HBV-specific CD8+ T cell responses. These results revealed a previously unappreciated role of antigen presentation by hepatocytes in the induction of HBV-specific CD8+ T cell responses.

  • Sirtuin 2 Isoform 1 Enhances Hepatitis B Virus RNA Transcription and DNA Synthesis through the AKT/GSK-3{beta}/{beta}-Catenin Signaling Pathway [Virus-Cell Interactions]

  • Sirtuin 2 (Sirt2), a NAD+-dependent protein deacetylase, is overexpressed in many hepatocellular carcinomas (HCCs) and can deacetylate many proteins, including tubulins and AKT, prior to AKT activation. Here, we found that endogenous Sirt2 was upregulated in wild-type hepatitis B virus (HBV WT)-replicating cells, leading to tubulin deacetylation; however, this was not the case in HBV replication-deficient-mutant-transfected cells and 1.3-mer HBV WT-transfected and reverse transcriptase inhibitor (entecavir or lamivudine)-treated cells, but all HBV proteins were expressed. In HBV WT-replicating cells, upregulation of Sirt2 induced AKT activation, which consequently downregulated glycogen synthase kinase 3bbeta; (GSK-3bbeta;) and increased bbeta;-catenin levels; however, downregulation of Sirt2 in HBV-nonreplicating cells impaired AKT/GSK-3bbeta;/bbeta;-catenin signaling. Overexpression of Sirt2 isoform 1 stimulated HBV transcription and consequently HBV DNA synthesis, which in turn activated AKT and consequently increased bbeta;-catenin levels, possibly through physical interactions with Sirt2 and AKT. Knockdown of Sirt2 by short hairpin RNAs (shRNAs), inhibition by 2-cyano-3-[5-(2,5-dichlorophenyl)-2-furanyl]-N-5-quinolinyl-2-propenamide (AGK2), or dominant negative mutant expression inhibited HBV replication, reduced AKT activation, and decreased bbeta;-catenin levels. Through HBV infection, we demonstrated that Sirt2 knockdown inhibited HBV replication from transcription. Although HBx itself activates AKT and upregulates bbeta;-catenin, Sirt2-mediated signaling and upregulated HBV replication were HBx independent. Since constitutively active AKT inhibits HBV replication, the results suggest that upregulated Sirt2 and activated AKT may balance HBV replication to prolong viral replication, eventually leading to the development of HCC. Also, the results indicate that Sirt2 inhibition may be a new therapeutic option for controlling HBV infection and preventing HCC.

    IMPORTANCE Even though Sirt2, a NAD+-dependent protein deacetylase, is overexpressed in many HCCs, and overexpressed Sirt2 promotes hepatic fibrosis and associates positively with vascular invasion by primary HCCs through AKT/GSK-3bbeta;/bbeta;-catenin signaling, the relationship between Sirt2, HBV, HBx, and/or HBV-associated hepatocarcinogenesis is unclear. Here, we show that HBV DNA replication, not HBV expression, correlates positively with Sirt2 upregulation and AKT activation. We demonstrate that overexpression of Sirt2 further increases HBV replication, increases AKT activation, downregulates GSK-3bbeta;, and increases bbeta;-catenin levels. Conversely, inhibiting Sirt2 decreases HBV replication, reduces AKT activation, and decreases bbeta;-catenin levels. Although HBx activates AKT to upregulate bbeta;-catenin, Sirt2-mediated effects were not dependent on HBx. The results also indicate that a Sirt2 inhibitor may control HBV infection and prevent the development of hepatic fibrosis and HCC.

  • Using a Virion Assembly-Defective Dengue Virus as a Vaccine Approach [Vaccines and Antiviral Agents]

  • Dengue virus (DENV) is the most prevalent mosquito-transmitted viral pathogen in humans. The recently licensed dengue vaccine has major weaknesses. Therefore, there is an urgent need to develop improved dengue vaccines. Here, we report a virion assembly-defective DENV as a vaccine platform. DENV containing an amino acid deletion (K188) in nonstructural protein 2A (NS2A) is fully competent in viral RNA replication but is completely defective in virion assembly. When trans-complemented with wild-type NS2A protein, the virion assembly defect could be rescued, generating pseudoinfectious virus (PIVNS2A) that could initiate single-round infection. The trans-complementation efficiency could be significantly improved through selection for adaptive mutations, leading to high-yield PIVNS2A production, with titers of ggt;107 infectious-focus units (IFU)/ml. Mice immunized with a single dose of PIVNS2A elicited strong T cell immune responses and neutralization antibodies and were protected from wild-type-virus challenge. Collectively, the results proved the concept of using assembly-defective virus as a vaccine approach. The study also solved the technical bottleneck in producing high yields of PIVNS2A vaccine. The technology could be applicable to vaccine development for other viral pathogens.

    IMPORTANCE Many flaviviruses are significant human pathogens that pose global threats to public health. Although licensed vaccines are available for yellow fever, Japanese encephalitis, tick-borne encephalitis, and dengue viruses, new approaches are needed to develop improved vaccines. Using dengue virus as a model, we developed a vaccine platform using a virion assembly-defective virus. We show that such an assembly-defective virus could be rescued to higher titers and infect cells for a single round. Mice immunized with the assembly-defective virus were protected from wild-type-virus infection. This vaccine approach could be applicable to other viral pathogens.

  • Alternative Strategy for a Quadrivalent Live Attenuated Influenza Virus Vaccine [Vaccines and Antiviral Agents]

  • Influenza virus infections continue to pose a major public health threat worldwide associated with seasonal epidemics and sporadic pandemics. Vaccination is considered the first line of defense against influenza. Live attenuated influenza virus vaccines (LAIVs) may provide superior responses compared to inactivated vaccines because the former can better elicit a combination of humoral and cellular responses by mimicking a natural infection. Unfortunately, during the 2013nndash;2014, 2014nndash;2015, and 2015nndash;2016 seasons, concerns emerged about the effectiveness of the only LAIV approved in the United States that prevented the Advisory Committee on Immunization Practices (ACIP) from recommending its use. Such drawbacks open up the opportunity for alternative LAIV strategies that could overcome such concerns. Previously, we developed a combined strategy of temperature-sensitive mutations in the PB2 and PB1 segments and an epitope tag in the C terminus of PB1 that effectively attenuates influenza A viruses of avian and mammalian origin. More recently, we adopted a similar strategy for influenza B viruses. The resulting attenuated (att) influenza A and B viruses were safe, immunogenic, and protective against lethal influenza virus challenge in a variety of animal models. In this report, we provide evidence of the potential use of our att strategy in a quadrivalent LAIV (QIV) formulation carrying H3N2 and H1N1 influenza A virus subtype viruses and two antigenic lineages of influenza B viruses. In naive DBA/2J mice, two doses of the QIV elicited hemagglutination inhibition (HI) responses with HI titers of gge;40 and effectively protected against lethal challenge with prototypical pandemic H1N1 influenza A and influenza B virus strains.

    IMPORTANCE Seasonal influenza viruses infect 1 billion people worldwide and are associated with ~500,000 deaths annually. In addition, the never-ending emergence of zoonotic influenza viruses associated with lethal human infections and of pandemic concern calls for the development of better vaccines and/or vaccination strategies against influenza virus. Regardless of the strategy, novel influenza virus vaccines must aim at providing protection against both seasonal influenza A and B viruses. In this study, we tested an alternative quadrivalent live attenuated influenza virus vaccine (QIV) formulation whose individual components have been previously shown to provide protection. We demonstrate in proof-of principle studies in mice that the QIV provides effective protection against lethal challenge with either influenza A or B virus.

  • Newly Distinguished Cell Punctures Associated with Transmission of the Semipersistent Phloem-Limited Beet Yellows Virus [Virus-Cell Interactions]

  • Here we report on plant penetration activities (probing) by the aphid Myzus persicae (Sulzer, 1776) in association with the transmission, acquisition, and inoculation of the semipersistent Beet yellows virus (BYV; Closterovirus) in sugar beet. During electrical penetration graph (EPG) recording of stylet pathways, standard intracellular stylet punctures occur which are called potential drop (pd) waveforms. In addition to the standard pd, there also appeared to be a unique type of intracellular stylet puncture that always preceded the phloem salivation phase (waveform E1). This type of pd, the phloem-pd, showed properties distinct from those of the standard pds and has never been described before. We manually ended EPG recordings during the acquisition and inoculation tests by removing aphids from the source or test plant after specific waveforms were recorded. Inoculation of BYV occurred at the highest rate when probing was interrupted just after a single or various phloem-pds. In contrast, BYV acquisition showed an intimate association with sustained phloem sap ingestion from phloem sieve elements (SEs) (E2 waveform). Our work shows for the first time that the inoculation of a phloem-limited virus occurs during specific intracellular stylet punctures and before phloem salivation (waveform E1). Further studies are needed to establish in what cells this novel phloem-pd occurs: phloem parenchyma, companion, or SE cells. The role of the different stylet activities in the acquisition and inoculation of BYV by M. persicae is discussed.

    IMPORTANCE We discovered the specific feeding activities of Myzus persicae (Sulzer, 1776) associated with the transmission of Beet yellows virus (BYV; Closterovirus). Our work strongly suggests that aphids can insert their stylets into the membranes of phloem cellsmmdash;visualized as a unique type of waveform that is associated with the inoculation of BYV. This intracellular puncture (3 to 5 s) occurs just before the phloem salivation phase and can be distinguished from other nonvascular stylet cell punctures. This is the first time that the transmission of a phloem-limited semipersistent virus has been shown to be associated with a unique type of intracellular puncture. Our work offers novel information and strongly contributes to the existing literature on the transmission of plant viruses. Here we describe a new kind of aphid behavioral pattern that could be key in further works, such as studying the transmission of other phloem-limited viruses (e.g., luteoviruses).

  • Exploring Crimean-Congo Hemorrhagic Fever Virus-Induced Hepatic Injury Using Antibody-Mediated Type I Interferon Blockade in Mice [Pathogenesis and Immunity]

  • Crimean-Congo hemorrhagic fever virus (CCHFV) can cause severe hepatic injury in humans. However, the mechanism(s) causing this damage is poorly characterized. CCHFV produces an acute disease, including liver damage, in mice lacking type I interferon (IFN-I) signaling due to either STAT-1 gene deletion or disruption of the IFN-I receptor 1 gene. Here, we explored CCHFV-induced liver pathogenesis in mice using an antibody to disrupt IFN-I signaling. When IFN-I blockade was induced within 24 h postexposure to CCHFV, mice developed severe disease with greater than 95% mortality by 6 days postexposure. In addition, we observed increased proinflammatory cytokines, chemoattractants, and liver enzymes in these mice. Extensive liver damage was evident by 4 days postexposure and was characterized by hepatocyte necrosis and the loss of CLEC4F-positive Kupffer cells. Similar experiments in CCHFV-exposed NOD-SCID- (NSG), Rag2-deficient, and perforin-deficient mice also demonstrated liver injury, suggesting that cytotoxic immune cells are dispensable for hepatic damage. Some apoptotic liver cells contained viral RNA, while other apoptotic liver cells were negative, suggesting that cell death occurred by both intrinsic and extrinsic mechanisms. Protein and transcriptional analysis of livers revealed that activation of tumor necrosis factor superfamily members occurred by day 4 postexposure, implicating these molecules as factors in liver cell death. These data provide insights into CCHFV-induced hepatic injury and demonstrate the utility of antibody-mediated IFN-I blockade in the study of CCHFV pathogenesis in mice.

    IMPORTANCE CCHFV is an important human pathogen that is both endemic and emerging throughout Asia, Africa, and Europe. A common feature of acute disease is liver injury ranging from mild to fulminant hepatic failure. The processes through which CCHFV induces severe liver injury are unclear, mostly due to the limitations of existing small-animal systems. The only small-animal model in which CCHFV consistently produces severe liver damage is mice lacking IFN-I signaling. In this study, we used antibody-mediated blockade of IFN-I signaling in mice to study CCHFV liver pathogenesis in various transgenic mouse systems. We found that liver injury did not depend on cytotoxic immune cells and observed extensive activation of death receptor signaling pathways in the liver during acute disease. Furthermore, acute CCHFV infection resulted in a nearly complete loss of Kupffer cells. Our model system provides insight into both the molecular and the cellular features of CCHFV hepatic injury.

  • Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein [Virus-Cell Interactions]

  • Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV can be sensed by host innate immunity to induce expression of interferons (IFNs) and a number of antiviral effectors. In this study, we found HCV infection induced the expression of neuralized E3 ubiquitin protein ligase 3 (NEURL3), a putative E3 ligase, in a manner that requires the involvement of innate immune sensing but is independent of the IFN action. Furthermore, we showed that NEURL3 inhibited HCV infection while it had little effect on other RNA viruses, including Zika virus (ZIKV), dengue virus (DENV), and vesicular stomatitis virus (VSV). Mechanistic studies demonstrated that NEURL3 inhibited HCV assembly by directly binding HCV envelope glycoprotein E1 to interfere with the E1/E2 heterodimerization, an important prerequisite for virion morphogenesis. Finally, we showed that knockout of NEURL3 significantly enhanced HCV infection. In summary, we identified NEURL3 as a novel inducible antiviral host factor that suppresses HCV assembly. Our results not only shed new insight into how host innate immunity acts against HCV but also revealed a new important biological function for NEURL3.

    IMPORTANCE The exact biological function of NEURL3, a putative E3 ligase, remains largely unknown. In this study, we found that NEURL3 could be upregulated upon HCV infection in a manner dependent on pattern recognition receptor-mediated innate immune response. NEURL3 inhibits HCV assembly by directly binding viral E1 envelope glycoprotein to disrupt its interaction with E2, an action that requires its Neuralized homology repeat (NHR) domain but not the RING domain. Furthermore, we found that NEURL3 has a pangenotypic anti-HCV activity and interacts with E1 of genotypes 2a, 1b, 3a, and 6a but does not inhibit other closely related RNA viruses, such as ZIKV, DENV, and VSV. To our knowledge, our study is the first report to demonstrate that NEURL3 functions as an antiviral host factor. Our results not only shed new insight into how host innate immunity acts against HCV, but also revealed a new important biological function for NEURL3.

  • Distinctive Roles for Type I and Type II Interferons and Interferon Regulatory Factors in the Host Cell Defense against Varicella-Zoster Virus [Virus-Cell Interactions]

  • Both type I and type II interferons (IFNs) have been implicated in the host defense against varicella-zoster virus (VZV), a common human herpesvirus that causes varicella and zoster. The purpose of this study was to compare their contributions to the control of VZV replication, to identify the signaling pathways that are critical for mediating their antiviral activity, and to define the mechanisms by which the virus counteracts their effects. Gamma interferon (IFN-) was much more potent than IFN-aalpha; in blocking VZV infection, which was associated with a differential induction of the interferon regulatory factor (IRF) proteins IRF1 and IRF9, respectively. These observations account for the clinical experience that while the formation of VZV skin lesions is initially controlled by local immunity, adaptive virus-specific T cell responses are required to prevent life-threatening VZV infections.

    IMPORTANCE While both type I and type II IFNs are involved in the control of herpesvirus infections in the human host, to our knowledge, their relative contributions to the restriction of viral replication and spread have not been assessed. We report that IFN- has more potent activity than IFN-aalpha; against VZV. Findings from this comparative analysis show that the IFN-aalpha;nndash;IRF9 axis functions as a first line of defense to delay the onset of viral replication and spread, whereas the IFN-nndash;IRF1 axis has the capacity to block the infectious process. Our findings underscore the importance of IRFs in IFN regulation of herpesvirus infection and account for the clinical experience of the initial control of VZV skin infection attributable to IFN-aalpha; production, together with the requirement for induction of adaptive IFN--producing VZV-specific T cells to resolve the infection.

  • The Endonucleolytic RNA Cleavage Function of nsp1 of Middle East Respiratory Syndrome Coronavirus Promotes the Production of Infectious Virus Particles in Specific Human Cell Lines [Virus-Cell Interactions]

  • Middle East respiratory syndrome coronavirus (MERS-CoV) nsp1 suppresses host gene expression in expressed cells by inhibiting translation and inducing endonucleolytic cleavage of host mRNAs, the latter of which leads to mRNA decay. We examined the biological functions of nsp1 in infected cells and its role in virus replication by using wild-type MERS-CoV and two mutant viruses with specific mutations in the nsp1; one mutant lacked both biological functions, while the other lacked the RNA cleavage function but retained the translation inhibition function. In Vero cells, all three viruses replicated efficiently with similar replication kinetics, while wild-type virus induced stronger host translational suppression and host mRNA degradation than the mutants, demonstrating that nsp1 suppressed host gene expression in infected cells. The mutant viruses replicated less efficiently than wild-type virus in Huh-7 cells, HeLa-derived cells, and 293-derived cells, the latter two of which stably expressed a viral receptor protein. In 293-derived cells, the three viruses accumulated similar levels of nsp1 and major viral structural proteins and did not induce IFN-bbeta; and IFN- mRNAs; however, both mutants were unable to generate intracellular virus particles as efficiently as wild-type virus, leading to inefficient production of infectious viruses. These data strongly suggest that the endonucleolytic RNA cleavage function of the nsp1 promoted MERS-CoV assembly and/or budding in a 293-derived cell line. MERS-CoV nsp1 represents the first CoV gene 1 protein that plays an important role in virus assembly/budding and is the first identified viral protein whose RNA cleavage-inducing function promotes virus assembly/budding.

    IMPORTANCE MERS-CoV represents a high public health threat. Because CoV nsp1 is a major viral virulence factor, uncovering the biological functions of MERS-CoV nsp1 could contribute to our understanding of MERS-CoV pathogenicity and spur development of medical countermeasures. Expressed MERS-CoV nsp1 suppresses host gene expression, but its biological functions for virus replication and effects on host gene expression in infected cells are largely unexplored. We found that nsp1 suppressed host gene expression in infected cells. Our data further demonstrated that nsp1, which was not detected in virus particles, promoted virus assembly or budding in a 293-derived cell line, leading to efficient virus replication. These data suggest that nsp1 plays an important role in MERS-CoV replication and possibly affects virus-induced diseases by promoting virus particle production in infected hosts. Our data, which uncovered an unexpected novel biological function of nsp1 in virus replication, contribute to further understanding of the MERS-CoV replication strategies.

  • Infection Dynamics of Hepatitis E Virus in Wild-Type and Immunoglobulin Heavy Chain Knockout JH-/- Gnotobiotic Piglets [Pathogenesis and Immunity]

  • Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important but incompletely understood pathogen causing high mortality during pregnancy and leading to chronic hepatitis in immunocompromised individuals. The underlying mechanisms leading to hepatic damage remain unknown; however, the humoral immune response is implicated. In this study, immunoglobulin (Ig) heavy chain JHnndash;/nndash; knockout gnotobiotic pigs were generated using CRISPR/Cas9 technology to deplete the B-lymphocyte population, resulting in an inability to generate a humoral immune response to genotype 3 HEV infection. Compared to wild-type gnotobiotic piglets, the frequencies of B lymphocytes in the Ig heavy chain JHnndash;/nndash; knockouts were significantly lower, despite similar levels of other innate and adaptive T-lymphocyte cell populations. The dynamic of acute HEV infection was subsequently determined in heavy chain JHnndash;/nndash; knockout and wild-type gnotobiotic pigs. The data showed that wild-type piglets had higher viral RNA loads in feces and sera compared to the JHnndash;/nndash; knockout pigs, suggesting that the Ig heavy chain JHnndash;/nndash; knockout in pigs actually decreased the level of HEV replication. Both HEV-infected wild-type and JHnndash;/nndash; knockout gnotobiotic piglets developed more pronounced lymphoplasmacytic hepatitis and hepatocellular necrosis lesions than other studies with conventional pigs. The HEV-infected JHnndash;/nndash; knockout pigs also had significantly enlarged livers both grossly and as a ratio of liver/body weight compared to phosphate-buffered saline-inoculated groups. This novel gnotobiotic pig model will aid in future studies into HEV pathogenicity, an aspect which has thus far been difficult to reproduce in the available animal model systems.

    IMPORTANCE According to the World Health Organization, approximately 20 million HEV infections occur annually, resulting in 3.3 million cases of hepatitis E and ggt;44,000 deaths. The lack of an efficient animal model that can mimic the full-spectrum of infection outcomes hinders our ability to delineate the mechanism of HEV pathogenesis. Here, we successfully generated immunoglobulin heavy chain JHnndash;/nndash; knockout gnotobiotic pigs using CRISPR/Cas9 technology, established a novel JHnndash;/nndash; knockout and wild-type gnotobiotic pig model for HEV, and systematically determined the dynamic of acute HEV infection in gnotobiotic pigs. It was demonstrated that knockout of the Ig heavy chain in pigs decreased the level of HEV replication. Infected wild-type and JHnndash;/nndash; knockout gnotobiotic piglets developed more pronounced HEV-specific lesions than other studies using conventional pigs, and the infected JHnndash;/nndash; knockout pigs had significantly enlarged livers. The availability of this novel model will facilitate future studies of HEV pathogenicity.

  • In Vivo Persistence of Chimeric Virus after Substitution of the Kaposi's Sarcoma-Associated Herpesvirus LANA DNA Binding Domain with That of Murid Herpesvirus 4 [Pathogenesis and Immunity]

  • The latency-associated nuclear antigen from Kaposi's sarcoma-associated herpesvirus (KSHV), kLANA, and its homolog from the murid herpesvirus 4 (MuHV-4), mLANA, are essential for viral latency. kLANA is nearly four times the size of mLANA, mainly due to an extensive central repeat region that is absent in mLANA. Both proteins harbor a C-terminal DNA binding domain (DBD). The DBD binds the terminal repeat (TR) DNA sequences of the viral genome to mediate persistence. Despite structural conservation, the kLANA and mLANA DBDs differ in sequence and mode of oligomerization. kLANA DBD oligomers are flexible and bent, while mLANA DBD oligomers bind DNA in a rigid, linear conformation. We previously reported that kLANA and mLANA acted reciprocally on TR sequences. Furthermore, a MuHV-4 expressing kLANA instead of mLANA (v-kLANA) established latency in mice, albeit at a lower magnitude than the wild-type (WT) virus. Here, we asked if kLANA can accommodate the mLANA DBD and generated a fusion protein which contains kLANA but with the mLANA C-terminal region in place of that of kLANA. We report a recombinant MuHV-4 (v-KM) encoding this LANA fusion protein instead of mLANA. The fusion protein was expressed in lytic infection in vitro and assembled nuclear LANA dots in infected splenocytes. Results demonstrated that kLANA functionally accommodated mLANA's mode of DNA binding, allowing MuHV-4 chimeric virus to establish latency in vivo. Notably, v-KM established latency in germinal center B cells more efficiently than did v-kLANA, although levels were reduced compared to WT MuHV-4.

    IMPORTANCE KSHV is a human oncogenic virus for which there is no tractable, immunocompetent animal model of infection. MuHV-4, a related rodent gammaherpesvirus, enables pathogenesis studies in mice. In latency, both viruses persist as extrachromosomal, circular genomes (episomes). LANA proteins encoded by KSHV (kLANA) and MuHV-4 (mLANA) contain a C-terminal DNA binding domain (DBD) that acts on the virus terminal repeats to enable episome persistence. mLANA is a smaller protein than kLANA. Their DBDs are structurally conserved but differ strikingly in the conformation of DNA binding. We report a recombinant, chimeric MuHV-4 which contains kLANA in place of mLANA, but in which the DBD is replaced with that of mLANA. Results showed that kLANA functionally accommodated mLANA's mode of DNA binding. In fact, the new chimeric virus established latency in vivo more efficiently than MuHV-4 expressing full-length kLANA.

  • Transcriptome Analysis of Infected and Bystander Type 2 Alveolar Epithelial Cells during Influenza A Virus Infection Reveals In Vivo Wnt Pathway Downregulation [Virus-Cell Interactions]

  • Influenza virus outbreaks remain a serious threat to public health. A greater understanding of how cells targeted by the virus respond to the infection can provide insight into the pathogenesis of disease. Here we examined the transcriptional profile of in vivo-infected and uninfected type 2 alveolar epithelial cells (AEC) in the lungs of influenza virus-infected mice. We show for the first time the unique gene expression profiles induced by the in vivo infection of AEC as well as the transcriptional response of uninfected bystander cells. This work allows us to distinguish the direct and indirect effects of infection at the cellular level. Transcriptome analysis revealed that although directly infected and bystander AEC from infected animals shared many transcriptome changes compared to AEC from uninfected animals, directly infected cells produce more interferon and express lower levels of Wnt signaling-associated transcripts, while concurrently expressing more transcripts associated with cell death pathways, than bystander uninfected AEC. The Wnt signaling pathway was downregulated in both in vivo-infected AEC and in vitro-infected human lung epithelial A549 cells. Wnt signaling did not affect type I and III interferon production by infected A549 cells. Our results reveal unique transcriptional changes that occur within infected AEC and show that influenza virus downregulates Wnt signaling. In light of recent findings that Wnt signaling is essential for lung epithelial stem cells, our findings reveal a mechanism by which influenza virus may affect host lung repair.

    IMPORTANCE Influenza virus infection remains a major public health problem. Utilizing a recombinant green fluorescent protein-expressing influenza virus, we compared the in vivo transcriptomes of directly infected and uninfected bystander cells from infected mouse lungs and discovered many pathways uniquely regulated in each population. The Wnt signaling pathway was downregulated in directly infected cells and was shown to affect virus but not interferon production. Our study is the first to discern the in vivo transcriptome changes induced by direct viral infection compared to mere exposure to the lung inflammatory milieu and highlight the downregulation of Wnt signaling. This downregulation has important implications for understanding influenza virus pathogenesis, as Wnt signaling is critical for lung epithelial stem cells and lung epithelial cell differentiation. Our findings reveal a mechanism by which influenza virus may affect host lung repair and suggest interventions that prevent damage or accelerate recovery of the lung.

  • The Human Cytomegalovirus Trimer and Pentamer Promote Sequential Steps in Entry into Epithelial and Endothelial Cells at Cell Surfaces and Endosomes [Virus-Cell Interactions]

  • Human cytomegalovirus (HCMV) infects a wide variety of human cell types by different entry pathways that involve distinct envelope glycoprotein complexes that include gH/gL, a trimer complex consisting of gHgL/gO, and a pentamer complex consisting of gH/gL/UL128/UL130/UL131. We characterized the effects of soluble forms of these proteins on HCMV entry. Soluble trimer and pentamer blocked entry of HCMV into epithelial and endothelial cells, whereas soluble gH/gL did not. Trimer inhibited HCMV entry into fibroblast cells, but pentamer and gH/gL did not. Both trimer and pentamer bound to the surfaces of fibroblasts and epithelial cells, whereas gH/gL did not bind to either cell type. Cell surface binding of trimer and pentamer did not involve heparin sulfate moieties. The ability of soluble trimer to block entry of HCMV into epithelial cells did not involve platelet-derived growth factor PDGFRaalpha;, which has been reported as a trimer receptor for fibroblasts. Soluble trimer reduced the amount of virus particles that could be adsorbed onto the surface of epithelial cells, whereas soluble pentamer had no effect on virus adsorption. However, soluble pentamer reduced the ability of virus particles to exit from early endosomes into the cytoplasm and then travel to the nucleus. These studies support a model in which both the trimer and pentamer are required for HCMV entry into epithelial and endothelial cells, with trimer interacting with cell surface receptors other than PDGFR and pentamer acting later in the entry pathway to promote egress from endosomes.

    IMPORTANCE HCMV infects nearly 80% of the world's population and causes significant morbidity and mortality. The current antiviral agents used to treat HCMV infections are prone to resistance and can be toxic to patients, and there is no current vaccine against HCMV available. The data in this report will lead to a better understanding of how essential HCMV envelope glycoproteins function during infection of biologically important cell types and will have significant implications for understanding HCMV pathogenesis for developing new therapeutics.

  • Correction for Wang et al., "Porcine MKRN1 Modulates the Replication and Pathogenesis of Porcine Circovirus Type 2 by Inducing Capsid Protein Ubiquitination and Degradation" [Author Correction]

  • Epstein-Barr Virus Nuclear Antigen 3C Inhibits Expression of COBLL1 and the ADAM28-ADAMDEC1 Locus via Interaction with the Histone Lysine Demethylase KDM2B [Genome Replication and Regulation of Viral Gene Expression]

  • Epstein-Barr virus nuclear antigen 3C (EBNA3C) is a well-defined repressor of host gene expression in B cells transformed by Epstein-Barr virus (EBV) that cooperates with various cellular factors. It is established that EBNA3C interacts with the cellular factor RBPJ (RBP-J or CBF1) through two distinct motifs: the TFGC motif, also called the homology domain (HD) motif, and the VWTP motif. In this study, we investigated the role of each motif in EBNA3C transcriptional repression activity by using two novel recombinant viruses with single RBPJ interaction motifs mutated (EBNA3C HDmut and EBNA3C W227S). Infection of primary B cells with either of these recombinant EBVs led to the successful establishment of lymphoblastoid cell lines (LCLs). Gene expression analysis showed that full repression of EBNA3C target genes is not achieved by EBNA3C HDmut compared to that with EBNA3C W227S or the EBNA3C wild type (WT). Focusing on the well-characterized EBNA3C-repressed genes COBLL1, ADAM28, and ADAMDEC1, we investigated the mechanism of EBNA3C-mediated transcriptional repression. Chromatin immunoprecipitation (ChIP) analysis indicated that EBNA3C HDmut is still able to recruit Polycomb proteins BMI1 and SUZ12 to COBLL1 as efficiently as EBNA3C WT does, leading to the full deposition of the repressive histone mark H3K27me3. However, we found that the activation-associated chromatin mark H3K4me3 is highly enriched at EBNA3C target genes in LCLs expressing EBNA3C HDmut. We show here that EBNA3C interacts with the histone lysine demethylase KDM2B and that this interaction is important for H3K4me3 removal and for the EBNA3C-mediated repression of COBLL1 and the ADAM28-ADAMDEC1 locus.

    IMPORTANCE EBV is a virus associated with human cancers and is well known for its ability to transform B lymphocytes into continuously proliferating lymphoblastoid cell lines. EBNA3C is considered an oncoprotein and has been shown to be essential for B cell transformation by EBV. EBNA3C is well characterized as a viral transcription factor, but very little is known about its mechanisms of action. In the present study, we demonstrate that removal of the activating histone mark H3K4me3 and deposition of the repressive mark H3K27me3 by EBNA3C on COBLL1 are achieved by at least two distinct mechanisms. Furthermore, we discovered that EBNA3C interacts with the lysine demethylase KDM2B and that this interaction is important for its transcriptional repressive function. The findings in this study provide new insights into the mechanism used by the oncoprotein EBNA3C to repress cellular target genes.

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    JVI Accepts: Articles Published Ahead of Print

  • Risk assessment of fifth-wave H7N9 influenza A viruses in mammalian models [Pathogenesis and Immunity]

  • The fifth-wave of the H7N9 influenza epidemic in China was distinguished by a sudden increase in human infections, an extended geographic distribution, and the emergence of highly pathogenic avian influenza (HPAI) viruses. Genetically, some H7N9 viruses from the fifth-wave have acquired novel amino acid changes at positions involved in mammalian adaptation, antigenicity, and HA cleavability. Here, several low pathogenic avian influenza (LPAI) and HPAI H7N9 human isolates from the fifth epidemic wave were assessed for their pathogenicity and transmissibility in mammalian models, as well as their ability to replicate in human airway epithelial cells. We found that a LPAI virus exhibited a similar capacity to replicate and cause disease in two animal species as viruses from previous waves. In contrast, HPAI H7N9 viruses possessed enhanced virulence, causing greater lethargy and mortality, with an extended tropism for brain tissues in both ferret and mouse models. These HPAI viruses also showed signs of adaptation to mammalian hosts by acquiring the ability to fuse at a lower pH threshold compared with other H7N9 viruses. All of the fifth-wave H7N9 viruses were able to transmit among cohoused ferrets, but exhibited a limited capacity to transmit by respiratory droplets and deep sequencing analysis revealed that the H7N9 viruses sampled after transmission showed a reduced amount of minor variants. Taken together, we conclude that the fifth-wave HPAI H7N9 viruses have gained the ability to cause enhanced disease in mammalian models, and with further adaptation may acquire the ability to cause an H7N9 pandemic.

    Importance

    The potential pandemic risk posed by avian influenza H7N9 viruses was heightened during the fifth epidemic wave in China due to the sudden increased number of human infections and the emergence of antigenically distinct LPAI and HPAI H7N9 viruses. In this study, a group of fifth-wave HPAI and LPAI viruses were evaluated for their ability to infect, cause disease, and transmit in small animal models. The ability of HPAI H7N9 viruses to cause more severe disease and to replicate in brain tissues in animal models as well as their ability to fuse at a lower pH threshold compared to LPAI H7N9 viruses suggest that the fifth-wave H7N9 viruses have evolved to acquire novel traits with the potential to pose a higher risk to humans. Although the fifth-wave H7N9 viruses have not yet gained the ability to transmit efficiently by air, continuous surveillance and risk assessment remain essential parts of our pandemic preparedness efforts.

  • Synergistic activation of bovine herpesvirus 1 productive infection and viral regulatory promoters by the progesterone receptor and Krüppel-like transcription factor 15. [Virus-Cell Interactions]

  • Bovine herpesvirus 1 (BoHV-1), including modified live vaccines, readily infect the fetus and ovaries, which can lead to reproductive failure. The BoHV-1 latency-reactivation cycle in sensory neurons may further complicate reproductive failure in pregnant cows. The immediate early transcription unit 1 (IEtu1) promoter drives expression of important viral transcriptional regulators (bICP0 and bICP4). This promoter contains two functional glucocorticoid receptor (GR) response elements (GREs) that have the potential to stimulate productive infection following stressful stimuli. Since progesterone and the progesterone receptor (PR) can activate many GREs, we hypothesized that the PR and/or progesterone regulates productive infection and viral transcription. New studies demonstrated that progesterone stimulated productive infection. Additional studies revealed the PR and Krüppel-like transcription factor 15 (KLF15) cooperated to stimulate productive infection and IEtu1 promoter activity. IEtu1 promoter activation required both GREs, which correlated with the ability of the PR to interact with wt GREs, but not mutant GREs. KLF15 also cooperated with the PR to trans-activate the bICP0 early promoter, a promoter that maintains bICP0 protein expression during productive infection. Intergenic viral DNA fragments (less than 400 bp) containing two GREs and putative KLF binding sites present within genes encoding unique long 52 (UL-52; component of DNA primase/helicase complex), Circ, bICP4, and IEtu2 were stimulated by KLF15 and the PR more than 10-fold suggesting additional viral promoters are activated by these transcription factors. Collectively, these studies suggest progesterone and the PR promote BoHV-1 spread to reproductive tissues, thus increasing the incidence of reproductive failure.

    IMPORTANCE

    Bovine herpesvirus 1 (BoHV-1) is the most frequently diagnosed cause of abortions in pregnant cows and can cause "abortion storms" in susceptible herds. Virulent field strains and even commercially available modified live vaccines can induce abortion, in part because BoHV-1 replicates efficiently in the ovary and corpus luteum. We now demonstrate that progesterone and the progesterone receptor (PR) stimulate productive infection. The BoHV-1 genome contains approximately 100 glucocorticoid receptor (GR) response elements (GREs). Interestingly, the PR can bind and activate many promoters that contain GREs. The PR and Krüppel-like transcription factor 15 (KLF15), which regulate key steps during embryo implantation, cooperate to stimulate productive infection and two viral promoters that drive expression of key viral transcriptional regulators. These studies suggest that the ability of progesterone and the PR to stimulate productive infection has the potential to promote virus spread in reproductive tissue and induce reproductive failure.

  • Genotypic and Mechanistic Characterization of Subtype-specific HIV Adaptation to Host Cellular Immunity [Genetic Diversity and Evolution]

  • The extent to which viral genetic context influences HIV adaptation to Human Leukocyte Antigen (HLA) class I-restricted immune pressures remains incompletely understood. The Ugandan HIV epidemic, where major pandemic group M subtypes A1 and D co-circulate in a single host population, provides an opportunity to investigate this question. We characterized plasma HIV RNA gag, pol and nef sequences, along with host HLA genotypes, in 464 antiretroviral-naïve individuals chronically infected with HIV subtypes A1 or D. Using phylogenetically-informed statistical approaches, we identified HLA-associated polymorphisms and formally compared their strengths of selection between viral subtypes. A substantial number (32%) of HLA-associated polymorphisms identified in subtypes A1 and/or D had previously been reported in subtypes B, C and/or Circulating Recombinant Form (CRF) 01_AE, confirming the shared nature of many HLA-driven escape pathways regardless of viral genetic context. Nevertheless, 34% of identified HLA-associated polymorphisms were significantly differentially selected between subtypes A1 and D. Experimental investigation of select examples of subtype-specific escape revealed distinct underlying mechanisms with important implications for vaccine design: whereas some were attributable to subtype-specific sequence variation that influenced epitope-HLA binding, others were attributable to differential mutational barriers to immune escape. Overall, our results confirm HIV genetic context as a key modulator of viral adaptation to host cellular immunity and highlight the power of combined bioinformatic and mechanistic studies, paired with knowledge of epitope immunogenicity, to identify appropriate viral regions for inclusion in subtype-specific and universal HIV vaccine strategies.

    Importance

    The identification of HIV polymorphisms reproducibly selected under pressure by specific HLA alleles, and the elucidation of their impact on viral function, can help identify immunogenic viral regions where immune escape incurs a fitness cost. However, our knowledge of HLA-driven escape pathways and their functional costs is largely limited to HIV subtype B, and to a lesser extent C. Our study represents the first characterization of HLA-driven adaptation pathways in HIV subtypes A1 and D, which dominate in East Africa, and the first statistically rigorous characterization of differential HLA-driven escape across viral subtypes. Results support a considerable impact of viral genetic context on HIV adaptation to host HLA, where HIV subtype-specific sequence variation influences both epitope-HLA binding and the fitness costs of escape. Integrated bioinformatic and mechanistic characterization of these and other instances of differential escape could aid rational CTL-based vaccine immunogen selection for both subtype-specific and universal HIV vaccines.

  • The interaction between ORF18 and ORF30 is required for late gene expression in Kaposis sarcoma-associated herpesvirus [Genome Replication and Regulation of Viral Gene Expression]

  • In the beta- and gammaherpesviruses, a specialized complex of viral transcriptional activators (vTAs) coordinate to direct expression of virus-encoded late genes, which are critical for viral assembly and whose transcription initiates only after the onset of viral DNA replication. The vTAs in Kaposirrsquo;s sarcoma-associated herpesvirus (KSHV) are ORF18, ORF24, ORF30, ORF31, ORF34, and ORF66. While the general organization of the vTA complex has been mapped, the individual roles of these proteins, and how they coordinate to activate late gene promoters, remains largely unknown. Here, we performed a comprehensive mutational analysis of the conserved residues in ORF18, which is a highly interconnected vTA component. Surprisingly, the mutants were largely selective for disrupting the interaction with ORF30 but not the other three ORF18 binding partners. Furthermore, disrupting the ORF18-ORF30 interaction weakened the vTA complex as a whole, and an ORF18 point mutant that failed to bind ORF30 was unable to complement an ORF18 null virus. Thus, contacts between individual vTAs are critical, as even small disruptions in this complex result in profound defects in KSHV late gene expression.

    Importance

    Kaposirrsquo;s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposirrsquo;s sarcoma and other B-cell cancers and remains a leading cause of death in immunocompromised individuals. A key step in the production of infectious virions is the transcription of viral late genes, which generates capsid and structural proteins and requires the coordination of six viral proteins that form a complex. The role of these proteins during transcription complex formation and the importance of protein-protein interactions are not well understood. Here, we focused on a central component of the complex, ORF18, and revealed that disruption of its interaction with even a single component of the complex (ORF30) prevents late gene expression and completion of the viral lifecycle. These findings underscore how individual interactions between the late gene transcription components are critical for both the stability and function of the complex.

  • Bone marrow-derived CD4+ T cells are depleted in SIV-infected macaques and contribute to the size of the replication competent reservoir on ART [Pathogenesis and Immunity]

  • The bone marrow (BM) is the key anatomic site for hematopoiesis and plays a significant role in the homeostasis of mature T cells. However, very little is known on the phenotype of BM-derived CD4+ T cells, their fate during SIV infection, and their contribution to viral persistence during antiretroviral therapy (ART). Here, we characterized the immunologic and virologic status of BM-derived CD4+ T cells in rhesus macaques prior to SIV infection, during the early chronic phase of infection, and after ART. We found that BM memory CD4+ T cells are significantly depleted following SIV infection, at levels that are similar to those measured in PB. In addition, BM-derived memory CD4+ T cells include a high frequency of cells that express the co-inhibitory receptors CTLA-4 and PD-1, two subsets previously shown to be enriched in viral reservoir; these cells appear to have a more resting phenotype then the same cells in the blood (PB). Finally, when we analyzed SIV-infected RMs in which viral replication was effectively suppressed by 12-months of ART-treatment, we found that BM CD4+ T cells harbor SIV-DNA and SIV-RNA at levels comparable to those of PB CD4+ T cells, including replication competent SIV. Thus, BM is a largely understudied anatomic site of the latent reservoir which contributes to viral persistence during ART and needs to be further characterized and targeted when designing therapies for a functional or sterilizing cure to HIV.

    Importance The latent viral reservoir is one of the major obstacles in purging the immune system of HIV. It is paramount that we elucidate which anatomic compartments harbor replication competent virus, that upon ART-treatment interruption results in viral rebound and pathogenesis. Here, using the rhesus macaque model of SIV infection and ART treatment, we examined the immunologic status of the BM and its role as a potential sanctuary for latent virus. We found that the BM compartment undergoes a similar depletion of memory CD4+ T cells as PB, and during ART treatment the BM-derived memory CD4+ T cells contain high levels of cells expressing CTLA-4 and PD-1, as well as comparable amounts of cell-associated SIV-DNA, SIV-RNA, and replication competent virus as PB. These results enrich our understanding of which anatomic compartments harbor replication virus and suggests that BM-derived CD4+ T cells need to be targeted by therapeutic strategies aimed at achieving an HIV cure.

  • Going the distance: optimizing RNA-Seq strategies for transcriptomic analysis of complex viral genomes [Gem]

  • Transcriptome profiling has become routine in studies of many biological processes. However, favored approaches such as short-read Illumina RNA sequencing are giving way to long-read sequencing platforms better suited to interrogating the complex transcriptomes typical of many RNA and DNA viruses. Here, we provide a guide - tailored to molecular virologists - to the ins-and-outs of viral transcriptome sequencing and discuss the strengths and weaknesses of the major RNA sequencing technologies as tools to analyze the abundance and diversity of viral transcripts made during infection.

  • Identification of anti-norovirus genes in human cells using genome-wide CRISPR activation screening [Virus-Cell Interactions]

  • Noroviruses (NoVs) are a leading cause of gastroenteritis world-wide, yet host factors that restrict NoV replication are not well understood. Here, we use a CRISPR activation (CRISPRa) genome-wide screening to identify host genes that can inhibit murine norovirus (MNoV) replication in human cells. Our screens identified with high confidence 49 genes that can inhibit MNoV infection when overexpressed. A significant number of these genes are in interferon and immune regulation signaling networks, but surprising, the majority of the genes identified are not associated with innate or adaptive immunity nor with any antiviral activity. Confirmatory studies of eight of the genes validate the initial screening data. Mechanistic studies on TRIM7 demonstrated a conserved role of the molecule in mouse and human cells in restricting MNoV in a step of infection after viral entry. Furthermore, we demonstrate that two isoforms of TRIM7 have differential antiviral activity. Taken together these data provide a resource for understanding norovirus biology and demonstrate a robust methodology for identifying new antiviral molecules.

    Importance: Norovirus is one of the leading causes of foodborne illness world-wide. Despite its prevalence, our understanding of norovirus biology is limited due to the difficulty in growing human norovirus in vitro and a lack of an animal model. Murine norovirus (MNoV) is a model norovirus system because MNoV replicates robustly in cell culture and in mice. To identify host genes that can restrict norovirus replication when overexpressed we performed genome-wide CRISPR activation (CRISPRa) screens to induce gene overexpression at the native locus through recruitment of transcriptional activators to individual gene promoters. We found 49 genes could block murine norovirus replication in human cells. Several of these genes are associated with classical immune signaling pathways, while many of the molecules we identified have not been previously associated with antiviral activity. Our data is a resource for those studying norovirus and we provide a robust approach to identify novel antiviral genes.

  • A 1000 year-old RNA virus [Genetic Diversity and Evolution]

  • Only a few RNA viruses have been discovered from archaeological samples, the oldest dating from about 750 years ago. Using ancient maize cobs from Antelope house, Arizona, dating from circa 1,000 CE, we discovered a novel plant virus with a double-stranded RNA genome. The virus is a member of the family Chrysoviridae that infect plants and fungi in a persistent manner. The extracted double-stranded RNA from 312 maize cobs was converted to cDNA and sequences were determined on Illumina HiSeq 2000. Assembled contigs from many samples showed similarity to Anthurium mosaic-associated virus and Persea americana chrysovirus, putative species in the Chrysovirus genus, and nearly complete genomes were found in three ancient maize samples. We named this new virus Zea mays chrysovirus 1. Using specific primers, we were able to recover sequences of a closely related virus from modern maize and obtained the nearly complete sequences of the three genomic RNAs. Comparing the nucleotide sequences of the three genomic RNAs of the modern and ancient viruses showed 98%, 96.7%, and 97.4% identity, respectively. Hence, in 1,000 years of maize cultivation, this virus has undergone about 3% divergence.

    Importance

    A virus related to plant chrysoviruses was found in numerous ancient samples of maize, with nearly complete genomes in three samples. The age of the ancient samples (about 1,000 years old) was confirmed by carbon dating. Chrysoviruses are persistent plant viruses. They infect their hosts from generation to generation by transmission through seeds, and can remain in their hosts for very long time periods. When modern corn samples were analyzed, a closely related chrysovirus was found with only about 3% divergence from the ancient sequences. This virus represents the oldest known plant virus.

  • P2X antagonists inhibit HIV-1 productive infection and inflammatory cytokines IL-10 and IL-1{beta} in a human tonsil explant model [Virus-Cell Interactions]

  • HIV-1 causes a persistent infection of the immune system that is associated with chronic comorbidities. The mechanisms that underlie this inflammation are poorly understood. Emerging literature has implicated pro-inflammatory purinergic receptors and downstream signaling mediators in HIV-1 infection. This study probed whether inhibitors of purinergic receptors would reduce HIV-1 infection and HIV-1 stimulated inflammation. A human ex vivo human tonsil histo-culture infection model was developed to support HIV-1 productive infection and stimulated inflammatory cytokine interleukin-1 beta (IL-1bbeta;) and immunosuppressive cytokine, interleukin-10 (IL-10). This study tests whether inhibitors of purinergic receptors would reduce HIV-1 infection and HIV-1 stimulated inflammation. The purinergic P2X1 receptor antagonist, NF449, the purinergic P2X7 receptor antagonists, A438079, and azidothymidine (AZT) were tested in HIV-1 infected human tonsil explants to compare inhibition of HIV-1 infection and HIV-stimulated inflammatory cytokine production. All drugs limited HIV-1 productive infection, but P2X-selective antagonists (NF449, and A438079) significantly lowered HIV-stimulated IL-10 and IL-1bbeta;. We further observed that P2X1- and P2X7-selective antagonists can act differentially as inhibitors of both HIV-1 infection and HIV-1-stimulated inflammation. Our findings highlight the differential effects of HIV-1 on inflammation in peripheral blood as compared to lymphoid tissue. For the first time, we demonstrate that P2X-selective antagonists act differentially as inhibitors of both HIV-1 infection and HIV-1-stimulated inflammation. Drugs that block these pathways can have independent inhibitory activities against HIV-1 infection and HIV-induced inflammation.

    IMPORTANCE:

    Patients who are chronically infected with HIV-1 experience sequelae related to chronic inflammation. The mechanisms of this inflammation have not been elucidated. Here we describe a class of drugs that target the P2X pro-inflammatory signaling receptors in a human tonsil explant model. This model highlights differences in HIV-1 stimulation of lymphoid tissue inflammation and peripheral blood. These drugs serve to both block HIV-1 infection and production of IL-10 and IL-1bbeta; in lymphoid tissue suggesting a novel approach to HIV-1 therapeutics in which both HIV-1 replication and inflammatory signaling are simultaneously targeted.

  • Defining HIV-1 Envelope N-glycan Microdomains Through Site-specific Heterogeneity Profiles [Structure and Assembly]

  • The HIV-1 envelope (Env) glycans shield the surface of Env from the immune system and form integral interactions important for a functional Env. To understand how individual N-glycosylation sites (NGS) coordinate to form a dynamic shield and evade the immune system through mutations, we tracked 20 NGS in Env from HIV transmitted/founder (T/F) and immune-escape variants and their mutants involving the N262 glycan. NGS were profiled in a site-specific manner using a high-resolution mass spectrometry (MS)-based workflow. Using this site-specific quantitative heterogeneity profiling, we empirically characterized the interdependent NGS of a microdomain in the high-mannose patch (HMP). The changes (shifts) in NGS heterogeneity between the T/F and immune-escape variants defined a range of NGS that we further probed for exclusive combinations of sequons in the HMP microdomain using the Los Alamos HIV sequence database. The resultant sequon combinations, including the highly conserved NGS N262, N448, and N301, created an immune-escape map of the conserved and variable sequons in the HMP microdomain. This report provides details on how some clustered NGS form microdomains that can be identified and tracked across Env variants. These microdomains have a limited number of N-glycan-sequon combinations that may allow the anticipation of immune-escape variants.

    Importance

    The Env of HIV is highly glycosylated and the sites of glycosylation can change as the virus mutates during immune evasion. Due to these changes, the glycan location and heterogeneity of surrounding N-glycosylation sites can be altered resulting in exposure of different glycan or proteoglycan surfaces while still producing a viable HIV variant. These changes present a need for vaccine developers to identify Env variants with epitopes most likely to induce durable protective responses. Here, we describe a means of anticipating HIV-1 immune evasion by dividing Env into N-glycan microdomains that have a limited number of N-glycan sequon combinations.

  • Structure-based classification defines the discrete conformational classes adopted by the arenaviral GP1 [Structure and Assembly]

  • The emergence of Old and New World arenaviruses from rodent reservoirs persistently threatens human health. The GP1 subunit of the envelope-displayed arenaviral glycoprotein spike complex, GPC, mediates host-cell recognition and is an important determinant of cross-species transmission. Previous structural analyses of Old World arenaviral GP1 glycoproteins, alone and in complex with a cognate GP2 subunit, have revealed that GP1 adopts two distinct conformational states, distinguished by differences in orientation of helical regions of the molecule. Here, through comparative study of the GP1 glycoprotein architectures of Old World Loei River virus and New World Whitewater Arroyo virus, we show that these rearrangements are restricted to Old World arenaviruses and are not solely induced by the pH change that is associated with virus endosomal trafficking. Our structure-based phylogenetic analysis of arenaviral GP1s provides a blueprint for understanding the discrete structural classes adopted by these therapeutically important targets.

    IMPORTANCE The genetically and geographically diverse group of viruses within the Arenaviridae family includes a number of zoonotic pathogens capable of causing fatal hemorrhagic fever. The multi-subunit GPC glycoprotein spike complex displayed on the arenavirus envelope is a key determinant of species tropism and the primary target of the host humoral immune response. Here, we show that the receptor-binding GP1 sub-component of the GPC spike from Old World but not New World arenaviruses adopts a distinct, pH-independent conformation in the absence of the cognate GP2. Our analysis provides a structure-based approach for understanding the discrete conformational classes sampled by these therapeutically important targets, informing strategies to develop arenaviral glycoprotein immunogens that resemble GPC, as presented on the mature virion surface.

  • Influenza A virus utilizes low affinity, high avidity interactions with the nuclear import machinery to ensure infection and immune evasion [Virus-Cell Interactions]

  • The incoming influenza A virus (IAV) genome must pass through two distinct barriers in order to establish infection in the cell- the plasma membrane and the nuclear membrane. A precise understanding of the challenges imposed by the nuclear barrier remain outstanding. Passage across is mediated by host karyopherins (KPNAs), which bind to the viral nucleoprotein (NP) via its N-terminal nuclear localization sequence (NLS). Binding affinity between the two molecules is low but NP is present in high copy number, which suggests that binding avidity plays a compensatory role during import. Using nanobody-based technology, we demonstrate that high binding avidity is required for infection though the absolute value differs between cell types and correlates with their relative susceptibility to infection. In addition, we demonstrate that increasing the affinity level caused a decrease in avidity requirements for some cell types but blocked infection in others. Finally, we show that genomes that become frustrated by low avidity and remain cytoplasmic trigger the Type-I interferon response. Based on these results, we conclude that IAV balances affinity and avidity considerations in order to overcome the nuclear barrier across a broad range of cell types. Furthermore, these results provide evidence to support the long-standing hypothesis that IAV's strategy of import and replication in the nucleus facilitates immune evasion.

    Importance

    We used intracellular nanobodies to block influenza virus infection at the step prior to nuclear import of its ribonucleoproteins. By doing so, we were able to answer an important but outstanding question that could not be addressed with conventional tools- how many of the ~500 available NLS motifs are needed to establish infection? Furthermore, by controlling the sub-cellular localization of the incoming vRNPs and measuring the cell's antiviral response, we were able to provide direct evidence for the longstanding hypothesis that influenza virus exploits nuclear localization to delay activation of the innate immune response.

  • Using antibodies and mutants to localize the presumptive gH/gL binding site on HSV gD [Virus-Cell Interactions]

  • HSV virus-cell and cell-cell fusion requires multiple interactions between four essential virion envelope glycoproteins, gD, gB, gH and gL, and between gD and a cellular receptor, nectin-1 or herpesvirus entry mediator (HVEM). Current models suggest that binding of gD to receptors induces a conformational change that leads to activation of gH/gL and consequent triggering of the pre-fusion form of gB to promote membrane fusion. Since protein-protein interactions guide each step of fusion, identifying the sites of interaction may lead to the identification of potential therapeutic targets that block this process. We have previously identified two "faces" on gD: one for receptor-binding and the other for its presumed interaction with gH/gL. We previously separated the gD monoclonal antibodies (Mabs) into five competition communities. Mabs from two communities (MC2 and MC5) neutralize virus infection and block cell-cell fusion but do not block receptor binding, suggesting that they block binding of gD to gH/gL. Using a combination of classical epitope mapping of gD mutants with fusion and entry assays we identified two residues (R67 and P54) on the presumed gH/gL interaction face of gD that allowed for fusion and viral entry, but were no longer sensitive to inhibition by MC2 or MC5, yet both were blocked by other Mabs. As neutralizing antibodies interfere with essential steps in the fusion pathway, our studies strongly suggest that these key residues block the interaction of gD with gH/gL.

    Importance Virus entry and cell-cell fusion mediated by HSV require gD, gH/gL, gB and a gD receptor. Neutralizing antibodies directed against any of these proteins bind to residues within key functional sites and interfere with an essential step in the fusion pathway. Thus, the epitopes of these Mabs identify critical, functional sites on their target proteins. Unlike many anti-gD Mabs, which block binding of gD to a cellular receptor, two, MC2 and MC5, block a separate, downstream step in the fusion pathway which is presumed to be the activation of the modulator of fusion, gH/gL. By combining epitope mapping of a panel of gD mutants with fusion and virus entry assays, we have identified residues that are critical in the binding and function of these two Mabs. This new information helps to define the site of the presumptive interaction of gD with gH/gL of which we have limited knowledge.

  • Engineered small molecule control of influenza A virus replication [Vaccines and Antiviral Agents]

  • Influenza A virus (IAV) remains a global health concern despite the availability of a seasonal vaccine. It is difficult to predict which strains will circulate during influenza season, and therefore extremely challenging to test novel vaccines in the human population. To overcome this obstacle, new vaccines must be tested in challenge studies. This approach poses significant safety problems, as current pharmacological interventions for IAV are poorly efficacious. New methods are needed to enhance the safety of these challenge studies. Here, we generate a virus expressing a small molecule-assisted shutoff (SMASh) tag as a safety switch for IAV replication. Addition of the SMASh tag to an essential IAV protein allows for small molecule-mediated inhibition of replication. Treatment with this drug controls SMASh-tagged virus replication in vitro and in vivo. This model for restriction of viral replication has potential for broad applications in vaccine studies, virotherapy, and basic virus research.

    Importance Influenza A virus (IAV) causes significant morbidity and mortality worldwide annually, despite the availability of new formulations of the vaccine each season. There is a critical need to develop more efficacious vaccines. However, testing novel vaccines in the human population in controlled studies is difficult due to the limited availability and efficacy of intervention strategies should the vaccine fail. There are also significant safety concerns for work with highly pathogenic IAV strains in the laboratory. Therefore, novel strategies are needed to improve the safety of vaccine studies and highly pathogenic IAV research. Here we developed an IAV strain engineered to contain a small molecule-mediated safety switch. This tag, when attached to an essential viral protein, allows for regulation of IAV replication in vitro and in vivo. This strategy provides a platform for regulation of virus replication without targeting viral proteins directly.

  • PI3K/Akt and MEK/ERK signaling pathways facilitate sapovirus trafficking and late endosomal acidification for viral uncoating in LLC-PK cells [Virus-Cell Interactions]

  • Sapovirus, an important cause of acute gastroenteritis in humans and animals, travels from the early to the late endosomes, and requires late endosomal acidification for viral uncoating. However, the signaling pathways responsible for these viral entry processes remain unknown. Here, we demonstrate the receptor-mediated early activation of PI3K/Akt and MEK/ERK signaling pathways involved in sapovirus entry processes. Both signaling pathways were activated during the early stage of porcine sapovirus (PSaV) infection. However, depletion of the cell surface carbohydrate receptors by pretreatment of sodium periodate or neuraminidase reduced the PSaV-induced early activation of these signaling pathways, indicating that PSaV-binding to the cell surface carbohydrate receptors triggered these cascades. Addition of bile acid, known to be essential for PSaV escape from late endosomes, was also found to exert a stiffening effect to stimulate both pathways. Inhibition of these signaling pathways by inhibitors specific for PI3K or MEK, or siRNAs against PI3K or MEK resulted in entrapment of PSaV particles in early endosomes and prevented their trafficking to late endosomes. Moreover, phosphorylated PI3K and ERK co-immunoprecipitated subunit E of the V-ATPase proton pump that is important for endosomal acidification. Our data conclude that receptor-binding of PSaV activates both PI3K/Akt and MEK/ERK signaling pathways, which in turn promote PSaV trafficking from early to late endosomes and acidification of late endosomes for PSaV uncoating. These signaling cascades may provide a target for potent therapeutics against infections by PSaV and other caliciviruses.

    IMPORTANCE Sapoviruses cause acute gastroenteritis in both humans and animals. However, the host signaling pathway(s) that facilitate host cell entry by sapoviruses remains largely unknown. Here we demonstrate that porcine sapovirus (PSaV) activates both PI3K/Akt and MEK/ERK cascades at early stage of infection. Removal of cell surface receptors decreased PSaV-induced early activation of both cascades. Moreover, blocking of PI3K/Akt and MEK/ERK cascades entrapped PSaV particles in early endosomes and prevented their trafficking to the late endosomes. PSaV-induced early activation of PI3K and ERK molecules further mediated V-ATPase-dependent late endosomal acidification for PSaV uncoating. This work unravels a new mechanism by which receptor-mediated early activation of both cascades could facilitate PSaV trafficking from early to late endosomes and late endosomal acidification for PSaV uncoating, which in turn can be a new target for treatment of sapovirus infection.

  • Amino acid residue 217 in the hemagglutinin glycoprotein is a key mediator of avian influenza H7N9 virus antigenicity [Genetic Diversity and Evolution]

  • Avian influenza viruses continue to evolve and acquire mutations that facilitate antigenic drift and virulence change. In 2017, low pathogenicity H7N9 avian influenza viruses evolved to a high pathogenicity phenotype in China. The comparative antigenic analysis between the low and high pathogenicity virus strains showed marked variability. In order to identify residues that may be linked to the antigenic change amongst the H7N9 viruses, we serially passaged the viruses in the presence of homologous ferret antisera. Progeny viruses able to overcome the neutralising capacity of the antisera were sequenced. The analysis showed that the emergent immune escaped viruses contained mutations A125T, A151T and L217Q in the hemagglutinin (HA) glycoprotein as early as passage 5 and these mutations persisted until passage 10. The results revealed that a single mutation L217Q in the HA of H7N9 virus led to 23- and 8-fold reductions in hemagglutination inhibition (HI) titre with ferret and chicken antisera, respectively. Further analysis showed that this change also contributed to antigenic differences between the low and high pathogenicity H7N9 viruses, therein, playing a major role in their antigenic diversification. Therefore, evolutionary changes at amino acid position 217 in the H7N9 viruses can serve as a genetic marker for virus antigenic diversity during vaccine seed matching and selection. The in vitro immune escape mutant selection method used in this study could also aid in the prediction of emerging antigenic variants in the naturally infected or immunised animals.

    IMPORTANCE Avian influenza H7N9 viruses circulating in poultry and wild birds continue to evolve and acquire important phenotypic changes. Mutations to the virus hemagglutinin (HA) glycoprotein can modulate virus antigenicity and facilitate virus escape from natural or vaccine-induced immunity. The focus of this study was to identify evolutionary markers in the HA of H7N9 that drive escape from antibody-based immunity. To achieve this, we propagated low pathogenicity H7N9 virus in the presence of polyclonal antisera derived from ferrets infected with the same strain of virus (homologous antisera). This selection process was repeated 10 times. The HA gene sequence of viruses recovered after the 5th passage showed that the viruses readily acquired mutation at three different amino acid positions (A125T, A151T and L217Q). Further functional analysis of these mutations confirmed that mutation at residue HA 217 was responsible for mediating changes to the immunological properties of the H7N9 virus.

  • Exosomes exploit the virus entry machinery and pathway to transmit IFN-{alpha}-induced antiviral activity. [Virus-Cell Interactions]

  • Interferon-aalpha; (IFN-aalpha;) induces the transfer of resistance to hepatitis B virus (HBV) from liver nonparenchymal cells (LNPCs) to hepatocytes via exosomes. However, little is known about the entry machinery and pathway involved in the transmission of IFN-aalpha;-induced antiviral activity. Here, we found that macrophage exosomes uniquely depend on T cell immunoglobulin and mucin receptor 1 (TIM-1), a hepatitis A virus (HAV) receptor, to enter hepatocytes for delivering IFN-aalpha;-induced anti-HBV activity. Moreover, two primary endocytic routes for virus infection, clathrin-mediated endocytosis (CME) and macropinocytosis, collaborate to permit exosome entry and anti-HBV activity transfer. Subsequently, lysobisphosphatidic acid (LBPA), an anionic lipid closely related to endosome penetration of virus, facilitates membrane fusion of exosomes in late endosomes/multivesicular bodies (LEs/MVBs) and the accompanying exosomal cargo uncoating. Together, this study provides comprehensive insights into the transmission route of macrophage exosomes to efficiently deliver IFN-aalpha;-induced antiviral substances and highlights the similarities between the entry mechanisms of exosomes and virus.

    Importance Our previous study showed that LNPC-derived exosomes could transmit IFN-aalpha;-induced antiviral activity to HBV replicating hepatocytes, but the concrete transmission mechanisms which include exosome entry and exosomal cargo release remain unclear. In this study, we found that virus entry machinery and pathway were also applied to exosome-mediated cell-to-cell antiviral activity transfer. Macrophage-derived exosomes distinctively exploit hepatitis A virus receptor for access to hepatocytes. Later, CME and macropinocytosis are utilized by exosomes which is followed by exosome-endosome fusion for efficient transfer of IFN-aalpha;-induced anti-HBV activity. We believe that understanding the cellular entry pathway of exosomes will be beneficial to designing exosomes as efficient vehicles for antiviral therapy.

  • The Nonstructural Protein 11-kDa of Human Parvovirus B19 Facilitates Viral DNA Replication by Interacting with Grb2 through Its Proline-rich Motifs [Virus-Cell Interactions]

  • Lytic infection of human parvovirus B19 (B19V) takes place exclusively in human erythroid progenitor cells of bone marrow and fetal liver, which disrupts erythropoiesis. During infection, B19V expresses three nonstructural proteins (NS1, 11-kDa, and 7.5-kDa) and two structural proteins (VP1 and VP2). While NS1 is essential for B19V DNA replication, the 11-kDa enhances viral DNA replication significantly. In this study, we confirmed the enhancement role of the 11-kDa in viral DNA replication and elucidated the underlying mechanism. We found that 11-kDa specially interacts with cellular growth factor receptor-bound protein 2 (Grb2) during virus infection and in vitro. We determined a high affinity interaction between 11-kDa and Grb2 that has an equilibrium dissociation constant (KD) value of 18.13 nM. In vitro, one proline-rich motif was sufficient for 11-kDa to sustain a strong interaction with Grb2. In consistence, in vivo during infection, one proline-rich motif was enough for 11-kDa to significantly reduce phosphorylation of extracellular signal-regulated kinase (ERK). Mutations of all three proline-rich motifs of the 11-kDa abolished its capability to reduce ERK activity, and accordingly, decreased viral DNA replication. Transduction of a lentiviral vector encoding an shRNA targeting Grb2 decreased the expression of Grb2 as well as the level of ERK phosphorylation, which results in an increase of B19V replication. These results, in concert, indicate that the B19V 11-kDa interacts with cellular Grb2 to downregulate ERK activity, which upregulates viral DNA replication.

    SIGNIFICANCE Human parvovirus B19 (B19V) infection causes hematological disorders and is the leading cause of non-immunological fetal hydrops during pregnancy. During infection, B19V expresses two structural proteins VP1/VP2 and three nonstructural proteins, NS1, 11-kDa, and 7.5-kDa. While NS1 is essential, 11-kDa plays an enhancing role in viral DNA replication. Here we elucidated a mechanism underlying the 11-kDa regulated B19V DNA replication. 11-kDa is tightly associated with cellular growth factor receptor-bound protein 2 (Grb2) during infection. In vitro, 11-kDa interacts with Grb2 at a high affinity through three proline-rich motifs, of which at least one is indispensable for the regulation of viral DNA replication. 11-kDa and Grb2 interaction disrupts the extracellular signal-regulated kinase (ERK) signaling, which mediates upregulation of B19V replication. Thus, our study reveals a novel mechanism of how a parvoviral small nonstructural protein regulates viral DNA replication through interacting with a host protein that is predominately expressed in the cytoplasm.

  • HSV-1 latency and the kinetics of reactivation are regulated by a complex network of interactions between HVEM, its ligands (gD, BTLA, LIGHT and CD160) and LAT [Pathogenesis and Immunity]

  • Recently, we reported that the herpesvirus entry mediator (HVEM; TNFRSF14: CD270) is upregulated by the latency-associated transcript (LAT) of HSV-1 and that the absence of HVEM affects latency-reactivation but not primary infection in ocularly infected mice. gD has been shown to bind to HVEM. LIGHT (TNFSF14), CD160 and BTLA (B- and T-lymphocyte attenuator) also interact with HVEM and can interfere with HSV gD binding. It was not known if LIGHT, CD160 or BTLA affected the level of latency-reactivation in the trigeminal ganglia (TG) of latently infected mice. To address this issue, we ocularly infected LIGHT-/-, CD160-/- and BTLA-/- mice with LAT(+) and LAT(-) viruses using similarly infected wild-type (WT) and HVEM-/- mice as controls. The amount of latency, as determined by the levels of gB DNA in the TG of the LIGHT-/-, CD160-/- and BTLA-/- mice infected with either LAT(+) or LAT(-) viruses was lower than that in WT mice infected with LAT(+) virus and was similar in WT mice infected with LAT(-) virus. The levels of LAT RNA in HVEM-/-, LIGHT-/-, CD160-/- and BTLA-/- mice infected with LAT(+) virus were similar and were lower than the levels of LAT RNA in WT mice. However, LIGHT-/-, CD160-/- and BTLA-/- mice, independent of the presence of LAT, had similar levels of reactivation as WT mice infected with LAT(+) virus. Faster reactivation correlated with the upregulation of HVEM transcript. The LIGHT-/-, CD160-/- and BTLA-/- mice had higher levels of HVEM expression and this, along with the absence of BTLA, LIGHT or CD160, may contribute to faster reactivation, while the absence of each molecule and independent of LAT may have contributed to lower latency. This study suggests that, in the absence of competition with gD for binding to HVEM, LAT RNA is important for WT levels of latency but not for WT levels of reactivation.

    Importance The effects of BTLA, LIGHT and CD160 on latency-reactivation are not known. We show here that in BTLA, LIGHT or CD160 null mice, latency is reduced; however, HVEM expression is upregulated as compared to WT mice and this upregulation is associated with higher reactivation that is independent of LAT but dependent on gD expression. Thus, one of the mechanisms by which BTLA, LIGHT and CD160 null mice enhance reactivation appears to be the increased expression of HVEM in the presence of gD. Thus, our results suggest that blockade of HVEM-LIGHT-BTLA-CD160 could contribute to reduced HSV-1 latency and reactivation.

  • Salinomycin Inhibits Influenza Virus Infection by Disrupting Endosomal Acidification and Viral Matrix Protein 2 Function [Vaccines and Antiviral Agents]

  • Screening of chemical libraries with 2,000 synthetic compounds identified salinomycin as a hit against influenza A and B viruses with 50% effective concentrations ranging from 0.4 to 4.3 mmu;M in cells. This compound is a carboxylic polyether ionophore that exchanges monovalent ions for protons across lipid bilayer membranes. Monitoring the time course of viral infection showed that salinomycin blocked nuclear migration of viral nuclear protein (NP) that is the most abundant component of the viral ribonucleoprotein (vRNP) complex. It caused cytoplasmic accumulation of NP, particularly within perinuclear endosomes, during virus entry. This was primarily associated with failure to acidify the endosomal-lysosomal compartments. Similar to amantadine (AMT), proton channel activity of viral matrix protein 2 (M2) was blocked by salinomycin. Using purified retroviral Gag-based virus-like particles (VLPs) with M2, it was proved that salinomycin directly affect the kinetics of a proton influx into the particles but in a different manner from that of AMT. Notably, oral administration of salinomycin together with the neuraminidase inhibitor oseltamivir phosphate (OSV-P) led to enhanced antiviral effect over either compound used alone in influenza A virus-infected mouse models. These results provide a new paradigm for developing antivirals and their combination therapy that control both host and viral factors.

    IMPORTANCE Influenza virus is a main cause of viral respiratory infection in humans as well as animals, occasionally with high mortality. Circulation of influenza viruses resistant to the matrix protein 2 (M2) inhibitor, amantadine, is highly prevalent. Moreover, detection frequency of viruses resistant to the neuraminidase inhibitors including oseltamivir-phosphate (OSV-P) or zanamivir is also increasing. These issues highlight the need for discovery of new antiviral agents with different mechanisms. Salinomycin as the monovalent cation-proton antiporter exhibited consistent inhibitory effects against influenza A and B viruses. It plays multifunctional roles by blocking endosomal acidification and by inactivating the proton transport function of M2, the key steps for influenza viral uncoating. Notably, salinomycin resulted in marked therapeutic effects in influenza virus-infected mice when combined with OSV-P, suggesting that its chemical derivatives could be developed as an adjuvant antiviral therapy to treat influenza infections resistant or less sensitive to existing drugs.

  • Dynamin is required for efficient cytomegalovirus maturation and envelopment [Structure and Assembly]

  • Cytomegalovirus secondary envelopment occurs in a virus-induced cytoplasmic assembly compartment (vAC) generated via a drastic reorganization of the membranes of the secretory and endocytic systems. Dynamin is a eukaryotic GTPase that is implicated in membrane remodeling and endocytic membrane fission events; however, the role of dynamin in cellular trafficking of viruses beyond virus entry is only partially understood. Mouse embryonic fibroblasts (MEF) engineered to excise all three isoforms of dynamin were infected with mouse cytomegalovirus (MCMV-K181). Immediate early (IE1; m123) viral protein was detected in these triple dynamin knockout (TKO) cells as well as in mock-induced parental MEF at early times post infection although levels were reduced in TKO cells, indicating that virus entry was affected but not eliminated. Levels of IE1 protein and another viral early protein (m04) were normalized by 48 hours post infection; however, late protein (m55; gB) expression was reduced in infected TKO cells compared to parental MEF. Ultrastructural analysis revealed intact stages of nuclear virus maturation in both cases with equivalent numbers of nucleocapsids containing packaged viral DNA (C-capsids) indicating successful viral DNA replication, capsid assembly and genome packaging. Most importantly, severe defects in virus envelopment were visualized in TKO cells but not in parental cells. Dynamin inhibitor (dynasore) treated MEF showed a phenotype similar to TKO cells upon MCMV infection confirming the role of dynamin in late maturation processes. In summary, dynamin-mediated endocytic pathways are critical for the completion of cytoplasmic stages of cytomegalovirus maturation.

    Importance

    Viruses are known to exploit specific cellular functions at different stages of their life cycle in order to replicate, avoid immune recognition by the host and to establish a successful infection. Cytomegalovirus (CMV) infected cells are characterized by a prominent cytoplasmic inclusion (virus assembly compartment; vAC) that is the site of virus maturation and envelopment. While endocytic membranes are known to be the functional components of vAC, knowledge of specific endocytic pathways implicated in CMV maturation and envelopment is lacking. Here we show that dynamin, which is an integral part of host endocytic machinery, is largely dispensable for early stages of CMV infection but is required at a late stage of CMV maturation. Studies on dynamin function in CMV infection will help us understand the host-virus interaction pathways amenable to targeting by conventional small molecules as well as by newer generation nucleotide-based therapeutics (e.g. siRNA, CRISPR/CAS gRNA, etc.).

  • Remodeling mTORC1 responsiveness to amino acids by the herpes simplex virus UL46 and Us3 gene products supports replication during nutrient insufficiency [Genome Replication and Regulation of Viral Gene Expression]

  • By sensing fundamental parameters including nutrient availability, activated mTORC1 suppresses catabolic outcomes and promotes anabolic processes needed for HSV-1 productive growth. While the virus-encoded Us3 ser/thr kinase is required to activate mTORC1, whether stress associated with amino acid insufficiency impacts mTORC1 activation in infected cells and virus reproduction was unknown. In contrast to uninfected cells where amino acid withdrawal inhibits mTORC1 activation, we demonstrate that mTORC1 activity is sustained in HSV-1-infected cells during amino acid insufficiency. In the absence of Us3, we show that the insensitivity of mTORC1 to amino acid withdrawal in infected cells was dependent on the host kinase Akt, and establish a role for the HSV-1 UL46 gene product, which stimulates PI-3kinase signaling. Significantly, virus reproduction during amino acid insufficiency was stimulated by the viral UL46 gene product. By synergizing with Us3, UL46 reprograms mTORC1 such that it is insensitive to amino acid withdrawal and supports sustained mTORC1 activation and virus reproduction during amino acid insufficiency. This identifies an unexpected function for UL46 in supporting virus reproduction during physiological stress and identifies a new class of virus-encoded mTORC1 regulators that selectively uncouple mTORC1 activation from amino acid sufficiency.

    IMPORTANCE The mechanistic target of rapamycin complex 1 (mTORC1) is a multisubunit cellular kinase that coordinates protein synthesis with changing amino acid levels. During amino acid insufficiency, mTORC1 is repressed in uninfected cells, dampening protein synthesis and potentially restricting virus reproduction. Here, we establish that HSV-1 alters the responsiveness of mTORC1 to metabolic stress resulting from amino acid insufficiency. Unlike uninfected cells, mTORC1 remains activated in HSV-1-infected cells deprived of amino acids. Synergistic action of the HSV-1 UL46 gene product, which stimulates PI-3 kinase, and the Us3 kinase supports virus reproduction during amino acid withdrawal. These results define how HSV-1, a medically important human pathogen associated with a range of diseases, uncouples mTORC1 activation from amino acid availability. Furthermore, they help explain how the virus reproduces during physiological stress. Reproduction triggered by physiological stress is characteristic of herpesvirus infections, where lifelong latency is punctuated by episodic reactivation events.

  • The abundant tegument protein pUL25 of human cytomegalovirus prevents proteasomal degradation of pUL26 and supports its suppression of ISGylation [Virus-Cell Interactions]

  • The tegument of HCMV virions contains proteins that interfere with both the intrinsic and the innate immunity. One protein with thus far unknown function is pUL25. The deletion of pUL25 in a viral mutant (Towne-UL25) had no impact on the release of virions and subviral dense bodies or on virion morphogenesis. Proteomic analyses showed little alterations in the overall protein composition of extracellular particles. A surprising result, however, was the almost complete absence of pUL26 in virions and dense bodies of Towne-UL25 and a reduction of the large isoform pUL26-p27 in mutant virus infected cells. The pUL26 was shown to inhibit protein conjugation with the interferon-stimulated-gene 15 protein (ISG15), thereby supporting HCMV replication. To test a functional relationship between pUL25 and pUL26, we addressed the steady-state levels of pUL26 and found them reduced in Towne-UL25-infected cells. Co-immunoprecipitation experiments proved an interaction between pUL25 and pUL26. Surprisingly, the overall protein ISGylation was enhanced in Towne-UL25-infected cells, thus mimicking the phenotype of a pUL26-deleted HCMV mutant. The functional relevance of this was confirmed by showing that the replication of Towne-UL25 was more sensitive to IFN-bbeta;. The increase of protein ISGylation was also seen in cells infected with a mutant lacking the tegument protein pp65. Upon retesting, we found that pUL26 degradation was also increased when pp65 was unavailable. Our experiments show that both pUL25 and pp65 regulate pUL26-degradation and pUL26-dependent reduction of ISGylation and add pUL25 as another HCMV tegument protein that interferes with the intrinsic immunity of the host cell.

    IMPORTANCE Human cytomegalovirus (HCMV) expresses a number of tegument proteins that interfere with the intrinsic and the innate defense mechanisms of the cell. Initial induction of the interferon-stimulated-gene 15 protein (ISG15) and conjugation of proteins with ISG15 (ISGylation) by HCMV infection is subsequently attenuated by the expression of the viral IE1, pUL50, and pUL26 proteins. This study adds pUL25 as another factor that contributes to suppression of ISGylation. The tegument protein interacts with pUL26 and prevents its degradation by the proteasome. By this it supports its restrictive influence on ISGylation. In addition, lack of pUL25 enhances levels of free ISG15, indicating that the tegument protein may interfere with the interferon response on levels other than interacting with pUL26. Knowledge obtained in this study widens our understanding of HCMV immune evasion and may also provide a new avenue for the use of pUL25 negative strains for vaccine production.

  • Mapping the nonstructural protein interaction network of porcine reproductive and respiratory syndrome virus [Structure and Assembly]

  • Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-stranded RNA virus belonging to the family Arteriviridae. Synthesis of the viral RNA is directed by replication/transcription complexes (RTC) that are composed of mainly a network of PRRSV nonstructural proteins (nsps) and likely cellular proteins. Here we mapped the interaction network among PRRSV nsps by using yeast two hybrid screening in conjunction with co-immunoprecipitation (Co-IP) and co-transfection assays. We identified a total of 24 novel interactions and found that the interactions were centered on ORF1b-encoded nsps that were mainly connected by the transmembrane proteins nsp2, nsp3 and nsp5. Interestingly, the interactions of the core enzymes nsp9 and nsp10 with transmembrane proteins did not occur in a straightforward manner, as they worked in the Co-IP assay but were poorly capable of finding each other within intact mammalian cells. Further proof that they can interact within cells required the engineering of N-terminal truncations of both nsp9 and nsp10. However, despite the poor co-localization relationship in co-transfected cells, both nsp9 and nsp10 came together with membrane proteins (e.g., nsp2) at the viral RTC in PRRSV-infected cells. Thus, our results indicate the existence of a complex interaction network among PRRSV nsps and raise the possibility that the recruitment of key replicase proteins to membrane-associated nsps may involve some regulatory mechanisms during infection.

    Importance

    Synthesis of PRRSV RNAs within host cells depends on the efficient and correct assembly of replication and transcription complexes (RTC) that takes places on modified intracellular membranes. As an important step toward dissecting this poorly understood event, we investigated the interaction network among PRRSV nsps. Our studies established a comprehensive interaction map for PRRSV nsps and revealed important players within the network. The results also highlight the likely existence of a regulated recruitment of PRRSV core enzymes nsp9 and nsp10 to viral membrane nsps during PRRSV RTC assembly.

  • Targeting of the nasal mucosa by Japanese encephalitis virus for non-vector-borne transmission [Pathogenesis and Immunity]

  • The mosquito-borne Japanese encephalitis virus (JEV) causes severe central nervous system diseases, and cycles between Culex mosquitoes and different vertebrates. For JEV and some other flaviviruses oronasal transmission is described, but the mode of infection is unknown. Using nasal mucosal tissue explants and primary porcine nasal epithelial cells (NEC) at the air-liquid interface (ALI) and macrophages as ex vivo and in vitro models, our results indicate that the nasal epithelium could represent the route of entry and exit for JEV in pigs. Porcine NEC at the ALI exposed to with JEV resulted in apical and basolateral virus shedding, release of monocyte recruiting chemokines, indicating infection and replication in macrophages. Moreover, macrophages stimulated by alarmins, including interleukin-25, interleukin-33 and thymic stromal lymphopoietin, were more permissive to the JEV infection. Altogether, our data are important to understand the mechanism of non-vector born direct transmission of Japanese encephalitis virus in pigs.

    Importance JEV, a main cause of severe viral encephalitis in humans, has a complex ecology composed of a mosquito-waterbird cycle and a cycle involving also pigs. Which amplifies virus transmission to mosquitoes, leading to increased human cases. JEV can be transmitted between pigs by contact in the absence of arthropod vectors. Moreover, virus or viral RNA is found in oronasal secretions and the nasal epithelium. Using nasal mucosa tissue explants and three-dimensional porcine nasal epithelial cells cultures and macrophages as ex vivo and in vitro models, this study indicates that the nasal epithelium could be a route of entry as well as exit for the virus. Infection of nasal epithelial cells resulted in apical and basolateral virus shedding, release of monocyte recruiting chemokines, therefore infection and replication in macrophages, which is favored by epithelial-cell derived cytokines. The results are relevant to understand the mechanism of non-vector borne direct transmission of JEV.

  • Non-pathogenic heterologous prions can interfere with prion infection in a strain dependent manner [Prions]

  • Co-occurrence of different prion strains into the same host has been recognized as a natural phenomenon for several sporadic Creutzfeldt-Jakob disease (sCJD) patients and natural scrapie cases. The final outcome of prion co-infection is not easily predictable. In addition to the usual factors that influence prion conversion, the replication of one strain may entail positive or negative consequences to the other.

    The main aim of this study was to gain insights into the prion co-infection and interference concepts and their potential therapeutic implications. Herein, different mouse models were challenged with several combinations of prion strains coupled on the basis of the length of their incubation periods and the existence/absence of species barrier in the tested animal model.

    We found that non-transmissible strains can interfere the replication of fully transmissible strains when there is a species transmission barrier involved, as it happened with the combination of a mouse (22L) and a human (sCJD) strain. However, this phenomenon seems to be strain-dependent since no interference was observed when the human strain co-inoculated was vCJD.

    For the other combinations tested in this study, the results suggest that both strains replicate independently without effect on the replication of one over the other. It is common that the strain with more favorable conditions (higher speed of disease development or absence of species barrier) ends being the only one detectable at the terminal stage of the disease. However, this does not exclude the replication of the least favored strain, leading to situations of co-existence of prion strains.

    IMPORTANCE As a general conclusion, the outcome of prion co-infection is strongly dependent on the strain combination and the model utilized and therefore, difficult to predict.

    Co-existence of several prion strains may remain undetected if one of the strains has more favorable conditions to replicate in the host. The use of several models (as transgenic mouse expressing PrP from different species) to analyze field prion isolates is recommended to avoid this situation.

    The inference effect exerted by non-replicative prion strains should be consider as an interesting tool to advance in new therapeutic strategies for treating prion diseases, or even be a proper therapeutic strategy.

  • Extracellular HBV RNAs are heterogeneous in length and circulate as capsid-antibody-complexes in addition to virions in chronic hepatitis B patients [Genome Replication and Regulation of Viral Gene Expression]

  • Extracellular HBV RNA has been detected in both HBV-replicating cell culture media and sera from chronic hepatitis B (CHB) patients, but its exact origin and composition remain controversial. Here, we demonstrated that extracellular HBV RNA species were of heterogeneous lengths, ranging from the length of pregenomic RNA to a few hundred nucleotides. In cell models, these RNAs were predominantly associated with naked capsids although virions also harbored a minority of them. Moreover, HBV RNAs in hepatitis B patientsrrsquo; blood circulation were localized in unenveloped capsids in the form of capsid-antibody-complexes (CACs) and in virions. Furthermore, we showed that extracellular HBV RNAs could serve as template for viral DNA synthesis. In conclusion, extracellular HBV RNAs mainly consist of pgRNA or the pgRNA species degraded by the RNase H domain of the polymerase in the process of viral DNA synthesis and circulate as CACs and virions. Their presence in the blood circulation of CHB patients may be exploited to develop novel biomarkers for HBV persistence.

    Importance Although increasing evidence suggests the presence of extracellular HBV RNA species, their origin and molecular forms are still under debate. In addition to the infectious virions, HBV is known to secrete several species of incomplete viral particles, including hepatitis B surface antigen (HBsAg) particles, naked capsids and empty virions during its replication cycle. Here, we demonstrated that extracellular HBV RNAs were associated with naked capsids and virions in HepAD38 cells. Interestingly, we found that unenveloped capsids circulate in the blood of hepatitis B patients in the form of capsids-antibody-complexes (CACs) and, together with virions, serve as vehicles carrying these RNAs molecules. Moreover, extracellular HBV RNAs are heterogeneous in length and represent either pregenomic RNA (pgRNA) or products of incomplete reverse transcription during viral replication. These findings provide a conceptual basis for further application of extracellular RNA species as novel biomarkers for HBV persistence.

  • Influenza Virus NS1 Protein RNA-Interactome Reveals Intron Targeting [Virus-Cell Interactions]

  • The NS1 protein of influenza A virus is a multifunctional virulence factor that inhibits cellular processes to facilitate viral gene expression. While NS1 is known to interact with RNA and proteins to execute these functions, the cellular RNAs that physically interact with NS1 have not been systematically identified. Here we reveal a NS1 protein-RNA interactome and show that NS1 primarily binds intronic sequences. Among this subset of pre-mRNAs is the RIG-I pre-mRNA, which encodes the main cytoplasmic antiviral sensor of influenza virus infection. This suggested that NS1 interferes with the antiviral response at a posttranscriptional level by virtue of its RNA binding properties. Indeed, we show that NS1 is necessary in the context of viral infection and sufficient upon transfection to decrease the rate of the RIG-I intron removal. This NS1 function requires a functional RNA binding domain and is independent of the NS1 interaction with the cleavage and polyadenylation specificity factor CPSF30. NS1 has been previously shown to abrogate RIG-I-mediated antiviral immunity by inhibiting its protein function. Our data further suggest that NS1 also posttranscriptionally alters RIG-I pre-mRNA processing by binding to the RIG-I pre-mRNA.

    IMPORTANCE

    A key virulence factor of influenza A virus is the NS1 protein, which inhibits various cellular processes to facilitate viral gene expression. NS1 protein is localized in the nucleus and in the cytoplasm during infection. In the nucleus, NS1 has functions related to inhibition of gene expression that involve protein-protein and protein-RNA interactions. While several studies have elucidated the protein interactome of NS1, we still lack a clear and systematic understanding of the NS1-RNA interactome. Here we reveal a nuclear NS1 RNA interactome and show that NS1 primarily binds intronic sequences within a subset of pre-mRNAs, including the RIG-I pre-mRNA that encodes the main cytoplasmic antiviral sensor of influenza virus infection. Our data here further suggest that NS1 is necessary and sufficient to impair intron processing of the RIG-I pre-mRNA. These findings support a posttranscriptional role for NS1 in the inhibition of RIG-I expression.

  • Virulent PRV infection induces a specific and lethal systemic inflammatory response in mice [Pathogenesis and Immunity]

  • Pseudorabies virus (PRV) is an alphaherpesvirus that infects the peripheral nervous system (PNS). The natural host of PRV is the swine, but it can infect most mammals, including cattle, rodents and dogs. In these non-natural hosts, PRV always causes a severe acute and lethal neuropathy called the "mad itch", which is uncommon in swine. So far, the pathophysiological and immunological processes leading to the development of the neuropathic itch, and death of the animal are unclear.

    Using a footpad inoculation model, we established that mice inoculated with PRV-Becker (virulent strain) develop a severe pruritus in the foot and become moribund at 82 hours post-inoculation (hpi). We found necrosis and inflammation with a massive neutrophil infiltration only in the footpad and DRGs by Haamp;E staining. PRV load was detected in the foot, PNS and CNS tissues by quantitative-RT-PCR. Infected mice had elevated plasma levels of pro-inflammatory cytokines (IL6 and G-CSF) and chemokines (Gro-1 and MCP-1). Significant IL6 and G-CSF levels were detected in several tissues at 82hpi. High plasma levels of C-reactive protein confirmed the acute inflammatory response to PRV-Becker infection. Moreover, mice inoculated with PRV-Bartha (attenuated, live vaccine strain), did not develop pruritus at 82hpi. PRV-Bartha also replicated in the PNS, infection spread further in the brain than PRV-Becker. PRV-Bartha infection did not induce the specific and lethal systemic inflammatory response seen with PRV-Becker. Overall, we demonstrated the importance of inflammation in the clinical outcome of PRV infection in mice and provide new insights into the process of PRV-induced neuroinflammation.

    Importance

    Pseudorabies virus (PRV) is an alphaherpesvirus related to human pathogens such as herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV). The natural host of PRV is the swine but it can infect most mammals. In susceptible animals other than pigs, PRV infection always causes a characteristic lethal pruritus known as the "mad itch". The role of the immune response in the clinical outcome of PRV infection is still poorly understood. Here, we show that a systemic host inflammatory response is responsible for the severe pruritus and acute death of mice infected with virulent PRV-Becker but not attenuated strain PRV-Bartha. In addition, we identified IL-6 and G-CSF as two main cytokines that play crucial roles in the regulation of this process. Our findings give new insights into neuroinflammatory diseases and strengthen further the similarities between VZV and PRV infections at the level of innate immunity.

  • Lysosomal proteases are a determinant of coronavirus tropism [Virus-Cell Interactions]

  • Cell entry of coronaviruses involves two principal steps: receptor binding and membrane fusion, the latter of which requires activation by host proteases, particularly lysosomal proteases. Despite the importance of lysosomal proteases in both coronavirus entry and cell metabolism, the correlation between lysosomal proteases and cell tropisms of coronaviruses has not been critically established. Here we examined the roles of lysosomal proteases in activating coronavirus-surface spike proteins for membrane fusion, using the spike proteins from SARS and MERS coronaviruses as the model system. To this end, we controlled the contributions from receptor binding and other host proteases, thereby attributing coronavirus entry solely or mainly to the efficiency of lysosomal proteases in activating coronavirus-spike-mediated membrane fusion. Our results showed that lysosomal proteases from bat cells support coronavirus-spike-mediated pseudovirus entry and cell-cell fusion more effectively than their counterparts from human cells. Moreover, purified lysosomal extracts from bat cells cleave cell-surface-expressed coronavirus spike proteins more efficiently than their counterparts from human cells. Overall, our study suggests that differential lysosomal protease activities from different host species and tissue cells are an important determinant of the species and tissue tropism of coronaviruses.

    Significance

    Coronaviruses are capable of colonizing new species, as evidenced by the recent emergence of SARS and MERS coronaviruses; they can also infect multiple tissues in the same species. Lysosomal proteases play critical roles in coronavirus entry by cleaving coronavirus-surface spike proteins and activating the fusion of host and viral membranes; they also play critical roles in cell physiology by processing cellular products. How do differential lysosomal protease activities from different cells impact coronavirus entry? Here we controlled the contributions from known factors that function in coronavirus entry, such that lysosomal protease activities became the only or main determinant of coronavirus entry. Using pseudovirus entry, cell-cell fusion, and biochemical assays, we showed that lysosomal proteases from bat cells activate coronavirus-spike-mediated membrane fusion more efficiently than their counterparts from human cells. Our study provides the first direct evidence supporting lysosomal proteases as a determinant of the species and tissue tropism of coronaviruses.

  • Comparison of the immunogenicity and cross-lineage efficacy of live attenuated peste des petits ruminants virus vaccines PPRV/Nigeria/75/1 and PPRV/Sungri/96. [Vaccines and Antiviral Agents]

  • Peste des petits ruminants (PPR) is a severe disease of goats and sheep that is widespread in Africa, the Middle East and Asia. Several effective vaccines exist for the disease, based on attenuated strains of the virus (PPRV) that causes PPR. While the efficacy of these vaccines has been established by use in the field, the nature of the protective immune response has not been determined. In addition, while the vaccine derived from PPRV/Nigeria/75/1 (N75) is used in many countries, those developed in India have never been tested for their efficacy outside that country. We have studied the immune response in goats to vaccination with either N75 or the main Indian vaccine, which is based on isolate PPRV/India/Sungri/96 (S96). In addition, we have compared the ability of these two vaccines, in parallel, to protect animals against challenge with pathogenic viruses from the four known genetic lineages of PPRV, representing viruses from different parts of Africa as well as Asia. These studies showed that, while N75 elicited a stronger antibody response than S96, as measured by both ELISA and virus neutralisation, S96 resulted in more pronounced cellular immune responses, as measured by virus antigen-induced proliferation and interferon gamma production. While both vaccines induced comparable numbers of PPRV-specific CD8+ T cells, S96 induced a higher number of CD4+ T cells specifically responding to virus. Despite these quantitative and qualitative differences in the immune responses following vaccination, both vaccines gave complete clinical protection against challenge with all four lineages of PPRV.

    Importance: Despite the widespread use of live attenuated PPRV vaccines, this is the first systematic analysis of the immune response elicited in small ruminants. These data will help in the establishment of the immunological determinants of protection, an important step in the development of new vaccines, especially DIVA vaccines using alternative vaccination vectors. This study is also the first controlled test of the ability of the two major vaccines used against virulent PPRV strains from all genetic lineages of the virus, showing conclusively the complete cross-protective ability of these vaccines.

  • Expression of Human Cytomegalovirus IE1 Leads to Accumulation of Mono-SUMOylated PML That Is Protected from Degradation by HSV-1 ICP0 [Virus-Cell Interactions]

  • To countermeasure the host cellular intrinsic defense, cytomegalovirus (CMV) and herpes simplex viruses (HSV) have evolved the ability to disperse nuclear domain 10 (ND10, aka PML body). However, mechanisms underlying their action on ND10 differ. HSV infection produces ICP0 that degrades ND10 forming protein, PML; human CMV (HCMV) infection expresses IE1 that deSUMOylates PML to result in dispersion of ND10. It has been demonstrated that HSV ICP0 degraded only the SUMOylated PML, so we hypothesized that HCMV IE1 can protect PML from degradation by ICP0. HCMV IE1-expressing cell lines (U-251 MG-IE1 and HELF-IE1) were used for infection of HSV-1 or transfection of ICP0-expressing plasmid. Multi-labelling by immunocytochemistry assay and protein examination by western blot assay were performed to determine the resultant fate of PML by ICP0 in the presence or absence of HCMV IE1. Here, we report that deSUMOylation of human PML (hPML) by HCMV IE1 was incomplete, as mono-SUMOylated PML remained in the IE1-expressing cells, which is consistent to the report by Schilling et al. (J. Virol. 2017). As expected, we found that IE1 protected PML from degradation by ICP0 or HSV-1 infection. An in vitro study found that IE1 with mutation of L174P failed to deSUMOylate PML and did not protect PML from degradation by ICP0; hence, we conclude that the deSUMOylation of PML is important for IE1 to protect PML from degradation by ICP0. In addition, we revealed that murine CMV failed to deSUMOylate and to protect the HSV-mediated degradation of hPML, and that HCMV failed to deSUMOylate and protect the HSV-mediated degradation of mouse PML. However, IE1-expressing cells did not enhance wild type HSV-1 replication, but significantly increased ICP0-defective HSV-1 replication at a low moi (multiplication of infection). Therefore, our results uncovered a host-virus functional interaction at the posttranslational level.

    Importance Our finding that HCMV IE1 protected hPML from the degradation by HSV ICP0 is important because the PML body (aka ND10) is believed to be the first line of host intrinsic defense against herpesviral infection. How the infected viruses overcome the nuclear defensive structure (PML body) has not been fully understood. Herpesviral proteins, ICP0 of HSV and IE1 of CMV, have been identified to interact with PML. Here, we report that HCMV IE1 incompletely deSUMOylated PML, resulting in the mono-SUMOylated PML, which is consistent to the report of Schilling et al. (J. Virol. 2017). The mono-SUMOylated PML was subjected to degradation by HSV ICP0. However, it was protected by IE1 from degradation by ICP0 or HSV-1 infection. In contrast, the IE1 with L174P mutation lost the function of deSUMOylating PML and failed to protect the degradation of the mono-SUMOylated PML. Whether the mono-SUMOylated PML has any defensive function against viral infection will be further investigated.

  • A novel class of viral Ankyrin proteins targeting the host E3 ubiquitin ligase Cullin-2 [Virus-Cell Interactions]

  • Ankyrin repeat (ANK) domains are one of the most abundant motifs in eukaryotic proteins. ANK proteins are rare amongst viruses with the exception of poxviruses, which presumably acquired them from the host via horizontal gene transfer. The architecture of poxvirus ANK proteins is however different from their cellular counterparts and this precludes a direct acquisition event. Here we combine bioinformatics analysis and quantitative proteomics to discovera new class of viral ANK proteins with a domain organisation that relates to cellular ANK proteins. These non-canonical viral ANK proteins,termed ANK/BC, interact with host Cullin-2 via a C-terminal BCbox resembling that of cellular Cullin-2 substrate adaptors such as the von Hippel-Lindau protein. Mutagenesis of the BC box-like sequence abrogatesbinding to Cullin-2, whereas fusion of this motif to an ANK-only protein confersCullin-2 association. We demonstrate that these viral ANK/BC proteins are potent immunomodulatory proteins suppressing the activation of the pro-inflammatory transcription factors NF-B and IRF-3 and the production of cytokines and chemokines including interferon, and that association with Cullin-2 is required for optimal inhibitory activity. ANK/BC proteins exist in several orthopoxvirusesand cluster into 2 closely relatedorthologue groups in a phylogenetic lineage that is separate from canonical ANK/F-box proteins. Given the existence of cellular proteins with similar architecture,viral ANK/BC proteins may be closely related to the original ANK gene acquired by an ancestral orthopoxvirus.These findingsuncover a novel viral strategy to antagonise innate immunity and shed light on the origin of the poxviral ANK protein family.

    IMPORTANCE

    Viruses encode multiple proteins aimed at modulating cellular homeostasis and antagonising the host anti-viral response. Most of these genes were originally acquired from the host and subsequently adapted to the virus advantage. ANK proteins are common in eukaryotes, but unusual amongst viruses with the exception of poxviruses where they represent one of the largest protein families. We discover here the existence of a new class of viral ANK proteins, termed ANK/BC, that provide new insights on the origin of poxvirus ANK proteins. ANK/BC proteins target the host E3 ubiquitin ligase Cullin-2 via a C-terminal BC box domain and are potent suppressors of the production of inflammatory cytokines including interferon. The existence of cellular ANK proteins with similar architecture suggests the acquisition of a host ANK/BC gene by an ancestral orthopoxvirus and its subsequent duplication and adaptation to widen the repertoire of immune evasion strategies.

  • Rotavirus induces formation of remodeled stress granules and P-bodies and their sequestration in viroplasms to promote progeny virus production [Virus-Cell Interactions]

  • Rotavirus replicates in unique virus-induced cytoplasmic inclusion bodies called viroplasms (VMs), the composition and structure of which are yet to be understood. Based on the analysis of a few proteins, earlier studies reported that rotavirus infection inhibits stress granule (SG) formation and disrupts P-bodies (PBs). However, the recent demonstration that rotavirus infection induces cytoplasmic re-localization and colocalization with VMs of several nuclear hnRNPs and AU-rich element-binding proteins (ARE-BPs), which are known components of SGs and PBs, suggested to the possibility of rotavirus-induced remodelling of SGs and PBs, prompting us to analyse a large number of the SG and PB components to understand the status of SGs and PBs in rotavirus-infected cells. Here, it is demonstrated that rotavirus infection induces molecular triage by selective exclusion of a few proteins of SGs (G3BP1, ZBP1) and PBs (DDX6, EDC4 and Pan3) and sequestration of the re-modelled/atypical cellular organelles containing majority of their components in the VM. The punctate SG and PB structures are seen at about 4 hpi, coinciding with the appearance of small VMs, many of which fuse to form mature large VMs with progression of infection. Employing siRNA-mediated knock-down and/or ectopic over-expression, majority of the SG and PB components, except ADAR1, were observed to inhibit viral protein expression and virus growth. In conclusion, this study demonstrates that VMs are highly complex supramolecular structures and that rotavirus employs a novel strategy of sequestration in the VM and harnessing the remodelled cellular RNA recycling bins to promote its growth.

    IMPORTANCE Rotavirus is known to replicate in specialised virus-induced cytoplasmic inclusion bodies called viroplasms (VMs) but their composition and structure are not yet understood. Here it is demonstrated that rotavirus interferes with normal SG and PB assembly but promotes formation of atypical SG-PB structures by selective exclusion of a few components and employs a novel strategy of sequestration of the remodelled SG-PB granules in the VMs to promote virus growth by modulating their negative influence on virus infection. Rotavirus VMs appear to be complex supramolecular structures formed by the union of the triad of viral replication complexes and remodelled SGs and PBs as well as other host factors, designed to promote productive virus infection. These observations have implications in planning future direction of research towards understanding the structure of the VM, mechanism of morphogenesis of the virus and the detailed roles of host proteins in rotavirus biology.

  • Visualization of Ca2+ loss from rotavirus during cell entry [Virus-Cell Interactions]

  • Bound calcium ions stabilize many non-enveloped virions. Loss of Ca2+ from these particles appears to be a regulated part of entry or uncoating. The outer layer of an infectious rotavirus triple-layered particle (TLP) comprises a membrane-interacting protein (VP4) anchored by a Ca2+ stabilized protein (VP7). Membrane coupled, conformational changes in VP4 (cleaved to VP8* and VP5*) and dissociation of VP4 and VP7 accompany penetration of the double layered, inner capsid particle (DLP) into the cytosol. Removal of Ca2+ in vitro strips away both outer-layer proteins; we and others have postulated that loss of Ca2+ triggers molecular events in viral penetration. We have now investigated, with the aid of a fluorescent Ca2+ sensor, the timing of Ca2+ loss from entering virions with respect to dissociation of VP4 and VP7. In live-cell imaging experiments, distinct fluorescent markers on the DLP and on VP7 report on outer-layer dissociation and DLP release. The Ca2+ sensor, placed on VP5*, monitors the Ca2+ concentration within the membrane-bound vesicle enclosing the entering particle. Slow (1 min duration) loss of Ca2+ precedes by about 2 min the onset of VP7 dissociation and by about 7 min, DLP release. Coupled with our previous results, showing that VP7 loss follows tight binding to the cell surface by about 5 min, these data indicate that Ca2+ loss begins as soon as the particle has become fully engulfed within the uptake vesicle. We discuss the implications of these findings for the molecular mechanism of membrane disruption during viral entry.

    Importance: Non-enveloped viruses penetrate into the cytosol of the cells they infect by disrupting the membrane of an intracellular compartment. The molecular mechanisms of membrane disruption remain largely undefined. Functional reconstitution of infectious rotavirus particles (TLPs) from RNA-containing core particles (DLPs) and the outer-layer proteins that deliver them into a cell makes these important pediatric pathogens particularly good models for studying non-enveloped virus entry. We report here how use of a fluorescent Ca2+ sensor, covalently linked to one of the viral proteins, allows us to establish, using live-cell imaging, the timing of Ca2+ loss from an entering particle and other molecular events in the entry pathway. Specific Ca2+ binding stabilizes many other viruses of eukaryotes, and Ca2+ loss appears to be a trigger for steps in penetration or uncoating. The experimental design we describe may be useful for studying entry of other viral pathogens.

  • Dynamin-like proteins of endocytosis in plants are co-opted by potyviruses to enhance virus infection [Virus-Cell Interactions]

  • Endocytosis and endosomal trafficking regulate the proteins targeted to the plasma membrane and play essential roles in diverse cellular processes including responses to pathogen attack. Here, we report the identification of an endocytosis dynamin-like protein 5A (GmSDL5A) associated with purified soybean mosaic virus (SMV) virions from soybean using a top-down proteomics approach. Knock-down of GmSDL5A and its homologous gene GmSDL12A inhibits SMV infection in soybean. The role of analogous dynamin-like proteins in potyvirus infection was further confirmed and investigated using the Arabidopsis/turnip mosaic virus (TuMV) pathosystem. We demonstrate that dynamin-related proteins 2A and 2B (AtDRP2A, AtDRP2B), homologs of GmSDL5A in Arabidopsis, are recruited to the virus replication complex (VRC) of TuMV. TuMV infection is inhibited in both atdrp2a and atdrp2b knock-out mutants. Overexpression of AtDRP2 promotes TuMV replication and intercellular movement. AtRDP2 interacts with TuMV VPg, CP, CI and 6K2. Of these viral proteins, VPg, CP and CI are essential for viral intercellular movement, and 6K2, VPg and CI are critical components of the VRC. We reveal that VPg and CI are present in the punctate structures labeled by the endocytic tracer FM4-64, suggesting that VPg and CI can be endocytosed. Treatment of plant leaves with a dynamin-specific inhibitor disrupts the delivery of VPg and CI to endocytic structures and suppresses TuMV replication and intercellular movement. Taken together these data suggest that dynamin-like proteins are novel host factors of potyviruses and endocytic processes are involved in potyvirus infection.

    IMPORTANCE

    It is well known that animal viruses enter host cells via endocytosis, whereas plant viruses require physical assistance such as human and inset activities to penetrate into the host cell to establish their infection. In this study, we report that the endocytosis pathway is also involved in virus infection in plants. We show that plant potyviruses recruit endocytosis dynamin-like proteins to support their infection. Depletion of them by knock-out of the corresponding genes suppresses virus replication, whereas overexpression of them enhances virus replication and intercellular movement. We also demonstrate that the dynamin-like proteins interact with several viral proteins that are essential for virus replication and cell-to-cell movement. We further show that treatment of a dynamin-specific inhibitor disrupts endocytosis and inhibits virus replication and intercellular movement. Therefore, the dynamin-like proteins are novel host factors of potyviruses. The corresponding genes may be manipulated using advanced biotechnology to control potyviral diseases.

  • CD4 T cells, CD8 T cells, and monocytes coordinate to prevent Rift Valley fever virus encephalitis [Pathogenesis and Immunity]

  • Rift Valley fever virus (RVFV) is an arbovirus that causes disease in livestock and humans in Africa and the Middle East. While human disease is typically mild and self-limiting, some individuals develop severe manifestations, such as hepatitis, hemorrhagic fever or encephalitis. Encephalitis occurs 2nndash;3 weeks after acute illness; therefore, we hypothesized that it was a result of an inadequate adaptive immunity. To test this hypothesis in vivo, we used an attenuated virus (DelNSsRVFV) that does not typically cause disease in mice. We first characterized the normal immune response to infection with DelNSsRVFV in immune-competent mice and noted expansion of natural killer cells and monocytes, as well as activation of both CD8 and CD4 T cells. Depleting C57BL/6 mice of CD4 T cells prior to DelNSsRVFV infection resulted in encephalitis in 30% of the mice; in encephalitic mice, we noted infiltration of T cells and inflammatory monocytes into the brain. CD4 and CD8 co-depletion in C57Bl/6 mice, as well as CD4 depletion in CCR2 knock-out mice increased the frequency of encephalitis, demonstrating that these cell types normally contributed to the prevention of disease. Encephalitic mice had similar viral RNA loads in the brain regardless of which cell types were depleted, suggesting that CD4 T cells, CD8 T cells, and inflammatory monocytes did little to control viral replication in the brain. CD4-depleted mice exhibited diminished humoral and T cell memory responses, suggesting that these immune mechanisms contributed to peripheral control of virus, thus preventing infection of the brain.

    Importance:

    RVFV is found in Africa and the Middle East and is transmitted by mosquitos or through contact with infected animals. Infected individuals can develop mild disease or more severe forms such as hepatitis or encephalitis. In order to understand why some individuals develop encephalitis, we first need to know which immune functions protect those who do not develop this form of disease. In this study, we used a mouse model of RVFV infection to demonstrate that CD4 T cells, CD8 T cells, and monocytes all contribute to prevention of encephalitis. Their likely mechanism of action is preventing RVFV from ever reaching the brain.

  • Analysis of fitness trade-offs in the host range expansion of an RNA virus, tobacco mild green mosaic virus [Genetic Diversity and Evolution]

  • The acquisition of new hosts provides a virus with more opportunities for transmission and survival, but may be limited by across-host fitness trade-offs. Major causes of across-hosts trade-offs are antagonistic pleiotropy, that is, host-differential phenotypic effects of mutations, a Genotype x Environment interaction, and epistasis, a Genotype x Genotype interaction. Here we analyse if there are trade-offs, and what are the causes, associated with the acquisition by tobacco mild green mosaic virus (TMGMV) of a new host. For this, the multiplication of sympatric field isolates of TMGMV from its wild reservoir host Nicotiana glauca and from pepper crops was quantified in the original and the heterologous hosts. TMGMV isolates from N. glauca were adapted to their host, but pepper isolates were not adapted to pepper, and the acquisition of this new host was associated with a fitness penalty in the original host. Analyses of the collection of field isolates and of mutant genotypes derived from biologically active cDNA clones showed a role of mutations in the coat protein and the 3rrsquo;UTR in determining within-host virus fitness. Fitness depended on host-specific effects of these mutations, on the genetic background in which they occurred, and on higher order interactions of the type Genotype x Genotype x Environment. These type of effects had been reported to generate across-hosts fitness trade-offs under experimental evolution. Our results show they may also operate in heterogeneous natural environments, and could explain that pepper isolates were not adapted to pepper and their lower fitness in N. glauca.

    Importance

    The acquisition of new hosts conditions virus epidemiology and emergence, hence the interest in understanding the mechanisms behind host-range expansions. Experimental evolution studies have identified antagonistic pleiotropy and epistasis as genetic mechanisms that limit host-range expansions, but studies from virus field populations are few. Here we compare the performance of isolates of tobacco mild green mosaic virus from its reservoir host, Nicotiana glauca and its new host, pepper, showing that acquisition of a new host was not followed by adaptation to it, but was associated with a fitness loss in the original host. Analysis of mutations determining host-specific virus multiplication identified antagonistic pleiotropy, epistasis and host-specific epistasis as mechanisms generating across-host fitness trade-offs that may prevent adaptation to pepper and cause a loss of fitness in N. glauca. Thus, mechanisms determining trade-offs, identified under experimental evolution, could also operate in the heterogeneous environment in which natural plant virus populations occur.

  • CVB3 nonstructural 2A protein modulates SREBP1a signaling via the MEK/ERK pathway [Virus-Cell Interactions]

  • Coxsackieviruse B3 (CVB3) is the predominant pathogen of viral myocarditis. In our previous study, we found that CVB3 caused abnormal lipid accumulation in host cells. However, the underlying mechanisms by which CVB3 disrupts and exploits the host lipid metabolism are not well understood. Sterol regulatory element-binding protein 1 (SREBP1) is the major transcriptional factor in lipogenic genes expression. In this study, we demonstrated that CVB3 infection and nonstructural 2A protein upregulated and activated SREBP1a at the transcriptional level. Deletion analysis of SREBP1a promoter revealed that two regions -1821/-1490 and -312/+217 in this promoter were both required for its activation by 2A. These promoter regions possessed several binding motifs for transcription factor SP1. Next, we used SP1-specific siRNAs to confirm that SP1 might be the essential factor in SREBP1a upregulation by 2A. Furthermore, we showed that MEK/ERK pathway was involved in the activation of SREBP1a by 2A, and blocking this signaling pathway with specific inhibitor U0126 attenuated SREBP1a activation and lipid accumulation by 2A. Finally, we showed that inhibition of SREBP1 with siRNAs attenuated lipid accumulation induced by CVB3 infection and reduced the virus replication. Moreover, inhibition of the MEK/ERK pathway also led to reduction of SREBP1a activation, lipid accumulation and virus replication during CVB3 infection. Taken together, these data demonstrate that CVB3 nonstructural 2A protein activates SREBP1a at the transcription level through a complex mechanism involving MEK/ERK signaling pathway and SP1 transcription factor, which promotes cellular lipid accumulation and benefits virus replication.

    IMPORTANCE Coxsackieviruse B3 (CVB3) infection is one of the leading causes of viral myocarditis, but effective vaccines and antiviral therapies against CVB3 infection are still lacking. It is important to understand the precise interactions between host and virus, for the rational design of effective therapies. During infection, CVB3 disrupts and exploits host lipid metabolism to promote excessive lipid accumulation, which benefits virus replication. SREBP1 is the master regulator of cellular lipid metabolism. Here, we report that one of the viral nonstructural proteins, 2A, upregulates and activates SREBP1a. Furthermore, we find that inhibition of SREBP1 decreases CVB3 virus replication. These results reveal the regulation of SREBP1a expression by 2A, and the roles of SREBP1 in lipid accumulation and viral replication during CVB3 infection. Our findings provide a new insight into CVB3-host interactions and inform a potential novel therapeutic strategy for this important pathogen.

  • Spatio-temporal distribution and evolution of the A/H1N1 2009 pandemic virus in pigs in France from 2009 to 2017: identification of a potential swine-specific lineage [Genetic Diversity and Evolution]

  • The H1N1 influenza virus responsible for the most recent pandemic in 2009 (H1N1pdm) has spread to swine populations worldwide while it replaced the previous seasonal H1N1 virus in humans. In France, surveillance of swine influenza A viruses in pig herds with respiratory outbreaks led to the detection of 44 H1N1pdm strains between 2009 and 2017, regardless of the season, and findings were not correlated to pig density. From these isolates, 17 whole genome sequences were obtained as well as 6 additional HA/NA sequences, in order to perform spatial and temporal analyses of the genetic diversity, and to compare evolutionary patterns of H1N1pdm in pigs to patterns for human strains. Following mutation accumulation and fixation over time, phylogenetic analyses revealed for the first time the divergence of a swine-specific genogroup within the H1N1pdm lineage. The divergence is thought to have occurred around 2011, although this was only demonstrated through strains isolated in 2015-2016 in the southern half of France. To date, these H1N1pdm swine strains have not been related to any increased virulence in swine herds and have not exhibited any antigenic drift as compared to seasonal human strains. However, further monitoring is encouraged as diverging evolutionary patterns in these two species, i.e. swine and humans, may lead to the emergence of viruses with a potentially higher risk for both animal and human health.

    Importance

    Pigs are a llsquo;mixing vesselrrsquo; for influenza A viruses (IAVs) because of their ability to be infected by avian and human IAVs, and their propensity to facilitate viral genomic reassortment events. Also, as IAVs may evolve differently in swine and humans, pigs can become a reservoir for old human strains against which the human population has become immunologically naïve. Thus, viruses from the novel swine-specific H1N1pdm genogroup may continue to diverge from seasonal H1N1pdm strains and/or from other H1N1pdm viruses infecting pigs and lead to the emergence of viruses that would not be covered by human vaccines and/or swine vaccines based on antigens closely related to the original H1N1pdm virus. This discovery confirms the importance of encouraging swine IAV monitoring because H1N1pdm swine viruses could carry an increased risk for both human and swine health in the future, as a whole H1N1pdm or gene provider in subsequent reassortant viruses.

  • Long noncoding RNA ANRIL supports proliferation of adult T-cell leukemia cells through cooperation with EZH2 [Transformation and Oncogenesis]

  • Adult T-cell leukemia (ATL) is a highly aggressive T-cell malignancy induced by human T-cell leukemia virus type 1 (HTLV-1) infection. Long noncoding RNA (lncRNA) plays a critical role in the development and progression of multiple human cancers. However, the function of lncRNA on HTLV-1-induced oncogenesis has not been elucidated. In the present study, we show that the expression of the lncRNA ANRIL was elevated in HTLV-1 infected cell lines and clinical ATL samples. E2F1 induced ANRIL transcription by enhancing its promoter activity. Knocking down of ANRIL in ATL cells repressed cellular proliferation and increased apoptosis in vitro and in vivo. As a mechanism for these actions, we found that ANRIL targeted EZH2, and activated the NF-B pathway in ATL cells. This activation was independent of the histone methyltransferase (HMT) activity of EZH2, but required the formation of an ANRIL/EZH2/p65 ternary complex. Chromatin immunoprecipitation assay revealed that ANRIL/EZH2 enhanced p65 DNA binding capability. In addition, we observed that ANRIL/EZH2 complex repressed p21/CDKN1A transcription through H3K27 trimethylation of the p21/CDKN1A promoter. Taken together, our results implicate that lncRNA ANRIL, by cooperating with EZH2, supports the proliferation of HTLV-1 infected cells, which is thought to be critical for oncogenesis.

    IMPORTANCE

    Human T-cell leukemia virus type 1 (HTLV-1) is the pathogen that causes adult T-cell leukemia (ATL), which is a unique malignancy of CD4+ T cells. A role for long noncoding RNA (lncRNA) in HTLV-1-mediated cellular transformation has not been described. In this study, we demonstrated that lncRNA ANRIL was important for maintaining proliferation of ATL cells in vitro and in vivo. ANRIL was shown to activate NF-B signaling through forming a ternary complex with EZH2 and p65. Further, epigenetic inactivation of p21/CDKN1A was involved in the oncogenic function of ANRIL. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA ANRIL in ATL and provides an important clue to prevent or treat HTLV-1 associated human diseases.

  • Characterization of a threonine-rich cluster in Hepatitis C virus nonstructural protein 5A and its contribution to hyperphosphorylation [Genome Replication and Regulation of Viral Gene Expression]

  • Hepatitis C virus (HCV) nonstructural protein (NS)5A is a phosphoprotein with key functions in regulating viral RNA replication and assembly. Two phospho-isoforms are discriminated by their different apparent molecular weight: a basally phosphorylated (p56) and a hyperphosphorylated (p58) variant. The precise mechanisms governing p58 synthesis and specific functions of the isoforms are poorly understood.

    Our study aimed at a deeper understanding of determinants involved in p58 synthesis. We analyzed two variants of p56 and p58 of isolate JFH-1 separately by mass spectrometry using an expression model and thereby identified a threonine-rich phosphopeptide exclusively found in the hyperphosphorylated variant. Individual exchange of possible phospho-acceptor sites to phospho-ablatant or nndash;mimetic residues had little impact on HCV replication or assembly in cell culture. A phospho-specific antibody recognizing pT242 revealed that this position was indeed phosphorylated only in p58 and depended on casein kinase Iaalpha;. Importantly, phospho-ablative mutations at positions T244 and S247 abrogated pT242 detection, without substantial effects on global p58 levels, whereas mutations in the preceding serine-rich cluster dramatically reduced total p58 levels but had minor impact on pT242 levels, suggesting the existence of distinct subspecies of hyperphosphorylated NS5A. Mass spectrometry analyses of different genotypes showed variable phosphorylation patterns across NS5A and suggested that the threonine rich region is also phosphorylated at T242 in gt4a and at S249 in gt1a, gt1b and gt4a.

    Our data therefore indicate that p58 is not a single homogenously phosphorylated protein species, but rather a population of various phospho-isoforms, with high variability between genotypes.

    Importance

    Hepatitis C virus infections affect 71 million people worldwide and cause severe chronic liver disease. Recently, efficient antiviral therapies have been established, with inhibitors of nonstructural protein NS5A as a cornerstone. NS5A is a central regulator of HCV replication and assembly, but still enigmatic in its molecular functions. It exists in two phospho-isoforms, p56 and p58. We identified now a phosphopeptide exclusively found in p58 and analyzed the determinants involved in phosphorylation of this region. We found evidence for very different phosphorylation patterns resulting in p58. These results challenge the concept of p58 being a homogenous species of NS5A molecules phosphorylated at the same positions, but argues for at least two independently phosphorylated variants showing the same electrophoretic mobility, likely serving different functions.

  • Human MX2/MxB: a potent interferon-induced post-entry inhibitor of herpesviruses and HIV-1 [Gem]

  • Interferons limit viral replication by inducing intracellular restriction factors such as the GTPase MxB (also designated MX2), which inhibits HIV-1 and, as recently shown, herpesviruses. Inhibition of these viruses occurs at ill-defined steps after viral entry and requires formation of MxB dimers or oligomers, but GTP hydrolysis is needed only for blocking herpesviruses. Together with previous findings on related MxA, the new research on MxB highlights the mechanistic diversity by which MX proteins interfere with viral replication.

  • Cellular Protein Kinase D Modulators Play a Role during Multiple Steps of Herpes Simplex Virus Type 1 Egress [Virus-Cell Interactions]

  • The assembly of new herpes simplex virus type 1 particles takes place in the nucleus. These particles then travel across the two nuclear membranes and acquire a final envelope from a cellular compartment. The contribution of the cell to the release of the virus is, however, little known. We previously demonstrated using a synchronized infection that the host protein kinase D and diacylglycerol, a lipid that recruits the kinase to the TGN, promote the release of the virus from that compartment. Given the role this cellular protein plays in the herpes simplex virus type 1 life cycle and the many molecules that modulate its activity, we aimed to determine to what extent this virus utilizes the protein kinase D pathway during a non-synchronized infection. Several molecular PKD regulators were targeted by RNA interference and viral production monitored. Surprisingly, many of these modulators negatively impacted the extracellular release of the virus. Overexpression studies, use of pharmacological reagents and assays to monitor intracellular lipids implicated in the biology of PKD suggested that these effects were oddly independent of total intracellular diacylglycerol levels. Instead, mapping of the viral intermediates by electron microscopy suggested that some of these modulators could modulate distinct steps along the viral egress pathway, notably nuclear egress. Altogether, this suggests a more complex contribution of PKD to HSV-1 egress than originally anticipated and new research avenues to explore.

    Importance

    Viruses are obligatory parasites that highjack numerous cellular functions. This is certainly true when it comes to transport viral particles within the cell. Herpesviruses share the unique property to travel through the two nuclear membranes by subsequent budding and fusion and to acquire their final envelope from a cellular organelle. Albeit disputed, the overall evidence from many laboratories points to the TGN as the source of that membrane. Moreover, past findings revealed that the host protein kinase D plays an important role at that stage, which is significant given the known implication of that protein in vesicular transport. The present findings suggest that the PKD machinery not only affects the late stages of herpes simplex virus type I egress but also modulates earlier steps such as nuclear egress. This opens up new means to control these viruses.

  • Sindbis virus infection causes cell death by nsP2-induced transcriptional shutoff or by nsP3-dependent translational shutoff. [Virus-Cell Interactions]

  • Sindbis virus (SINV) is a representative member of the Alphavirus genus in Togaviridae family. The hallmark of SINV replication in vertebrate cells is a rapid development of the cytopathic effect (CPE), which usually occurs within 24 h post infection. Mechanistic understanding of CPE might lead to development of new prophylactic vaccines and therapeutic means against alphavirus infections. However, development of noncytopathic SINV variants and those of other Old World alphaviruses was always highly inefficient and usually resulted in selection of mutants demonstrating poor replication of the viral genome and transcription of subgenomic RNA. This likely caused a nonspecific negative effect on the rates of CPE development. The results of this study demonstrate that CPE induced by SINV and likely by other Old World alphaviruses is a multicomponent process, in which transcriptional and translational shutoffs are the key contributors. Inhibition of cellular transcription and translation are determined by SINV nsP2 and nsP3 proteins, respectively. Defined mutations in the nsP2-specific peptide between aa 674nndash;688 prevent virus-induced degradation of the catalytic subunit of cellular DNA-dependent RNA polymerase II, transcription inhibition, and make SINV a strong type I IFN inducer without affecting its replication rates. Mutations in the nsP3 macrodomain, which were demonstrated to inhibit its mono-ADP-ribosylhydrolase activity, downregulate the second component of CPE development, inhibition of cellular translation, and also have no effect on virus replication rates. Only the combination of both nsP2- and nsP3-specific mutations in the SINV genome has a dramatic negative effect on virus ability to induce CPE.

    IMPORTANCE Alphaviruses are a group of important human and animal pathogens with worldwide distribution. Their characteristic feature is a highly cytopathic phenotype in cells of vertebrate origin. The molecular mechanism of CPE remains poorly understood. In this study, by using Sindbis virus (SINV) as a model of the Old World alphaviruses, we demonstrate that SINV-specific CPE is redundantly determined by viral nsP2 and nsP3 proteins. NsP2 induces the global transcriptional shutoff, and this nuclear function can be abolished by the mutations of the small, surface exposed peptide in the nsP2 protease domain. NsP3, in turn, determines the development of translational shutoff, and this activity depends on nsP3 macrodomain-associated mono-ADP-ribosylhydrolase activity. A combination of defined mutations in nsP2 and nsP3, which abolish SINV-induced transcription and translation inhibition, in the same viral genome does not affect SINV replication rates, but make it noncytopathic and potent inducer of type I interferon.

  • A novel chimeric oncolytic virus vector for improved safety and efficacy in hepatocellular carcinoma [Virus-Cell Interactions]

  • Oncolytic viruses represent an exciting new aspect of the evolving field of cancer immunotherapy. We have engineered a novel hybrid vector comprising vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV), named rVSV-NDV, wherein the VSV backbone is conserved, but its glycoprotein has been replaced by the hemagglutinin-neuraminidase (HN) and the modified, hyperfusogenic fusion (F) envelope proteins of recombinant NDV. As opposed to wild-type VSV, which kills cells through a classical cytopathic effect, the recombinant virus is able to induce tumor-specific syncytia formation, allowing for efficient cell-to-cell spread of the virus and a rapid onset of immunogenic cell death. Furthermore, the glycoprotein exchange substantially abrogates the off-target effects in brain and liver tissue associated with wildtype VSV, resulting in a substantially enhanced safety profile, even in immune-deficient NOD-SCID mice which are highly susceptible to wild-type VSV. Although NDV causes severe pathogenicity in its natural avian hosts, the incorporation of the envelope proteins in the chimeric rVSV-NDV vector is avirulent in embryonated chicken eggs. Finally, systemic administration of rVSV-NDV in orthotopic HCC-bearing immune-competent mice resulted in significant survival prolongation. This strategy, therefore, combines the beneficial properties of the rapidly replicating VSV platform with the highly efficient spread and immunogenic cell death of a fusogenic virus, without risking the safety and environmental threats associated with either parental vector. Taken together, rVSV-NDV represents an attractive vector platform for clinical translation as a safe and effective oncolytic virus.

    Importance The therapeutic efficacy of oncolytic viral therapy often comes as a trade-off with safety, such that potent vectors are often associated with toxicity, while safer viruses tend to have attenuated therapeutic effects. Despite promising preclinical data, the development of VSV as a clinical agent has been substantially hampered by the fact that severe neuro- and hepatotoxicity have been observed in rodents and nonhuman primates in response to treatment with wild-type VSV. Although NDV has been shown to have an attractive safety profile in humans with promising oncolytic effects, its further development has been severely restricted due to the environmental risks it poses. The hybrid rVSV-NDV vector, therefore, represents an extremely promising vector platform in that it has been rationally designed to be safe, both to the recipient and the environment, while being simultaneously effective, both through its direct oncolytic actions and via induction of immunogenic cell death.

  • Molecular characterization of the viroporin function of foot-and-mouth disease virus non-structural protein 2B [Structure and Assembly]

  • The non-structural protein 2B of foot-and-mouth disease virus (FMDV) is comprised of a small hydrophobic, 154 amino acid protein. Structure-function analyses demonstrated that FMDV 2B is an ion channel forming protein. Infrared spectroscopy measurements using partially overlapping peptides that spanned regions between amino acids 28nndash;147 demonstrated the adoption of helical conformations in two putative transmembrane regions between residues 60nndash;78 and 119nndash;147, and a third transmembrane region between residues 79nndash;106, adopting a mainly extended structure. Using synthetic peptides, ion-channel activity measurements in planar lipid bilayers and imaging of single Giant Unilamellar Vesicles (GUVs) revealed the existence of two sequences endowed with membrane-porating activity: one spanning FMDV 2B residues 55nndash;82, and the other spanning the C-terminal region of 2B residues 99nndash;147. Mapping the latter sequence identified residues 119nndash;147 as responsible for the activity. Experiments to assess the degree of insertion on the synthetic peptides in bilayers, and the inclination angle adopted by each peptide regarding the membrane plane normal confirm the residues 55nndash;82 and 119-147 of 2B actively insert as transmembrane helices. Using reverse genetics, a panel of thirteen FMD recombinant mutant viruses was designed harboring non-conservative as well as alanine substitutions in critical amino acid residues in the area between amino acid residues 28 and 147. Alterations to any of these structures interfered with pore-channel activity, and the capacity of the protein to permeabilize the ER to calcium, and were lethal for virus replication. Thus, FMDV 2B emerges as the first member of the viroporin family containing two distinct pore domains.

    IMPORTANCE FMDV non-structural protein 2B is able to insert itself into cellular membranes to form a pore. This pore allows for the passage of ions and small molecules through the membrane. In this study we were able to show that both current and small molecules are able to pass though the pore made by 2B. We also discovered for the first time a virus with a pore forming protein that contains two independent functional pores. By making mutations in our infectious clone of FMDV, we determined that mutations in either pore resulted in non-viable virus. This suggests that both pore forming functions are independently required during FMDV infection.

  • Redundant late domain functions of tandem VP2 YPX3L motifs in nonlytic cellular egress of quasi-enveloped hepatitis A virus [Virus-Cell Interactions]

  • The quasi-envelopment of hepatitis A virus (HAV) capsids in exosome-like virions (eHAV) is an important but incompletely understood aspect of the hepatovirus life cycle. This process is driven by recruitment of newly assembled capsids to endosomal vesicles into which they bud to form multi-vesicular bodies with intraluminal vesicles that are later released at the plasma membrane as eHAV. The endosomal sorting complexes required for transport (ESCRT) are key to this process, as is the ESCRT-III associated protein, ALIX, which also contributes to membrane budding of conventional enveloped viruses. YPX1or3L late domains in the structural proteins of these viruses mediate interactions with ALIX, and two such domains exist in the HAV VP2 capsid protein. Mutational studies of these domains are confounded by the fact that the Tyr residues (important for interactions of YPX1or3L peptides with ALIX) are required for efficient capsid assembly. However, single Leu-to-Ala substitutions within either VP2 YPX3L motif (L1-A and L2-A mutants) were well tolerated, albeit associated with significantly reduced eHAV release. In contrast, simultaneous substitutions in both motifs (L1,2-A) eliminated virus release, but did not inhibit assembly of infectious intracellular particles. Immunoprecipitation experiments suggested that the loss of eHAV release was associated with a loss of ALIX recruitment. Collectively, these data indicate that HAV YPX3L motifs function as redundant late domains during quasi-envelopment and viral release. Since these motifs present little solvent accessible area in the crystal structure of the naked extracellular capsid, the capsid structure may be substantially different during quasi-envelopment.

    IMPORTANCE Nonlytic release of hepatitis A virus (HAV) as exosome-like quasi-enveloped virions is a unique but incompletely understood aspect of the hepatovirus life cycle. Several lines of evidence indicate that the host protein ALIX is essential for this process. Tandem YPX3L llsquo;late domainsrrsquo; in the VP2 capsid protein could be sites of interaction with ALIX, but they are not accessible on the surface of an X-ray model of the extracellular capsid, raising doubts about this putative late domain function. Here we describe YPX3L domain mutants that assemble capsids normally, but fail to bind ALIX and be secreted as quasi-enveloped eHAV. Our data support late domain function for the VP2 YPX3L motifs, and raise questions about the structure of the HAV capsid prior to and following quasi-envelopment.

  • Robust human and murine hepatocyte culture models of hepatitis B virus infection and replication [Genome Replication and Regulation of Viral Gene Expression]

  • Hepatitis B virus (HBV) is a major cause of chronic liver diseases including hepatitis, cirrhosis, and hepatocellular carcinoma. HBV research has been hampered by the lack of robust cell culture and small animal models of HBV infection. The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor has been a landmark advance in HBV research in recent years. Ectopic expression of NTCP in nonpermissive HepG2, Huh7, and AML12 cell lines confers HBV susceptibility. However, HBV replication in these human and murine hepatocytes cell lines appeared suboptimal. In present study, we have constructed stable NTCP-expressing HepG2 and AML12 cell lines and found that HBV permissiveness is correlated with NTCP expression. More significantly, we have developed robust HBV cell culture models by treating the HBV-infected cells with dimethyl sulfoxide (DMSO) and hydrocortisone, which significantly promoted HBV replication and production. Mechanistic studies suggested that hydrocortisone significantly enhanced the transcription and expression of PGC1aalpha; and HNF4aalpha; which are known to promote HBV transcription and replication. These new human and murine hepatocyte culture systems of HBV infection and replication will accelerate the determination of molecular aspects underlying HBV infection, replication, and morphogenesis in human and murine hepatocytes. We anticipate that our HBV cell culture models will also facilitate the discovery and development of antiviral drugs towards the ultimate eradication of chronic hepatitis B.

    IMPORTANCE

    HBV research has been greatly hampered by the lack of robust cell culture and small animal models of HBV infection and propagation. The discovery of NTCP as an HBV receptor has greatly impacted the field of HBV research. Although HBV infection of the NTCP-expressing human and murine hepatocyte cell lines has been demonstrated, its replication in cell culture appeared inefficient. To further improve cell culture systems of HBV infection and replication, we have constructed NTCP-expressing HepG2 and AML-12 cell lines that are highly permissive to HBV infection. More significantly, we have found that DMSO and hydrocortisone markedly enhanced HBV transcription and replication in human and murine hepatocytes when added to cell culture medium. These new cell culture models of HBV infection and replication will facilitate HBV research and antiviral drug discovery towards ultimate elimination of chronic hepatitis B.

  • Structure-guided identification of a non-human morbillivirus with zoonotic potential [Virus-Cell Interactions]

  • Morbilliviruses infect a broad range of mammalian hosts including ruminants, carnivores and humans. The recent eradication of rinderpest virus (RPV), as well as active campaigns for the human-specific measles virus (MeV), have raised significant concerns that the remaining morbilliviruses may emerge in so-called vacated ecological niches. Seeking to assess the zoonotic-potential of non-human morbilliviruses within human populations we identified that peste des petits ruminants virus (PPRV) - the small ruminant morbillivirus - is restricted at the point of entry into human cells due to deficient interactions with human SLAMF1 nndash; the immune cell receptor for morbilliviruses. Using a structure-guided approach, we characterised a single amino acid change, mapping to the receptor-binding domain in the PPRV Haemagglutinin (H) protein, which overcomes this restriction. The same mutation allowed escape from some cross-protective, human-patient, anti-MeV antibodies, raising concerns that PPRV is a pathogen with zoonotic potential. Analysis of natural variation within human and ovine SLAMF1 also identified polymorphisms that could correlate with disease resistance. Finally, the mechanistic nature of the PPRV restriction was also investigated, identifying charge incompatibility and steric hindrance between PPRV H and human SLAMF1 proteins. Importantly, this research was performed entirely using surrogate virus entry assays, negating the requirement for in situ derivation of a human-tropic PPRV and illustrating alternative strategies for identifying gain-of-function mutations in viral pathogens.

    Importance

    A significant proportion of viral pandemics occur following zoonotic transmission events, where animal-associated viruses jump species into human populations. In order to forewarn against the emergence of these viruses it is necessary to develop a better understanding of what determines virus host-range, often at the genetic and structural level. In this research paper, we demonstrate that the small ruminant morbillivirus, a close relative of measles, is unable to use human receptors to enter cells; however, a change of a single amino acid in the virus is sufficient to overcome this restriction. This information will be important for monitoring this virusrrsquo;s evolution in the field. Of note, this study was undertaken in vitro, without generation of a fully infectious virus with this phenotype.

  • Toll Like Receptor-7 and -9 agonists improve Hepatitis C Virus replication and infectivity inhibition by plasmacytoid dendritic cells [Cellular Response to Infection]

  • Plasmacytoid dendritic cells (pDCs) are innate immune cells with high antiviral activity triggered by Toll like receptor (TLR)-7 and -9 stimulation. Moreover, they are important mediators between innate and adaptive immunity. Although nowadays there is available an effective therapeutic arsenal against hepatitis C virus (HCV), a protective vaccine is not available. We have analyzed the pDCs response to HCV infection in a HCVnndash;Huh7.5 virus-cell system, which allows completing the virus infectious cycle. pDCs were cocultured following human immunodeficiency virus (HIV) aldrithiol-2 inactivated (AT-2) (TLR-7 agonist) and CpG (TLR-9 agonist) stimulation. We employed three virus derivatives, a wild type Jc1, an interferon-resistant virus IR, and a high replicative fitness virus P100, in order to explore additional IFN-aalpha;-related virus inhibition mechanisms. pDCs inhibited HCV infectivity and replication, and produced IFN-aalpha;. After TLR-7 and TLR-9 stimulation, inhibition of infectivity and IFN-aalpha; production by pDCs were enhanced. TLR-7 stimulation drove a higher TNF-related apoptosis-inducing ligand (TRAIL) expression in pDCs. Additionally, TLR-7 and TLR-9 stimulated pDCs exhibited a mature phenotype, improving the antigen presentation and lymph node homing related markers. In conclusion, pDCs could serve as a drug target against HCV in order to improve antiviral activity and as an enhancer of viral immunization.

    IMPORTANCE We implemented a coculture system of pDCs with HCV-infected hepatoma cell line, Huh7.5. We used three HCV derivatives in order to gain insight into pDCs behavior against HCV and associated antiviral mechanisms. The results with this cell coculture system support the capacity of pDCs to inhibit HCV replication and infectivity mainly via IFN-aalpha;, but also through additional mechanisms associated with pDC maturation. We provided evidence that TLR agonists can enhance antiviral pDCs function and can induce phenotypic changes that may facilitate the interplay with other immune cells. These findings suggest the possibility of including TLR agonists in the strategies of HCV vaccine development.

  • NSs protein of Sandfly fever Sicilian phlebovirus counteracts interferon induction by masking the DNA-binding domain of interferon regulatory factor 3 [Virus-Cell Interactions]

  • Sandfly fever Sicilian virus (SFSV) is one of the most widespread and frequent members of the genus Phlebovirus (order Bunyavirales, family Phenuiviridae) infecting humans. Being transmitted by Phlebotomus sandflies, SFSV causes a self-limiting acute, often incapacitating febrile disease ("sandfly fever", "pappataci fever" or "dog disease") that is known at least since the beginning of the 20th century. We show that, similar to other pathogenic phleboviruses, SFSV suppresses the induction of the antiviral type I interferon (IFN) system in an NSs-dependent manner. SFSV NSs interfered with the TBK1-IRF3 branch of the RIG-I signalling pathway, but not with NF-B activation. Consistently, we identified interferon regulatory factor 3 (IRF3) as host interactor of SFSV NSs. In contrast to IRF3, neither the IFN master regulator IRF7, nor the related transcription factors IRF2, IRF5, or IRF9 were bound by SFSV NSs. In spite of this specificity for IRF3, NSs inhibited neither its phosphorylation, dimerization, nor nuclear accumulation, and the interaction was independent of the IRF3 activation or multimerization state. In further studies we identified the DNA-binding domain of IRF3 (amino acids 1 to 113) as sufficient for NSs binding, and found that SFSV NSs prevented the association of activated IRF3 with the IFN-bbeta; promoter. Thus, unlike highly virulent phleboviruses which either destroy antiviral host factors or sequester whole signalling chains into inactive aggregates, SFSV modulates type I IFN induction by directly masking the DNA-binding domain of IRF3.

    Significance Phleboviruses are receiving increased attention due to the constant discovery of new species, and the ongoing spread of long-known members of the genus. Outbreaks of Sandfly fever were reported in the nineteenth century, in World War I, and in World War II. Nowadays, SFSV is recognized as being one of the most widespread phleboviruses, exhibiting high seroprevalence rates in humans and domestic animals and causing a self-limiting but incapacitating disease predominantly in immunologically naïve troops and travellers. We show how the non-structural protein NSs of SFSV counteracts the upregulation of the antiviral interferon (IFN) system. SFSV NSs specifically inhibits promoter binding by the IFN transcription factor IRF3, a molecular strategy which is unique among phleboviruses and, to our knowledge, human pathogenic RNA viruses in general. This IRF3-specific and stoichiometric mechanism, much distinct from the ones exhibited by the highly virulent phleboviruses, correlates with the intermediate level of pathogenicity of SFSV.

  • Host Enzymes Heparanase and Cathepsin L Promote Herpes Simplex Virus-2 Release from Cells [Virus-Cell Interactions]

  • Herpes simplex virus-2 (HSV-2) can productively infect many different cell types of human and non-human origin. Here we demonstrate interconnected roles for two host enzymes, heparanase (HPSE) and cathepsin L in HSV-2 release from cells. In vaginal epithelial cells HSV-2 causes heparan sulfate shedding and upregulation in HPSE levels during the productive phase of infection. We also noted increased levels of cathepsin L and show that regulation of HPSE by cathepsin L via cleavage of HPSE proenzyme is important for infection. Furthermore, inhibition of HPSE by a specific inhibitor, OGT 2115, dramatically reduces HSV-2 release from vaginal epithelial cells. Likewise, we show evidence that the inhibition of cathepsin L is detrimental to the infection. The HPSE increase after infection is mediated by an increased NF-kB nuclear localization and a resultant activation of HPSE transcription. Together these mechanisms contribute to the removal of heparan sulfate from the cell surface, and thus facilitate virus release from cells.

    Importance

    Genital infections by HSV-2 represent one of the most common sexually transmitted viral infections. The virus causes painful lesions, and sores around the genitals or rectum. Intermittent release of the virus from infected tissues during sexual activities is the most common cause of transmission. At the molecular level, cell surface heparan sulfate (HS) is known to provide attachment sites for HSV-2. While the removal of HS during HSV-1 release has been shown, not much is known about the host factors and their regulators that contribute to HSV-2 release from natural target cell types. Here we suggest a role for the host enzyme heparanase in HSV-2 release. Our work reveals that in addition to the regulation of transcription by NF-kB, HPSE is also regulated post-translationally by cathepsin L and that inhibition of heparanase activity directly affects HSV-2 release. We provide unique insights into the host mechanisms controlling HSV-2 egress and spread.

  • T5 exonuclease hydrolysis of Hepatitis B Virus replicative intermediates allows reliable quantification and fast drug efficacy testing of covalently closed circular DNA by PCR [Genome Replication and Regulation of Viral Gene Expression]

  • Chronic infection with the human Hepatitis B Virus (HBV) is a major health problem. Virus persistence requires the establishment and maintenance of covalently closed circular (ccc) DNA, the episomal virus template in the nucleus of infected hepatocytes. Compared to replicative DNA intermediates (relaxed circular (rc) DNA), copy numbers of cccDNA in infected hepatocytes are low. Accordingly, accurate analyses of cccDNA require enrichment of nuclear fractions and southern blotting or selective qPCR methods allowing discrimination of cccDNA and rcDNA.

    In this report, we analyzed cccDNA-specific primer pairs for their ability to amplify cccDNA selectively. Using mixtures of defined forms of HBV and genomic DNA, we determined the potential of different nucleases to targeted digestion of the open/relaxed circular DNA forms in the absence and presence of genomic DNA without affecting cccDNA. We found that the combination of T5 exonuclease with a primer set amplifying an approximate 1 kb fragment permits reliable quantification of cccDNA without the requirement of prior nuclei enrichment or Hirt extraction. We tested this method in four different in vitro infection systems and quantified cccDNA copy numbers at increasing multiplicity of inoculated genome equivalents. We further analyzed the kinetics of cccDNA formation and the effect of drugs (interferon, entry inhibitors, and capsid inhibitors) on cccDNA. Our method allows reliable cccDNA quantification at early stages of infection in the presence of a high excess of input virus and replicative intermediates and is thereby suitable for drug screening and investigation of cccDNA formation and maintenance.

    Importance

    cccDNA elimination is a major goal in future curative regimens for chronic HBV patients. However, PCR-based assays for cccDNA quantification show a principally constrained specificity when high levels of input virus or replicative intermediates are present. Here, we characterized T5 exonuclease as a suitable enzyme for medium throughput in vitro assays that preserves cccDNA but efficiently removes rcDNA prior to PCR-based quantification. We compared T5 exonuclease with the previously described exonuclease III and show that both nucleases are suitable for reliable quantification of cccDNA by PCR. We substantiated the applicability of our method through examination of early cccDNA formation and stable accumulation in several in vitro infection models and analyzed cccDNA stability after administration of anti-HBV drugs. Our results support the use of T5 exonuclease for fast and convenient rcDNA removal especially for early cccDNA quantification and rapid drug testing in in vitro studies.

  • Characterisation of Antibody Interactions with the G Protein of Vesicular Stomatitis Virus Indiana Strain and Other Vesiculovirus G Proteins [Vaccines and Antiviral Agents]

  • Vesicular stomatitis virus Indiana strain G protein (VSVind.G) is the most commonly used envelope glycoprotein to pseudotype lentiviral vectors (LV) for experimental and clinical applications. Recently, G proteins derived from other vesiculoviruses (VesG), for example Cocal virus, have been proposed as alternative LV envelopes with possible advantages compared to VSVind.G. Well-characterised antibodies that recognise VesG will be useful for vesiculovirus research, development of G protein-containing advanced therapy medicinal products (ATMPs), and deployment of VSVind-based vaccine vectors. Here we show that one commercially available monoclonal antibody, 8G5F11, binds to and neutralises G proteins from three strains of VSV as well as Cocal, and Maraba viruses, whereas the other commercially available monoclonal anti-VSVind.G antibody, IE9F9, binds to and neutralises only VSVind.G. Using a combination of G protein chimeras and site-directed mutations, we mapped the binding epitopes of IE9F9 and 8G5F11 on VSVind.G. IE9F9 binds close to the receptor binding site and competes with soluble low-density lipoprotein receptor (LDLR) for binding to VSVind.G, explaining its mechanism of neutralisation. In contrast, 8G5F11 binds close to a region known to undergo conformational changes when the G protein moves to its post-fusion structure, and we propose that 8G5F11 cross-neutralises VesGs by inhibiting this.

    IMPORTANCE VSVind.G is currently regarded as the gold-standard envelope to pseudotype lentiviral vectors. However, recently other G proteins derived from vesiculoviruses have been proposed as alternative envelopes. Here, we investigated two commercially available anti-VSVind.G monoclonal antibodies for their ability to cross-react with other vesiculovirus G proteins, and identified the epitopes they recognise, and explored their neutralisation activity. We have identified 8G5F11, for the first time, as a cross-neutralising antibody against several vesiculovirus G proteins. Furthermore, we elucidated the two different neutralisation mechanisms employed by these two monoclonal antibodies. Understanding how cross-neutralising antibodies interact with other G proteins may be of interest in the context of host-pathogen interaction and co-evolution as well as providing the opportunity to modify the G proteins and improve G protein-containing medicinal products and vaccine vectors.

  • Attenuation of influenza A virus disease severity by viral co-infection in a mouse model [Pathogenesis and Immunity]

  • Influenza viruses and rhinoviruses are responsible for a large number of acute respiratory viral infections in human populations and are detected as co-pathogens within hosts. Clinical and epidemiological studies suggest that co-infection by rhinovirus and influenza virus may reduce disease severity and that they may also interfere with each other's spread within a host population. To determine how co-infection by these two unrelated respiratory viruses affects pathogenesis, we established a mouse model using a minor serogroup rhinovirus (RV1B) and mouse-adapted influenza A virus (PR8). Infection of mice with RV1B two days before PR8 reduced the severity of infection by a low or medium, but not high, dose of PR8. Disease attenuation was associated with an early inflammatory response in the lungs and enhanced clearance of PR8. However, co-infection by RV1B did not reduce PR8 viral loads early in infection or inhibit replication of PR8 within respiratory epithelia or in vitro. Inflammation in co-infected mice remained focal, in comparison to diffuse inflammation and damage in the lungs of mice infected by PR8. The timing of RV1B co-infection was a critical determinant of protection, suggesting that sufficient time is needed to induce this response. Finally, disease attenuation was not unique to RV1B: dose-dependent co-infection by a murine coronavirus (MHV-1) also reduced the severity of PR8 infection. Unlike RV1B, co-infection with MHV-1 reduced early PR8 replication, which was associated with up-regulation of IFN-bbeta; expression. This model is critical for understanding the mechanisms responsible for influenza disease attenuation during co-infection by unrelated respiratory viruses.

    Importance Viral infections in the respiratory tract can cause severe disease and are responsible for a majority of pediatric hospitalizations. Molecular diagnostics have revealed that approximately 20% of these patients are infected by more than one unrelated viral pathogen. To understand how viral co-infection affects disease severity, we inoculated mice with a mild viral pathogen (rhinovirus or murine coronavirus) followed two days later by a virulent viral pathogen (influenza A virus). This model demonstrated that rhinovirus can reduce the severity of influenza A virus, which corresponded with an early but controlled inflammatory response in the lungs and early clearance of influenza A virus. We further determined the dose and timing parameters that were important for effective disease attenuation and showed that influenza disease is also reduced by co-infection with a murine coronavirus. These findings demonstrate that co-infecting viruses can alter immune responses and pathogenesis in the respiratory tract.

  • Natural Secretory Immunoglobulins Promote Enteric Viral Infections [Pathogenesis and Immunity]

  • Noroviruses are enteric pathogens causing significant morbidity, mortality and economic losses worldwide. Secretory immunoglobulins (SIg) are a first line of mucosal defense against enteric pathogens. They are secreted into the intestinal lumen via the polymeric immunoglobulin receptor (pIgR), where they bind to antigens. However, whether natural SIg protect against norovirus infection remains unknown. To determine if natural SIg alter murine norovirus (MNV) pathogenesis, we infected pIgR knockout (KO) mice, which lack SIg in mucosal secretions. Acute MNV infection was significantly reduced in pIgR KO mice compared to controls, despite increased MNV target cells in the Peyer's patch. Natural SIg did not alter MNV binding to, or crossing, of the follicle-associated epithelium (FAE) into the lymphoid follicle. Instead, naïve pIgR KO mice have enhanced levels of the antiviral inflammatory molecules interferon gamma (IFN) and inducible nitric oxide synthase (iNOS) in the ileum compared to controls. Strikingly, depletion of the intestinal microbiota in pIgR KO and control mice resulted in comparable IFN and iNOS levels, as well as MNV infectious titers. IFN treatment of WT mice and neutralization of IFN in pIgR KO mice reduced MNV titers, implicating this antiviral cytokine in the phenotype. Reduced gastrointestinal infection in pIgR KO mice was also observed with another enteric virus, reovirus. Collectively, our findings suggest that natural SIg are not protective during enteric virus infection, but rather that SIg promote enteric viral infection through alterations in microbial immune responses.

  • Recombinant Infectious Bronchitis Viruses expressing chimaeric spike glycoproteins induce partial protective immunity against homologous challenge despite limited replication in vivo [Vaccines and Antiviral Agents]

  • Vaccination regimes against Infectious bronchitis virus, which are based on a single virus serotype, often induce insufficient levels of cross-protection against serotypes and two or more antigenically diverse vaccines are used in attempt to provide broader protection. Amino acid differences in the surface protein, spike (S), in particular the S1 subunit, are associated with poor cross-protection. Here, homologous vaccination trials with recombinant IBVs, based on the apathogenic strain, BeauR, were conducted to elucidate the role of S1 in protection. A single vaccination of SPF-chickens with rIBV expressing S1 of virulent strains M41 or QX, BeauR-M41(S1) and BeauR-QX(S1), gave incomplete protection against homologous challenge, based on ciliary activity and clinical signs. There could be conformational issues with the spike if heterologous S1 and S2 are linked, suggesting a homologous S2 might be essential. To address this, a homologous vaccination-challenge trial incorporating rIBVs expressing full spike from M41, BeauR-M41(S), and S2 subunit from M41, BeauR-M41(S2) was conducted. All chimaeric viruses grew to similar titres in vitro, induced virus-specific partial protective immunity, evident by cellular infiltrations, reductions in viral RNA load in the trachea and conjunctiva and higher serum anti-IBV titres. Collectively, these show that vaccination with rIBVs primed the birds for challenge but the viruses were cleared rapidly from the mucosal tissues in the head. Chimaeric S1 and S2 viruses did not protect as effectively as BeauR-M41(S) based on ciliary activity and clinical signs. Booster vaccinations and a rIBV with improved in vivo replication may improve the levels of protection.

    IMPORTANCE Infectious bronchitis virus causes an acute, highly contagious respiratory disease, responsible for significant economic losses to the poultry industry. Amino acid differences in the surface protein, spike (S), in particular the S1 subunit, have been associated with poor cross-protection. Available vaccines give poor cross-protection and rationally designed live attenuated vaccines, based on apathogenic BeauR, could address these. Here, to determine the role of S1 in protection, a series of homologous vaccination trials with rIBVs were conducted. Single vaccinations with chimaeric rIBVs induced virus-specific partial protective immunity, characterised by reduction in viral load and serum antibody titres. However, BeauR-M41(S) was the only vaccination to improve the level of protection against clinical signs and the loss of tracheal ciliary activity. Growth characteristics show all of the rIBVs replicated in vitro to similar levels. Booster vaccinations and a rIBV with improved in vivo replication may improve the levels of protection.

  • A single point mutation in the rhinovirus 2B protein reduces the requirement for phosphatidylinositol 4-kinase class 3beta in viral replication [Cellular Response to Infection]

  • Rhinoviruses (RVs) replicate on cytoplasmic membranes derived from the Golgi apparatus. They encode membrane-targeted proteins 2B, 2C and 3A, which control trafficking and lipid composition of the replication membrane. The virus recruits host factors for replication, such as the phosphatidylinositol 4 (PI4)-kinase 3beta (PI4K3b), which boosts PI4-phosphate (PI4P) levels, and drives lipid counter-current exchange of PI4P against cholesterol at endoplasmic reticulum-Golgi membrane contact sites through the lipid shuttling protein oxysterol binding protein (OSBP) 1. We identified a PI4K3b-inhibitor resistant RV-A16 variant with a single point mutation in the conserved 2B protein near the cytosolic carboxy-terminus, isoleucine 92 to threonine [I92T]. The mutation did not confer resistance to cholesterol sequestering compounds or OSBP1 inhibition, suggesting invariant dependency on the PI4P/cholesterol lipid counter-currents. In presence of PI4K3b-inhibitor, Golgi reorganization and PI4P lipid induction occurred in RV-A16 2B[I92], but not wild-type infection. The knock-out of PI4K3b abolished the replication of both 2B[I92T] mutant and wild-type. Doxycyclin-inducible expression of PI4K3b in PI4K3b knock-out cells efficiently rescued the 2B[I92T] mutant, and less effectively wild-type virus infection. Ectopic expression of 2B[I92T] or 2B was less efficient than 3A in recruiting PI4K3b to perinuclear membranes, suggesting a supportive rather than decisive role of 2B in recruiting PI4K3b. The data suggest that 2B tunes the recruitment of PI4K3b to the replication membrane, and allows the virus to adapt to cells with low levels of PI4K3b, yet maintaining the PI4P/cholesterol counter-current for establishing Golgi-derived RV replication membranes.

    Importance

    Human rhinoviruses (RVs) are the major cause of common cold worldwide. They cause asthmatic exacerbations and chronic obstructive pulmonary disease. Despite recent advances, the development of antivirals and vaccines has proven difficult due to the high number and variability of RV types. The identification of critical host factors and their interactions with viral proteins and membrane lipids for the establishment of viral replication is a basis for drug development strategies. Our findings here shed new light on the interactions between nonstructural viral membrane proteins and class III phosphatidylinositol 4 kinases from the host, and highlight the importance of phosphatidyl-inositol 4 phosphate for RV replication.

  • HOIL1 is essential for the induction of type I and III interferons by MDA5 and regulates persistent murine norovirus infection [Cellular Response to Infection]

  • The Linear Ubiquitin Chain Assembly Complex (LUBAC), composed of heme-oxidized IRP2 ubiquitin ligase-1 (HOIL1), HOIL-1-interacting protein (HOIP) and SHANK-associated RH-domain-interacting protein (SHARPIN), is a crucial regulator of multiple immune signaling pathways. In humans, HOIL1- or HOIP-deficiency is associated with an immune disorder involving auto-inflammation, immunodeficiency and inflammatory bowel disease (IBD)-like symptoms. During viral infection, LUBAC is reported to inhibit the induction of interferon (IFN) by the cytosolic RNA sensor, RIG-I. Surprisingly, we found that HOIL1 is essential for the induction of both type I and type III IFNs, as well as the phosphorylation of IFN regulatory factor (IRF3), during murine norovirus (MNoV) infection in cultured dendritic cells. The RIG-I-like receptor, MDA5, is also required for IFN induction and IRF3 phosphorylation during MNoV infection. Furthermore, HOIL1 and MDA5 were required for IFN induction after Theilerrrsquo;s murine encephalomyelitis virus infection and poly(I:C) transfection, but not Sendai virus or vesicular stomatitis virus infection, indicating that HOIL1 and LUBAC are required specifically for MDA5 signaling. Moreover, Hoil1-/- mice exhibited defective control of acute and persistent murine norovirus (MNoV) infection, and defective regulation of MNoV persistence by the microbiome as also observed previously for IFN- receptor-, STAT1- and IRF3-deficient mice. These data indicate that LUBAC plays a critical role in IFN induction to control RNA viruses sensed by MDA5.

    IMPORTANCE Human noroviruses are a leading cause of gastroenteritis throughout the world, but are challenging to study in vivo and in vitro. Murine norovirus (MNoV) provides a tractable genetic and small animal model to study norovirus biology and immune responses. Interferons are critical mediators of anti-viral immunity, but excessive expression can dysregulate the immune system. IFN- plays an important role at mucosal surfaces including the gastrointestinal tract, and both IFN- and commensal enteric bacteria are important modulators of persistent MNoV infection. The LUBAC complex, of which HOIL1 is a component, is reported to inhibit type I IFN induction after RIG-I stimulation. We show, in contrast, that HOIL1 is critical for type I and III IFN induction during infection with MNoV, a virus that preferentially activates MDA5. Moreover, HOIL1 regulates MNoV infection in vivo. These data reveal distinct functions for LUBAC in these closely related signaling pathways and in modulation of IFN expression.

  • Human host-range restriction of the vaccinia virus C7/K1 double deletion mutant is mediated by an atypical mode of translation inhibition [Virus-Cell Interactions]

  • Replication of vaccinia virus in human cells depends on the viral C7 or K1 protein. A previous human genome-wide siRNA screen with a C7/K1 double deletion mutant revealed SAMD9 as a principal host-range restriction factor plus additional candidates including WDR6 and FTSJ1. To compare their abilities to restrict replication, the cellular genes were individually inactivated by CRISPR/Cas9 mutagenesis. The C7/K1 deletion mutant exhibited enhanced replication in each knock-out (KO) cell line but reached wild-type levels only in SAMD9 KO cells. SAMD9 was not depleted in either WDR6 or FTSJ1 KO cells, suggesting less efficient alternative rescue mechanisms. Using the SAMD9 KO cells as controls, we verified a specific block in host and viral intermediate and late protein synthesis in HeLa cells and demonstrated that the inhibition could be triggered by events preceding viral DNA replication. Inhibition of cap-dependent and -independent protein synthesis occurred primarily at the translational level, as supported by DNA and mRNA transfection experiments. Concurrent with collapse of polyribosomes, viral mRNA was predominantly in 80s and lighter ribonucleoprotein fractions. We confirmed the accumulation of cytoplasmic granules in HeLa cells infected with the C7/K1 deletion mutant and further showed that viral mRNA was sequestered with SAMD9. RNA granules were still detected in G3BP KO U2OS cells, which remained non-permissive for the C7/K1 deletion mutant. Inhibition of cap-dependent and internal ribosome entry site mediated translation, sequestration of viral mRNA, and failure of PKR, RNase L or G3BP KO cells to restore protein synthesis support an unusual mechanism of host restriction.

    IMPORTANCE A dynamic relationship exists between viruses and their hosts in which each ostensibly attempts to exploit the otherrrsquo;s vulnerabilities. A window is opened into the established condition, which evolved over millennia, if loss-of-function mutations occur in either the virus or host. Thus, the inability of viral host-range mutants to replicate in specific cells can be overcome by identifying and inactivating the opposing cellular gene. Here, we investigated a C7/K1 host range mutant of vaccinia virus in which the cellular gene SAMD9 serves as the principal host restriction factor. Host restriction was triggered early in infection and manifested as a block in translation of viral mRNAs. Features of the block include inhibition of cap-dependent and internal ribosome entry site-mediated translation, sequestration of viral RNA and inability to overcome the inhibition by inactivation of protein kinase R, ribonuclease L or G3 binding proteins, suggesting a novel mechanism of host restriction.

  • Global analysis of baculovirus AcMNPV gene expression in the midgut of the lepidopteran host, Trichoplusia ni [Virus-Cell Interactions]

  • The baculovirus Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) is a large dsDNA virus that encodes approximately 156 genes and is highly pathogenic to a variety of larval lepidopteran insects in nature. Oral infection of larval midgut cells is initiated by the occlusion derived virus (ODV), while secondary infection of other tissues is mediated by the budded virus (BV). Global viral gene expression has been studied in detail in BV-infected cell cultures, but studies of ODV-infection in the larval midgut are limited. In this study, we examined expression of the ~156 AcMNPV genes in Trichoplusia ni midgut tissue using a transcriptomic approach. We analyzed expression profiles of viral genes in the midgut, and compared them with profiles from a T. ni cell line (Tnms42). Several viral genes (p6.9, orf76, orf75, pp31, Ac-bro, odv-e25, and odv-ec27) had high expression levels in the midgut throughout the infection. Also, the expression of genes associated with occlusion bodies (polh and p10) appeared to be delayed in the midgut in comparison with the cell line. Comparisons of viral gene expression profiles revealed remarkable similarities between the midgut and cell line for most genes, although substantial differences were observed for some viral genes. These included genes associated with high level BV production (fp-25k), acceleration of systemic infection (v-fgf), and enhancement of viral movement (arif-1/orf20). These differential expression patterns appear to represent specific adaptations for virus infection and transmission through the polarized cells of the lepidopteran midgut.

    IMPORTANCE Baculoviruses such as AcMNPV are pathogens that are natural regulators of certain insect populations. Baculovirus infections are biphasic, with a primary phase initiated by oral infection of midgut epithelial cells by occlusion derived virus (ODV) virions, and a secondary phase in which other tissues are infected by budded virus (BV) virions. While AcMNPV infections in cultured cells have been studied extensively, comparatively little detail is known regarding primary infection in the midgut. In these studies, we identified gene expression patterns associated with ODV-mediated infection of the midgut in Trichoplusia ni, and compared those results with prior results from BV-infected cultured cells, which simulate secondary infection. These studies provide a detailed analysis of viral gene expression patterns in the midgut, which likely represent specific viral strategies to: a) overcome or avoid host defenses in the gut, and b) rapidly move infection from the midgut, into the hemocoel to facilitate systemic infection.

  • Hepatitis B spliced protein (HBSP) suppresses Fas-mediated hepatocyte apoptosis via activation of PI3K/Akt signaling [Pathogenesis and Immunity]

  • Hepatitis B spliced protein (HBSP) is known to associate with viral persistence and pathogenesis, however, its biological and clinical significance remains poorly defined. Acquired resistance to Fas-mediated apoptosis is thought as one of the major promotors for hepatitis B virus (HBV) chronicity and malignancy. The purpose of this study was to investigate whether HBSP could protect hepatocytes against Fas-initiated apoptosis. We showed here that HBSP mediated resistance of hepatoma cells or primary human hepatocytes (PHH) to agonistic anti-Fas antibody (CH11)- or FasL-induced apoptosis. Under Fas signaling stimulation, expression of HBSP inhibited Fas aggregation and prevented recruitment of the adaptor molecule Fas-associated death domain (FADD) and procaspase-8 (or FADD like interleukin 1 bbeta;-converting enzyme, FLICE) into the death-inducing signaling complex (DISC), while increasing recruitment of cellular FLICE-inhibitory protein L (FLIPL) into the DISC. Those effects may be mediated through activation of PI3K/AKT pathway as evidenced by increased cellular PIP3 content and PI3K activity, and enhanced phosphorylation of mTORC2 and PDPK1 as well as Akt itself. Confirmedly, inhibition of PI3K by LY294002 reversed the effect of HBSP on Fas aggregation, FLIPL expression and cellular apoptosis. These results indicate that HBSP functions to prevent hepatocytes from Fas-induced apoptosis by enhancing PI3K/Akt activity, which may contribute to the survival and persistence of infected hepatocytes during chronic infection.

    Importance: Our study revealed a previously unappreciated role of HBSP in Fas-mediated apoptosis. The anti-apoptotic activity of HBSP is important for understanding hepatitis B pathogenesis. In particular, HBV variants associated with hepatoma carcinoma may down-regulate apoptosis of hepatocytes through enhanced HBSP expression. Our study also found that AKT is centrally involved in Fas-induced hepatocyte apoptosis and revealed interventions directed at inhibiting the activation or functional activity of AKT may be of therapeutic value in this process.

  • Interplay between the poly(A) tail, poly(A)-binding protein and coronavirus nucleocapsid protein regulates gene expression of the coronavirus and host cell [Genome Replication and Regulation of Viral Gene Expression]

  • In the present study, we investigated the roles of interactions among poly(A) tail, coronavirus nucleocapsid (N) protein and poly(A)-binding protein (PABP) in the regulation of coronavirus gene expression. Through dissociation constant (Kd) comparison, we found that the coronavirus N protein can bind to the poly(A) tail with high affinity, establishing N protein as a PABP. A subsequent analysis with UV cross-linking and immunoprecipitation revealed that the N protein is able to bind to the poly(A) tail in infected cells. Further examination demonstrated that poly(A) tail binding by the N protein negatively regulates translation of coronaviral RNA and host mRNA both in vitro and in cells. Although the N protein can interact with PABP and eIF4G, the poor interaction efficiency between the poly(A)-bound N protein and eIF4E may explain the observed decreased translation efficiency. In addition to interaction with translation factor eIF4G, the N protein is able to interact with coronavirus nonstructural protein 9 (nsp9), a replicase protein required for replication. Together, the study demonstrates interactions among the poly(A) tail, N protein and PABP both in vitro and in infected cells. Of the interactions, binding of poly(A) tail to N protein decreases the interaction efficiency between the poly(A) tail and eIF4E, leading to translation inhibition. The poly(A)-dependent translation inhibition by N protein has not been previously demonstrated and thus extends our understanding of coronavirus gene expression.

    IMPORTANCE Gene expression in coronavirus is a complicated and dynamic process. In this study, we demonstrate coronavirus N protein is able to bind to the poly(A) tail with high affinity, establishing N protein as a PABP. We also show how the interplay between coronavirus 3'-poly(A) tail, PABP and N protein regulates gene expression of the coronavirus and host cell. Of the interactions, poly(A) tail binding by the N protein negatively regulates translation and, to our knowledge, this inhibition of translation by binding of the N protein to poly(A) tail has not been previously studied. Accordingly, the study provides fundamental molecular details regarding coronavirus infection and expands our knowledge of coronavirus gene expression.

  • Modulation of Vaccine-induced CD4 T Cell Functional Profiles by Changes in Components of HIV Vaccine Regimens in Humans [Cellular Response to Infection]

  • To date, six vaccine strategies have been evaluated in clinical trials for their efficacy to induce protective immune responses against HIV infection. However, only ALVAC-HIV/AIDSVAXrreg; B/E (RV144) has demonstrated protection, albeit modestly (31%, p=0.03). One potential correlate of protection was a low-frequency HIV-specific CD4 T cell population with diverse functionality. Although CD4 T cells, particularly T follicular helper cells (Tfh) are critical for effective antibody responses, most studies involving HIV vaccines focused on humoral immunity or CD8 T cell effector responses, while little is known about functionality and frequency of vaccine-induced CD4 T cells. We therefore assessed responses from several phase I/II clinical trials and compared them to natural HIV-1 infection. We found that all vaccines induced a lower magnitude of HIV-specific CD4 T cell responses compared to chronic infection. Responses differed in functionality, with a CD40L-dominated response and more Tfh cells after vaccination, whereas chronic HIV infection provoked TNFaalpha;-dominated responses. Vaccine delivery route further impacted CD4 T cells, showing a stronger Th1 polarization after dendritic cell delivery compared to intramuscular vaccination. In prime/boost regimens, the choice of prime and boost influenced the functional profile of CD4 T cells to induce more or less polyfunctionality. In summary, vaccine-induced CD4 T cell responses differ remarkably between vaccination strategies, modes of delivery and boosts and do not resemble those in chronic HIV infection. Understanding functional profiles of CD4 T cells that best facilitate protective antibody responses will be critical when CD4 T cell responses are considered as a clinical trial go/no-go criterion.

    IMPORTANCE Only one HIV-1 candidate vaccine strategy has shown protection, albeit marginal (31%), against HIV-1 acquisition and correlates of protection suggested that a multifunctional CD4 T cell immune response may be important for this protective effect. Therefore, the functional phenotype of HIV-specific CD4 T cell responses induced by different phase I and phase II clinical trials was assessed to better understand how different vaccine strategies influence the phenotype and function of HIV specific CD4 T cell immune responses. The significance of this research lies in our comprehensive comparison of the composition of the T cell immune responses to different HIV vaccine modalities. Specifically, our work allows for the evaluation of vaccination strategies in terms of their success in inducing Tfh cell populations.

  • Type I interferon signaling prevents hepatitis B virus-specific T cell responses by reducing antigen expression [Pathogenesis and Immunity]

  • Robust virus-specific CD8+ T cell responses are required for the clearance of hepatitis B virus (HBV). However, the factors that determine the magnitude of HBV-specific CD8+ T cell responses are poorly understood. To examine the impact of genetic variations of HBV on HBV-specific CD8+ T cell responses, we introduced three HBV clones (Aa, C22, D60) that express varying amounts of HBV antigens into the livers of C57BL/6 (B6) mice (H-2b) and B10.D2 mice (H-2d). In B6 mice, clone C22 barely induced HBV-specific CD8+ T cell responses and persisted the longest, while clone D60 elicited strong HBV-specific CD8+ T cell responses and was rapidly cleared. These differences between HBV clones largely diminished in B10.D2 mice (H-2d). Interestingly, the magnitude of HBV-specific CD8+ T cell responses in B6 mice was associated with the HB core antigen expression level during the early phase of HBV transduction. Surprisingly, robust HBV-specific CD8+ T cell responses to clone C22 were induced in interferon-aalpha;/bbeta; receptor-deficient (IFN-aalpha;bbeta;R-/-) mice (H-2b). The induction of HBV-specific CD8+ T cell responses to C22 in IFN-aalpha;bbeta;R-/- mice reflects enhanced HBV antigen expression because the suppression of antigen expression by HBV-specific siRNA attenuated HBV-specific T cell responses in IFN-aalpha;bbeta;R-/- mice and prolonged HBV expression. Collectively, these results suggest that HBV genetic variation and type I interferon signaling determine the magnitude of HBV-specific CD8+ T cell responses by regulating the initial antigen expression levels.

    IMPORTANCE Hepatitis B virus (HBV) causes acute and chronic infection, and approximately 240 million people are chronically infected with HBV worldwide. It is generally believed that virus-specific CD8+ T cell responses are required for the clearance of HBV. However, the relative contribution of genetic variation and innate immune responses to the induction of HBV-specific CD8+ T cell responses are not fully understood. In this study, we discovered that different clearance rate between HBV clones after hydrodynamic transduction was associated with the magnitude of HBV-specific CD8+ T cell responses and initial HB core antigen expression. Surprisingly, type I interferon signaling negatively regulated HBV-specific CD8+ T cell responses by reducing early HBV antigen expression. These results show that the magnitude of HBV-specific CD8+ T cell response is primarily regulated by the initial antigen expression level.

  • The nucleolar protein LYAR facilitates ribonucleoprotein assembly of influenza A virus [Virus-Cell Interactions]

  • Influenza A viral ribonucleoprotein (vRNP) is responsible for transcription and replication of the viral genome in infected cells and depend on host factors for its functions. Identification of the host factors interacting with vRNP not only improves understanding of virus-host interactions, but also provides insights into novel mechanisms of viral pathogenicity and the development of new antiviral strategies. Here, we have identified eighty host factors that co-purified with vRNP using affinity purification followed by mass spectrometry. LYAR, a cell growth-regulating nucleolar protein, has been shown to be important for influenza A virus replication. During influenza A virus infection, LYAR expression is increased and it partly translocates from the nucleolus to nucleoplasm and cytoplasm. Furthermore, LYAR interacts with RNP subunits, resulting in enhancing viral RNP assembly, thereby facilitating viral RNA synthesis. Taken together, our studies identify a novel vRNP binding host partner important for influenza A virus replication, and further reveal the mechanism of LYAR regulating influenza A viral RNA synthesis by facilitating viral RNP assembly.

    IMPORTANCE Influenza A virus (IAV) must utilize the host cell machinery to replicate, but many of the mechanisms of the IAV-host interaction remain poorly understood. Improved understanding of interactions between host factors and vRNP not only increases our basic knowledge of the molecular mechanisms of virus replication and pathogenicity, but also provides insights into possible novel antiviral targets that are necessary due to the widespread emergence of drug-resistant IAV strains. Herein, we have identified LYAR, a cell growth-regulating nucleolar protein, which interacts with viral RNP components and is important for efficient replication of IAVs, and whose role in the IAV life cycle has never been reported. In addition, we further reveal the role of LYAR in viral RNA synthesis. Our results extend and improve the current knowledge on the mechanisms of IAV transcription and replication.

  • Epstein-Barr Virus miR-BART5-3p inhibits p53 expression [Transformation and Oncogenesis]

  • Epstein-Barr virus (EBV) is the first human virus found to encode many microRNAs. It is etiologically linked to nasopharyngeal carcinoma and EBV-associated gastric carcinoma. During the latent infection period, there are only a few EBV proteins expressed, whereas EBV microRNAs, such as BART microRNAs, are highly expressed. However, how these BART miRNAs precisely regulate the tumor growth in nasopharyngeal carcinoma and gastric carcinoma remains obscure. Here, we report that up-regulation of EBV-miR-BART5-3p promotes the growth of nasopharyngeal carcinoma and gastric carcinoma cells. BART5-3p directly targets the tumor suppressor gene TP53 on its 3'-UTR and consequently down-regulates CDKN1A, BAX and FAS expression, leading to acceleration of the cell cycle progress and inhibition of cell apoptosis. BART5-3p contributes to the resistance to chemotherapeutic drugs and ionizing irradiation-induced p53 increase. Moreover, BART5-3p also facilitates degradation of p53 proteins. BART5-3p is the first EBV-microRNA to be identified to inhibiting p53 expression and function, which suggests a novel mechanism underlying the strategies employed by EBV to maintain latent infection and promote the development of EBV-associated carcinomas.

    IMPORTANCE EBV encodes 44 mature microRNAs, which have been proven to promote EBV-associated diseases by targeting host genes and self-viral genes. In EBV-associated carcinomas, the expression of viral protein is limited but the expression of BART microRNAs is extremely high, suggesting that they could be major factors in the contribution of EBV-associated tumorigenesis. p53 is a critical tumor suppressor. Unlike in most human solid tumors, TP53 mutations are rare in nasopharyngeal carcinoma and EBV associated gastric carcinoma tissues, suggesting a possibility that some EBV-encoded products suppress the functions of p53. This study provides the first evidence that a BART microRNA can suppress p53 expression by directly targeting its 3'-UTR. This study implies that EBV can use its BART microRNAs to modulate the expression of p53, thus maintaining its latency and contributing to tumorigenesis.

  • Reovirus Neurotropism and Virulence Are Dictated by Sequences in the Head Domain of the Viral Attachment Protein [Pathogenesis and Immunity]

  • Viral capsid components that bind cellular receptors mediate critical functions in viral tropism and disease pathogenesis. Mammalian orthoreoviruses (reoviruses) spread systemically in newborn mice to cause serotype-specific disease in the central nervous system (CNS). Serotype 1 (T1) reovirus infects ependymal cells to cause non-lethal hydrocephalus, whereas serotype 3 (T3) reovirus infects neurons to cause fulminant and lethal encephalitis. This serotype-dependent difference in tropism and concomitant disease is attributed to the 1 viral attachment protein, which is composed of head, body, and tail domains. To identify 1 sequences that contribute to tropism for specific cell types in the CNS, we engineered a panel of viruses expressing chimeric 1 proteins in which discrete 1 domains have been reciprocally exchanged. Parental and chimeric 1 viruses were compared for replication, tropism, and disease induction following intracranial inoculation of newborn mice. Viruses expressing T1 1 head sequences infect the ependyma, produce relatively lower titers in the brain, and do not cause significant disease. In contrast, viruses expressing T3 1 head sequences efficiently infect neurons, replicate to relatively higher titers in the brain, and cause a lethal encephalitis. Additionally, T3 1 head-expressing viruses display enhanced infectivity of cultured primary cortical neurons compared with T1 1 head-expressing viruses. These results indicate that T3 1 head-domain sequences promote infection of neurons, likely by interaction with a neuron-specific receptor, and dictate tropism in the CNS and induction of encephalitis.

    IMPORTANCE Viral encephalitis is a serious and often life-threatening inflammation of the central nervous system (CNS). Mammalian orthoreoviruses are promising oncolytic therapeutics for humans but establish virulent, serotype-dependent disease in the CNS of many young mammals. Serotype 1 reoviruses infect ependymal cells and produce hydrocephalus, whereas serotype 3 reoviruses infect neurons and cause encephalitis. Reovirus neurotropism is hypothesized to be dictated by the filamentous 1 viral attachment protein. However, it is not apparent how this protein mediates disease. We discovered that sequences forming the most virion-distal domain of T1 and T3 1 coordinate infection of either ependyma or neurons, respectively, leading to mutually exclusive patterns of tropism and disease in the CNS. These studies contribute new knowledge about how reoviruses target cells for infection in the brain and inform the rational design of improved oncolytic therapies to mitigate difficult-to-treat tumors of the CNS.

  • Distribution, diversity and evolution of endogenous retroviruses in perissodactyl genomes. [Genetic Diversity and Evolution]

  • The evolution of mammalian genomes has been shaped by interactions with endogenous retroviruses (ERVs). In this study, we investigated the distribution and diversity of ERVs in the mammalian order Perissodactyla, with a view to understanding their impact on the evolution of modern equids (family Equidae). We characterize the major ERV lineages in the horse genome in terms of their genomic distribution, ancestral genome organization and time of activity. Our results show that subsequent to their ancestral divergence from rhinos and tapirs, equids acquired four novel ERV lineages. We show that two of these proliferated extensively in the lineage leading to modern horses, and one contains loci that are actively transcribed in specific tissues. In addition, we show that the white rhinoceros has resisted germline colonisation by retroviruses for over 54 million years - longer than any other extant mammalian species. The map of equine ERVs that we provide here will be of great utility to future studies aiming to investigate the potential functional roles of equine ERVs, and their impact on equine evolution.

    IMPORTANCE ERVs in the host genome are highly informative about the long-term interactions of retroviruses and hosts. They are also interesting because they have influenced the evolution of mammalian genomes in various ways. In this study, we derive a calibrated timeline describing the process through which ERV diversity has been generated in the equine germline. We determined the distribution and diversity of perissodactyl ERV lineages and inferred their retrotranspositional activity during evolution, thereby gaining insight into the long-term co-evolutionary history of retroviruses and mammals. Our study provides a platform for future investigations to identify equine ERV loci involved in physiological processes and/or pathological conditions.

  • Susceptibility to Neutralization by Broadly Neutralizing Antibodies Generally Correlates with Infected Cell Binding for a Panel of Clade B HIV Reactivated from Latent Reservoirs [Pathogenesis and Immunity]

  • Efforts to HIV cure are obstructed by reservoirs of latently infected CD4+ T-cells that can re-establish viremia. Broadly neutralizing HIV-specific antibodies (bNAbs), defined by unusually high neutralization breadths against globally diverse viruses, may contribute to the elimination of these reservoirs by binding to reactivated cells, targeting them for immune clearance. However, the relationship between neutralization of reservoir isolates and binding to corresponding infected primary CD4+ T-cells has not been determined. Thus, the extent to which neutralization breadths and potencies can be used to infer the corresponding parameters of infected-cell binding is currently unknown. We assessed the breadths and potencies of bNAbs against 36 viruses reactivated from peripheral blood CD4+ T-cells of ARV-treated HIV-infected individuals, using paired neutralization and infected-cell binding assays. Single antibody breadths ranged from 0nndash;64% for neutralization (IC80lle;10mmu;g/ml) and 0nndash;89% for binding, with two-antibody combinations (antibody combinations are theoretical/predicted) reaching 0-83% and 50-100%, respectively. Infected-cell binding correlated with virus neutralization for 10 out of 14 antibodies (e.g. 3BNC117, r=0.82, pllt;0.0001). Heterogeneity was observed, however, with a lack of significant correlations for 2G12, CAP256.VRC26.25, 2F5, and 4E10. Our results provide guidance on the selection of bNAbs for interventional cure studies; both by providing a direct assessment of intra- and inter-individual variability in neutralization and infected cell binding in a novel cohort, and by defining the relationships between these parameters for a panel of bNAbs.

    Importance Although anti-retroviral therapies have improved the lives of people who are living with HIV, they do not cure infection. Efforts are being directed towards harnessing the immune system to eliminate the virus that persists, potentially resulting in virus-free remission without medication. HIV-specific antibodies hold promise for such therapies owing to their abilities to both prevent the infection of new cells (neutralization), and also to direct the killing of infected cells. We isolated 36 HIV strains from individuals whose virus was suppressed by medication, and tested 14 different antibodies for neutralization of these viruses and for binding to cells infected with the same viruses (critical for engaging natural killer cells). For both neutralization and infected-cell binding, we observed variation both between individuals, and amongst different viruses within an individual. For most antibodies, neutralization activity correlated with infected cell binding. These data provide guidance on the selection of antibodies for clinical trials.

  • The human cytomegalovirus US27 gene product constitutively activates antioxidant response element (ARE)-mediated transcription through G{beta}{gamma}, phosphoinositide 3-kinase (PI3K), and nuclear respiratory factor 1 (NRF-1) [Virus-Cell Interactions]

  • Human cytomegalovirus (HCMV) is a widespread pathogen that modulates host chemokine signaling during persistent infection in the host. HCMV encodes four proteins with homology to the chemokine receptor family of G protein-coupled receptors (GPCRs): US27, US28, UL33, and UL78. Each of the four receptors modulates host CXCR4 signaling. US28, UL33 and UL78 impair CXCR4 signaling outcomes while US27 enhances signaling, as evidenced by increased calcium mobilization and cell migration to CXCL12. To investigate effects of US27 on CXCR4 during virus infection, fibroblasts were infected with BAC-derived clinical strain HCMV TB40/E-mCherry (WT) or mutants lacking US27 (TB40/E-mCherry-US27) or all four GPCRs (TB40 E-mCherry-all), or expressing only US27, but not US28, UL33, or UL78 (TB40/E-mCherry-US27wt). CXCR4 gene expression was significantly higher in WT and US27wt-infected fibroblasts. This effect was evident at 3 hours post-infection, suggesting that US27 derived from the parental virion enhanced CXCR4 expression. Reporter gene assays demonstrated US27 increased transcriptional activity regulated by the antioxidant response element (ARE), and siRNA treatment indicated this effect was mediated by NRF-1, the primary transcription factor for CXCR4. Increased translocation of NRF-1 into the nucleus of WT-infected cells compared to mock- or US27-infected cells was confirmed by immunofluorescence microscopy. Chemical inhibitors targeting Gbbeta; and phosphoinositide 3-kinase (PI3K) ablated the increase in ARE-driven transcription, implicating these proteins as mediators of US27-stimulated gene transcription. This work identifies the first signaling pathway activated by HCMV US27 and may reveal a novel regulatory function for this orphan viral receptor in stimulating stress response genes during infection.

    IMPORTANCE Human cytomegalovirus (HCMV) is the most common congenital infection worldwide, causing deafness, blindness, and other serious birth defects. CXCR4 is a human chemokine receptor that is crucial for both fetal development and immune responses. We found that the HCMV protein US27 stimulates increased expression of CXCR4 through activation of the transcription factor nuclear respiratory factor 1 (NRF-1). NRF-1 regulates stress response genes that contain the antioxidant response element (ARE), and HCMV infection is associated with increased expression of many stress response genes when US27 is present. Our results show that the US27 protein activates the NRF-1/ARE pathway, stimulating higher expression of CXCR4 and other stress response genes, which is likely to be beneficial for virus replication and/or immune evasion.

  • Herpes simplex virus-1 specifically targets human CD1d antigen presentation to enhance its pathogenicity [Pathogenesis and Immunity]

  • Herpes simplex virus-1 (HSV-1) is one of the most prevalent herpes viruses in humans and represents a constant health threat to aged and immune-compromised populations. It is only partially known how HSV-1 interacts with the host immune system to efficiently establish their infection and latency. CD1d-restricted NKT cells are a critical arm of host innate immune system and play potent roles in anti-infection and anti-tumor immune responses. We previously discovered that upon infection, HSV-1 rapidly and efficiently down-regulates CD1d cell surface expression and suppresses the function of NKT cells. Furthermore, we identified that the viral serine/threonine protein kinase, US3, is a major viral factor down-regulating CD1d during infection. Interestingly, neither HSV-1, nor its US3 protein, efficiently inhibits mouse CD1d expression, suggesting that HSV-1 has co-evolved with human immune system to specifically suppress human CD1d and NKT cell function for its pathogenesis. This is consistent with the fact that wild-type mice are mostly resistant to HSV-1 infection. On the other hand, in-vivo infection of CD1d-humanized mouse (human CD1d-knockin) showed that HSV-1 indeed can evade human CD1d function and establish infection in these mice. We also reported here that US3-deficient viruses can not efficiently infect hCD1d-knockin mice but infect mice lacking all NKT cells at a higher efficiency. Together these studies supported that HSV-1 evasion of human CD1d and NKT cell function as an important pathogenic factor for the virus. Our results also validated the potent roles of NKT cells in anti-herpesviral immune responses and pointed to the potentials of NKT cell ligands as adjuvants for future vaccine development.

    Importance Herpes simplex virus-1 (HSV-1) is among the most common human pathogens. Little is known regarding the exact mechanism how this virus evades human immune system, particularly the innate immune system. We previously reported that HSV-1 employs its protein kinase US3 to modulate the expression of the key antigen-presenting molecule, CD1d, to evade the antiviral function of NKT cells. Here we demonstrated that the virus has co-evolved with human CD1d and NKT cell system and the NKT cells indeed play potent roles in anti-HSV immune responses. These studies point to the great potential of exploring NKT cell ligands as adjuvants for HSV vaccines.

  • Development of a Pan-H1 Influenza Vaccine [Vaccines and Antiviral Agents]

  • The efficacy of current seasonal influenza vaccines varies greatly, depending on the match to circulating viruses. Though most vaccines elicit strain-specific responses, some present cross-reactive epitopes that elicit antibodies against diverse viruses and remain unchanged and effective for several years (1). To determine whether combinations of specific H1 HA antigens stimulate immune responses that protect against diverse H1 influenza, we evaluated the antibody responses elicited by HA-ferritin nanoparticles derived from six evolutionarily divergent H1 sequences and two computationally optimized broadly reactive antigen (COBRA) HA antigens. Humoral responses were assessed against a panel of 16 representative influenza virus strains from the past 80 years. HAs from the strains A/NewCaledonia/20/1999 (NC99), A/California/04/2009 (CA09), A/HongKong/117/1977 (HK77) or COBRA-X6 and P1 elicited neutralization against diverse strains, and a combination of these three wildtype HA- or two COBRA HA-nanoparticles conferred significant additional breadth beyond that observed with any individual strain. Therefore, combinations of H1 HAs may potentially constitute a pan-H1 influenza vaccine.

    IMPORTANCE Seasonal influenza vaccines elicit strain-specific immune responses designed to protect against circulating viruses. Because these vaccines often show limited efficacy, the search for a broadly protective seasonal vaccine remains a priority. Among different influenza virus subtypes, H1N1 has long been circulating in humans and has caused pandemic outbreaks. In order to assess the potential of a multivalent HA combination vaccine to improve the breadth of protection against divergent H1N1 viruses, HA-ferritin nanoparticles were made and evaluated in mice against a panel of historical and contemporary influenza virus strains. Trivalent combinations of H1-nanoparticles improved the breadth of immunity against divergent H1 influenza viruses.

  • Comparison of adjuvanted-whole inactivated virus and live-attenuated virus vaccines against challenge with contemporary, antigenically distinct swine H3N2 influenza A viruses [Pathogenesis and Immunity]

  • Influenza A virus in swine (IAV-S) circulating in the United States of America are phylogenetically and antigenically distinct. A human H3 hemagglutinin (HA) was introduced in the IAV-S gene pool in the late 1990s, sustained continued circulation, and evolved into five monophyletic genetic clades after 2009, H3 IVA-E. Across these phylogenetic clades, distinct antigenic clusters were identified, with three clusters (cyan, red and green) among the most frequently detected antigenic phenotypes. Although it was demonstrated that antigenic diversity of H3N2 IAV-S was associated with changes at a few amino acid positions in the head of the HA, the implications of this diversity on vaccine efficacy was not tested. Using antigenically representative H3N2 viruses, we compared whole inactivated virus (WIV) and live attenuated influenza vaccine (LAIV) vaccines for protection against challenge with antigenically distinct H3N2 viruses in pigs. WIV provided partial protection against antigenically distinct viruses, but did not prevent virus replication in the upper respiratory tract. In contrast, LAIV provided complete protection from disease and virus was not detected after challenge with antigenically distinct viruses.

    IMPORTANCE Due to the rapid evolution of the influenza A virus, vaccines require continuous strain updates. Additionally, the platform used to deliver the vaccine can have an impact on the breadth of protection. Currently, there are various vaccine platforms available to prevent influenza A virus infection in swine, and we experimentally tested two: adjuvanted-whole inactivated virus and live attenuated virus. When challenged with an antigenically distinct virus, adjuvanted-whole inactivated virus provided partial protection while live attenuated virus provided effective protection. Additional strategies are required to broaden the protective properties of inactivated virus vaccines given the dynamic antigenic landscape of co-circulating strains in North America, whereas live attenuated vaccines may require less frequent strain updates based on demonstrated cross-protection. Enhancing vaccine efficacy to control influenza infections in swine will help reduce the impact it has on swine production and reduce the risk of swine-to-human transmission.

  • A proteomic atlas of the African swine fever virus particle [Structure and Assembly]

  • African swine fever virus (ASFV) is a large and complex DNA virus that causes a highly lethal swine disease for which there is no vaccine available. The ASFV particle, with an icosahedral multilayered structure, contains multiple polypeptides whose identity is largely unknown. Here, we analyzed by mass spectroscopy the protein composition of highly purified extracellular ASFV particles and performed immunoelectron microscopy to localize several of the detected proteins. The proteomic analysis identified 68 viral proteins, which account for 39% of the genome coding capacity. The ASFV proteome includes essentially all the previously described virion proteins and, interestingly, 44 newly identified virus-packaged polypeptides, half of them of unknown function. A great proportion of the virion proteins are committed to the virus architecture, including two new structural proteins, p5 and p8, which are derived from the core polyproteins pp220 and pp62. In addition, the virion contains a full complement of enzymes and factors involved in the viral transcription, various enzymes implicated in DNA repair and protein modification and some proteins concerned with virus entry and host defense evasion. Finally, 21 host proteins were reproducibly detected in the ASFV particle, many of them localized at the cell surface and related to the cortical actin cytoskeleton. Immunoelectron microscopy strongly supports that these host membrane-associated proteins are recruited during the virus budding at actin-dependent membrane protrusions. Altogether, this study provides a comprehensive model of the ASFV architecture that integrates both compositional and structural information.

    IMPORTANCE African swine fever virus causes a highly contagious and lethal disease of swine that currently affects many countries of Sub-Saharan Africa, the Caucasus, the Russian Federation and Eastern Europe and has very recently spread to China. Despite extensive research, effective vaccines or anti-viral strategies are still lacking and many basic questions on the molecular mechanisms underlying the infective cycle remain. One of such gaps regards the composition and structure of the infectious virus particle. In this report, we have identified the set of viral and host proteins that compose the virion and determined or inferred the localization of many of them. This information significantly increases our understanding of the biological and structural features of an infectious African swine fever virus particle and will help direct future research efforts.

  • Qualitative differences in capsidless L-particles released as a by-product of bovine herpesvirus 1 and herpes simplex virus 1 infections [Structure and Assembly]

  • Despite differences in the pathogenesis and host range of alphaherpesviruses, many stages of their morphogenesis are thought to be conserved. Here, an ultrastructural study of bovine herpesvirus 1 (BoHV-1) envelopment revealed similar profiles to those previously found for HSV-1, with BoHV-1 capsids associating with endocytic tubules. Consistent with the similarity of their genomes and envelopment strategies, the proteomic composition of BoHV-1 and HSV-1 virions was also comparable. However, BoHV-1 morphogenesis exhibited a diversity in envelopment events. First, heterogeneous primary envelopment profiles were readily detectable at the inner nuclear membrane of BoHV-1 infected cells. Second, the BoHV-1 progeny comprised not just full virions, but also an abundance of capsidless, non-infectious light (L)-particles that were released from the infected cell in similar numbers to virions, and in the absence of DNA replication. Proteomic analysis of BoHV-1 L-particles and the much less abundant HSV-1 L-particles revealed that they contained the same complement of envelope proteins as virions but showed variations in tegument content. In the case of HSV-1, the UL46 tegument protein was reproducibly found to be more than 6-fold enriched in HSV-1 L-particles. More strikingly, the tegument proteins UL36, UL37, UL21 and UL16 were depleted in BoHV-1 but not HSV-1 L-particles. We propose that these combined differences reflect the presence of a truly segregated "inner" and "outer" tegument in BoHV-1, making it a critical system for studying the structure and process of tegumentation and envelopment.

    IMPORTANCE The alphaherpesvirus family includes viruses that infect humans and animals. Hence, not only do they have a significant impact on human health but also a substantial economic impact on the farming industry. While the pathogenic manifestations of the individual viruses differ from host to host, their relative genetic composition suggests similarity at the molecular level. This study provides a side-by-side comparison of the particle output from the major human pathogen HSV-1 and the veterinary pathogen BoHV-1. Ultrastructural and proteomic analyses have revealed that both viruses have broadly similar morphogenesis profiles and infectious virus composition. However, the demonstration that BoHV-1 has the capacity to generate vast numbers of capsidless enveloped particles that differ to those produced by HSV-1 in composition, imply a divergence in the cell biology of these viruses that impacts on our general understanding of alphaherpesvirus morphogenesis.

  • Antibody-mediated CD4 depletion induces homeostatic CD4+ T cell proliferation without detectable virus reactivation in ART-treated SIV-infected macaques [Pathogenesis and Immunity]

  • A major barrier to HIV eradication is the long-term persistence of latently-infected CD4+ T-cells harboring integrated replication-competent virus. It has been proposed that the homeostatic proliferation of these cells drives long-term reservoir persistence in absence of virus reactivation, thus avoiding cell death due to either virus-mediated cytopathicity or immune effector mechanisms. Here, we conducted an experimental depletion of CD4+ T-cells in eight ART-treated, SIV-infected rhesus macaques (RMs) to determine whether the homeostatically-driven CD4+ T-cell proliferation that follows CD4+ T-cell depletion results in reactivation of latent virus and/or expansion of the virus reservoir. After administration of the CD4R1 antibody, we observed a CD4+ T-cell depletion of 65-89% in peripheral blood and 20-50% in lymph nodes, followed by a significant increase in CD4+ T-cell proliferation during CD4+ T-cell reconstitution. However, this CD4+ T-cell proliferation was not associated with detectable increases in viremia, indicating that the homeostatic activation of CD4+ T-cells is not sufficient to induce virus reactivation from latently infected cells. Interestingly, the homeostatic reconstitution of the CD4+ T-cell pool was not associated with significant changes in the number of circulating cells harboring SIV DNA as compared to the first post-depletion time point. This study indicates that, in ART-treated SIV-infected RMs, the homeostasis-driven CD4+ T-cell proliferation that follows experimental CD4+ T-cell depletion occurs in absence of detectable reactivation of latent virus and does not increase the size of the virus reservoir as measured in circulating cells.

    IMPORTANCE Despite successful suppression of HIV replication with antiretroviral therapy, current treatments are unable to eradicate the latent virus reservoir and treatment interruption almost invariably results in the reactivation of HIV even after decades of virus suppression. Homeostatic proliferation of latently infected cells is one mechanism that could maintain the latent reservoir. To understand the impact of homeostatic mechanisms on virus reactivation and reservoir size, we experimentally depleted CD4+ T cells in ART-treated SIV-infected rhesus macaques and followed their homeostatic rebound. We find that depletion-induced proliferation of CD4+ T cells is insufficient to reactivate the viral reservoir in vivo. Furthermore, the proportion of SIV-DNA+ CD4+ T cells remains unchanged during reconstitution, suggesting that the reservoir is resistant to this mechanism of expansion at least in this experimental system. Understanding how T cell homeostasis impacts latent reservoir longevity could lead to the development of new treatment paradigms aimed at curing HIV infection.

  • Exchange Proteins Directly Activated by cAMP and Their Roles in Respiratory Syncytial Virus Infection [Cellular Response to Infection]

  • Respiratory syncytial virus (RSV) is the leading cause of respiratory infection in young children and high-risk adults. However, the specific treatment for this viral infection is not currently available. In this study, we discovered that an exchange protein directly activated by cyclic AMP (EPAC) can serve as a potential therapeutic target for RSV. In both lower and upper epithelial cells, the treatment of EPAC inhibitor (ESI-09), but not PKA inhibitor (H89), significantly inhibits the RSV replication and pro-inflammatory cytokine/chemokine induction. In addition, RSV-activated transcriptional factors belonging to the NF-B and IRF families are also suppressed by ESI-09. Through isoform-specific gene knock down, we found that EPAC2, but not EPAC1, plays a dominant role in controlling RSV replication and viral-induced host responses. Experiments using both EPAC2 knock out and EPAC2-specific inhibitor support such roles of EPAC2. Therefore, EPAC2 could be a promising therapeutic target to regulate RSV replication and associated inflammation.

    IMPORTANCE Respiratory syncytial virus (RSV) is a serious public health problem as it is associated with bronchiolitis, pneumonia, and asthma exacerbations. Currently no effective treatment or vaccine is currently available, yet many molecular mechanisms regarding RSV-induced lung disease are still significantly unknown. This project aims to elucidate an important and novel function of a protein called EPAC2 in RSV replication innate inflammatory responses. Our results should provide an important insight to the development of new pharmacologic strategies against RSV infection, therefore reducing RSV-associated morbidity and mortality.

  • Large-scale synonymous substitutions in the cucumber mosaic virus RNA 3 facilitate amino acid mutations in the coat protein [Genetic Diversity and Evolution]

  • The effect of large-scale synonymous substitutions in a small icosahedral, single-stranded RNA viral genome on virulence, viral titer, and protein evolution were analyzed. The coat protein (CP) gene of the Fny stain of Cucumber mosaic virus (CMV) was modified. We created four CP mutants in which all the codons of nine amino acids in the 5'- or 3'-half of the CP were replaced by either the most frequently or the least frequently used synonymous codons in monocot plants. When the dicot host (Nicotiana benthamiana) was inoculated with these four CP mutants, viral RNA titers in uninoculated symptomatic leaves decreased, while all mutants eventually showed mosaic symptoms similar to wildtype. The codon adaptation index of these four CP mutants against dicots genes was similar to those of the wild type CP gene, indicating that the reduction of viral RNA titer was due to deleterious changes of the RNA secondary structure of RNA 3 and 4. When two 5'-mutants were serial-passaged in N. benthamiana, viral RNA titers were rapidly restored but competitive fitness remained decreased. Although no nucleic acid changes were observed in the passaged wildtype CMV, one to three amino acid changes were observed in the synonymously-mutated CP of each passaged virus, which were involved in recovery of viral RNA titer of 5'-mutants. Thus, we demonstrated that deleterious effects of the large-scale synonymous substitutions in the RNA viral genome facilitated the rapid amino acid mutation(s) in the CP to restore the viral RNA titer.

    IMPORTANCE Recently, it has been known that the synonymous substitutions in RNA virus genes affect viral pathogenicity and competitive fitness by alteration of global or local RNA secondary structure of the viral genome. We confirmed that the large-scale synonymous substitutions in the CP gene of CMV resulted in decreased viral RNA titer. Importantly, when viral evolution was stimulated by serial-passage inoculation, viral RNA titer was rapidly restored concurrent with a few amino acid changes in the CP. This novel finding indicates that the deleterious effects of large-scale nucleic acid mutations on viral RNA secondary structure is readily tolerated by structural changes in the CP, demonstrating a novel part of the adaptive evolution of an RNA viral genome. In addition, our serial inoculation experimental system of large-scale synonymous mutants could uncover the role of new amino acid residues in the viral protein that have not been observed in the wildtype virus strains.

  • PJA1 Coordinates with the SMC5/6 Complex to Restrict DNA Viruses and Episomal Genes through Interferon-independent Manner [Virus-Cell Interactions]

  • Viral and episomal DNAs, as signs of infections and dangers, induce series of immune responses in the host, and cells must sense foreign DNAs to eliminate the invaders. Cell nucleus is not "immune privileged" and exerts intrinsic mechanisms to control nuclear replicating DNA viruses. Thus, it is important to understand the action of viral DNA sensing in the cell nucleus. Here, we reveal a mechanism of restriction of DNA viruses and episomal plasmids mediated by PJA1, a RING-H2 E3 ubiquitin ligase. PJA1 restricts DNA viruses, hepatitis B virus (HBV) and herpes simplex virus-1 (HSV-1), but not RNA viruses, enterovirus 71 (EV71) and vesicular stomatitis virus (VSV). Similarly, PJA1 inhibits episomal plasmids, but not chromosome integrated reporters or endogenous genes. In addition, PJA1 has no effect on endogenous type I and II interferons (IFNs) and interferon stimulated genes (ISGs), suggesting that PJA1 silences DNA viruses independent of the IFN pathways. Interestingly, PJA1 interacts with the SMC5/6 complex (a complex essential for chromosome maintenance and HBV restriction) to facilitate the complex to bind viral and episomal DNAs in the cell nucleus. Moreover, treatment with inhibitors of DNA topoisomerases (Tops) and knock-down of Tops release PJA1-mediated silence of viral and extrachromosomal DNAs. Taken together, this work demonstrates that PJA1 interacts with SMC5/6 and facilitates the complex to bind and eliminate viral and episomal DNAs through DNA Tops, and thus reveals a distinct mechanism underlying restriction of DNA viruses and foreign genes in the cell nucleus.

    IMPORTANCE DNA viruses, including hepatitis B virus and herpes simplex virus, induce series of immune responses in the host and lead to human public health concerns worldwide. In addition to cytokines in the cytoplasm, restriction of viral DNA in the nucleus is an important approach of host immunity. However, the mechanism of foreign DNA recognition and restriction in the cell nucleus is largely unknown. This work demonstrates that an important cellular factor (PJA1) suppresses DNA viruses and transfected plasmids independent of type I and II interferon (IFN) pathways. Instead, PJA1 interacts with the chromosome maintenance complex (SMC5/6) and facilitates the complex to recognize and bind viral and episomal DNAs and recruits DNA topoisomerases to restrict the foreign molecules. These results reveal a distinct mechanism underlying the silence of viral and episomal invaders in the cell nuclei, and suggest that PJA1 acts as a potential agent to preventing infectious and inflammatory diseases.

  • Coronavirus TGEV Evades the Type I Interferon Response through IRE1{alpha}-Mediated Manipulation of the miR-30a-5p/SOCS1/3 Axis [Virus-Cell Interactions]

  • In host innate immunity, type I interferons (IFN-I) are major antiviral molecules, and coronaviruses have evolved diverse strategies to counter the IFN-I response during infection. Transmissible gastroenteritis virus (TGEV), a member of the alphacoronavirus family, induces endoplasmic reticulum (ER) stress and significant IFN-I production after infection. However, how TGEV evades the IFN-I antiviral response despite the marked induction of endogenous IFN-I has remained unclear. IRE1aalpha;, a highly conserved ER stress sensor with both kinase and RNase activities, is involved in the IFN response. In this study, IRE1aalpha; facilitated TGEV replication via downmodulating the host miR-30a-5p abundance. miR-30a-5p normally enhances IFN-I antiviral activity by directly targeting the negative regulators of JAK-STAT, SOCS1 and SOCS3. Furthermore, TGEV infection increased SOCS1 and SOCS3 expression, which dampened IFN-I antiviral response and facilitated TGEV replication. Importantly, compared with mock infection, TGEV infection in vivo resulted in decreased miR-30a-5p levels and significantly elevated SOCS1 and SOCS3 expression in piglet ileum. Taken together, our data reveal a new strategy used by TGEV to escape the IFN-I response by engaging the IRE1aalpha;-miR-30a-5p-SOCS1/3 axis, thus improving our understanding of how TGEV escapes host innate immune defenses.

    Importance: Type I interferons (IFN-I) play essential roles in restricting viral infections. Coronavirus infection induces ER stress and the interferon response, which reflects different adaptive cellular processes. An understanding of how coronavirus-elicited ER stress is actively involved in viral replication and manipulates the host IFN-I response has remained elusive. Here, TGEV inhibited host miR-30a-5p via the ER stress sensor IRE1aalpha;, which led to the increased expression of negative regulators of JAK-STAT signaling cascades, namely, SOCS1 and SOCS3. Increased SOCS1 or SOCS3 expression impaired the IFN-I antiviral response, promoting TGEV replication. These findings enhance our understanding of the strategies used by coronaviruses to antagonize IFN-I innate immunity via IRE1aalpha;-mediated manipulation of the miR-30a-5p-SOCS axis, highlighting the crucial role of IRE1aalpha; in innate antiviral resistance and the potential of IRE1aalpha; as a novel target against coronavirus infection.

  • The RNA epitranscriptome of DNA viruses [Gem]

  • RNA modifications have generated much interest in the virology field as recent works have shown that many viruses harbor these marks and modify cellular marks. The most abundant mRNA modification in eukaryotic cells, N6-methyladenosine (m6A), has been extensively examined in genome-wide scale in both cellular and viral contexts. This review discusses the role of m6A in gene regulation, followed by recent advancements in Kaposi's sarcoma-associated herpesvirus (KSHV) and simian virus 40 (SV40). We provide insights on future research related to m6A in DNA viruses.

  • Persistent replication of HIV, HCV and HBV results in distinct gene expression profiles by human NK cells [Cellular Response to Infection]

  • NK cells during chronic viral infection have been well studied in the past. We performed an unbiased next-generation RNA-sequencing approach to identify commonalities or differences of the effect of HIV, HCV and HBV viremia on NK cell transcriptomes. Using cell sorting, we obtained CD3-CD56+ NK cells from blood of 6 HIV, 8 HCV, and 32 HBV infected patients without treatment. After library preparation and sequencing, we used an in-house analytic pipeline to compare expression levels with matched healthy controls. In NK cells from HIV, HCV and HBV patients, transcriptome analysis identified 272, 53, and 56 differentially expressed genes, respectively (fold change ggt;1.5, q-value 0.2). Interferon stimulated genes were induced in NK cells from HIV/HCV patients, but not during HBV infection. HIV viremia downregulated ribosome assembly genes in NK cells. In HBV, viral load and ALT variation had little effect on genes related to NK effector function. In conclusion, we compare, for the first time, NK cell transcripts of viremic HIV, HCV and HBV patients. We clearly demonstrate distinctive NK cell gene signatures in 3 different populations, suggestive for a different degree of functional alterations of the NK cell compartment as compared to healthy individuals.

    Importance Three viruses exist that can result in persistently high viral loads in immune competent humans: HIV, hepatitis C and hepatitis B. In the last decades, using flow cytometry and in vitro assays on NK cells from patients with these diseases, several impairments have been established, particularly during and possibly contributing to HIV viremia. However, the background of NK cell impairments in viremic patients is not well understood. In this study we describe the NK cell transcriptome of patients with high viral loads of different etiologies. We clearly demonstrate distinctive NK cell gene signatures, with regard to ISG induction, expression of genes coding for activation markers or proteins involved in cytotoxic action, as well immunological genes. This study provides important details necessary to uncover the origin of functional and phenotypical differences between viremic patients and healthy subjects, and provides many leads that can be confirmed using future in vitro manipulation experiments.

  • Potent neutralizing human monoclonal antibodies preferentially target mature dengue virus particles: implication for novel strategy of dengue vaccine [Pathogenesis and Immunity]

  • The four serotypes of dengue virus (DENV) cause the most important mosquito-borne viral disease in humans. The envelope (E) protein is the major target of neutralizing antibodies and contains 3 domains (DI, DII and DIII). Recent studies reported that human monoclonal antibodies (mAbs) recognizing the DIII, D1/DII hinge, the E-dimer epitope or a quaternary epitope involving DI/DII/DIII are more potently neutralizing compared with those recognizing the fusion-loop (FL) of DII. Due to inefficient cleavage of premembrane protein, DENV suspensions consist of a mixture of mature, immature and partially immature particles. We investigated the neutralization and binding of 22 human mAbs to DENV1 virions with differential maturation status. Compared with FL mAbs, DIII, DI/DII hinge and E-dimer epitope mAbs showed higher maximum binding and avidity to mature particles relative to immature particles; this feature may contribute to the strong neutralizing potency of such mAbs. FL-specific mAbs required 57 to 87% occupancy on mature particles to achieve half maximal neutralization (NT50), whereas the potently neutralizing mAbs achieved NT50 states at 20 to 38% occupancy. Analysis of the mAbs repertoire and polyclonal sera from patients with primary DENV1 infection supports the immunodominance of cross-reactive anti-E antibodies over type-specific antibodies. After depletion with viral particles from a heterologous DENV serotype, the type-specific neutralizing antibodies remained and showed binding features shared by potent neutralizing mAbs. Taken together, these findings suggest that the use of homogenous mature DENV particles as an immunogen may induce more potent neutralizing antibodies against DENV than immature or mixed particles.

    IMPORTANCE With an estimated 390 million infections per year, the four serotypes of dengue virus (DENV) cause the most important mosquito-borne viral disease in humans. The dengue vaccine, Dengvaxia, was licensed, however its low efficacy among dengue-naïve individuals and increased risk of severe dengue in children highlight the need for a better understanding of the role of human antibodies in immunity against DENV. DENV suspensions contain mature, immature and partially immature particles. We investigated the binding of 22 human monoclonal antibodies (mAbs) to DENV envelope protein on particles with differing maturation status. Compared with weakly neutralizing mAbs, potently neutralizing mAbs had higher relative maximum binding and avidity to mature particles. This was supported by analysis of mAbs repertoires and polyclonal sera from patients with primary DENV infection. Together, these findings suggest that mature particles may be the optimal form of presenting envelope protein to induce more potent neutralizing antibodies against DENV.

  • Comparative in vitro and in vivo analysis of H1N1 and H1N2 variant influenza viruses isolated from humans between 2011 and 2016. [Pathogenesis and Immunity]

  • Influenza A virus pandemics are rare events caused by novel viruses which have the ability to spread in susceptible human populations. With respect to H1 subtype viruses, swine H1N1 and H1N2 viruses occasionally cross the species barrier to cause human infection. Recently isolated from humans (termed variants), swine viruses were shown to display great genetic and antigenic diversity, hence posing considerable public health risk. Here, we utilized in vitro and in vivo approaches to provide characterization of H1 subtype variant viruses isolated since the 2009 pandemic and discuss the findings in context with previously studied H1 subtype human isolates. The variant viruses were well adapted to replicate in human respiratory cell line, Calu-3, and the respiratory tracts of mice and ferrets. However, with respect to HA activation pH, the variant viruses had fusion pH thresholds closer to that of most classical swine and triple reassortant H1 isolates rather than viruses that had adapted to humans. Consistent with previous observations for swine isolates, the tested variant viruses were capable of efficient transmission between co-housed ferrets but could transmit via respiratory droplets to differing degrees. Overall, this investigation demonstrates that swine H1 viruses that infected humans possess adaptations required for robust replication and, in some cases, efficient respiratory droplet transmission in a mammalian model, and therefore, need to be closely monitored for additional molecular changes that could facilitate transmission among humans. This work highlights the need for risk assessments of emerging H1 viruses as they continue to evolve and cause human infections.

    IMPORTANCE Influenza A virus is a continuously evolving respiratory pathogen. Endemic in swine, H1 and H3 subtype viruses sporadically cause human infections. As each zoonotic infection represents an opportunity for human adaptation, the emergence of a transmissible influenza virus to which there is little or no pre-existing immunity is an ongoing threat to public health. Recently isolated variant H1 subtype viruses were shown to display extensive genetic diversity and in many instances were antigenically distinct from seasonal vaccine strains. In this study, we provide characterization of representative H1N1v and H1N2v viruses isolated since the 2009 pandemic. Our results show that, although recent variant H1 viruses possess some adaptation markers of concern, these viruses have not fully adapted to humans and require further adaptation to present a pandemic threat. This investigation highlights the need for close monitoring of emerging variant influenza viruses for molecular changes that could facilitate efficient transmission among humans.

  • Influenza A virus M2 protein apical targeting is required for efficient virus replication [Structure and Assembly]

  • The influenza A virus (IAV) M2 protein is a multifunction protein with critical roles in virion entry, assembly and budding. M2 is targeted to the apical plasma membrane of polarized epithelial cells, and the interaction of the viral proteins M2, M1, HA, and NA near glycolipid rafts in the apical plasma membrane is hypothesized to coordinate the assembly of infectious virus particles. To determine the role of M2 protein apical targeting in IAV replication, a panel of M2 proteins with basolateral plasma membrane (M2-Baso) or endoplasmic reticulum (M2-ER) targeting sequences was generated. MDCK II cells stably expressing M2-Baso, but not M2-ER, complemented the replication of M2-stop viruses. However, in primary human nasal epithelial cell (hNEC) cultures, viruses encoding M2-Baso and M2-ER replicated to negligible titers compared to WT virus. M2-Baso replication was negatively correlated with cell polarization. These results demonstrate that M2 apical targeting is essential for IAV replication: targeting M2 to the ER results in a strong, cell type independent inhibition of virus replication, and targeting M2 to the basolateral membrane has greater effects in hNECs than in MDCK cells.

    IMPORTANCE Influenza A virus assembly and particle release occurs at the apical membrane of polarized epithelial cells. The integral membrane proteins encoded by the virus nndash; HA, NA and M2 nndash; are all targeted to the apical membrane and believed to recruit the other structural proteins to sites of virus assembly. By targeting M2 to the basolateral or endoplasmic reticulum membranes, influenza A virus replication was significantly reduced. Basolateral targeting of M2 reduced the infectious virus titers with minimal effects on virus particle release while targeting to the endoplasmic reticulum resulted in reduced infectious and total virus particle release. Therefore, altering the expression and the intracellular targeting of M2 has major effects on virus replication.

  • Role of Capsid anchor in the morphogenesis of Zika virus [Structure and Assembly]

  • The flavivirus capsid protein (C) is separated from the downstream pre-membrane (PrM) protein by a hydrophobic sequence named capsid anchor (Ca). During polyprotein processing, Ca is sequentially cleaved by the viral NS2B/NS3 protease on the cytosolic side and by signal peptidase on the luminal side of the ER. To date, Ca is considered important mostly for directing translocation of PrM into the ER lumen. In this study, the role of Ca in the assembly and secretion of ZIKV was investigated using a pseudovirus-based approach. Our results show that, while Ca-mediated anchoring of C to the ER membrane is not needed for the production of infective particles, Ca expression in cis with respect to PrM is strictly required to allow proper assembly of infectious particles. Finally, we show that the presence of a heterologous, but not the homologous, Ca induces degradation of E through the autophagy/lysosomal pathway.

    IMPORTANCE The capsid anchor (Ca) is a single pass transmembrane domain at the C-terminus of the capsid protein (C) known to function as a signal for the translocation of PrM into the ER lumen. The objective of this study was to further understand the role of Ca in ZIKV life cycle, whether involved in the formation of nucleocapsid through association with C or in the formation of viral envelope. In this study, we show that Ca has a function beyond the one of translocation signal, controlling protein E stability and therefore its availability for assembly of infectious particles.

  • Human Immunodeficiency Virus Type-1 gp120 and Tat Induce Mitochondrial Fragmentation and Incomplete Mitophagy in Human Neurons [Virus-Cell Interactions]

  • HIV enters the central nervous system (CNS) during the early stages of infection and can cause neurological dysfunction including neurodegeneration and neurocognitive impairment. Specific autophagy responsible for removal of damaged mitochondria (mitophagy) and mitochondrial dynamics constitute neuronal mitochondrial quality control mechanisms, and are impaired in neurodegenerative disorders and numerous other diseases. Release of HIV proteins, gp120 and Tat, from infected cells is thought to play an important role in HIV-associated neurocognitive disorders (HAND), but the mechanism(s) leading to impairment are poorly understood. Here, we report that exposure of human primary neurons (HPNs) to HIV gp120 and Tat accelerate the balance of mitochondrial dynamics toward fission (fragmented mitochondria) and induce perinuclear aggregation of mitochondria and dynamin related protein (DRP1) mitochondrial translocation, leading to neuronal mitochondrial fragmentation. HIV gp120 and Tat increased LC3 protein expression, and induced Parkin/SQSTM1 selective recruitment to the damaged mitochondria. Using a dual fluorescence reporter system expressing mito-mRFP-EGFP or a tandem LC3 vector (mRFP-EGFP-LC3), both HIV proteins were found to inhibit mitophagic flux in human primary neurons. HIV gp120 and Tat induced mitochondrial damage and altered mitochondrial dynamics by decreasing mitochondrial membrane potential (m). These findings indicate that HIV gp120 and Tat initiate activation and recruitment of mitophagy markers to damaged mitochondria in neurons, but impair the delivery of mitochondria to the lysosomal compartment. Altered mitochondrial dynamics associated with HIV infection and incomplete neuronal mitophagy may play a significant role in the development of HAND and accelerated aging associated with HIV infection.

    IMPORTANCE Despite viral suppression by antiretrovirals, HIV proteins continue to be detected in infected cells and neurologic complications remain common in infected people. Although HIV is unable to infect neurons, viral proteins including gp120 and Tat, can enter neurons and can cause neuronal degeneration and neurocognitive impairment. Neuronal health is dependent on the functional integrity of mitochondria and damaged mitochondria are subjected to mitochondrial control mechanisms. Multiple lines of evidence suggest that specific elimination of damaged mitochondria through mitophagy and mitochondrial dynamics play an important role in CNS diseases. Here, we show that in human primary neurons, gp120 and Tat favor the balance of mitochondrial dynamics toward enhanced fragmentation through the activation of mitochondrial translocation of DRP1 to the damaged mitochondria. However, mitophagy fails to go to completion leading to neuronal damage. These findings support a role for altered mitophagy in HIV-associated neurological disorders and provide novel targets for potential intervention.

  • An N-glycosylated form of SERINC5 is specifically incorporated into HIV-1 virions [Virus-Cell Interactions]

  • SERINC5 is an inhibitor of retroviral infectivity that is counteracted by viral proteins, including HIV-1 Nef. Inhibition of infectivity by SERINC5 is associated with its incorporation into virions. Nef counteracts this inhibition, presumably by removing SERINC5 from sites of virion assembly at the plasma membrane. While evaluating the virion-incorporation of SERINC5, we observed that a relatively high molecular weight form was preferentially present in virions. We used various glycosidases to establish that virion-associated SERINC5 is modified by N-linked, complex glycans, whereas the majority of SERINC5 in cells is of relatively low molecular weight and is modified by high-mannose glycans. Sequence alignment of SERINC family proteins led us to identify a conserved N-glycosylation site, N294, in SERINC5. We mutated this site to evaluate its effect on glycosylation, the restrictive activity of SERINC5, and the sensitivity of SERINC5 to antagonism by Nef. Our results demonstrate that N294 is the major site of N-glycosylation in SERINC5. Although N-glycosylation was neither required for restrictive activity nor for sensitivity to Nef per se, we observed a decrease in the steady state expression of glycosylation-deficient SERINC5 (the N294A mutant) compared to the wild type protein. Expression of this mutant was partly restored by treatment of cells with MG132 (a proteasome-inhibitor) but not with bafilomycin A1 (a lysosomal inhibitor). We conclude that although not required for restrictive-activity or Nef-sensitivity, N-linked glycosylation is important for maintaining the steady-state expression of SERINC5 and that non-glycosylated SERINC5 is likely subjected to a quality-control mechanism that induces its proteasomal degradation.

    IMPORTANCE SERINC5 is a member of a family of multi-pass transmembrane proteins that inhibit the infectivity of retroviruses including HIV-1. These proteins are incorporated into virions and inhibit infection of target cells unless counteracted by viral antagonists such as HIV-1 Nef. The only other biological function with which these proteins have been associated is the formation of serine-containing membrane lipids. Here we show that SERINC5 is a glycosylated protein and that N-glycosylation is important for its steady state expression. In the absence of N-glycosylation, SERINC5 is prone to proteasomal degradation. Nonetheless, N-glycosylation per se is neither required for the ability of SERINC5 to inhibit HIV-1 infectivity nor for its sensitivity to antagonism by Nef.

  • KSHV de-regulates host cellular replication during lytic reactivation by disrupting the MCM complex through ORF59 [Genome Replication and Regulation of Viral Gene Expression]

  • Mini Chromosome Maintenance proteins (MCMs) play an important role in DNA replication by binding to the origins as helicase and recruiting polymerases for DNA synthesis. During the S phase, MCM complex is loaded once to limit DNA replication once per cell-cycle. We identified MCMs as ORF59 binding partners in our protein pull down assays, which led us to hypothesize that this interaction may influence DNA replication. ORF59's interactions with MCMs were confirmed in both endogenous and overexpression systems, which showed its association with MCM 3, 4, 5 and 6. Interestingly, MCM6 interacted with both the N and C terminal domains of ORF59 and its depletion in BCBL-1 and BC3 cells led to an increase in viral genome copies, viral late gene transcripts and virion production compared to the control cells following reactivation. MCMs perform its function by loading onto the replication competent DNA and one means of regulating chromatin loading/unloading, in addition to enzymatic activity of the MCM complex, is by post-translational modifications including phosphorylation of these factors. Interestingly, a hypo-phosphorylated form of MCM3, which is associated with reduced loading onto the chromatin, was detected during lytic reactivation and correlated with its inability to associate with histones in reactivated cells. Additionally, chromatin immunoprecipitation showed lower levels of MCM3 and MCM4 association at cellular origins of replication and decreased levels of cellular DNA synthesis in cells undergoing reactivation. Taken together, this suggests a mechanism in which KSHV ORF59 disrupts the assembly and functions of MCM complex to stall cellular DNA replication and promote viral replication.

    Importance

    KSHV is the causative agent of various lethal malignancies affecting immunocompromised individuals. Both lytic and latent phases of the viral lifecycle contribute to the progression of these cancers. A better understanding of how viral proteins disrupt functions of a normal healthy cell to cause oncogenesis is warranted. One crucial lytic protein produced early during lytic reactivation is the multi-functional ORF59. In this report, we elucidated an important role of ORF59 in manipulating the cellular environment conducive for viral DNA replication by deregulating the normal functions of the host MCM proteins. ORF59 binds to specific MCMs and sequesters them away from replication origins in order to sabotage cellular DNA replication. Blocking cellular DNA replication ensures that cellular resources are utilized for transcription and replication of viral DNA.

  • Inhibition of MALT1 decreases neuroinflammation and pathogenicity of virulent rabies virus in mice [Pathogenesis and Immunity]

  • Rabies virus is a neurovirulent RNA virus, which causes about 59000 human deaths each year. Treatment for rabies does not exist due to incomplete understanding of the pathogenesis. MALT1 mediates activation of several immune cell types and is involved in the proliferation and survival of cancer cells. MALT1 acts as a scaffold protein for NF-B signaling and a cysteine protease that cleaves substrates, leading to the expression of immunoregulatory genes. Here, we examined the impact of genetic or pharmacological MALT1 inhibition in mice on disease development after infection with the virulent rabies virus strain CVS-11. Morbidity and mortality were significantly delayed in Malt1-/- compared to Malt1+/+ mice, which was associated with a lower viral load, proinflammatory gene expression and infiltration and activation of immune cells in brain. Specific deletion of Malt1 in T cells also delayed disease development while deletion in myeloid cells, neuronal cells or NK cells had no effect. Disease development was also delayed in mice treated with the MALT1 protease inhibitor mepazine, and in knock-in mice expressing a catalytically inactive MALT1 mutant protein, showing an important role of MALT1 proteolytic activity. The described protective effect of MALT1 inhibition against infection with a virulent rabies virus is the precise opposite of the sensitizing effect of MALT1 inhibition that we previously observed in case of infection with an attenuated rabies virus strain. Together, these data demonstrate that the role of immunoregulatory responses in rabies pathogenicity is dependent on virus virulence, and reveal the potential of MALT1 inhibition for therapeutic intervention.

    IMPORTANCE Rabies virus is a neurotropic RNA virus that causes encephalitis and still poses an enormous challenge to animal and public health. Efforts to establish reliable therapeutic strategies have been unsuccessful and are hampered by gaps in the understanding of virus pathogenicity. MALT1 is an intracellular protease that mediates the activation of several innate and adaptive immune cells in response to multiple receptors, and therapeutic MALT1 targeting is believed to be a valid approach for autoimmunity and MALT1-addicted cancers. Here, we study the impact of MALT1 deficiency on brain inflammation and disease development in response to infection of mice with the highly virulent CVS-11 rabies virus. We demonstrate that pharmacological or genetic MALT1 inhibition decreases neuroinflammation and extends the survival of CVS-11 infected mice, providing new insights in the biology of MALT1 and rabies virus infection.

  • Acid/base character of the Japanese Encephalitis Virus E protein residue 138 alters neurovirulence in mice [Pathogenesis and Immunity]

  • The Japanese Encephalitis virus (JEV) envelope (E) protein, as one of mediators for virus entry into host cells plays critical role in determinants of virulence. The Glu-to-Lys mutation of residue 138 in E protein (E138) played important role in attenuating JEV vaccine strain SA14-14-2. However, it is not clear how E138 attenuate JEV. Here, we demonstrate that the Glu-to-Arg mutation of E138 also determines attenuation of JEV strain 10S3. Likewise, for its parent strain (HEN0701), a virulence strain, the mutations of E138 are responsible for virulence alteration. Furthermore, we demonstrated that mutation with alkaline residues in E138 contributed to attenuation of neurovirulence; in contrast, mutation with acidic residues enhanced the neurovirulence of those strains. Moreover, acidity in residue E47 had similar effect on neurovirulence. Furthermore, the alkaline E138 residue enhanced the suscepbility of heparin inhibition in vitro and limited JEV diffuse in mouse brain. These results suggest that the acidity/alkalinity of E138 residue plays important role in neurovirulence determinants.

    IMPORTANCE The E protein is the only glycoprotein in mature JEV and plays an important role in viral neurovirulence. E protein mutations attenuate JEV neurovirulence through unclear mechanisms. Here, we discovered that E138 is a predominant determinant for JEV neurovirulence. We demonstrated that the alkalinity/acidity of E138 and its interaction amino acid E47 determines JEV neurovirulence. These data contribute to the characterization of the E protein and the rational development of novel JEV vaccines.

  • Mutations in the nucleocapsid protein were complemented by mutations in the L protein to restore viral RNA synthesis [Genome Replication and Regulation of Viral Gene Expression]

  • During viral RNA synthesis by the viral RNA-dependent RNA polymerase (vRdRp) of vesicular stomatitis virus, the sequestered RNA genome must be released from the nucleocapsid in order to serve as the template. Unveiling the sequestered RNA by interactions of vRdRp proteins, the large subunit (L) and the phosphoprotein (P), with the nucleocapsid protein (N) must not disrupt the nucleocapsid assembly. We noticed that a flexible structural motif composed of an aalpha;-helix and a loop in the N protein may act as the access gate to the sequestered RNA. This suggests that local conformational changes in this structural motif may be induced by interactions with the polymerase to unveil the sequestered RNA, without disrupting the nucleocapsid assembly. Mutations of several residues in this structural motif, Glu169, Phe171 and Leu174, to Ala resulted in loss of viral RNA synthesis in a minigenome assay. After implementing these mutations in the viral genome, mutant viruses were recovered by reverse genetics and serial passages. Sequencing the genomes of the mutant viruses revealed that compensatory mutations in L, P and N were required to restore the viral viability. Corresponding mutations were introduced in L, P and N, and their complementarity to the N mutations was confirmed by the minigenome assay. Introduction of the corresponding mutations is also sufficient to rescue the mutant viruses. These results suggested that the interplay of the N structural motif with the L protein may play a role in accessing the nucleotide template without disrupting the overall structure of the nucleocapsid.

    IMPORTANCE

    During viral RNA synthesis of a negative strand RNA virus, the viral RNA-dependent RNA polymerase (vRdRp) must gain access to the sequestered RNA in the nucleocapsid to use it as the template, but at the same time may not disrupt the nucleocapsid assembly. Our structural and mutagenesis studies showed that a flexible structural motif acts as a potential access gate to the sequestered RNA and plays an essential role in viral RNA synthesis. Interactions of this structural motif within the vRdRp may be required for unveiling the sequestered RNA. This mechanism of action allows the sequestered RNA to be released locally without disrupting the overall structure of the nucleocapsid. Since this flexible structural motif is present in the N proteins of many NSVs, release of the sequestered RNA genome by local conformational changes in the N protein may be a general mechanism in NSV viral RNA synthesis.

  • The Human Bocavirus 1 (HBoV1) NP1 protein is a multifunctional regulator of viral RNA processing [Genome Replication and Regulation of Viral Gene Expression]

  • Human bocavirus 1 (HBoV1) encodes a genus-specific protein, NP1, which regulates viral alternative pre-mRNA processing. Similar to NP1 of the related bocavirus minute virus of canine (MVC), HBoV1 NP1 suppressed cleavage and polyadenylation of RNAs at the viral internal polyadenylation site (pA)p. HBoV1 (pA)p is a complex region. It contains 5 significant cleavage and polyadenylation sites, and NP1 was found to only regulate the three of these sites which are governed by canonical AAUAAA hexamer signals. HBoV1 NP1 also facilitated splicing of the upstream intron adjacent to (pA)p. Alternative polyadenylation and splicing of the upstream intron were independent of each other, functioned efficiently within an isolated transcription unit, and were responsive independently to NP1. Characterization of HBoV1 NP1 generalizes its function within the genus Bocaparvovirus, and uncovers important differences and provides important comparisons with MVC NP1 for mechanistic and evolutionary considerations.

    IMPORTANCE

    The Parvovirinae are small non-enveloped icosahedral viruses that are important pathogens in many animal species including humans. The NP1 protein of human bocavirus 1 (HBoV1), similar to NP1 of the bocavirus minute virus of canine (MVC), regulates viral alternative RNA processing by both suppressing polyadenylation at an internal site, (pA)p, and facilitating splicing of an upstream adjacent intron. These effects allow both extension into the capsid gene and splicing of the viral pre-mRNA that correctly registers the capsid gene open reading frame. Characterization of HBoV1 NP1 generalizes this central mode of parvovirus gene regulation to another member of the bocavirus genus, and uncovers both important similarities and differences in function compared to MVC NP1 that will be important for future comparative studies.

  • Structural and biochemical characterization of endoribonuclease Nsp15 encoded by Middle East respiratory syndrome coronavirus [Structure and Assembly]

  • Non-structural protein 15 (Nsp15) encoded by coronavirus (CoV) is a uridylate specific endoribonuclease (NendoU) that plays an essential role in the life cycle of the virus. Structural information of this crucial protein from the Middle East respiratory syndrome (MERS) CoV, which is lethally pathogenic and has caused severe respiratory diseases worldwide, is lacking. Here, we report the crystal structure of MERS-CoV Nsp15 at a 2.7 AAring; resolution and perform the relevant biochemical assays to study how NendoU activity is regulated. Although the overall structure is conserved, MERS-CoV Nsp15 shows unique and novel features compared to its homologs. Serine substitution of residue F285, which harbors an aromatic side chain that disturbs RNA binding compared with other homologs, increases catalytic activity. Mutations of residues residing on the oligomerization interfaces that distort hexamerization, namely, N38A, Y58A and N157A, decrease thermostability, decrease binding affinity with RNA and reduce the NendoU activity of Nsp15. In contrast, mutant D39A exhibits increased activity and a higher substrate binding capacity. Importantly, Nsp8 is found to interact with both monomeric and hexameric Nsp15. The Nsp7/Nsp8 complex displays a higher binding affinity for Nsp15. Furthermore, Nsp8 and the Nsp7/Nsp8 complex also enhance the NendoU activity of hexameric Nsp15 in vitro. Taken together, this work first provides evidence on how the activity of Nsp15 may be functionally mediated by catalytic residues, oligomeric assembly, RNA binding efficiency or the possible association with other non-structural proteins.

    IMPORTANCE

    The lethally pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV) and the severe acute respiratory syndrome coronavirus (SARS-CoV) pose serious threats to humans. Endoribonuclease Nsp15 encoded by coronavirus plays an important role in viral infection and pathogenesis. This study determines the structure of MERS-CoV Nsp15 and demonstrates how the catalytic activity of this protein is potentially mediated, thereby providing structural and functional evidence for developing antiviral drugs. We also hypothesize that the primase-like protein Nsp8 and Nsp7/Nsp8 complex may interact with Nsp15 and affect enzymatic activity. This contributes to the understanding of the association of Nsp15 with the viral replication and transcription machinery.

  • Polymorphisms in Rhesus Macaque Tetherin are Associated with Differences in Acute Viremia in SIV {Delta}Nef-Infected Animals [Genetic Diversity and Evolution]

  • Tetherin (BST-2 or CD317) is an interferon-inducible transmembrane protein that inhibits virus release from infected cells. To determine the extent of sequence variation and the impact of polymorphisms in rhesus macaque tetherin on SIV infection, tetherin alleles were sequenced from 146 rhesus macaques, including 68 animals infected with wild-type SIVmac239 and 47 animals infected with SIVmac239nef. Since Nef is the viral gene product of SIV that counteracts restriction by tetherin, these groups afford a comparison of the effects of tetherin polymorphisms on SIV strains that are, and are not, resistant to tetherin. We identified 15 alleles of rhesus macaque tetherin with dimorphic residues at 9 positions. The relationship between these alleles and plasma viral loads was compared during acute infection, prior to the onset of adaptive immunity. Acute viremia did not differ significantly among the wild-type SIV-infected animals; however, differences in acute viral loads were associated with polymorphisms in tetherin among the animals infected with SIVnef. In particular, polymorphisms at positions 43 and 111 (P43 and H111) were associated with lower acute phase viral loads for SIVnef infection. These observations reveal extensive polymorphism in rhesus macaque tetherin, maintained perhaps as a consequence of variability in the selective pressure of diverse viral pathogens, and identify tetherin alleles that may have an inherently greater capacity to restrict SIV replication in the absence of Nef.

    IMPORTANCE

    As a consequence of ongoing evolutionary conflict with viral pathogens, tetherin has accumulated numerous species-specific differences that represent important barriers to the transmission of viruses between species. This study reveals extensive polymorphism in rhesus macaque tetherin and identifies specific alleles that are associated with lower viral loads during the first few weeks after the infection with nef-deleted SIV. These observations suggest that the variable selective pressure of viral pathogens, in addition to driving the diversification of tetherin among species, may also operate within certain species to maintain sequence variation in tetherin.

  • Localization and regulation of the T1 unimolecular spanin [Virus-Cell Interactions]

  • Spanins are bacteriophage lysis proteins responsible for disruption of the outer membrane, the final step of Gram-negative host lysis. The absence of spanins results in a terminal phenotype of fragile spherical cells. The phage T1 employs a unimolecular spanin gp11 that has an N-terminal lipoylation signal and a C-terminal transmembrane domain. Upon maturation and localization, gp11 ends up as an outer membrane lipoprotein with a C-terminal transmembrane domain embedded in the inner membrane, thus connecting both the membranes as a covalent polypeptide chain. Unlike the two-component spanins encoded by most of the other phages, including lambda, the unimolecular spanins have not been studied extensively. In this work, we show that the gp11 mutants lacking either membrane localization signal were non-functional and conferred a partially dominant phenotype. Translation from internal start sites within the gp11 coding sequence generated a shorter product which exhibited a negative regulatory effect on gp11 function. Fluorescence spectroscopy time-lapse videos of gp11-GFP expression showed gp11 accumulated in distinct punctate foci, suggesting localized clusters assembled within the peptidoglycan meshwork. In addition, gp11 was shown to mediate lysis in the absence of holin and endolysin function when peptidoglycan density was depleted by starvation for murein precursors. This result indicates the peptidoglycan is a negative regulator of gp11 function. This supports a model in which gp11 acts by fusing the inner and outer membranes, a mode of action analogous to, but mechanistically distinct from that proposed for the two-component spanin systems.

    Importance Spanins have been proposed to fuse the cytoplasmic and outer membranes during phage lysis. Recent work with the lambda spanins Rz-Rz1, which are similar to class I viral fusion proteins, has thrown light on the functional domains and requirements for two-component spanin function. Here we report, for the first time, a genetic and biochemical approach to characterize unimolecular spanins which are structurally and mechanistically different from two-component spanins. Considering similar predicted secondary structures within the ectodomains, unimolecular spanins can be regarded as a prokaryotic version of type II viral membrane fusion proteins. This work not only adds to our understanding of regulation of phage lysis at various levels but also provides a prokaryotic genetically tractable platform for interrogating class II-like membrane fusion proteins.

  • Sequence variation of Epstein-Barr virus: viral types, geography, codon usage and diseases [PublishAheadOfPrint]

  • 138 new Epstein-Barr virus (EBV) genome sequences have been determined. 125 of these and 116 from previous reports were combined to produce a multiple sequence alignment of 241 EBV genomes, which we have used to analyze variation within the viral genome. The type 1/type2 classification of EBV remains the major form of variation and is defined mostly by EBNA2 and EBNA3, but the type 2 SNPs at the EBNA3 locus extend into the adjacent gp350 and gp42 genes, whose products mediate infection of B cells by EBV. A small insertion within the BART miRNA region of the genome was present in 21 EBV strains. EBV from saliva of USA patients with chronic active EBV infection aligned with the wild type EBV genome, with no evidence of WZhet rearrangements. The V3 polymorphism in the Zp promoter for BZLF1 was found to be frequent in nasopharyngeal carcinoma cases both from Hong Kong and Indonesia. Codon usage was found to differ between latent and lytic cycle EBV genes and the main forms of variation of the EBNA1 protein have been identified.

    IMPORTANCE Epstein-Barr virus causes most cases of infectious mononucleosis and post-transplant lymphoproliferative disease. It contributes to several types of cancer including Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large B cell lymphoma, nasopharyngeal carcinoma and gastric carcinoma. EBV genome variation is important because some of the diseases associated with EBV have very different incidences in different populations and geographic regions nndash; differences in the EBV genome might contribute to these diseases. Some specific EBV genome alterations that appear to be significant in EBV associated cancers are already known and current efforts to make an EBV vaccine and antiviral drugs should also take account of sequence differences in the proteins used as targets.

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