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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

  • Recombinant Porcine Reproductive and Respiratory Syndrome Virus Expressing Membrane-Bound Interleukin-15 as an Immunomodulatory Adjuvant Enhances NK and {gamma}{delta} T Cell Responses and Confers Heterologous Protection [Vaccines and Antiviral Agents]

  • Cytokines are often used as adjuvants to improve vaccine immunogenicity, since they are important in initiating and shaping the immune response. The available commercial modified live-attenuated vaccines (MLVs) against porcine reproductive and respiratory syndrome virus (PRRSV) are unable to mount sufficient heterologous protection, as they typically induce weak innate and inadequate T cell responses. In this study, we investigated the immunogenicity and vaccine efficacy of recombinant PRRSV MLVs incorporated with the porcine cytokine interleukin-15 (IL-15) or IL-18 gene fused to a glycosylphosphatidylinositol (GPI) modification signal that can anchor the cytokines to the cell membrane. We demonstrated that both cytokines were successfully expressed on the cell membrane of porcine alveolar macrophages after infection with recombinant MLVs. Pigs vaccinated with recombinant MLVs or the parental Suvaxyn MLV had significantly reduced lung lesions and viral RNA loads in the lungs after heterologous challenge with the PRRSV NADC20 strain. The recombinant MLVs SUV-IL-15 and SUV-IL-18 recovered the inhibition of the NK cell response seen with Suvaxyn MLV. The recombinant MLV SUV-IL-15 significantly increased the numbers of gamma interferon (IFN-)-producing cells in circulation at 49 days postvaccination (dpv), especially for IFN--producing CD4nndash; CD8+ T cells and T cells, compared to the Suvaxyn MLV and SUV-IL-18. Additionally, MLV SUV-IL-15-vaccinated pigs also had elevated levels of T cell responses observed at 7 dpv, 49 dpv, and 7 days postchallenge. These data demonstrate that the recombinant MLV expressing membrane-bound IL-15 enhances NK and T cell immune responses after vaccination and confers improved heterologous protection, although this was not statistically significant compared to the parental MLV.

    IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS) has arguably been the most economically important global swine disease, causing immense economic losses worldwide. The available commercial modified live-attenuated vaccines (MLVs) against PRRS virus (PRRSV) are generally effective against only homologous or closely related virus strains but are ineffective against heterologous strains, partially due to the insufficient immune response induced by the vaccine virus. To improve the immunogenicity of MLVs, in this study, we present a novel approach of using porcine IL-15 or IL-18 as an adjuvant by directly incorporating its encoding gene into a PRRSV MLV and expressing it as an adjuvant. Importantly, we directed the expression of the incorporated cytokines to the cell membrane surface by fusing the genes with a membrane-targeting signal from CD59. The recombinant MLV virus expressing the membrane-bound IL-15 cytokine greatly enhanced NK cell and T cell responses and also conferred improved protection against heterologous challenge with the PRRSV NADC20 strain.

  • HIV Protease-Generated Casp8p41, When Bound and Inactivated by Bcl2, Is Degraded by the Proteasome [Vaccines and Antiviral Agents]

  • HIV protease is known to cause cell death, which is dependent upon cleavage of procaspase 8. HIV protease cleavage of procaspase 8 generates Casp8p41, which directly binds Bak with nanomolar affinity, causing Bak activation and consequent cell death. Casp8p41 can also bind Bcl2 with nanomolar affinity, in which case cell death is averted. Central memory CD4 T cells express high levels of Bcl2, possibly explaining why those cells do not die when they reactivate HIV. Here, we determine that the Casp8p41-Bcl2 complex is polyubiquitinated and degraded by the proteasome. Ixazomib, a proteasome inhibitor in clinical use, blocks this pathway, increasing the abundance of Casp8p41 and causing more cells to die in a Casp8p41-dependent manner.

    IMPORTANCE The Casp8p41 pathway of cell death is unique to HIV-infected cells yet is blocked by Bcl2. Once bound by Bcl2, Casp8p41 is polyubiquitinated and degraded by the proteasome. Proteasome inhibition blocks degradation of Casp8p41, increasing Casp8p41 levels and causing more HIV-infected cells to die.

  • A Novel Marsupial Hepatitis A Virus Corroborates Complex Evolutionary Patterns Shaping the Genus Hepatovirus [Genetic Diversity and Evolution]

  • The discovery of highly diverse nonprimate hepatoviruses illuminated the evolutionary origins of hepatitis A virus (HAV) ancestors in mammals other than primates. Marsupials are ancient mammals that diverged from other Eutheria during the Jurassic. Viruses from marsupials may thus provide important insight into virus evolution. To investigate Hepatovirus macroevolutionary patterns, we sampled 112 opossums in northeastern Brazil. A novel marsupial HAV (MHAV) in the Brazilian common opossum (Didelphis aurita) was detected by nested reverse transcription-PCR (RT-PCR). MHAV concentration in the liver was high, at 2.5 x 109 RNA copies/g, and at least 300-fold higher than those in other solid organs, suggesting hepatotropism. Hepatovirus seroprevalence in D. aurita was 26.6% as determined using an enzyme-linked immunosorbent assay (ELISA). Endpoint titers in confirmatory immunofluorescence assays were high, and marsupial antibodies colocalized with anti-HAV control sera, suggesting specificity of serological detection and considerable antigenic relatedness between HAV and MHAV. MHAV showed all genomic hallmarks defining hepatoviruses, including late-domain motifs likely involved in quasi-envelope acquisition, a predicted C-terminal pX extension of VP1, strong avoidance of CpG dinucleotides, and a type 3 internal ribosomal entry site. Translated polyprotein gene sequence distances of at least 23.7% from other hepatoviruses suggested that MHAV represents a novel Hepatovirus species. Conserved predicted cleavage sites suggested similarities in polyprotein processing between HAV and MHAV. MHAV was nested within rodent hepatoviruses in phylogenetic reconstructions, suggesting an ancestral hepatovirus host switch from rodents into marsupials. Cophylogenetic reconciliations of host and hepatovirus phylogenies confirmed that host-independent macroevolutionary patterns shaped the phylogenetic relationships of extant hepatoviruses. Although marsupials are synanthropic and consumed as wild game in Brazil, HAV community protective immunity may limit the zoonotic potential of MHAV.

    IMPORTANCE Hepatitis A virus (HAV) is a ubiquitous cause of acute hepatitis in humans. Recent findings revealed the evolutionary origins of HAV and the genus Hepatovirus defined by HAV in mammals other than primates in general and in small mammals in particular. The factors shaping the genealogy of extant hepatoviruses are unclear. We sampled marsupials, one of the most ancient mammalian lineages, and identified a novel marsupial HAV (MHAV). The novel MHAV shared specific features with HAV, including hepatotropism, antigenicity, genome structure, and a common ancestor in phylogenetic reconstructions. Coevolutionary analyses revealed that host-independent evolutionary patterns contributed most to the current phylogeny of hepatoviruses and that MHAV was the most drastic example of a cross-order host switch of any hepatovirus observed so far. The divergence of marsupials from other mammals offers unique opportunities to investigate HAV species barriers and whether mechanisms of HAV immune control are evolutionarily conserved.

  • Editorial Board [Masthead]

  • Inefficient HIV-1 trans Infection of CD4+ T Cells by Macrophages from HIV-1 Nonprogressors Is Associated with Altered Membrane Cholesterol and DC-SIGN [Pathogenesis and Immunity]

  • Professional antigen-presenting cells (APC; myeloid dendritic cells [DC] and macrophages [M]; B lymphocytes) mediate highly efficient HIV-1 infection of CD4+ T cells, termed trans infection, that could contribute to HIV-1 pathogenesis. We have previously shown that lower cholesterol content in DC and B lymphocytes is associated with a lack of HIV-1 trans infection in HIV-1-infected nonprogressors (NP). Here, we assessed whether HIV-1 trans infection mediated by another major APC, M, is deficient in NP due to altered cholesterol metabolism. When comparing healthy HIV-1 seronegatives (SN), rapid progressors (PR), and NP, we found that monocyte-derived M from NP did not mediate HIV-1 trans infection of autologous CD4+ T cells, in contrast to efficient trans infection mediated by SN and PR M. M trans infection efficiency was directly associated with the number of DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN)-expressing M. Significantly fewer NP M expressed DC-SIGN. Unesterified (free) cholesterol in M cell membranes and lipid rafting was significantly lower in NP than PR, as was virus internalization in early endosomes. Furthermore, simvastatin (SIMV) decreased the subpopulation of DC-SIGN+ M as well as cis and trans infection. Notably, SIMV decreased cell membrane cholesterol and led to lipid raft dissociation, effectively mimicking the incompetent APC trans infection environment characteristic of NP. Our data support that DC-SIGN and membrane cholesterol are central to M trans infection, and a lack of these limits HIV-1 disease progression. Targeting the ability of M to drive HIV-1 dissemination in trans could enhance HIV-1 therapeutic strategies.

    IMPORTANCE Despite the success of combination antiretroviral therapy, neither a vaccine nor a cure for HIV infection has been developed, demonstrating a need for novel prophylactic and therapeutic strategies. Here, we show that efficiency of M-mediated HIV trans infection of CD4+ T cells is a unique characteristic associated with control of disease progression, and it is impaired in HIV-infected NP. In vitro treatment of M from healthy donors with SIMV lowers their cholesterol content, which results in a strongly reduced trans infection ability, similar to the levels of M from NP. Taken together, our data support the hypothesis that M-mediated HIV-1 trans infection plays a role in HIV infection and disease progression and demonstrate that the use of SIMV to decrease this mechanism of virus transfer should be considered for future HIV therapeutic development.

  • Discovery of Novel Bat Coronaviruses in South China That Use the Same Receptor as Middle East Respiratory Syndrome Coronavirus [Genetic Diversity and Evolution]

  • Middle East respiratory syndrome coronavirus (MERS-CoV) has represented a human health threat since 2012. Although several MERS-related CoVs that belong to the same species as MERS-CoV have been identified from bats, they do not use the MERS-CoV receptor, dipeptidyl peptidase 4 (DPP4). Here, we screened 1,059 bat samples from at least 30 bat species collected in different regions in south China and identified 89 strains of lineage C betacoronaviruses, including Tylonycteris pachypus coronavirus HKU4, Pipistrellus pipistrellus coronavirus HKU5, and MERS-related CoVs. We sequenced the full-length genomes of two positive samples collected from the great evening bat, Ia io, from Guangdong Province. The two genomes were highly similar and exhibited genomic structures identical to those of other lineage C betacoronaviruses. While they exhibited genome-wide nucleotide identities of only 75.3 to 81.2% with other MERS-related CoVs, their gene-coding regions were highly similar to their counterparts, except in the case of the spike proteins. Further protein-protein interaction assays demonstrated that the spike proteins of these MERS-related CoVs bind to the receptor DPP4. Recombination analysis suggested that the newly discovered MERS-related CoVs have acquired their spike genes from a DPP4-recognizing bat coronavirus HKU4. Our study provides further evidence that bats represent the evolutionary origins of MERS-CoV.

    IMPORTANCE Previous studies suggested that MERS-CoV originated in bats. However, its evolutionary path from bats to humans remains unclear. In this study, we discovered 89 novel lineage C betacoronaviruses in eight bat species. We provide evidence of a MERS-related CoV derived from the great evening bat that uses the same host receptor as human MERS-CoV. This virus also provides evidence for a natural recombination event between the bat MERS-related CoV and another bat coronavirus, HKU4. Our study expands the host ranges of MERS-related CoV and represents an important step toward establishing bats as the natural reservoir of MERS-CoV. These findings may lead to improved epidemiological surveillance of MERS-CoV and the prevention and control of the spread of MERS-CoV to humans.

  • Electrostatic Interactions between Hendra Virus Matrix Proteins Are Required for Efficient Virus-Like-Particle Assembly [Structure and Assembly]

  • Hendra virus (HeV) is a zoonotic paramyxovirus belonging to the genus Henipavirus. HeV is highly pathogenic, and it can cause severe neurological and respiratory illnesses in both humans and animals, with an extremely high mortality rate of up to 70%. Among the genes that HeV encodes, the matrix (M) protein forms an integral part of the virion structure and plays critical roles in coordinating viral assembly and budding. Nevertheless, the molecular mechanism of this process is not fully elucidated. Here, we determined the crystal structure of HeV M to 2.5-AAring; resolution. The dimeric structural configuration of HeV M is similar to that of Newcastle disease virus (NDV) M and is fundamental to protein stability and effective virus-like-particle (VLP) formation. Analysis of the crystal packing revealed a notable interface between the aalpha;1 and aalpha;2 helices of neighboring HeV M dimers, with key residues sharing degrees of sequence conservation among henipavirus M proteins. Structurally, a network of electrostatic interactions dominates the aalpha;1-aalpha;2 interactions, involving residues Arg57 from the aalpha;1 helix and Asp105 and Glu108 from the aalpha;2 helix. The disruption of the aalpha;1-aalpha;2 interactions using engineered charge reversal substitutions (R57E, R57D, and E108R) resulted in significant reduction or abrogation of VLP production. This phenotype was reversible with an R57E E108R mutant that was designed to partly restore salt bridge contacts. Collectively, our results define and validate previously underappreciated regions of henipavirus M proteins that are crucial for productive VLP assembly.

    IMPORTANCE Hendra virus is a henipavirus associated with lethal infections in humans. It is classified as a biosafety level 4 (BSL4) agent, and there are currently no preventive or therapeutic treatments available against HeV. Vital to henipavirus pathogenesis, the structural protein M has been implicated in viral assembly and budding, as well as host-virus interactions. However, there is no structural information available for henipavirus M, and the basis of M-driven viral assembly is not fully elucidated. We demonstrate the first three-dimensional structure of a henipavirus M protein. We show the dimeric organization of HeV M as a basic unit for higher-order oligomerization. Additionally, we define key regions/residues of HeV M that are required for productive virus-like-particle formation. These findings provide the first insight into the mechanism of M-driven assembly in henipavirus.

  • E1B-55K-Mediated Regulation of RNF4 SUMO-Targeted Ubiquitin Ligase Promotes Human Adenovirus Gene Expression [Virus-Cell Interactions]

  • Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB-associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOylated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.

    IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4- and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention.

  • Subclinical Cytomegalovirus Infection Is Associated with Altered Host Immunity, Gut Microbiota, and Vaccine Responses [Virus-Cell Interactions]

  • Subclinical viral infections (SVI), including cytomegalovirus (CMV), are highly prevalent in humans, resulting in lifelong persistence. However, the impact of SVI on the interplay between the host immunity and gut microbiota in the context of environmental exposures is not well defined. We utilized the preclinical nonhuman primate (NHP) model consisting of SVI-free (specific-pathogen-free [SPF]) rhesus macaques and compared them to the animals with SVI (non-SPF) acquired through natural exposure and investigated the impact of SVI on immune cell distribution and function, as well as on gut microbiota. These changes were examined in animals housed in the outdoor environment compared to the controlled indoor environment. We report that SVI are associated with altered immune cell subsets and gut microbiota composition in animals housed in the outdoor environment. Non-SPF animals harbored a higher proportion of potential butyrate-producing Firmicutes and higher numbers of lymphocytes, effector T cells, and cytokine-producing T cells. Surprisingly, these differences diminished following their transfer to the controlled indoor environment, suggesting that non-SPFs had increased responsiveness to environmental exposures. An experimental infection of indoor SPF animals with CMV resulted in an increased abundance of butyrate-producing bacteria, validating that CMV enhanced colonization of butyrate-producing commensals. Finally, non-SPF animals displayed lower antibody responses to influenza vaccination compared to SPF animals. Our data show that subclinical CMV infection heightens host immunity and gut microbiota changes in response to environmental exposures. This may contribute to the heterogeneity in host immune response to vaccines and environmental stimuli at the population level.

    IMPORTANCE Humans harbor several latent viruses that modulate host immunity and commensal microbiota, thus introducing heterogeneity in their responses to pathogens, vaccines, and environmental exposures. Most of our understanding of the effect of CMV on the immune system is based on studies of children acquiring CMV or of immunocompromised humans with acute or reactivated CMV infection or in ageing individuals. The experimental mouse models are genetically inbred and are completely adapted to the indoor laboratory environment. In contrast, nonhuman primates are genetically outbred and are raised in the outdoor environment. Our study is the first to report the impact of long-term subclinical CMV infection on host immunity and gut microbiota, which is evident only in the outdoor environment but not in the indoor environment. The significance of this study is in highlighting the impact of SVI on enhancing host immune susceptibility to environmental exposures and immune heterogeneity.

  • Human Cytomegalovirus Protein pUL38 Prevents Premature Cell Death by Binding to Ubiquitin-Specific Protease 24 and Regulating Iron Metabolism [Virus-Cell Interactions]

  • Human cytomegalovirus (HCMV) protein pUL38 has been shown to prevent premature cell death by antagonizing cellular stress responses; however, the underlying mechanism remains unknown. In this study, we identified the host protein ubiquitin-specific protease 24 (USP24) as an interaction partner of pUL38. Mutagenesis analysis of pUL38 revealed that amino acids TFV at positions 227 to 230 were critical for its interaction with USP24. Mutant pUL38 TFV/AAA protein did not bind to USP24 and failed to prevent cell death induced by pUL38-deficient HCMV infection. Knockdown of USP24 suppressed the cell death during pUL38-deficient HCMV infection, suggesting that pUL38 achieved its function by antagonizing the function of USP24. We investigated the cellular pathways regulated by USP24 that might be involved in the cell death phenotype by testing several small-molecule compounds known to have a protective effect during stress-induced cell death. The iron chelators ciclopirox olamine and Tiron specifically protected cells from pUL38-deficient HCMV infection-induced cell death, thus identifying deregulated iron homeostasis as a potential mechanism. Protein levels of nuclear receptor coactivator 4 (NCOA4) and lysosomal ferritin degradation, a process called ferritinophagy, were also regulated by pUL38 and USP24 during HCMV infection. Knockdown of USP24 decreased NCOA4 protein stability and ferritin heavy chain degradation in lysosomes. Blockage of ferritinophagy by genetic inhibition of NCOA4 or Atg5/Atg7 prevented pUL38-deficient HCMV infection-induced cell death. Overall, these results support the hypothesis that pUL38 binds to USP24 to reduce ferritinophagy, which may then protect cells from lysosome dysfunction-induced cell death.

    IMPORTANCE Premature cell death is considered a first line of defense against various pathogens. Human cytomegalovirus (HCMV) is a slow-replicating virus that encodes several cell death inhibitors, such as pUL36 and pUL37x1, which allow it to overcome both extrinsic and intrinsic mitochondrion-mediated apoptosis. We previously identified HCMV protein pUL38 as another virus-encoded cell death inhibitor. In this study, we demonstrated that pUL38 achieved its activity by interacting with and antagonizing the function of the host protein ubiquitin-specific protease 24 (USP24). pUL38 blocked USP24-mediated ferritin degradation in lysosomes, which could otherwise be detrimental to the lysosome and initiate cell death. These novel findings suggest that iron metabolism is finely tuned during HCMV infection to avoid cellular toxicity. The results also provide a solid basis for further investigations of the role of USP24 in regulating iron metabolism during infection and other diseases.

  • Kaposi's Sarcoma-Associated Herpesvirus mRNA Accumulation in Nuclear Foci Is Influenced by Viral DNA Replication and Viral Noncoding Polyadenylated Nuclear RNA [Virus-Cell Interactions]

  • Kaposi's sarcoma-associated herpesvirus (KSHV), like other herpesviruses, replicates within the nuclei of its human cell host and hijacks host machinery for expression of its genes. The activities that culminate in viral DNA synthesis and assembly of viral proteins into capsids physically concentrate in nuclear areas termed viral replication compartments. We sought to better understand the spatiotemporal regulation of viral RNAs during the KSHV lytic phase by examining and quantifying the subcellular localization of select viral transcripts. We found that viral mRNAs, as expected, localized to the cytoplasm throughout the lytic phase. However, dependent on active viral DNA replication, viral transcripts also accumulated in the nucleus, often in foci in and around replication compartments, independent of the host shutoff effect. Our data point to involvement of the viral long noncoding polyadenylated nuclear (PAN) RNA in the localization of an early, intronless viral mRNA encoding ORF59-58 to nuclear foci that are associated with replication compartments.

    IMPORTANCE Late in the lytic phase, mRNAs from Kaposi's sarcoma-associated herpesvirus accumulate in the host cell nucleus near viral replication compartments, centers of viral DNA synthesis and virion production. This work contributes spatiotemporal data on herpesviral mRNAs within the lytic host cell and suggests a mechanism for viral RNA accumulation. Our findings indicate that the mechanism is independent of the host shutoff effect and splicing but dependent on active viral DNA synthesis and in part on the viral noncoding RNA, PAN RNA. PAN RNA is essential for the viral life cycle, and its contribution to the nuclear accumulation of viral messages may facilitate propagation of the virus.

  • SUN1 Regulates HIV-1 Nuclear Import in a Manner Dependent on the Interaction between the Viral Capsid and Cellular Cyclophilin A [Virus-Cell Interactions]

  • Human immunodeficiency virus type 1 (HIV-1) can infect nondividing cells via passing through the nuclear pore complex. The nuclear membrane-imbedded protein SUN2 was recently reported to be involved in the nuclear import of HIV-1. Whether SUN1, which shares many functional similarities with SUN2, is involved in this process remained to be explored. Here we report that overexpression of SUN1 specifically inhibited infection by HIV-1 but not that by simian immunodeficiency virus (SIV) or murine leukemia virus (MLV). Overexpression of SUN1 did not affect reverse transcription but led to reduced accumulation of the 2-long-terminal-repeat (2-LTR) circular DNA and integrated viral DNA, suggesting a block in the process of nuclear import. HIV-1 CA was mapped as a determinant for viral sensitivity to SUN1. Treatment of SUN1-expressing cells with cyclosporine (CsA) significantly reduced the sensitivity of the virus to SUN1, and an HIV-1 mutant containing CA-G89A, which does not interact with cyclophilin A (CypA), was resistant to SUN1 overexpression. Downregulation of endogenous SUN1 inhibited the nuclear entry of the wild-type virus but not that of the G89A mutant. These results indicate that SUN1 participates in the HIV-1 nuclear entry process in a manner dependent on the interaction of CA with CypA.

    IMPORTANCE HIV-1 infects both dividing and nondividing cells. The viral preintegration complex (PIC) can enter the nucleus through the nuclear pore complex. It has been well known that the viral protein CA plays an important role in determining the pathways by which the PIC enters the nucleus. In addition, the interaction between CA and the cellular protein CypA has been reported to be important in the selection of nuclear entry pathways, though the underlying mechanisms are not very clear. Here we show that both SUN1 overexpression and downregulation inhibited HIV-1 nuclear entry. CA played an important role in determining the sensitivity of the virus to SUN1: the regulatory activity of SUN1 toward HIV-1 relied on the interaction between CA and CypA. These results help to explain how SUN1 is involved in the HIV-1 nuclear entry process.

  • Bovine Herpesvirus 1 UL49.5 Interacts with gM and VP22 To Ensure Virus Cell-to-Cell Spread and Virion Incorporation: Novel Role for VP22 in gM-Independent UL49.5 Virion Incorporation [Virus-Cell Interactions]

  • Alphaherpesvirus envelope glycoprotein N (gN) and gM form a covalently linked complex. Bovine herpesvirus type 1 (BHV-1) UL49.5 (a gN homolog) contains two predicted cysteine residues, C42 and C78. The C42 is highly conserved among the alphaherpesvirus gN homologs (e.g., herpes simplex virus 1 and pseudorabies virus). To identify which cysteine residue is required for the formation of the UL49.5/gM complex and to characterize the functional significance of the UL49.5/gM complex, we constructed and analyzed C42S and C78S substitution mutants in either a BHV-1 wild type (wt) or BHV-1 UL49.5 cytoplasmic tail-null (CT-null) virus background. The results demonstrated that BHV-1 UL49.5 residue C42 but not C78 was essential for the formation of the covalently linked functional UL49.5/gM complex, gM maturation in the Golgi compartment, and efficient cell-to-cell spread of the virus. Interestingly, the C42S and CT-null mutations separately did not affect mutant UL49.5 virion incorporation. However, when both of the mutations were introduced simultaneously, the UL49.5 C42S/CT-null protein virion incorporation was severely reduced. Incidentally, the anti-VP22 antibody coimmunoprecipitated the UL49.5 C42S/CT-null mutant protein at a noticeably reduced level compared to that of the individual UL49.5 C42S and CT-null mutant proteins. As expected, in a dual UL49.5 C42S/VP22 virus with deletion of VP22 (VP22), the UL49.5 C42S virion incorporation was also severely reduced while in a gM virus, UL49.5 virion incorporation was affected only slightly. Together, these results suggested that UL49.5 virion incorporation is mediated redundantly, by both UL49.5/gM functional complex and VP22, through a putative gM-independent novel UL49.5 and VP22 interaction.

    IMPORTANCE Bovine herpesvirus 1 (BHV-1) envelope protein UL49.5 is an important virulence determinant because it downregulates major histocompatibility complex class I (MHC-I). UL49.5 also forms a covalently linked complex with gM. The results of this study demonstrate that UL49.5 regulates gM maturation and virus cell-to-cell spread since gM maturation in the Golgi compartment depends on covalently linked UL49.5/gM complex. The results also show that the UL49.5 residue cysteine 42 (C42) mediates the formation of the covalently linked UL49.5-gM interaction. Furthermore, a C42S mutant virus in which UL49.5 cannot interact with gM has defective cell-to-cell spread. Interestingly, UL49.5 also interacts with the tegument protein VP22 via its cytoplasmic tail (CT). The putative UL49.5 CT-VP22 interaction is essential for a gM-independent UL49.5 virion incorporation and is revealed when UL49.5 and gM are not linked. Therefore, UL49.5 virion incorporation is mediated by UL49.5-gM complex interaction and through a gM-independent interaction between UL49.5 and VP22.

  • Characterization of Three Novel Linear Neutralizing B-Cell Epitopes in the Capsid Protein of Swine Hepatitis E Virus [Structure and Assembly]

  • Hepatitis E virus (HEV) causes liver disease in humans and is thought to be a zoonotic infection, with domestic animals, including swine and rabbits, being a reservoir. One of the proteins encoded by the virus is the capsid protein. This is likely the major immune-dominant protein and a target for vaccination. Four monoclonal antibodies (MAbs), three novel, 1E4, 2C7, and 2G9, and one previously characterized, 1B5, were evaluated for binding to the capsid protein from genotype 4 swine HEV. The results indicated that 625DFCP628, 458PSRPF462, and 407EPTV410 peptides on the capsid protein comprised minimal amino acid sequence motifs recognized by 1E4, 2C7, and 2G9, respectively. The data suggested that 2C7 and 2G9 epitopes were partially exposed on the surface of the capsid protein. Truncated genotype 4 swine HEV capsid protein (sp239, amino acids 368 to 606) can exist in multimeric forms. Preincubation of swine HEV with 2C7, 2G9, or 1B5 before addition to HepG2 cells partially blocked sp239 cell binding and inhibited swine HEV infection. The study indicated that 2C7, 2G9, and 1B5 partially blocked swine HEV infection of rabbits better than 1E4 or normal mouse IgG. The cross-reactivity of antibodies suggested that capsid epitopes recognized by 2C7 and 2G9 are common to HEV strains infecting most host species. Collectively, MAbs 2C7, 2G9, and 1B5 were shown to recognize three novel linear neutralizing B-cell epitopes of genotype 4 HEV capsid protein. These results enhance understanding of HEV capsid protein structure to guide vaccine and antiviral design.

    IMPORTANCE Genotype 3 and 4 HEVs are zoonotic viruses. Here, genotype 4 HEV was studied due to its prevalence in human populations and pig herds in China. To improve HEV disease diagnosis and prevention, a better understanding of the antigenic structure and neutralizing epitopes of HEV capsid protein are needed. In this study, the locations of three novel linear B-cell recognition epitopes within genotype 4 swine HEV capsid protein were characterized. Moreover, the neutralizing abilities of three MAbs specific for this protein, 2C7, 2G9, and 1B5, were studied in vitro and in vivo. Collectively, these findings reveal structural details of genotype 4 HEV capsid protein and should facilitate development of applications for the design of vaccines and antiviral drugs for broader prevention, detection, and treatment of HEV infection of diverse human and animal hosts.

  • A Single Amino Acid in the Polymerase Acidic Protein Determines the Pathogenicity of Influenza B Viruses [Pathogenesis and Immunity]

  • Influenza B virus (IBV) is one of the human respiratory viruses and one of the targets of seasonal vaccination. However, the bifurcation of two antigenically distinct lineages of IBVs makes it difficult to arrange proper medical countermeasures. Moreover, compared with pathogenicity-related molecular markers known for influenza A virus, little has been known for IBVs. To understand pathogenicity caused by IBVs, we investigated the molecular determinants of IBV pathogenicity in animal models. After serial lung-to-lung passages of Victoria lineage B/Brisbane/60/2008 (Vc_BR60) and Yamagata lineage B/Wisconsin/01/2010 (Ym_WI01) viruses in BALB/c mice, we identified the mouse-adapted Vc_BR60 (maVc_BR60) and Ym_WI01 (maYm_WI01) viruses, respectively. To find a molecular clue(s) to the increased pathogenicity of maVc_BR60 and maYm_WI01, we determined their genetic sequences. Several amino acid mutations were identified in the PB2, PB1, PA, BM2, and/or NS1 protein-coding regions, and one concurrent lysine (K)-to-arginine (R) mutation in PA residue 338 (PA K338R) was found in both maVc_BR60 and maYm_WI01 viruses. When analyzed using viruses rescued through reverse genetics, it was shown that PA K338R alone could increase the pathogenicity of both IBVs in mice and viral replication in the respiratory tracts of ferrets. In a subsequent minireplicon assay, the effect of PA K338R was highlighted by the enhancement of viral polymerase complex activity of both Vc_BR60 and Ym_WI01 viruses. These results suggest that the PA K338R mutation may be a molecular determinant of IBV pathogenicity via modulating the viral polymerase function of IBVs.

    IMPORTANCE To investigate molecular pathogenic determinants of IBVs, which are one of the targets of seasonal influenza vaccines, we adapted both Victoria and Yamagata lineage IBVs independently in mice. The recovered mouse-adapted viruses exhibited increased virulence, and of the various mutations identified from both mouse-adapted viruses, a concurrent amino acid mutation was found in the PA protein-coding region. When analyzed using viruses rescued through reverse genetics, the PA mutation alone appeared to contribute to viral pathogenicity in mice within the compatible genetic constellation between the IBV lineages and to the replication of IBVs in ferrets. Regarding the potential mechanism of increased viral pathogenicity, it was shown that the PA mutation could upregulate the viral polymerase complex activity of both IBV lineages. These results indicate that the PA mutation could be a newly defined molecular pathogenic determinant of IBVs that substantiates our understanding of the viral pathogenicity and public health risks of IBVs.

  • Truncated CPSF6 Forms Higher-Order Complexes That Bind and Disrupt HIV-1 Capsid [Virus-Cell Interactions]

  • Cleavage and polyadenylation specificity factor 6 (CPSF6) is a human protein that binds HIV-1 capsid and mediates nuclear transport and integration targeting of HIV-1 preintegration complexes. Truncation of the protein at its C-terminal nuclear-targeting arginine/serine-rich (RS) domain produces a protein, CPSF6-358, that potently inhibits HIV-1 infection by targeting the capsid and inhibiting nuclear entry. To understand the molecular mechanism behind this restriction, the interaction between CPSF6-358 and HIV-1 capsid was characterized using in vitro and in vivo assays. Purified CPSF6-358 protein formed oligomers and bound in vitro-assembled wild-type (WT) capsid protein (CA) tubes, but not CA tubes containing a mutation in the putative binding site of CPSF6. Intriguingly, binding of CPSF6-358 oligomers to WT CA tubes physically disrupted the tubular assemblies into small fragments. Furthermore, fixed- and live-cell imaging showed that stably expressed CPSF6-358 forms cytoplasmic puncta upon WT HIV-1 infection and leads to capsid permeabilization. These events did not occur when the HIV-1 capsid contained a mutation known to prevent CPSF6 binding, nor did they occur in the presence of a small-molecule inhibitor of capsid binding to CPSF6-358. Together, our in vitro biochemical and transmission electron microscopy data and in vivo intracellular imaging results provide the first direct evidence for an oligomeric nature of CPSF6-358 and suggest a plausible mechanism for restriction of HIV-1 infection by CPSF6-358.

    IMPORTANCE After entry into cells, the HIV-1 capsid, which contains the viral genome, interacts with numerous host cell factors to facilitate crucial events required for replication, including uncoating. One such host cell factor, called CPSF6, is predominantly located in the cell nucleus and interacts with HIV-1 capsid. The interaction between CA and CPSF6 is critical during HIV-1 replication in vivo. Truncation of CPSF6 leads to its localization to the cell cytoplasm and inhibition of HIV-1 infection. Here, we determined that truncated CPSF6 protein forms large higher-order complexes that bind directly to HIV-1 capsid, leading to its disruption. Truncated CPSF6 expression in cells leads to premature capsid uncoating that is detrimental to HIV-1 infection. Our study provides the first direct evidence for an oligomeric nature of truncated CPSF6 and insights into the highly regulated process of HIV-1 capsid uncoating.

  • Neutralizing Antibody Responses following Long-Term Vaccination with HIV-1 Env gp140 in Guinea Pigs [Vaccines and Antiviral Agents]

  • A vaccination regimen capable of eliciting potent and broadly neutralizing antibodies (bNAbs) remains an unachieved goal of the HIV-1 vaccine field. Here, we report the immunogenicity of longitudinal prime/boost vaccination regimens with a panel of HIV-1 envelope (Env) gp140 protein immunogens over a period of 200 weeks in guinea pigs. We assessed vaccine regimens that included a monovalent clade C gp140 (C97ZA012 [C97]), a tetravalent regimen consisting of four clade C gp140s (C97ZA012, 459C, 405C, and 939C [4C]), and a tetravalent regimen consisting of clade A, B, C, and mosaic gp140s (92UG037, PVO.4, C97ZA012, and Mosaic 3.1, respectively [ABCM]). We found that the 4C and ABCM prime/boost regimens were capable of eliciting greater magnitude and breadth of binding antibody responses targeting variable loop 2 (V2) over time than the monovalent C97-only regimen. The longitudinal boosting regimen conducted over more than 2 years increased the magnitude of certain tier 1 NAb responses but did not increase the magnitude or breadth of heterologous tier 2 NAb responses. These data suggest that additional immunogen design strategies are needed to induce broad, high-titer tier 2 NAb responses.

    IMPORTANCE The elicitation of potent, broadly neutralizing antibodies (bNAbs) remains an elusive goal for the HIV-1 vaccine field. In this study, we explored the use of a long-term vaccination regimen with different immunogens to determine if we could elicit bNAbs in guinea pigs. We found that longitudinal boosting over more than 2 years increased tier 1 NAb responses but did not increase the magnitude and breadth of tier 2 NAb responses. These data suggest that additional immunogen designs and vaccination strategies will be necessary to induce broad tier 2 NAb responses.

  • Morphologic and Genomic Analyses of New Isolates Reveal a Second Lineage of Cedratviruses [Genetic Diversity and Evolution]

  • Giant viruses have been isolated and characterized in different environments, expanding our knowledge about the biology of these unique microorganisms. In the last 2 years, a new group was discovered, the cedratviruses, currently composed of only two isolates and members of a putative new family, "Pithoviridae," along with previously known pithoviruses. Here we report the isolation and biological and genomic characterization of two novel cedratviruses isolated from samples collected in France and Brazil. Both viruses were isolated using Acanthamoeba castellanii as a host cell and exhibit ovoid particles with corks at either extremity of the particle. Curiously, the Brazilian cedratvirus is ~20% smaller and presents a shorter genome of 460,038 bp, coding for fewer proteins than other cedratviruses. In addition, it has a completely asyntenic genome and presents a lower amino acid identity of orthologous genes (~73%). Pangenome analysis comprising the four cedratviruses revealed an increase in the pangenome concomitant with a decrease in the core genome with the addition of the two novel viruses. Finally, phylogenetic analyses clustered the Brazilian virus in a separate branch within the group of cedratviruses, while the French isolate is closer to the previously reported Cedratvirus lausannensis. Taking all together, we propose the existence of a second lineage of this emerging viral genus and provide new insights into the biodiversity and ubiquity of these giant viruses.

    IMPORTANCE Various giant viruses have been described in recent years, revealing a unique part of the virosphere. A new group among the giant viruses has recently been described, the cedratviruses, which is currently composed of only two isolates. In this paper, we describe two novel cedratviruses isolated from French and Brazilian samples. Biological and genomic analyses showed viruses with different particle sizes, genome lengths, and architecture, revealing the existence of a second lineage of this new group of giant viruses. Our results provide new insights into the biodiversity of cedratviruses and highlight the importance of ongoing efforts to prospect for and characterize new giant viruses.

  • CD4 T Cell Epitope Specificity and Cytokine Potential Are Preserved as Cells Transition from the Lung Vasculature to Lung Tissue following Influenza Virus Infection [Cellular Response to Infection]

  • Pulmonary CD4 T cells are critical in respiratory virus control, both by delivering direct effector function and through coordinating responses of other immune cells. Recent studies have shown that following influenza virus infection, virus-specific CD4 T cells are partitioned between pulmonary vasculature and lung tissue. However, very little is known about the peptide specificity or functional differences of CD4 T cells within these two compartments. Using a mouse model of influenza virus infection in conjunction with intravascular labeling in vivo, the cell surface phenotype, epitope specificity, and functional potential of the endogenous polyclonal CD4 T cell response was examined by tracking nine independent CD4 T cell epitope specificities. These studies revealed that tissue-localized CD4 cells were globally distinct from vascular cells in expression of markers associated with transendothelial migration, residency, and micropositioning. Despite these differences, there was little evidence for remodeling of the viral epitope specificity or cytokine potential as cells transition from vasculature to the highly inflamed lung tissue. Our studies also distinguished cells in the pulmonary vasculature from peripheral circulating CD4 T cells, providing support for the concept that the pulmonary vasculature does not simply reflect circulating cells that are trapped within the narrow confines of capillary vessels but rather is enriched in transitional cells primed in the draining lymph node that have specialized potential to enter the lung tissue.

    IMPORTANCE CD4 T cells convey a multitude of functions in immunity to influenza, including those delivered in the lymph node and others conveyed by CD4 T cells that leave the lymph node, enter the blood, and extravasate into the lung tissue. Here, we show that the transition of recently primed CD4 cells detected in the lung vasculature undergo profound changes in expression of markers associated with tissue localization as they establish residence in the lung. However, this transition does not edit CD4 T cell epitope specificity or the cytokine potential of the CD4 T cells. Thus, CD4 T cells that enter the infected lung can convey diverse functions and have a sufficiently broad viral antigen specificity to detect the complex array of infected cells within the infected tissue, offering the potential for more effective protective function.

  • Effects of Adjuvants on HIV-1 Envelope Glycoprotein SOSIP Trimers In Vitro [Vaccines and Antiviral Agents]

  • Native-like, soluble, recombinant SOSIP trimers of various designs and based on several env genes of human immunodeficiency virus type 1 (HIV-1) are being tested as immunogens in different animal models. These experiments almost always involve coformulating the trimers with an adjuvant to boost the magnitude of the immune responses. One factor relevant to the choice of an adjuvant is that it should not physically damage the immunogen or impede its ability to present relevant epitopes. As examples, an adjuvant formulation that includes harsh detergents could disrupt the structural integrity of a trimer, and any charged compounds in the formulation could bind to countercharged regions of the trimer and physically occlude nearby epitopes. While a few adjuvants have been tested for their potential effects on SOSIP trimers in vitro, there has been no systematic study. Here, we have assessed how nine different adjuvants of various compositions affect SOSIP trimers of the BG505 and B41 genotypes. We used negative-stain electron microscopy, thermal denaturation, and gel electrophoresis to evaluate effects on trimer integrity and immunoassays to measure effects on the presentation of various epitopes. We conclude that most of the tested adjuvants are benign from these perspectives, but some raise grounds for concern. An acidified alum formulation is highly disruptive to trimer integrity, and a DNA-based polyanionic CpG oligodeoxynucleotide adjuvant binds to trimers and occludes the trimer apex epitope for the PGT145 neutralizing antibody. The methods described here should be generalizable to protein subunit vaccines targeting various pathogens.

    IMPORTANCE Adjuvant formulations increase the magnitude of immune responses to vaccine antigens. They are critically important for formulation of HIV-1 envelope glycoprotein (Env) vaccines intended to induce antibody production, as Env proteins are otherwise only very weakly immunogenic. The HIV-1 vaccine field now uses the well-defined structures of trimeric Env glycoproteins, like SOSIPs, to present multiple known epitopes for broad and potent neutralizing human antibodies in a native-like conformation. Successful adjuvant formulations must not disrupt how the trimers are folded, as that could adversely affect their performance as immunogens. We studied whether the various adjuvants most commonly used in animal experiments affect the integrity of two different SOSIP trimers in vitro. Most adjuvant classes are not problematic, but an aluminum sulfate formulation is highly damaging, as it exposes trimers to acidic pH and a nucleic acid-based adjuvant can bind to the trimer and block access to a key neutralizing epitope.

  • A GXXXA Motif in the Transmembrane Domain of the Ebola Virus Glycoprotein Is Required for Tetherin Antagonism [Virus-Cell Interactions]

  • The interferon-induced antiviral host cell protein tetherin can inhibit the release of several enveloped viruses from infected cells. The Ebola virus (EBOV) glycoprotein (GP) antagonizes tetherin, but the domains and amino acids in GP that are required for tetherin antagonism have not been fully defined. A GXXXA motif within the transmembrane domain (TMD) of EBOV-GP was previously shown to be important for GP-mediated cellular detachment. Here, we investigated whether this motif also contributes to tetherin antagonism. Mutation of the GXXXA motif did not impact GP expression or particle incorporation and only modestly reduced EBOV-GP-driven entry. In contrast, the GXXXA motif was required for tetherin antagonism in transfected cells. Moreover, alteration of the GXXXA motif increased tetherin sensitivity of a replication-competent vesicular stomatitis virus (VSV) chimera encoding EBOV-GP. Although these results await confirmation with authentic EBOV, they indicate that a GXXXA motif in the TMD of EBOV-GP is important for tetherin antagonism. Moreover, they provide the first evidence that GP can antagonize tetherin in the context of an infectious EBOV surrogate.

    IMPORTANCE The glycoprotein (GP) of Ebola virus (EBOV) inhibits the antiviral host cell protein tetherin and may promote viral spread in tetherin-positive cells. However, tetherin antagonism by GP has so far been demonstrated only with virus-like particles, and it is unknown whether GP can block tetherin in infected cells. Moreover, a mutation in GP that selectively abrogates tetherin antagonism is unknown. Here, we show that a GXXXA motif in the transmembrane domain of EBOV-GP, which was previously reported to be required for GP-mediated cell rounding, is also important for tetherin counteraction. Moreover, analysis of this mutation in the context of vesicular stomatitis virus chimeras encoding EBOV-GP revealed that GP-mediated tetherin counteraction is operative in infected cells. To our knowledge, these findings demonstrate for the first time that GP can antagonize tetherin in infected cells and provide a tool to study the impact of GP-dependent tetherin counteraction on EBOV spread.

  • Neuronal Subtype Determines Herpes Simplex Virus 1 Latency-Associated-Transcript Promoter Activity during Latency [Pathogenesis and Immunity]

  • Herpes simplex virus (HSV) latency in neurons remains poorly understood, and the heterogeneity of the sensory nervous system complicates mechanistic studies. In this study, we used primary culture of adult trigeminal ganglion (TG) mouse neurons in microfluidic devices and an in vivo model to examine the subtypes of sensory neurons involved in HSV latency. HSV-infected neurofilament heavy-positive (NefH+) neurons were more likely to express latency-associated transcripts (LATs) than infected neurofilament heavy-negative (NefHnndash;) neurons. This differential expression of the LAT promoter correlated with differences in HSV-1 early infection that manifested as differences in the efficiency with which HSV particles reached the cell body following infection at the distal axon. In vivo, we further identified a specific subset of NefH+ neurons which coexpressed calcitonin gene-related peptide aalpha; (NefH+ CGRP+ neurons) as the sensory neuron subpopulation with the highest LAT promoter activity following HSV-1 infection. Finally, an early-phase reactivation assay showed HSV-1 reactivating in NefH+ CGRP+ neurons, although other sensory neuron subpopulations were also involved. Together, these results show that sensory neurons expressing neurofilaments exhibit enhanced LAT promoter activity. We hypothesize that the reduced efficiency of HSV-1 invasion at an early phase of infection may promote efficient establishment of latency in NefH+ neurons due to initiation of the antiviral state preceding arrival of the virus at the neuronal cell body. While the outcome of HSV-1 infection of neurons is determined by a broad variety of factors in vivo, neuronal subtypes are likely to play differential roles in modulating the establishment of latent infection.

    IMPORTANCE Two pivotal properties of HSV-1 make it a successful pathogen. First, it infects neurons, which are immune privileged. Second, it establishes latency in these neurons. Together, these properties allow HSV to persist for the lifetime of its host. Neurons are diverse and highly organized cells, with specific anatomical, physiological, and molecular characteristics. Previous work has shown that establishment of latency by HSV-1 does not occur equally in all types of neurons. Our results show that the kinetics of HSV infection and the levels of latency-related gene expression differ in certain types of neurons. The neuronal subtype infected by HSV is therefore a critical determinant of the outcome of infection and latency.

  • Complex and Dynamic Interactions between Parvovirus Capsids, Transferrin Receptors, and Antibodies Control Cell Infection and Host Range [Virus-Cell Interactions]

  • Antibody and receptor binding are key virus-host interactions that control host range and determine the success of infection. Canine and feline parvovirus capsids bind the transferrin receptor type 1 (TfR) to enter host cells, and specific structural interactions appear necessary to prepare the stable capsids for infection. Here, we define the details of binding, competition, and occupancy of wild-type and mutant parvovirus capsids with purified receptors and antibodies. TfR-capsid binding interactions depended on the TfR species and varied widely, with no direct relationship between binding affinity and infection. Capsids bound feline, raccoon, and black-backed jackal TfRs at high affinity but barely bound canine TfRs, which mediated infection efficiently. TfRs from different species also occupied capsids to different levels, with an estimated 1 to 2 feline TfRs but 12 black-backed jackal TfRs binding each capsid. Multiple alanine substitutions within loop 1 on the capsid surface reduced TfR binding but substitutions within loop 3 did not, suggesting that loop 1 directly engaged the TfR and loop 3 sterically affected that interaction. Binding and competition between different TfRs and/or antibodies showed complex relationships. Both antibodies 14 and E competed capsids off TfRs, but antibody E could also compete capsids off itself and antibody 14, likely by inducing capsid structural changes. In some cases, the initial TfR or antibody binding event affected subsequent TfR binding, suggesting that capsid structure changes occur after TfR or antibody binding and may impact infection. This shows that precise, host-specific TfR-capsid interactions, beyond simple attachment, are important for successful infection.

    IMPORTANCE Host receptor binding is a key step during viral infection and may control both infection and host range. In addition to binding, some viruses require specific interactions with host receptors in order to infect, and anti-capsid antibodies can potentially disrupt these interactions, leading to neutralization. Here, we examine the interactions between parvovirus capsids, the receptors from different hosts, and anti-capsid antibodies. We show that interactions between parvovirus capsids and host-specific TfRs vary in both affinity and in the numbers of receptors bound, with complex effects on infection. In addition, antibodies binding to two sites on the capsids had different effects on TfR-capsid binding. These experiments confirm that receptor and antibody binding to parvovirus capsids are complex processes, and the infection outcome is not determined simply by the affinity of attachment.

  • Arenaviral Nucleoproteins Suppress PACT-Induced Augmentation of RIG-I Function To Inhibit Type I Interferon Production [Pathogenesis and Immunity]

  • RIG-I is a major cytoplasmic sensor of viral pathogen-associated molecular pattern (PAMP) RNA and induces type I interferon (IFN) production upon viral infection. A double-stranded RNA (dsRNA)-binding protein, PACT, plays an important role in potentiating RIG-I function. We have shown previously that arenaviral nucleoproteins (NPs) suppress type I IFN production via their RNase activity to degrade PAMP RNA. We report here that NPs of arenaviruses block the PACT-induced enhancement of RIG-I function to mediate type I IFN production and that this inhibition is dependent on the RNase function of NPs, which is different from that of a known mechanism of other viral proteins to abolish the interaction between PACT and RIG-I. To understand the biological roles of PACT and RIG-I in authentic arenavirus infection, we analyze growth kinetics of recombinant Pichinde virus (PICV), a prototypical arenavirus, in RIG-I knockout (KO) and PACT KO mouse embryonic fibroblast (MEF) cells. Wild-type (WT) PICV grew at higher titers in both KO MEF lines than in normal MEFs, suggesting the important roles of these cellular proteins in restricting virus replication. PICV carrying the NP RNase catalytically inactive mutation could not grow in normal MEFs but could replicate to some extent in both KO MEF lines. The level of virus growth was inversely correlated with the amount of type I IFNs produced. These results suggest that PACT plays an important role in potentiating RIG-I function to produce type I IFNs in order to restrict arenavirus replication and that viral NP RNase activity is essential for optimal viral replication by suppressing PACT-induced RIG-I activation.

    IMPORTANCE We report here a new role of the nucleoproteins of arenaviruses that can block type I IFN production via their specific inhibition of the cellular protein sensors of virus infection (RIG-I and PACT). Our results suggest that PACT plays an important role in potentiating RIG-I function to produce type I IFNs in order to restrict arenavirus replication. This new knowledge can be exploited for the development of novel antiviral treatments and/or vaccines against some arenaviruses that can cause severe and lethal hemorrhagic fever diseases in humans.

  • Incomplete Downregulation of CD4 Expression Affects HIV-1 Env Conformation and Antibody-Dependent Cellular Cytotoxicity Responses [Virus-Cell Interactions]

  • HIV-1-infected cells expressing envelope glycoproteins (Env) in the CD4-bound conformation on their surfaces are targeted by antibody-dependent cellular cytotoxicity (ADCC) mediated by CD4-induced (CD4i) antibodies and sera from HIV-1-infected individuals (HIV+ sera). By downregulating the surface expression of CD4, Nef prevents Env-CD4 interaction, thus protecting HIV-1-infected cells from ADCC. HIV-1 infectious molecular clones (IMCs) are widely used to measure ADCC. In order to facilitate the identification of infected cells and high-throughput ADCC analysis, reporter genes (e.g., the Renilla luciferase [LucR] gene) are often introduced into IMC constructs. We evaluated the susceptibility of HIV-1-infected CD4+ T lymphocytes to ADCC using a panel of parental IMCs and derivatives that expressed the LucR reporter gene, utilizing different molecular strategies, including one specifically designed to retain Nef expression. We found that in some of these constructs, Nef expression in CD4+ T cells was suboptimal, and consequently, CD4 downregulation was incomplete. CD4 molecules remaining on the cell surface resulted in the exposure of ADCC-mediating CD4i epitopes on Env and a dramatic increase in the susceptibility of the infected cells to ADCC. Strikingly, protection from ADCC was observed when cells were infected with the parental IMC, which exhibited strong CD4 downregulation. This discrepancy between the parental and Nef-impaired viruses was independent of the strains of Env expressed, but rather, it was correlated with the levels of CD4 surface expression. Overall, our results indicate that caution should be taken when selecting IMCs for ADCC measurements and that CD4 downregulation needs to be carefully monitored when drawing conclusions about the nature and magnitude of ADCC.

    IMPORTANCE In-depth understanding of the susceptibility of HIV-1-infected cells to ADCC might help establish correlates of vaccine protection and guide the development of HIV-1 vaccine strategies. Different ADCC assays have been developed, including those using infectious molecular clones (IMCs) carrying a LucR reporter gene that greatly facilitates large-scale quantitative analysis. We previously reported different molecular strategies for introducing LucR while maintaining Nef expression and function and, consequently, CD4 surface downregulation. Here, we demonstrate that utilizing IMCs that exhibit impaired Nef expression can have undesirable consequences due to incomplete CD4 downregulation. CD4 molecules remaining on the cell surface resulted in the exposure of ADCC-mediating CD4i epitopes on Env and a dramatic increase in the susceptibility of the infected cells to ADCC. Overall, our results indicate that CD4 downregulation needs to be carefully monitored when drawing conclusions about the nature and magnitude of ADCC.

  • Evolution-Guided Structural and Functional Analyses of the HERC Family Reveal an Ancient Marine Origin and Determinants of Antiviral Activity [Virus-Cell Interactions]

  • In humans, homologous to the E6-AP carboxyl terminus (HECT) and regulator of chromosome condensation 1 (RCC1)-like domain-containing protein 5 (HERC5) is an interferon-induced protein that inhibits replication of evolutionarily diverse viruses, including human immunodeficiency virus type 1 (HIV-1). To better understand the origin, evolution, and function of HERC5, we performed phylogenetic, structural, and functional analyses of the entire human small-HERC family, which includes HERC3, HERC4, HERC5, and HERC6. We demonstrated that the HERC family emerged ggt;595 million years ago and has undergone gene duplication and gene loss events throughout its evolution. The structural topology of the RCC1-like domain and HECT domains from all HERC paralogs is highly conserved among evolutionarily diverse vertebrates despite low sequence homology. Functional analyses showed that the human small HERCs exhibit different degrees of antiviral activity toward HIV-1 and that HERC5 provides the strongest inhibition. Notably, coelacanth HERC5 inhibited simian immunodeficiency virus (SIV), but not HIV-1, particle production, suggesting that the antiviral activity of HERC5 emerged over 413 million years ago and exhibits species- and virus-specific restriction. In addition, we showed that both HERC5 and HERC6 are evolving under strong positive selection, particularly blade 1 of the RCC1-like domain, which we showed is a key determinant of antiviral activity. These studies provide insight into the origin, evolution, and biological importance of the human restriction factor HERC5 and the other HERC family members.

    IMPORTANCE Intrinsic immunity plays an important role as the first line of defense against viruses. Studying the origins, evolution, and functions of proteins responsible for effecting this defense will provide key information about virus-host relationships that can be exploited for future drug development. We showed that HERC5 is one such antiviral protein that belongs to an evolutionarily conserved family of HERCs with an ancient marine origin. Not all vertebrates possess all HERC members, suggesting that different HERCs emerged at different times during evolution to provide the host with a survival advantage. Consistent with this, two of the more recently emerged HERC members, HERC5 and HERC6, displayed strong signatures of having been involved in an ancient evolutionary battle with viruses. Our findings provide new insights into the evolutionary origin and function of the HERC family in vertebrate evolution, identifying HERC5 and possibly HERC6 as important effectors of intrinsic immunity in vertebrates.

  • Generation of Recombinant Rotaviruses Expressing Fluorescent Proteins by Using an Optimized Reverse Genetics System [Genome Replication and Regulation of Viral Gene Expression]

  • An entirely plasmid-based reverse genetics system for rotaviruses was established very recently. We improved the reverse genetics system to generate recombinant rotavirus by transfecting only 11 cDNA plasmids for its 11 gene segments under the condition of increasing the ratio of the cDNA plasmids for NSP2 and NSP5 genes. Utilizing this highly efficient system, we then engineered infectious recombinant rotaviruses expressing bioluminescent (NanoLuc luciferase) and fluorescent (enhanced green fluorescent protein [EGFP] and mCherry) reporters. These recombinant rotaviruses expressing reporters remained genetically stable during serial passages. Our reverse genetics approach and recombinant rotaviruses carrying reporter genes will be great additions to the tool kit for studying the molecular virology of rotavirus and for developing future next-generation vaccines and expression vectors.

    IMPORTANCE Rotavirus is one of the most important pathogens causing severe gastroenteritis in young children worldwide. In this paper, we describe a robust and simple reverse genetics system based on only rotavirus cDNAs and its application for engineering infectious recombinant rotaviruses harboring bioluminescent (NanoLuc) and fluorescent (EGFP and mCherry) protein genes. This highly efficient reverse genetics system and recombinant group A rotaviruses expressing reporters could be powerful tools for the study of different aspects of rotavirus replication. Furthermore, they may be useful for next-generation vaccine production for this medically important virus.

  • The S Gene Is Necessary but Not Sufficient for the Virulence of Porcine Epidemic Diarrhea Virus Novel Variant Strain BJ2011C [Pathogenesis and Immunity]

  • The recently emerged highly virulent variants of porcine epidemic diarrhea virus (PEDV) have caused colossal economic losses to the worldwide swine industry. In this study, we investigated the viral virulence determinants by constructing a series of chimeric mutants between the highly virulent strain BJ2011C and the avirulent strain CHM2013. When tested in the 2-day-old piglet model, wild-type (WT) BJ2011C caused severe diarrhea and death of the piglets within 72 h. In contrast, its chimeric derivative carrying the S gene from CHM2013 (BJ2011C-SCHM) was avirulent to the piglets. Moreover, reciprocal substitution of the BJ2011C S gene (CHM2013-SBJ) did not enable CHM2013 to gain any virulence. However, when the whole structural protein-coding region of BJ2011C (CHM2013-SPBJ) was swapped, CHM2013 started to gain the ability to efficiently colonize the intestinal tract and caused diarrhea in piglets. A further gain of virulence required additional acquisition of the 3' untranslated region (UTR) of BJ2011C, and the resultant virus (CHM2013-SP + 3UTRBJ) caused more severe diarrhea and death of piglets. Together, our findings suggest that the virulence of PEDV epidemic strains is a multigenic event and that the S gene is only one of the necessary determinants.

    IMPORTANCE The recently emerged highly virulent PEDV variants are the major cause of the global porcine epidemic diarrhea (PED) pandemic. The S gene of the variants undergoes remarkable variations and has been thought to be the virulence determinant for the enhanced pathogenesis. Our studies here showed that the S gene is only part of the story and that full virulence requires cooperation from other genes. Our findings provide insight into the pathogenic mechanism of the highly virulent PEDV variants and have implications for future vaccine development.

  • Comparative Analysis of UL16 Mutants Derived from Multiple Strains of Herpes Simplex Virus 2 (HSV-2) and HSV-1 Reveals Species-Specific Requirements for the UL16 Protein [Structure and Assembly]

  • Orthologs of the herpes simplex virus (HSV) UL16 gene are conserved throughout the Herpesviridae. Because of this conservation, one might expect that the proteins perform similar functions for all herpesviruses. Previous studies on a UL16-null mutant derived from HSV-2 strain 186 revealed a roughly 100-fold replication defect and a critical role for UL16 in the nuclear egress of capsids. These findings were in stark contrast to what has been observed with UL16 mutants of HSV-1 and pseudorabies virus, where roughly 10-fold replication deficiencies that were accompanied by defects in the secondary envelopment of cytoplasmic capsids were reported. One possible explanation for this discrepancy is that HSV-2 strain 186 is not representative of the HSV-2 species. To address this possibility, multiple UL16-null mutants were constructed in multiple HSV-2 and HSV-1 strains by CRISPR/Cas9 mutagenesis, and their phenotypes were characterized side by side. This analysis showed that all the HSV-2 UL16 mutants had 50- to 100-fold replication deficiencies that were accompanied by defects in the nuclear egress of capsids, as well as defects in the secondary envelopment of cytoplasmic capsids. By contrast, most HSV-1 UL16 mutants had 10-fold replication deficiencies that were accompanied by defects in secondary envelopment of cytoplasmic capsids. These findings indicated that UL16 has HSV species-specific functions. Interestingly, HSV-1 UL16 could promote the nuclear egress of HSV-2 UL16-null strains, suggesting that, unlike HSV-1, HSV-2 lacks an activity that can promote nuclear egress in the absence of UL16.

    IMPORTANCE HSV-2 and HSV-1 are important human pathogens that cause distinct diseases in their hosts. A complete understanding of the morphogenesis of these viruses is expected to reveal vulnerabilities that can be exploited in the treatment of HSV disease. UL16 is a virion structural component that is conserved throughout the Herpesviridae and functions in virus morphogenesis; however, previous studies have suggested different roles for UL16 in the morphogenesis of HSV-2 and HSV-1. This study sought to resolve this apparent discrepancy by analyzing multiple UL16 mutant viruses derived from multiple strains of HSV-2 and HSV-1. The data indicate that UL16 has HSV species-specific functions, as HSV-2 has a requirement for UL16 in the escape of capsids from the nucleus whereas both HSV-2 and HSV-1 require UL16 for final envelopment of capsids at cytoplasmic membranes.

  • Hepatitis C Virus NS5A Protein Promotes the Lysosomal Degradation of Hepatocyte Nuclear Factor 1{alpha} via Chaperone-Mediated Autophagy [Virus-Cell Interactions]

  • Hepatitis C virus (HCV) infection is closely associated with type 2 diabetes. We reported that HCV infection induces the lysosomal degradation of hepatocyte nuclear factor 1 alpha (HNF-1aalpha;) via interaction with HCV nonstructural protein 5A (NS5A) protein, thereby suppressing GLUT2 gene expression. The molecular mechanisms of selective degradation of HNF-1aalpha; caused by NS5A are largely unknown. Chaperone-mediated autophagy (CMA) is a selective lysosomal degradation pathway. Here, we investigated whether CMA is involved in the selective degradation of HNF-1aalpha; in HCV-infected cells and observed that the pentapeptide spanning from amino acid (aa) 130 to aa 134 of HNF-1aalpha; matches the rule for the CMA-targeting motif, also known as KFERQ motif. A cytosolic chaperone protein, heat shock cognate protein of 70 kDa (HSC70), and a lysosomal membrane protein, lysosome-associated membrane protein type 2A (LAMP-2A), are key components of CMA. Immunoprecipitation analysis revealed that HNF-1aalpha; was coimmunoprecipitated with HSC70, whereas the Q130A mutation (mutation of Q to A at position 130) of HNF-1aalpha; disrupted the interaction with HSC70, indicating that the CMA-targeting motif of HNF-1aalpha; is important for the association with HSC70. Immunoprecipitation analysis revealed that increasing amounts of NS5A enhanced the association of HNF-1aalpha; with HSC70. To determine whether LAMP-2A plays a role in the degradation of HNF-1aalpha; protein, we knocked down LAMP-2A mRNA by RNA interference; this knockdown by small interfering RNA (siRNA) recovered the level of HNF-1aalpha; protein in HCV J6/JFH1-infected cells. This result suggests that LAMP-2A is required for the degradation of HNF-1aalpha;. Immunofluorescence study revealed colocalization of NS5A and HNF-1aalpha; in the lysosome. Based on our findings, we propose that HCV NS5A interacts with HSC70 and recruits HSC70 to HNF-1aalpha;, thereby promoting the lysosomal degradation of HNF-1aalpha; via CMA.

    IMPORTANCE Many viruses use a protein degradation system, such as the ubiquitin-proteasome pathway or the autophagy pathway, for facilitating viral propagation and viral pathogenesis. We investigated the mechanistic details of the selective lysosomal degradation of hepatocyte nuclear factor 1 alpha (HNF-1aalpha;) induced by hepatitis C virus (HCV) NS5A protein. Using site-directed mutagenesis, we demonstrated that HNF-1aalpha; contains a pentapeptide chaperone-mediated autophagy (CMA)-targeting motif within the POU-specific domain of HNF-1aalpha;. The CMA-targeting motif is important for the association with HSC70. LAMP-2A is required for degradation of HNF-1aalpha; caused by NS5A. We propose that HCV NS5A interacts with HSC70, a key component of the CMA machinery, and recruits HSC70 to HNF-1aalpha; to target HNF-1aalpha; for CMA-mediated lysosomal degradation, thereby facilitating HCV pathogenesis. We discovered a role of HCV NS5A in CMA-dependent degradation of HNF-1aalpha;. Our results may lead to a better understanding of the role of CMA in the pathogenesis of HCV.

  • Articles of Significant Interest in This Issue [Spotlight]

  • Amino Acid Substitutions within HLA-B*27-Restricted T Cell Epitopes Prevent Recognition by Hepatitis Delta Virus-Specific CD8+ T Cells [Pathogenesis and Immunity]

  • Virus-specific CD8 T cell response seems to play a significant role in the outcome of hepatitis delta virus (HDV) infection. However, the HDV-specific T cell epitope repertoire and mechanisms of CD8 T cell failure in HDV infection have been poorly characterized. We therefore aimed to characterize HDV-specific CD8 T cell epitopes and the impacts of viral mutations on immune escape. In this study, we predicted peptide epitopes binding the most frequent human leukocyte antigen (HLA) types and assessed their HLA binding capacities. These epitopes were characterized in HDV-infected patients by intracellular gamma interferon (IFN-) staining. Sequence analysis of large hepatitis delta antigen (L-HDAg) and HLA typing were performed in 104 patients. The impacts of substitutions within epitopes on the CD8 T cell response were evaluated experimentally and by in silico studies. We identified two HLA-B*27-restricted CD8 T cell epitopes within L-HDAg. These novel epitopes are located in a relatively conserved region of L-HDAg. However, we detected molecular footprints within the epitopes in HLA-B*27-positive patients with chronic HDV infections. The variant peptides were not cross-recognized in HLA-B*27-positive patients with resolved HDV infections, indicating that the substitutions represent viral escape mutations. Molecular modeling of HLA-B*27 complexes with the L-HDAg epitope and its potential viral escape mutations indicated that the structural and electrostatic properties of the bound peptides differ considerably at the T cell receptor interface, which provides a possible molecular explanation for the escape mechanism. This viral escape from the HLA-B*27-restricted CD8 T cell response correlates with a chronic outcome of hepatitis D infection. T cell failure resulting from immune escape may contribute to the high chronicity rate in HDV infection.

    IMPORTANCE Hepatitis delta virus (HDV) causes severe chronic hepatitis, which affects 20 million people worldwide. Only a small number of patients are able to clear the virus, possibly mediated by a virus-specific T cell response. Here, we performed a systematic screen to define CD8 epitopes and investigated the role of CD8 T cells in the outcome of hepatitis delta and how they fail to eliminate HDV. Overall the number of epitopes identified was very low compared to other hepatotropic viruses. We identified, two HLA-B*27-restricted epitopes in patients with resolved infections. In HLA-B*27-positive patients with chronic HDV infections, however, we detected escape mutations within these identified epitopes that could lead to viral evasion of immune responses. These findings support evidence showing that HLA-B*27 is important for virus-specific CD8 T cell responses, similar to other viral infections. These results have implications for the clinical prognosis of HDV infection and for vaccine development.

  • Dynamics of Simian Immunodeficiency Virus Two-Long-Terminal-Repeat Circles in the Presence and Absence of CD8+ Cells [Pathogenesis and Immunity]

  • CD8+ cells play a key role in human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection, but their specific mechanism(s) of action in controlling the virus is unclear. Two-long-terminal-repeat (2-LTR) circles are extrachromosomal products generated upon failed integration of HIV/SIV. To understand the specific effects of CD8+ cells on infected cells, we analyzed the dynamics of 2-LTR circles in SIVmac251-infected rhesus macaques (RMs) treated with an integrase inhibitor (INT). Twenty RMs underwent CD8+ cell depletion and received raltegravir (RAL) monotherapy or a combination of both. Blood, lymph nodes (LNs), and gut biopsy specimens were routinely sampled. Plasma viral loads (pVLs) and 2-LTR circles from peripheral blood mononuclear cells (PBMCs) and LN lymphocytes were measured with quantitative reverse transcription-PCR (qRT-PCR). In the CD8 depletion group, an ~1-log increase in pVLs and a slow increase in PBMC 2-LTRs occurred following depletion. In the INT group, a strong decline in pVLs upon treatment initiation and no change in 2-LTR levels were observed. In the INT and CD8+ cell depletion group, an increase in pVLs following CD8 depletion similar to that in the CD8 depletion group was observed, with a modest decline following INT initiation, and 2-LTR circles significantly increased in PBMCs and LNs. Analyzing the 2-LTR data across all treatment groups with a mathematical model indicates that the data best support an effect of CD8+ cells in killing cells prior to viral integration. Sensitivity analyses of these results confirm that effect but also allow for additional effects, which the data do not discriminate well. Overall, we show that INT does not significantly increase the levels of 2-LTR circles. However, CD8+ cell depletion increases the 2-LTR levels, which are enhanced in the presence of an INT.

    IMPORTANCE CD8+ T cells play an essential role in controlling HIV and SIV infection, but the specific mechanisms involved remain poorly understood. Due to failed viral infection, HIV and SIV can form 2-LTR extrachromosomal circles that can be quantified. We present novel data on the dynamics of these 2-LTR forms in a SIV-infected macaque model under three different treatment conditions: depletion of CD8+ cells, administration of the integrase inhibitor in a monotherapy, which favors the formation of 2-LTR circles, and a combination of the two treatments. We used a new mathematical model to help interpret the data, and the results suggest that CD8+ cells exert a killing effect on infected cells prior to virus integration. These results provide new insights into the mechanisms of action of CD8+ cells in SIV infection. Confirmation of our results would be an important step in understanding immune control of HIV.

  • Osteopontin Regulates Hepatitis C Virus (HCV) Replication and Assembly by Interacting with HCV Proteins and Lipid Droplets and by Binding to Receptors {alpha}V{beta}3 and CD44 [Virus-Cell Interactions]

  • Hepatitis C virus (HCV) replication and assembly occur at the specialized site of endoplasmic reticulum (ER) membranes and lipid droplets (LDs), respectively. Recently, several host proteins have been shown to be involved in HCV replication and assembly. In the present study, we demonstrated the important relationship among osteopontin (OPN), the ER, and LDs. OPN is a secreted phosphoprotein, and overexpression of OPN in hepatocellular carcinoma (HCC) tissue can lead to invasion and metastasis. OPN expression is also enhanced in HCV-associated HCC. Our recent studies have demonstrated the induction, proteolytic cleavage, and secretion of OPN in response to HCV infection. We also defined the critical role of secreted OPN in human hepatoma cell migration and invasion through binding to receptors integrin aalpha;Vbbeta;3 and CD44. However, the role of HCV-induced OPN in the HCV life cycle has not been elucidated. In this study, we showed a significant reduction in HCV replication, assembly, and infectivity in HCV-infected cells transfected with small interfering RNA (siRNA) against OPN, aalpha;Vbbeta;3, and CD44. We also observed the association of endogenous OPN with HCV proteins (NS3, NS5A, NS4A/B, NS5B, and core). Confocal microscopy revealed the colocalization of OPN with HCV NS5A and core in the ER and LDs, indicating a possible role for OPN in HCV replication and assembly. Interestingly, the secreted OPN activated HCV replication, infectivity, and assembly through binding to aalpha;Vbbeta;3 and CD44. Collectively, these observations provide evidence that HCV-induced OPN is critical for HCV replication and assembly.

    IMPORTANCE Recently, our studies uncovered the critical role of HCV-induced endogenous and secreted OPN in migration and invasion of hepatocytes. However, the role of OPN in the HCV life cycle has not been elucidated. In this study, we investigated the importance of OPN in HCV replication and assembly. We demonstrated that endogenous OPN associates with HCV NS3, NS5A, NS5B, and core proteins, which are in close proximity to the ER and LDs. Moreover, we showed that the interactions of secreted OPN with cell surface receptors aalpha;Vbbeta;3 and CD44 are critical for HCV replication and assembly. These observations provide evidence that HCV-induced endogenous and secreted OPN play pivotal roles in HCV replication and assembly in HCV-infected cells. Taken together, our findings clearly demonstrate that targeting OPN may provide opportunities for therapeutic intervention of HCV pathogenesis.

  • Hepatitis B Virus Core Protein Dephosphorylation Occurs during Pregenomic RNA Encapsidation [Genome Replication and Regulation of Viral Gene Expression]

  • Hepatitis B virus (HBV) core protein consists of an N-terminal assembly domain and a C-terminal domain (CTD) with seven conserved serines or threonines that are dynamically phosphorylated/dephosphorylated during the viral replication cycle. Sulfamoylbenzamide derivatives are small molecular core protein allosteric modulators (CpAMs) that bind to the heteroaryldihydropyrimidine (HAP) pocket between the core protein dimer-dimer interfaces. CpAM binding alters the kinetics and pathway of capsid assembly and can result in the formation of morphologically "normal" capsids devoid of viral pregenomic RNA (pgRNA) and DNA polymerase. In order to investigate the mechanism underlying CpAM inhibition of pgRNA encapsidation, we developed an immunoblotting assay that can resolve core protein based on its phosphorylation status and demonstrated, for the first time, that core protein is hyperphosphorylated in free dimers and empty capsids from both mock-treated and CpAM-treated cells but is hypophosphorylated in pgRNA- and DNA-containing nucleocapsids. Interestingly, inhibition of pgRNA encapsidation by a heat shock protein 90 (HSP90) inhibitor prevented core protein dephosphorylation. Moreover, core proteins with point mutations at the wall of the HAP pocket, V124A and V124W, assembled empty capsids and nucleocapsids with altered phosphorylation status. The results thus suggest that core protein dephosphorylation occurs in the assembly of pgRNA and that interference with the interaction between core protein subunits at dimer-dimer interfaces during nucleocapsid assembly alters not only capsid structure, but also core protein dephosphorylation. Hence, inhibition of pgRNA encapsidation by CpAMs might be due to disruption of core protein dephosphorylation during nucleocapsid assembly.

    IMPORTANCE Dynamic phosphorylation of HBV core protein regulates multiple steps of viral replication. However, the regulatory function was mainly investigated by phosphomimetic mutagenesis, which disrupts the natural dynamics of core protein phosphorylation/dephosphorylation. Development of an immunoblotting assay capable of resolving hyper- and hypophosphorylated core proteins allowed us to track the phosphorylation status of core proteins existing as free dimers and the variety of intracellular capsids and to investigate the role of core protein phosphorylation/dephosphorylation in viral replication. Here, we found that disruption of core protein interaction at dimer-dimer interfaces during nucleocapsid assembly (by CpAMs or mutagenesis) inhibited core protein dephosphorylation and pgRNA packaging. Our work has thus revealed a novel function of core protein dephosphorylation in HBV replication and the mechanism by which CpAMs, a class of compounds that are currently in clinical trials for treatment of chronic hepatitis B, induce the assembly of empty capsids.

  • Interferon-Stimulated Gene (ISG)-Expression Screening Reveals the Specific Antibunyaviral Activity of ISG20 [Virus-Cell Interactions]

  • Bunyaviruses pose a significant threat to human health, prosperity, and food security. In response to viral infections, interferons (IFNs) upregulate the expression of hundreds of interferon-stimulated genes (ISGs), whose cumulative action can potently inhibit the replication of bunyaviruses. We used a flow cytometry-based method to screen the ability of ~500 unique ISGs from humans and rhesus macaques to inhibit the replication of Bunyamwera orthobunyavirus (BUNV), the prototype of both the Peribunyaviridae family and the Bunyavirales order. Candidates possessing antibunyaviral activity were further examined using a panel of divergent bunyaviruses. Interestingly, one candidate, ISG20, exhibited potent antibunyaviral activity against most viruses examined from the Peribunyaviridae, Hantaviridae, and Nairoviridae families, whereas phleboviruses (Phenuiviridae) largely escaped inhibition. Similar to the case against other viruses known to be targeted by ISG20, the antibunyaviral activity of ISG20 is dependent upon its functional RNase activity. Through use of an infectious virus-like particle (VLP) assay (based on the BUNV minigenome system), we confirmed that gene expression from all 3 viral segments is strongly inhibited by ISG20. Using in vitro evolution, we generated a substantially ISG20-resistant BUNV and mapped the determinants of ISG20 sensitivity/resistance. Taking all the data together, we report that ISG20 is a broad and potent antibunyaviral factor but that some bunyaviruses are remarkably ISG20 resistant. Thus, ISG20 sensitivity/resistance may influence the pathogenesis of bunyaviruses, many of which are emerging viruses of clinical or veterinary significance.

    IMPORTANCE There are hundreds of bunyaviruses, many of which cause life-threatening acute diseases in humans and livestock. The interferon (IFN) system is a key component of innate immunity, and type I IFNs limit bunyaviral propagation both in vitro and in vivo. Type I IFN signaling results in the upregulation of hundreds of IFN-stimulated genes (ISGs), whose concerted action generates an "antiviral state." Although IFNs are critical in limiting bunyaviral replication and pathogenesis, much is still unknown about which ISGs inhibit bunyaviruses. Using ISG-expression screening, we examined the ability of ~500 unique ISGs to inhibit Bunyamwera orthobunyavirus (BUNV), the prototypical bunyavirus. Using this approach, we identified ISG20, an interferon-stimulated exonuclease, as a potent inhibitor of BUNV. Interestingly, ISG20 possesses highly selective antibunyaviral activity, with multiple bunyaviruses being potently inhibited while some largely escape inhibition. We speculate that the ability of some bunyaviruses to escape ISG20 may influence their pathogenesis.

  • Developing and Evaluating Inhibitors against the RNase H Active Site of HIV-1 Reverse Transcriptase [Vaccines and Antiviral Agents]

  • We tested three compounds for their ability to inhibit the RNase H (RH) and polymerase activities of HIV-1 reverse transcriptase (RT). A high-resolution crystal structure (2.2 AAring;) of one of the compounds showed that it chelates the two magnesium ions at the RH active site; this prevents the RH active site from interacting with, and cleaving, the RNA strand of an RNA-DNA heteroduplex. The compounds were tested using a variety of substrates: all three compounds inhibited the polymerase-independent RH activity of HIV-1 RT. Time-of-addition experiments showed that the compounds were more potent if they were bound to RT before the nucleic acid substrate was added. The compounds significantly inhibited the site-specific cleavage required to generate the polypurine tract (PPT) RNA primer that initiates the second strand of viral DNA synthesis. The compounds also reduced the polymerase activity of RT; this ability was a result of the compounds binding to the RH active site. These compounds appear to be relatively specific; they do not inhibit either Escherichia coli RNase HI or human RNase H2. The compounds inhibit the replication of an HIV-1-based vector in a one-round assay, and their potencies were only modestly decreased by mutations that confer resistance to integrase strand transfer inhibitors (INSTIs), nucleoside analogs, or nonnucleoside RT inhibitors (NNRTIs), suggesting that their ability to block HIV replication is related to their ability to block RH cleavage. These compounds appear to be useful leads that can be used to develop more potent and specific compounds.

    IMPORTANCE Despite advances in HIV-1 treatment, drug resistance is still a problem. Of the four enzymatic activities found in HIV-1 proteins (protease, RT polymerase, RT RNase H, and integrase), only RNase H has no approved therapeutics directed against it. This new target could be used to design and develop new classes of inhibitors that would suppress the replication of the drug-resistant variants that have been selected by the current therapeutics.

  • The Pathway To Establishing HIV Latency Is Critical to How Latency Is Maintained and Reversed [Virus-Cell Interactions]

  • HIV infection requires lifelong antiretroviral therapy because of the persistence of latently infected CD4+ T cells. The induction of virus expression from latently infected cells occurs following T cell receptor (TCR) activation, but not all latently infected cells respond to TCR stimulation. We compared two models of latently infected cells using an enhanced green fluorescent protein (EGFP) reporter virus to infect CCL19-treated resting CD4+ (rCD4+) T cells (preactivation latency) or activated CD4+ T cells that returned to a resting state (postactivation latency). We isolated latently infected cells by sorting for EGFP-negative (EGFPnndash;) cells after infection. These cells were cultured with antivirals and stimulated with anti-CD3/anti-CD28, mitogens, and latency-reversing agents (LRAs) and cocultured with monocytes and anti-CD3. Spontaneous EGFP expression was more frequent in postactivation than in preactivation latency. Stimulation of latently infected cells with monocytes/anti-CD3 resulted in an increase in EGFP expression compared to that for unstimulated controls using the preactivation latency model but led to a reduction in EGFP expression in the postactivation latency model. The reduced EGFP expression was not associated with reductions in the levels of viral DNA or T cell proliferation but depended on direct contact between monocytes and T cells. Monocytes added to the postactivation latency model during the establishment of latency reduced spontaneous virus expression, suggesting that monocyte-T cell interactions at an early time point postinfection can maintain HIV latency. This direct comparison of pre- and postactivation latency suggests that effective strategies needed to reverse latency will depend on how latency is established.

    IMPORTANCE One strategy being evaluated to eliminate latently infected cells that persist in HIV-infected individuals on antiretroviral therapy (ART) is to activate HIV expression or production with the goal of inducing virus-mediated cytolysis or immune-mediated clearance of infected cells. The gold standard for the activation of latent virus is T cell receptor stimulation with anti-CD3/anti-CD28. However, this stimulus activates only a small proportion of latently infected cells. We show clear differences in the responses of latently infected cells to activating stimuli based on how latent infection is established, an observation that may potentially explain the persistence of noninduced intact proviruses in HIV-infected individuals on ART.

  • RIG-I-Like Receptor and Toll-Like Receptor Signaling Pathways Cause Aberrant Production of Inflammatory Cytokines/Chemokines in a Severe Fever with Thrombocytopenia Syndrome Virus Infection Mouse Model [Pathogenesis and Immunity]

  • Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by a tick-borne phlebovirus of the family Bunyaviridae, SFTS virus (SFTSV). Wild-type and type I interferon (IFN-I) receptor 1-deficient (IFNAR1nndash;/nndash;) mice have been established as nonlethal and lethal models of SFTSV infection, respectively. However, the mechanisms of IFN-I production in vivo and the factors causing the lethal disease are not well understood. Using bone marrow-chimeric mice, we found that IFN-I signaling in hematopoietic cells was essential for survival of lethal SFTSV infection. The disruption of IFN-I signaling in hematopoietic cells allowed an increase in viral loads in serum and produced an excess of multiple inflammatory cytokines and chemokines. The production of IFN-I and inflammatory cytokines was abolished by deletion of the signaling molecules IPS-1 and MyD88, essential for retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) and Toll-like receptor (TLR) signaling, respectively. However, IPS-1nndash;/nndash; MyD88nndash;/nndash; mice exhibited resistance to lethal SFTS with a moderate viral load in serum. Taken together, these results indicate that adequate activation of RLR and TLR signaling pathways under low to moderate levels of viremia contributed to survival through the IFN-I-dependent antiviral response during SFTSV infection, whereas overactivation of these signaling pathways under high levels of viremia resulted in abnormal induction of multiple inflammatory cytokines and chemokines, causing the lethal disease.

    IMPORTANCE SFTSV causes a severe infectious disease in humans, with a high fatality rate of 12 to 30%. To know the pathogenesis of the virus, we need to clarify the innate immune response as a front line of defense against viral infection. Here, we report that a lethal animal model showed abnormal induction of multiple inflammatory cytokines and chemokines by an uncontrolled innate immune response, which triggered the lethal SFTS. Our findings suggest a new strategy to target inflammatory humoral factors to treat patients with severe SFTS. Furthermore, this study may help the investigation of other tick-borne viruses.

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    JVI Accepts: Articles Published Ahead of Print

  • The cellular coactivator HCF-1 is required for glucocorticoid receptor-mediated transcription of bovine herpesvirus 1 immediate early genes [PublishAheadOfPrint]

  • Following productive infection, Bovine herpesvirus-1 (BoHV-1) establishes latency in sensory neurons. Like other aalpha;-herpesviruses, expression of BoHV-1 Immediate Early (IE) genes are regulated by an enhancer complex containing the viral IE activator VP16, the cellular transcription factor Oct-1, and transcriptional coactivator HCF-1 that is assembled on an IE enhancer core element (TAATGARAT). Expression of the IE transcription unit that encodes the viral IE activators bICP0 and bICP4 may also be induced by the activated glucocorticoid receptor via two Glucocorticoid Response Elements (GREs) located upstream of the enhancer core. Strikingly, lytic infection and reactivation from latency are consistently enhanced by glucocorticoid treatment in vivo. As the coactivator HCF-1 is essential for IE gene expression of alpha-herpesviruses and recruited by multiple transcription factors, we tested whether HCF-1 is required for glucocorticoid-induced IE gene expression. Depletion of HCF-1 reduced GR-mediated activation of the IE promoter in mouse neuroblastoma cells (Neuro-2A). More importantly, HCF-1-mediated GR activation of the promoter was dependent on the presence of GRE sites but independent of the TAATGARAT enhancer core element. HCF-1 was also recruited to the GRE region of a promoter lacking the enhancer core, consistent with a direct role of the coactivator in mediating GR-induced transcription. Similarly, during productive lytic infection, HCF-1 and GR occupied the IE region containing the GREs. These studies indicate HCF-1 is critical for GR activation of the viral IE genes and suggests that glucocorticoid induction of viral reactivation may proceed via an HCF-1-GR mechanism in the absence of the viral IE activator VP16.

    IMPORTANCE Bovine herpesvirus 1 (BoHV-1) transcription is rapidly activated during stress-induced reactivation from latency. The immediate early transcription unit 1 (IEtu1) promoter is regulated by the glucocorticoid receptor (GR) via two glucocorticoid receptor-binding sites (GRE). The IEtu1 promoter regulates expression of two viral transcriptional regulatory proteins, infected cell proteins 0 (bICP0 and bICP4), and thus must be stimulated during reactivation. This study demonstrates that activation of the IEtu1 promoter by the synthetic corticosteroid dexamethasone requires host cell factor 1 (HCF-1). Interestingly, the GRE sites, but not the IE enhancer core element (TAATGARAT), were required for HCF-1-mediated GR promoter activation. The GR and HCF-1 were recruited to the IEtu1 promoter in transfected and infected cells. Collectively, these studies indicate that HCF-1 is critical for GR activation of the viral IE genes and suggest that an HCF-1-GR complex can stimulate the IEtu1 promoter in the absence of the viral IE activator VP16.

  • Screening and Identification of Lassa Virus Entry Inhibitors from an FDA-Approved Drugs Library [PublishAheadOfPrint]

  • Lassa virus (LASV) belongs to the Mammarenavirus genus (family Arenaviridae) and causes severe hemorrhagic fever in humans. At present, there are no Food and Drug Administration (FDA)-approved drugs or vaccines specific for LASV. Herein, high-throughput screening of an FDA-approved drug library was performed against LASV entry by using pseudotype virus bearing LASV envelope glycoprotein (GPC). Two hit compounds, lacidipine and phenothrin, were identified as LASV entry inhibitors in the micromolar range. A mechanistic study revealed that both compounds inhibited LASV entry by blocking low-pH-induced membrane fusion. Accordingly, lacidipine showed virucidal effects on the pseudotype virus of LASV. Adaptive mutant analyses demonstrated that replacement of T40, located in the ectodomain of the stable-signal peptide (SSP), with lysine (K) conferred LASV resistance to lacidipine. Furthermore, lacidipine showed antiviral activity against LASV, the closely related Mopeia virus (MOPV), and the new world arenavirus Guanarito virus (GTOV). Drug-resistant variants indicated that the V36M in ectodomain of SSP mutant and V436A in the transmembrane domain of GP2 mutant conferred GTOV resistance to lacidipine, suggesting the interface between SSP and GP2 is the target of lacidipine. This study shows that lacidipine is a candidate for LASV therapy, reinforcing the notion that the SSP-GP2 interface provides an entry-targeted platform for arenavirus inhibitors design.

    IMPORTANCE Currently, there is no approved therapy to treat Lassa fever; therefore, repurposing of approved drugs will accelerate the development of a therapeutic stratagem. In this study, we screened an FDA-approved library of drugs and identified two compounds, lacidipine and phenothrin, which inhibited Lassa virus entry by blocking low-pH-induced membrane fusion. Additionally, both compounds extended their inhibition against the entry of Guanarito virus, and the viral targets were identified as the SSP-GP2 interface.

  • Broadly-reactive human monoclonal antibodies elicited following pandemic H1N1 influenza virus exposure protect mice from highly pathogenic H5N1 challenge [PublishAheadOfPrint]

  • Broadly cross-reactive antibodies that recognize conserved epitopes within the influenza virus hemagglutinin (HA) stalk domain are of particular interest for their potential use as therapeutic and prophylactic agents against multiple influenza virus subtypes including zoonotic virus strains. Here, we characterized four human HA stalk-reactive monoclonal antibodies (mAbs) for their binding breadth and affinity, in vitro neutralization capacity, and in vivo protective potential against an highly pathogenic avian influenza virus. The monoclonal antibodies were isolated from individuals shortly following infection with (70-1F02 and 1009-3B05) or vaccination against (05-2G02 and 09-3A01) A(H1N1)pdm09. Three of the mAbs bound HAs from multiple strains of group 1 viruses, and one mAb, 05-2G02, bound to both group 1 and group 2 influenza A HAs. All four antibodies prophylactically protected mice against a lethal challenge with the highly pathogenic A/Vietnam/1203/04 (H5N1) strain. Two mAbs, 70-1F02 and 09-3A01, were further tested for their therapeutic efficacy against the same strain and showed good efficacy in this setting as well. One mAb, 70-1F02, was co-crystallized with H5 HA and showed similar heavy chain only interactions as a the previously described anti-stalk antibody CR6261. Finally, we showed that antibodies that compete with these mAbs are prevalent in serum from an individual recently infected with A(H1N1)pdm09 virus. The antibodies described here can be developed into broad-spectrum antiviral therapeutics that could be used to combat infections with zoonotic or emerging pandemic influenza viruses.

    IMPORTANCE The rise in zoonotic infections of humans with emerging influenza viruses is a worldwide public health concern. The majority of recent zoonotic human influenza cases were caused by H7N9 and H5Nx viruses and were associated with high morbidity and mortality. In addition, seasonal influenza viruses are estimated to cause up to 650,000 deaths annually worldwide. Currently available anti-viral treatment options include only neuraminidase inhibitors, but some influenza viruses are naturally resistant to these drugs, and others quickly develop resistance-conferring mutations. Alternative therapeutics are urgently needed. Broadly protective antibodies that target the conserved llsquo;stalkrrsquo; domain of the hemagglutinin represent potential potent antiviral prophylactic and therapeutic agents that can assist pandemic preparedness. Here, we describe four human monoclonal antibodies that target conserved regions of influenza HA and characterize their binding spectrum, as well as their protective capacity in prophylactic and therapeutic settings against a lethal challenge with a zoonotic influenza virus.

  • TRIM41-Mediated Ubiquitination of Nucleoprotein Limits Influenza A Virus Infection [PublishAheadOfPrint]

  • Influenza A virus (IAV) is a highly transmissible respiratory pathogen and a major cause of morbidity and mortality around the world. Nucleoprotein (NP) is an abundant IAV protein essential for multiple steps of viral life cycle. Our recent proteomic study of the IAV-host interaction network found that the tripartite motif containing 41 (TRIM41), a ubiquitin E3 ligase, interacted with NP. However, the role of TRIM41 in IAV infection is unknown. Here, we report that TRIM41 interacts with NP through its SPRY domain. Furthermore, TRIM41 is constitutively expressed in lung epithelial cells and overexpression of TRIM41 inhibits IAV infection. Conversely, RNA interference (RNAi) and knockout of TRIM41 increase host susceptibility to IAV infection. As a ubiquitin E3 ligase, TRIM41 ubiquitinates NP in vitro and in cells. The TRIM41 mutant lacking E3 ligase activity fails to inhibit IAV infection, suggesting that the E3 ligase activity is indispensable for TRIM41 antiviral function. Mechanistic analysis further revealed that the polyubiquitination leads to NP protein degradation and viral inhibition. Taken together, TRIM41 is a constitutively expressed intrinsic IAV restriction factor that targets NP for ubiquitination and protein degradation.

    IMPORTANCE

    Influenza control strategies rely on annual immunization and require frequent updates of the vaccine, which are not always a foolproof process. Furthermore, the current antivirals are also losing effectiveness as new viral strains are often refractory to conventional treatments. Thus, there is an urgent need to find new antiviral mechanisms and develop therapeutic drugs based on these mechanisms. Targeting the virus-host interface is an emerging new strategy because host factors controlling viral replication activity will be ideal candidates and cellular proteins are less likely to mutate under drug-mediated selective pressure. Here, we show that the ubiquitin E3 ligase TRIM41 is an intrinsic host restriction factor to IAV. TRIM41 directly binds the viral nucleoprotein and targets it for ubiquitination and proteasomal degradation, thereby limiting viral infection. Exploitation of this natural defense pathway may open new avenues to develop influenza antivirals.

  • Multiple Host Factors Interact with Hypervariable Domain of Chikungunya Virus nsP3 and Determine Viral Replication in Cell-Specific Mode. [PublishAheadOfPrint]

  • Alphaviruses are widely distributed in both hemispheres and circulate between mosquitoes and amplifying vertebrate hosts. Geographically separated alphaviruses have adapted to replication in particular organisms. The accumulating data suggest that this adaptation is determined not only by changes in their glycoproteins, but also by the amino acid sequence of the hypervariable domain (HVD) of the alphavirus nsP3 protein. We performed a detailed investigation of chikungunya virus (CHIKV) nsP3 HVD interactions with host factors and their roles in viral replication in vertebrate and mosquito cells. The results demonstrate that CHIKV HVD is intrinsically disordered and binds several distinctive cellular proteins. These host factors include two members of the G3BP family and their mosquito homolog Rin, two members of the NAP1 family and several SH3 domain-containing proteins. Interaction with G3BP proteins or Rin is an absolute requirement for CHIKV replication, although it is insufficient to solely drive it in either vertebrate or mosquito cells. To achieve a detectable level of virus replication, HVD needs to bind members of at least one more protein family in addition to G3BPs. Interaction with NAP1L1 and NAP1L4 plays a more pro-viral role in vertebrate cells, while binding of SH3 domain-containing proteins to a proline-rich fragment of HVD is more critical for virus replication in the cells of mosquito origin. Modifications of binding sites in CHIKV HVD allow manipulation of the cell specificity of CHIKV replication. Similar changes may be introduced into HVDs of other alphaviruses to alter their replication in particular cells or tissues.

    IMPORTANCE Alphaviruses utilize a broad spectrum of cellular factors for efficient formation and function of replication complexes (RCs). Our data demonstrate for the first time that the hypervariable domain (HVD) of chikungunya virus nonstructural protein 3 (nsP3) is intrinsically disordered. It binds at least 3 families of cellular proteins, which play an indispensable role in viral RNA replication. The proteins of each family demonstrate functional redundancy. We provide a detailed map of the binding sites on CHIKV nsP3 HVD and show that mutations in these sites or the replacement of CHIKV HVD by heterologous HVD change cell specificity of viral replication. Such manipulations with alphavirus HVDs open an opportunity for development of new irreversibly attenuated vaccine candidates. To date, the disordered protein fragments have been identified in the nonstructural proteins of many other viruses. They may also interact with variety of cellular factors that determine critical aspects of virus-host interactions.

  • Regulation of Herpes Simplex Virus 2 Protein Kinase UL13 by Phosphorylation and Its Role in Viral Pathogenesis [PublishAheadOfPrint]

  • UL13 proteins are serine/threonine protein kinases encoded by herpes simplex virus HSV-1 and HSV-2. Although the downstream effects of the HSV protein kinases, mostly those of HSV-1 UL13, have been reported, there is a lack of information on how these viral protein kinases are regulated in HSV-infected cells. In this study, we used a large-scale phosphoproteomic analysis of HSV-2-infected cells to identify a physiological phosphorylation site in HSV-2 UL13 (i.e., Ser-18), and investigated the significance of phosphorylation of this site in HSV-2-infected cell cultures and mice. Our results were as follows. (i) An alanine substitution at UL13 Ser-18 (S18A) significantly reduced HSV-2 replication and cell-cell spread in U2OS cells to a level similar to the UL13 null and kinase-dead mutations. (ii) The UL13 S18A mutation significantly impaired phosphorylation of a cellular substrate of this viral protein kinase in HSV-2-infected U2OS cells. (iii) Following vaginal infection of mice, the UL13 S18A mutation significantly reduced mortality, HSV-2 replication in the vagina and development of vaginal disease to levels similar to the UL13 null and the kinase-dead mutations. (iv) A phosphomimetic substitution at UL13 Ser-18 significantly restored the phenotype observed with the UL13 S18A mutation in U2OS cells and mice. Collectively, our results suggested that phosphorylation of UL13 Ser-18 regulated UL13 function in HSV-2-infected cells and this regulation was critical for the functional activity of HSV-2 UL13 in vitro and in vivo, and also for HSV-2 replication and pathogenesis.

    IMPORTANCE: Based on studies on cellular protein kinases, it is obvious that the regulatory mechanisms of protein kinases are as crucial as their functional consequences. Herpesviruses each encode at least one protein kinase, but the mechanism by which these kinases are regulated in infected cells remains to be elucidated with a few exceptions, although information on their functional effects has been accumulating. In this study, we have shown that phosphorylation of the HSV-2 UL13 protein kinase at Ser-18 regulated its function in infected cells, and this regulation was critical for HSV-2 replication and pathogenesis in vivo. UL13 is conserved in all members of the family Herpesviridae, and this is the first report clarifying the regulatory mechanism of a conserved herpesvirus protein kinase that is involved in viral replication and pathogenesis in vivo. Our study may provide insight into the regulatory mechanisms of the other conserved herpesvirus protein kinases.

  • A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism is Independent of Integrins {alpha}3{beta}1, {alpha}V{beta}3, and {alpha}V{beta}5 [PublishAheadOfPrint]

  • Host receptor usage by KSHV has been best studied using primary microvascular endothelial and fibroblast cells, although the virus infects a wide variety of cell types in culture and in natural infections. In these two infection models, KSHV adheres to the cell though heparan sulfate (HS) binding, then interacts with a complex of EphA2, xct, and integrins aalpha;3bbeta;1, aalpha;Vbbeta;3, aalpha;Vbbeta;5 to catalyze viral entry. We dissected this receptor complex at the genetic level with CRISPR-Cas9 to precisely determine receptor usage in two epithelial cell lines. Surprisingly, we discovered an infection mechanism that requires HS and EphA2 but is independent of aalpha;V- and bbeta;1-family integrin expression. Furthermore, infection appears to be independent of the EphA2 intracellular domain. We also demonstrated that while two other endogenous Eph receptors were dispensable for KSHV infection, transduced EphA4 and EphA5 significantly enhanced infection of cells lacking EphA2.

    IMPORTANCE Our data reveals an integrin-independent route of KSHV infection and suggests that multiple Eph receptors besides EphA2 can promote and regulate infection. Since integrins and Eph receptors are large protein families with diverse expression patterns across cells and tissues, we propose that KSHV may engage with several proteins from both families in different combinations to negotiate successful entry into diverse cell types.

  • A dual motif in the hemagglutinin of H5N1 Goose/Guangdong-like HPAIV is conserved from their early evolution and increases both membrane fusion pH and virulence. [PublishAheadOfPrint]

  • Zoonotic highly pathogenic avian influenza viruses (HPAIV) have raised serious public health concerns of a novel pandemic. These strains emerge from low-pathogenic precursors by acquisition of a polybasic hemagglutinin (HA) cleavage site, the prime virulence determinant. However, required co-adaptations of the HA early in HPAIV evolution remained uncertain. To address this question, we generated several HA1/HA2 chimeras and point mutants of an H5N1 clade 2.2.2 HPAIV and an H5N1 low-pathogenic strain. Initial surveys of 3385 HPAIV H5 HA sequences revealed frequencies of 0.5% for the single amino acids 123R or 124I each, in dual combination however at 97.5%. This highly conserved dual motif is still retained in contemporary H5 HPAIV including the novel H5NX reassortants carrying neuraminidases of different subtypes like the H5N8 and the zoonotic H5N6 strains. Remarkably, the earliest Asian H5N1 HPAIV, the Goose/Guangdong strains from 1996/97 carried 123R only, whereas 124I appeared later in 1997. Experimental reversion in the HPAIV HA to the two residues 123S/124T, characteristic in low-pathogenic strains, prevented virus rescue while the single substitutions attenuated the virus both in chicken and mice considerably, accompanied by a decreased HA fusion pH. This increased pH sensitivity of H5 HPAIV enables HA-mediated membrane fusion at a higher endosomal pH. Therefore, this HA adaptation may permit infection of cells with less acidic endosomes, e.g. within the respiratory tract, resulting in an extended organ tropism. Taken together, HA co-adaptation to increased acid sensitivity promoted early evolution of H5 Goose/Guangdong-like HPAIV and is still required for their zoonotic potential.

    IMPORTANCE Zoonotic highly pathogenic avian influenza viruses (HPAIV) have raised serious public health concerns of a novel pandemic. Their prime virulence determinant is the polybasic hemagglutinin (HA) cleavage site. However, required co-adaptations in the HA (and other genes) remained uncertain. Here, we identified the dual motif 123R/124I in the HA head that increases the activation pH of HA-mediated membrane fusion, essential for virus genome release into the cytoplasm. This motif is extremely predominant in H5 HPAIV and emerged already in the earliest 1997 H5N1 HPAIV. Reversion to 123S or 124T, characteristic in low-pathogenic strains, attenuated the virus in chicken and mice, accompanied by a decreased HA activation pH. This increased pH sensitivity of H5 HPAIV extends the viral tropism to cells with less acidic endosomes, e.g. within the respiratory tract. Therefore, early HA adaptation to increased acid sensitivity promoted emergence of H5 Goose/Guangdong-like HPAIV and is required for their zoonotic potential.

  • Design of Novel HIV-1/2 Fusion Inhibitors with Efficient Therapeutic Efficacy in Rhesus Monkey Models [PublishAheadOfPrint]

  • T-20 (enfuvirtide) is the only approved viral fusion inhibitor, which is used for treatment of HIV-1 infection; however, it has relatively low antiviral activity and easily induces drug resistance. We recently reported a T-20-based lipopeptide fusion inhibitor (LP-40) showing improved anti-HIV activity (Ding et al. J Virol, 2017). In this study, we designed LP-50 and LP-51 by refining the structure and function of LP-40. Two new lipopeptides showed dramatically enhanced secondary structures and binding stability and were exceptionally potent inhibitors of HIV-1, HIV-2, simian immunodeficiency virus (SIV), and simian-human chimeric virus (SHIV), with mean IC50 values at very low picomolar concentrations. They also exhibited dramatically increased potencies in inhibiting a panel of T-20 and LP-40-resistant mutant viruses. In line with their in vitro data, LP-50 and LP-51 exhibited extremely potent and long-lasting ex vivo anti-HIV activities in rhesus monkeys, with serum dilution peaks that inhibited 50% virus infection being ggt;15,200-fold higher than T-20 and LP-40. A low-dose, short-term monotherapy of LP-51 could sharply reduce viral loads to undetectable levels in acutely and chronically SHIV-infected monkey models. In our knowledge, LP-50 and LP-51 are the most potent and broad HIV-1/2 and SIV fusion inhibitors, which can be developed for clinical use and serve as tools to explore the mechanisms of viral entry and inhibition.

    IMPORTANCE T-20 remains the only membrane fusion inhibitor available for treatment of viral infection, but its relatively low anti-HIV and genetic barrier for drug resistance have significantly limited its clinical application. Herein, we report two new lipopeptide-based fusion inhibitors (LP-50 and LP-51) showing extremely potent inhibitory activities against diverse HIV-1, HIV-2, SIV, and T-20-resistant variants. Promisingly, both inhibitors exhibited a potent and long-lasting ex vivo anti-HIV activity and could efficiently suppress viral loads at undetectable levels in SHIV-infected monkey models. We believe that they are the most potent and broad-spectrum fusion inhibitors known to date and thus have high potential for clinical development.

  • Viral discovery in the invasive Australian cane toad (Rhinella marina) using metatranscriptomic and genomic approaches [PublishAheadOfPrint]

  • Cane toads are a notorious invasive species, inhabiting over 1.2 million km2 of Australia and threatening native biodiversity. Release of pathogenic cane toad viruses is one possible biocontrol strategy yet is currently hindered by the poorly-described cane toad virome. Metatranscriptomic analysis of 16 cane toad livers revealed the presence of a novel and full-length picornavirus, Rhimavirus A (RhiV-A), a member of a reptile and amphibian specific-cluster of the Picornaviridae basal to the Kobuvirus-like group. In the combined liver transcriptome, we also identified a complete genome sequence of a distinct epsilonretrovirus, R. marina endogenous retrovirus (RMERV). The recently sequenced cane toad genome contains eight complete RMERV proviruses, as well as 21 additional truncated insertions. The oldest full length RMERV provirus was estimated to have inserted 1.9 MYA. To screen for these viral sequences in additional toads, we analysed publicly available transcriptomes from six diverse Australian locations. RhiV-A transcripts were identified in toads sampled from three locations across 1,000 km of Australia, stretching to the current Western Australia (WA) invasion front, whilst RMERV transcripts were observed at all six sites. Lastly, we scanned the cane toad genome for non-retroviral endogenous viral elements, finding three sequences related to small DNA viruses in the family Circoviridae. This shows ancestral circoviral infection with subsequent genomic integration. The identification of these current and past viral infections enriches our knowledge of the cane toad virome, an understanding of which will facilitate future work on infection and disease in this important invasive species.

    Importance Cane toads are poisonous amphibians which were introduced to Australia in 1935 for insect control. Since then, their population has increased dramatically, and they now threat many native Australian species. One potential method to control the population is to release a cane toad virus with high mortality, yet few cane toad viruses have been characterised. This study samples cane toads from different Australian locations and uses an RNA sequencing and computational approach to find new viruses. We report novel complete picornavirus and retrovirus sequences which were genetically similar to viruses infecting frogs, reptiles and fish. Using data generated in other studies, we show that these viral sequences are present in cane toads from distinct Australian locations. Three sequences related to circoviruses were also found in the toad genome. The identification of new viral sequences will aid future studies which investigate their prevalence and potential as agents for biocontrol.

  • The influenza B virus hemagglutinin head domain is less tolerant to transposon mutagenesis than that of the influenza A virus [PublishAheadOfPrint]

  • Influenza A and B viruses can continuously evade humoral immune responses by developing mutations in the globular head of the hemagglutinin (HA) that prevent antibody binding. However, the influenza B virus HA over time displays less antigenic variation despite being functionally and structurally similar to the influenza A virus HA. To determine if the influenza B virus HA is under constraints that limit its antigenic variation, we performed a transposon screen to compare the mutational tolerance of the currently circulating influenza A virus HAs (H1 and H3 subtypes) and influenza B virus HAs (B/Victoria87 and B/Yamagata88 antigenic lineages). A library of insertional mutants for each HA was generated and deep sequenced after passaging to determine where insertions were tolerated in replicating viruses. The head domains of both viruses tolerated transposon mutagenesis, but the influenza A virus head was more tolerant to insertions than the influenza B virus head domain. Furthermore, all five of the known antigenic sites of the influenza A virus HA were tolerant of 15 nucleotide insertions, while insertions were detected in only two of the four antigenic sites in the influenza B virus head domain. Our analysis demonstrated that the influenza B virus HA is inherently less tolerant of transposon mediated insertions than the influenza A virus HA. The reduced insertional tolerance of the influenza B virus HA may reveal genetic restrictions resulting in a lower capacity for antigenic evolution.

    IMPORTANCE Influenza viruses cause seasonal epidemics and result in significant human morbidity and mortality. Influenza viruses persist in the human population through generating mutations in the hemagglutinin head domain that prevent antibody recognition. Despite the similar selective pressures on influenza A and B viruses, influenza A virus displays a higher rate and breadth of antigenic variability than influenza B virus. A transposon mutagenesis screen was used to examine if the reduced antigenic variability of influenza B virus was due to inherent differences in mutational tolerance. This study demonstrates that the influenza A virus head domain and the individual antigenic sites targeted by humoral responses are more tolerant to insertions than those of influenza B virus. This finding sheds light on the genetic factors controlling the antigenic evolution of influenza viruses.

  • Dissecting the herpesvirus architecture by targeted proteolysis [PublishAheadOfPrint]

  • Herpesvirus particles have a complex architecture consisting of an icosahedral capsid that is surrounded by a lipid envelope. Connecting these two components is a layer of tegument that consists of varying amounts of twenty or more proteins. The arrangement of proteins within the tegument cannot easily be assessed and instead is inferred from tegument interactions identified in reductionist models. To better understand the tegument architecture, we have developed an approach to probe capsid-tegument interactions of extracellular viral particles by encoding tobacco etch virus (TEV) protease sites in viral structural proteins, along with distinct fluorescent tags in capsid and tegument components. In this study, TEV sites were engineered within the pUL36 large tegument protein: a critical structural element that is anchored directly on the capsid surface. Purified pseudorabies virus extracellular particles were permeabilized and TEV protease was added to selectively cleave the exposed pUL36 backbone. Interactions with the capsid were assessed in situ by monitoring the fate of the fluorescent signals following cleavage. Although several regions of pUL36 are proposed to bind capsids, pUL36 was found stably anchored to the capsid exclusively at its carboxyl terminus. Two additional tegument proteins, pUL37 and pUS3, were tethered to the capsid via pUL36 whereas the pUL16, pUL47, pUL48, and pUL49 tegument proteins were not stably bound to the capsid.

    IMPORTANCE: Neuroinvasive alphaherpesviruses produce diseases of clinical and economic significance in humans and veterinary animals, but are predominantly associated with less serious recurrent disease. Like all viruses, herpesviruses assemble a metastable particle that selectively dismantles during initial infection. This process is made more complex by the presence of a tegument layer that resides between the capsid surface and envelope. Components of the tegument are essential for particle assembly and also serve as critical effectors that promote infection upon entry into cells. How this dynamic network of protein interactions is arranged within virions is largely unknown. We present a molecular approach to dissect the tegument and with it, begin to tease apart the protein interactions that underlie this complex layer of the virion architecture.

  • Cross-Reactivity Against Multiple HIV-1 Epitopes Is Characteristic of HIV-1-Specific Cytotoxic T Lymphocyte Clones [PublishAheadOfPrint]

  • Although a high level of promiscuity for heterologous epitopes is believed to exist for cellular immunity, limited data explore this issue for Human Immunodeficiency Virus type 1 (HIV-1)-specific CD8+ T lymphocyte (CTL) responses. Here we found an unexpected degree of heterologous cross-reactivity against HIV-1 epitopes in addition to the targeted index epitope. Most CTL clones screened cross-reacted against other known HIV-1 epitopes of the same major histocompatibility type I (MHC-I) restriction, up to 40% of tested non-index epitopes in some cases. The observed cross-reactivity was universally lower avidity than recognition of the index epitope when examined for several A*02- and B*57- restricted CTL clones, demonstrating that high concentrations of exogenous epitope typically used for screening of CTL responses are prone to detect such cross-reactivity spuriously. In agreement, we found that these cross-reactive responses do not appear to mediate CTL activity against HIV-1-infected cells. Overall, our data indicate that low-level cross-reactivity is remarkably common for HIV-1-specific CTLs. The role for this phenomenon is unclear, but low avidity interactions have been shown to foster homeostatic proliferation of memory cells of T cells.

    IMPORTANCE This study raises two issues related to HIV-1-specific CTL responses. These are key immune responses that retard disease progression in infected persons, which are highly relevant to immunotherapies and vaccines for HIV-1. First, we make the novel observation that these responses are promiscuous, and that CTLs targeting one epitope may cross-recognize other completely distinct epitopes in the virus. While these are low avidity interactions that do not appear to contribute directly to the antiviral activity of CTLs, this raises interesting biologic implications regarding the purpose of the phenomenon, such as providing a stimulus for these responses to persist long term. Second, the data raise a technical caveat to detection of CTL responses against particular epitopes, suggesting that some methodologies may unintentionally detect cross-reactivity and overestimate responses against an epitope.

  • Immunization by replication-competent controlled herpesvirus vectors [PublishAheadOfPrint]

  • Replication-competent controlled virus vectors were derived from virulent HSV-1 wildtype strain 17syn+ by placing one or two replication-essential genes under the stringent control of a gene switch that is co-activated by heat and an antiprogestin. Upon activation of the gene switch, the vectors replicate in infected cells with an efficacy that approaches that of the wildtype virus from which they were derived. Essentially no replication occurs in the absence of activation. When administered to mice, localized application of a transient heat treatment in the presence of systemic antiprogestin results in efficient but limited virus replication at the site of administration. The immunogenicity of these viral vectors was tested in a mouse footpad lethal challenge model. Unactivated viral vectors - which may be regarded as equivalents of inactivated vaccines - induced detectable protection against lethality caused by wildtype virus challenge. Single activation of the viral vectors at the site of administration (rear footpads) greatly enhanced protective immune responses, and second immunization resulted in complete protection. Once activated vectors also induced far better neutralizing antibody and HSV-1-specific cellular immune responses than unactivated vectors. To find out whether the immunogenicity of a heterologous antigen was also enhanced in the context of efficient transient vector replication, a virus vector constitutively expressing an equine influenza virus hemagglutinin was constructed. Immunization of mice with this recombinant induced detectable antibody-mediated neutralization of equine influenza virus as well as a hemagglutinin-specific cellular immune response. Single activation of viral replication resulted in a several-fold enhancement of these immune responses.

    IMPORTANCE We hypothesized that vigorous replication of a pathogen may be critical for eliciting the most potent and balanced immune response against it. Hence, attenuation/inactivation (as in conventional vaccines) should be avoided. Instead, necessary safety should be provided by placing replication of the pathogen under stringent control and of activating time-limited replication of the pathogen strictly in an administration region in which pathology cannot develop. Immunization will then occur in the context of highly efficient pathogen replication and uncompromised safety. We found that localized activation in mice of efficient but limited replication of a replication-competent controlled herpesvirus vector resulted in a greatly enhanced immune response to the virus or an expressed heterologous antigen. This finding supports the above hypothesis as well as suggests that the vectors may be promising novel agents worth exploring for the prevention/mitigation of infectious diseases for which efficient vaccination is lacking, in particular in immunocompromised patients.

  • Porcine reproductive and respiratory syndrome virus infection induces both eIF2{alpha}-phosphorylation-dependent and -independent host translation shutoff [PublishAheadOfPrint]

  • Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has caused tremendous economic losses in the global swine industry since it was discovered in the late 1980s. Inducing host translation shutoff is a strategy used by many viruses to optimize their replication and spread. Here, we demonstrate that PRRSV infection causes host translation suppression, which is strongly dependent on viral replication. By screening PRRSV-encoded nonstructural proteins (nsps), we found that nsp2 participates in the induction of host translation shutoff and that its transmembrane (TM) domain is required for this process. Nsp2-induced translation suppression is independent of protein degradation pathways and the phosphorylation of eukaryotic initiation factor 2aalpha; (eIF2aalpha;). However, the overexpression of nsp2 or its TM domain significantly attenuated the mammalian target of rapamycin (mTOR) signaling pathway, an alternative pathway for modulating host gene expression. PRRSV infection also attenuated the mTOR signaling pathway, and PRRSV-induced host translation shutoff could be partly reversed when the attenuated mTOR phosphorylation was reactivated by an activator of the mTOR pathway. PRRSV infection still negatively regulated the host translation when the effects of eIF2aalpha; phosphorylation were completely reversed. Taken together, our results demonstrate that PRRSV infection induces host translation shutoff and that nsp2 is associated with this process. Both eIF2aalpha; phosphorylation and the attenuation of the mTOR signaling pathway contribute to PRRSV-induced host translation arrest.

    IMPORTANCE Viruses are obligate parasites, and the production of progeny viruses relies strictly on the host translation machinery. Therefore, the efficient modulation of host mRNA translation benefits viral replication, spread, and evolution. In this study, we provide evidence that porcine reproductive and respiratory syndrome virus (PRRSV) infection induces host translation shutoff and that the viral nonstructural protein nsp2 is associated with this process. Many viruses induce host translation shutoff by phosphorylating eukaryotic initiation factor 2aalpha; (eIF2aalpha;). However, PRRSV nsp2 does not induce eIF2aalpha; phosphorylation but attenuates the mTOR signaling pathway, another pathway regulating the host cell translational machinery. We also found that PRRSV-induced host translation shutoff was partly reversed by dephosphorylating eIF2aalpha; or reactivating the mTOR pathway, indicating that PRRSV infection induces both eIF2aalpha;-phosphorylation-dependent and -independent host translation shutoff.

  • Directed evolution of an influenza reporter virus to restore replication and virulence and enhance non-invasive bioluminescence imaging in mice [PublishAheadOfPrint]

  • Reporter viruses provide a powerful tool to study infection, yet incorporating a non-essential gene often results in virus attenuation and genetic instability. Here, we used directed evolution of a luciferase-expressing pandemic H1N1 (pH1N1) 2009 influenza A virus in mice to restore replication kinetics and virulence, increase the bioluminescence signal, and maintain reporter gene expression. An unadapted pH1N1 virus with NanoLuc luciferase inserted into the 5' end of the PA gene segment grew to titers 10-fold less than wild-type in MDCK cells and in DBA/2 mice and was less virulent. For twelve rounds, we propagated DBA/2 lung samples having the highest ratios of bioluminescence-to-titer. Every three rounds, we compared in vivo replication, weight loss, mortality, and bioluminescence. Mouse-adapted virus after 9 rounds (MA-9) had the highest relative bioluminescence signal and had wild-type-like fitness and virulence in DBA/2 mice. Using reverse genetics, we discovered fitness was restored in virus rPB2-MA9/PA-D479N by a combination of PA-D479N and PB2-E158G amino-acid mutations and PB2 non-coding mutations C1161T and C1977T. rPB2-MA9/PA-D479N has increased mRNA transcription, which helps restore wild-type-like phenotypes in DBA/2 and BALB/c mice. Overall, the results demonstrate directed evolution that maximizes foreign-gene expression while maintaining genetic stability is an effective method to restore wild-type-like in vivo fitness of a reporter virus. Virus rPB2-MA9/PA-D479N is expected to be a useful tool for noninvasive imaging of pH1N1 influenza virus infection and clearance while analyzing virus-host interactions and developing new therapeutics and vaccines.

    IMPORTANCE Influenza viruses contribute to 290,000-650,000 deaths globally each year. Infection is studied in mice to learn how it causes sickness and to develop new drugs and vaccines. During experiments, scientists have needed to euthanize groups of mice at different times to measure the amount of infectious virus in mouse tissues. By inserting a foreign gene into the virus that causes infected cells to light up, scientists could now see infection spread in living mice. Unfortunately, adding an extra gene not needed by the virus slowed it down and made it weaker. Here, we used a new strategy to restore the fitness and lethality of an influenza reporter virus; we adapted it to mouse lungs and selected for variants that had the greatest light signal. The adapted virus can be used to study influenza infection, immunology, and disease in living mice. The strategy can also be used to adapt other viruses.

  • Lnc-ISG20 inhibits influenza A virus replication by enhancing ISG20 expression [PublishAheadOfPrint]

  • LncRNAs are involved in many aspects of cellular processes, including antiviral immune response. To identify influenza A virus (IAV)-related lncRNAs, we performed RNA-deep sequencing to compare the profiles of lncRNAs in A549 and 293T cells with or without IAV infection. We identified an IAV-upregulated lncRNA named lnc-ISG20 because it shares most of its sequence with ISG20. We found that lnc-ISG20 is an interferon-stimulated gene similar to ISG20. Overexpression of lnc-ISG20 inhibited IAV replication, while lnc-ISG20 knockdown favored viral replication, suggesting that ln-ISG20 is inhibitory to IAV replication. Further study indicated that overexpression of lnc-ISG20 enhances ISG20 protein levels, while knockdown of lnc-ISG20 reduced ISG20 protein levels in A549 cells induced with polyI:C and Sendai virus. We demonstrated that lnc-ISG20 inhibits IAV replication in an ISG20-dependent manner. As lnc-ISG20 did not affect the mRNA level of ISG20, we postulated that lnc-ISG20 may function as competing endogenous RNA to ISG20 to enhance its translation. Indeed, we identified that miR-326 is a mutual miRNA for both ISG20 and lnc-ISG20 that targets the 3'UTR of ISG20 mRNA to inhibit its translation. We confirmed that lnc-ISG20 can bind miR-326, which in turn decreased the amount of miR-326 bound to ISG20 mRNA. In conclusion, we identified that the IAV-upregulated lnc-ISG20 is a novel interferon-stimulating gene that elicits its inhibitory effect on IAV replication by enhancing ISG20 expression. We demonstrated that lnc-ISG20 functions as a ceRNA to bind miR-326 to reduce its inhibition on ISG20 translation. Our results revealed the mechanism by which lnc-ISG20 inhibits IAV replication.

    Importance

    The replication of Influenza A virus is regulated by host factors. However, the mechanisms by which lncRNAs regulate IAV infection are not well understood. We identified that lnc-ISG20 is up-regulated during IAV infection and is also an interferon-stimulated gene. We demonstrated that lnc-ISG20 can enhance ISG20 expression, which in turn inhibits IAV replication. Our studies indicate that lnc-ISG20 functions as a competing endogenous RNA that binds miR-326 and reduces its inhibitory effect on ISG20. Taken together, our findings reveal the mechanistic details of lnc-ISG20 negatively regulating IAV replication. These findings indicate that lnc-ISG20 plays an important role during the host antiviral immune response.

  • Human Asymptomatic Epitope Peptide/CXCL10-Based Prime/Pull Vaccine Induces Herpes Simplex Virus-Specific Gamma Interferon-Positive CD107+ CD8+ T Cells That Infiltrate the Cornea and Trigeminal Ganglia of Humanized HLA Transgenic Rabbits and Protect against Ocular Herpes Challenge [PublishAheadOfPrint]

  • Herpes simplex virus 1 (HSV-1) is a prevalent human pathogen that infects the cornea causing potentially blinding herpetic disease. A clinical herpes vaccine is still lacking. In the present study, a novel prime/pull vaccine was tested in Human Leukocyte Antigen- (HLA-) transgenic rabbit model of ocular herpes (HLA Tg rabbit). Three asymptomatic (ASYMP) peptide epitopes were selected from the HSV-1 membrane glycoprotein C (UL44400-408), the DNA replication binding helicase (UL9196-204), and the tegument protein (UL25572-580), all preferentially recognized by CD8+ T cells from "naturally protected" HSV-1-seropositive healthy ASYMP individuals (who never had recurrent corneal herpetic disease). HLA Tg rabbits were immunized with a mixture of these three ASYMP CD8+ T cell peptide epitopes (UL44400-408, UL9196-204 and UL25572-580), delivered subcutaneously with CpG2007 adjuvant (prime). Fifteen days later, half of the rabbits received a topical ocular treatment with a recombinant neurotropic AAV8 vector, expressing the T cell-attracting CXCL10 chemokine (pull). The frequency, function of HSV-specific CD8+ T cells induced by the prime/pull vaccine were assessed in peripheral blood, cornea, and trigeminal ganglia (TG). Compared to peptides alone, the peptides/CXCL10 prime/pull vaccine generated frequent polyfunctional gamma interferon-positive (IFN-+) CD107+ CD8+ T cells that infiltrated both the cornea and TG. CD8+ T cells mobilization into cornea and TG of prime/pull- vaccinated rabbits was associated with a significant reduction in corneal herpes infection and disease following an ocular HSV-1 challenge (McKrae). These findings draw attention to the novel prime/pull vaccine strategy to mobilize anti-viral CD8+ T cells into tissues protecting them against herpes infection and disease.

    IMPORTANCE

    There is an urgent need for a vaccine against widespread herpes simplex virus infections. The present study demonstrates that immunization of HLA transgenic rabbits with a peptide/CXCL10 prime/pull vaccine triggered mobilization of HSV-specific CD8+ T cells locally in the cornea and TG, the sites of acute and latent herpes infections. Mobilization of antiviral CD8+ T cells into cornea and TG of rabbits that received the prime/pull vaccine was associated with protection against ocular herpes infection and disease following an ocular HSV-1 challenge. These results highlight the importance of the prime/pull vaccine strategy to bolster the number and function of protective CD8+ T cells within infected tissues.

  • Efficient genome engineering of a virulent Klebsiella bacteriophage using CRISPR-Cas9 [PublishAheadOfPrint]

  • Klebsiella pneumoniae is one of the most common nosocomial opportunistic pathogens usually with multiple drug-resistance. Phage therapy, a potential new therapeutics to replace or supplement antibiotics, has attracted much attention. However, very few Klebsiella phages have been well-characterized as the lack of efficient genome editing tools. Here, Cas9 from Streptococcus pyogenes and a single guide RNA (sgRNA) were used to modify a virulent Klebsiella bacteriophage phiKpS2. We firstly evaluated the distribution of sgRNA activity in phages and proved that it's largely inconsistent with the predicted activity from current models trained on eukaryotic cell datasets. A simple CRISPR-based phage genome editing procedure was developed based on the discovery that homologous arms as short as 30-60 bp was sufficient to introduce point mutation, gene deletion and swap. We also demonstrated that weak sgRNAs could be used for precise phage genome editing but failed to select random recombinants, possibly because inefficient cleavage can be tolerated through continuous repair by homologous recombination with the uncut genomes. Small frameshift deletion was proved to be an efficient way to evaluate the essentiality of phage genes. By using the above strategies, a putative promoter and nine genes of phiKpS2 were successfully deleted. Interestingly, the holin gene can be deleted with little effect on phiKpS2 infection, but the reason is not yet clear. This study established an efficient, time-saving, and cost-effective procedure for phage genome editing, which is expected to significantly promote the development of bacteriophage therapy.

    IMPORTANCE

    In the present study, we have addressed an efficient, time-saving and cost-effective CRISPR-based phage genome editing of Klebsiella phage, which has the potential to significantly expand our knowledge of phage-host interactions and to promote the applications of phage therapy. The distribution of sgRNA activity was first evaluated in phages. Short homologous arms were proved enough to introduce point mutation, small frameshift deletion, gene deletion and swap into phages, and weak sgRNAs were proved useful for precise phage genome editing but failed to select random recombinants, which all make the CRISPR-based phage genome editing easier to use.

  • Pharmacologic depletion of microglia increases viral load in the brain and enhances mortality in murine models of Flavivirus-induced encephalitis [PublishAheadOfPrint]

  • Flaviviruses account for most arthropod-borne cases of human encephalitis in the world. However the exact mechanisms of injury to the central nervous system (CNS) during Flavivirus infections remain poorly understood. Microglia are the resident immune cell of the CNS and are important for multiple functions, including control of viral pathogenesis. Utilizing a pharmacologic method of microglia depletion (PLX5622, Plexxikon Inc, an inhibitor of colony stimulating factor 1 receptor) we sought to determine the role of microglia in Flaviviral pathogenesis. Depletion of microglia resulted in increased mortality and viral titer in the brain following infection with either West Nile virus (WNV) or Japanese encephalitis virus (JEV). Interestingly, microglial depletion did not prevent virus-induced increases in the expression of relevant cytokines and chemokines at the mRNA level. In fact, the expression of several pro-inflammatory genes was increased in virus-infected, microglial-depleted mice compared to virus-infected, untreated controls. In contrast, and as expected, expression of the macrophage marker triggering receptor expressed on myeloid cells 2 (TREM-2) was decreased in virus-infected, PLX5622-treated mice, compared to virus-infected, controls.

    Importance

    As CNS invasion by Flaviviruses is a rare but life-threatening event it is critical to understand how brain-resident immune cells elicit protection or injury during disease progression. Microglia have been shown to be important in viral clearance, but may also contribute to CNS injury as part of the neuroinflammatory process. By utilizing a microglial depletion model we can begin to parse out the exact roles of microglia during Flaviviral pathogenesis with hopes of understanding specific mechanisms as potential targets for therapeutics.

  • LncITPRIP-1 Positively Regulates Innate Immune Response through Promoting Oligomerization and Activation of MDA5 [PublishAheadOfPrint]

  • Emerging evidence indicates that long non-coding RNAs (lncRNAs) regulate various biological processes, especially innate and adaptive immunity. However, the relationship between lncRNAs and interferon (IFN) pathway remains largely unknown. Here, we report that lncRNA ITPRIP-1 (lncITPRIP-1) is involved in viral infection and plays a crucial role in virus-triggered IFN signaling pathway through targeting MDA5. LncITPRIP-1 can be induced by viral infection, which is not entirely dependent on IFN signal. Besides, there is no coding potential found in lncITPRIP-1 transcript. LncITPRIP-1 binds to the C-terminal of MDA5 and it possesses the ability to boost oligomerization of both full length and 2CARD domains of MDA5 in a K63-linked-polyubiquitination-independent manner. Amazingly, we also find that MDA5 could suppress HCV replication independent of IFN signaling through its C-terminal deficient domain bound to viral RNA, in which lncITPRIP-1 plays as an assistant. In addition, the expression of lncITPRIP-1 is highly consistent with MDA5 expression, indicating that lncITPRIP-1 may function as a cofactor of MDA5. All the data suggest that lncITPRIP-1 enhances innate immune response to viral infection through promoting oligomerization and activation of MDA5. Our study discovers the first lncRNA ITPRIP-1 involved in MDA5 activation.

    SIGNIFICANCE Hepatitis C virus infection causes a global health issue and there is still no available vaccine, which makes it urgent to reveal the underlying mechanisms of HCV and host factors. Although RIG-I has been recognized as the leading cytoplasmic sensor against HCV for a long time, recent findings of MDA5 regulating IFN response to HCV have emerged. Our work validates the significant role of MDA5 in IFN signaling and HCV infection, and proposes the first lncRNA inhibiting HCV replication by promoting the activation of MDA5 and mediating the association between MDA5 and HCV RNA, which may shed light on MDA5 function study and the treatment for hepatitis C patients. Our suggested model of how lncITPRIP-1 can orchestrate signal transduction for IFN production illustrates the essential role of lncRNAs in virus elimination.

  • A single mutation at position 156 in envelope protein of Tembusu virus is responsible for virus tissue tropism and transmissibility in ducks [PublishAheadOfPrint]

  • Duck Tembusu virus (TMUV), like other mosquito-borne flaviviruses such as Japanese encephalitis virus (JEV), West Nile virus (WNV) and Bagaza virus (BAGV), is able to transmit vector-independently. To date, why these flaviviruses can be transmitted without mosquito vectors remains poorly understood. To explore the key molecular basis of flavivirus transmissibility, we compared virus replication and transmissibility of an early and a recent TMUV in ducks. The recent TMUV strain FX2010 replicated systemically and transmitted efficiently in ducks while the replication of early strain MM1775 was limited and did not transmit among ducks. The TMUV envelope protein and its domain I were responsible for tissue tropism and transmissibility. The mutation S156P in the domain I resulted in disruption of N-linked glycosylation at amino acid 154 of the E protein and changed the conformation of "150 loop" of the E protein, which reduced virus replication in lungs and abrogated transmission in ducks. These data indicate that the 156S in the envelope protein is critical for TMUV tissue tropism and transmissibility in ducks in the absence of mosquitos. Our findings provide novel insights on understanding TMUV transmission among ducks.

    Author summary

    Tembusu virus, similar to other mosquito-borne flaviviruses such as WNV, JEV, and BAGV, can be transmitted without the presence of mosquito vectors. We demonstrate that the envelope protein of TMUV and its amino acid (S) at position 156 is responsible for tissue tropism and transmission in ducks. The mutation S156P results in disruption of N-linked glycosylation at amino acid 154 of the E protein and changes the conformation of "150 loop" of the E protein, which induces limited virus replication in lungs and abrogates transmission between ducks. Our findings provide new knowledge about TMUV transmission among ducks.

  • Expression of a structural protein of the mycovirus FgV-ch9 negatively affects the transcript level of a novel symptom alleviation factor and causes virus-infection like symptoms in Fusarium graminearum [PublishAheadOfPrint]

  • Infections of fungi by mycoviruses are often symptomless but sometimes also fatal as they perturb sporulation, growth, and, if applicable, virulence of the fungal host. Hypovirulence-inducing mycoviruses, therefore, represent a powerful mean to defeat fungal epidemics on crop plants. Infection with Fusarium graminearum virus China 9 (FgV-ch9), a dsRNA chrysovirus-like mycovirus, debilitates Fusarium graminearum, the causal agent of Fusarium Head Blight. In search for potential symptom alleviation or aggravation factors in F. graminearum, we consecutively infected a custom-made F. graminearum mutant collection with FgV-ch9 and found a mutant with constantly elevated expression of a gene coding for a putative mRNA-binding protein that did not show any disease symptoms despite harboring high amounts of virus. Deletion of this gene, named virus response 1 (vr1), resulted in phenotypes identical to those observed in the virus-infected wild type with respect to growth, reproduction, and virulence. Similarly, the viral structural protein coded on segment 3 (P3) caused virus-infection like symptoms when expressed in the wild-type but not in the vr1-overexpression mutant. Gene expression analysis revealed a drastic downregulation of vr1 in the presence of virus and in mutants expressing P3. We conclude that symptom development and severity correlate with gene expression levels of vr1. This was confirmed by comparative transcriptome analysis showing a large transcriptional overlap between the virus-infected wild type, the vr1 deletion mutant and the P3-expressing mutant. Hence, vr1 represents a fundamental host factor for the expression of virus-related symptoms and helps to understand the underlying mechanism of hypovirulence.

    IMPORTANCE Virus infections of phytopathogenic fungi occasionally impair growth, reproduction, and virulence, a phenomenon referred to as hypovirulence. Hypovirulence-inducing mycoviruses, therefore, represent a powerful mean to defeat fungal epidemics on crop plants. However, the poor understanding of the molecular basis of hypovirulence induction limits their application. Using the devastating fungal pathogen on cereal crops, Fusarium graminearum, we identified an mRNA binding protein (named virus response 1, vr1) which is involved in symptom expression. Downregulation of vr1 in the virus-infected fungus and vr1 deletion evoke virus-infection like symptoms while constitutive expression overrules the cytopathic effects of the virus infection. Intriguingly, the presence of a specific viral structural protein is sufficient to trigger the fungal response, i.e. vr1 downregulation, and symptom development similar to virus infection. The advancements in understanding fungal infection and response may aid biological pest control approaches using mycoviruses or viral proteins to prevent future Fusarium epidemics.

  • Secretome Screening Reveals Fibroblast Growth Factors as Novel Inhibitors of Viral Replication [PublishAheadOfPrint]

  • Cellular antiviral programs can efficiently inhibit viral infection. These programs are often initiated through signaling cascades induced by secreted proteins such as type I interferons, IL-6 or TNF-aalpha;. Here, we generated an arrayed library of 756 human secreted proteins to perform a secretome screen focused on the discovery of novel modulators of viral entry and/or replication. The individual secreted proteins were tested for their capacity to inhibit infection by two replication-competent recombinant vesicular stomatitis viruses (VSV) with distinct glycoproteins utilizing different entry pathways. Fibroblast growth factor 16 (FGF16) was identified and confirmed as the most prominent novel inhibitor of both VSVs and therefore of viral replication and not entry. Importantly, an antiviral interferon signature was completely absent in FGF16 treated cells. Nevertheless, the antiviral effect of FGF16 is broad as it was evident on multiple cell types and also on infection of Coxsackievirus. In addition, other members of the FGF family also inhibited viral infection. Thus, our unbiased secretome screen revealed a novel protein family capable of inducing a cellular antiviral state. This previously unappreciated role of the FGF family may have implications for the development of new antivirals and the efficacy of oncolytic virus therapy.

    Importance

    Viruses infect human cells in order to replicate, while human cells aim to resist infection. Several cellular antiviral programs have therefore evolved to resist infection. Knowledge of these programs is essential for the design of antiviral therapeutics in the future. The induction of antiviral programs is often initiated by secreted proteins such as interferons. We hypothesized that other secreted proteins may also promote resistance to viral infection. Thus we tested 756 human secreted proteins for their capacity to inhibit two pseudotypes of vesicular stomatitis virus (VSV). In this first secretome screen on viral infection we identified fibroblast growth factor 16 (FGF16) as a novel antiviral against multiple VSV pseudotypes as well as Coxsackievirus. Subsequent testing of other FGF family members revealed that FGF signaling generally inhibits viral infection. This finding may lead to the development of new antivirals and may also be applicable to enhance oncolytic virus therapy.

  • The Role of Microtubules and Microtubule Associated Proteins in HIV-1 Infection [PublishAheadOfPrint]

  • Recent studies show HIV-1 can utilize microtubules and their associated proteins to complete key post fusion steps during infection. This includes associating with both dynein and kinesin motors, as well as proteins, which enhance infection by altering microtubule dynamics during infection. In this article, we will discuss these findings on how dynein and kinesin motors, as well as other microtubule associated proteins, influence HIV-1 trafficking, viral core uncoating and nuclear import of the viral RNP.

  • Modulation of Cellular CpG DNA Methylation by Kaposi's Sarcoma Associated Herpesvirus [PublishAheadOfPrint]

  • Kaposi's sarcoma associated herpesvirus (KSHV, HHV-8) is a gamma herpesvirus associated with several human malignancies. DNA methylation at CpG dinucleotides is an epigenetic mark dysregulated in many cancer types, and in KSHV infected cells. Several previous studies have analyzed in detail the CpG methylation of the KSHV episomal genomes, but little is known about the impact of KSHV on the human genome. Our knowledge of cellular CpG methylation in the context of KSHV infection is currently limited to four hyper-methylated human gene promoters. Therefore, we undertook a comprehensive CpG methylation analysis of the human methylome in KSHV-infected cells and KSHV-associated primary effusion lymphoma (PEL). We performed Infinium HumanMethylation 450K and EPIC BeadChip arrays and identified panels of hyper and hypo-methylated cellular promoters in KSHV infected cells. We combined our genome wide methylation analysis with RNA-sequencing (RNA-seq) to add functional outcomes to the virally induced methylation changes. We were able to correlate many downregulated genes with promoter hyper-methylation, and upregulated genes with hypo-methylation. In addition, we show that treating the cells with a de-methylating agent leads to re-expression of these downregulated genes, indicating that indeed DNA methylation plays a role in the repression of these human genes. Comparison between de-novo infection and PEL, suggests that the virus induces initial hyper-methylation followed with a slow increase in genome wide hypo-methylation. This study extends our understanding of the relationship between epigenetic changes induced by KSHV infection and tumorigenesis.

    Importance

    In cancer cells, certain promoters become aberrantly methylated, contributing to the phenotype of the tumor. KSHV infection seems to modify cellular CpG methylation, but only a handful of methylated promoters have been identified in KSHV infected cells. Here we investigated the CpG methylation of the human genome in KSHV-associated primary effusion lymphoma (PEL) and KSHV infected cells. We have identified many hyper and hypo-methylated gene promoters and correlated their methylation with cellular gene expression. These differentially methylated cellular promoters can distinguish KSHV-positive from uninfected cells, and may serve the foundation for the use of these differentially methylated regions as potential biomarkers for KSHV associated malignancies. Drugs that reverse these cancerous methylation patterns have the potential to inhibit tumor growth. Here we show that treating PEL cells with a de-methylating drug (5-aza-2'-deoxycytidine) led to inhibition of cell growth, raising the possibility of testing this drug for the treatment of PEL.

  • The murine polyomavirus microRNA locus is required to promote viruria during the acute phase of infection [PublishAheadOfPrint]

  • Polyomaviruses (PyVs) can cause serious disease in immunosuppressed hosts. Several pathogenic PyVs encode microRNAs (miRNAs), small RNAs that regulate gene expression via RNA silencing. Despite recent advances in understanding the activities of PyV miRNAs, the biological functions of PyV miRNAs during in vivo infections are mostly unknown. Studies presented here use murine polyomavirus (MuPyV) as a model to assess the roles of the PyV miRNAs in a natural host. This analysis reveals that a MuPyV mutant that is unable to express miRNAs has enhanced viral DNA loads in select tissues at late times after infection. This is consistent with the PyV miRNAs functioning to reduce viral replication during the persistent phase of infection in a natural host. Additionally, the MuPyV miRNA locus promotes viruria during the acute phase of infection as evidenced by a defect in shedding during infection with the miRNA mutant virus. The viruria defect of the miRNA mutant virus could be rescued by infecting Rag2-/- mice. These findings implicate the miRNA locus as functioning in both the persistent and acute phases of infection and suggest a role for MuPyV miRNA in evading the adaptive immune response.

    IMPORTANCE MicroRNAs are expressed by diverse viruses, but for only a few is there any understanding of their in vivo function. PyVs can cause serious disease in immunocompromised hosts. Therefore, increased knowledge of how these viruses interact with the immune response is of clinical relevance. Here we show a novel activity for a viral miRNA locus in promoting virus shedding. This work indicates that in addition to any role for the PyV miRNA locus in long-term persistence, that it also has biological activity during the acute phase. As this mutant phenotype is alleviated by infection of mice lacking an adaptive immune response, our work also connects the in vivo activity of the PyV miRNA locus to the immune response. Given that PyV-associated disease is associated with alterations in the immune response, our findings help to better understand how the balance between PyVs and the immune response becomes altered in pathogenic states.

  • CD8 T CELL RESPONSES TO AN IMMUNODOMINANT EPITOPE WITHIN THE NON-STRUCTURAL PROTEIN NS1 PROVIDES WIDE IMMUNOPROTECTION AGAINST BLUETONGUE VIRUS IN IFNAR(-/-) MICE [PublishAheadOfPrint]

  • The development of vaccines against Bluetongue, a prevalent livestock disease, has been focused on surface antigens that induce strong neutralizing antibody responses. Because their antigenic variability, these vaccines are usually serotype restricted. We now show that a single highly conserved non-structural protein, NS1, expressed in a modified vaccinia Ankara virus (MVA) vector can provide multiserotype protection in IFNAR(-/-) 129 mice against Bluetongue virus that is largely dependent on CD8 T cell responses. We found that the protective antigenic capacity of NS1 resides within the N-terminus of the protein and is provided in the absence of neutralizing antibodies. The protective CD8 T cell response requires the presence of a specific peptide within the N-terminus of NS1, since its deletion ablates the efficacy of the vaccine formulation. These data reveal the importance of the non-structural protein NS1 in CD8 T cell-mediated protection against multiple BTV serotypes when vectorized as a recombinant MVA vaccine.

    IMPORTANCE Conventional vaccines have controlled or limited BTV expansion in the past but they cannot address the need for cross-protection among serotypes and do not allow to distinguish between infected and vaccinated animals (DIVA strategy). There is a need to develop universal vaccines that induce effective protection against multiple BTV serotypes. In this work we have shown the importance of the non-structural protein NS1, conserved among all the BTV serotypes, in CD8 T cell-mediated protection against multiple BTV serotypes when vectorized as a recombinant MVA vaccine.

  • Kaposi's sarcoma-associated herpesvirus ORF68 is a DNA binding protein required for viral genome cleavage and packaging [PublishAheadOfPrint]

  • Herpesviral DNA packaging into nascent capsids requires multiple conserved viral proteins that coordinate genome encapsidation. Here, we investigated the role of the ORF68 protein of Kaposi's sarcoma-associated herpesvirus (KSHV), a protein required for viral DNA encapsidation whose function remains largely unresolved across the herpesviridae. We found that KSHV ORF68 is expressed with early kinetics and localizes predominantly to viral replication compartments, although it is dispensable for viral DNA replication and gene expression. However, in agreement with its proposed role in viral DNA packaging, KSHV-infected cells lacking ORF68 failed to cleave viral DNA concatemers, accumulated exclusively immature B-capsids, and released no infectious progeny virions. ORF68 has no predicted domains aside from a series of putative zinc finger motifs. However, in vitro biochemical analyses of purified ORF68 protein revealed that it robustly binds DNA and is associated with nuclease activity. These activities provide new insights into the role of KSHV ORF68 in viral genome encapsidation.

    IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma and several B-cell cancers, causing significant morbidity and mortality in immunocompromised individuals. A critical step in the production of infectious viral progeny is the packaging of the newly replicated viral DNA genome into the capsid, which involves coordination between at least seven herpesviral proteins. While the majority of these packaging factors have been well studied in related herpesviruses, the role of the KSHV ORF68 protein and its homologs remains unresolved. Here, using a KSHV mutant lacking ORF68, we confirm its requirement for viral DNA processing and packaging in infected cells. Furthermore, we show that the purified ORF68 protein directly binds DNA and is associated with a metal-dependent cleavage activity on double stranded DNA in vitro. These activities suggest a novel role for ORF68 in herpesviral genome processing and encapsidation.

  • In vivo Knock-down of the HSV-1 Latency-Associated Transcript Reduces Reactivation from Latency [PublishAheadOfPrint]

  • During Herpes Simplex Virus (HSV) latency, most viral genes are silenced with the exception of one region of the genome encoding the latency-associated transcript (LAT). This long non-coding RNA was originally described as having a role in enhancing HSV-1 reactivation. However, subsequent evidence showing that the LAT blocked apoptosis and promoted efficient establishment of latency suggested that its effects on reactivation were secondary to establishment. Here, we utilize an Adeno-associated Virus (AAV) vector to deliver a LAT-targeting hammerhead ribozyme to HSV-1-infected neurons of rabbits after the establishment of HSV-1 latency. The rabbits were then induced to reactivate latent HSV-1. Using this model, we show that decreasing LAT levels in neurons following the establishment of latency reduced the ability of the virus to reactivate. This demonstrates that the HSV-1 LAT RNA has a role in reactivation that is independent of its function in establishment of latency. In addition these results suggest the potential of AAV vectors expressing LAT-targeting ribozymes as a potential therapy for recurrent HSV disease such as herpes stromal keratitis, a leading cause of infectious blindness.

    Importance Herpes Simplex Virus (HSV) establishes a life long infection and remains dormant (latent) in our nerve cells. Occasionally HSV reactivates to cause disease, with HSV-1 typically causing cold sores whereas HSV-2 is the most common cause of genital herpes. The details of how HSV reactivates are largely unknown. Most of HSV's genes are silent during latency with the exception of RNAs made from the latency-associated transcript (LAT) region. While viruses that make less LAT do not reactivate efficiently, these viruses also do not establish latency as efficiently. Here we deliver a ribozyme that can degrade the LAT to the nerve cells of latently infected rabbits using a gene therapy vector. We show that this treatment blocks reactivation in the majority of the rabbits. This work shows that the LAT RNA is important for reactivation and the suggests the potential of this treatment as a therapy for treating HSV infections.

  • HIV-1 Protease Evolvability is Affected by Synonymous Nucleotide Recoding [PublishAheadOfPrint]

  • One unexplored aspect of HIV-1 genetic architecture is how codon choice influences population diversity and evolvability. Here we compared the development of HIV-1 resistance to protease inhibitors (PIs) between wild-type (WT) virus and a synthetic virus (MAX) carrying a codon-pair re-engineered protease sequence including 38 (13%) synonymous mutations. WT and MAX viruses showed indistinguishable replication in MT-4 cells or PBMCs. Both viruses were subjected to serial passages in MT-4 cells with selective pressure from the PIs atazanavir (ATV) and darunavir (DRV). After 32 successive passages, both the WT and MAX viruses developed phenotypic resistance to PIs (IC50 14.6 pplusmn; 5.3 and 21.2 pplusmn; 9 nM for ATV, and 5. 9 pplusmn; 1.0 and 9.3 pplusmn; 1.9 for DRV, respectively). Ultra-deep sequence clonal analysis revealed that both viruses harbored previously described resistance mutations to ATV and DRV. However, the WT and MAX virus proteases showed different resistance variant repertoires, with the G16E and V77I substitutions observed only in WT, and the L33F, S37P, G48L, Q58E/K, and L89I substitutions detected only in MAX. Remarkably, G48L and L89I are rarely found in vivo in PI-treated patients. The MAX virus showed significantly higher nucleotide and amino acid diversity of the propagated viruses with and without PIs (P llt; 0.0001), suggesting higher selective pressure for change in this recoded virus. Our results indicate that HIV-1 protease position in sequence space delineates the evolution of its mutant spectra. Nevertheless, the investigated synonymously recoded variant showed mutational robustness and evolvability similar to the WT virus.

    IMPORTANCE

    Large-scale synonymous recoding of virus genomes is a new tool for exploring various aspects of virus biology. Synonymous virus genome recoding can be used to investigate how a virus's position in sequence space defines its mutant spectrum, evolutionary trajectory, and pathogenesis. In this study, we evaluated how synonymous recoding of the human immunodeficiency virus type 1 (HIV-1) protease impacts the development of protease inhibitor (PI) resistance. HIV-1 protease is a main target of current antiretroviral therapies. Our present results demonstrate that the wild-type (WT) virus and the virus with the recoded protease exhibited different patterns of resistance mutations after PI treatment. Nevertheless, the developed PI resistance phenotype was indistinguishable between the recoded virus and the WT virus, suggesting that the synonymously recoded protease HIV-1 and the WT protease virus were equally robust and evolvable.

  • A Single Locus Controls Interferon Gamma-Independent Antiretroviral Neutralizing Antibody Responses [PublishAheadOfPrint]

  • An essential step in the development of effective antiviral humoral responses is cytokine-triggered class switch recombination resulting in the production of antibodies of a specific isotype. Most viral and parasitic infections in mice induce predominantly IgG2a specific antibody responses that are stimulated by interferon- (IFN-). However, in some mice deficient in IFN-, class-switching to IgG2a antibodies is relatively unaffected, indicating that other signal(s) can be generated upon viral or parasitic infections that trigger this response. Here, we found that a single recessive locus, provisionally called IFN- independent IgG2a (igii), which confers the ability to produce IFN-independent production of IgG2a antibodies upon retroviral infection. The igii locus was mapped to Chromosome 9 and was found to function in the radiation resistant compartment. Thus, our data implicate non-hematopoietic cells in activation of antiviral antibody responses in the absence of IFN.

    IMPORTANCE Understanding the signals that stimulate antibody production and class-switch recombination to specific antibody isotypes is crucial for the development of novel vaccines and adjuvants. While an interferon--mediated switch to the IgG2a isotype upon viral infection in mice has been well established, this investigation reveals a non-canonical, interferon--independent pathway for anti-retroviral antibody production and IgG2a class-switch recombination that is controlled by a single recessive locus. Furthermore, this study indicates that the radiation-resistant compartment can direct antiviral antibody responses, suggesting that detection of infection by non-hematopoietic cells is involved is stimulating adaptive immunity.

  • Mamu-B*17+ rhesus macaques vaccinated with env, vif, and nef manifest early control of SIVmac239 replication [PublishAheadOfPrint]

  • Certain major histocompatibility complex class-I (MHC-I) alleles are associated with spontaneous control of viral replication in human immunodeficiency virus (HIV)-infected people and simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs). These cases of "elite" control of HIV/SIV replication are often immune-mediated, thereby providing a framework for studying anti-lentiviral immunity. Here we examined how vaccination impacts SIV replication in RMs expressing the MHC-I allele Mamu-B*17. Approximately 21% of Mamu-B*17+ and 50% of Mamu-B*08+ RMs control chronic phase viremia after SIVmac239 infection. Because CD8+ T-cells targeting Mamu-B*08-restricted SIV epitopes have been implicated in virologic suppression in Mamu-B*08+ RMs, we investigated whether this might also be true for Mamu-B*17+ RMs. Two groups of Mamu-B*17+ RMs were vaccinated with genes encoding Mamu-B*17-restricted epitopes in Vif and Nef. These genes were delivered by themselves (Group 1) or together with env (Group 2). Group 3 included MHC-I-matched RMs and served as the control group. Surprisingly, the Group 1 vaccine regimen had little effect on viral replication compared to Group 3, suggesting that, unlike Mamu-B*08+ RMs, pre-existing SIV-specific CD8+ T-cells alone do not facilitate long term virologic suppression in Mamu-B*17+ RMs. Remarkably, however, 5/8 Group 2 vaccinees controlled viremia to llt;15 viral RNA copies/mL soon after infection. No serological neutralizing activity against SIVmac239 was detected in Group 2, although vaccine-elicited gp140-binding antibodies correlated inversely with nadir viral loads. Collectively, these data shed new light into the unique mechanism of elite control in Mamu-B*17+ RMs and implicate vaccine-induced, non-neutralizing anti-Env antibodies in the containment of immunodeficiency virus infection.

    Importance

    A better understanding of the immune correlates of protection against HIV might facilitate the development of a prophylactic vaccine. Therefore, we investigated simian immunodeficiency virus (SIV) infection outcomes in rhesus macaques expressing the major histocompatibility complex class I allele Mamu-B*17. Approximately 21% of Mamu-B*17+ macaques spontaneously control chronic phase viremia after SIV infection, an effect that may involve CD8+ T-cells targeting Mamu-B*17-restricted SIV epitopes. We vaccinated Mamu-B*17+ macaques with genes encoding immunodominant epitopes in Vif and Nef alone (Group 1) or together with env (Group 2). Although neither vaccine regimen prevented SIV infection, 5/8 Group 2 vaccinees controlled viremia to below detection limits shortly after infection. This outcome, which was not observed in Group 1, was associated with vaccine-induced, non-neutralizing Env-binding antibodies. Together, these findings suggest a limited contribution of Vif- and Nef-specific CD8+ T-cells for virologic control in Mamu-B*17+ macaques and implicate anti-Env antibodies in containment of SIV infection.

  • The surface-exposed PA51-72-loop of the influenza A virus polymerase is required for viral genome replication [PublishAheadOfPrint]

  • The heterotrimeric influenza A virus RNA-dependent RNA polymerase complex, composed of PB1, PB2 and PA subunits, is responsible for transcribing and replicating the viral RNA genome. The N-terminal endonuclease domain of the PA subunit performs endonucleolytic cleavage of capped host RNAs to generate capped RNA primers for viral transcription. A surface-exposed flexible loop (PA51-72-loop) in the PA endonuclease domain has been shown to be dispensable for endonuclease activity. Interestingly, the PA51-72-loop was found to form different intramolecular interactions depending on the conformational arrangement of the polymerase. In this study, we show that a PA subunit lacking the PA51-72-loop assembles into a heterotrimeric polymerase with PB1 and PB2. We demonstrate that in a cellular context the PA51-72-loop is required for RNA replication but not transcription by the viral polymerase. In agreement, recombinant viral polymerase lacking the PA51-72-loop is able to carry out cap-dependent transcription but is inhibited in de novo replication initiation in vitro. Furthermore, vRNA synthesis is also restricted during ApG-primed extension, indicating that the PA51-72-loop is required not only for replication initiation but also for elongation on a cRNA template. We propose that the PA51-72-loop plays a role in the stabilisation of the replicase conformation of the polymerase. Together, these results further our understanding of influenza virus RNA genome replication in general and highlight a role of the PA endonuclease domain in polymerase function in particular.

    IMPORTANCE Influenza A viruses are a major global health threat, not only causing significant morbidity and mortality every year, but also having the potential to cause severe pandemic outbreaks like the 1918 influenza pandemic. The viral polymerase is a protein complex which is responsible for transcription and replication of the viral genome and therefore is an attractive target for antiviral drug development. For that purpose it is important to understand the mechanisms of how the virus replicates its genome and how the viral polymerase works on a molecular level. In this report, we characterise the role of the flexible surface-exposed PA51-72-loop in polymerase function and offer new insights into the replication mechanism of influenza A viruses.

  • HIV-1 gp41 Residues Modulate CD4-Induced Conformational Changes in the Envelope Glycoprotein and Evolution of a Relaxed Conformation of gp120 [PublishAheadOfPrint]

  • Entry of the human immunodeficiency virus type 1 (HIV-1) into host cells is mediated by conformational changes in the envelope glycoprotein (Env) that are triggered by Env binding to cellular CD4 and chemokine receptors. These conformational changes involve opening of the gp120 surface subunit, exposure of the fusion peptide in the gp41 transmembrane subunit, and refolding of the gp41 N- and C-terminal heptad-repeat regions (HR1 and HR2) first into an extended pre-hairpin intermediate, then into a compact 6-helix bundle (6HB) that facilitates fusion between viral and host cell membranes. Previously, we reported that Envs resistant to HR1 peptide fusion inhibitors acquired key resistance mutations in either HR1 or HR2 that increased 6HB stability. Here, we identify residues in HR1 that not only contribute to fusion inhibitor resistance and 6HB stability, but also to reduced reactivity to CD4-induced conformational changes that lead to 6HB formation. While all Envs show increased neutralization sensitivity to mimetic CD4 (mCD4), Envs with either the E560K or Q577R HR1 mutation reduced conformational reactivity to CD4 that resisted viral inactivation and triggering to the 6HB. Using a panel of monoclonal antibodies (mAbs) we further determined that Envs from both HR1 and HR2 resistance pathways exhibit a relaxed trimer conformation due to gp120 adaptive mutations in different regions of Env that segregate by resistance pathway. These findings highlight regions of cross-talk between gp120 and gp41 and identify HR1 residues that play important roles in regulating CD4-induced conformational changes in Env.

    IMPORTANCE Binding of the HIV envelope glycoprotein (Env) to cellular CD4 and chemokine receptors triggers conformational changes in Env that mediate virus entry, but premature triggering of Env conformational changes leads to virus inactivation. Currently, we have a limited understanding of the network of residues that regulate Env conformational changes. Here, we identify residues in the HR1 of gp41 that modulate conformational changes in response to gp120 binding to CD4 and show that the mutations in HR1 and HR2 that confer resistance to fusion inhibitors are associated with gp120 mutations in different regions of Env that confer a more open conformation. These findings contribute to our understanding of the regulation of Env conformational changes and efforts to design new entry inhibitors and stable Env vaccine immunogens.

  • Batf3 Dependent Dendritic Cells Promote Optimal CD8 T Cell Responses Against Respiratory Poxvirus Infection [PublishAheadOfPrint]

  • Respiratory infection with vaccinia virus (VacV) elicits robust CD8+ T cell responses that play an important role in host resistance. In the lung, VacV encounters multiple tissue resident APC populations, but which cell plays a dominant role in priming of virus-specific CD8+ effector T cell responses remains poorly defined. We used Batf3-/- mice to investigate the impact of CD103+ DC and CD8aalpha;+ DC deficiency on anti-VacV CD8+ T cell responses. We found that Batf3-/- mice were more susceptible to VacV infection, exhibiting profound weight loss, which correlated with impaired accumulation of IFN--producing CD8+ T cell in the lungs. This was largely due to defective priming since early in the response, antigen-specific CD8+ T cells in the draining lymph nodes of Batf3-/- mice expressed significantly reduced levels of Ki67, CD25 and T-bet. These results underscore a specific role for Batf3 dependent DCs in regulating priming and expansion of effector CD8+ T cells necessary for host resistance against acute respiratory VacV infection.

    IMPORTANCE During respiratory infection with vaccinia virus (VacV), a member of Poxviridae family, CD8+ T cells play important role in resolving the primary infection. Effector CD8+ T cells clear the virus by accumulating in the infected lungs in high numbers and secreting molecules such as IFN- that kill virally infected cells. However, precise cell-types that regulate the generation of effector CD8+ T cells in the lungs are not well defined. Dendritic cells (DCs) are heterogeneous population of immune cells that are recognized as key initiators and regulators of T cell mediated immunity. In this study, we reveal that a specific subset of DCs that are dependent on the transcription factor, Batf3, for their development, regulate the magnitude of CD8+ T cell effector responses in the lungs, thereby providing protection during pulmonary VacV infection.

  • Persistent Replication of a Chikungunya Virus Replicon in Human Cells is Associated with Presence of Stable Cytoplasmic Granules Containing Non-structural Protein 3 [PublishAheadOfPrint]

  • Chikungunya virus (CHIKV), a mosquito-borne human pathogen, causes a disabling disease characterized by severe joint pain that can persist for weeks, months or even years in patients. The non-structural protein 3 (nsP3) plays essential roles during acute infection, but little is known about the function of nsP3 during chronic disease. Here, we used sub-diffraction multi-color microscopy for spatial and temporal analysis of CHIKV nsP3 within human cells that persistently replicate replicon RNA. Round cytoplasmic granules of various sizes (i) contained nsP3 and stress granule assembly factors 1 and 2 (G3BP1/2); (ii) were next to double-stranded RNA foci and nsP1-positive structures; and (iii) were close to the nuclear membrane and the nuclear pore complex protein Nup98. Analysis of protein turnover and mobility by live-cell microscopy revealed that granules could persist for hours to days, accumulated newly synthesized protein, and moved through the cytoplasm at varying speeds. Granules also had a static internal architecture and were stable in cell lysates. Refractory cells that had cleared the non-cytotoxic replicon regained the ability to respond to arsenite-induced stress. In summary, nsP3 can form uniquely stable granular structures that persist long-term within the host cell. This continued presence of viral and cellular protein-complexes has implications for the study of the pathogenic consequences of lingering CHIKV infection and the development of strategies to mitigate the burden of chronic musculoskeletal disease brought about by a medically important arthropod-borne virus (arbovirus).

    Importance

    Chikungunya virus (CHIKV) is a re-emerging alphavirus transmitted by mosquitos and causes transient sickness but also chronic disease affecting muscles and joints. No approved vaccines or antivirals are available. Thus, a better understanding of the viral life cycle and the role of viral proteins can aid in identifying new therapeutic targets. Advances in microscopy and development of non-cytotoxic replicons (Utt, Das, Varjak, Lulla, Lulla, Merits, J Virol 89:3145-62, 2015, doi:10.1128/JVI.03213-14) have allowed researchers to study viral proteins within controlled laboratory environments over extended durations. Here we established human cells that stably replicate replicon RNA and express tagged non-structural protein 3. The ability to track nsP3 within the host cell and during persistent replication can benefit fundamental research efforts to better understand long-term consequences of the persistence of viral protein complexes and thereby provide the foundation for new therapeutic targets to control CHIKV infection and treat chronic disease symptoms.

  • Multiple functions of c-FLIP are essential for replication of hepatitis B virus [PublishAheadOfPrint]

  • Hepatitis B virus (HBV) infection is a leading cause of liver diseases; however, the host factors which facilitate the replication and persistence of HBV are largely unidentified. Cellular FLICE inhibitory protein (c-FLIP) is a typical anti-apoptotic protein. In many cases of liver diseases, the expression level of c-FLIP is altered, which affects the fate of hepatocytes. We previously found that c-FLIP and its cleaved form interact with HBV X protein (HBx), which is essential for HBV replication, and regulate diverse cellular signals. In this study, we investigated the role of endogenous c-FLIP in HBV replication and its underlying mechanisms.

    The knock-down of endogenous c-FLIP revealed that this protein regulates HBV replication through two different mechanisms: 1) c-FLIP interacts with HBx and protects it from ubiquitin-dependent degradation. The N-terminal DED1 domain of c-FLIP is required for HBx stabilization. 2) c-FLIP regulates the expression or stability of hepatocyte nuclear factors (HNFs), which have critical roles in HBV transcription and maintenance of hepatocytes. c-FLIP regulates the stability of HNFs through physical interactions. We verified our findings in three HBV infection systems: HepG2-NTCP cells, differentiated HepaRG cells, and primary human hepatocytes.

    In conclusion, our results identify c-FLIP as an essential factor in HBV replication. c-FLIP regulates viral replication through its multiple effects on viral and host proteins that have critical roles in HBV replication.

    IMPORTANCE Although the chronic hepatitis B virus (HBV) infection still poses a major health concern, the host factors which are required for the replication of HBV are largely uncharacterized. Our studies identify cellular FLICE inhibitory protein (c-FLIP) as an essential factor in HBV replication. We found the dual roles of c-FLIP in regulation of HBV replication: c-FLIP interacts with HBx and enhances its stability, and regulates the expression or stability of hepatocyte nuclear factors which are essential for transcription of HBV genome. Our findings may provide a new target for intervention in persistent HBV infection.

  • Ulk1 Governs Nerve Growth Factor/TrkA Signaling by Mediating Rab5 GTPase Activation in Porcine Hemagglutinating Encephalomyelitis Virus-Induced Neurodegenerative Disorders [PublishAheadOfPrint]

  • Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurovirulent coronavirus and causes neurological dysfunction in the central nervous system (CNS), but the neuropathological mechanism of PHEV remains poorly understood. We report that Unc51-like kinase 1 (Ulk1/Unc51.1) is a pivotal regulator of PHEV-induced neurological disorders and functions to selectively control the initiation of NGF/TrkA endosome trafficking. We first identified the function of Ulk1 by histopathologic evaluation in PHEV-infected mouse model where neuronal loss was accompanied by the suppression of Ulk1 expression. Morphogenesis assessments in the primary cortical neurons revealed that overexpression or mutations of Ulk1 modulated neurite outgrowth, collateral sprouting, and endosomal transport. Likewise, Ulk1 expression was decreased following PHEV infection, suggesting that there was a correlation between the neurodegeneration and functional Ulk1 deficiency. We then showed that Ulk1 forms a multiprotein complex with TrkA and the early endosome marker Rab5 and that Ulk1 defects lead to either blocking of NGF/TrkA endocytosis or premature degradation of pTrkA via constitutive activation of the Rab5 GTPase. Further investigation determined that the ectopic expression of Rab5 mutants induces aberrant endosomal accumulation of activated pTrkA, proving that targeting of Ulk1-TrkA-NGF signaling to the retrograde transport route in the neurodegenerative process that underlies PHEV infection is dependent on Rab5 GTPase activity. Therefore, we described a long-distance signaling mechanism of PHEV-driven deficits in neurons and suggested that such Ulk1 repression may result in limited NGF/TrkA retrograde signaling within activated Rab5 endosomes, explaining the progressive failure of neurite outgrowth and survival.

    IMPORTANCE Porcine hemagglutinating encephalomyelitis virus (PHEV) is neurotropic coronavirus and targets neurons in the nervous system for proliferation, frequently leaving behind grievous neurodegeneration. Structural plasticity disorders occur in the axons, dendrites, and dendritic spines of PHEV-infected neurons, and dysfunction of this neural process may contribute to neurologic pathologies, but the mechanisms remain undetermined. Further understanding of the neurological manifestations underlying PHEV infection in the CNS may provide insights into both neurodevelopmental and neurodegenerative diseases that may be necessarily conducive to targeted approaches for treatment. The significance of our research is in identifying an Ulk1-related neurodegenerative mechanism, focusing on the regulatory functions of Ulk1 in the transport of long-distance trophic signaling endosomes, thereby explaining the progressive failure of neurite outgrowth and survival associated with PHEV aggression. This is the first report to define a mechanistic link between alterations in signaling from endocytic pathways and the neuropathogenesis of PHEV-induced CNS disease.

  • Multiple incursions and recurrent epidemic fade-out of H3N2 canine influenza A virus in the United States [PublishAheadOfPrint]

  • Avian-origin H3N2 canine influenza virus (CIV) transferred to dogs in Asia around 2005, becoming enzootic throughout China and Korea before reaching the USA in early 2015. To understand the post-transfer evolution and epidemiology of this virus, particularly the cause of recent and ongoing increases in incidence in the USA, we performed an integrated analysis of whole-genome sequence data from 64 newly sequenced viruses and comprehensive surveillance data. This reveals that the circulation of H3N2 CIV within the USA is typified by recurrent epidemic burst-fadeout dynamics driven by multiple introductions of virus from Asia. Although all major viral lineages displayed similar rates of genomic sequence evolution, H3N2 CIV consistently exhibited proportionally more non-synonymous substitutions per site compared to avian reservoir viruses, indicative of a large-scale change in selection pressures. Despite these genotypic differences, we found no evidence of adaptive evolution or increased viral transmission, with epidemiological models indicating a basic reproductive number, R0, of between 1 and 1.5 across nearly all USA outbreaks, consistent with maintained, but heterogeneous circulation. We propose that CIV's mode of viral circulation may have resulted in evolutionary cul-de-sacs, in which there is little opportunity for the selection of the more transmissible H3N2 CIV phenotypes necessary to enable circulation through a general dog population characterized by widespread contact heterogeneity. CIV must therefore rely on metapopulations of high host density (notably animal shelters) within the greater dog population and reintroduction from other populations or face complete epidemic extinction.

    IMPORTANCE The relatively recent appearance of influenza A virus (IAV) epidemics in dogs expands our understanding of IAV host-range and ecology, providing useful and relevant models for understanding critical factors involved in viral emergence. Here, we integrate viral whole-genome sequence analysis and comprehensive surveillance data to examine the evolution of the emerging avian-origin H3N2 canine influenza virus (CIV), particularly the factors driving ongoing circulation and recent increase in incidence of the virus within the USA. Our results provide a detailed understanding of how H3N2 CIV achieves sustained circulation within the USA, despite widespread host contact heterogeneity and recurrent epidemic fade-out. Moreover, our findings suggest that the types and intensity of selection pressures an emerging virus experiences are highly dependent on host population structure and ecology, and may inhibit an emerging virus from acquiring sustained epidemic or pandemic circulation.

  • Divergent human origin influenza viruses detected in Australian swine populations [PublishAheadOfPrint]

  • Global swine populations infected with influenza A viruses pose a persistent pandemic risk. With the exception of a few countries, our understanding of the genetic diversity of swine influenza viruses is limited, hampering control measures and pandemic risk assessment. Here we report the genomic characteristics and evolutionary history of influenza A viruses isolated in Australia during 2012nndash;2016 from two geographically isolated swine populations in the states of Queensland and Western Australia. Phylogenetic analysis with an expansive human and swine influenza virus dataset comprising ggt;40,000 sequences sampled globally, revealed evidence of the pervasive introduction and long-term establishment of gene segments derived from several human influenza viruses of past seasons, including H1N1/1977, H1N1/1995, H3N2/1968, H3N2/2003, and H1N1pdm09, and a genotype that contained gene segments derived from the past three pandemics (1968, re-emerged 1977 and 2009). Of the six human-derived gene lineages only one comprising two viruses isolated in Queensland during 2012 was closely related to swine viruses detected from other regions, indicating a previously undetected circulation of Australian swine lineages for approximately 3nndash;44 years. Although the date of introduction of these lineages into Australian swine populations could not be accurately ascertained, we found evidence of sustained transmission of two lineages in swine during 2012nndash;2016. The continued detection of human-origin influenza virus lineages in swine over several decades with little or unpredictable antigenic drift indicates that isolated swine populations can act as llsquo;antigenic archivesrrsquo; of human influenza, raising the risk of re-emergence in humans when sufficient susceptible populations arise.

    IMPORTANCE We described the evolutionary origins and antigenic properties of influenza A viruses isolated from two separate Australian swine populations during 2012nndash;2016, showing that these viruses were distinct to each other and to those isolated from swine globally. Whole genome sequencing of virus isolates revealed a high genotypic diversity that had been generated exclusively through the introduction and establishment of human influenza viruses that circulated in past seasons. We detected six reassortants with gene segments derived from human H1N1/H1N1pdm09 and various human H3N2 viruses that circulated at various periods since 1968. We also found that these swine viruses were not related to swine viruses collected elsewhere indicating independent circulation. The detection of unique lineages and genotypes in Australia suggests that isolated swine populations that are sufficiently large can sustain influenza for extensive periods, we showed direct evidence of a sustained transmission for at least 4 years between 2012nndash;2016.

  • Arabidopsis Histone Reader EMSY-LIKE 1 Binds H3K36 and Suppresses Geminivirus Infection [PublishAheadOfPrint]

  • Histone post-translational modifications (PTMs) impart information that regulates chromatin structure and activity. Their effects are mediated by histone reader proteins that bind specific PTMs to modify chromatin and/or recruit appropriate effectors to alter the chromatin landscape. Despite their crucial juxtaposition between information and functional outcome, relatively few plant histone readers have been identified, and nothing is known about their impact on viral chromatin and pathogenesis. We used the geminivirus Cabbage leaf curl virus (CaLCuV) as a model to functionally characterize two recently identified reader proteins, EMSY-LIKE 1 and 3 (EML1 and EML3), which contain Tudor-like Agenet domains predictive of histone PTM binding function. Here, we show that mutant Arabidopsis plants exhibit contrasting hypersusceptible (eml1) and tolerant (eml3) responses to CaLCuV infection, and that EML1 deficiency correlates with RNA polymerase II (Pol II) enrichment on viral chromatin and upregulated viral gene expression. Consistent with reader activity, EML1 and EML3 associate with nucleosomes and with CaLCuV chromatin, suggesting a direct impact on pathogenesis. We also demonstrate that EML1 and EML3 bind peptides containing histone H3 lysine 36 (H3K36), a PTM usually associated with active gene expression. The interaction encompasses multiple H3K36 PTMs, including methylation and acetylation, suggesting nuanced regulation. Further, EML1 and EML3 associate with similar regions of viral chromatin, implying possible competition between the two readers. Regions of EML1 and EML3 association correlate with sites of trimethylated H3K36 (H3K36me3) enrichment, consistent with regulation of geminivirus chromatin by direct EML targeting.

    IMPORTANCE

    Histone PTMs convey information that regulates chromatin compaction and DNA accessibility. Histone reader proteins bind specific PTMs and translate their effects by modifying chromatin and/or by recruiting effectors that alter chromatin structure or activity. In this study, CaLCuV was used to characterize the activities of two Arabidopsis Agenet domain histone readers, EML1 and EML3. We show that eml1 mutants are hypersusceptible to CaLCuV, whereas eml3 plants are more tolerant of infection than wild type plants. We also demonstrate that EML1 and EML3 associate with histones and viral chromatin in planta, and that both proteins bind peptides containing H3K36, a PTM associated with active gene expression. Consistent with antiviral activity, EML1 suppresses CaLCuV gene expression and reduces Pol II access to viral chromatin. By linking EML1 and EML3 to pathogenesis, these studies have expanded our knowledge of histone reader proteins and uncovered an additional level of viral chromatin regulation.

  • Translational Model of Zika Virus Disease in Baboons [PublishAheadOfPrint]

  • Zika virus (ZIKV) is an emerging mosquito-borne flavivirus with devastating outcomes seen recently in the Americas due to the association of maternal ZIKV infection with fetal microcephaly and other fetal malformations not previously associated with flavivirus infections. Here, we have developed the olive baboon (Papio anubis) as a non-human primate (NHP) translational model for the study of ZIKV pathogenesis and associated disease outcomes to contrast and compare with humans and other major NHPs such as macaques. Following subcutaneous inoculation of adult male and non-pregnant female baboons, viremia was detected at 3 and 4 days post infection (dpi) with the concordant presentation of a visible rash and conjunctivitis similar to human ZIKV infection. Furthermore, virus was detected in the mucosa and cerebrospinal fluid. A robust ZIKV-specific IgM and IgG antibody response was also observed in all animals. These data show striking similarity between humans and the olive baboon following infection with ZIKV suggesting our model as a suitable translational NHP model to study ZIKV pathogenesis and potential therapeutics.

    IMPORTANCE ZIKV was first identified in 1947 in a sentinel rhesus monkey in Uganda and subsequently spread to Southeast Asia. Until 2007, only a small number of cases were reported, and ZIKV infection was relatively minor until the South Pacific and Brazilian outbreaks where more severe outcomes were reported. Here we present the baboon as a non-human primate model for contrast and comparison to other published animal models of ZIKV such as the mouse and macaque species. Baboons breed year around and are not currently a primary non-human primate species used in biomedical research marking them more readily available for studies other than Human Immunodeficiency virus studies, which many macaque species are designated for. This taken together with the similarities baboons have with humans, such as immunology, reproduction, genetics and size, makes the baboon an attractive NHP model for ZIKV studies in comparison to other non-human primates.

  • Insights into circovirus host range from the genomic fossil record [PublishAheadOfPrint]

  • A diverse range of DNA sequences derived from circoviruses (family Circoviridae) have been identified in samples obtained from humans and domestic animals, often in association with pathological conditions. In the majority of cases, however, little is known about the natural biology of the viruses from which these sequences are derived. Endogenous circoviral elements (CVe) are DNA sequences derived from circoviruses that occur in animal genomes and provide a useful source of information about circovirus-host relationships. In this study we screened genome assemblies of 675 animal species and identified numerous circovirus-related sequences, including the first examples of CVe derived from cycloviruses. We confirmed the presence of these CVe in the germline of the elongate twig ant (Pseudomyrmex gracilis), thereby establishing that cycloviruses infect insects. We examined the evolutionary relationships between CVe and contemporary circoviruses, showing that CVe from ants and mites group relatively closely with cycloviruses in phylogenies. Furthermore, the relatively random interspersal of CVe from insect genomes with cyclovirus sequences recovered from vertebrate samples, suggested that contamination might be an important consideration in studies reporting these viruses. Our study demonstrates how endogenous viral sequences can inform metagenomics-based virus discovery. In addition, it raises doubts about the role of cycloviruses as pathogens of humans and other vertebrates.

    Importance

    Advances DNA sequencing have dramatically increased the rate at which new viruses are being identified. However, the host species associations of most virus sequences identified in metagenomic samples are difficult to determine. Our analysis indicates that viruses proposed to infect vertebrates (in some cases being linked to human disease) may in fact be restricted to arthropod hosts. The detection of these sequences in vertebrate samples may reflect their widespread presence in the environment as viruses of parasitic arthropods.

  • TIM-1 Mediates Dystroglycan-Independent Entry of Lassa Virus [PublishAheadOfPrint]

  • Lassa virus (LASV) is an Old World arenavirus responsible for hundreds of thousands of infections in West Africa every year. LASV entry into a variety of cell types is mediated by interactions with glycosyltransferase LARGE-modified O-linked glycans present on the ubiquitous receptor, aalpha;-dystroglycan (aalpha;DG). Yet, cells lacking aalpha;DG are permissive to LASV infection, suggesting that alternative receptors exist. Previous studies demonstrate that phosphatidylserine (PtdSer)-binding receptors, Axl and Tyro3 along with C-type lectin receptors, mediate aalpha;DG-independent entry. Here, we demonstrate that another PtdSer receptor, TIM-1, mediates LASV glycoprotein (GP) pseudotyped virions entry into aalpha;DG knocked out HEK 293T and wild-type (WT) Vero which express aalpha;DG lacking appropriate glycosylation. To investigate the mechanism by which TIM-1 mediates enhancement of entry, we demonstrate that mutagenesis of the TIM-1 IgV domain PtdSer-binding pocket abrogated transduction. Further, the human TIM-1 IgV domain binding monoclonal antibody, ARD5, blocked transduction of pseudovirions bearing LASV GP in a dose-dependent manner. Finally, as we showed previously for other viruses that use TIM-1 for entry, a chimeric TIM-1 protein that substitutes the proline rich region (PRR) from murine leukemia virus envelope (Env) for the mucin-like domain served as a competent receptor. These studies provide evidence that, in the absence of a functional aalpha;DG, TIM-1 mediates entry of LASV pseudoviral particles through interactions of virions with the IgV PtdSer binding pocket of TIM-1.

    Importance

    PtdSer receptors, such as TIM-1, are emerging as critical entry factors for many enveloped viruses. Most recently, Hepatitis C virus and Zika virus have been added to a growing list. PtdSer receptors engage with enveloped viruses through binding of PtdSer embedded in the viral envelope, defining them as GP-independent receptors. This GP-independent entry mechanism should effectively mediate entry of all enveloped viruses, yet LASV GP pseudotyped viruses were previously found to be unresponsive to PtdSer receptor enhancement in HEK 293T cells. Here we demonstrate that LASV pseudovirions can utilize the PtdSer receptor TIM-1, but only in the absence of appropriately glycosylated aalpha;-dystroglycan (aalpha;DG), the high affinity cell surface receptor for LASV. Our studies shed light on LASV receptor utilization and explain why earlier studies performed in aalpha;-DG-expressing cells did not find that LASV pseudovirions utilize PtdSer receptors for virus uptake.

  • The dual tropism of noroviruses [PublishAheadOfPrint]

  • Noroviruses are highly prevalent enteric RNA viruses. Human noroviruses (HuNoVs) cause significant morbidity, mortality and economic losses worldwide. Infections also occur in other mammalian species, including mice. Despite the discovery of the first norovirus in 1972, the viral tropism has long remained an enigma. A long-held assumption was that these viruses infect intestinal epithelial cells. Recent data support a more complex cell tropism of epithelial and non-epithelial cell types.

  • Human cytomegalovirus productively replicates in vitro in undifferentiated oral epithelial cells. [PublishAheadOfPrint]

  • Human cytomegalovirus (HCMV) productive replication in vitro is most often studied in fibroblasts. In vivo, fibroblasts amplify viral titers, but transmission and pathogenesis requires the infection of other cell types, most notably epithelial cells. In vitro, the study of HCMV infection of epithelial cells has been almost exclusively restricted to ocular epithelial cells. Here we present oral epithelial cells with relevance for viral inter-host transmission as an in vitro model system to study HCMV infection. We discovered that HCMV productively replicates in normal oral keratinocytes (NOKs) and telomerase-immortalized gingival cells (hGETs). Our work introduces oral epithelial cells for the study of HCMV productive infection, drug screening, and vaccine development.

    IMPORTANCE The ocular epithelial cells currently used to study HCMV infections in vitro have historical significance based upon their role in retinitis, an HCMV disease most often seen in AIDS patients. However, with the successful implementation of HAART regimens, the incidence of HCMV retinitis has rapidly declined, and therefore the relevance of studying ocular epithelial cell HCMV infection has decreased as well. Our introduction here of oral epithelial cells provides two alternative in vitro models for the study of HCMV infection that complement and extend the physiologic relevance of the ocular system currently in use.

  • Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals [PublishAheadOfPrint]

  • Chronic infection with hepatitis B virus (HBV) is a major cause of liver disease and cancer in humans. HBVs (family Hepadnaviridae) have been associated with mammals for millions of years. Recently, the Smc5/6 complex, known for its essential housekeeping functions in genome maintenance, was identified as an antiviral restriction factor of human HBV. The virus has, however, evolved to counteract this defense mechanism by degrading the complex via its regulatory HBx protein. Whether the antiviral activity of the Smc5/6 complex against hepadnaviruses is an important and evolutionarily conserved function is unknown. Here, we used an evolutionary and functional approach to address this question. We first performed phylogenetic and positive selection analyses of the Smc5/6 complex subunits and found that they have been conserved in primates and mammals. Yet, Smc6 showed marks of adaptive evolution, potentially reminiscent of virus-host "arms-race". We then functionally tested the HBx from six divergent hepadnaviruses naturally infecting primates, rodents, and bats. Despite little sequence homology, we demonstrate that these HBx efficiently degraded mammalian Smc5/6 complexes, independently of the host species and of the sites under positive selection. Importantly, all HBx also rescued the replication of an HBx-deficient HBV in primary human hepatocytes. These findings point to an evolutionarily-conserved requirement for Smc5/6 inactivation by HBx, showing that the Smc5/6 antiviral activity has been an important defense mechanism against hepadnaviruses in mammals. It would be interesting to investigate whether Smc5/6 may further be a restriction factor of other yet unidentified viruses that may have driven some of its adaptation.

    Importance Infection with hepatitis B virus (HBV) led to 887000 human deaths in 2015. HBV has been co-evolving with mammals for millions of years. Recently, the Smc5/6 complex, which has essential housekeeping functions, was identified as a restriction factor of human HBV antagonized by the regulatory HBx protein. Here, we address whether the antiviral activity of Smc5/6 is an important evolutionarily conserved function. We found that all six subunits of Smc5/6 have been conserved in primates with only Smc6 showing signatures of "evolutionary arms-race". Using evolution-guided functional analyses that include infections of primary human hepatocytes, we demonstrate that HBx from very divergent mammalian HBVs could all efficiently antagonize Smc5/6, independently of the host species and sites under positive selection. These findings show that the Smc5/6 antiviral activity against HBV is an important function in mammals. They also raise the intriguing possibility that Smc5/6 may restrict other, yet unidentified viruses.

  • VP1 amino acid residue 145 of enterovirus 71 is a key residue for its receptor attachment and resistance to neutralizing antibody during cynomolgus monkey infection [PublishAheadOfPrint]

  • Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and sometimes causes severe or fatal neurological complications. The amino acid at VP1-145 determines virological characteristics of EV71. Viruses with glutamic acid (E) at VP1-145 (VP1-145E) are virulent in neonatal mice and transgenic mice expressing human scavenger receptor B2, whereas those with glutamine (Q) or glycine (G) are not. However, the contribution of this variation to pathogenesis in humans is not fully understood. We compared the virulence of VP1-145E and VP1-145G viruses of Isehara and C7/Osaka backgrounds in cynomolgus monkeys. VP1-145E, but not VP1-145G, viruses induced neurological symptoms. VP1-145E viruses were frequently detected in the tissues of infected monkeys. VP1-145G viruses were detected less frequently and disappeared quickly. Instead, mutants that had a G to E mutation at VP1-145 emerged, suggesting that VP1-145E viruses have a replication advantage in the monkeys. This is consistent with our hypothesis proposed in the accompanying paper that the VP1-145G virus is attenuated due to its adsorption by heparan sulfate. Monkeys infected with both viruses produced neutralizing antibodies before the onset of the disease. Interestingly, VP1-145E viruses were more resistant to neutralizing antibodies than VP1-145G viruses in vitro. A small amount of neutralizing antibody raised in the early phase of infection may not be sufficient to block the dissemination of VP1-145E viruses. The different resistance of the VP1-145 variants to neutralizing antibodies may be one of the reasons for the difference in virulence.

    IMPORTANCE The contribution of VP1-145 variants in humans is not fully understood. In some reports, VP1-145G/Q viruses were more frequently isolated from severely affected than from mildly affected patients, suggesting that VP1-145G/Q viruses are more virulent. In the accompanying paper, we showed that VP1-145E viruses are more virulent than VP1-145G viruses in human SCARB2 transgenic mice. Heparan sulfate acts as a decoy to specifically trap the VP1-145G viruses and leads to abortive infection. Here, we demonstrated that VP1-145G was attenuated in cynomolgus monkeys, suggesting that this hypothesis is also true in a non-human primate model. VP1-145E viruses, but not VP1-145G viruses, were highly resistant to neutralizing antibodies. We propose the difference in resistance against neutralizing antibodies as another mechanism of EV71 virulence. In summary, VP1-145 contributes to virulence determination by controlling attachment receptor usage and antibody sensitivity.

  • Amino acid variation at VP1-145 of enterovirus 71 determines attachment receptor usage and neurovirulence in human scavenger receptor B2 transgenic mice. [PublishAheadOfPrint]

  • Infection of enterovirus 71 (EV71) is affected by cell surface receptors, including the scavenger receptor B2, which are required for viral uncoating, and attachment receptors, such are heparan sulfate (HS), which bind virus, but do not support uncoating. Amino acid residue 145 of the capsid protein VP1 affects viral binding to HS, and virulence in mice. However, the contribution of this amino acid to pathogenicity in humans is not known. We produced EV71 having glycine (VP1-145G) or glutamic acid (VP1-145E) at position 145. VP1-145G, but not VP1-145E, enhanced viral infection in cell culture in an HS-dependent manner. However, VP1-145G showed an attenuated phenotype in wild-type suckling mice and in a transgenic mouse model expressing human scavenger receptor B2 (hSCARB2), while VP1-145E virus showed a virulent phenotype in both models. Thus HS-binding property and in vivo virulence are negatively correlated. Immunohistochemical analyses showed that HS is highly expressed in vascular endothelial cells and some other cell types where hSCARB2 is expressed at low or undetectable levels. VP1-145G virus bound to tissue homogenate of both hSCARB2 transgenic and non-transgenic mice in vitro, and viral titer was reduced in the bloodstream immediately after intravenous inoculation. Furthermore, VP1-145G virus failed to disseminate well in the mouse organs. These data suggest that VP1-145G virus is adsorbed by attachment receptors such as HS, during circulation in vivo, leading to abortive infection of HS-positive cells. This trapping effect is thought to be a major mechanism of attenuation of the VP1-145G virus.

    IMPORTANCE Attachment receptors expressed on the host cell surface are thought to enhance EV71 infection by increasing the chance of encountering true receptors. Although this has been confirmed using cell culture for some viruses, the importance of attachment receptors in vivo is unknown. This report provides an unexpected answer to this question. We demonstrated that the VP1-145G virus binds to HS and shows an attenuated phenotype in an hSCARB2-dependent animal infection model. HS is highly expressed in cells that express hSCARB2 at low or undetectable levels. Our data indicate that HS binding directs VP1-145G virus towards abortive infection, and keeps virus away from hSCARB2-positive cells. Thus, although the ability of VP1-145G virus to use HS might be an advantage in replication in certain cultured cells, it becomes a serious disadvantage in replication in vivo. This adsorption is thought to be a major mechanism of attenuation associated with attachment receptor usage.

  • Pathogenesis and transmission of genetically diverse swine-origin H3N2v influenza A viruses from multiple lineages isolated in the United States, 2011-2016 [PublishAheadOfPrint]

  • While several swine-origin influenza A H3N2 variant (H3N2v) viruses isolated from humans prior to 2011 have been previously characterized for their virulence and transmissibility in ferrets, recent genetic and antigenic divergence of H3N2v viruses warrants an updated assessment of their pandemic potential. Here, four contemporary H3N2v viruses isolated during 2011-2016 were evaluated for their replicative ability in both in vitro and in vivo mammalian models, as well as their transmissibility among ferrets. We found that all four H3N2v viruses possessed similar or enhanced replication capacity in a human bronchial epithelium cell line (Calu-3) compared to a human seasonal influenza virus, suggestive of strong fitness in human respiratory tract cells. The majority of H3N2v viruses examined in our study were mildly virulent in mice and capable of replicating in mouse lungs with different degrees of efficiency. In ferrets, all four H3N2v viruses caused moderate morbidity and exhibited comparable titers in the upper respiratory tract, but only 2 of the 4 viruses replicated in the lower respiratory tract in this model. Furthermore, despite efficient transmission among cohoused ferrets, recently isolated H3N2v viruses displayed considerable variance in their ability to transmit by respiratory droplets. The lack of a full understanding of the molecular correlates of virulence and transmission underscores the need for close genotypic and phenotypic monitoring of H3N2v viruses and the importance of continued surveillance to improve pandemic preparedness.

    Importance: Swine-origin influenza viruses of the H3N2 subtype, with the HA and NA derived from historic human seasonal influenza viruses, continue to cross species barriers and cause human infections, posing an indelible threat to public health. To help us better understand the potential risk associated with swine-origin H3N2v viruses that emerged in the U.S between 2011-2016 influenza seasons, we use both in vitro and in vivo models to characterize the ability of these viruses to replicate, caused disease, and transmit in mammalian hosts. The efficient respiratory droplet transmission exhibited by some of the H3N2v viruses in the ferret model combined with the existing evidence of low immunity against such viruses in young children and older adults highlights their pandemic potential. Extensive surveillance and risk assessment of H3N2v viruses should continue to be an essential component of our pandemic preparedness strategy.

  • The HPV E6 PDZ Binding Motif links DNA Damage Response Signaling to E6 Inhibition of p53 Transcriptional Activity. [PublishAheadOfPrint]

  • The presence of a PDZ binding motif (PBM) in the HPV E6 oncoprotein appears to be a characteristic marker of high oncogenic potential and confers interaction with a number of different cellular PDZ domain-containing substrates. The E6 PBM is also subject to phosphorylation, resulting in an inhibition of E6 PDZ binding activity and instead allowing E6 to associate with 14-3-3 proteins. In this study, we have analyzed the conditions under which the E6 PBM is phosphorylated. We demonstrate that in normal cycling cells the levels of E6 phosphorylation are very low. However, following exposure of cells to oxidative stress or the induction of DNA damage, there is a striking increase in the levels of E6 phosphorylation. Depending on the specific stimulus, this phosphorylation of E6 can involve the ATM/ATR pathway and is performed primarily through Chk1, although the Chk2 pathway is also involved indirectly through activation of PKA. To understand the biological relevance of these phospho-modifications of E6, we analysed their effects upon the ability of E6 to inhibit p53 transcriptional activity. We show that an intact E6 phospho-acceptor site plays an essential role in the ability of E6 to inhibit p53 transcriptional activity on a subset of p53-responsive promoters, in a manner that is independent from E6's ability to direct p53 degradation. These results are, to our knowledge, the first example of a DNA damage response controlling PBM-PDZ recognition. This study also provides links between the DNA damage response, the regulation of E6 PBM function, and the inhibition of p53 activity, and begins to explain how HPV-infected cells remain within the cell cycle, despite activation of DNA damage response pathways during productive virus infections.

    IMPORTANCE The cancer-causing HPV E6 oncoproteins all possess a PDZ binding motif at their extreme carboxy-termini. Depending upon whether this motif is phosphorylated, E6 can recognise PDZ domain-containing proteins or members of the 14-3-3 family of proteins. We show here that DNA damage response pathways directly signal to the E6 PBM, resulting in Chk1 and Chk2-driven phosphorylation. This phosphorylation is particularly pronounced following treatment of cells with a variety of different chemotherapeutic drugs. A direct functional consequence of this signaling is to confer an enhanced ability upon E6 to inhibit p53 transcriptional activity in a proteasome-independent but phosphorylation-dependent manner. These results are the first example of DNA damage signaling pathways regulating PBM-PDZ interactions, and provide the mechanistic link between E6 PBM function and perturbation of p53 activity.

  • Structural and Molecular Characterization of the Hemagglutinin from the Fifth Epidemic Wave A(H7N9) Influenza Viruses [PublishAheadOfPrint]

  • The avian influenza A(H7N9) virus continues to cause human infections in China and is a major ongoing public health concern. Five epidemic waves of A(H7N9) infection have occurred since 2013, and the recent fifth epidemic wave saw the emergence of two distinct lineages with elevated numbers of human infection cases and broader geographic distribution of viral diseases compared to the first four epidemic waves. Moreover, highly pathogenic avian influenza (HPAI) A(H7N9) viruses were also isolated during the fifth epidemic wave. Here, we present a detailed structural and biochemical analysis of the surface hemagglutinin (HA) antigen from viruses isolated during this recent epidemic wave. Results highlight that when compared to the 2013 virus HAs, the fifth wave virus HAs remained a weak binder to human glycan receptor analogs. We also studied three mutations, V177K-K184T-G219S, that were recently reported to switch a 2013 A(H7N9)HA to human-type receptor specificity. Our results indicate that these mutations could also switch the H7 HA receptor preference to a predominantly human binding specificity for both fifth wave H7 HAs analyzed in this study.

    IMPORTANCE The A(H7N9) viruses circulating in China are of great public health concern. Herein, we report a molecular and structural study of the major surface proteins from several recent A(H7N9) influenza viruses. Our results improve the understanding of these evolving viruses and provide important information on their receptor preference that is central to ongoing pandemic risk assessment.

  • The SMC5/6 complex interacts with the papillomavirus E2 protein and influences maintenance of viral episomal DNA [PublishAheadOfPrint]

  • The papillomavirus E2 protein executes numerous essential functions related to viral transcription, replication of viral DNA, and viral genome maintenance. Because E2 lacks enzymatic activity, many of these functions are mediated by interactions with host cellular proteins. Unbiased proteomics approaches have successfully identified a number of E2-host protein interactions. We have extended such studies and have identified and validated the cellular proteins SMC5 and SMC6 as interactors of the viral E2 protein. These two proteins make up the core components of the SMC5/6 complex. The SMC5/6 complex is a member of the conserved Structural Maintenance of Chromosomes (SMC) family of proteins, which are essential for genome maintenance. We have examined the role of SMC5/6 in various E2 functions. Our data suggest that SMC6 is not required for E2-mediated transcriptional activation, E1/E2-mediated transient replication, or differentiation-dependent amplification of viral DNA. Our data however suggest a role for SMC5/6 in viral genome maintenance.

    IMPORTANCE The high-risk human papillomaviruses are the etiological cause of cervical cancer and the most common sexually transmitted infection. While the majority of infections may be asymptomatic or only cause benign lesions, persistent infection with the oncogenic high-risk HPV types may lead to serious diseases, such as cervical cancer, anogenital carcinoma, or head and neck oropharyngeal squamous cell carcinoma. The identification of virus-host protein interactions provides insights into the mechanisms of viral DNA persistence, viral genome replication, and cellular transformation. Elucidating the mechanism of early events in the virus replication cycle as well as of integration of viral DNA into host chromatin may present novel antiviral strategies and targets for counteracting persistent infection. The E2 protein is an important viral regulatory protein whose functions are mediated through interactions with host cell proteins. Here we explore the interaction of E2 with SMC5/6 and the functional consequences.

  • Partial inactivation of the chromatin remodelers SMARCA2 and SMARCA4 in virus-infected cells by caspase-mediated cleavage. [PublishAheadOfPrint]

  • The BAF-chromatin remodeling complex with its mutually exclusive ATPases SMARCA2 and SMARCA4 is essential for the transcriptional activation of numerous genes, including a subset of interferon stimulated genes (ISGs). Here we show that C-terminally truncated forms of both SMARCA2 and SMARCA4 accumulate in cells infected with different RNA or DNA viruses. The levels of truncated SMARCA2 or SMARCA4 strongly correlate with the degree of cell damage and death observed after virus infection. The use of a pan-caspase inhibitor and genetically modified cell lines unable to undergo apoptosis revealed that the truncated forms result from the activity of caspases downstream of the activated intrinsic apoptotic pathway. C-terminally cleaved SMARCA2 and SMARCA4 lack potential nuclear localization signals as well as the bromo- and SnAC domain, the latter two domains believed to be essential for chromatin association and remodeling. Consistently, C-terminally truncated SMARCA2 was partially relocated to the cytoplasm. However, the remaining nuclear protein was sufficient to induce ISG expression and inhibit the replication of vesicular stomatitis virus and influenza A virus. This suggests that virus-induced apoptosis does not occur at the expense of an intact interferon-mediated antiviral response pathway.

    IMPORTANCE Efficient induction of interferon stimulated genes (ISGs) prior to infection is known to effectively convert a cell into an antiviral state, blocking viral replication. Additionally, cells can undergo caspase-mediated apoptosis to control viral infection. Here, we identify SMARCA2 and SMARCA4 to be essential for the efficient induction of ISGs, but to be also targeted by cellular caspases downstream of the intrinsic apoptotic pathway. We find that C-terminally cleaved SMARCA2 and SMARCA4 accumulate at late stages of infection when cell damage already had occurred. Cleavage of the C-terminus removes domains important for nuclear localization and chromatin binding of SMARCA2 and SMARCA4. Consequently, the cleaved forms are unable to efficiently accumulate in the cell nucleus. Intriguingly, the remaining nuclear C-terminally-truncated SMARCA2 still induced ISG expression, although to lower levels. These data suggest that in virus-infected cells caspase-mediated cell death does not completely inactivate the SMARCA2 and SMARCA4 dependent interferon signaling pathway.

  • Herpes simplex virus 1 tegument protein VP22 abrogates cGAS/STING-mediated antiviral innate immunity [PublishAheadOfPrint]

  • Cytosolic DNA arising from intracellular pathogens is sensed by cyclic GMP-AMP synthase (cGAS), and triggers a powerful innate immune response. However, herpes simplex virus type 1 (HSV-1), a double-strand DNA virus, has developed multiple mechanisms to attenuate host antiviral machinery and facilitate viral infection and replication. In the present study, we found that HSV-1 tegument protein VP22 acted as an inhibitor of cGAS/STING-mediated production of interferon (IFN) and its downstream antiviral genes. Our results showed that ectopic expression of VP22 decreased cGAS/STING-mediated IFN-bbeta; promoter activation and IFN-bbeta; production. The infection of wild-type HSV-1 (WT), but not VP22-deficient virus (VP22), inhibited immunostimulatory DNA (ISD) induced activation of IFN signaling pathway. Further study showed that VP22 interacted with cGAS and inhibited the enzymatic activity of cGAS. In addition, stably knockdown of cGAS facilitated the replication of VP22, but not WT. In summary, our findings indicated that HSV-1 VP22 acted as an antagonist of IFN signaling to persistently evade host innate antiviral responses.

    IMPORTANCE cGAS is very important for the host to defense viral infection, many viruses have evolved ways to target cGAS and successfully evade the attack by the immune system of their susceptible host. This study demonstrated that HSV-1 tegument protein VP22 counteracts cGAS/STING mediated DNA sensing antiviral innate immunity signal pathway by inhibiting the enzymatic activity of cGAS. Findings in this study will expand our understanding of the interaction between HSV-1 replication and host DNA sensing signal pathway.

  • Defining the role of stress granules in innate immune suppression by the HSV-1 endoribonuclease VHS [PublishAheadOfPrint]

  • In response to virus-induced shut-off of host protein synthesis, dynamic aggregates containing mRNA, RNA-binding proteins and translation factors termed stress granules (SGs) often accumulate within the cytoplasm. SGs typically form following phosphorylation and inactivation of the eukaryotic translation initiation factor 2 alpha, a substrate of the double-stranded (ds) RNA-activated kinase PKR. The detection of innate immune sensors and effectors like PKR at SGs is suggestive of role in pathogen nucleic acid sensing. However, the functional importance of SGs in host innate responses is unclear and has primarily been examined in response to infection with select RNA viruses. During infection with the DNA virus herpes simplex virus-1 (HSV-1), the virus-encoded endoribonuclease VHS is required to restrict interferon production, PKR activation, and SG formation although the relationship between these activities remains incompletely understood. Here, we show that in cells infected with a VHS-deficient HSV-1 (VHS) dsRNA accumulated and localized to SGs. Surprisingly, formation of dsRNA and its concentration at SGs was not required for interferon bbeta; mRNA induction, indicating that suppression of type I interferon induction by VHS does not stem from its control of dsRNA accumulation. Instead, STING-signaling downstream of cGAS-dependent DNA-sensing is required for interferon bbeta; induction. By contrast, significantly less PKR activation is observed when SG assembly is disrupted by ISRIB, an inhibitor of phosphorylated eIF2aalpha;-mediated translation repression, or depleting SG scaffolding proteins G3BP1 or TIA1. This demonstrates that PKR activation is intimately linked to SG formation and that SGs form important hubs to potentiate PKR activation during infection.

    IMPORTANCE Formation of cytoplasmic stress granules that are enriched for innate immune sensors and effectors is suppressed during many viral infections. It is unclear, however, to what extent this is a side effect of viral efforts to maintain protein synthesis or intentional disruption of a hub for innate immune sensing. In this study, we utilize a herpes simplex virus 1 mutant lacking the RNA nuclease VHS which upon infection induces SGs, PKR activation and interferon bbeta; to address this question. We show that dsRNA is localized to SGs and SGs can function to promote PKR activation in the context of a DNA virus infection, but find no evidence to support their importance for interferon induction during HSV-1 infection.

  • N-linked glycosylation is not essential for sodium taurocholate cotransporting polypeptide to mediate hepatitis B virus infection in vitro [PublishAheadOfPrint]

  • Sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a hepatitis B virus (HBV) receptor, and its overexpression in HepG2 cell line leads to efficient secretion of hepatitis B e antigen (HBeAg) following challenge with a large dose of cell culture-derived HBV (cHBV) particles. However, NTCP-reconstituted HepG2 cells are inefficiently infected by patient serum-derived HBV (sHBV), and release very little hepatitis B surface antigen (HBsAg) following cHBV infection in comparison to differentiated HepaRG cells, which are naturally susceptible to both cHBV and sHBV particles. Here we investigated whether NTCP could explain the different behaviors between the two cell types. Endogenous NTCP protein from differentiated HepaRG cells was unglycosylated despite wild-type coding sequence. HepaRG cells stably transfected with epitope-tagged NTCP expression construct displayed higher sHBV but not cHBV susceptibility than cells transfected with the null mutant. Tagged NTCP introduced to both HepG2 and HepaRG cells was glycosylated, with N5 and N11 being sites of N-linked glycosylation. Mutating N5, N11, or both did not alter cell surface availability of NTCP or its subcellular localization, with both the singly glycosylated and nonglycosylated forms still capable of mediating cHBV infection in HepG2 cells. In conclusion, nonglycosylated NTCP is expressed by differentiated HepaRG cells and capable of mediating cHBV infection in HepG2 cells, but it cannot explain differential susceptibility of HepaRG and HepG2/NTCP cells to cHBV vs. sHBV infection and different HBsAg/HBeAg ratios following cHBV infection. The responsible host factor(s) remain to be identified.

    IMPORTANCE HBV can infect differentiated HepaRG cells and also HepG2 cells overexpressing NTCP, the currently accepted HBV receptor. However, HepG2/NTCP cells remain poorly susceptible to patient serum-derived HBV particles and release very little hepatitis B surface antigen following infection by cell culture-derived HBV. We found differentiated HepaRG cells expressed nonglycosylated NTCP despite a wild-type coding sequence. NTCP introduced to HepG2 cells was glycosylated at two N-linked glycosylation sites, but mutating either or both sites failed to prevent infection by cell culture-derived HBV or to confer susceptibility to serum-derived HBV. Overexpressing NTCP in HepRG cells did not increase infection by cell culture-derived HBV or distort the ratio between the two viral antigens. These findings suggest that host factors unique to HepaRG cells are required for efficient infection by serum-derived HBV, and factors other than NTCP contribute to balanced viral antigen production following infection by cell culture-derived HBV.

  • Canine Distemper Virus Spread and Transmission to Naïve Ferrets: Selective Pressure on SLAM-Dependent Entry [PublishAheadOfPrint]

  • Upon infection, morbilliviruses such as measles, rinderpest, and canine distemper virus (CDV) initially target immune cells via the signalling lymphocyte activation molecule (SLAM) before spreading to respiratory epithelia through the adherens junction protein nectin-4. However, the roles of these receptors in transmission from infected to naïve hosts have not yet been formally tested. Towards experimentally addressing this question, we established a model of CDV contact transmission between ferrets. We show here that transmission of wild type CDV sometimes precedes the onset of clinical disease. In contrast, transmission was not observed in most animals infected with SLAM- or nectin-4-blind CDVs, even though all animals infected with the nectin-4-blind virus developed sustained viremia. There was an unexpected case of transmission of a nectin-4-blind virus, possibly due to biting. Another unprecedented event was transient viremia in an infection with a SLAM-blind virus. We identified three compensatory mutations within or near its SLAM-binding surface of the attachment protein. A recombinant CDV expressing the mutated attachment protein regained the ability to infect ferret lymphocytes in vitro, but its replication was not as efficient as wild-type CDV. Ferrets infected with this virus developed transient viremia and fever, but there was no transmission to naïve contacts. Our study supports the importance of epithelial cell infection, and of sequential CDV H protein interactions first with SLAM and then nectin-4 receptors for transmission to naïve hosts. It also highlights the in vivo selection pressure on the H protein interactions with SLAM.

    IMPORTANCE Morbilliviruses such as measles, rinderpest, and canine distemper virus (CDV) are highly contagious. Despite extensive knowledge of how morbilliviruses interact with their receptors, little is known about how those interactions influence viral transmission to naive hosts. In a ferret model of CDV contact transmission, we show that sequential use of the signaling lymphocytic activation molecule (SLAM) and nectin-4 receptors is essential for transmission. In one animal infected with a SLAM-blind CDV, we documented mild viremia due to the acquisition of three compensatory mutations within or near its SLAM-binding surface. The interaction, however, was not sufficient to cause disease or sustain transmission to naive contacts. This work confirms the sequential roles of SLAM and nectin-4 in morbillivirus transmission, and highlights the selective pressure directed toward productive interactions with SLAM.

  • Frequencies of circulating Th1-biased T follicular helper cells in acute HIV-1 infection correlate with the development of HIV-specific antibody responses and lower set point viral load [PublishAheadOfPrint]

  • Despite decades of focused research, the field has yet to develop a prophylactic vaccine for HIV-1 infection. In the RV144 vaccine trial, non-neutralizing antibody responses were identified as a correlate for prevention of HIV acquisition. However, factors that predict the development of such antibodies are not fully elucidated. We sought to define the contribution of circulating T follicular helper (cTfh) cell subsets to the development of non-neutralizing antibodies in HIV-1 clade C infection. Study participants were recruited from an acute HIV-1 clade C infection cohort. Plasma anti-gp41, -gp120, -p24 and -p17 antibodies were screened using a customized multivariate Luminex assay. Phenotypic and functional characterization of cTfh were performed using HLA class II tetramers and intracellular cytokine staining. In this study, we found that acute HIV-1 clade C infection skewed differentiation of functional cTfh subsets towards increased Tfh1 (p=0.02) and Tfh2 (pllt;0.0001) subsets, with a concomitant decrease in overall Tfh1-17 (that shares both Tfh1 and Tfh17 properties) (p=0.01) and Tfh17 subsets (pllt;0.0001) compared to HIV negative subjects. Interestingly, the frequencies of Tfh1 during acute infection (5.0-8.0 weeks post-infection) correlated negatively with set point viral load (p=0.03, r=-60) and were predictive of p24-specific plasma IgG titers at one year of infection (p=0.003, r=0.85). Taken together, our results suggest that the circulating Tfh1 subset plays an important role in the development of anti-HIV antibody responses and contributes to HIV suppression during acute HIV-1 infection. These results have implications for vaccine studies aimed at inducing long lasting anti-HIV antibody responses.

    IMPORTANCE The HIV epidemic in southern Africa accounts for almost half of the global HIV burden with HIV-1 clade C being the predominant strain. It is therefore important to define immune correlates of clade C HIV control that might have implications for vaccine design in this region. T follicular helper (Tfh) cells are critical for the development of HIV-specific antibody responses and could play a role in viral control. Here we showed that the early induction of circulating Tfh1 cells during acute infection correlated positively with the magnitude of p24-specific IgG and was associated with lower set point viral load. This study highlights a key Tfh cell subset that could limit HIV replication by enhancing antibody generation. This study underscores the importance of circulating Tfh cells in promoting non-neutralizing antibodies during HIV-1 infection.

  • Function of the non-conserved N-terminal domain of Pseudorabies Virus pUL31 in nuclear egress [PublishAheadOfPrint]

  • Nuclear egress of herpesvirus capsids is mediated by the conserved nuclear egress complex (NEC) composed of the membrane anchored pUL34 and its nucleoplasmic interaction partner, pUL31. The recently solved crystal structures of the NECs from different herpesviruses showed a high structural similarity with the pUL34 homologs building a platform recruiting pUL31 to the inner nuclear membrane. Both proteins possess a central globular fold while the conserved N-terminal portion of pUL31 forms an extension reaching around the core of pUL34. However, the extreme N-terminus of the pUL31 homologs, which is highly variable in length and amino acid composition, had to be removed for crystallization. Several pUL31 homologs contain a classical nuclear localization signal (NLS) within this part mediating efficient nuclear import. In addition, membrane-binding activity, blocking premature interaction with pUL34, nucleocapsid trafficking, and regulation of NEC assembly and disassembly via phosphorylation were assigned to the extreme pUL31 N-terminus. To test the functional importance in the alphaherpesvirus pseudorabies virus (PrV) pUL31 N-terminal truncations and site-specific mutations were generated and the resulting proteins were tested for intracellular localization, interaction with pUL34, and functional complementation of PrV-UL31. Our data show that neither the bipartite NLS nor the predicted phosphorylation sites are essential for pUL31 function during nuclear egress. Moreover, nearly the complete variable N-terminal part was dispensable for function as long as a stretch of basic amino acids was retained. Phosphorylation of this domain controls efficient nucleocapsid release from the perinuclear space.

    IMPORTANCE Nuclear egress of herpesvirus capsids is a unique vesicle-mediated nucleo-cytoplasmic transport. Crystal structures of the heterodimeric nuclear egress complexes (NEC) from different herpesviruses provided important details of this viral nuclear membrane deformation and scission machinery but excluded the highly variable N-terminus of the pUL31 component. We present here a detailed mutagenesis study of this important portion of pUL31 and show that basic amino acid residues within this domain play an essential role for proper targeting, complex formation and function during nuclear egress while phosphorylation modulates efficient release from the perinuclear space. Thus, our data complement previous structure-function assignments of the nucleocapsid-interacting component of the NEC.

  • Similar epitope specificities of IgG and IgA antibodies elicited by Ad26 vector prime, Env protein boost immunizations in rhesus monkeys [PublishAheadOfPrint]

  • Vaccine-elicited immunoglobulin G (IgG) has been shown to be important for protection against simian/human immunodeficiency virus (SHIV) infection in rhesus monkeys. However, it remains unclear whether vaccine-elicited IgA responses are beneficial or detrimental for protection. In this study, we evaluated the kinetics, magnitude, breadth, and linear epitope specificities of vaccine-elicited IgG and IgA responses in serum and mucosal secretions following intramuscular immunization with Ad26 prime, Env protein boost vaccination regimens. The systemic and mucosal antibody responses exhibited similar kinetics but lower titers than serum antibody responses. Moreover, IgG and IgA responses were correlated, both in terms of the magnitude of responses and in terms of antibody specificities against linear HIV-1 Env, Gag and Pol epitopes. These data suggest that IgG and IgA responses are highly coordinated in both peripheral blood and mucosal compartments following Ad26/Env vaccination in rhesus monkeys.

    IMPORTANCE Vaccine-elicited IgG responses are important for protection against simian/human immunodeficiency virus (SHIV) infection in non-human primates. However, much less is known about the role and function of IgA, despite it being the predominant antibody in mucosal sites. There is debate as to whether HIV-1-specific IgA responses are beneficial or detrimental, since serum anti-Env IgA titers have been shown to be inversely correlated with protection in the RV144 clinical trial. We thus assessed vaccine-elicited IgG and IgA antibody responses in peripheral blood and mucosal secretions following vaccination with the Ad26/Env vaccine.

  • Nsp1{alpha} of PRRSV strain BB0907 impairs the function of monocyte-derived dendritic cells via the release of soluble CD83 [PublishAheadOfPrint]

  • Porcine reproductive and respiratory syndrome virus (PRRSV), a virulent pathogen of swine, suppresses the innate immune response and induces persistent infection. One mechanism used by viruses to evade the immune system is to cripple the antigen processing machinery in monocyte-derived dendritic cells (MoDCs). In this study, we show that MoDCs infected by PRRSV express lower levels of the MHC-peptide complex proteins TAP1 and ERp57, are impaired in their ability to stimulate T cell proliferation, and increase their production of CD83. Neutralization of sCD83 removes the inhibitory effects of PRRSV on MoDCs. When MoDCs are incubated with exogenously added sCD83 protein, TAP1 and ERp57 expression decreases and T lymphocyte activation is impaired. PRRSV non-structural protein 1aalpha; (Nsp1aalpha;) enhances CD83 promoter activity. Mutations in the ZF domain of Nsp1aalpha; abolish its ability to activate the CD83 promoter. We generated recombinant PRRSVs with mutations in Nsp1aalpha; and the corresponding repaired PRRSVs. Viruses with Nsp1aalpha; mutations did not decrease levels of TAP1 and ERp57, impair the ability of MoDCs to stimulate T cell proliferation, or increase levels of sCD83. We show that the ZF domain of Nsp1aalpha; stimulates the secretion of CD83, which in turn inhibits MoDC function. Our study provides new insights into the mechanisms of immune suppression by PRRSV.

    Importance PRRSV has a severe impact on the swine industry throughout the world. Understanding the mechanisms by which PRRSV infection suppresses the immune system is essential for a robust and sustainable swine industry. Here, we demonstrated that PRRSV infection manipulates MoDCs by interfering with their ability to produce proteins in the MHC-peptide complex. The virus also impairs the ability MoDCs to stimulate cell proliferation, due in large part to the enhanced release of soluble CD83 from PRRSV-infected MoDCs. The viral non-structural protein 1 (Nsp1) is responsible for up-regulating CD83 promoter activity. Amino acids in the ZF domain of Nsp1aalpha; (L5-2A, rG45A, G48A, L61-6A) are essential for CD83 promoter activation. Viruses with mutations at these sites no longer inhibit MoDC-mediated T cell proliferation. These findings provide novel insights into the mechanism by which the adaptive immune response is suppressed during PRRSV infection.

  • Varicella Zoster Virus ORF9p binding to cellular adaptor protein complex-1 is important for viral infectivity [PublishAheadOfPrint]

  • ORF9p (homologous to HSV-1 VP22) is a varicella-zoster virus (VZV) tegument protein essential for viral replication. Even though its precise functions are far from being fully described, a role in the secondary envelopment of the virus has long been suggested. We performed a yeast two-hybrid screen to identify cellular proteins interacting with ORF9p that might be important for this function. We found thirty-one ORF9p interaction partners, among which AP1M1, the mmu; subunit of the adaptor protein complex-1 (AP-1). AP-1 is a heterotetramer involved in intracellular vesicle-mediated transport and regulates the shuttling of cargo proteins between endosomes and the TGN via clathrin-coated vesicles. We confirmed that AP-1 interacts with ORF9p in infected cells and mapped potential interaction motifs within ORF9p. We generated VZV mutants in which each of these motifs is individually impaired and identified leucine 231 in ORF9p as critical to interact with AP-1. Disrupting ORF9p binding to AP-1 by mutating leucine 231 to alanine in ORF9p strongly impaired viral growth, most likely by preventing efficient secondary envelopment of the virus. Leucine 231 is part of a di-leucine motif conserved among alphaherpesviruses and we showed that VP22 of MDV and HSV-2 also interact with AP-1. This indicates that the function of this interaction in the secondary envelopment might be conserved as well.

    IMPORTANCE Herpesviruses are responsible for infections that, especially in immunocompromised patients, can lead to severe complications, including neurological symptoms and strokes. The constant emergence of viral strains resistant to classical antivirals (mainly acyclovir and its derivatives) pleads for the identification of new targets for future antiviral treatments. Cellular adaptor protein complexes (AP) have been implicated in the correct addressing of herpesvirus glycoproteins in infected cells and the discovery that a major constituent of varicella-zoster virus tegument is interacting with AP-1 reveals a previously unsuspected role of this tegument protein. Unraveling the complex mechanisms leading to virion production will certainly be an important step in the discovery of future therapeutic targets.

  • SAMHD1 Impairs HIV-1 Gene Expression and Negatively Modulates Reactivation of Viral Latency in CD4+ T-cells [PublishAheadOfPrint]

  • Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in non-dividing cells by degrading intracellular deoxynucleoside triphosphates (dNTPs). SAMHD1 is highly expressed in resting CD4+ T-cells that are important for the HIV-1 reservoir and viral latency; however, whether SAMHD1 affects HIV-1 latency is unknown. Recombinant SAMHD1 binds HIV-1 DNA or RNA fragments in vitro, but the function of this binding remains unclear. Here we investigate the effect of SAMHD1 on HIV-1 gene expression and reactivation of viral latency. We found that endogenous SAMHD1 impaired HIV-1 LTR activity in monocytic THP-1 cells and HIV-1 reactivation in latently infected primary CD4+ T-cells. Overexpression of wild-type (WT) SAMHD1 suppressed HIV-1 long terminal repeat (LTR)-driven gene expression at a transcriptional level. Tat co-expression abrogated SAMHD1-mediated suppression of HIV-1 LTR-driven luciferase expression. SAMHD1 overexpression also suppressed LTR activity of human T-cell leukemia virus type 1 (HTLV-1), but not from murine leukemia virus (MLV), suggesting specific suppression of retroviral LTR-driven gene expression. WT SAMHD1 bound to proviral DNA and impaired reactivation of HIV-1 gene expression in latently infected J-Lat cells. In contrast, a nonphosphorylated mutant (T592A) and a dNTP triphosphohydrolase (dNTPase) inactive mutant (H206D/R207N, or HD/RN) of SAMHD1 failed to efficiently suppress HIV-1 LTR-driven gene expression and reactivation of latent virus. Purified recombinant WT SAMHD1, but not T592A and HD/RN mutants, bound to fragments of the HIV-1 LTR in vitro. These findings suggest that SAMHD1-mediated suppression of HIV-1 LTR-driven gene expression potentially regulates viral latency in CD4+ T-cells.

    IMPORTANCE A critical barrier to developing a cure for HIV-1 infection is the long-lived viral reservoir that exists in resting CD4+ T-cells, the main targets of HIV-1. The viral reservoir is maintained through a variety of mechanisms, including regulation of the HIV-1 LTR promoter. The host protein SAMHD1 restricts HIV-1 replication in non-dividing cells, but its role in HIV-1 latency remains unknown. Here we report a new function of SAMHD1 in regulating HIV-1 latency. We found that SAMHD1 suppressed HIV-1 LTR promoter-driven gene expression and reactivation of viral latency in cell lines and primary CD4+ T-cells. Furthermore, SAMHD1 bound to the HIV-1 LTR in vitro and in a latently infected CD4+ T-cell line, suggesting that the binding may negatively modulate reactivation of HIV-1 latency. Our findings indicate a novel role for SAMHD1 in regulating HIV-1 latency, which enhances our understanding of the mechanisms regulating proviral gene expression in CD4+ T-cells.

  • Control of Heterologous SIVsmE660 infection by DNA and Protein Co-immunization Regimens Combined with Different Toll-like Receptor-4 (TLR-4) Based Adjuvants in Macaques [PublishAheadOfPrint]

  • We developed a method of simultaneous vaccination with DNA and protein resulting in robust and durable cellular and humoral immune responses with efficient dissemination to mucosal sites and protection against SIV infection. To further optimize the DNA+Protein co-immunization regimen, we tested a SIVmac251-based vaccine formulated with either of two TLR-4 ligand-based liposomal adjuvant formulations (TLR-4+TLR-7 or TLR-4+QS21) in macaques. Although both vaccines induced humoral responses of similar magnitude, they differed in their functional quality, including broader neutralizing activity and effector functions in the TLR4+7 group. Upon repeated heterologous SIVsmE660 challenge, a trend of delayed viral acquisition was found in vaccinees compared to controls, which reached statistical significance in animals with the TRIM-5aalpha; resistant allele. Vaccinees were preferentially infected by SIVsmE660 transmitted founder virus carrying neutralization resistant A/K mutations at residues 45/47 in Env, demonstrating a strong vaccine-induced sieve effect. In addition, delay in virus acquisition directly correlated with SIVsmE660-specific neutralizing antibodies. Presence of mucosal V1V2 IgG binding antibodies correlated with significantly decreased risk of virus acquisition in both TRIM-5aalpha; R and M/S animals, although this vaccine effect was more prominent in animals with the TRIM-5aalpha; R allele. These data support the combined contribution of immune responses and genetic background to vaccine efficacy. Humoral responses targeting V2 and SIV-specific T-cell responses correlated with viremia control. In conclusion, combination of DNA and gp120 Env protein vaccine regimens using two different adjuvants induced durable and potent cellular and humoral responses contributing to lower risk of infection by heterologous SIV challenge.

    IMPORTANCE An effective AIDS vaccine continues to be of paramount importance for the control of the pandemic, and it has been proven an elusive target. Vaccine efficacy trials and macaque challenge studies indicate that protection may be the result of combinations of many parameters. We show that a combination of DNA and protein vaccination applied at the same time provides rapid and robust cellular and humoral immune responses and evidence for reduced risk of infection. Vaccine-induced neutralizing antibodies and Env V2-specific antibodies at mucosal sites contribute to the delay of SIVsmE660 acquisition and genetic make-up (TRIM-5aalpha;) affects the effectiveness of the vaccine. These data are important for the design of better vaccines and may also affect other vaccine platforms.

  • RESTRICTION OF {gamma}34.5-DELETED ONCOLYTIC HERPES SIMPLEX VIRUS REPLICATION IN GLIOBLASTOMA STEM-LIKE CELLS [PublishAheadOfPrint]

  • Oncolytic viruses, including herpes simplex viruses (HSVs), are a new class of cancer therapeutic engineered to infect and kill cancer cells, while sparing normal tissue. To ensure that oncolytic HSV (oHSV) is safe in the brain, all oHSVs in clinical trial for glioma lack the 34.5 genes responsible for neurovirulence. However, loss of 34.5 attenuates growth in cancer cells. Glioblastoma (GBM) is a lethal brain tumor that is heterogeneous and contains a subpopulation of cancer stem cells, termed GBM stem-like cells (GSCs), that likely promote tumor progression and recurrence. GSCs and matched serum-cultured GBM cells (ScGCs), representative of bulk or differentiated tumor cells, were isolated from the same patient tumor specimens. ScGCs are permissive to 34.5-deleted oHSV replication and cell killing, while patient-matched GSCs were not, implying an underlying biological difference between stem and bulk cancer cells. GSCs specifically restrict the synthesis of HSV1 true late (TL) proteins, without affecting viral DNA replication or transcription of TL genes. A global shutoff of cellular protein synthesis also occurs late after 34.5-deleted oHSV infection of GSCs, but does not affect the synthesis of early and leaky late viral proteins. Levels of phosphorylated eIF2aalpha; and eIF4E do not correlate with cell permissivity. Expression of Us11 in GSCs rescues replication of 34.5-deleted oHSV. The difference in permissivity between GSCs and ScGCs to 34.5-deleted oHSV illustrates a selective translational regulatory pathway in GSCs that may be operative in other stem-like cells and has implications for creating oHSVs.

    IMPORTANCE Herpes simplex virus (HSV) can be genetically engineered to endow cancer selective replication and oncolytic activity. 34.5, a key neurovirulence gene, has been deleted in all oncolytic HSVs in clinical trial for glioma. Glioblastoma stem-like cells (GSCs) are a subpopulation of tumor cells thought to drive tumor heterogeneity and therapeutic resistance. GSCs are non-permissive for 34.5-deleted HSV, while non-stem-like cancer cells from the same patient tumors are permissive. GSCs restrict true late protein synthesis, despite normal viral DNA replication and transcription of all kinetic classes. This is specific for true late translation, as early and leaky late transcripts are translated late in infection, notwithstanding shutoff of cellular protein synthesis. Expression of Us11 in GSCs rescues the replication of 34.5-deleted HSV. We have identified a cell type specific innate response to HSV1 that limits oncolytic activity in glioblastoma.

  • Differential responses by human respiratory epithelial cell lines to respiratory syncytial virus reflect distinct patterns of infection control [PublishAheadOfPrint]

  • RSV infects small foci of respiratory epithelial cells via infected droplets. Infection induces expression of types I and III interferons (IFNs) and pro-inflammatory cytokines, the balance of which may restrict viral replication and affect disease severity. We explored this balance by infecting two respiratory epithelial cell lines with low doses of recombinant RSV expressing green fluorescent protein (rgRSV). A549 cells were highly permissive whereas BEAS-2B cells restricted infection to individual cells or small foci. After infection, A549 cells expressed higher levels of IFN-bbeta;, IFN-, and NF-B inducible pro-inflammatory cytokines. In contrast, BEAS-2B cells expressed higher levels of anti-viral interferon-stimulated genes, pattern recognition receptors, and other signaling intermediaries constitutively and after infection. Transcriptome analysis revealed that constitutive expression of antiviral and pro-inflammatory genes predicted responses by each cell line. These two cell lines provide a model for elucidating critical mediators of local control of viral infection in respiratory epithelial cells.

    Importance Airway epithelium is both the primary target and the first defense against respiratory syncytial virus (RSV). Whether RSV replicates and spreads to adjacent epithelial cells depends on the quality of their innate immune response. A549 and BEAS-2B are alveolar and bronchial epithelial cell lines, respectively, that are often used to study RSV infection. We show that A549 cells are permissive to RSV infection and express genes characteristic of a pro-inflammatory response. By contrast, BEAS-2B cells restrict infection and express genes characteristic of an antiviral response associated with expression of types I and III interferons. Transcriptome analysis of constitutive gene expression revealed patterns that may predict the response of each cell line to infection. This study suggests that restrictive and permissive cell lines may provide a model towards identifying critical mediators of local control of infection, and stresses the importance of the constitutive antiviral state towards the response to viral challenge.

  • Protein Phosphatase 1{alpha} Interacts with Venezuelan Equine Encephalitis Virus Capsid Protein and Regulates Viral Replication through Modulation of Capsid Phosphorylation [PublishAheadOfPrint]

  • Protein Phosphatase 1 (PP1) is a serine/threonine phosphatase which has been implicated in the regulation of a number of viruses including HIV-1, Ebolavirus and Rift Valley fever virus. Catalytic subunits of PP1 (PP1aalpha;, PP1bbeta;, or PP1) interact with a host of regulatory subunits and target a wide variety of cellular substrates through a combination of short binding motifs, including an RVxF motif present in the majority of PP1 regulatory subunits. Targeting the RVxF interacting site on PP1 with the small molecule, 1E7-03 inhibits HIV-1, Ebolavirus, and Rift Valley fever virus replication. Here we determined the effect of PP1 on Venezuelan equine encephalitis virus (VEEV) replication. Treatment of VEEV infected cells with 1E7-03 decreased viral replication by over 2 logs (EC50=0.6 mmu;M). 1E7-03 treatment reduced viral titers starting at 8 hours post infection. Viral replication was also decreased after treatment with PP1aalpha;-targeting siRNA. Confocal microscopy demonstrated that PP1aalpha; shuttles toward the cytosol during infection with VEEV and that PP1aalpha; colocalizes with VEEV capsid. Co-immunoprecipitation experiments confirmed VEEV capsid interacting with PP1aalpha;. Furthermore, immunoprecipitation and mass spectrometry data showed that VEEV capsid is phosphorylated and that phosphorylation is moderated by PP1aalpha;. Finally, less viral RNA is associated with capsid after treatment with 1E7-03. Coupled with data that shows 1E7-03 inhibits several alphaviruses, this study indicates that inhibition of the PP1aalpha; RVxF binding pocket is a promising therapeutic target and provides novel evidence that PP1aalpha; modulation of VEEV capsid phosphorylated influences viral replication.

    Importance

    Venezuelan equine encephalitis virus (VEEV) causes moderate flu-like symptoms and can lead to severe encephalitic disease and potentially death. There are no currently FDA approved therapeutics or vaccines for human use and understanding the molecular underpinning of host:virus interactions can aid in the rational design of intervention strategies. The significance of our research is in identifying the interaction between Protein Phosphatase 1 (PP1) and the viral capsid protein. This interaction is important for viral replication as inhibition of PP1 results in decrease viral replication. Inhibition of PP1 also inhibited multiple biomedically important alphaviruses, indicating PP1 may be a potential therapeutic target for alphavirus induced disease.

  • Mutational Analysis of the Bovine Hepacivirus Internal Ribosome Entry Site [PublishAheadOfPrint]

  • In recent years, hepatitis C virus (HCV)-related viruses were identified in several species including dogs, horses, bats and rodents. Additionally, a novel virus of the genus Hepacivirus has been discovered in bovine samples and was termed bovine hepacivirus (BovHepV). Prediction of the BovHepV IRES (internal ribosome entry site) structure revealed strong similarities to the HCV IRES structure comprising domains II, IIIabcde, pseudoknot IIIf and domain IV with the initiation codon AUG. Unlike HCV, only one microRNA-122 (miR-122) binding site could be identified in the BovHepV 5' NTR. In this study, we analyzed the necessity of BovHepV IRES domains to initiate translation and investigated possible interactions between the IRES and core coding sequences by using a dual luciferase reporter assay. Our results suggest that such long-range interactions within the viral genome can affect IRES-driven translation. Moreover, the significance of a possible miR-122 binding to the BovHepV IRES was investigated. When analyzing translation in human Huh-7 cells with high amounts of endogenous miR-122, introduction of point mutations to the miR-122 binding site resulted in reduced translation efficiency. Similar results were observed in HeLa cells after substitution of miR-122. Nevertheless, absence of pronounced effects in a bovine hepatocyte cell line, expressing hardly any miR-122 as well suggests additional functions of this host factor in virus replication.

    Importance

    Several members of the family Flaviviridae including HCV have adapted cap-independent translation strategies to overcome canonical eukaryotic translation pathways and use cis-acting RNA-elements, designated as viral internal ribosome entry sites (IRES) to initiate translation. Although novel hepaciviruses have been identified in different animal species, only limited information is available on their biology on molecular level. Therefore, our aim was a fundamental analysis of BovHepV IRES functions. The findings which show that functional IRES elements are also crucial for BovHepV translation expand our knowledge on molecular mechanism of hepacivirus propagation. In the course of that, we also studied possible effects of one major host factor implicated in HCV pathogenesis, miR-122. The results of mutational analyses suggested that miR-122 enhances virus translation mediated by BovHepV IRES.

  • Rab5, Rab7, and Rab11 are required for caveolae-dependent endocytosis of classic swine fever virus in porcine alveolar macrophages [PublishAheadOfPrint]

  • The members of Flaviviridae utilize several endocytic pathways to enter a variety of host cells. Our previous work showed that classical swine fever virus (CSFV) enters porcine kidney (PK-15) cells through a clathrin-dependent pathway that requires Rab5 and Rab7. The entry mechanism for CSFV into other cell lines remains unclear, for instance, porcine alveolar macrophages (3D4/21 cells). More importantly, the trafficking of CSFV within endosomes controlled by Rab GTPases is unknown in 3D4/21 cells. In this study, entry and post-internalization of CSFV were analyzed using chemical inhibitors, RNA interference, and dominant negative (DN) mutants. Our data demonstrated that CSFV entry into 3D4/21 cells depends on caveolae, dynamin, and cholesterol, but not clathrin or macropinocytosis. The effects of DN mutants and knockdown of four Rab proteins that regulate endosomal trafficking were examined on CSFV infection, respectively. The results showed that Rab5, Rab7, and Rab11, but not Rab9, regulate CSFV endocytosis. Confocal microscopy showed that virus particles colocalize with Rab5, Rab7, or Rab11 within 30 min after virus entry, and further with lysosomes, suggesting that after internalization CSFV moves to early, late, and recycling endosomes, and then into lysosomes before the release of the viral genome. Our findings provide insights into the life cycle of pestiviruses in macrophages.

    Importance

    Classical swine fever (CSF), is caused by classical swine fever virus (CSFV). The disease is notifiable to the OIE in most countries and causes significant financial losses to the pig industry globally. Understanding the processes of CSFV endocytosis and post-internalization will advance our knowledge of the disease and provide potential novel drug targets against CSFV. With this objective, we used systematic approaches to dissect these processes in CSFV-infected 3D4/21 cells. The data presented here demonstrate for the first time to our knowledge that CSFV is able to enter cells via caveolae-mediated endocytosis that requires Rab5, Rab7 and Rab11, in addition to the previously described classical clathrin- dependent pathway that requires Rab5 and Rab7. The characterization of CSFV entry will further promote our current understanding of Pestivirus cellular entry pathways and provide novel targets for antiviral drug development.

  • The major tegument protein of bovine herpesvirus-1, VP8, interacts with DNA damage response proteins and induces apoptosis. [PublishAheadOfPrint]

  • VP8, the UL47 gene product in bovine herpes virus-1 (BoHV-1), is a major tegument protein, essential for virus replication in vivo. The major DNA damage response protein, ataxia telangiectasia mutated (ATM), phosphorylates Nijmegen breakage syndrome (NBS1) and structural maintenance of chromosome-1 (SMC1) proteins during the DNA damage response. VP8 was found to interact with ATM and NBS1 during transfection and BoHV-1 infection. However, VP8 did not interfere with phosphorylation of ATM in transfected or BoHV-1-infected cells. In contrast, VP8 inhibited phosphorylation of both NBS1 and SMC1 in transfected cells, as well as in BoHV-1-infected cells, but not in cells infected with a VP8 deletion mutant (BoHV-1UL47). Inhibition of NBS1 and SMC1 phosphorylation was observed at 4 h post infection by nuclear VP8. Furthermore, ultraviolet light (UV)-induced cyclobutane pyrimidine dimer (CPD) repair was reduced in the presence of VP8, and VP8 in fact enhanced etoposide or UV-induced apoptosis. This suggests that VP8 blocks the ATM/NBS1/SMC1 pathway and inhibits DNA repair. VP8 induced apoptosis in VP8-transfected cells through caspase-3 activation. The fact that BoHV-1 is known to induce apoptosis through caspase-3 activation is in agreement with this observation. The role of VP8 was confirmed by the observation that BoHV-1 induced significantly more apoptosis than BoHV-1UL47. These data reveal a potential role of VP8 in the modulation of the DNA damage response pathway and induction of apoptosis during BoHV-1 infection.

    IMPORTANCE To our knowledge, the effect of BoHV-1 infection on the DNA damage response has not been characterized. Since BoHV-1UL47 was previously shown to be avirulent in vivo, VP8 is critical for the progression of viral infection. We demonstrated that VP8 interacts with DNA damage response proteins and disrupts the ATM-NBS1-SMC1 pathway by inhibiting phosphorylation of DNA repair proteins, NBS1 and SMC1. Furthermore, interference of VP8 with DNA repair was correlated to decreased cell viability and increased DNA damage-induced apoptosis. These data show that BoHV-1 VP8 developed a novel strategy to interrupt the ATM signaling pathway and to promote apoptosis. These results further enhance our understanding of the functions of VP8 during BoHV-1 infection and provide an additional explanation for the reduced virulence of BoHV-1UL47.

  • Porcine deltacoronavirus accessory protein NS6 antagonizes IFN-{beta} production by interfering with the binding of RIG-I/MDA5 to double-stranded RNA [PublishAheadOfPrint]

  • Porcine deltacoronavirus (PDCoV) has recently emerged as an enteric pathogen that can cause serious vomiting and diarrhea in suckling piglets. The first outbreak of PDCoV occurred in the United States in 2014 and was followed by reports of PDCoV in South Korea, China, Thailand, Lao people's Democratic Republic, and Vietnam, leading to economic losses for pig farms and posing considerable threat to the swine industry worldwide. Our previous studies have shown that PDCoV encodes three accessory proteins, NS6, NS7, and NS7a, but the functions of these proteins in viral replication, pathogenesis, and immune regulation remain unclear. Here, we found that ectopic expression of accessory protein NS6 significantly inhibits Sendai virus-induced interferon-bbeta; (IFN-bbeta;) production, as well as the activation of transcription factors IRF3 and NF-B. Interestingly, NS6 does not impede the IFN-bbeta; promoter activation mediated via key molecules in the RIG-I-like receptor (RLR) signaling pathway, specifically RIG-I, MDA5, and their downstream molecules MAVS, TBK1, IKK, and IRF3. Further analyses revealed that NS6 is not a RNA-binding protein; however, it interacts with RIG-I/MDA5. This interaction attenuates the binding of double-stranded RNA by RIG-I/MDA5, resulting in the reduction of RLR-mediated IFN-bbeta; production. Taken together, our results demonstrate that ectopic expression of NS6 antagonizes IFN-bbeta; production by interfering with the binding of RIG-I/MDA5 to double-stranded RNA, revealing a new strategy employed by PDCoV accessory proteins to counteract the host innate antiviral immune response.

    IMPORTANCE

    Coronavirus accessory proteins are species-specific, and they perform multiple functions in viral pathogenicity and immunity, such as acting as interferon (IFN) antagonists and cell death inducers. Our previous studies have shown that porcine deltacoronavirus (PDCoV) encodes three accessory proteins. Here, we demonstrated for the first time that PDCoV accessory protein NS6 antagonizes IFN-bbeta; production by interacting with RIG-I and MDA5 to impede their association with double-stranded RNA. This is an efficient strategy of antagonizing type I IFN production by disrupting the binding of host pattern recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs). These findings deepen our understanding of the function of accessory protein NS6 and may direct us toward novel therapeutic targets and lead to the development of more effective vaccines against PDCoV infection.

  • The HTLV-1 basic leucine zipper factor (HBZ) attenuates repair of double-stranded DNA breaks via non-homologous end joining (NHEJ) [PublishAheadOfPrint]

  • Adult T-cell leukemia (ATL) is a fatal malignancy of CD4+ T-cells infected with human T-cell leukemia virus type I (HTLV-1). ATL cells often exhibit random gross chromosomal rearrangements that are associated with the induction and improper repair of double-stranded DNA breaks (DSBs). The viral oncoprotein Tax has been reported to impair DSB repair, but is not shown to be consistently expressed throughout all phases of infection. The viral oncoprotein HTLV-1 basic leucine zipper factor (HBZ) is consistently expressed prior to and throughout disease progression, but it is unclear whether it also influences DSB repair. We report that HBZ attenuates DSB repair by non-homologous end joining (NHEJ), in a manner dependent upon the basic leucine zipper (bZIP) domain. HBZ was found to interact with two vital members of the NHEJ core machinery, Ku70 and Ku80, and to be recruited to DSBs in a bZIP-dependent manner in vitro. We observed that HBZ expression also resulted in a bZIP-dependent delay in DNA-PK activation following treatment with etoposide. Though Tax is reported to interact with Ku70, we did not find Tax expression to interfere with HBZ:Ku complex formation. However, as Tax was reported to saturate NHEJ, we found this effect masked the attenuation of NHEJ by HBZ. Overall, these data suggest that DSB repair mechanisms are impaired not only by Tax, but also by HBZ, and show that HBZ expression may significantly contribute to the accumulation of chromosomal abnormalities during HTLV-1 mediated oncogenesis.

    IMPORTANCE Human T-cell leukemia virus type 1 (HTLV-1) infects 15-20 million people worldwide. Approximately 90% of infected individuals are asymptomatic and may remain undiagnosed, increasing the risk that they will unknowingly transmit the virus. About 5% of the HTLV-1 positive population to develop Adult T-cell Leukemia (ATL), a fatal disease that is not highly responsive to treatment. Though ATL development remains poorly understood, two viral proteins, Tax and HBZ, have been implicated in driving disease progression by manipulating host cell signaling and transcriptional pathways. Unlike Tax, HBZ expression is consistently observed in all infected individuals, making it important to elucidate the specific role of HBZ in disease progression. Here, we present evidence that HBZ could promote the accumulation of double-stranded DNA breaks (DSBs) through the attenuation of the non-homologous end joining (NHEJ) repair pathway. This effect may lead to genome instability, ultimately contributing to the development of ATL.

  • Glycoprotein 3 of porcine reproductive and respiratory syndrome virus exhibits an unusual hairpin-like membrane topology [PublishAheadOfPrint]

  • The glycoprotein GP3 of the Arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) consists of a cleaved signal peptide, a highly glycosylated domain, a short hydrophobic region and an unglycosylated C-terminal domain. GP3 is supposed to form a complex with GP2 and GP4 in virus particles, but secretion of GP3 from cells has also been reported.

    We analyzed the membrane topology of GP3 from various PRRSV strains. A fraction of the protein is secreted from transfected cells; GP3 from PRRSV-1 strains to a greater extent than GP3 from PRRSV-2 strains. This secretion behavior is reversed after exchange of the variable C-terminal domain. A fluorescence protease protection assay shows that the C-terminus of GP3, fused to GFP, is resistant against proteolytic digestion in permeabilized cells. Furthermore, glycosylation sites inserted into the C-terminal part of GP3 are used. Both experiments indicate that the C-terminus of GP3 is translocated into the lumen of the endoplasmic reticulum. Deletion of the conserved hydrophobic region greatly enhances secretion of GP3 and fusion of this domain to GFP promotes membrane anchorage. Bioinformatics suggests that the hydrophobic region might form an amphipathic helix. Accordingly, exchanging only a few amino acids in its hydrophilic face prevents and in its hydrophobic face enhances secretion of GP3. Exchanging the latter amino acids in the context of the viral genome did not affect release of virions, but released particles were not infectious. In sum, GP3 exhibits an unusual hairpin-like membrane topology that might explain why a fraction of the protein is secreted.

    IMPORTANCE

    The porcine reproductive and respiratory syndrome virus (PRRSV) is the most important pathogen in the pork industry. It causes persistent infections that lead to reduced weight gain of piglets; highly pathogenic strains even kill 90% of an infected pig population. PRRSV cannot be eliminated from pig farms by vaccination due to the large amino acid variability between the existing strains, especially in the glycoproteins. Here we analyzed basic structural features of glycoprotein 3 (GP3) from various PRRSV strains. We show that the protein exhibits an unusual hairpin-like membrane topology; membrane anchoring might occur via an amphipathic helix. This rather weak membrane anchor explains why a fraction of the protein is secreted from cells. Interestingly, PRRSV-1 strains secrete more GP3 than PRRSV-2. We speculate that secreted GP3 might play a role during PRRSV infection of pigs; it might serve as a decoy to distract antibodies away from virus particles.

  • Cytoplasmic re-localization and colocalization with viroplasms of host cell proteins, and their role in rotavirus infection [PublishAheadOfPrint]

  • Rotavirus replicates in the cytoplasm of infected cells in unique virus-induced cytoplasmic inclusion bodies called viroplasms (VM), which are nucleated by two essential viral non-structural proteins NSP2 and NSP5. However, the precise composition of the VM, intracellular localization of host proteins during virus infection, their association with VM or role in rotavirus growth remained largely unexplored. Mass-spectrometry analyses revealed the presence of several host heterogeneous nuclear ribonucleoproteins (hnRNPs) and AU-rich element-binding proteins (ARE-BPs) and cytoplasmic proteins from uninfected MA104 cell extracts in the pull-down (PD) complexes of purified viroplasmic proteins NSP2 and NSP5. Immunoblot analyses of the PD complexes from RNase-treated and untreated cell extracts, co-immunoprecipitation complexes using RNase-treated infected cell lysates and direct binding assays using purified recombinant proteins further demonstrated that the interactions of the majority of the hnRNPs and ARE-BPs with viroplasmic proteins are RNA-independent. Time-course immunoblot analysis of the nuclear and cytoplasmic fractions from rotavirus-infected and mock-infected cells and immunofluorescence confocal microscopy analyses of virus-infected cells revealed surprising sequestration of the majority of the re-localized host proteins in viroplasms. Ectopic over-expression and siRNA-mediated down-regulation of expression analyses revealed that host proteins either promote or inhibit viral protein expression and progeny virus production in virus-infected cells. This study demonstrates that rotavirus induces cytoplasmic re-localization and sequestration of a large number of nuclear and cytoplasmic proteins in viroplasms, subverting essential cellular processes in both the compartments to promote rapid virus growth, and reveals that the composition of rotavirus viroplasms is much more complex than that is currently understood.

    IMPORTANCE Rotavirus replicates exclusively in the cytoplasm. Knowledge on re-localization of nuclear proteins to the cytoplasm or the role(s) of host proteins in rotavirus infection is very limited. In this study, it is demonstrated that rotavirus infection induces cytoplasmic re-localization of a large number of nuclear RNA-binding proteins (hnRNPs and AU-rich element-binding proteins). Except for a few, most nuclear hnRNPs and ARE-BPs, nuclear transport proteins and some cytoplasmic proteins directly interacted with viroplasmic proteins NSP2 and NSP5 in an RNA-independent manner and become sequestered in the viroplasms in infected cells. The host proteins differentially affected viral gene expression and virus growth. This study demonstrates that rotavirus induces re-localization and sequestration of a large number of host proteins in viroplasms, affecting the host processes in both the compartments and generating conditions conducive for virus growth in the cytoplasm in infected cells.

  • Bag2 is a component of a cytosolic extraction machinery that promotes membrane penetration of a nonenveloped virus [PublishAheadOfPrint]

  • During entry, the nonenveloped polyomavirus (PyV) SV40 traffics from the cell surface to the endoplasmic reticulum (ER) where it penetrates the ER membrane to reach the cytosol; the virus then transports into the nucleus to cause infection. Although a coherent understanding of SV40's host entry is emerging, how the virus is ejected from the ER into the cytosol remains mysterious. Our previous analyses revealed that the cytosolic Hsc70-SGTA-Hsp105 complex binds to and extracts SV40 from the ER into the cytosol. We now report that the nucleotide exchange factor (NEF) Bag2 stimulates SV40 release from Hsc70, thereby enabling successful virus arrival to the cytosol that leads to infection. Hsp105, another NEF of Hsc70, displays overlapping function with Bag2, underscoring the importance of this release reaction. Our findings identify a new component of an extraction machinery essential during membrane penetration of a nonenveloped virus, and provide further mechanistic insights into this process.

    IMPORTANCE How a nonenveloped virus penetrates a biological membrane to cause infection is a mystery. For the nonenveloped polyomavirus SV40, transport across the ER membrane to reach the cytosol is an essential virus infection step. Here we identify a novel component of a cytosolic Hsc70-dependent chaperone complex called Bag2 that extracts SV40 from the ER into the cytosol. Bag2 does so by triggering SV40 release from Hsc70, thus ensuring that the virus reaches the cytosol en route for productive infection.

  • Efficient delivery of HCMV T cell antigens by attenuated Sendai virus vectors [PublishAheadOfPrint]

  • Human Cytomegalovirus (HCMV) represents a major cause of clinical complications during pregnancy as well as immunosuppression and the licensing of a protective HCMV vaccine remains an unmet global need. Herein, we designed and validated novel Sendai virus (SeV) vectors delivering T cell immunogens IE-1 and pp65. To enhance vector safety, we used a replication-deficient strain (rdSeV) that infects target cells in a non-productive manner while retaining viral gene expression. In this study, we explored the impact that transduction with rdSeV has on human dendritic cells (DCs) by comparing it to the parental, replication-competent Sendai virus strain (rcSeV) as well as the poxvirus strain Modified Vaccinia Ankara (MVA). We found that wild-type SeV is capable of replicating to high titers in DCs while rdSeV infects cells abortively. Due to the higher degree of attenuation, IE-1 and pp65 protein levels mediated by rdSeV after infection of DCs were markedly reduced compared to the parental Sendai virus recombinants, but antigen-specific restimulation of T cell clones was not negatively affected by this. Importantly, rdSeV showed reduced cytotoxic effects compared to rcSeV and MVA and was capable of mediating DC maturation as well as secretion of IFNaalpha; and IL-6. Finally, in a challenge model with a murine Cytomegalovirus (MCMV) strain carrying an HCMV pp65 peptide, we found that viral replication was restricted if mice were previously vaccinated with rdSeV-pp65. Taken together, these data demonstrate that rdSeV has great potential as a vector system for the delivery of HCMV immunogens.

    Importance

    Cytomegalovirus (HCMV) is a highly prevalent bbeta;-Herpesvirus that establishes life-long latency after primary infection. Congenital HCMV infection is the most common viral complication in newborns causing a number of late sequelae ranging from impaired hearing to mental retardation. At the same time, managing HCMV reactivation during immunosuppression remains a major hurdle in post-transplant care. Since options for the treatment of HCMV infection are still limited, the development of a vaccine to confine HCMV-related morbidities is urgently needed. We generated new vaccine candidates in which the main targets of T cell immunity during natural HCMV infection, IE-1 and pp65, are delivered by a replication-deficient, Sendai virus-based vector system. In addition to classical prophylactic vaccine concepts, these vectors could also be used for therapeutic applications, thereby expanding preexisting immunity in high-risk groups such as transplant recipients or for immunotherapy of glioblastomas expressing HCMV antigens.

  • Reovirus Nonstructural Protein {sigma}NS Acts as an RNA-Stability Factor Promoting Viral Genome Replication [PublishAheadOfPrint]

  • Viral nonstructural proteins, which are not packaged into virions, are essential for replication of most viruses. Reovirus, a nonenveloped, double-stranded RNA (dsRNA) virus, encodes three nonstructural proteins that are required for viral replication and dissemination in the host. Reovirus nonstructural protein NS is a single-stranded RNA (ssRNA)-binding protein that must be expressed in infected cells for production of viral progeny. However, activities of NS during individual steps of the reovirus replication cycle are poorly understood. We explored the function of NS by disrupting its expression during infection using cells expressing a small interfering RNA (siRNA) targeting the NS-encoding S3 gene and found that NS is required for viral genome replication. Using complementary biochemical assays, we determined that NS forms complexes with viral and nonviral RNAs. We also discovered that NS increases RNA half-life using in vitro and cell-based RNA degradation experiments. Cryo-electron microscopy revealed that NS and ssRNAs organize into long, filamentous structures. Collectively, our findings indicate that NS functions as an RNA-binding protein that increases viral RNA half-life. These results suggest that NS forms RNA-protein complexes in preparation for genome replication.

    IMPORTANCE Following infection, viruses synthesize nonstructural proteins that mediate viral replication and promote dissemination. Viruses from the Reoviridae family encode nonstructural proteins that are required for the formation of progeny viruses. Although nonstructural proteins of different Reoviridae family viruses are diverged in primary sequence, these proteins are functionally homologous and appear to facilitate conserved mechanisms of dsRNA virus replication. Using in vitro and cell-culture approaches, we found that the mammalian reovirus nonstructural protein NS binds and stabilizes viral RNA and is required for genome synthesis. This work contributes new knowledge about basic mechanisms of dsRNA virus replication and provides a foundation for future studies to determine how viruses in the Reoviridae family assort and replicate their genomes.

  • Microglia Are Critical in Host Defense Against Prion Disease [PublishAheadOfPrint]

  • Microglial cells in the central nervous system play important roles in neurodevelopment and resistance to infection, yet microglia can become neurotoxic under some conditions. An early event during prion infection is the activation of microglia and astrocytes in the brain prior to damage or death of neurons. Previous prion disease studies using two different strategies to manipulate signaling through the microglial receptor CSF-1R reported contrary effects on survival from prion disease. However, in these studies, reduction of microglial numbers and function were variable, thus confounding interpretation of the results. In the present work, we used oral treatment with a potent inhibitor of CSF-1R, PLX5622, to eliminate 78 to 90% of microglia from cortex shortly after and during the course of prion infection. Oral drug treatment early after infection with the RML scrapie strain significantly accelerated vacuolation, astrogliosis and deposition of disease-associated prion protein. Furthermore, drug-treated mice had advanced clinical disease requiring euthanasia 31 days earlier than untreated control mice. Similarly, PLX5622 treatment during the preclinical phase at 80 days post-infection with RML scrapie also accelerated disease and resulted in euthanasia of mice 33 days earlier than infected controls. PLX5622 also accelerated clinical disease after infection with scrapie strains ME7 and 22L. Thus, microglia are critical in host defense during prion disease. The early accumulation of PrPSc in the absence of microglia suggested that microglia may function by clearing PrPSc resulting in longer survival.

    IMPORTANCE Microglia contribute to many aspects of health and disease. When activated, microglia can be beneficial by repairing damage in the CNS or they can turn harmful by becoming neurotoxic. In prion and prion-like diseases, the involvement of microglia in disease is unclear. Previous studies suggest that microglia can either speed up or slow down disease. In this study, we infected mice with prions and depleted microglia from the brains of mice using PLX5622, an effective CSF-1R tyrosine kinase inhibitor. Microglia were markedly reduced in brains, and prion disease was accelerated, so that mice needed to be euthanized 20 to 33 days earlier than infected control mice due to advanced clinical disease. Similar results occurred when mice were treated with PLX5622 at 80 days after infection, which was just prior to the start of clinical signs. Thus, microglia are important for removing prions, and the disease is faster when microglia are depleted.

  • Entry of Herpes Simplex Virus 1 into Epidermis and Dermal Fibroblasts is Independent of the Scavenger Receptor MARCO [PublishAheadOfPrint]

  • To enter host cells, herpes simplex virus 1 (HSV-1) initially attaches to cell surface glycosaminoglycans followed by requisite binding to one of several cellular receptors leading to viral internalization. Although virus-receptor interactions have been studied in various cell lines, the contributions of individual receptors to uptake into target tissues such as mucosa, skin or cornea are not well understood. We demonstrated that nectin-1 acts as major receptor for HSV-1 entry into murine epidermis, while HVEM can serve as alternative receptor. Recently, the macrophage receptor with collagenous structure (MARCO) has been described to mediate adsorption of HSV-1 to epithelial cells. Here, we investigated the impact of MARCO on the entry process of HSV-1 into the two major cell types of skin, keratinocytes in the epidermis and fibroblasts in the underlying dermis. Using ex vivo infection of murine epidermis, we showed that HSV-1 entered basal keratinocytes of MARCO-/- epidermis as efficiently as those of control epidermis. In addition, entry into dermal fibroblasts was not impaired in the absence of MARCO. When we treated epidermis, primary keratinocytes or fibroblasts with polyinosinic acid (Poly(I)), a ligand for class A scavenger receptors, HSV-1 entry was strongly reduced. As we observed reducing effects of Poly(I) also in the absence of both MARCO and scavenger receptor A1, we concluded that the inhibitory effects of Poly(I) on HSV-1 infection are not directly linked to class A scavenger receptors. Overall, our results support that HSV-1 entry into skin cells is independent of MARCO.

    IMPORTANCE During entry into its host cells, the human pathogen herpes simplex virus (HSV) interacts with various cellular receptors. Initially, receptor interaction can mediate cellular adsorption followed by receptor binding that triggers viral internalization. The intriguing question is, which receptors are responsible for the various steps during entry into the natural target tissues of HSV. Previously, we demonstrated the role of nectin-1 as a major and of HVEM as an alternative receptor for HSV-1 to invade murine epidermis. As MARCO has been described to promote infection in skin, we explored the predicted role of MARCO as a receptor that mediates adsorption to epithelial cells. Our infection studies of murine skin cells indicate that the absence of MARCO does not interfere with the efficiency of HSV-1 entry and that the inhibitory effect on viral adsorption by Poly(I), a ligand of MARCO, is independent of MARCO.

  • Cotesia congregata Bracovirus circles encoding PTP and Ankyrin genes integrate into the DNA of parasitized Manduca sexta hemocytes. [PublishAheadOfPrint]

  • Polydnaviruses (PDVs) are essential for the parasitism success of tens of thousands of species of parasitoid wasps. PDVs are present in wasp genomes as proviruses, which serve as template for the production of double stranded circular viral DNA carrying virulence genes that are injected into lepidopteran hosts. PDV circles do not contain genes coding for particle production hence impeding viral replication in caterpillar hosts during parasitism. Here we investigated the fate of PDV circles of Cotesia congregata bracovirus during parasitism of the tobacco hornworm, Manduca sexta, by the wasp Cotesia congregata. Sequences sharing similarities with Host integration Motifs (HIM) involved in integration into DNA of Microplitis demolitor bracovirus (MdBV) circles, could be identified in 12 CcBV circles, which encode PTP and VANK gene families involved in host immune disruption. A PCR approach performed on a subset of these circles indicated that they persisted in parasitized M. sexta hemocytes as linear forms, possibly integrated in host DNA. Furthermore by using a primer extension capture method based on these HIM motifs, and high through-put sequencing we could show that 8 over 9 circles tested were integrated in M. sexta hemocyte genomic DNA and that integration had occurred specifically using the HIM motif, indicating that a HIM-mediated specific mechanism was involved in their integration. Investigation of BV circle insertion sites at the genome scale revealed that certain genomic regions appeared to be enriched in BV insertions, but no specific M. sexta target site could be identified.

    Importance

    The identification of a specific and efficient integration mechanism shared by several bracovirus species opens the question of its role in Braconid parasitoid wasp parasitism success. Indeed results obtained here show massive integration of bracovirus DNA in somatic immune cells at each parasitism event of a caterpillar host. Given that bracoviruses do not replicate in infected cells, integration of viral sequences in host DNA might allow the production of PTP and VANK virulence proteins, within newly dividing cells of caterpillar hosts, that continue to develop during parasitism. Furthermore, this integration process could serve as a basis to understand how PDVs mediate the recently identified gene flux between parasitoid wasps and Lepidoptera and the frequency of these horizontal transfer events in nature.

  • Avian influenza viruses in wild birds: virus evolution in a multi-host ecosystem [PublishAheadOfPrint]

  • Wild ducks and gulls are the major reservoirs for avian influenza A viruses (AIVs). The mechanisms that drive AIV evolution are complex at sites where various duck and gull species from multiple flyways breed, winter or stage. The Republic of Georgia is located at the intersection of three migratory flyways: Central Asian Flyway, East Asian/East African Flyway and Black Sea/Mediterranean Flyway. For six consecutive years (2010-2016), we collected AIV samples from various duck and gull species that breed, migrate and overwinter in Georgia. We found substantial subtype diversity of viruses that varied in prevalence from year to year. Low pathogenic (LP)AIV subtypes included H1N1, H2N3, H2N5, H2N7, H3N8, H4N2, H6N2, H7N3, H7N7, H9N1, H9N3, H10N4, H10N7, H11N1, H13N2, H13N6, H13N8, H16N3, plus two H5N5 and H5N8 highly pathogenic (HP)AIVs belonging to clade 2.3.4.4. Whole genome phylogenetic trees showed significant host species lineage restriction for nearly all gene segments and significant differences for LPAIVs among different host species in observed reassortment rates, as defined by quantification of phylogenetic incongruence, and in nucleotide diversity. Hemagglutinin clade 2.3.4.4 H5N8 viruses, circulated in Eurasia during 2014-2015 did not reassort, but analysis after its subsequent dissemination during 2016-2017 revealed reassortment in all gene segments except NP and NS. Some virus lineages appeared to be unrelated to AIVs in wild bird populations in other regions with maintenance of local AIV viruses in Georgia, whereas other lineages showed considerable genetic inter-relationship with viruses circulating in other parts of Eurasia and Africa, despite relative under-sampling in the area.

    Importance

    Waterbirds (e.g., gulls/ducks) are natural reservoirs of avian influenza viruses (AIVs) and have been shown to mediate dispersal of AIV at inter-continental scales during seasonal migration. The segmented genome of influenza viruses enables viral RNA from different lineages to mix or re-assort when two viruses infect the same host. Such reassortant viruses have been identified in most major human influenza pandemics and several poultry outbreaks. Despite their importance, we have only recently begun to understand AIV evolution and reassortment in their natural host reservoirs. This comprehensive study illustrates of AIV evolutionary dynamics within a multi-host ecosystem at a stop-over site where three major migratory flyways intersect. Our analysis of this ecosystem over a six-year period provides a snapshot of how these viruses are linked to global AIV populations. Understanding the evolution of AIVs in the natural host is imperative to both mitigating the risk of incursion into domestic poultry and potential risk to mammalian hosts including humans.

  • Identification of plant virus receptor candidates in the stylets of their aphid vectors [PublishAheadOfPrint]

  • Plant viruses transmitted by insects cause tremendous losses in most important crops around the world. The identification of receptors of plant viruses within their insect vectors is a key challenge to understanding the mechanisms of transmission, and offers an avenue for future alternative control strategies to limit viral spread. We here report the identification of two cuticular proteins within aphid mouthparts, and we provide experimental support for the role of one of them in the transmission of a non-circulative virus. These two proteins, named Stylin-01 and Stylin-02, belong to the RR-1 cuticular protein subfamily, and are highly conserved among aphid species. Using an immunolabeling approach, they were localized in the maxillary stylets of the pea aphid Acyrthosiphon pisum and the green peach aphid Myzus persicae, in the acrostyle, an organ earlier shown to harbor receptors of a non-circulative virus. A peptide motif present at the C-terminus of both Stylin-01 and Stylin-02 is readily accessible all over the surface of the acrostyle. Competition for in vitro binding to the acrostyle was observed between an antibody targeting this peptide and the helper component protein P2 of Cauliflower mosaic virus. Furthermore, silencing stylin-01 but not stylin-02 gene through RNA interference decreased the efficiency of Cauliflower mosaic virus transmission by Myzus persicae. These results identify the first cuticular proteins ever reported within arthropod mouthparts and distinguish Stylin-01 as the best candidate receptor for the aphid transmission of non-circulative plant viruses.

    IMPORTANCE Most non-circulative plant viruses transmitted by insect vectors bind to their mouthparts. They are acquired and inoculated within seconds when insects hop from plant to plant. The receptors involved remains totally elusive due to long-standing technical bottleneck in working with insect cuticle. Here we characterize the role of the two first cuticular proteins ever identified in arthropod mouthparts. A domain of these proteins is directly accessible at the surface of the cuticle of the acrostyle, an organ at the tip of aphid stylets. The acrostyle has been shown to bind a plant virus and we consistently demonstrate that one of the identified proteins is involved in viral transmission. Our findings provide an approach to identify proteins in insect mouthparts and points at unprecedented gene candidate for plant virus receptor.

  • The PERK Arm of the Unfolded Protein Response Negatively Regulates Transmissible Gastroenteritis Virus Replication by Suppressing Protein Translation and Promoting Type I IFN Production [PublishAheadOfPrint]

  • Coronavirus replication is closely associated with the endoplasmic reticulum (ER), the primary cellular organelle for protein synthesis, folding, and modification. ER stress is a common consequence in coronavirus-infected cells. However, how the virus-induced ER stress influences coronavirus replication and pathogenesis remains controversial. Here, we demonstrated that infection with the alphacoronavirus transmissible gastroenteritis virus (TGEV) induced ER stress and triggered the unfolded protein response (UPR) in vitro and in vivo, and ER stress negatively regulated TGEV replication in vitro. Although TGEV infection activated all three UPR pathways (ATF6, IRE1, and PERK), the virus-triggered UPR suppressed TGEV replication in both ST and IPEC-J2 cells primarily through activation of the PERK-eIF2aalpha; axis, as shown by functional studies with overexpression, siRNA, or specific chemical inhibitors. Moreover, we demonstrated that PERK-eIF2aalpha; axis-mediated inhibition of TGEV replication is through phosphorylated eIF2aalpha;-induced overall attenuation of protein translation. In addition to direct inhibition of viral production, the PERK-eIF2aalpha; pathway activated NF-B and then facilitated type I IFN production, resulting in TGEV suppression. Taken together, our results suggest that the TGEV-triggered PERK-eIF2aalpha; pathway negatively regulates TGEV replication and represents a vital aspect of host innate responses to invading pathogens.

    IMPORTANCE The induction of ER stress is a common outcome in cells infected with coronaviruses. The UPR initiated by ER stress is actively involved in viral replication and modulates the host innate responses to the invading viruses, but these underlying mechanisms remain incompletely understood. We show here that infection with the alphacoronavirus TGEV elicited ER stress in vitro and in vivo, and the UPR PERK-eIF2aalpha; branch was predominantly responsible for the suppression of TGEV replication by ER stress. Furthermore, the PERK-eIF2aalpha; axis inhibited TGEV replication through direct inhibition of viral proteins due to global translation inhibition and type I IFN induction. These findings highlight a critical role of the UPR PERK-eIF2aalpha; pathway in modulating the host innate immunity and coronavirus replication.

  • Discovery and sequence analysis of four deltacoronaviruses from birds in the Middle East suggest interspecies jumping and recombination as potential mechanism for avian-to-avian and avian-to-mammalian transmission [PublishAheadOfPrint]

  • The emergence of Middle East respiratory syndrome showed once again that coronaviruses (CoVs) in animals are potential source for epidemics in humans. To explore the diversity of deltacoronaviruses in animals in the Middle East, we tested fecal samples from 1,356 mammals and birds in Dubai. Four novel deltacoronaviruses were detected from eight birds of four species by RTnndash;PCR: FalCoV UAE-HKU27 from a falcon, HouCoV UAE-HKU28 from a houbara bustard, PiCoV UAE-HKU29 from a pigeon and QuaCoV UAE-HKU30 from five quails. Complete genome sequencing showed that FalCoV UAE-HKU27, HouCoV UAE-HKU28 and PiCoV UAE-HKU29 belong to the same CoV species, suggesting recent interspecies transmission between falcons and their preys, houbara bustards and pigeons possibly along the food chain. Western blot detected specific anti-FalCoV UAE-HKU27 antibodies in 33 (75%) of 44 falcon serum samples, supporting genuine infection in falcons after virus acquisition. QuaCoV UAE-HKU30 belongs to the same CoV species as PorCoV HKU15 and SpCoV HKU17 discovered previously from swine and tree sparrows respectively, supporting avian-to-swine transmission. Recombination involving the spike protein is common among deltacoronaviruses, which may facilitate cross-species transmission. FalCoV UAE-HKU27, HouCoV UAE-HKU28 and PiCoV UAE-HKU29 were originated from recombination between WECoV HKU16 and MRCoV HKU18; QuaCoV UAE-HKU30 from recombination between PorCoV HKU15/SpCoV HKU17 and MunCoV HKU13, and PorCoV HKU15 from recombination between SpCoV HKU17 and BuCoV HKU11. Birds in the Middle East are hosts for diverse deltacoronaviruses with potential for interspecies transmission.

    IMPORTANCE

    During an attempt to explore the diversity of deltacoronaviruses among mammals and birds in Dubai, four novel deltacoronaviruses were detected in fecal samples from eight birds of four different species: FalCoV UAE-HKU27 from a falcon, HouCoV UAE-HKU28 from a houbara bustard, PiCoV UAE-HKU29 from a pigeon and QuaCoV UAE-HKU30 from five quails. Genome analysis revealed evidence of recent interspecies transmission between falcons and their preys, houbara bustards and pigeons possibly along the food chain, as well as avian-to-swine transmission. Recombination, which is known to occur frequently in some coronaviruses, was also common among these deltacoronaviruses and predominantly occurred at the spike region. Such recombination, involving the receptor binding protein, may contribute to the emergence of new viruses capable of infecting new hosts. Birds in the Middle East are hosts for diverse deltacoronaviruses with potential for interspecies transmission.

  • HIV-1 activation of innate immunity depends strongly on the intracellular level of TREX1 and sensing of incomplete reverse transcription products [PublishAheadOfPrint]

  • TREX1 has been reported to degrade cytosolic immune-stimulatory DNA, including viral DNA generated during HIV-1 infection, but the dynamic range of its capacity to suppress innate immune stimulation is unknown and its full role in the viral life cycle remains unclear. A main purpose of our study was to determine how the intracellular level of TREX1 affects HIV-1 activation and avoidance of innate immunity. Using stable over-expression and CRISPR-mediated gene disruption, we engineered a range of TREX1 levels in human THP-1 monocytes. Increasing the level of TREX1 dramatically suppressed HIV-1 induction of interferon-stimulated genes (ISGs). Productive infection and integrated proviruses were equal to increased. Knocking out TREX1 impaired viral infectivity, increased early viral cDNA and caused ten-fold or greater increases in HIV-1 ISG induction. Knockout of cyclic GMP-AMP synthase (cGAS) abrogated all ISG induction. Moreover, cGAS knockout produced no increase in single cycle infection, establishing that HIV-1 DNA-triggered signaling is not rapid enough to impair the initial ISG-triggering infection cycle. Disruption of the HIV-1 capsid by PF74 also induced ISGs and this was TREX1 level-dependent, required reverse transcriptase catalysis, and was eliminated by cGAS gene knockout. Thus, the intracellular level of TREX1 pivotally modulates innate immune induction by HIV-1. Partial HIV-1 genomes are the TREX1 target and are sensed by cGAS. The nearly complete lack of innate immune induction despite equal to increased viral integration observed when the TREX1 protein level is experimentally elevated indicates that integration-competent genomes are shielded from cytosolic sensor-effectors during uncoating and transit to the nucleus.

    IMPORTANCE

    Much remains unknown about how TREX1 influences HIV-1 replication, whether it targets full-length viral DNA versus partial intermediates, how intracellular TREX1 protein levels correlate with ISG induction, and whether TREX1 digestion of cytoplasmic DNA and subsequent cGAS pathway activation affects both initial and subsequent cycles of infection. To answer these questions, we experimentally varied the intracellular level of TREX1 and show that this strongly determines the innate immunogenicity of HIV-1. In addition, several lines of evidence including time of addition experiments with drugs that impair reverse transcription or capsid integrity showed that the pathogen-associated molecular patterns sensed after viral entry contain DNA, are TREX1 and cGAS substrates, and are derived from incomplete RT products. In contrast, the experiments demonstrate that full-length integration competent viral DNA is immune to TREX1. Treatment approaches that reduce TREX1 levels or facilitate release of DNA intermediates may advantageously combine enhanced innate immunity with antiviral effects.

  • A novel strategy to adapt SHIV-E1 carrying env from an RV144 volunteer to rhesus macaques: coreceptor switch and final recovery of a pathogenic virus with exclusive R5 tropism [PublishAheadOfPrint]

  • The Phase III RV144 HIV vaccine trial conducted in Thailand remains the only study to show efficacy in decreasing HIV acquisition risk. In Thailand, circulating recombinant forms of HIV clades A/E (CRF01_AE) predominate; in such viruses, env originates from clade E (HIV-E). We constructed a simian-human immunodeficiency virus (SHIV) chimera carrying env isolated from an RV144 placebo recipient in the SHIV-1157ipd3N4 backbone. The latter contains long terminal repeats (LTRs) with duplicated NF-B sites, thus resembling HIV LTRs. We devised a novel strategy to adapt the parental infectious molecular clone (IMC), R5 SHIV-E1, to rhesus macaques: simultaneous depletion of B and CD8+ cells followed by intramuscular inoculation of proviral DNA and repeated administrations of cell-free virus. High viremia and CD4+ T-cell depletion ensued. Passage 3 virus unexpectedly caused acute, irreversible CD4+ T-cell loss; the partially adapted SHIV had become dual-tropic. Virus and IMCs with exclusive R5 tropism were reisolated from earlier passages, combined, and used to complete the adaptation through additional macaques. The final isolate, SHIV-E1p5, remained solely R5-tropic. It had a tier 2 neutralization phenotype, was mucosally transmissible, and pathogenic. Deep sequencing revealed 99% Env amino acid sequence conservation; X4-only and dual-tropic strains had evolved independently from an early branch of parental SHIV-E1. To conclude, our primate model data reveal that SHIV-E1p5 recapitulates important aspects of HIV transmission and pathobiology in humans.

    IMPORTANCE Understanding the protective principles that lead to a safe, effective vaccine against HIV in non-human primate (NHP) models requires test viruses that allow evaluation of anti-HIV envelope responses. Reduced HIV acquisition risk in RV144 has been linked to non-neutralizing IgG antibodies with a range of effector activities. Definitive experiments to decipher the mechanisms of the partial protection observed in RV144 require passive immunization studies in NHPs with a relevant test virus. We have generated such a virus by inserting env from an RV144 placebo recipient into a SHIV backbone with HIV-like LTRs. The final SHIV-E1p5 isolate, grown in rhesus monkey peripheral blood mononuclear cells, was mucosally transmissible and pathogenic. Earlier SHIV-E passages showed coreceptor switch, again mimicking HIV biology in humans. Thus, our series of SHIV-E strains mirrors HIV transmission and disease progression in humans. SHIV-E1p5 represents a biologically relevant tool to assess prevention strategies.

  • The HIV-1 Tat protein enhances splicing at the major splice donor site [PublishAheadOfPrint]

  • Transcription of the HIV-1 proviral DNA and subsequent processing of the primary transcript results in the production of a large set of unspliced and differentially spliced viral RNAs. The major splice donor site (5'ss) that is located in the untranslated leader of the HIV-1 transcript is used for the production of all spliced RNAs and splicing at this site has to be tightly regulated to allow the balanced production of all viral RNAs and proteins. We demonstrate that the viral Tat protein, which is known to activate viral transcription, also stimulates splicing at the major 5'ss. Like for the transcription effect, Tat requires the viral long terminal repeat (LTR) promoter and the trans-acting responsive (TAR) RNA hairpin for splicing regulation. These results indicate that HIV-1 transcription and splicing are tightly coupled processes through the coordinated action of the essential Tat protein.

    IMPORTANCE The HIV-1 proviral DNA encodes a single RNA transcript that is used as RNA genome and packaged into newly assembled virus particles. This full-length RNA is also used as mRNA for the production of structural and enzymatic proteins. Production of other essential viral proteins depends on alternative splicing of the primary transcript, which yields a large set of differentially spliced mRNAs. Optimal virus replication requires a balanced production of all viral RNAs, which means that the splicing process has to be strictly regulated. We show that the HIV-1 Tat protein, a factor that is well known for its transcription activating function, also stimulates splicing. Thus, Tat does not only control the level of the viral RNAs, but also the balance between spliced and unspliced RNAs.

  • Orsay {delta} protein is required for non-lytic viral egress [PublishAheadOfPrint]

  • Non-enveloped gastrointestinal viruses such as human rotavirus can exit infected cells from the apical surface without cell lysis. The mechanism of such non-lytic exit is poorly understood. The non-enveloped Orsay virus is an RNA virus infecting the intestine cells of the nematode Caenorhabditis elegans. Dye staining results suggested that Orsay exits from the intestine of infected worms in a non-lytic manner. Therefore, the Orsay-C. elegans system provides an excellent in vivo model to study viral exit. The Orsay genome encodes three proteins: RNA-dependent RNA polymerase, capsid protein (CP), and a nonstructural protein . can also be expressed as a structural CP- fusion. We generated an ATG-to-CTG mutant virus that had normal CP- fusion but could not produce free due to lack of the start codon. This mutant virus showed a viral exit defect without obvious phenotypes in other steps of viral infection, suggesting that is involved in viral exit. Ectopically expressed free localized near the apical membrane of intestine cells in C. elegans and co-localized with ACT-5, an intestine-specific actin that is a component of the terminal web. Orsay infection rearranged ACT-5 apical localization. Reduction of ACT-5 level via RNAi significantly exacerbated the viral exit defect of the mutant virus, suggesting that and ACT-5 functionally interact to promote Orsay exit. Together, these data support a model that the viral protein interacts with the actin network at the apical side of host intestine cells to mediate polarized, non-lytic egress of the Orsay virus.

    Importance An important step of the viral life cycle is how viruses exit from host cells to spread to other cells. Certain non-enveloped viruses can exit cultured cells in non-lytic ways, however, such non-lytic exit has not been demonstrated in vivo. In addition, it is not clear how such non-lytic exit is achieved mechanistically in vivo. Orsay is a non-enveloped RNA virus that infects the intestine cells of the nematode C. elegans. It is currently the only virus known to naturally infect C. elegans. Using this in vivo model, we show that the protein encoded by Orsay facilitates the non-lytic exit of the virus, possibly by interacting with host actin on the apical side of the worm intestine cells.

  • Comparative Characterization of the Sindbis Virus Proteome from Mammalian and Invertebrate Hosts Identifies nsP2 as a Component of the Virus Nucleocapsid and Sorting Nexin 5 as a Significant Host Factor for Alphavirus Replication [PublishAheadOfPrint]

  • Recent advances in mass spectrometry methods and instrumentation now allow for more accurate identification of proteins in low abundance. This technology was applied to Sindbis virus, the prototypical alphavirus to investigate the viral proteome. To determine if host proteins are specifically packaged into alphavirus virions, Sindbis virus (SINV) was grown in multiple host cells representing vertebrate and mosquito hosts and total protein content of purified virions was determined. This analysis identified host factors not previously associated with alphavirus entry, replication, or egress. One host protein, sorting nexin 5 (SNX5), was shown to be critical for the replication of three different alphaviruses, Sindbis, Mayaro and Chikungunya virus. The most significant finding was that in addition to the host proteins, SINV non-structural protein 2 (nsP2) was detected within virions grown in all host cells examined. The protein and RNA-interacting capabilities of nsP2 coupled with its presence in the virion support a role for nsP2 during packaging and/or entry of progeny virus. This function has not been identified for this protein. Taken together, this strategy identified at least one host factor integrally involved in alphavirus replication. Identification of other host proteins provides insight into alphavirus-host interactions during viral replication in both vertebrate and invertebrate hosts. This method of virus proteome analysis may also be useful for the identification of protein candidates for host-based therapeutics.

    IMPORTANCE Pathogenic Alphaviruses, such as Chikungunya and Mayaro virus, continue to plague public health in developing and developed countries alike. Alphaviruses belong to a group of viruses vectored in nature by hematophagous (blood-feeding) insects and are termed Arboviruses (arthropod-borne viruses). This group of viruses contains many human pathogens such as dengue fever, West Nile and Yellow fever viruses. With few exceptions there are no vaccines or prophylactics for these agents leaving one third of the world population at risk of infection. Identifying effective antivirals has been a long term goal for combating these diseases not only because of the lack of vaccines but also because they are effective during an ongoing epidemic. Mass spectrometry-based analysis of the Sindbis virus proteome can be effective in identifying host genes involved in virus replication and novel functions for virus proteins. Identification of these factors is invaluable for the prophylaxis of this group of viruses.

  • Dengue virus induces increased activity of the complement alternative pathway in infected cells [PublishAheadOfPrint]

  • Severe dengue virus (DENV) infection is associated with overactivity of the complement alternative pathway (AP) in patient studies. Here, the molecular changes in components of the AP during DENV infection in vitro are investigated. mRNA for factor H (FH) a major negative regulator of the AP, is significantly increased in DENV-infected endothelial cells (EC) and macrophages but in contrast production of extracellular FH protein is not. This discord is not seen for the AP activator, factor B (FB), with DENV induction of both FB mRNA and protein, nor with Toll-like receptor 3 or 4 stimulation of EC and macrophages, which induces both FH and FB mRNA and protein. Surface bound and intracellular FH protein is however induced by DENV, but only in DENV antigen-positive cells, while in two other DENV-susceptible immortalised cell lines (ARPE-19 and HREC) FH protein is induced both intracellularly and extracellularly by DENV infection. Regardless of the cell type, there is an imbalance in AP components and an increase in markers of complement AP activity associated with DENV-infected cells nndash; with lower FH relative to FB protein, increased ability to promote AP-mediated lytic activity and increased deposition of complement component C3b on the surface of DENV-infected cells. For EC in particular, these changes are predicted to result in higher complement activity in the local cellular microenvironment, with the potential to induce functional changes that may result in increased vascular permeability, a hallmark of dengue disease.

    IMPORTANCE Dengue virus (DENV) is a significant human viral pathogen with global medical and economic impact. DENV may cause serious and life-threatening disease with increased vascular permeability and plasma leakage. The pathogenic mechanisms underlying these features remain unclear; however overactivity of the complement alternative pathway has been suggested to play a role. In this study we investigate the molecular events that may be responsible for this observed alternative pathway overactivity and provide novel findings of changes in the complement system in response to DENV infection in primary cell types that are a major target for DENV infection (macrophages) and pathogenesis (endothelial cells) in vivo. Our results suggest a new dimension of cellular events that may influence endothelial cell barrier function during DENV infection that could expand strategies for developing therapeutics to prevent or control DENV-mediated vascular disease.

  • Five Residues in the Apical Loop of the Respiratory Syncytial Virus Fusion Protein F2 Subunit are Critical for its Fusion Activity [PublishAheadOfPrint]

  • The respiratory syncytial virus (RSV) fusion (F) protein is a trimeric, membrane-anchored glycoprotein capable of mediating both viralnndash;target cell membrane fusion to initiate infection and cellnndash;cell fusion, even in the absence of the attachment glycoprotein. The F protein is initially expressed in a precursor form, whose functional capabilities are activated by proteolysis at two sites between the F1 and F2 subunits. This cleavage results in expression of the metastable and high-energy prefusion conformation. To mediate fusion, the F protein is triggered by an unknown stimulus, causing the F1 subunit to refold dramatically while F2 changes minimally. Hypothesizing that the most likely site for interaction with a target-cell component would be the top, or apex, of the protein, we determined the importance of the residues in the apical loop of F2 by scanning mutagenesis. Five residues were not important, two were of intermediate importance, and all four lysines and one isoleucine were essential. Alanine replacement did not result in the loss of pre-F conformation for any of these mutants. Each of the four lysines required its specific charge for fusion function. Alanine replacement of the three essential lysines on the ascent to the apex hindered fusion following a forced fusion event, suggesting they are involved in refolding. Alanine mutation at Ile64, also on the ascent to the apex, and Lys75, did not prevent fusion following forced triggering, suggesting they are not involved in refolding and may instead be involved in the natural triggering of the F protein.

    IMPORTANCE RSV infects virtually every child by the age of 3, causing nearly 33 million acute lower respiratory infections (ALRI) worldwide each year in children younger than 5 (Nair H, et al. 2010. Lancet 375:1545nndash;55). RSV is also the second leading cause of respiratory related death in the elderly (Falsey AR, Walsh EE. 2005. Drugs Aging 22:577nndash;87; Falsey AR, Hennessey PA, Formica MA, Cox C, Walsh EE. 2005. N Engl J Med 352:1749nndash;1759). The monoclonal antibody palivizumab is approved for prophylactic use in some at-risk infants, but healthy infants remain unprotected. Furthermore, its expense limits its use primarily to developed countries. No vaccine or effective small-molecule drug is approved for preventing disease or treating infection (Costello HM, Ray W, Chaiwatpongsakorn S, Peeples ME. 2012. 12:110nndash;128). The essential residues identified in the apical domain of F2 are adjacent to the apical portion of F1 which, upon triggering, refolds into the long heptad repeat A (HRA) with the fusion peptide at its N-terminus. These essential residues in F2 are likely involved in triggering and/or refolding of the F protein and as such may be ideal targets for antiviral drug development.

  • Expression levels of glycoprotein O (gO) vary between strains of human cytomegalovirus, influencing the assembly of gH/gL complexes and virion infectivity. [PublishAheadOfPrint]

  • Tropism of human cytomegalovirus (HCMV) is influenced by the envelope glycoprotein complexes gH/gL/gO and gH/gL/UL128-131. During virion assembly, gO and the UL128-131 proteins compete for binding to gH/gL in the ER. This assembly process clearly differs among strains since Merlin (ME) virions contain abundant gH/gL/UL128-131 and little gH/gL/gO, whereas TR contains much higher levels of total gH/gL, mostly in the form of gH/gL/gO, but much less gH/gL/UL128-131 than ME. Remaining questions include 1) what are the mechanisms behind these assembly differences, and 2) do differences reflect in vitro culture adaptations or natural genetic variations? Since the UL74(gO) ORF differs by 25% of amino acids between TR and ME, we analyzed recombinant viruses in which the UL74(gO) ORF was swapped. TR virions were ggt;40-fold more infectious than ME. Transcriptional repression of UL128-131 enhanced infectivity of ME to the level of TR, despite still far lower levels of gH/gL/gO. Swapping the UL74(gO) ORF had no effect on either TR or ME. A quantitative immunoprecipitation approach revealed that gH/gL expression was within 4-fold between TR and ME, but gO expression was 20-fold less by ME, and suggested differences in mRNA transcription, translation or rapid ER-associated degradation of gO. Trans-complementation of gO expression during ME replication gave 6-fold enhancement of infectivity beyond the 40-fold effect of UL128-131 repression alone. Overall, strain variations in assembly of gH/gL complexes result from differences in expression of gO and UL128-131, and selective advantages for reduced UL128-131 expression during fibroblast propagation are much stronger than for higher gO expression.

    IMPORTANCE Specific genetic differences between independently isolated HCMV strains may result from purifying selection on de novo mutations arising during propagation in culture, or random sampling among the diversity of genotypes present in clinical specimens. Results presented indicate that while reduced UL128-131 expression may confer a powerful selective advantage during cell-free propagation of HCMV in fibroblast cultures, selective pressures for increased gO expression are much weaker. Thus, variation in gO expression among independent strains may represent natural genotype variability present in vivo. This may have important implications for virus-host interactions such as immune recognition, and underscores the value of studying molecular mechanisms of replication using multiple HCMV strains.

  • Roles of Fc domain and exudation in L2 antibody-mediated protection against human papillomavirus [PublishAheadOfPrint]

  • To address how L2-specific antibodies prevent human papillomavirus (HPV) infection of the genital tract we generated neutralizing monoclonal antibodies (MAbs) WW1, a rat IgG2a that binds L2 residues 17-36 (like mouse MAb RG1), and JWW3, a mouse IgG1 derivative of Mab24 specific to L2 residues 58-64. By Western blot, WW1 recognized L2 of 29/34 HPV genotypes tested, compared to only 13/34 for RG1, and 25/34 for JWW3. WW1 IgG and (Fab')2 bound HPV16 pseudovirions similarly, however whole IgG provided better protection against HPV vaginal challenge. Passive transfer of WW1 IgG was similarly protective in wild type and FcRn-deficient mice, suggesting that protection by WW1 IgG is not mediated by FcRn-dependent transcytosis. Rather, local epithelial disruption required for genital infection and induced by either brushing or nonoxynol-9 treatment released serum IgG in the genital tract, suggesting Fc-independent exudation. Depletion of neutrophils and macrophages reduced protection of mice upon passive transfer of whole WW1 or JWW3 IgGs. Similarly, IgG-mediated protection by L2 MAbs WW1, JWW3 and RG1 was reduced in Fc-receptor knockout compared to wild type mice. However, in vitro neutralization by WW1 IgG was similar in TRIM21 knockout and wild type cells indicating that the Fc does not contribute to antibody-dependent intracellular neutralization (ADIN). In conclusion, the Fc domain of L2-specific IgGs is not active for ADIN, but it opsonizes bound extracellularpseudovirions for phagocytes in protecting mice from intra-vaginal HPV challenge. Systemically administered neutralizing IgG can access the site of infection in an abrasion via exudation without need for FcRn-mediated transcytosis.

    IMPORTANCE At least fifteen alpha HPVs are causative agents for 5% of all cancers worldwide, and beta types have been implicated in non-melanoma skin cancer, whereas others produce benign papillomas such as genital warts associated with considerable morbidity and health systems costs. Vaccines targeting minor capsid protein L2 have the potential to provide broad spectrum immunity against medically relevant HPVs of divergent genera via induction of broadly cross-neutralizing serum IgG. Here we examine the mechanisms by which L2-specific serum IgG reach the viral inoculum in the genital tract to effect protection. Abrasion of the vaginal epithelium allows the virus to access and infect basal keratinocytes, and our findings suggest this also permits local exudation of neutralizing IgG and vaccine-induced sterilizing immunity. We also demonstrate the importance of Fc-mediated phagocytosis of L2 antibody-virion complexes to humoral immunity, a protective mechanism that is not detected by current in vitro neutralization assays.

  • Inhibitory effects of HIV-2 Vpx on replication of HIV-1 [PublishAheadOfPrint]

  • The human immunodeficiency viruses type 1 and 2 share a striking genomic resemblance, however, variability in the genetic sequence accounts for the presence of unique accessory genes; such as viral protein x (vpx) in HIV-2. Dual infection with both viruses has long been described in the literature, yet the molecular mechanism of how dually infected patients tend to do better than those who are mono-infected with HIV-1 has not yet been explored. We hypothesized that in addition to extracellular mechanisms, an HIV-2 accessory gene is the culprit, and interference at viral accessory/regulatory protein level is perhaps responsible for the attenuated pathogenicity of HIV-1 observed in dually infected patients. Following simulation of dual infection in cell culture experiments, we found that pre-transduction of cells with HIV-2 significantly protects against HIV-1 transduction. Importantly, we have found that this dampening of HIV-1's infectivity was a result of inter-viral interference carried out by the viral protein X of HIV-2, resulting in a severe hindrance to HIV-1's replication dynamics, influencing both its early- and late-phase of the viral life-cycle. Our findings shed light on potential intracellular interactions between the two viruses, and broaden our understanding of the observed clinical spectrum in dually infected patients, highlighting HIV-2 Vpx as a potential candidate worth exploring in the fight against HIV-1.

    Importance Dual infection with the human immunodeficiency viruses type 1 and 2 is relatively common in endemic areas. For yet unclarified reasons, patients dually infected were shown to have lower viral loads and generally a slower rate of progression to AIDS than those who are mono-infected. We have aimed to explore dual infection in cell culture, to elucidate possible mechanisms by which HIV-2 may be able to exert such an effect. Our results indicate that on the cellular level, pre-transduction of cells with HIV-2 significantly protects against HIV-1 transduction, which was found to be a result of inter-viral interference carried out by the viral protein X of HIV-2. These findings broaden our knowledge of inter-viral interactions on the cellular level, and may provide an explanation for the decreased pathogenicity of HIV-1 in dually infected patients, highlighting HIV-2 Vpx as a potential candidate worth exploring in the fight against HIV.

  • Equine Myxovirus Resistance Protein 2 Restricts Lentiviral Replication by Blocking Nuclear Uptake of Capsid Protein [PublishAheadOfPrint]

  • Human Myxovirus resistance 2 (huMxB) has been shown to be a determinant type I interferon-induced host factor involved in the inhibition of HIV-1 as well as many other primate lentiviruses. This blocking occurs after the reverse transcription of viral RNA and ahead of the integration into the host DNA, which is closely connected to the ability of the protein to bind the viral capsid. To date, Mx2s derived from non-primate animals have shown no capacity for HIV-1 suppression. In this study, we examined the restrictive effect of equine Mx2 (eqMx2) on both the equine infectious anemia virus (EIAV) and HIV-1 and investigated possible mechanisms for its specific function. We demonstrated that IFNaalpha;/bbeta; upregulates the expression of eqMx2 in equine monocyte-derived macrophages (eMDMs). Overexpression of eqMx2 significantly suppresses the replication of EIAV, HIV-1, and SIVs, but not that of MLV. Knockdown of eqMx2 transcription weakens the inhibition of EIAV replication by type I interferon. Interestingly, immunofluorescence assays suggest that the subcellular localization of eqMx2 changes following virus infection, from being dispersed in the cytoplasm to being accumulated at the nuclear envelope. Furthermore, eqMx2 blocks the nuclear uptake of the proviral genome by binding to the viral capsid. The N-truncated mutant of eqMx2 lost the ability to bind the viral capsid as well as the restriction effect for lentiviruses. These results improve our understanding of the Mx2 protein in non-primate animals.

    IMPORTANCE Previous research has shown that the antiviral ability of Mx2s is confined to primates, particularly humans. EIAV has been shown to be insensitive to the restriction by human MxB. Here, we described the function of equine Mx2. This protein plays an important role in the suppression of EIAV, HIV-1, and SIVs. The antiviral activity of eqMx2 depends on its subcellular location as well as its capsid binding capacity. Our results showed that following viral infection, eqMx2 changes its original cytoplasmic location and accumulates at the nuclear envelope where it binds to the viral capsid and blocks the nuclear entry of reverse transcribed proviral DNAs. In contrast, huMxB does not bind to the EIAV capsid and shows no EIAV restriction effect. These studies expand our understanding of the function of the equine Mx2 protein.

  • Regulation of virus-associated lymphoma growth and gene expression by bacterial quorum sensing molecules [PublishAheadOfPrint]

  • Kaposi sarcoma-associated herpesvirus (KSHV) can cause several human cancers including primary effusion lymphoma (PEL), which frequently occur in immunocompromised patients. KSHV infected patients often suffer from polymicrobial infections caused by opportunistic bacterial pathogens. Therefore, it is crucial to understand how these co-infected microorganisms or their secreted metabolites may affect KSHV infection and virus-associated malignancies pathogenesis. Quorum sensing (QS), a cell density-based intercellular communication system, employs extracellular diffusible signaling molecules to regulate bacterial virulence mechanisms in a wide range of bacterial pathogens, such as Pseudomonas aeruginosa, which is one of mostly common opportunistic microorganisms found in immunocompromised individuals. In this study, we evaluated and compared the influence of PEL growth and host/viral interactome by the major QS signal molecules (OdDHL, BHL and PQS), the conditioned medium from wild type (wt) and QS mutants of laboratory strains as well as clinical isolates of P. aeruginosa. Our data indicate that P. aeruginosa co-infection may facilitate virus dissemination, establishment of new infection and further promote tumor development, through effectively inducing viral lytic gene expression by its QS systems.

    IMPORTANCE Currently, most studies about KSHV infection and/or virus-associated malignancies depend on pure culture system or immunodeficiency animal models. However, the real situation should be much more complicated in KSHV-infected immunocompromised patients due to frequent polymicrobial infections. It is important to understand the interaction of KSHV and co-infected microorganisms, especially those opportunistic bacterial pathogens. Here we for the first time report that P. aeruginosa and its quorum sensing signaling molecules display a complicated impact on KSHV-associated lymphoma growth as well as intracellular host/viral gene expression profile. Our data imply that targeting co-infected pathogens is probably necessary when treatment of virus-associated malignancies in these immunocompromised patients.

  • Transmembrane protein pUL50 of human cytomegalovirus inhibits ISGylation by downregulating UBE1L [PublishAheadOfPrint]

  • Interferon-stimulated gene (ISG) 15 encodes a ubiquitin-like protein that can be conjugated to proteins via an enzymatic cascade involving the E1, E2, and E3 enzymes. ISG15 expression and protein ISGylation modulate viral infection; however, the viral mechanisms regulating the function of ISG15 and ISGylation are not well understood. We recently showed that ISGylation suppresses the growth of human cytomegalovirus (HCMV) at multiple steps of the virus life cycle, and that the virus-encoded pUL26 protein inhibits protein ISGylation. In this study, we demonstrate that the HCMV UL50-encoded transmembrane protein, a component of the nuclear egress complex, also inhibits ISGylation. pUL50 interacted with UBE1L, an E1-activating enzyme for ISGylation, and to a lesser extent ISG15, as did pUL26. However, unlike pUL26, pUL50 caused proteasomal degradation of UBE1L. The UBE1L level induced in human fibroblast cells by interferon-bbeta; treatment or virus infection was reduced by pUL50 expression. This activity of pUL50 involved the transmembrane (TM) domain within its C-terminal region, although pUL50 could interact with UBE1L independent of the TM domain. Consistently, colocalization of pUL50 with UBE1L was observed in cells treated with a proteasome inhibitor. Furthermore, we found that RNF170, an ER-associated ubiquitin E3 ligase, interacted with pUL50 and promoted pUL50-mediated UBE1L degradation via ubiquitination. Our results demonstrate a novel role for the pUL50 transmembrane protein of HCMV in the regulation of protein ISGylation.

    IMPORTANCE Proteins can be covalently conjugated by ubiquitin or ubiquitin-like proteins such as SUMO and ISG15. ISG15 is highly induced in viral infection, and ISG15 conjugation, termed ISGylation, plays important regulatory roles in viral growth. Although ISGylation has been shown to negatively affect many viruses including human cytomegalovirus (HCMV), viral countermeasures that might modulate ISGylation are not well understood. In the present study, we show that the transmembrane protein encoded by HCMV UL50 inhibits ISGylation by causing proteasomal degradation of the E1-activating enzyme UBE1L for ISGylation. This pUL50 activity requires membrane targeting. Supporting this, RNF170, an ER-associated ubiquitin E3 ligase, interacts with pUL50 and promotes UL50-mediated UBE1L ubiquitination and degradation. Our results provide the first evidence that viruses can regulate ISGylation by directly targeting the ISGylation E1 enzyme.

  • Adenovirus 5 E1A-mediated suppression of p53 via FUBP1 [PublishAheadOfPrint]

  • The Far Upstream Element Binding Protein 1 (FUBP1) was first identified as a regulator of the oncogene c-Myc via binding to the Far Upstream Element (FUSE) within the c-Myc promoter and activating expression of the gene. Recent studies have identified FUBP1 as a regulator of transcription, translation, and splicing via its DNA and RNA binding activities. Here we report the identification of FUBP1 as a novel binding partner of E1A. FUBP1 binds directly to E1A via the N-terminus (residues 1-82) and Conserved Region 3 (residues 139 to 204) of adenovirus 5 E1A. Depletion of FUBP1 via short interfering RNAs (siRNA) reduces virus growth and drives upregulation of cellular stress response by activating expression of p53-regulated genes. During infection FUBP1 is relocalized within the nucleus and it is recruited to viral promoters together with E1A while at the same time being lost from the FUSE upstream of the c-Myc promoter. Depletion of FUBP1 affects viral and cellular gene expression. Importantly, in FUBP1-depleted cells p53-responsive genes are upregulated, p53 occupancy on target promoters is enhanced and histone H3 lysine 9 is hyperacetylated. This is likely due to the loss of FUBP1-mediated suppression of p53 DNA binding. We also observed that E1A stabilizes FUBP1-p53 complex, preventing p53 promoter binding. Together our results identify, for the first time, FUBP1 as a novel E1A binding protein that participates in aspects of viral replication and is involved in E1A-mediated suppression of p53 function.

    IMPORTANCE Viral infection triggers innate cellular defence mechanisms that have evolved to block virus replication. To overcome this, viruses have counter-evolved mechanisms that ensure that cellular defences are either disarmed or not activated to guarantee successful replication. One of the key regulators of cellular stress is the tumour suppressor p53 that responds to a variety of cellular stress stimuli and safeguards integrity of the genome. During infection, many viruses target the p53 pathway in order to deactivate it. Here we report that human adenovirus 5 co-opts the cellular protein FUBP1 to prevent activation of the p53 stress response pathway that would block viral replication. This finding adds to our understanding of p53 deactivation by adenovirus and highlights its importance in infection and innate immunity.

  • T cell-derived Lymphotoxin is Essential for anti-HSV-1 Humoral Immune Response [PublishAheadOfPrint]

  • B cell-derived lymphotoxin (LT) is required for the development of follicular dendritic cell clusters for the formation of primary and secondary lymphoid follicles, but the role of T cell-derived LT in antibody response has not been well demonstrated. We observed that lymphotoxin-bbeta;-receptor (LTbbeta;R) signaling is essential for optimal humoral immune response and protection against an acute HSV-1 infection. Blocking the LTbbeta;R pathway caused poor maintenance of germinal center B (GC-B) cells and follicular helper T (Tfh) cells. Using bone marrow chimeric mice and adoptive transplantation, we determined that T cell-derived LT played an indispensable role in the humoral immune response to HSV-1. Up-regulation of IFN by the LTbbeta;R-Ig blockade impairs the sustainability of Tfh-like cells, thus leading to an impaired humoral immune response. Our findings have identified a novel role of T cell-derived LT in the humoral immune response against HSV-1 infection.

    IMPORTANCE Immunocompromised people are susceptible for HSV-1 infection and lethal recurrence, which could be inhibited by anti-HSV-1 humoral immune response in the host. This study sought to explore the role of T cell-derived LT in the anti-HSV-1 humoral immune response using LT-LTbbeta;R signaling deficient mice and the LTbbeta;R-Ig blockade. The data indicate that the T cell-derived LT may play an essential role in sustaining Tfh-like cells and ensure Tfh-like cells' migration into primary or secondary follicles for further maturation. This study provides insights for vaccine development against infectious diseases.

  • A Novel gp41-Binding Adnectin with Potent Anti-HIV Activity Is Highly Synergistic when Linked to a CD4-Binding Adnectin [PublishAheadOfPrint]

  • The N17 region of gp41 in HIV-1 is the most conserved region in gp160. mRNA selection technologies were used to identify an Adnectin that binds to this region and inhibits gp41-induced membrane fusion. Additional selection conditions were used to optimize the Adnectin to greater potency (5.4 pplusmn; 2.6 nM) to HIV-1 and improved binding affinity to a N17-containing helical trimer (0.8 pplusmn; 0.4 nM). Resistance to this Adnectin mapped to a single Glu to Arg change within the N17 coding region. The optimized Adnectin (6200_A08) exhibited high potency and broad spectrum against 123 envelope proteins and multiple clinical isolate viruses, although certain envelope proteins did exhibit reduced susceptibility against 6200_A08 alone. The reduced potency could not be correlated with sequence changes in the target region and was thought to be the result of faster kinetics of fusion mediated by these envelope proteins. Optimized linkage of 6200_A08 with a previously characterized Adnectin targeting CD4 produced a highly synergistic molecule, with potency of the tandem measured at 37 pplusmn; 1 pM. In addition, these tandem molecules now exhibited little potency differences against the same panel of envelope proteins with reduced susceptibility to 6200_A08 alone, providing evidence that they did not have intrinsic resistance to 6200_A08 and that coupling 6200_A08 to the anti-CD4 Adnectin may provide a faster effective on-rate for gp41 target engagement.

    IMPORTANCE There continue to be significant unmet medical needs for patients with HIV-1 infection. One way to improve adherence and decrease the likelihood of drug-drug interactions in HIV-1 infected patients is through the development of long acting biologic inhibitors. This study describes the development and properties of an Adnectin molecule that targets the most conserved region of the gp41 protein and inhibits HIV-1 with good potency. Moreover, when fused to a similar Adnectin targeted to the human CD4 protein, the receptor for HIV-1, significant synergies in potency and efficacy are observed. These inhibitors are part of an effort to develop a larger biologic molecule that function as a long acting self-administered regimen for patients with HIV-1 infection.

  • Herpes Simplex Virus 1 Inhibits TANK-Binding Kinase 1 through Formation of the Us11-Hsp90 Complex [PublishAheadOfPrint]

  • The Us11 protein of herpes simplex virus 1 (HSV-1) is an accessory factor with multiple functions. In virus-infected cells, it inhibits double-stranded RNA dependent protein kinase PKR, 2',5'-oligoadenylate synthetase, RIG-I and MDA-5. However, its precise role is incompletely defined. By screening human cDNA library, we show that the Us11 protein targets heat shock protein 90 (Hsp90), which inactivates TANK binding kinase 1 (TBK1) and antiviral immunity. When ectopically expressed, HSV-1 Us11 precludes the access of TBK1 to Hsp90 and IFN promoter activation. Consistently, upon HSV infection the Us11 protein suppresses the expression of IFN-bbeta;, RANTES, and interferon stimulated genes. This is mirrored by a blockade in the phosphorylation of interferon regulatory factor 3. Mechanistically, the Us11 protein associates with endogenous Hsp90 to disrupt the Hsp90-TBK1 complex. Furthermore, Us11 induces destabilization of TBK1 through a proteasome dependent pathway. Accordingly, Us11 expression facilitates HSV growth. Conversely, TBK1 expression restricts viral replication. These results suggest that control of TBK1 by Us11 promotes HSV-1 infection.

    IMPORTANCE TANK binding kinase 1 plays a key role in antiviral immunity. Although multiple factors are thought to participate in this process, the picture is obscure in herpes simplex virus infection. We demonstrate that the Us11 protein of HSV-1 forms a complex with heat shock protein 90, which inactivates TANK binding kinase 1 and IFN induction. As a result, expression of the Us11 protein promotes HSV replication. These experimental data provide a new insight into the molecular network of virus-host interactions.

  • Influenza A virus infection triggers pyroptosis and apoptosis of respiratory epithelial cells through type I IFN signaling pathway in a mutually exclusive manner [PublishAheadOfPrint]

  • Respiratory epithelial cell death by influenza virus infection is responsible for induction of inflammatory responses, but the exact cell death mechanism is not understood. Here we showed that influenza virus infection induces apoptosis and pyroptosis in normal or precancerous human bronchial epithelial cells. Apoptosis was induced only in malignant tumor cells infected with influenza virus. In PL16T human precancerous respiratory epithelial cells, the number of apoptotic cells increased at early phases of infection, but pyroptotic cells were observed at late phases of infection. These findings suggest that apoptosis is induced at early phases of infection, but the cell death pathway is shifted to pyroptosis at late phases of infection. We also found that the type I IFN-mediated JAK-STAT signaling pathway promotes the switch from apoptosis to pyroptosis by inhibiting apoptosis possibly through the induced expression of Bcl-xL anti-apoptotic gene. Further, the inhibition of JAK-STAT signaling repressed pyroptosis, but enhanced apoptosis in infected PL16T cells. Collectively, we propose that type I IFN signaling pathway triggers pyroptosis but not apoptosis in the respiratory epithelial cells in a mutually exclusive manner to initiate pro-inflammatory responses against influenza virus infection.

    IMPORTANCE Respiratory epithelium functions as a sensor of infectious agents to initiate inflammatory responses along with cell death. However, the exact cell death mechanism responsible for inflammatory responses by influenza virus infection is still unclear. We showed that influenza virus infection induced apoptosis and pyroptosis in normal or precancerous human bronchial epithelial cells. Apoptosis was induced at early phases of infection, but the cell death pathway was shifted to pyroptosis at late phases of infection under the regulation of type I IFN signaling to promote pro-inflammatory cytokines production. Taken together, type I IFN signaling pathway plays an important role to induce pyroptosis but repressed apoptosis in the respiratory epithelial cells to initiate pro-inflammatory responses against influenza virus infection.

  • The chromatin remodeling factor SPOC1 acts as a cellular restriction factor against human cytomegalovirus by repressing the major immediate-early promoter [PublishAheadOfPrint]

  • The cellular protein SPOC1 (survival time-associated PHD finger protein in ovarian cancer 1) acts as a regulator of chromatin structure and DNA damage response. It binds H3K4me2/3 containing chromatin and promotes DNA condensation by recruiting corepressors such as KAP-1 and H3K9 methyltransferases. Previous studies identified SPOC1 as a restriction factor against human adenovirus (HAdV) infection that is antagonized by E1B-55K/E4orf6-dependent proteasomal degradation. Here, we demonstrate that, in contrast to HAdV-infected cells, SPOC1 is transiently upregulated during the early phase of HCMV replication. We show that expression of the immediate-early protein 1 (IE1) is sufficient and necessary to induce SPOC1. Additionally, we discovered that during later stages of infection SPOC1 is downregulated in a GSK-3bbeta;-dependent manner. We provide evidence that SPOC1 overexpression severely impairs HCMV replication by repressing the initiation of viral immediate early (IE) gene expression. Consistently, we observed that SPOC1-depleted primary human fibroblasts displayed augmented initiation of viral IE gene expression. This occurs in a MOI-dependent manner, a defining hallmark of intrinsic immunity. Interestingly, repression requires the presence of high SPOC1 levels at the start of infection while a later upregulation had no negative impact suggesting distinct temporal roles of SPOC1 during the HCMV replicative cycle. Mechanistically, we observed a highly specific association of SPOC1 with the major immediate-early promoter (MIEP) strongly suggesting that SPOC1 inhibits HCMV replication by MIEP binding and subsequent recruitment of heterochromatin building factors. Thus, our data add SPOC1 as a novel factor to the endowment of a host cell to restrict cytomegalovirus infections.

    IMPORTANCE Accumulating evidence indicates that during millennia of co-evolution host cells have developed a sophisticated compilation of cellular factors to restrict cytomegalovirus infections. Defining this equipment is important to understand cellular barriers against viral infection and to develop strategies to utilize these factors for antiviral approaches. So far, constituents of PML nuclear bodies and the interferon gamma inducible protein 16 (IFI16) were known to mediate intrinsic immunity against HCMV. In this study, we identify the chromatin modulator SPOC1 as a novel restriction factor against HCMV. We show that pre-existing high SPOC1 protein levels mediate a silencing of HCMV gene expression via a specific association with an important viral cis-regulatory element, the major immediate-early promoter. Since SPOC1 expression varies between cell-types, this factor may play an important role in the tissue-specific defense against HCMV.

  • TRIM21 Promotes Innate Immune Response to RNA Viral Infection through Lys27-linked Polyubiquitination of MAVS [PublishAheadOfPrint]

  • Human innate immunity responds to viral infection by activating the production of interferons (IFNs) and proinflammatory cytokines. The mitochondrial adaptor molecule MAVS plays a critical role in innate immune response to viral infection. In this study, we show that TRIM21 interacts with MAVS to positively regulate innate immunity. Under viral infection, TRIM21 is upregulated through the IFN/JAK/STAT signaling pathway. Knockdown of TRIM21 dramatically impairs innate immune response to viral infection. Moreover, TRIM21 interacts with MAVS and catalyzes the K27-linked polyubiquitination of MAVS, thereby promoting the recruitment of TBK1 to MAVS. Specifically, the PRY-SPRY domain of TRIM21 is the key domain for its interaction with MAVS, while the RING domain of TRIM21 facilitates the polyubiquitination chains of MAVS. In addition, MAVS-mediated innate immune response is enhanced by both of the PRY-SPRY and RING domain of TRIM21. Mutation analyses on all lysine residues of MAVS further reveal that Lys325 of MAVS is catalyzed by TRIM21 for the K27-linked polyubiquitination. Overall, this study reveals a novel mechanism that TRIM21 promotes the K27-linked polyubiquitination of MAVS to positively regulate innate immune response, thereby inhibiting viral infection.

    IMPORTANTCE Activation of innate immunity is essential for host cells to restrict the spread of invading viruses and other pathogens. MAVS plays a critical role in innate immune response to RNA viral infection. In this study, we demonstrated that TRIM21 targets MAVS to positively regulate the innate immunity. Notably, TRIM21 targets and catalyzes K27-linked polyubiquitination of MAVS, and then promotes the recruitment of TBK1 to MAVS, leading to upregulation of innate immunity. Our study outlines a novel mechanism that IFN signaling pathway blocks RNA virus to escape the immune elimination.

  • STAT2-dependent immune responses ensure host survival despite the presence of a potent viral antagonist [PublishAheadOfPrint]

  • Pathogen encounter induces interferons which signal via Janus kinases and STAT transcription factors to establish an antiviral state. However, host and pathogens are situated in a continuous arms race which shapes host evolution towards optimized immune responses and the pathogens towards enhanced immune evasive properties.

    Mouse cytomegalovirus (MCMV) counteracts interferon responses by pM27-mediated degradation of STAT2 which directly affects the signaling of type I as well as type III interferons. Using MCMV mutants lacking M27 and mice lacking STAT2, we studied the opposing relationship between antiviral activities and viral antagonism in a natural host-pathogen pair in vitro and in vivo. In contrast to wt-MCMV, M27-MCMV was efficiently cleared from all organs within a few days in BALB/c, C57BL/6, and 129 mice, highlighting the general importance of STAT2 antagonism for MCMV replication. Despite this effective and relevant STAT2 antagonism, wt and STAT2-deficient mice exhibited fundamentally different susceptibilities to MCMV infections. MCMV replication was increased in all assessed organs (e.g. liver, spleen, lungs, and salivary glands) of STAT2-deficient mice, resulting in mortality during the first week after infection.

    Taken together, our study reveals the importance of cytomegaloviral interferon antagonism for viral replication as well as a pivotal role of the remaining STAT2 activity for host survival. This mutual influence establishes a stable evolutionary stand-off situation with fatal consequences when the equilibrium is disturbed.

    IMPORTANCE The host limits viral replication by interferons which signal via STAT proteins. Several viruses evolved antagonists targeting STATs to antagonize IFNs (e.g. cytomegaloviruses, Zika virus, Dengue virus, and several paramyxoviruses). We analyzed infections of mouse CMV expressing or lacking the STAT2 antagonist pM27 in STAT2-deficient and control mice to evaluate their importance for host and virus in vitro and in vivo. The inability to counteract STAT2 directly translates into exaggerated IFN susceptibility in vitro and pronounced attenuation in vivo. Thus, the antiviral activity mediated by IFNs via STAT2-dependent signaling drove the development of a potent MCMV-encoded STAT2 antagonist which became indispensable for efficient virus replication and spread to organs required for dissemination. Despite this clear impact of viral STAT2 antagonism, the host critically required the remaining STAT2 activity to prevent overt disease and mortality upon MCMV infection. Our findings highlight a remarkably delicate balance between host and virus.

  • Common and Distinct Capsid and Surface Protein Requirements for Secretion of Complete and Genome-free Hepatitis B Virions [PublishAheadOfPrint]

  • During the morphogenesis of hepatitis B virus (HBV), an enveloped virus, two types of virions are secreted: (1) a minor population of complete virions containing a mature nucleocapsid with the characteristic, partially double-stranded, relaxed circular DNA genome and (2) a major population containing an empty capsid with no DNA or RNA (empty virions). Secretion of both types of virions requires interactions between the HBV capsid or core protein (HBc) and the viral surface or envelope proteins. We have studied the requirements from both HBc and envelope proteins for empty virion secretion, in comparison with those for secretion of complete virions. Substitutions within the N-terminal domain of HBc that block secretion of DNA-containing virions reduced but did not prevent secretion of empty virions. The HBc C-terminal domain was not essential for empty virion secretion. Among the three viral envelope proteins, the smallest, S, alone was sufficient for empty virion secretion at a basal level. The largest protein, L, essential for complete virion secretion, was not required for, but could stimulate empty virion secretion. Also, substitutions in L that eliminate secretion of complete virions reduced but did not eliminate empty virion secretion. S mutations that block secretion of the hepatitis D virus (HDV), an HBV satellite, did not block secretion of either empty or complete HBV virions. Together, these results indicate that both common and distinct signals on empty capsids vs. mature nucleocapsids interact with the S and L proteins during the formation of complete vs. empty virions.

    IMPORTANCE Hepatitis B virus (HBV) is a major cause of severe liver diseases including cirrhosis and cancer. In addition to the complete infectious virion particle, which contains an outer envelope layer and an interior capsid that, in turn, encloses a DNA genome, HBV infected cells also secrete non-infectious, incomplete viral particles in large excess over the complete virions. In particular, the empty (or genome-free) virion share with the complete virion the outer envelope and interior capsid but contain no genome. We have carried out a comparative study on the capsid and envelope requirements for the secretion of these two types of virion particles and uncovered both shared and distinct determinants on the capsid and envelope for their secretion. These results provide new information on HBV morphogenesis, and have implications for efforts to develop empty HBV virions as a novel biomarker and a new generation of HBV vaccine.

  • Breadth and functionality of varicella zoster virus glycoprotein-specific antibodies identified after Zostavax vaccination in humans [PublishAheadOfPrint]

  • Herpes zoster (HZ/shingles) is the clinical manifestation of varicella zoster virus (VZV) reactivation. HZ typically develops as people age due to decreased cell-mediated immunity. However, the importance of antibodies for immunity against HZ prevention remains to be understood. The goal of this study was to examine the breadth and functionality of VZV-specific antibodies after vaccination with a live attenuated HZ vaccine (Zostavax). Direct enumeration of VZV-specific antibody secreting cells (ASC) via ELISPOT showed that Zostavax can induce both IgG and IgA ASCs seven days after vaccination, but not IgM ASCs. The VZV-specific ASCs range from 33-55% of the total IgG ASCs. 25 human VZV-specific monoclonal antibodies (mAbs) were cloned and characterized from single-cell sorted ASCs of five subjects (ggt; 60 years old) who received Zostavax. These mAbs had an average of ~20 somatic hypermutations per VH gene, similar to those seen after seasonal influenza vaccination. Fifteen of the 25 mAbs were gE-specific, whereas the remaining mAbs were gB-, gH- or gI-specific. The most potent neutralizing antibodies were gH-specific and were also able to inhibit cell-to-cell spread of the virus in vitro. Most gE-specific mAbs were able to neutralize VZV, but they required the presence of complement and were unable to block cell-to-cell spread. These data indicate that Zostavax induces a memory B cell recall response characterized by anti-gEggt;gIggt;gBggt;gH antibodies. While antibodies to gH could be involved in limiting the spread of VZV upon reactivation, the contribution of anti-gE antibodies towards protective immunity after Zostavax needs further evaluation.

    Importance Varicella zoster virus (VZV) is the causative agent of chickenpox and shingles. Following infection with VZV, the virus becomes latent and resides in nerve cells. Age-related declines in immunity/immunosuppression can result in reactivation of this latent virus causing shingles. It has been shown that waning T cell immunity correlates with an increased incidence of VZV reactivation. Interestingly, serum with high levels of VZV-specific antibodies (VariZIG; IV immunoglobulin) has been administered to high-risk populations, e.g. immunocompromised children, newborns and pregnant women, after exposure to VZV and has shown some protection against chickenpox. However, the relative contribution of antibodies against individual surface glycoproteins towards protection from shingles in the elderly/immunocompromised individuals has not been established. Here, we examined the breadth and functionality of VZV-specific antibodies after vaccination with a live attenuated VZV vaccine Zostavax in humans. This study will add to our understanding of the role of antibodies in protection against shingles.

  • A Screen for Epstein-Barr Virus Proteins that Inhibit the DNA Damage Response Reveals a Novel Histone Binding Protein [PublishAheadOfPrint]

  • To replicate and persist in human cells, linear double-stranded (ds) DNA viruses, such as Epstein-Barr virus (EBV), must overcome the host DNA damage response (DDR) that is triggered by the viral genomes. Since this response is necessary to maintain cellular genome integrity, its inhibition by EBV is likely an important factor in the development of cancers associated with EBV infection, including gastric carcinoma. Here we present the first extensive screen of EBV proteins that inhibit dsDNA break signaling. We identify the BKRF4 tegument protein as a DDR inhibitor that interferes with histone ubiquitylation at dsDNA breaks and recruitment of the RNF168 histone ubiquitin ligase. We further show that BKRF4 binds directly to histones through an acidic domain that targets BKRF4 to cellular chromatin and is sufficient to inhibit dsDNA break signaling. BKRF4 transcripts were detected in EBV-positive gastric carcinoma cells (AGS-EBV) and these increased in lytic infection. Silencing of BKRF4 in both latent and lytic AGS-EBV cells (but not in EBV-negative AGS cells) resulted in increased dsDNA break signaling, confirming a role for BKRF4 in DDR inhibition in the context of EBV infection and suggesting that BKRF4 is expressed in latent cells. BKRF4 was also found to be consistently expressed in EBV-positive gastric tumours in the absence of a full lytic infection. The results suggest that BKRF4 plays a role in inhibiting the cellular DDR in latent and lytic EBV infection, and that the resulting accumulation of DNA damage might contribute to development of gastric carcinoma.

    IMPORTANCE Epstein-Barr virus (EBV) infects most people worldwide and is causatively associated with several types of cancer, including ~10% of gastric carcinoma. EBV encodes ~80 proteins many of which are believed to manipulate cellular regulatory pathways but are poorly characterized. The DNA damage response (DDR) is one such pathway that is critical for maintaining genome integrity and preventing cancer-associated mutations. Here a screen for EBV proteins that inhibit the DDR identified BKRF4 as a DDR inhibitor that binds histones and block their ubiquitylation at the DNA damage sites. We also present evidence that BKRF4 is expressed in both latent and lytic forms of EBV infection, where it down-regulates the DDR, as well as in EBV-positive gastric tumours. The results suggest that BKRF4 could contribute to the development of gastric carcinoma through its ability to inhibit the DDR.

  • The Antiviral Activity of the Cellular Glycoprotein LGALS3BP/90K is Species-Specific [PublishAheadOfPrint]

  • Cellular antiviral proteins interfere with distinct steps of replication cycles of viruses. The galectin 3 binding protein (LGALS3BP, also known as 90K) was previously shown to lower the infectivity of nascent HIV-1 virions when expressed in virus-producing cells. This antiviral effect was accompanied by impaired gp160Env processing and reduced viral incorporation of mature Env glycoproteins. Here, we examined the ability of 90K orthologs from primate species to reduce particle infectivity of distinct lentiviruses. We show that 90K's ability to diminish the infectivity of lentiviral particles is conserved within primate species, with the notable exception of 90K from rhesus macaque. Comparison of active and inactive 90K orthologs and variants uncovered that inhibition of processing of the HIV-1 Env precursor and reduction of cell surface expression of HIV-1 Env gp120 are required, but not sufficient for 90K-mediated antiviral activity. On the contrary, 90K-mediated reduction of virion-associated gp120 coincided with antiviral activity, suggesting that 90K impairs the incorporation of HIV-1 Env into budding virions. We show that a single "humanizing" amino acid exchange in the BTB (broad complex, tramtrack, and bric-a-brac)/POZ (poxvirus and zinc finger) domain is sufficient to fully rescue antiviral activity of a shortened version of rhesus macaque 90K, but not of the full-length protein. Comparison of the X-ray structures of the BTB/POZ domain of 90K from both species point towards a slightly larger hydrophobic patch at the surface of the rhesus macaque BTB domain that may modulate a direct interaction with either a second 90K domain or a different protein.

    IMPORTANCE The cellular 90K protein was shown to diminish the infectivity of nascent HIV-1 particles. When produced in 90K-expressing cells, particles bear lower amounts of the HIV-1 Env glycoprotein that is essential for attaching to and entering new target cells in the subsequent infection round. However, whether the antiviral function of 90K is conserved across primates is unknown. Here, we found that 90K orthologs from most primate species, but surprisingly not from rhesus macaques, inhibit HIV-1. Introduction of a single amino acid exchange in a short version of the rhesus macaque 90K protein, consisting of the two intermediate domains of 90K, resulted in full restoration of antiviral activity. Structural elucidation of the respective domain suggests that the absence of antiviral activity in the rhesus macaque factor may be linked to a subtle change in protein-protein interaction.

  • Intrastructural Help: Harnessing T-Helper Cells Induced by Licensed Vaccines for Improvement of HIV Env Antibody Responses to Virus-Like Particle Vaccines [PublishAheadOfPrint]

  • Induction of persistent antibody responses by vaccination is generally thought to depend on efficient help by T follicular helper cells. Since the T helper cell response to HIV Env may not be optimal, we explored the possibility to improve the HIV Env antibody response to virus-like particle (VLP) vaccines by recruiting T helper cells induced by a commonly-used licensed vaccines to provide help for Env-specific B cells. B cells specific for the surface protein of a VLP can internalize the entire VLP and thus present peptides derived from the surface and core proteins on their MHC-II molecules. This allows T helper cells specific for the core protein to provide intrastructural help for B cells recognizing the surface protein. Consistently, priming mice with an adjuvanted Gag protein vaccine enhanced the HIV Env antibody response to subsequent booster immunizations with HIV VLPs. To harness T helper cells induced by the licensed Tetanolrreg;pur vaccines, HIV VLPs were generated that contained T helper cell epitopes of tetanus-toxoid. Tetanol-immunized mice raised stronger antibody responses to immunizations with VLPs containing tetanus-toxoid T helper cell epitopes, but not to VLPs lacking these epitopes. Depending on the priming immunization, the IgG subtype response to HIV Env after the VLP immunization could also be modified. Thus, harnessing T helper cells induced by other vaccines appears to be a promising approach to improve the HIV Env antibody response to VLP vaccines.

    IMPORTANCE Induction of HIV Env antibodies at sufficient levels with optimal Fc effector functions for durable protection remains a challenge. Efficient T cell help may be essential to induce such a desirable antibody response. Here, we provide proof-of-concept that T helper cells induced by a licensed vaccine can be harnessed to provide help for HIV Env-specific B cells and to modulate the Env-specific IgG subtype response.

  • The complexities of viral mutation rates [PublishAheadOfPrint]

  • Many viruses evolve rapidly. This is due, in part, to their high mutation rates. Mutation rate estimates for over 25 viruses are currently available. Here, we review the population genetics of virus mutation rates. We specifically cover the topics of mutation rate estimation, the forces that drive the evolution of mutation rates, and how the optimal mutation rate can be context-dependent.

  • Herpes Simplex Virus 2 Latency-Associated Transcript (LAT) region mutations do not identify a role for LAT-Associated Micro RNAs in viral reactivation in the Guinea Pig Genital Model [PublishAheadOfPrint]

  • Despite the long-standing observation that herpes simplex virus (HSV) Latency-Associated Transcript (LAT) promoter-deletion viruses show impaired recurrence phenotypes in relevant animal models, the mechanism by which these sequences exert this phenotypic effect is unknown. We constructed and evaluated four mutant HSV-2 viruses with targeted mutations in the LAT promoter and LAT-associated miRNAs affecting (1) the LAT TATA box, (2) the LAT ICP4-binding site, (3) miR-I and miR-II (miR-I/II), which both target ICP34.5, and (4) miR-III, which targets ICP0. While the LAT-TATA box mutant caused milder acute infections than wild-type (WT), there was no difference in recurrence phenotype between these viruses. LAT and miRNA expression during latency were not impaired by this mutation, suggesting that other promoter elements may be more important for latent HSV-2 LAT expression. Mutation of the LAT ICP4-binding site also did not cause an in vivo phenotypic difference between mutant and WT viruses. Acute infection and reactivation from latency of the miR-I/II mutant was similar to that of its rescuant, although slightly reduced in severity relative to the wild-type virus. The miR-III mutant also exhibited WT phenotypes in acute and recurrent phases of infection. While not ruling out an effect of these elements in human latency or reactivation, these findings do not identify a specific role for LAT or LAT-associated miRNAs in the HSV-2 LAT promoter deletion phenotype in guinea pigs. Thus, other sequences in this region may play a more important role in the long-studied LAT-associated phenotype in animals.

    IMPORTANCE While it has been known for several decades that specific HSV-1 and HSV-2 sequences near the LAT promoter are required for efficient viral reactivation in animal models, the mechanism is still not known. We constructed four mutant viruses with the goal of identifying critical sequence elements and of specifically testing the hypothesis that microRNAs that are expressed during latency play a role. Determination that specific LAT promoter sequences and miRNA sequences do not influence viral reactivation of HSV-2 helps to narrow down the search for the mechanism by which the virus controls its latency and recurrence phenotype.

  • Glycan Binding Specificity and Mechanism of Human and Porcine P[6]/P[19] Rotavirus VP8*s [PublishAheadOfPrint]

  • Rotaviruses (RVs), which cause severe gastroenteritis in infants and children, recognize glycan ligands in a genotype-dependent manner via the distal VP8* head of the spike protein VP4. However, the glycan binding mechanisms remain elusive for the P[II] genogroup RVs, including the widely prevalent human RVs (P[8], P[4], and P[6]) and a rare P[19] RV. In this study, we characterized the glycan binding specificity of human and porcine P[6]/P[19] RV VP8* and found that the P[II] genogroup RV VP8*s could commonly interact with mucin core 2 which may play an important role in the RV evolution and cross-species transmission. We determined the first P[6] VP8* structure, as well as the complex structures of human P[19] VP8* with core 2 and lacto-N-tetraose (LNT). A glycan binding site was identified in human P[19] VP8*. Structural superimposition and sequence alignment revealed the conservation of the glycan binding site in the P[II] genogroup RV VP8*s. Our data provide significant insight into the glycan binding specificity and glycan binding mechanism of the P[II] genogroup RV VP8*s, which would help understanding of RV evolution, transmission, epidemiology, and vaccine approach.

    IMPORTANCE

    Rotaviruses (RVs), belonging to the family Reoviridae, are double-stranded RNA viruses causing acute gastroenteritis in children and animals worldwide. Depending on phylogeny of the VP8* sequences, P[6] and P[19] RVs are grouped into the genogroup II together with P[4] and P[8] that are widely prevalent in humans. In this study, we characterized the glycan binding specificity of human and porcine P[6]/P[19] RV VP8*s, determined the crystal structure of P[6] VP8*, and uncovered the glycan binding pattern in P[19] VP8*, revealing a conserved glycan binding site in the VP8*s of P[II] genogroup RVs by structural superimposition and sequence alignment. Our data suggested that mucin core 2 may play an important role in the P[II] RV evolution and cross-species transmission. These data provide insight into cell attachment, infection, epidemiology, and evolution of P[II] genogroup RVs, which would help to develop control and prevention strategies against RVs.

  • Antiviral Activity of HIV gp120 Targeting Bispecific T Cell Engager (BiTE(R)) Antibody Constructs [PublishAheadOfPrint]

  • Today's gold standard in HIV therapy is the combined antiretroviral therapy (cART). It requires strict adherence by patients and life-long medication, which can lower the viral load below detection limits and prevent HIV-associated immunodeficiency, but cannot cure patients. The bispecific T cell engaging (BiTErreg;) antibody technology has demonstrated long-term relapse-free outcomes in patients with relapsed and refractory acute lymphocytic leukemia. We here generated BiTErreg; antibody constructs that target the HIV-1 envelope protein gp120 (HIV gp120) using either the scFv B12 or VRC01, the first two extracellular domains (1+2) of human CD4 alone or joined to the single chain variable fragment (scFv) of the antibody 17b fused to an anti-human CD3 scFv. These engineered human BiTErreg; antibody constructs showed engagement of T cells for redirected lysis of HIV gp120-transfected CHO cells. Furthermore, they substantially inhibited HIV-1 replication in PBMCs as well as in macrophages co-cultured with autologous CD8+ T-cells, the most potent being the human CD4(1+2) BiTErreg; antibody construct and the CD4(1+2)L17b BiTErreg; antibody construct. The CD4(1+2) h BiTErreg; antibody construct promoted HIV infection of human CD4-/CD8+ T cells. In contrast, the neutralizing B12 and the VRC01 BiTErreg; antibody constructs as well as the CD4(1+2)L17b BiTErreg; antibody construct did not. Thus, BiTErreg; antibody constructs targeting HIV gp120 are very promising for constraining HIV and warrant further development as novel antiviral therapy with curative potential.

    Importance

    HIV is a chronic infection well controlled with the current cART. However, we lack cure of HIV, and the HIV pandemic goes on. Here we showed in vitro and ex vivo that a bispecific T-cell engaging (BiTErreg;) antibody construct targeting HIV gp120 resulted in substantially reduced HIV replication. In addition, these BiTErreg; antibody constructs display efficient killing of gp120 expressing cells and inhibited replication in ex vivo HIV-infected PBMCs or macrophages. We believe that BiTErreg; antibody constructs recognizing HIV gp120 could be a very valuable strategy for a cure of HIV in combination with cART and compounds, which reverse latency.

  • A Crystal Structure of Classical Swine Fever Virus NS5B Reveals a Novel N-terminal Domain [PublishAheadOfPrint]

  • Classical swine fever virus (CSFV) is the ringleader of Classical swine fever (CSF). The non-structural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp) that is a key enzyme initiating viral RNA replication by a de novo mechanism. It is also an attractive target for the development of anti-CSFV drugs. To gain a better understanding on the mechanism of CSFV RNA synthesis, here we solved the first crystal structure of CSFV-NS5B. Our studies show that the CSFV-NS5B RdRp contains characteristic fingers, palm domain and thumb domain as well as a unique N-terminal domain (NTD) that had never been observed. Mutagenesis studies on NS5B validated the importance of NTD in the catalytic activity of this novel RNA-dependent RNA polymerase. Moreover, our results shed light on the understanding of CSFV infection.

    IMPORTANCE Pigs are important domestic animal. However, a highly contagious viral disease named Classical swine fever (CSF) causes devastating economic losses. Classical swine fever virus (CSFV) is the primary culprit of CSF, which is a positive-sense single-stranded RNA virus belonging to the Pestivirus genus, Flaviviridae family. Genome replication of CSFV depends on RNA-dependent RNA polymerase known as NS5B. However, the structure of CSFV-NS5B has never been reported, and the mechanism of CSFV replication is poorly understood. Here, we solved the first crystal structure of CSFV-NS5B, analyzed the function of characteristic fingers, palm, and thumb domains. Additionally, our structure also revealed the presence of a novel N-terminal domain (NTD). Biochemical studies demonstrated that the NTD of CSFV-NS5B is very important for RNA-dependent RNA polymerase (RdRp) activity. Collectively, our studies provide a structural basis for future rational design of anti-CSFV drugs which is critically important as no effective anti-CSFV drugs have been developed.

  • Short-term pIFN-{alpha}2a treatment does not significantly reduce the viral reservoir of SIV-infected, ART-treated rhesus macaques [PublishAheadOfPrint]

  • The major obstacle to HIV-1 eradication is a reservoir of latently infected cells that persists despite long-term antiretroviral therapy (ART) and causes rapid viral rebound if treatment is interrupted. Type I interferons are immunomodulatory cytokines that induce antiviral factors and have been evaluated for the treatment of HIV-infected individuals, resulting in moderate reduction of viremia and inconclusive data about their effect on reservoir size. Here, we assessed the potential of pegylated IFN-aalpha;2a (pIFN-aalpha;2a) to reduce the viral reservoir in SIV-infected, ART-treated rhesus macaques (RMs). We found that pIFN-aalpha;2a treatment of animals in which virus replication is effectively suppressed with ART is safe and well-tolerated as no major clinical side effects were observed. By monitoring the cellular immune response during this intervention, we established that pIFN-aalpha;2a administration is not associated with either CD4+ T cell depletion or increased immune activation. Importantly, we found that Interferon Stimulated Genes (ISGs) were significantly up-regulated in IFN-treated RMs when compared to control animals, confirming that pIFN-aalpha;2a is bioactive in vivo. To evaluate the effect of pIFN-aalpha;2a administration on the viral reservoir in CD4+ T cells, we performed cell-associated proviral SIV DNA measurements in multiple tissues and assessed levels of replication-competent virus by a quantitative viral outgrowth assay (QVOA). These analyses failed to reveal any significant difference in reservoir size between IFN-treated and control animals. In summary, our data suggest that short-term type I interferon treatment in combination with suppressive ART is not sufficient to induce a significant reduction of the viral reservoir in SIV-infected RMs.

    IMPORTANCE The potential of type I interferons to reduce the viral reservoir has been recently studied in clinical trials in HIV-infected humans. However, given the lack of mechanistic data and the potential for safety concerns, a more comprehensive testing of IFN treatment in vivo in SIV-infected rhesus macaques (RM) is critical to provide rationale for further development of this intervention in humans. Utilizing the SIV/RM model in which virus replication is suppressed with ART, we addressed experimental limitations of previous human studies, in particular the lack of a control group and specimen sampling limited to blood. Here, we show by rigorous testing of blood and lymphoid tissues that virus replication and reservoir size was not significantly affected by pIFN-aalpha;2a treatment in SIV-infected, ART-treated RMs. This suggests that intensified and/or prolonged IFN treatment regimens, possibly in combination with other anti-latency agents, might be necessary to effectively purge the HIV/SIV reservoir under ART.

  • Secretion of the non-structural protein 1 of dengue virus from infected mosquito cells: facts and speculations [PublishAheadOfPrint]

  • The dengue virus NS1 is a multifunctional glycoprotein. For decades, the notion in the field was that NS1 was secreted exclusively from vertebrate, but not mosquito, cells. However, recent evidence shows that mosquito cells also secrete NS1 efficiently. In this review, we discuss the evidence for dengue, and other flaviviruses, NS1 secretion from mosquito cells, differences between NS1 secreted from mosquito and vertebrate cells, and possible roles of soluble NS1 in the insect flavivirus vector.

  • Small bottleneck size in a highly multipartite virus during a complete infectious cycle [PublishAheadOfPrint]

  • Multipartite viruses package their genomic segments independently and thus incur the risk of being unable to transmit their entire genome during host-to-host transmission if they undergo severe bottlenecks. In this paper we estimated the bottleneck size during one infection cycle of Faba bean necrotic stunt virus (FBNSV), an octopartite nanovirus whose segments have been previously shown to converge to particular and unequal relative frequencies within host plants and aphid vectors. Two methods were used to derive this estimate: one based on the probability of transmission of the virus and the other based on the temporal evolution of the relative frequency of markers for two genomic segments, one frequent and one rare (segment N and S respectively) both in plants and vectors. Our results show that FBNSV undergoes severe bottlenecks during aphid transmission. Further, even though the bottlenecks are always narrow under our experimental conditions, they slightly widen up with the number of transmitting aphids. In particular, when several aphids are used for transmission the bottleneck size of the segments is also affected by within-plant processes and, importantly, significantly differs across segments. These results indicate that genetic drift must not only be an important process affecting the evolution of these viruses, but also that these effects may vary across genomic segments, and thus across viral genes, a rather unique and intriguing situation. We further discuss the potential consequences of our findings for the transmission of multipartite viruses.

    Importance

    Multipartite viruses package their genomic segments in independent capsids. The most obvious cost of such genomic structure is the risk of losing at least one segment during host-to-host transmission. A theoretical study has shown that for nanoviruses, composed of 6 to 8 segments, hundreds of copies of each segment need to be transmitted to ensure that at least one copy of each segment was present in the host. These estimations seem to be very high compared to the size of the bottlenecks measured with other viruses. Here we estimated the bottleneck size during one infection cycle of FBNSV, an octopartite nanovirus. We show that these bottlenecks are always narrow (few viral particles) and slightly widen up with the number of transmitting aphids. These results contrast with theoretical predictions and illustrate the fact that a new conceptual framework is probably needed to understand the transmission of highly multipartite viruses.

  • Seminal SIV in chronically-infected cynomolgus macaques is dominated by virus originating from multiple genital organs [PublishAheadOfPrint]

  • The sexual transmission of viruses is responsible for the spread of multiple infectious diseases. Although the HIV/AIDS pandemic remains fueled by sexual contacts with infected semen, the origin of virus in semen is still unknown. In a substantial number of HIV- infected men, viral strains present in semen differ from the ones in blood, suggesting that HIV is locally produced within the genital tract. Such local production may be responsible for the persistence of HIV in semen despite effective antiretroviral therapy. Here we use single genome amplification, amplicon sequencing (env gene) and phylogenetic analyses to compare the genetic structure of SIV populations across all the male genital organs and blood in intravenously inoculated cynomolgus macaques in the chronic stage of infection. Examination of the virus populations present in the male genital tissues of the macaques revealed compartmentalized SIV populations in testis, epididymis, vas deferens, seminal vesicles and urethra. We found genetic similarities between the viral strains present in semen and those in epididymis, vas deferens and seminal vesicles. The contribution of male genital organs to virus shedding in semen varied among individuals and could not be predicted based on their infection or pro-inflammatory cytokine mRNA levels. These data indicate that, rather than a single source, multiple genital organs are involved in the release of free virus and infected cells into semen. These findings have important implications for our understanding of systemic virus shedding and persistence in semen and for the design of eradication strategies to access viral reservoirs.

    IMPORTANCE

    Semen is instrumental for the dissemination of viruses through sexual contacts. Worryingly, a number of systemic viruses such as HIV can persist in this body fluid in the absence of viremia. The local source(s) of virus in semen, however, remain unknown. To elucidate the anatomic origin(s) of the virus released in semen, we compared viral populations present in semen with those in the male genital organs and blood of the Asian macaque model, using single genome amplification, amplicon sequencing (env gene) and phylogenetic analysis. Our results show that multiple genital tissues harbor compartmentalized strains, some of them (i.e. epididymis, vas deferens and seminal vesicle) displaying genetic similarities with the viral populations present in semen. This study is the first to uncover local genital sources of viral populations in semen, providing a new basis for innovative targeted strategies to prevent and eradicate HIV in the male genital tract.

  • Lack of Transmission of Chronic Wasting Disease to Cynomolgus Macaques [PublishAheadOfPrint]

  • Chronic wasting disease (CWD) is a fatal prion disease that can infect deer, elk and moose. CWD was first recognized in captive deer kept in wildlife facilities in Colorado from 1967-1979. CWD has now been detected in 25 states of the USA, 2 Canadian provinces, South Korea, Norway and Finland. It is currently unknown if humans are susceptible to CWD infection. Understanding the health risk from consuming meat and/or products from CWD-infected cervids is a critical human health concern. Prior research using transgenic mouse models and in vitro conversion assays suggest that a significant species barrier exists between CWD and humans. To date, published epidemiologic studies of humans consuming cervids in CWD endemic areas have found no evidence to confirm CWD transmission to humans. Previously, we reported data from ongoing cross-species CWD transmission studies using two species of non-human primates as models. Squirrel monkeys (SM) and Cynomolgus macaques (CM) were inoculated by either intracerebral or oral routes with brain homogenates from CWD-infected deer and elk containing high levels of infectivity. SM were highly susceptible to CWD infection while CM were not. In the current study, we present new data for seven CWD-inoculated CM euthanized from 11-13 years post CWD-inoculation and eight additional uninoculated control CM. New and archival CM tissues were screened for prion infection using the ultrasensitive RT-QuIC assay, immunohistochemistry and immunoblot. In this study, there was no clinical, pathological or biochemical evidence suggesting that CWD was transmitted from cervids to CM.

    IMPORTANCE Chronic wasting disease (CWD) is a fatal prion disease found in deer, elk and moose. Since first discovered in the late 1960's, CWD has now spread to at least 25 states of the USA, 2 Canadian provinces, South Korea, Norway and Finland. Eradication of CWD from endemic areas is very unlikely and additional spread will occur. As the range and prevalence of CWD increases, so will the potential for human exposure to CWD prions. It is currently unknown if CWD poses a risk to human health. However, determining this risk is critical to prevent a similar scenario as what occurred when mad cow disease was found to be transmissible to humans. In the current study, we used cynomolgus macaque monkeys as a surrogate model for CWD transmission to humans. After 13 years, no evidence for CWD transmission to macaques was detected clinically or using highly sensitive prion disease screening assays.

  • Mapping and Engineering Functional Domains of the Assembly Activating Protein of Adeno-Associated Viruses [PublishAheadOfPrint]

  • Adeno-associated viruses (AAV) encode a unique assembly activating protein (AAP) within their genome that is essential for capsid assembly. Studies to date have focused on establishing the role of AAP as a chaperone that mediates stability, nucleolar transport, and assembly of AAV capsid proteins. Here, we map structure-function correlates of AAP using secondary structure analysis followed by deletion and substitutional mutagenesis of specific domains, namely, the hydrophobic N-terminal domain (HR), conserved core (CC), proline-rich region (PRR), threonine/serine rich region (T/S) and basic region (BR). First, we establish that the centrally located PRR and T/S regions are flexible linker domains that can either be deleted completely or replaced by heterologous functional domains that enable ancillary functions such as fluorescent imaging or increased AAP stability. We also demonstrate that the C-terminal BR domains can be substituted with heterologous nuclear or nucleolar localization sequences that display varying ability to support AAV capsid assembly. Further, by replacing the BR domain with immunoglobulin (IgG) Fc domains, we assessed AAP complexation with AAV capsid subunits and demonstrate that the hydrophobic region (HR) and the conserved core (CC) in the AAP N-terminus are the sole determinants for viral protein (VP) recognition. However, VP recognition alone is not sufficient for capsid assembly. Our study sheds light on the modular structure-function correlates of AAP and provides multiple approaches to engineer AAP that might prove useful towards understanding and controlling AAV capsid assembly.

    Importance: Adeno-associated viruses (AAV) encode a unique assembly activating protein (AAP) within their genome that is essential for capsid assembly. Understanding how AAP acts as a chaperone for viral assembly could help improve efficiency and potentially control this process. Our studies reveal that AAP has a modular architecture, with each module playing a distinct role and can be engineered for carrying out new functions.

  • Structural and Functional Features of the Reovirus {sigma}1 Tail [PublishAheadOfPrint]

  • Mammalian orthoreovirus attachment to target cells is mediated by outer-capsid protein 1, which projects from the virion surface. The 1 protein is a homotrimer consisting of a filamentous tail, which is partly inserted into the virion, a body domain constructed from bbeta;-spiral repeats, and a globular head with receptor-binding properties. The 1 tail is predicted to form an aalpha;-helical coiled coil. Although 1 undergoes a conformational change during cell entry, the nature of this change and its contributions to viral replication are unknown. Electron micrographs of 1 molecules released from virions identified three regions of flexibility, including at the midpoint of the molecule, that may be involved in its structural rearrangement. To enable a detailed understanding of essential 1 tail organization and properties, we determined high-resolution structures of the reovirus type 1 Lang (T1L) and type 3 Dearing (T3D) 1 tail domains. Both molecules feature extended aalpha;-helical coiled coils, with T1L 1 harboring central chloride ions. Each molecule displays a discontinuity (stutter) within the coiled coil and an unexpectedly seamless transition to the body domain. The transition region features conserved interdomain interactions and appears rigid rather than highly flexible. Functional analyses of reoviruses containing engineered 1 mutations suggest that conserved residues predicted to stabilize the coiled coil-to-body junction are essential for 1 folding and encapsidation, whereas central chloride ion coordination and the stutter are dispensable for efficient replication. Together, these findings enable modeling of full-length reovirus 1 and provide insight into the stabilization of a multi-domain viral attachment protein.

    IMPORTANCE While it is established that different conformational states of attachment proteins of enveloped viruses mediate receptor binding and membrane fusion, less is understood about how such proteins mediate attachment and entry in nonenveloped viruses. The filamentous reovirus attachment protein, 1, binds cellular receptors, contains regions of predicted flexibility including at the fiber midpoint, and undergoes a conformational change during cell entry. Neither the nature of the structural change nor its contribution to viral infection is understood. We determined crystal structures of large 1 fragments for two different reovirus serotypes. We observed an unexpectedly tight transition between two domains spanning the fiber midpoint, which allows for little flexibility. Studies of reoviruses with engineered changes near the 1 midpoint suggest that the stabilization of this region is critical for function. Together with a previously determined structure, we now have a complete model of the full-length, elongated reovirus 1 attachment protein.

  • Critical Role of the HTLV-1 Capsid N-Terminal Domain for Gag-Gag Interactions and Virus Particle Assembly [PublishAheadOfPrint]

  • The retroviral Gag protein is the main structural protein responsible for virus particle assembly and release. Like human immunodeficiency virus type 1 (HIV-1) Gag, human T-cell leukemia virus type 1 (HTLV-1) has a structurally conserved capsid (CA) domain, including a bbeta;-hairpin turn and a centralized coiled-coil-like structure of six aalpha; helices in the CA amino-terminal domain (NTD) as well as four aalpha;-helices in the CA carboxy-terminal domain (CTD). CA drives Gag oligomerization, which is critical for both immature Gag lattice formation and particle production. The HIV-1 CA CTD has previously been shown to be a primary determinant for CA-CA interactions, and while both the HTLV-1 CA NTD and CTD have been implicated in Gag-Gag interactions, our recent observations have implicated the HTLV-1 CA NTD as encoding key determinants that dictate particle morphology. Here, we have conducted alanine-scanning mutagenesis in the HTLV-1 CA NTD nucleotide-encoding sequences spanning the loop regions and amino acids at the beginning and ends of aalpha;-helices due to their structural dissimilarity from the HIV-1 CA NTD structure. We analyzed both Gag subcellular distribution and efficiency of particle production for these mutants. We discovered several important residues (i.e., M17, Q47/F48, and Y61). Modeling implicated that these residues reside at the dimer interface (i.e., M17 and Y61) or at the trimer interface (i.e., Q47/F48). Taken together, these observations highlight the critical role of the HTLV-1 CA NTD in Gag-Gag interactions and particle assembly, which is, to the best of our knowledge, in contrast to HIV-1 and other retroviruses.

    Importance

    Retrovirus particle assembly and release from infected cells is driven by the Gag structural protein. Gag-Gag interactions, which form an oligomeric lattice structure at a particle budding site, are essential to the biogenesis of an infectious virus particle. The capsid (CA) domain of Gag is generally thought to possess the key determinants for Gag-Gag interactions, and the present study has discovered several critical amino acid residues in the CA domain of human T-cell leukemia virus type 1 (HTLV-1) Gag, an important cancer-causing human retrovirus, which are distinct from that of human immunodeficiency virus type 1 (HIV-1) as well as other retroviruses studied to date. Altogether, our results provide important new insights into a poorly understood aspect of HTLV-1 replication, which significantly enhances our understanding of the molecular nature of Gag-Gag interaction determinants crucial for virus particle assembly.

  • KRAB-ZFP repressors enforce quiescence of oncogenic human herpesviruses [PublishAheadOfPrint]

  • Cancer-causing herpesviruses infect nearly every human and persist indefinitely in B lymphocytes in a quiescent state known as latency. A hallmark of this quiescence or latency is the presence of extrachromosomal viral genomes with highly restricted expression of viral genes. Silencing of viral genes ensures both immune evasion by the virus and limited pathology to the host; yet, how multiple genes on multiple copies of viral genomes are simultaneously silenced is a mystery. In a unifying theme, we report that both cancer-causing human herpesviruses, despite having evolved independently, are silenced through the activities of two members of the KRAB-ZFP epigenetic silencing family, revealing a novel STAT3-KRAB-ZFP axis of viral latency. This dual-edged anti-viral strategy restricts the destructive ability of the lytic phase while promoting the cancer-causing latent phase. These findings also unveil roles for KRAB-ZFPs in silencing multi-copy foreign genomes with the promise of evicting herpesviruses to kill viral cancers bearing clonal viral episomes.

    Importance

    Despite robust immune responses, cancer-causing viruses EBV and KSHV persist for life. This persistence is accomplished partly through a stealth mechanism that keeps extrachromosomal viral genomes quiescent. Quiescence, or latency, ensures that not every cell harboring viral genomes is killed directly through lytic activation or indirectly via the immune response, thereby evicting virus from host. For the host, quiescence limits pathology. Thus, both virus and host benefit from quiescence; yet, how quiescence is maintained through silencing of a large set of viral genes on multiple viral genomes is not well-understood. Our studies reveal that members of a gene-silencing family, the KRAB-ZFP proteins, promote quiescence of both cancer-causing human viruses through simultaneous silencing of multiple genes on multi-copy extrachromosomal viral genomes.

  • Regulation of Apolipoprotein E Trafficking by Hepatitis C Virus-induced Autophagy [PublishAheadOfPrint]

  • Apolipoprotein E (ApoE) plays an important role in the maturation and infectivity of hepatitis C virus (HCV). By analyzing the subcellular localization of ApoE in Huh7 hepatoma cells that harbored an HCV subgenomic RNA replicon, we found that ApoE colocalized with autophagosomes. This colocalization was marginally detected in HCV-infected cells apparently due to the depletion of ApoE by HCV, as the treatment with bafilomycin A1 (BafA1), a vacuolar ATPase inhibitor that inhibits autophagic protein degradation, partially restored the ApoE level and enhanced its colocalization with autophagosomes in HCV-infected cells. The role of HCV-induced autophagy in the degradation of ApoE was further supported by the observations that nutrient starvation, which induces autophagic protein degradation, led to the loss of ApoE in HCV subgenomic RNA replicon cells, and the knockdown of ATG7, a protein essential for the formation of autophagic vacuoles, increased the ApoE level in cells with productive HCV replication. Interesting, the inhibition of autophagy by ATG7 knockdown reduced the colocalization of ApoE with the HCV E2 envelope protein and the HCV titers released from cells. In contrast, the treatment of cells with BafA1 enhanced the colocalization of ApoE and HCV E2 and increased both intracellular and extracellular HCV titers. These results indicated that autophagy played an important role in the trafficking of ApoE in HCV-infected cells. While it led to autophagic degradation of ApoE, it also promoted the interaction between ApoE and HCV E2 to enhance the production of infectious progeny viral particles.

    Importance

    Hepatitis C virus (HCV) is one of the most important human pathogens. Its virion is associated with apolipoprotein E (ApoE), which enhances its infectivity. HCV induces autophagy to enhance its replication. In this report, we demonstrate that autophagy plays an important role in the trafficking of ApoE in HCV-infected cells. This leads to the degradation of ApoE by autophagy. However, if the autophagic protein degradation is inhibited, ApoE is stabilized and colocalized with autophagosomes. This leads to its enhanced colocalization with the HCV E2 envelope protein and increased production of infectious progeny viral particles. If autophagy is inhibited by suppressing the expression of ATG7, a gene essential for the formation of autophagosomes, the colocalization of ApoE with E2 is reduced, resulting in the reduction of progeny viral titers. These results indicate an important role of autophagy in the transport of ApoE to promote the production of infectious HCV particles.

  • A Chikungunya virus trans-replicase system reveals the importance of delayed non-structural polyprotein processing for efficient replication complex formation in mosquito cells. [PublishAheadOfPrint]

  • Chikungunya virus (CHIKV) is a medically important alphavirus that is transmitted by Aedes aegypti and Aedes albopictus mosquitoes. The viral replicase complex consists of four non-structural proteins (nsPs) expressed as polyprotein precursor and encompasses all enzymatic activities required for viral RNA replication. nsPs interact with host components of which most are still poorly understood, especially in mosquitos. A CHIKV trans-replicase system that allows the uncoupling of RNA replication and nsPs expression was adapted to mosquito cells and subsequently used for analysis of universal and host-specific effects of 17 different non-structural (ns) polyprotein mutations. It was found that mutations blocking nsP enzymatic activities as well as insertions of EGFP into different nsPs had similar effects on trans-replicase activity regardless of the host (i.e. mammalian or mosquito). Mutations that slow down or accelerate ns-polyprotein processing generally had no effect or reduced trans-replicase activity in mammalian cells while in mosquito cells most of them increased trans-replicase activity prominently. Increased RNA replication in mosquito cells was counteracted by an antiviral RNAi response. Substitution of the W258 residue in the membrane binding peptide of nsP1 resulted in a temperature sensitive defect, both in the context of the trans-replicase and infectious CHIKV. The defect was compensated for by secondary mutations selected during passaging of mutant CHIKV. These findings demonstrate the value of alphavirus trans-replicase systems for studies of viral RNA replication and virus-host interactions.

    IMPORTANCE

    Chikungunya virus is an important mosquito-transmitted human pathogen. This virus actively replicates in mosquitoes but the underlying molecular mechanisms and interactions of viral and host components are poorly understood. This is partly due to the lack of reliable systems for functional analysis of viral non-structural (ns) polyproteins and -proteins (nsPs) in mosquito cells. Adaption of a CHIKV trans-replicase system allowed to study the effects of mutations in the ns-polyprotein on RNA replication in cells derived from mammalian and mosquito hosts. We found that a slowdown of ns-polyprotein processing facilitates replication complex formation and/or functioning in mosquito cells and that this process is antagonized by the natural RNAi defense system present in mosquito cells. The mosquito adapted CHIKV trans-replicase system represents a valuable tool to study alphavirus-mosquito interactions at the molecular level and to develop advanced anti-viral strategies.

  • Timeliness of proteolytic events is prerequisite for efficient functioning of the alphaviral replicase [PublishAheadOfPrint]

  • Polyprotein processing has an important regulatory role in the lifecycle of positive-strand RNA viruses. In the case of alphaviruses, sequential cleavage of non-structural (ns) polyprotein at three sites eventually yields four mature ns-proteins that continue working in complex to replicate viral genomic RNA and transcribe subgenomic RNA. Recognition of cleavage sites by viral nsP2 protease is guided by short sequences upstream of the scissile bond and, more importantly, by the spatial organization of the replication complex. In this study, we analyzed the consequences of the artificially accelerated processing of Semliki Forest virus ns-polyprotein. It was found that in mammalian cells not only the order but also the correct timing of the cleavage events is essential for the success of viral replication. Analysis of the effects of compensatory mutations in rescued viruses as well as in vitro translation and trans-replicase assays corroborated our findings and revealed the importance of the V515 residue in nsP2 for recognizing the P4 position in the nsP1/nsP2 cleavage site. We also extended our conclusions to the Sindbis virus by analyzing the properties of the hyperprocessive variant carrying the N614D mutation in nsP2. We conclude that the sequence of the nsP1/nsP2 site in alphaviruses is under selective pressure to avoid the presence of sequences that are recognized too efficiently and would otherwise lead to premature cleavage at this site before completion of essential tasks of RNA synthesis or virus-induced replication complex formation. Even subtle changes in the ns polyprotein processing pattern appear to lead to virus attenuation.

    IMPORTANCE

    Polyprotein expression strategy is a cornerstone of alphavirus replication. Three sites within ns-polyprotein are recognized by the viral nsP2 protease and cleaved in a defined order. Specific substrate targeting is achieved by the recognition of the short sequence upstream of the scissile bond and a correct macromolecular assembly of ns polyprotein. Here, we highlighted the importance of the timeliness of proteolytic events, as an additional layer of regulation of efficient virus replication. We conclude that, somewhat counterintuitively, the cleavage site sequences at nsP1/nsP2 and nsP2/nsP3 junctions are evolutionarily selected to be recognized by protease inefficiently, to avoid premature cleavages that would be detrimental for the assembly and functionality of replication complex. Understanding the causes and consequences of viral polyprotein processing events is important for predicting the properties of mutant viruses and should be helpful for the development of better vaccine candidates and understanding potential resistance mechanisms to protease inhibitors.

  • Molecular basis of unusually high neutralization resistance in tier 3 HIV-1 strain 253-11 [PublishAheadOfPrint]

  • Understanding the mechanisms used by HIV-1 to evade antibody neutralization may contribute to the design of a high-coverage vaccine. The tier 3 virus 253-11, is poorly neutralized by subtype-matched and subtype C sera, even when compared to other tier 3 viruses, and is also recognized poorly by V3/glycan targeting monoclonal antibodies. We found that sequence polymorphism in the V3 loop and N-linked glycosylation sites only minimally contribute to the high neutralization resistance of 253-11. Interestingly, the 253-11 membrane proximal external region (MPER) is rarely recognized by sera in the context of the wild-type virus, but is commonly recognized in the context of an HIV-2 chimeric virus, suggesting steric or kinetic hindrance of binding to MPER in the native Env. Mutations in the 253-11 MPER mmdash; which were previously reported to increase the lifetime of the pre-fusion Envelope (Env) conformation mmdash; affected the resistance of 253-11 to antibodies targeting various epitopes on HIV-1 Env, presumably destabilizing its otherwise stable, closed trimer structure. To gain insight into the structure of 253-11, we constructed and crystallized a recombinant 253-11 SOSIP trimer. The resulting structure revealed that the heptad repeat helices in gp41 are drawn in close proximity to the trimer axis and that gp120 protomers also showed a relatively compact disposition around the trimer axis. These observations give substantial insight into the molecular features of an envelope spike from a tier 3 virus and into possible mechanisms that may contribute to its unusually high neutralization resistance.

    IMPORTANCE HIV-1 isolates that are highly resistant to broadly neutralizing antibodies could limit the efficacy of an antibody-based vaccine. We studied 253-11, which is highly resistant to commonly-elicited neutralizing antibodies. To further understand its resistance, we made mutations that are known to delay fusion and thus increase the time the virus spends in the open conformation following CD4-binding. Interestingly, we found that these mutations affect the 253-11 Envelope (Env) spike before CD4 binding, presumably by destabilizing the trimer structure. To gain further information about the structure of the 253-11 Env trimer, we generated a recombinant 253-11 SOSIP trimer. The crystal structure of the SOSIP trimer revealed that the gp41 helices and the gp120 protomers were drawn in towards the center of the molecule compared to most solved HIV-1 Env structures. These observations provide insight into the distinct molecular features of a Tier 3 envelope spike.

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