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

Table of Contents for this page:

  • Current Issue
  • Advanced Online Publications Articles
  • Current Issue of Journal of Virology

    JVI Current Issue

  • Editorial Board [Masthead]

  • Low-Bias RNA Sequencing of the HIV-2 Genome from Blood Plasma [Genetic Diversity and Evolution]

  • Accurate determination of the genetic diversity present in the HIV quasispecies is critical for the development of a preventative vaccine: in particular, little is known about viral genetic diversity for the second type of HIV, HIV-2. A better understanding of HIV-2 biology is relevant to the HIV vaccine field because a substantial proportion of infected people experience long-term viral control, and prior HIV-2 infection has been associated with slower HIV-1 disease progression in coinfected subjects. The majority of traditional and next-generation sequencing methods have relied on target amplification prior to sequencing, introducing biases that may obscure the true signals of diversity in the viral population. Additionally, target enrichment through PCR requires a priori sequence knowledge, which is lacking for HIV-2. Therefore, a target enrichment free method of library preparation would be valuable for the field. We applied an RNA shotgun sequencing (RNA-Seq) method without PCR amplification to cultured viral stocks and patient plasma samples from HIV-2-infected individuals. Libraries generated from total plasma RNA were analyzed with a two-step pipeline: (i) de novo genome assembly, followed by (ii) read remapping. By this approach, whole-genome sequences were generated with a 28x to 67x mean depth of coverage. Assembled reads showed a low level of GC bias, and comparison of the genome diversities at the intrahost level showed low diversity in the accessory gene vpx in all patients. Our study demonstrates that RNA-Seq is a feasible full-genome de novo sequencing method for blood plasma samples collected from HIV-2-infected individuals.

    IMPORTANCE An accurate picture of viral genetic diversity is critical for the development of a globally effective HIV vaccine. However, sequencing strategies are often complicated by target enrichment prior to sequencing, introducing biases that can distort variant frequencies, which are not easily corrected for in downstream analyses. Additionally, detailed a priori sequence knowledge is needed to inform robust primer design when employing PCR amplification, a factor that is often lacking when working with tropical diseases localized in developing countries. Previous work has demonstrated that direct RNA shotgun sequencing (RNA-Seq) can be used to circumvent these issues for hepatitis C virus (HCV) and norovirus. We applied RNA-Seq to total RNA extracted from HIV-2 blood plasma samples, demonstrating the applicability of this technique to HIV-2 and allowing us to generate a dynamic picture of genetic diversity over the whole genome of HIV-2 in the context of low-bias sequencing.

  • Influenza A Virus Utilizes Low-Affinity, High-Avidity Interactions with the Nuclear Import Machinery To Ensure Infection and Immune Evasion [Virus-Cell Interactions]

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

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

  • Structure-Based Classification Defines the Discrete Conformational Classes Adopted by the Arenaviral GP1 [Structure and Assembly]

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

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

  • Naturally Occurring Single Mutations in Ebola Virus Observably Impact Infectivity [Pathogenesis and Immunity]

  • Sequencing of Ebola virus (EBOV) genomes during the 2014nndash;2016 epidemic identified several naturally occurring, dominant mutations potentially impacting virulence or tropism. In this study, we characterized EBOV variants carrying one of the following substitutions: A82V in the glycoprotein (GP), R111C in the nucleoprotein (NP), or D759G in the RNA-dependent RNA polymerase (L). Compared with the wild-type (WT) EBOV C07 isolate, NP and L mutants conferred a replication advantage in monkey Vero E6, human A549, and insectivorous bat Tb1.Lu cells, while L mutants displayed a disadvantage in human Huh7 cells. The replication of the GP mutant was significantly delayed in Tb1.Lu cells and similar to that of the WT in other cells. The L mutant was less virulent, as evidenced by increased survival for mice and a significantly delayed time to death for ferrets, but increased lengths of the period of EBOV shedding may have contributed to the prolonged epidemic. Our results show that single substitutions can have observable impacts on EBOV pathogenicity and provide a framework for the study of other mutations.

    IMPORTANCE During the Ebola virus (EBOV) disease outbreak in West Africa in 2014nndash;2016, it was discovered that several mutations in the virus emerged and became prevalent in the human population. This suggests that these mutations may play a role impacting viral fitness. We investigated three of these previously identified mutations (in the glycoprotein [GP], nucleoprotein [NP], or RNA-dependent RNA polymerase [L]) in cell culture, as well as in mice and ferrets, by generating recombinant viruses (based on an early West African EBOV strain) each carrying one of these mutations. The NP and L mutations appear to decrease virulence, whereas the GP mutation slightly increases virulence but mainly impacts viral tropism. Our results show that these single mutations can impact EBOV virulence in animals and have implications for the rational design of efficacious antiviral therapies against these infections.

  • Defining HIV-1 Envelope N-Glycan Microdomains through Site-Specific Heterogeneity Profiles [Structure and Assembly]

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

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

  • P2X Antagonists Inhibit HIV-1 Productive Infection and Inflammatory Cytokines Interleukin-10 (IL-10) and IL-1{beta} in a Human Tonsil Explant Model [Virus-Cell Interactions]

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

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

  • A 1,000-Year-Old RNA Virus [Genetic Diversity and Evolution]

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

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

  • Human MxB Inhibits the Replication of Hepatitis C Virus [Virus-Cell Interactions]

  • Type I interferon (IFN) inhibits viruses by inducing the expression of antiviral proteins. The IFN-induced myxovirus resistance B (MxB) protein has been reported to inhibit a limited number of viruses, including HIV-1 and herpesviruses, but its antiviral coverage remains to be explored further. Here we show that MxB interferes with RNA replication of hepatitis C virus (HCV) and significantly inhibits viral replication in a cyclophilin A (CypA)-dependent manner. Our data further show that MxB interacts with the HCV protein NS5A, thereby impairing NS5A interaction with CypA and NS5A localization to the endoplasmic reticulum, two events essential for HCV RNA replication. Interestingly, we found that MxB significantly inhibits two additional CypA-dependent viruses of the Flaviviridae family, namely, Japanese encephalitis virus and dengue virus, suggesting a potential link between virus dependence on CypA and virus susceptibility to MxB inhibition. Collectively, these data have identified MxB as a key factor behind IFN-mediated suppression of HCV infection, and they suggest that other CypA-dependent viruses may also be subjected to MxB restriction.

    IMPORTANCE Viruses of the Flaviviridae family cause major illness and death around the world and thus pose a great threat to human health. Here we show that IFN-inducible MxB restricts several members of the Flaviviridae, including HCV, Japanese encephalitis virus, and dengue virus. This finding not only suggests an active role of MxB in combating these major pathogenic human viruses but also significantly expands the antiviral spectrum of MxB. Our study further strengthens the link between virus dependence on CypA and susceptibility to MxB restriction and also suggests that MxB may employ a common mechanism to inhibit different viruses. Elucidating the antiviral functions of MxB advances our understanding of IFN-mediated host antiviral defense and may open new avenues to the development of novel antiviral therapeutics.

  • Identification of Antinorovirus Genes in Human Cells Using Genome-Wide CRISPR Activation Screening [Virus-Cell Interactions]

  • Noroviruses (NoVs) are a leading cause of gastroenteritis worldwide, yet host factors that restrict NoV replication are not well understood. Here, we use a CRISPR activation genome-wide screening to identify host genes that can inhibit murine norovirus (MNoV) replication in human cells. Our screens identified with high confidence 49 genes that can inhibit MNoV infection when overexpressed. A significant number of these genes are in interferon and immune regulation signaling networks, but surprisingly, the majority of the genes identified are neither associated with innate or adaptive immunity nor associated with any antiviral activity. Confirmatory studies of eight of the genes validate the initial screening data. Mechanistic studies on TRIM7 demonstrated a conserved role of the molecule in mouse and human cells in restricting MNoV in a step of infection after viral entry. Furthermore, we demonstrate that two isoforms of TRIM7 have differential antiviral activity. Taken together, these data provide a resource for understanding norovirus biology and demonstrate a robust methodology for identifying new antiviral molecules.

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

  • Contribution of DNA Replication to the FAM111A-Mediated Simian Virus 40 Host Range Phenotype [Virus-Cell Interactions]

  • Host range (HR) mutants of simian virus 40 (SV40) containing mutations in the C terminus of large T antigen fail to replicate efficiently or form plaques in restrictive cell types. HR mutant viruses exhibit impairments at several stages of the viral life cycle, including early and late gene and protein expression, DNA replication, and virion assembly, although the underlying mechanism for these defects is unknown. Host protein FAM111A, whose depletion rescues early and late gene expression and plaque formation for SV40 HR viruses, has been shown to play a role in cellular DNA replication. SV40 viral DNA replication occurs in the nucleus of infected cells in viral replication centers where viral proteins and cellular replication factors localize. Here, we examined the role of viral replication center formation and DNA replication in the FAM111A-mediated HR phenotype. We found that SV40 HR virus rarely formed viral replication centers in restrictive cells, a phenotype that could be rescued by FAM111A depletion. Furthermore, while FAM111A localized to nucleoli in uninfected cells in a cell cycle-dependent manner, FAM111A relocalized to viral replication centers after infection with SV40 wild-type or HR viruses. We also found that inhibition of viral DNA replication through aphidicolin treatment or through the use of replication-defective SV40 mutants diminished the effects of FAM111A depletion on viral gene expression. These results indicate that FAM111A restricts SV40 HR viral replication center formation and that viral DNA replication contributes to the FAM111A-mediated effect on early gene expression.

    IMPORTANCE SV40 has served as a powerful tool for understanding fundamental viral and cellular processes; however, despite extensive study, the SV40 HR mutant phenotype remains poorly understood. Mutations in the C terminus of large T antigen that disrupt binding to the host protein FAM111A render SV40 HR viruses unable to replicate in restrictive cell types. Our work reveals a defect of HR mutant viruses in the formation of viral replication centers that can be rescued by depletion of FAM111A. Furthermore, inhibition of viral DNA replication reduces the effects of FAM111A restriction on viral gene expression. Additionally, FAM111A is a poorly characterized cellular protein whose mutation leads to two severe human syndromes, Kenny-Caffey syndrome and osteocraniostenosis. Our findings regarding the role of FAM111A in restricting viral replication and its localization to nucleoli and viral replication centers provide further insight into FAM111A function that could help reveal the underlying disease-associated mechanisms.

  • Going the Distance: Optimizing RNA-Seq Strategies for Transcriptomic Analysis of Complex Viral Genomes [Gem]

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

  • Bone Marrow-Derived CD4+ T Cells Are Depleted in Simian Immunodeficiency Virus-Infected Macaques and Contribute to the Size of the Replication-Competent Reservoir [Pathogenesis and Immunity]

  • The bone marrow (BM) is the key anatomic site for hematopoiesis and plays a significant role in the homeostasis of mature T cells. However, very little is known on the phenotype of BM-derived CD4+ T cells, their fate during simian immunodeficiency virus (SIV) infection, and their contribution to viral persistence during antiretroviral therapy (ART). In this study, we characterized the immunologic and virologic status of BM-derived CD4+ T cells in rhesus macaques prior to SIV infection, during the early chronic phase of infection, and during ART. We found that BM memory CD4+ T cells are significantly depleted following SIV infection, at levels that are similar to those measured in the peripheral blood (PB). In addition, BM-derived memory CD4+ T cells include a high frequency of cells that express the coinhibitory receptors CTLA-4 and PD-1, two subsets previously shown to be enriched in the viral reservoir; these cells express Ki-67 at levels similar to or higher than the same cells in PB. Finally, when we analyzed SIV-infected RMs in which viral replication was effectively suppressed by 12 months of ART, we found that BM CD4+ T cells harbor SIV DNA and SIV RNA at levels comparable to those of PB CD4+ T cells, including replication-competent SIV. Thus, BM is a largely understudied anatomic site of the latent reservoir which contributes to viral persistence during ART and needs to be further characterized and targeted when designing therapies for a functional or sterilizing cure to HIV.

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

  • High-Resolution Structural Characterization of a New Adeno-associated Virus Serotype 5 Antibody Epitope toward Engineering Antibody-Resistant Recombinant Gene Delivery Vectors [Structure and Assembly]

  • Adeno-associated virus serotype 5 (AAV5) is being developed as a gene delivery vector for several diseases, including hemophilia and Huntingtonrrsquo;s disease, and has a demonstrated efficient transduction in liver, lung, skeletal muscle, and the central nervous system. One limitation of AAV gene delivery is preexisting neutralizing antibodies, which present a significant challenge for vector effectiveness in therapeutic applications. Here, we report the cryo-electron microscopy (cryo-EM) and image-reconstructed structure of AAV5 in complex with a newly generated monoclonal antibody, HL2476, at 3.1-AAring; resolution. Unlike other available anti-AAV5 capsid antibodies, ADK5a and ADK5b, with epitopes surrounding the 5-fold channel of the capsid, HL2476 binds to the 3-fold protrusions. To elucidate the capsid-antibody interactions, the heavy and light chains were sequenced and their coordinates, along with the AAV5 viral protein, assigned to the density map. The high resolution of the complex enabled the identification of interacting residues at the 3-fold protrusions of the capsid, including R483, which forms two hydrogen bonds with the light chain of HL2476. A panel of AAV5 variants was generated and analyzed by native dot immunoblot and transduction assays. This identified variants with antibody escape phenotypes that maintain infectivity.

    IMPORTANCE Biologics based on recombinant AAVs (rAAVs) are increasingly becoming attractive human gene delivery vehicles, especially after the approval of Glybera in Europe and Luxturna in the United States. However, preexisting neutralizing antibodies against the AAV capsids in a large percentage of the human population limit wide-spread utilization of these vectors. To circumvent this problem, stealth vectors must be generated that are undetectable by these antibodies. This study details the high-resolution characterization of a new antigenic region on AAV5, a vector being developed for numerous delivery applications. The structure of AAV5 complexed with HL2476, a novel antibody, was determined by cryo-EM to 3.1-AAring; resolution. The resolution of the density map enabled the identification of interacting residues between capsid and antibody and the determinants of neutralization. Thus, the information obtained from this study can facilitate the generation of host immune escape vectors.

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

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

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

  • {alpha}v{beta}3 Integrin Is Required for Efficient Infection of Epithelial Cells with Human Adenovirus Type 26 [Virus-Cell Interactions]

  • Human adenoviruses (HAdVs) are being explored as vectors for gene transfer and vaccination. Human adenovirus type 26 (HAdV26), which belongs to the largest subgroup of adenoviruses, species D, has a short fiber and a so-far-unknown natural tropism. Due to its low seroprevalence, HAdV26 has been considered a promising vector for the development of vaccines. Despite the fact that the in vivo safety and immunogenicity of HAdV26 have been extensively studied, the basic biology of the virus with regard to receptor use, cell attachment, internalization, and intracellular trafficking is poorly understood. In this work, we investigated the roles of the coxsackievirus and adenovirus receptor (CAR), CD46, and aalpha;v integrins in HAdV26 infection of human epithelial cell lines. By performing different gain- and loss-of-function studies, we found that aalpha;vbbeta;3 integrin is required for efficient infection of epithelial cells by HAdV26, while CAR and CD46 did not increase the transduction efficiency of HAdV26. By studying intracellular trafficking of fluorescently labeled HAdV26 in A549 cells and A549-derived cell clones with stably increased expression of aalpha;vbbeta;3 integrin, we observed that HAdV26 colocalizes with aalpha;vbbeta;3 integrin and that increased aalpha;vbbeta;3 integrin enhances internalization of HAdV26. Thus, we conclude that HAdV26 uses aalpha;vbbeta;3 integrin as a receptor for infecting epithelial cells. These results give us new insight into the HAdV26 infection pathway and will be helpful in further defining HAdV-based vector manufacturing and vaccination strategies.

    IMPORTANCE Adenovirus-based vectors are used today for gene transfer and vaccination. HAdV26 has emerged as a promising candidate vector for development of vaccines due to its relatively low seroprevalence and its ability to induce potent immune responses against inserted transgenes. However, data regarding the basic biology of the virus, like receptor usage or intracellular trafficking, are limited. In this work, we found that efficient infection of human epithelial cell lines by HAdV26 requires the expression of the aalpha;vbbeta;3 integrin. By studying intracellular trafficking of fluorescently labeled HAdV26 in a cell clone with stably increased expression of aalpha;vbbeta;3 integrin, we observed that HAdV26 colocalizes with aalpha;vbbeta;3 integrin and confirmed that aalpha;vbbeta;3 integrin expression facilitates efficient HAdV26 internalization. These results will allow further improvement of HAdV26-based vectors for gene transfer and vaccination.

  • Multiple Introductions and Antigenic Mismatch with Vaccines May Contribute to Increased Predominance of G12P[8] Rotaviruses in the United States [Genetic Diversity and Evolution]

  • Rotavirus is the leading global cause of diarrheal mortality for unvaccinated children under 5 years of age. The outer capsid of rotavirus virions consists of VP7 and VP4 proteins, which determine viral G and P types, respectively, and are primary targets of neutralizing antibodies. Successful vaccination depends upon generating broadly protective immune responses following exposure to rotaviruses presenting a limited number of G- and P-type antigens. Vaccine introduction resulted in decreased rotavirus disease burden but also coincided with the emergence of uncommon G and P genotypes, including G12. To gain insight into the recent predominance of G12P[8] rotaviruses in the United States, we evaluated 142 complete rotavirus genome sequences and metadata from 151 clinical specimens collected in Nashville, TN, from 2011 to 2013 through the New Vaccine Surveillance Network. Circulating G12P[8] strains were found to share many segments with other locally circulating strains but to have distinct constellations. Phylogenetic analyses of G12 sequences and their geographic sources provided evidence for multiple separate introductions of G12 segments into Nashville, TN. Antigenic epitopes of VP7 proteins of G12P[8] strains circulating in Nashville, TN, differ markedly from those of vaccine strains. Fully vaccinated children were found to be infected with G12P[8] strains more frequently than with other rotavirus genotypes. Multiple introductions and significant antigenic mismatch may in part explain the recent predominance of G12P[8] strains in the United States and emphasize the need for continued monitoring of rotavirus vaccine efficacy against emerging rotavirus genotypes.

    IMPORTANCE Rotavirus is an important cause of childhood diarrheal disease worldwide. Two immunodominant proteins of rotavirus, VP7 and VP4, determine G and P genotypes, respectively. Recently, G12P[8] rotaviruses have become increasingly predominant. By analyzing rotavirus genome sequences from stool specimens obtained in Nashville, TN, from 2011 to 2013 and globally circulating rotaviruses, we found evidence of multiple introductions of G12 genes into the area. Based on sequence polymorphisms, VP7 proteins of these viruses are predicted to present themselves to the immune system very differently than those of vaccine strains. Many of the sick children with G12P[8] rotavirus in their diarrheal stools also were fully vaccinated. Our findings emphasize the need for continued monitoring of circulating rotaviruses and the effectiveness of the vaccines against strains with emerging G and P genotypes.

  • Broad Recognition of Circulating HIV-1 by HIV-1-Specific Cytotoxic T-Lymphocytes with Strong Ability to Suppress HIV-1 Replication [Cellular Response to Infection]

  • HIV-1-specific cytotoxic T-lymphocytes (CTLs) with strong abilities to suppress HIV-1 replication and recognize most circulating HIV-1 strains are candidates for effector T cells for cure treatment and prophylactic AIDS vaccine. Previous studies demonstrated that the existence of CTLs specific for 11 epitopes was significantly associated with good clinical outcomes in Japan, although CTLs specific for one of these epitopes select for escape mutations. However, it remains unknown whether the CTLs specific for the remaining 10 epitopes suppress HIV-1 replication in vitro and recognize circulating HIV-1. Here, we investigated the abilities of these CTLs to suppress HIV-1 replication and to recognize variants in circulating HIV-1. CTL clones specific for 10 epitopes had strong abilities to suppress HIV-1 replication in vitro. The ex vivo and in vitro analyses of T-cell responses to variant epitope peptides showed that the T cells specific for 10 epitopes recognized mutant peptides which are detected in 84.1% to 98.8% of the circulating HIV-1 strains found in HIV-1-infected Japanese individuals. In addition, the T cells specific for 5 epitopes well recognized target cells infected with 7 mutant viruses that had been detected in ggt;5% of tested individuals. Taken together, these results suggest that CTLs specific for the 10 epitopes effectively suppress HIV-1 replication and broadly recognize the circulating HIV-1 strains in the HIV-1-infected individuals. This study suggests the use of these T cells in clinical trials.

    IMPORTANCE In recent T-cell AIDS vaccine trials, the vaccines did not prevent HIV-1 infection, although HIV-1-specific T cells were induced in the vaccinated individuals, suggesting that the T cells have a weak ability to suppress HIV-1 replication and fail to recognize circulating HIV-1. We previously demonstrated that the T-cell responses to 10 epitopes were significantly associated with good clinical outcome. However, there is no direct evidence that these T cells have strong abilities to suppress HIV-1 replication and recognize circulating HIV-1. Here, we demonstrated that the T cells specific for the 10 epitopes had strong abilities to suppress HIV-1 replication in vitro. Moreover, the T cells cross-recognized most of the circulating HIV-1 in HIV-1-infected individuals. This study suggests the use of T cells specific for these 10 epitopes in clinical trials of T-cell vaccines as a cure treatment.

  • The Interaction between ORF18 and ORF30 Is Required for Late Gene Expression in Kaposi's Sarcoma-Associated Herpesvirus [Genome Replication and Regulation of Viral Gene Expression]

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

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

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

  • The extent to which viral genetic context influences HIV adaptation to human leukocyte antigen (HLA) class I-restricted immune pressures remains incompletely understood. The Ugandan HIV epidemic, where major pandemic group M subtypes A1 and D cocirculate in a single host population, provides an opportunity to investigate this question. We characterized plasma HIV RNA gag, pol, and nef sequences, along with host HLA genotypes, in 464 antiretroviral-naive individuals chronically infected with HIV subtype A1 or D. Using phylogenetically informed statistical approaches, we identified HLA-associated polymorphisms and formally compared their strengths of selection between viral subtypes. A substantial number (32%) of HLA-associated polymorphisms identified in subtype A1 and/or D had previously been reported in subtype B, C, and/or circulating recombinant form 01_AE (CRF01_AE), confirming the shared nature of many HLA-driven escape pathways regardless of viral genetic context. Nevertheless, 34% of the identified HLA-associated polymorphisms were significantly differentially selected between subtypes A1 and D. Experimental investigation of select examples of subtype-specific escape revealed distinct underlying mechanisms with important implications for vaccine design: whereas some were attributable to subtype-specific sequence variation that influenced epitope-HLA binding, others were attributable to differential mutational barriers to immune escape. Overall, our results confirm that HIV genetic context is a key modulator of viral adaptation to host cellular immunity and highlight the power of combined bioinformatic and mechanistic studies, paired with knowledge of epitope immunogenicity, to identify appropriate viral regions for inclusion in subtype-specific and universal HIV vaccine strategies.

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

  • Synergistic Activation of Bovine Herpesvirus 1 Productive Infection and Viral Regulatory Promoters by the Progesterone Receptor and Krüppel-Like Transcription Factor 15 [Virus-Cell Interactions]

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

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

  • The C Terminus of Rotavirus VP4 Protein Contains an Actin Binding Domain Which Requires Cooperation with the Coiled-Coil Domain for Actin Remodeling [Virus-Cell Interactions]

  • The interactions between viruses and actin cytoskeleton have been widely studied. We showed that rotaviruses remodel microfilaments in intestinal cells and demonstrated that this was due to the VP4 spike protein. Microfilaments mainly occur in the apical domain of infected polarized enterocytes and favor the polarized apical exit of viral progeny. The present work aims at the identification of molecular determinants of actin-VP4 interactions. We used various deletion mutants of VP4 that were transfected into Cos-7 cells and analyzed interactions by immunofluorescence confocal microscopy. It has been established that the C-terminal part of VP4 is embedded within viral particles when rotavirus assembles. The use of specific monoclonal antibodies demonstrated that VP4 is expressed in different forms in infected cells: classically as spike on the outer layer of virus particles, but also as free soluble protein in the cytosol. The C terminus of free VP4 was identified as interacting with actin microfilaments. The VP4 actin binding domain is unable to promote microfilament remodeling by itself; the coiled-coil domain is also required in this process. This actin-binding domain was shown to dominate a previously identified peroxisomal targeting signal, located in the three last amino acids of VP4. The newly identified actin-binding domain is highly conserved in rotavirus strains from species A, B, and C, suggesting that actin binding and remodeling is a general strategy for rotavirus exit. This provides a novel mechanism of protein-protein interactions, not involving cell signaling pathways, to facilitate rotavirus exit.

    IMPORTANCE Rotaviruses are causal agents of acute infantile viral diarrhea. In intestinal cells, in vitro as well as in vivo, virus assembly and exit do not imply cell lysis but rely on an active process in which the cytoskeleton plays a major role. We describe here a novel molecular mechanism by which the rotavirus spike protein VP4 drives actin remodeling. This relies on the fact that VP4 occurs in different forms. Besides its structural function within the virion, a large proportion of VP4 is expressed as free protein. Here, we show that free VP4 possesses a functional actin-binding domain. This domain, in coordination with a coiled-coil domain, promotes actin cytoskeleton remodeling, thereby providing the capacity to destabilize the cell membrane and allow efficient rotavirus exit.

  • Amino Acid Residue 217 in the Hemagglutinin Glycoprotein Is a Key Mediator of Avian Influenza H7N9 Virus Antigenicity [Genetic Diversity and Evolution]

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

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

  • Structural Adaptations of Norovirus GII.17/13/21 Lineage through Two Distinct Evolutionary Paths [Virus-Cell Interactions]

  • Human noroviruses (huNoVs), which cause epidemic acute gastroenteritis, recognize histo-blood group antigens (HBGAs) as host attachment factors affecting host susceptibility. HuNoVs are genetically diverse, containing at least 31 genotypes in the two major genogroups (genogroup I [GI] and GII). Three GII genotypes, GII genotype 17 (GII.17), GII.13, and GII.21, form a unique genetic lineage, in which the GII.17 genotype retains the conventional GII HBGA binding site (HBS), while the GII.13/21 genotypes acquire a completely new HBS. To understand the molecular bases behind these evolutionary changes, we solved the crystal structures of the HBGA binding protruding domains of (i) an early GII.17 variant (the 1978 variant) that does not bind or binds weakly to HBGAs, (ii) the new GII.17 variant (the 2014/15 variant) that binds A/B/H antigens strongly via an optimized GII HBS, and (iii) a GII.13 variant (the 2010 variant) that binds the Lewis a (Lea) antigen via the new HBS. These serial, high-resolution structural data enable a comprehensive structural comparison to understand the evolutionary changes of the GII.17/13/21 lineage, including the emergence of the new HBS of the GII.13/21 sublineage and the possible HBS optimization of the recent GII.17 variant for an enhanced HBGA binding ability. Our study elucidates the structural adaptations of the GII.17/13/21 lineage through distinct evolutionary paths, which may allow a theory explaining huNoV adaptations and evolutions to be put forward.

    IMPORTANCE Our understanding of the molecular bases behind the interplays between human noroviruses and their host glycan ligands, as well as their evolutionary changes over time with alterations in their host ligand binding capability and host susceptibility, remains limited. By solving the crystal structures of the glycan ligand binding protruding (P) domains with or without glycan ligands of three representative noroviruses of the GII.17/13/21 genetic lineage, we elucidated the molecular bases of the human norovirus-glycan interactions of this special genetic lineage. We present solid evidence on how noroviruses of this genetic lineage evolved via different evolutionary paths to (i) optimize their glycan binding site for higher glycan binding function and (ii) acquire a completely new glycan binding site for new ligands. Our data shed light on the mechanism of the structural adaptations of human noroviruses through different evolutionary paths, facilitating our understanding of human norovirus adaptations, evolutions, and epidemiology.

  • Engineered Small-Molecule Control of Influenza A Virus Replication [Vaccines and Antiviral Agents]

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

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

  • Risk Assessment of Fifth-Wave H7N9 Influenza A Viruses in Mammalian Models [Pathogenesis and Immunity]

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

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

  • Articles of Significant Interest in This Issue [Spotlight]

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    JVI Accepts: Articles Published Ahead of Print

  • The 13th International Double-Stranded RNA Virus Symposium, Houffalize, Belgium, 24 to 28 September 2018 [Meeting Review]

  • The triennial International Double-Stranded RNA Virus Symposium, this year organised by J Matthijnssens, JSL Parker, P Danthi and P Van Damme in Belgium, gathered over 200 scientists to discuss novel observations and hypotheses in the field. The keynote lecture on functional interactions of bacteria and viruses in the gut microbiome was presented by Julie Pfeiffer. Workshops were held on viral diversity, molecular epidemiology, molecular virology, immunity and pathogenesis, virus structure, the viral use and abuse of cellular pathways, and applied dsRNA virology. The establishment of a plasmid only-based reverse genetics system for rotaviruses by several Japanese research groups in 2017 has now been reproduced by various other research groups and was discussed in detail. The visualization of dsRNA virus replication steps in living cells received much attention. Mechanisms of the cellular innate immune response to virus infection and of viral pathogenesis were explored. Knowledge of the gut microbiomerrsquo;s influence on specific immune responses has increased rapidly, also due to the availability of relevant animal models of virus infection. The method of cryo-EM tomography has elucidated various asymmetric structures in viral particles. The use of orthoreoviruses for oncolytic virotherapy was critically assessed. The application of llama-derived single chain nanobodies for passive immunotherapy was considered as attractive. In a satellite symposium the introduction, impact and further developments of rotavirus vaccines were reviewed. The Jean Cohen Lecturer of this meeting was Harry B Greenberg who presented aspects of his research on rotaviruses over a period of more than 40 years. He was also interviewed at the meeting by Vincent Racaniello for the 513th session of This Week in Virology.

  • Human papillomavirus 16 E2 regulates keratinocyte gene expression relevant to cancer and the viral life cycle [Cellular Response to Infection]

  • Human papillomaviruses (HPV) are causative agents in ano-genital and oropharyngeal cancers. The virus must reprogram host gene expression to promote infection, and E6 and E7 contribute to this via targeting of cellular transcription factors including p53 and pRb, respectively. The HPV16 E2 protein regulates host gene expression in U2OS cells and in this study we extend these observations into TERT immortalized oral keratinocytes (NOKs) that are capable of supporting late stages of the HPV16 life cycle. We observed repression of innate immune genes by E2 that are also repressed by the intact HPV16 genome in NOKs. RNA-seq data identified 167 up and 395 downregulated genes by E2; there was a highly significant overlap of the E2 regulated genes with those regulated by the intact HPV16 genome in the same cell type. siRNA targeting of E2 reversed repression of E2 targeted genes. The ability of E2 to repress innate immune genes was confirmed in an ano-genital immortalized keratinocyte cell line, N/Tert-1. We present analysis of data from The Cancer Genome Atlas (TCGA) for HPV16 positive and negative head and neck cancers (HNC) suggesting that E2 plays a role in regulation of the host genome in cancers. Patients with HPV16 positive HNC with a loss of E2 expression exhibit a worse clinical outcome and we discuss how this could, at least partially, be related to the loss of E2 host gene regulation.

    IMPORTANCE HPV16 positive tumors that retain expression of E2 have a better clinical outcome than those that have lost E2 expression. It has been suggested that this is due to a loss of E2 repression of E6 and E7 expression but this is not supported by data from tumors where there is not more E6 and E7 expression in the absence of E2. Here we report that E2 regulates host gene expression and place this regulation in context of the HPV16 life cycle and HPV16 positive head and neck cancers (the majority of which retain E2 expression). We propose that this E2 function may play an important part in the increased response of HPV16 positive cancers to radiation therapy. Therefore, host gene regulation by E2 may be important for promotion of the HPV16 life cycle, and also for the response of HPV16 positive tumors to radiation therapy.

  • Inhibition of DNA-sensing pathway by Mareks disease virus VP23 protein through suppression of interferon regulatory factor 7 activation [Virus-Cell Interactions]

  • The type I interferon (IFN) response is the first line of host innate immune defense against viral infection; however, viruses have developed multiple strategies to antagonize host IFN responses for efficient infection and replication. Here, we report that Marekrrsquo;s disease virus (MDV), an oncogenic herpesvirus, encodes VP23 protein as a novel immune modulator to block IFN-bbeta; activation induced by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) in chicken fibroblasts and macrophages. VP23 overexpression markedly reduces viral DNA-triggered IFN-bbeta; production and promotes viral replication, while knockdown of VP23 during MDV infection enhances the IFN-bbeta; response and suppresses viral replication. VP23 selectively inhibits IFN regulatory factor 7 (IRF7) but not nuclear factor B (NF-B) activation. Furthermore, we found that VP23 interacts with IRF7 and blocks its binding to TANK-binding kinase 1 (TBK1), thereby inhibiting IRF7 phosphorylation and nuclear translocation, resulting in reduced IFN-bbeta; production. These findings expand our knowledge of DNA sensing in chickens and reveal a mechanism through which MDV antagonizes the host IFN response.

    IMPORTANCE Despite widespread vaccination, Marekrrsquo;s disease (MD) continues to pose major challenges for the poultry industry worldwide. MDV causes immunosuppression and deadly lymphomas in chickens, suggesting that this virus has developed a successful immune evasion strategy. However, little is known regarding the initiation and modulation of the host innate immune response during MDV infection. This study demonstrates that the cGAS-STING DNA-sensing pathway is critical for the induction of the IFN-bbeta; response against MDV infection in chicken fibroblasts and macrophages. An MDV protein, VP23, was found to efficiently inhibit the cGAS-STING pathway. VP23 selectively inhibits IRF7 but not NF-B activation. VP23 interacts with IRF7 and blocks its binding to TBK1, thereby suppressing IRF7 activation and resulting in inhibition of the DNA-sensing pathway. These findings expand our knowledge of DNA sensing in chickens and reveal a mechanism through which MDV antagonizes the host IFN response.

  • The Herpesviridae conserved multifunctional infected cell protein 27 (ICP27) is important, but not required for replication and oncogenicity of Mareks disease alphaherpesvirus (MDV) [Genome Replication and Regulation of Viral Gene Expression]

  • The Herpesviridae conserved infected cell protein 27 (ICP27) is essential for cell culture-based replication of most herpesviruses studied. For members of the Alphaherpesvirinae, ICP27 regulates the expression of many viral genes, including expression of pUL44 (gC), pUL47 (VP13/14), and pUL48 (VP16). These three viral proteins are dysregulated during Marekrrsquo;s disease alphaherpesvirus (MDV) replication in cell culture. MDV replicates in a highly cell-associated manner in cell culture, producing little to no infectious virus. In contrast, infectious cell-free MDV is produced in specialized feather follicle epithelial (FFE) cells of infected chickens where expression of these three genes are abundantly expressed. This led us to hypothesize that MDV ICP27, encoded by gene UL54, is a defining factor for the dysregulation of gC, pUL47, and pUL48 and ultimately, ineffective virus production in cell culture. To address ICP27rrsquo;s role in MDV replication, we generated recombinant MDV with ICP27 deleted (v54). Interestingly, v54 replicated, but plaque sizes were significantly reduced compared to parental viruses. The reduced cell-to-cell spread was due to ICP27 since plaque sizes were restored in rescued viruses, as well as when v54 was propagated in cells expressing ICP27 in trans. In chickens, v54 replicated, induced disease, and was oncogenic, but was unable to transmit from chicken-to-chicken. To our knowledge, this is the first report in which the Herpesviridae conserved ICP27 protein is dispensable for replication and disease induction in its natural host.

    IMPORTANCE Marekrrsquo;s disease (MD) is a devastating oncogenic disease that affects the poultry industry and is caused by MD alphaherpesvirus (MDV). Current vaccines block induction of disease, but do not block chicken-to-chicken transmission. There is a knowledge gap in our understanding of how MDV spreads from chicken-to-chicken. Here, we studied the Herpesviridae conserved ICP27 regulatory protein in cell culture and during MDV infection in chickens. We determined MDV ICP27 is important, but not required for replication in both cell culture and chickens. In addition, MDV ICP27 was not required for disease induction or oncogenicity, but was required for chicken-to-chicken transmission. This study is important because it addresses the role of ICP27 during infection in the natural host and provides important information for the development of therapies to protect chickens against MD.

  • Mapping the pH sensors critical for host cell entry by a complex non-enveloped virus [Virus-Cell Interactions]

  • Bluetongue virus (BTV), family Reoviridae, is an insect-borne, double-capsid virus causing haemorrhagic disease in livestock around the world. Here we elucidate how outer capsid proteins VP2 and VP5 coordinate cell entry of BTV. To identify key functional residues, we used atomic-level structural data to guide mutagenesis of VP2 and VP5, and a series of biological and biochemical approaches, including site-directed mutagenesis, reverse genetics-based virus recovery, expression and characterization of individual recombinant mutant proteins and various in vitro and in vivo assays. We demonstrate the dynamic nature of the conformational change process, revealing that a unique zinc finger (CCCH) in VP2 acts as the major low pH sensor, coordinating VP2 detachment, subsequently allowing VP5 to sense low pH via specific histidine residues at key positions. We show that single substitution of only certain histidine residues has lethal effect, indicating location of histidine in VP5 is critical to inducing changes in VP5 conformation that facilitates membrane penetration. Further, we show VP5 anchoring domain alone recapitulates sensing of low pH. Our data reveals a novel, multi-conformational process that overcomes entry barriers faced by this multi-capsid nonenveloped virus.

    IMPORTANCE Virus entry into a susceptible cell is the first step of infection and a significant point at which infection can be prevented. To enter effectively viruses must sense the cellular environment and, when appropriate, initiate a series of changes that eventually jettison the protective shell and deposit virus genes into the cytoplasm. Many viruses sense pH but how this happens and the events that follow are often poorly understood. Here we address this question for a large multi-layered Bluetongue virus. We show key residues in outer capsid proteins, a pH sensing histidine of a zinc finger within the receptor-binding VP2 protein and certain histidine residues in the membrane-penetrating VP5 protein that detect cellular pH leading to irreversible changes and propel the virus through the cell membrane. Our data reveals a novel mechanism of cell entry for a nonenveloped virus and highlight mechanisms, which may also be used by other viruses.

  • HDAC6 restricts influenza A virus by deacetylation of the RNA polymerase PA subunit [Virus-Cell Interactions]

  • The life cycle of the influenza A virus (IAV) is modulated by various cellular host factors. Although earlier studies indicated that IAV infection is controlled by HDAC6, the deacetylase involved in the regulation of PA remained unknown. Here, we demonstrated that HDAC6 acts as a negative regulator of IAV infection through destabilizing PA. HDAC6 binds to and deacetylates PA, thereby promoting the proteasomal degradation of PA. Through mass spectrometric analysis, Lys(664) of PA can be deacetylated by HDAC6 and this residue is crucial for PA protein stability. The deacetylase activity of HDAC6 is required for anti-IAV activity, because IAV infection was enhanced due to elevated IAV RNA polymerase activity upon HDAC6 depletion and HDAC6 deacetylase dead mutant (HDAC6-DM, H216A;H611A). Finally, we also demonstrated that overexpression of HDAC6 suppresses the IAV RNA polymerase activity, but HDAC6-DM does not. Taken together, our findings provide the initial evidence that HDAC6 plays a negative role in IAV RNA polymerase activity via deacetylating PA and thus restricts IAV RNA transcription and replication.

    IMPORTANCE Influenza A virus (IAV) continues to threat global public health due to drug resistance and the emergency of frequently mutated strains of IAV. Thus, it is critical to find new strategies to control IAV infection. Here we discovered one host protein, HDAC6, which can inhibit viral RNA polymerase activity through deacetylating PA and thus suppresses virus RNA replication and transcription. Previously, it was reported that IAV can utilize the HDAC6-dependent aggresome formation mechanism to promote virus uncoating, but HDAC6-mediated deacetylation ofaalpha;-tubulin inhibits viral protein trafficking at late stages of the virus life cycle. These findings together will contribute to a better understanding of the role of HDAC6 in regulating IAV infection. Understanding the molecular mechanisms of HDAC6 in various periods of viral infection may illuminate novel strategies for developing antiviral drugs.

  • ANTIBODY-MEDIATED PROTECTIVE MECHANISMS INDUCED BY A TRIVALENT PARAINFLUENZA-VECTORED EBOLAVIRUS VACCINE [Vaccines and Antiviral Agents]

  • Ebolaviruses Zaire (EBOV), Bundibugyo (BDBV) and Sudan (SUDV) cause human disease with high case fatality rates. Experimental monovalent vaccines, which all utilize the sole envelope glycoprotein (GP), do not protect against heterologous ebolaviruses. Human parainfluenza virus type 3-vectored vaccines offer benefits including needle-free administration and induction of mucosal responses in the respiratory tract. Multiple approaches were taken to induce a broad protection against the three ebolaviruses. While GP consensus-based antigens failed to elicit neutralizing antibodies, polyvalent immunization induced neutralizing responses to all three ebolaviruses and protected animals from death and disease caused by EBOV, SUDV and BDBV. As immunization with a cocktail of antigenically-related antigens can skew the responses and change the epitope hierarchy, we performed comparative analysis of antibody repertoire and Fc-mediated protective mechanisms in animals immunized with monovalent versus polyvalent vaccines. Compared to the monovalent vaccines, sera from trivalent vaccinated guinea pigs bound and neutralized EBOV and SUDV at equivalent levels and BDBV at only slightly reduced level. Peptide microarrays revealed a preponderance of binding to amino acids 389nndash;403, 397-415 and 477-493, representing three linear epitopes in the mucin-like domain known to induce a protective antibody response. Competition binding assays with monoclonal antibodies isolated from human ebolavirus survivors demonstrated that the immune sera block binding of antibodies specific for the GP glycan cap, GP1-GP2 interface, the mucin-like domain, and the membrane-proximal external region. Thus, cocktail administration of three ebolavirus vaccines induces a desirable broad antibody response, without skewing of the response toward preferential recognition of a single virus.

    IMPORTANCE Symptoms of the disease caused by ebolaviruses Ebola, Bundibugyo and Sudan are similar, and their endemic areas overlap. However, because of the limited antigenic relatedness of ebolavirus glycoprotein (GP) used in all candidate vaccines against these viruses, they protect only against homologous but not heterologous ebolaviruses. Therefore, a broadly specific pan-ebolavirus vaccine is required, which might be achieved by administration of a cocktail of vaccines. The effects of cocktail administration of ebolavirus vaccines on the antibody repertoire remain unknown. Here in-depth analysis of the antibody responses to cocktail administration of human parainfluenza type 3-vectored vaccines against individual ebolaviruses was performed, which included analysis of binding to GP, neutralization of individual ebolaviruses, epitope specificity, Fc-mediated functions, and protection against the three ebolaviruses. The results demonstrated potent and balanced responses against individual ebolaviruses and no significant reduction of the responses, compared to that induced by individual vaccines.

  • Histidine-rich glycoprotein inhibits HIV-1 infection in a pH-dependent manner [Pathogenesis and Immunity]

  • Histidine-rich glycoprotein (HRG) is an abundant plasma protein with a multidomain structure allowing its interaction with many ligands including phospholipids, plasminogen, fibrinogen, IgG antibodies, and heparan sulfate. HRG has shown to regulate different biological responses such as angiogenesis, coagulation and fibrinolysis. Here, we found that HRG almost completely abrogated the infection of GHOST, Jurkat, CD4+ T cells, and macrophages by HIV-1 at low pH (range 6.5 to 5.5) but not at neutral pH. HRG was shown to interact with heparan sulfate expressed by target cells inhibiting an early post-binding step associated to HIV-1 infection. More importantly, by acting on the viral particle itself, HRG induced a deleterious effect which reduces viral infectivity. Because cervicovaginal secretions in healthy women show low pH values, even after semen deposition, our observations suggest that HRG might represent a constitutive defence mechanism in the vaginal mucosa. Of note, low pH also enabled HRG to inhibit the infection of Hep-2 cells and Vero cells by respiratory syncytial virus (RSV) and herpes simplex virus-type 2 (HSV-2), respectively, suggesting that HRG might display a broad antiviral activity under acidic conditions.

    IMPORTANCE. Vaginal intercourse represents a high-risk route for HIV-1 transmission. The efficiency of male-to-female HIV-1 transmission has been estimated as 1 in every 1,000 episodes of sexual intercourse, reflecting a high degree of protection conferred by the genital mucosa. However, the contribution of different host factors to the protection against HIV-1 at mucosal surfaces remains poorly defined. Here, we report for the first time that acidic values of pH enable the plasma protein histidine-rich glycoprotein (HRG) to strongly inhibit HIV-1 infection. Because cervicovaginal secretions usually show low pH values, our observations suggest that HRG might represent a constitutive antiviral mechanism in the vaginal mucosa. Interestingly, infection by other viruses such as respiratory syncytial virus and herpes simplex virus-type 2 was also markedly inhibited by HRG at low pH values, suggesting that extracellular acidosis enables HRG to display a broad antiviral activity.

  • CRISPR/Cas9-mediated knock-out of DNAJC14 verifies this chaperone as a pivotal host factor for RNA replication of Pestiviruses [Genome Replication and Regulation of Viral Gene Expression]

  • Pestiviruses like bovine viral diarrhea virus (BVDV) are a thread to livestock. For pestiviruses cytopathogenic (cp) and non-cytopathogenic (noncp) strains are distinguished in cell culture. The noncp biotype of BVDV is capable of establishing persistent infections which is a major problem in disease control. The noncp biotype rests on temporal control of viral RNA replication, mediated by regulated cleavage of nonstructural protein 2-3 (NS2-3). This cleavage is catalyzed by the autoprotease in NS2 which depends in its activity on its cellular cofactor DNAJC14. Since this chaperone is available in low amounts and binds tightly to NS2, NS2-3 translated later in infection is no longer cleaved. As NS3 is an essential constituent of the viral replicase, this shift in polyprotein processing correlates with downregulation of RNA replication. In contrast, cpBVDV strains arising mostly by RNA recombination, show highly variable genome structures and display unrestricted NS3 release. The functional importance of DNAJC14 for noncp pestiviruses was established so far only for BVDV-1. It was therefore enigmatic whether replication of other noncp pestiviruses is also DNAJC14-dependent. By generating bovine and porcine DNAJC14 knock-out cells we could show that (i) replication of 6 distinct noncp pestivirus species (A-D, F and G) depends on DNAJC14; (ii) the pestiviral replicase NS3-5B can assemble into functional complexes in the absence of DNAJC14; (iii) all cp pestiviruses replicate their RNA and generate infectious progeny independent of host DNAJC14. Together, these findings confirm DNAJC14 as pivotal cellular cofactor for the replication and maintenance of the noncp biotype of pestiviruses.

    IMPORTANCE Only non-cytopathogenic (noncp) pestivirus strains are capable of establishing life-long persistent infections to generate the virus reservoir in the field. The molecular basis for this biotype is only partially understood and only investigated in depth for BVDV-1 strains. Temporal control of viral RNA replication correlates with the noncp biotype and is mediated by limiting amounts of cellular DNAJC14 that activates the viral NS2 protease to catalyze the release of the essential replicase component NS3. Here we demonstrate that several species of noncp pestiviruses depend in their RNA replication on DNAJC14. Moreover, all cp pestiviruses, in sharp contrast to their noncp counterparts, replicate independently of DNAJC14. The generation of a cp BVDV in the persistently infected animal is causative for onset of mucosal disease. Therefore, the observed strict biotype-specific difference in DNAJC14-dependency should be further examined for its role in cell type/tissue tropism and the pathogenesis of this lethal disease.

  • Intrahost dynamics of human cytomegalovirus variants acquired by seronegative glycoprotein B vaccinees [Genetic Diversity and Evolution]

  • Human cytomegalovirus (HCMV) is the most common congenital infection worldwide, and a frequent cause of hearing loss and debilitating neurologic disease in newborn infants. Thus, a vaccine to prevent HCMV-associated congenital disease is a public health priority. One potential strategy is vaccination of women of child-bearing age to prevent maternal HCMV acquisition during pregnancy. The glycoprotein B (gB) + MF59 adjuvant subunit vaccine is the most efficacious tested clinically to date, demonstrating 50% protection against primary HCMV infection in a phase 2 clinical trial. Yet, the impact of gB/MF59-elicited immune responses on the population of viruses acquired by trial participants has not been assessed. In this analysis, we employed quantitative PCR as well as multiple sequencing methodologies to interrogate the magnitude and genetic composition of HCMV populations infecting gB/MF59 vaccinees and placebo recipients. We identified several differences between the viral dynamics of acutely-infected vaccinees and placebo recipients. First, viral load was reduced in the saliva of gB vaccinees, though not in whole blood, vaginal fluid, or urine. Additionally, we observed possible anatomic compartmentalization of gB variants in the majority of vaccinees compared to only a single placebo recipient. Finally, we observed reduced acquisition of genetically-related gB1, gB2, and gB4 genotype "supergroup" HCMV variants among vaccine recipients, suggesting that the gB1 genotype vaccine construct may have elicited partial protection against HCMV viruses with antigenically-similar gB sequences. These findings suggest that gB immunization had a measurable impact on viral intrahost population dynamics and support future analysis of a larger cohort.

    Importance: Though not a household name like Zika virus, human cytomegalovirus (HCMV) causes permanent neurologic disability in one newborn child every hour in the United States nndash; more than Down syndrome, fetal alcohol syndrome, and neural tube defects combined. There are currently no established effective measures to prevent viral transmission to the infant following HCMV infection of a pregnant mother. However, the gB/MF59 vaccine, which aims to prevent pregnant women from acquiring HCMV, is the most successful HCMV vaccine tested clinically to date. Here, we used viral DNA isolated from patients enrolled in a gB vaccine trial who acquired HCMV, and identified several impacts that this vaccine had on the size, distribution, and composition of the in vivo viral population. These results have increased our understanding of why the gB/MF59 vaccine was partially efficacious, and such investigations will inform future rational design of a vaccine to prevent congenital HCMV.

  • Directed Nucleosome Sliding in SV40 Minichromosomes During the Formation of the Virus Particle Exposes DNA Sequences Required for Early Transcription [Genome Replication and Regulation of Viral Gene Expression]

  • Simian Virus 40 (SV40) exists as chromatin throughout its life cycle, and undergoes typical epigenetic regulation mediated by changes in nucleosome location and associated histone modifications. In order to investigate the role of epigenetic regulation during the encapsidation of late stage minichromosomes into virions, we have mapped the location of nucleosomes containing acetylated or methylated lysines in the histone tails of H3 and H4 present in the chromatin from 48-hour post-infection minichromosomes and disrupted virions. In minichromosomes obtained late in infection, nucleosomes were found carrying various histone modifications primarily in the regulatory region with a major nucleosome located within the enhancer and other nucleosomes at the early and late transcriptional start sites. The nucleosome found in the enhancer would be expected to repress early transcription by blocking access to part of the SP1 binding sites and the left side of the enhancer in late stage minichromosomes while also allowing late transcription. In chromatin from virions, the principal nucleosome located in the enhancer was shifted ~ 70 bases in the late direction from what was found in minichromosomes and the level of modified histones was increased throughout the genome. The shifting of the enhancer-associated nucleosome to the late side would effectively serve as a switch to relieve the repression of early transcription found in late minichromosomes while likely also repressing late transcription by blocking access to necessary regulatory sequences. This epigenetic switch appeared to occur during the final stage of virion formation.

    IMPORTANCE For a virus to complete an infection, it must produce a new virus particle in which the genome is able to support a new infection. This is particularly important for viruses like Simian Virus 40 (SV40) which exist as chromatin throughout their life cycle, since chromatin structure plays a major role in the regulation of their life cycle. In order to determine the role of SV40 chromatin structure late in infection, we have mapped the location of nucleosomes and their histone tail modifications in SV40 minichromosomes and in the SV40 chromatin found in virions using ChIP-Seq. We have identified a novel viral transcriptional control mechanism in which a nucleosome found in the regulatory region of SV40 minichromosome is directed to slide during the formation of the virus particle exposing transcription factor binding sites required for early transcription that were previously blocked by the presence of the nucleosome.

  • The Need for Speed: Run-On Oligomer Filament Formation Provides Maximum Speed with Maximum Sequestration of Activity [Cellular Response to Infection]

  • Herein we investigate an unusual anti-viral mechanism developed in the bacterium Streptomyces griseus. SgrAI is a type II restriction endonuclease which forms run-on oligomer filaments when activated, and which possesses both accelerated DNA cleavage activity and expanded DNA sequence specificity. Mutations disrupting the run-on oligomer filament eliminate the robust anti-phage activity of wild type SgrAI, and the observation that even relatively modest disruptions completely abolish this anti-viral activity shows that the greater speed imparted by the run-on oligomer filament mechanism is critical to its biological function. Simulations of DNA cleavage by SgrAI uncover the origins of the kinetic advantage of this newly described mechanism of enzyme regulation over more conventional mechanisms, as well as the origin of the sequestering effect responsible for the protection of the host genome against the damaging DNA cleavage activity of activated SgrAI.

    IMPORTANCE This work is motivated by the interest in understanding the characteristics and advantages of a relatively newly discovered enzyme mechanism involving filament formation. SgrAI is an enzyme responsible for protecting against viral infections in its host bacterium, and was one of the first such enzymes shown to utilize such a mechanism. In this work, filament formation by SgrAI is disrupted and the effects on the speed of the purified enzyme as well as its function in cells are measured. It was found that even small disruptions, which weaken but do not destroy filament formation, eliminate the ability of SgrAI to protect cells from viral infection, its normal biological function. Simulations of enzyme activity were also performed and show how filament formation can greatly speed up an enzymerrsquo;s activation compared to other known mechanisms, as well as better localize its action to molecules of interest such as invading phage DNA.

  • Hemagglutinin stalk-reactive antibodies interfere with influenza virus neuraminidase activity by steric hindrance [Vaccines and Antiviral Agents]

  • Hemagglutinin (HA) stalk-reactive antibodies are the basis of several current "one-shot" universal influenza vaccine efforts because they protect against a wide spectrum of influenza virus strains. The appreciated mechanism of protection by HA-stalk antibodies is to inhibit HA stalk reconfiguration, blocking viral fusion and entry. This study shows that HA stalk-reactive antibodies also inhibit neuraminidase (NA) enzymatic activity, prohibiting viral egress. NA inhibition (NI) is evident for an attached substrate but not for unattached small molecule cleavage of sialic acid. This suggests that the antibodies inhibit NA enzymatic activity through steric hindrance, thus limiting NA access to sialic acids when adjacent to HA on whole virions. Consistently, F(abrrsquo;)2 fragments that occupy reduced area without loss of avidity or disrupted HA/NA interactions show significantly reduced NI activity. Notably, HA stalk binding antibodies lacking NI activity were unable to neutralize viral infection via microneutralization assays. This work suggests that NI activity is an important component of HA-stalk antibody mediated protection.

    IMPORTANCE This study reports a new mechanism of protection that is mediated by influenza hemagglutinin-stalk reactive antibodies: inhibition of neuraminidase activity by steric hindrance, blocking access of neuraminidase to sialic acids when it is abutted next to hemagglutinin on whole virions.

  • Spatio-temporal differences in presentation of CD8 T cell epitopes during HBV infection. [Virus-Cell Interactions]

  • Distinct populations of hepatocytes infected with HBV or only harboring HBV-DNA integrations coexist within an HBV chronically infected liver. These hepatocytes express HBV antigens at different levels and with different intracellular localizations but it is not known whether this heterogeneity of viral antigen expression could result in an uneven hepatic presentation of distinct HBV epitopes/HLA class-I complexes triggering different level of activation of HBV-specific CD8+ T cells.

    Using antibodies specific to two distinct HLAnndash;A*02:01/HBV epitope complexes of HBV nucleocapsid and envelope proteins, we mapped their topological distribution in liver biopsies of two anti-HBe+ chronic HBV (CHB) patients. We demonstrated that the core and envelope CD8+T cell epitopes were not uniformly distributed in the liver parenchyma but preferentially located in distinct and sometimes mutually exclusive hepatic zones. The efficiency of HBV epitope presentation was then tested in vitro utilizing HLA-A*02:01/HBV epitope-specific antibodies and the corresponding CD8+ T cells, in primary human hepatocyte and hepatoma cell lines either infected with HBV or harboring HBV-DNA integration. We confirmed the existence of a marked variability in the efficiency of HLA-class I/HBV epitope presentation among the different targets that was influenced by presence of IFN- and availability of newly-translated viral antigens. In conclusion, HBV antigen presentation can be heterogeneous within an HBV-infected liver. As a consequence, CD8+ T cells of different HBV specificities might have different antiviral efficacy.

    Importance The inability of patients with chronic HBV infection to clear HBV is associated with defective HBV-specific CD8+ T cells. Hence, the majority of immunotherapy developments focus on HBV specific T cell function restoration. However, knowledge of whether distinct HBV-specific T cells can equally target all the HBV-infected hepatocytes of a chronically infected liver are lacking. In this work, analysis of CHB patient liver parenchyma and in vitro HBV infection models shows a non-uniform distribution of HBV CD8+ T cells epitopes that is influenced by presence of IFN- and availability of newly-translated viral antigens. These results suggest that CD8+ T cells recognizing different HBV epitopes can be necessary for efficient immune therapeutic control of chronic HBV infection.

  • Neutralization Synergy between HIV-1 Attachment Inhibitor Fostemsavir and Anti-CD4 Binding Site Broadly Neutralizing Antibodies against HIV [Vaccines and Antiviral Agents]

  • Attachment inhibitor (AI) BMS-626529 (Fostemsavir) represents a novel class of antiretrovirals which targets human immunodeficiency virus type 1 (HIV-1) gp120 and blocks CD4-induced conformational changes required for viral entry. It is now in Phase III clinical trials and is expected to be approved by the United States Food and Drug Administration (FDA) in the near future. Although Fostemsavir is very potent against HIV in vitro and in vivo, there are already a number of resistant mutants identified. Broadly neutralizing HIV antibodies (bNAbs) can potently inhibit a wide range of HIV-1 strains by binding to viral Env and are very promising candidates for HIV-1 prevention and therapy. Since both target viral Env to block viral entry, we decided to investigate the relationship between these two inhibitors. Our data show that Env mutants resistant to BMS-626529 remained susceptibility to bNAbs. A single treatment of bNAb NIH45-46G54W completely inhibited the replication of these escape mutants. Remarkable synergy was observed between BMS-626529 and CD4 binding site (bs)-targeting bNAbs in neutralizing HIV-1 strains at low concentrations. This synergistic effect was enhanced against virus harboring resistance mutations to BMS-626529. The mechanistic basis of the observed synergy is likely enhanced inhibition of CD4 binding to HIV-1 Env trimer by the combination of BMS-626529 and CD4bs-targeting bNAbs. This work highlights the potential for positive interplay between small and large molecule therapeutics on HIV viral entry, which may prove useful as these agents enter clinical use.

    IMPORTANCE As the worldwide HIV pandemic continues, there is a continued need for novel drugs and therapies. A new class of drug, Attachment Inhibitor, will soon be approved for the treatment of HIV. Broadly neutralizing antibodies are also promising candidates for HIV prevention and therapy. We investigated how this drug might work with these exciting antibodies that are very potent in blocking HIV infection of cells. These antibodies worked against virus known to be resistant to the new drug. In addition, a specific type of antibody worked really well with the new drug in blocking virus infection of cells. This work has implications for both the new drug and the antibodies that are poised to be used against HIV.

  • Identification and functional characterization of a novel Fc gamma-binding glycoprotein in Rhesus Cytomegalovirus [Pathogenesis and Immunity]

  • Receptors recognizing the Fc part of immunoglobulin G (FcRs) are key determinants in antibody-mediated immune responses. Members of the Herpesviridae interfere with this immune regulatory network by expressing viral FcRs (vFcRs). Human cytomegalovirus (HCMV) encodes four distinct vFcRs that differ with respect to their IgG-subtype specificity and their impact on antibody-mediated immune function in vitro. The impact of vFcRs on HCMV pathogenesis and immunomodulation in vivo is not known. The evolutionary closest animal model of HCMV is rhesus CMV (RhCMV) infection of rhesus macaques. To enable the characterization of vFcR function in this model, we studied IgG binding by RhCMV. We show that lysates of RhCMV-infected cells contain an IgG-binding protein of 30kDa encoded by the gene Rh05 that is a predicted type I glycoprotein belonging to the RL11 gene family. Upon deletion of Rh05, IgG-Fc binding by RhCMV strain 68-1 is lost whereas ectopic expression of Rh05 results in IgG binding to transfected cells consistent with Rh05 being a vFcR. Using a set of reporter cell lines stably expressing human and rhesus FcRs we further demonstrate that Rh05 antagonizes host FcR activation. Compared to Rh05-intact RhCMV, RhCMVRh05 showed an increased activation of host FcR upon exposure of infected cells to IgG from RhCMV-seropositive animals suggesting that Rh05 protects infected cells from opsonization and IgG-dependent activation of host FcRs. However, antagonizing host FcR activation by Rh05 was not required for the establishment and maintenance of infection of RhCMV, even in a seropositive host, as shown by the induction of T cell responses to heterologous antigens expressed by RhCMV lacking the gene region encoding Rh05. In contrast to viral evasion of NK cells or T cell recognition, the evasion of antibody-mediated effects does not seem to be absolutely required for infection or re-infection. The identification of the first vFcR that efficiently antagonizes host FcR activation in the RhCMV genome will thus permit more detailed studies of this immunomodulatory mechanism in promoting viral dissemination in the presence of natural or vaccine-induced humoral immunity.

    IMPORTANCE Rhesus cytomegalovirus (RhCMV) offers a unique model for studying human cytomegalovirus (HCMV) pathogenesis and vaccine development. RhCMV infection of non-human primates greatly broadened the understanding of mechanisms by which CMVs evade or re-program T cell and NK cell responses in vivo. However, the role of humoral immunity and viral modulation of anti-CMV antibodies has not been studied in this model. There is evidence from in vitro studies that HCMVs can evade humoral immunity. By gene mapping and with the help of a novel cell-based reporter assay system we characterized the first RhCMV encoded IgG-Fc binding glycoprotein as a potent antagonist of rhesus FcR activation. We further demonstrate that, unlike evasion of T cell immunity, this viral Fc receptor is not required to overcome anti-CMV immunity to establish secondary infections. These findings enable more detailed studies of the in vivo consequences of CMV evasion from IgG responses in non-human primate models.

  • Novel mutations in nsP2 abolish chikungunya virus-induced transcriptional shutoff and make virus less cytopathic without affecting its replication rates [Virus-Cell Interactions]

  • Alphavirus infections are characterized by global inhibition of cellular transcription and rapid induction of a cytopathic effect (CPE) in cells of vertebrate origin. Transcriptional shutoff impedes the cellular response to alphavirus replication and prevents establishment of an antiviral state. Chikungunya virus (CHIKV) is a highly pathogenic alphavirus representative and its nonstructural protein 2 (nsP2) plays critical roles in both inhibition of transcription and CPE development. Previously, we have identified a small peptide in Sindbis virus (SINV) nsP2 (VLoop) that determined the proteinrrsquo;s transcriptional inhibition function. It is located in the surface-exposed loop of the carboxy-terminal domain of nsP2 and exhibits high variability between members of different alphavirus serocomplexes. In this study, we found that SINV-specific mutations could not be directly applied to CHIKV. However, after applying a new selection approach, we identified a variety of new VLoop variants that made CHIKV and its replicons incapable of inhibiting cellular transcription and dramatically less cytopathic. Importantly, the mutations had no negative effect on RNA and viral replication rates. In contrast to parental CHIKV, the developed VLoop mutants were unable to block induction of type I interferon. Consequently, they were cleared from IFN-competent cells without CPE development. Alternatively, in murine cells that have defects in type I IFN production or signaling, the VLoop mutants established persistent, noncytopathic replication. The mutations in nsP2 VLoop may be used for development of new vaccine candidates against alphavirus infections and vectors for expression of heterologous proteins.

    IMPORTANCE Chikungunya virus is an important human pathogen, which now circulates in both the Old and New Worlds. As in the case of other Old World alphaviruses, CHIKV nsP2 not only has enzymatic functions in viral RNA replication, but is also a critical inhibitor of the antiviral response and one of the determinants of CHIKV pathogenesis. In this study, we have applied a new strategy to select a variety of CHIKV nsP2 mutants that no longer exhibited transcription inhibitory functions. The designed CHIKV variants became potent type I interferon inducers and acquired a less cytopathic phenotype. Importantly, they demonstrated the same replication rates as the parental CHIKV. Mutations in the same identified peptide of nsP2 proteins derived from other Old World alphaviruses also abolished their nuclear functions. Such mutations can be further exploited for development of new attenuated alphaviruses.

  • Predicting Intraserotypic Recombination in Enterovirus 71 [Genetic Diversity and Evolution]

  • Enteroviruses are well known for their ability to cause neurological damage and paralysis. The model enterovirus is poliovirus (PV), the causative agent of poliomyelitis, a condition characterized by acute flaccid paralysis. A related virus, enterovirus 71 (EV-A71), causes similar clinical outcomes in recurrent outbreaks throughout Asia. Retrospective phylogenetic analysis has shown that recombination between circulating strains of EV-A71 produces the outbreak-associated strains which exhibit increased virulence and/or transmissibility. While studies on the mechanism(s) of recombination in PV are ongoing in several laboratories, little is known about factors that influence recombination in EV-A71. We have developed a cell-based assay to study recombination of EV-A71 based upon previously reported assays for poliovirus recombination. Our results show that: (1) EV-A71 strain-type and RNA sequence diversity impacts recombination frequency in a predictable manner that mimics the observations found in nature; (2) recombination is primarily a replicative process mediated by the RNA-dependent RNA polymerase (RdRp); (3) a mutation shown to reduce recombination in PV (L420A) similarly reduces EV-A71 recombination suggesting conservation in mechanism(s); and (4) sequencing of intraserotypic recombinant genomes indicates that template-switching is by a mechanism that may require some sequence homology at the recombination junction and that the triggers for template-switching may be sequence independent. The development of this recombination assay will permit further investigation on the interplay between replication, recombination and disease.

    IMPORTANCE Recombination is a mechanism that contributes to genetic diversity. We describe the first assay to study EV-A71 recombination. Results from this assay mimic what is observed in nature and can be used by others to predict future recombination events within the enterovirus species A group. In addition, our results highlight the central role played by the viral RdRp in the recombination process. Further, our results show that changes to a conserved residue in the RdRp from different species groups have a similar impact on viable recombinant virus yields, indicative of conservation in mechanism.

  • Induction of porcine dermatitis and nephropathy syndrome in piglets by infection with porcine circovirus type 3 [Pathogenesis and Immunity]

  • Porcine circovirus type 3 (PCV3) is an emerging porcine circovirus that has been associated with porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs, reproductive failure, cardiac pathologies and multisystemic inflammation in piglets and sows. Many aspects of PCV3 infection biology and pathogenesis, however, remain unknown. Here, we used a PCV3 virus stock from rescue of an infectious PCV3 DNA clone to intranasally inoculate 4- and 8-week-old specific-pathogen-free piglets for evaluation of PCV3 pathogenesis. For 4-week-old piglets, typical clinical signs resembling those of PDNS-like disease were observed when inoculated with PCV3 alone or PCV3 combined with immunostimulation by keyhole limpet hemocyanin, with a mortality of 40% (2/5) for both types of inoculated piglets during a 28-day observation period postinoculation. Both types of inoculated piglets showed a similar progressive increase in viral load in the sera and had seroconverted to PCV3 capsid antibody after inoculation. Pathological lesions and PCV3-specific antigen were detected in various tissues and organs, including the lung, heart, kidney, lymph nodes, spleen, liver, and small intestine, in both types of inoculated piglets. Levels of proinflammatory cytokines and chemokines, including IL-1bbeta;, IL-6, IL-23aalpha;, IFN-, TNF-aalpha;, and CCL5, were significantly upregulated in both groups of inoculated piglets. 8-week-old piglets also exhibited a similar PDNS-like disease but without death after PCV3 inoculation as evidenced by pathological lesions and PCV3 antigen in various tissues and organs. These results show for the first time successful reproduction of PDNS-like disease by PCV3 infection and further provide significant information regarding the pathogenesis of PCV3 in piglets.

    IMPORTANCE Porcine circovirus type 3 (PCV3), an emerging porcine circovirus, is considered the cause of porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs and other systemic diseases in piglets and sows. To evaluate the pathogenesis of PCV3 infection in vivo, we used a PCV3 virus stock from rescue of an infectious PCV3 DNA clone to intranasally inoculate 4- and 8-week-old specific-pathogen-free piglets and demonstrated successful reproduction of PDNS-like disease in animals that were inoculated with PCV3 alone or PCV3 combined with immunostimulation by keyhole limpet hemocyanin. Both 4- and 8-week-old PCV3-inoculated piglets showed a similar increase in viral load in the sera and had seroconverted to PCV3 capsid antibody. Pathological lesions and PCV3-specific antigen were detected in various tissues and organs, while numerous proinflammatory cytokines and chemokines in the sera were significantly upregulated after PCV3 inoculation. These results will provide significant information regarding the pathogenesis of PCV3 in piglets.

  • Hepatitis C virus escape studies for human antibody AR3A reveals a high barrier to resistance and novel insights on viral antibody evasion mechanisms [Virus-Cell Interactions]

  • Yearly ~2 million people become hepatitis C virus (HCV) infected, resulting in elevated life-time risk for severe liver-related chronic illnesses. Characterizing epitopes of broadly neutralizing antibodies (NAbs), such as AR3A, is critical to guide vaccine development. Previously identified alanine substitutions reducing AR3A binding to expressed H77-envelope were introduced into chimeric cell-culture-infectious HCVcc, H77(Core-NS2)/JFH1. Substitutions G523A, G530A and D535A greatly reduced fitness and S424A, P525A and N540A, though viable, conferred only low-level AR3A-resistance. Using highly NAb-sensitive hypervariable region 1 (HVR1)-deleted HCVcc, H77/JFH1HVR1 and J6(Core-NS2)/JFH1HVR1, we previously reported a low barrier to developing AR5A NAb resistance substitutions. Here, we cultured Huh7.5 cells infected with H77/JFH1, H77/JFH1HVR1 or J6/JFH1HVR1 with AR3A. We identified the resistance envelope substitutions M345T in H77/JFH1, L438S and F442Y in H77/JFH1HVR1 and D431G in J6/JFH1HVR1. M345T increased infectivity and conferred low-level AR3A-resistance to H77/JFH1, but not H77/JFH1HVR1. L438S and F442Y conferred high-level AR3A-resistance to H77/JFH1HVR1, but abrogated infectivity of H77/JFH1. D431G conferred AR3A-resistance to J6/JFH1HVR1, but not J6/JFH1. This was possibly because D431G conferred broadly increased neutralization sensitivity to J6/JFH1D431G, but not J6/JFH1HVR1/D431G, while decreasing scavenger receptor class B, type I co-receptor dependency. Common substitutions at positions 431 and 442 did not confer high-level resistance in other 2a recombinants (JFH1 or T9(Core-NS2)/JFH1). Although the data indicate that AR3A has a high barrier to resistance, our approach permitted identification of low-level resistance substitutions. Also, the HVR1-dependent effects of AR3A resistance substitutions suggest a complex role of HVR1 in virus escape and receptor usage with important implications for HCV vaccine development.

    IMPORTANCE Hepatitis C virus (HCV) is a leading cause of liver-related mortality and limited treatment accessibility makes vaccine development a high priority. The vaccine-relevant cross-genotype reactive antibody, AR3A, has shown high potency, but the ability of the virus to rapidly escape by mutating the AR3A epitope (barrier to resistance) remains unexplored. Here, we only succeeded in inducing low-level AR3A resistance, indicating a higher barrier to resistance than what we have previously reported for AR5A. Furthermore, we identify AR3A resistance substitutions that have hypervariable region 1 (HVR1)-dependent effects on HCV viability and on broad neutralization sensitivity. One of these substitutions increased envelope breathing and decreased scavenger receptor class B, type I HCV co-receptor dependency, both in an HVR1-dependent fashion. Thus, we identify novel AR3A-specific resistance substitutions and shed important light on the role of HVR1 in protecting HCV from antibodies with importance for future HCV vaccine development.

  • Cell cycle arrest in G2/M phase enhances replication of interferon-sensitive cytoplasmic RNA viruses via inhibition of antiviral gene expression [Virus-Cell Interactions]

  • Vesicular stomatitis virus (VSV, a rhabdovirus) and its variant VSV-M51 are widely used model systems to study mechanisms of virus-host interactions. Here, we investigated how cell cycle affects replication of these viruses using an array of cell lines with different levels of impairment of antiviral signaling, and a panel of chemical compounds arresting cell cycle at different phases. We observed that all compounds inducing cell cycle arrest in G2/M phase strongly enhanced replication of VSV-M51 in cells with functional antiviral signaling. G2/M arrest strongly inhibited type I and type III IFN production, as well as expression of IFN-stimulated genes in response to exogenously-added IFN. Moreover, G2/M arrest enhanced replication of Sendai virus (a paramyxovirus) that is also highly sensitive to type I IFN response but did not stimulate replication of a wild-type VSV that is more effective at evading antiviral responses. In contrast, the positive effect of G2/M arrest on virus replication was not observed in cells defective in IFN signaling. Altogether, our data show that replication of IFN-sensitive cytoplasmic viruses can be strongly stimulated during G2/M phase as a result of inhibition of antiviral gene expression, likely due to mitotic inhibition of transcription, a global repression of cellular transcription during G2/M phase. The G2/M phase thus that could represent an "Achilles heel" of the infected cell, a phase when the cell is inadequately protected. This model could explain at least one of the reasons why many viruses have been shown to induce G2/M arrest.

    IMPORTANCE Vesicular stomatitis virus (VSV, a rhabdovirus) and its variant VSV-M51 are widely used model systems to study mechanisms of virus-host interactions. Here, we investigated how cell cycle affects replication of VSV and VSV-M51. We show that the G2/M cell cycle arrest strongly enhances replication of VSV-M51 (but not of wild-type VSV) and Sendai virus (a paramyxovirus) via inhibition of antiviral gene expression, likely due to Mitotic Inhibition of Transcription, a global repression of cellular transcription during G2/M phase. Our data suggest that the G2/M phase could represent an "Achilles heel" of the infected cell, a phase when the cell is inadequately protected. This model could explain at least one of the reasons why many viruses have been shown to induce G2/M arrest, and it has important implications for oncolytic virotherapy, suggesting that frequent cell cycle progression in cancer cells could make them more permissive to viruses.

  • Unique Type I Interferon, Expansion/Survival Cytokines and JAK/STAT Gene Signatures of Multi-Functional HSV-Specific Effector Memory CD8+ TEM Cells Are Associated with Asymptomatic Ocular Herpes in Humans [Vaccines and Antiviral Agents]

  • A large proportion of the world population harbors herpes simplex virus type 1 (HSV-1) a major cause of infectious corneal blindness. HSV-specific CD8+ T cells protect from herpes infection and disease. However, the genomic, phenotypic and functional characteristics of CD8+ T cells associated with the protection seen in asymptomatic (ASYMP) individuals, who despite being infected, never experienced any recurrent herpetic disease, remains to be fully elucidated. In this investigation, we compared the phenotype, function, and level of expression of a comprehensive panel of 579 immune genes of memory CD8+ T cells, sharing the same HSV-1 epitope-specificities, and freshly isolated peripheral blood from well-characterized cohorts of protected ASYMP and non-protected symptomatic (SYMP) individuals, with a history of numerous episodes of recurrent herpetic disease using high throughput digital NanoString nCounterttrade; system and Flow Cytometry. Interestingly, our results demonstrated memory CD8+ T cells from ASYMP individuals expressed a unique set of genes involved in expansion and survival, type I interferon (IFN-I) and JAK/STAT pathways. Frequent multi-functional HSV-specific effector memory CD62LlowCD44highCD8+ TEM cells were detected in ASYMP individuals compared to more of mono-functional central memory CD62LhighCD44highCD8+ TCM cells in SYMP individuals. Shedding light on the genotype, phenotype, and function of antiviral CD8+ T cells from "naturally protected" ASYMP individuals will help design future T-cell based ocular herpes immunotherapeutic vaccine.

    IMPORTANCE A staggering number of the world population harbors herpes simplex virus type 1 (HSV-1) potentially leading to blinding recurrent herpetic disease. While the majority are asymptomatic (ASYMP) individuals who never experienced any recurrent herpetic disease, symptomatic (SYMP) individuals have a history of numerous episodes of recurrent ocular herpetic disease. This study elucidates the phenotype, the effector function, and the gene signatures of memory CD8+ T cell populations associated with protection seen in ASYMP individuals. Frequent multi-functional HSV-specific effector memory CD8+ TEM cells were detected in ASYMP individuals. In contrast, non-protected SYMP individuals had more of central memory CD8+ TCM cells. The memory CD8+ TEM cells from ASYMP individuals expressed unique gene signatures characterized by higher levels of type I interferon (IFN), expansion and expansion/survival cytokines and JAK/STAT pathways. Future studies on the genotype, phenotype, and function of antiviral CD8+ T cells from "naturally protected" ASYMP individuals will help in the potential design of T-cell based ocular herpes vaccines.

  • Restriction of human cytomegalovirus infection by galectin-9 [Pathogenesis and Immunity]

  • Human cytomegalovirus (HCMV) is a ubiquitous human herpesvirus. While HCMV infection is generally asymptomatic in the immunocompetent, it can have devastating consequences in those with compromised or under-developed immune systems, including transplant recipients and neonates. Galectins are a widely expressed protein family that have been demonstrated to modulate both anti-viral immunity and regulate direct host-virus interactions. The potential for galectins to directly modulate HCMV infection has not previously been studied, and our results reveal that galectin-9 (Gal-9) can potently inhibit HCMV infection. Gal-9 mediated inhibition of HCMV was dependent upon its carbohydrate recognition domains, and thus dependent on glycan interactions. Temperature-shift studies revealed that Gal-9 specific inhibition was mediated primarily at the level of virus-cell fusion, and not binding. Additionally, we found that during reactivation of HCMV in hematopoietic stem cell transplant (HSCT) patients soluble Gal-9 is upregulated. This study provides the first evidence for Gal-9 functioning as a potent, antiviral defense effector molecule against HCMV infection, and identify it as a potential clinical candidate to restrict HCMV infections.

    IMPORTANCE Human cytomegalovirus (HCMV) continues to cause serious and often life-threatening disease in those with impaired or under-developed immune systems. This virus is able to infect and replicate in a wide range of human cell types, which enables virus spread to other individuals in a number of settings. Current anti-viral drugs are associated with a significant toxicity profile and there is no vaccine, and these highlight a need to identify additional targets for the development of anti-HCMV therapies. We demonstrate for the first time that secretion of a member of the galectin family of proteins, galectin-9 (Gal-9), is upregulated during natural HCMV reactivated infection and that this soluble cellular protein possesses a potent capacity to block HCMV infection by inhibiting virus entry into the host cell. Our findings support the possibility of harnessing the anti-viral properties of Gal-9 to prevent HCMV infection and disease.

  • Analysis of a subacute sclerosing panencephalitis (SSPE) Genotype B3 virus from the 2009/10 South African measles epidemic shows hyperfusogenic F proteins contribute to measles virus infection in the brain. [Virus-Cell Interactions]

  • During a measles virus (MeV) epidemic in 2009 in South Africa, measles inclusion body encephalitis (MIBE) was identified in several HIV-infected patients. Years later, children are presenting with subacute sclerosing panencephalitis (SSPE). To investigate the features of established MeV neuronal infections, viral sequences were analysed from brain tissue samples of a single SSPE case and compared with MIBE sequences previously obtained from patients infected during the same epidemic. Both the SSPE and the MIBE viruses had amino acid substitutions in the ectodomain of the F protein that confer enhanced fusion properties. Functional analysis of the fusion complexes confirmed that both MIBE and SSPE F protein mutations promoted fusion with less dependence on interaction by the viral receptor-binding protein with known MeV receptors. While the SSPE F required the presence of a homotypic attachment protein MeV H in order to fuse, the MIBE F did not. Both F proteins had decreased thermal stability compared to the corresponding wild-type F protein. Finally, recombinant viruses expressing MIBE or SSPE fusion complexes spread in the absence of known MeV receptors, with MIBE F-bearing viruses causing large syncytia in these cells. Our results suggest that alterations to the MeV fusion complex that promote fusion and cell-to-cell spread in the absence of known MeV receptors is a key property for infection of the brain.

    IMPORTANCE Measles virus can invade the central nervous system (CNS) and cause severe neurological complications such as MIBE and SSPE. However, mechanisms by which MeV enters the CNS and triggers the disease remain unclear. We analysed viruses from brain tissue of individuals with MIBE or SSPE, infected during the same epidemic, after the onset of neurological disease. Our findings indicate that the emergence of hyper-fusogenic MeV F proteins may be associated with infection of the brain. We also demonstrate that hyper-fusogenic F proteins permit MeV to enter cells and spread without the need to engage nectin-4 or CD150, known receptors for MeV that are not present on neural cells.

  • Closing and opening holes in the glycan shield of HIV-1 envelope glycoprotein SOSIP trimers can redirect the neutralizing antibody response to the newly unmasked epitopes [Vaccines and Antiviral Agents]

  • In HIV-1 vaccine research, native-like, soluble envelope glycoprotein SOSIP trimers are widely used for immunizing animals. The epitopes of autologous neutralizing antibodies (NAbs) induced by the BG505 and B41 SOSIP trimers in rabbits and macaques have been mapped to a few holes in the glycan shields that cover most of the protein surfaces. For BG505 trimers, the dominant autologous NAb epitope in rabbits involves residues that line a cavity caused by the absence of a glycan at residue 241. Here, we blocked this epitope in BG505 SOSIPv4.1 trimer immunogens by knocking-in an N-linked glycan at residue 241. We then opened holes elsewhere on the trimer by knocking-out single N-linked glycans at residues 197, 234, 276, 332 or 355, and found that NAb responses induced by the 241-glycan-bearing BG505 trimers were frequently re-directed to the newly opened sites. The strongest evidence for redirection of the NAb response to neo-epitopes, through the opening and closing of glycan holes, was obtained from trimer immunogen groups with the highest occupancy of the N241 site. We also attempted to knock-in the N289-glycan to block the sole autologous NAb epitope on the B41 SOSIP.v4.1 trimer. Although a retrospective analysis showed that the new N289-glycan site was substantially under-occupied, we found some evidence for redirection of the NAb response to a neo-epitope when this site was knocked-in and the N356-glycan site knocked-out. In neither study, however, was redirection associated with increased neutralization of heterologous Tier-2 viruses.

    IMPORTANCE Engineered SOSIP-trimers mimic envelope-glycoprotein spikes, which stud the surface of HIV-1 particles and mediate viral entry into cells. When used for immunizing test animals they elicit antibodies that neutralize resistant sequence-matched HIV-1 isolates. These neutralizing antibodies recognize epitopes in holes in the glycan shield that covers the trimer. Here, we added glycans to block the most immunogenic neutralization epitopes on BG505 and B41 SOSIP trimers. In addition, we removed selected other glycans to open new holes that might expose new immunogenic epitopes. We immunized rabbits with the various glycan-modified trimers and then dissected the specificities of the antibody responses. Thus, in principle, the antibody response might be diverted from one site to a more cross-reactive one, which would help in the induction of broadly neutralizing antibodies by HIV-1 vaccines based on envelope glycoproteins.

  • Mechanisms of abrupt loss of virus control in a cohort of previous HIV controllers. [Pathogenesis and Immunity]

  • Elite and Viremic HIV controllers are able to control their HIV infection and maintain undetectable or low-level viremia in the absence of antiretroviral treatment. Despite extensive studies, the immune factors responsible for such exclusive control remain poorly defined. We identified a cohort of 14 HIV controllers that suffered an abrupt loss of HIV control (LoC) to investigate possible mechanisms and virological and immunological events related to the sudden loss of control. The in-depth analysis of these subjects involved the study of cell-tropism of circulating virus, evidence for HIV super-infection, cellular immune responses to HIV as well as an examination of viral adaptation to host immunity by Gag sequencing. Our data demonstrate that a poor capacity of T cells to mediate in-vitro viral suppression, even in the context of protective HLA alleles, predicts loss of viral control. In addition, the data suggest that inefficient viral control may be explained by an increase of CD8-T-cell activation and exhaustion before LoC. Furthermore, we detected a switch from C5 to X4-tropic viruses in 4 individuals after loss of control, suggesting that tropism shift might also contribute to disease progression in HIV controllers. The significantly reduced inhibition of in vitro viral replication and increased expression of activation and exhaustion markers preceding the abrupt loss of viral control may help to identify untreated HIV controllers that are at risk of losing control and may offer a useful tool for monitoring individuals during treatment interruption phases in therapeutic vaccine trials.

    IMPORTANCE Few individuals can control HIV infection without the need for antiretroviral treatment and are referred to as "HIV controllers". We have studied such HIV controllers who suddenly loose this ability and present with high in vivo viral replication and decays in their CD4+ T-cell counts, to identify potential immune and virological factors that were responsible for initial virus control. We identify in-vitro determined reductions in the ability of CD8 T cells to suppress viral control and the presence of PD-1 expressing CD8+ T-cells with a naïve immune phenotype as potential predictors of in vivo loss of virus control. The findings could be important for the clinical management of HIV controller individuals and may offer an important tool to anticipate viral rebound in individuals in clinical studies that include cART treatment interruptions and which, if not treated quickly, could pose a significant risk to the trial participants.

  • The dengue virus non-structural protein 1 (NS1) is secreted from mosquito cells in association with the intracellular cholesterol transporter chaperone caveolin complex [Virus-Cell Interactions]

  • Dengue virus (DENV) is a mosquito-borne virus of the family Flaviviridae. The RNA viral genome encodes three structural and seven non-structural proteins. The non-structural protein 1 (NS1) is a multifunctional protein actively secreted in vertebrate and mosquito cells during infection. In mosquito cells, NS1 is secreted in a caveolin-1 dependent manner by an unconventional route. The caveolin chaperone complex (CCC) is a cytoplasmic complex formed by caveolin-1 and the chaperones FKBP52, Cy40 and CyA responsible for the cholesterol traffic inside the cell. In this work, we demonstrate that in mosquito cells, but not in vertebrate cells, NS1 associates and relies on the CCC for secretion. Treatment of mosquito cells with classic secretion inhibitors such as brefeldin A, golgicide A and Fli-06 showed no effect on NS1 secretion, but significant reductions in recombinant luciferase secretion and virion release. Silencing the expression of CAV1, FKBP52 with siRNAs or the inhibition of CyA by cyclosporine A resulted in significant decrease in NS1 secretion, again without affecting virion release. Co-localization, co-inmunoprecipitation and proximity ligation assays, indicated that NS1 co-localizes and interacts with all the proteins of the CCC. In addition, CAV-1 and FKBP52 expression was found augmented in DENV infected cells. Results obtained with zika virus infected cells suggest that in mosquito cells, ZIKV NS1 also follows the same secretory pathway observed for DENV NS1. These results uncover important differences in the dengue virus-cell interactions between the vertebrate host and the mosquito vector, as well as novel functions for the chaperone caveolin complex.

    IMPORTANCE The dengue virus protein NS1 is secreted efficiently from both infected vertebrate and mosquito cells. Previously, our group reported that NS1 secretion in mosquito cells follows an unconventional secretion pathway dependent on Caveolin-1. In this work, we demonstrate that in mosquito cells, but not in vertebrate cells, NS1 secretion takes place in association with the chaperone caveolin complex, a complex formed by caveolin-1 and the chaperones FKBP52, CyA and Cy40, in charge of cholesterol transport inside the cell. Results obtained with ZIKV infected mosquito cells suggest that also ZIKV NS1 is released following an unconventional secretory route in association with the chaperone caveolin complex. These results uncover important differences in the virus-cell interactions between the vertebrate host and the mosquito vector, as well as novel functions for the chaperone caveolin complex. Moreover, manipulation of the NS1 secretory route may prove a valuable strategy to combat these two mosquito borne diseases.

  • Genetic compatibility of reassortants between avian H5N1 and H9N2 influenza viruses with higher pathogenicity in mammals [Pathogenesis and Immunity]

  • The co-circulation of H5N1 and H9N2 avian influenza viruses in birds in Egypt provides reassortment opportunities between these two viruses. However, little is known about the emergence potential of reassortants derived from Egyptian H5N1 and H9N2 viruses and about the biological properties of such reassortants. To evaluate the potential public health risk of reassortants of these viruses, we used reverse genetics to generate the 63 possible reassortants derived from contemporary Egyptian H5N1 and H9N2 viruses, containing the H5N1 surface gene segments and combinations of the H5N1 and H9N2 internal gene segments, and analyzed their genetic compatibility, replication ability and virulence in mice. Genes in the reassortants showed remarkably high compatibility. Replication of most reassortants was higher than the parental H5N1 virus in human cells. Six reassortants were thought to emerge in birds under neutral or positive selective pressure, and four of them had higher pathogenicity in vivo than the parental H5N1 and H9N2 viruses. Our results indicated that H5N1-H9N2 reassortants could be transmitted efficiently to mammals with significant public health risk if they emerge in Egypt, although the viruses might not emerge frequently in birds.

    IMPORTANCE

    Close interaction between avian influenza (AI) viruses and humans in Egypt appears to have resulted in many of the worldwide cases of human infections by both H5N1 and H9N2 AI viruses. Egypt is regarded as a hot spot of AI virus evolution. Although no natural reassortant of H5N1 and H9N2 AI viruses has been reported so far, their co-circulation in Egypt may allow emergence of reassortants that may present a significant public health risk. Using reverse genetics, we report here the first comprehensive data showing that H5N1-N9N2 reassortants have fairly high genetic compatibility and possibly higher pathogenicity in mammals, including humans, than the parental viruses. Our results provide insight into the emergence potential of avian H5N1-H9N2 reassortants that may pose a high public health risk.

  • Efficient inhibition of avian and seasonal influenza A viruses by a virus-specific Dicer-substrate siRNA swarm in human monocyte-derived macrophages and dendritic cells [Vaccines and Antiviral Agents]

  • Influenza A viruses (IAVs) are viral pathogens that cause epidemics and occasional pandemics of significant mortality. The generation of efficacious vaccines and antiviral drugs remains a challenge due to the rapid appearance of new influenza types and antigenic variants. Consequently, novel strategies for the prevention and treatment of IAV infections are needed given the limitations of the presently available antivirals. Here, we used enzymatically produced IAV-specific double-stranded RNA (dsRNA) molecules and Giardia intestinalis Dicer for the generation of a swarm of small interfering RNA (siRNA) molecules. The siRNAs target multiple conserved genomic regions of the IAVs. In mammalian cells, the produced 25nndash;27 nucleotide long siRNA molecules are processed by endogenous Dicer into 21-nucleotide siRNAs and are thus designated as Dicer-substrate siRNAs (DsiRNAs). We evaluated the efficacy of the above DsiRNA swarm at preventing IAV infections in human primary monocyte-derived macrophages and dendritic cells. The replication of different IAV strains, including avian influenza H5N1 and H7N9 viruses, was significantly inhibited by pre-transfection of the cells with the IAV-specific DsiRNA swarm. Up to 7 orders of magnitude inhibition of viral RNA expression was observed, which led to a dramatic inhibition of IAV protein synthesis and virus production. The IAV-specific DsiRNA swarm inhibited virus replication directly through the RNA interference pathway although a weak induction of innate interferon responses was detected. Our results provide direct evidence for the feasibility of the siRNA strategy and the potency of DsiRNA swarms in the prevention and treatment of influenza, including the highly pathogenic avian influenza viruses.

    IMPORTANCE In spite of the enormous amount of research, influenza virus is still one of the major challenges for medical virology due to its capacity to generate new variants, which potentially lead to severe epidemics and pandemics. We demonstrated here that a swarm of small interfering RNA (siRNA) molecules, including more than one hundred different antiviral RNA molecules targeting the most conserved regions of influenza A virus genome, could efficiently inhibit the replication of all tested avian and seasonal influenza A variants in human primary monocyte-derived macrophages and dendritic cells. The wide antiviral spectrum makes the virus-specific siRNA swarm a potentially efficient treatment modality against both avian and seasonal influenza viruses.

  • A Nanobody Targeting Viral Non-Structural Protein 9 Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication [Vaccines and Antiviral Agents]

  • Porcine Reproductive and Respiratory Syndrome (PRRS) is a great concern to the swine industry due to the pandemic outbreak of the disease, current ineffective vaccinations, and lack of efficient antiviral strategies. In our previous study, a PRRSV Nsp9-specific nanobody Nb6 was successfully isolated and the intracellularly expressed Nb6 could dramatically inhibit PRRSV replication in MARC-145 cells. However, despite its small size, the application of Nb6 protein in infected cells is greatly limited as the protein itself cannot enter the cells physically. In this study, a trans-activating transduction (TAT) peptide was fused with Nb6 to promote the protein entry into cells. TAT-Nb6 was expressed as an inclusion body in E.coli, and indirect ELISA and pulldown assays showed that E.coli-expressed TAT-Nb6 maintained the binding ability to E.coli-expressed or PRRSV-encoded Nsp9. We demonstrated that TAT delivered Nb6 into MARC-145 cells and PAMs in a dose-and time-dependent manner and TAT-Nb6 efficiently inhibited the replication of several PRRSV genotype 2 strains as well as a genotype 1 strain. Using a yeast two-hybrid assay, Nb6 recognition sites were identified in the C-terminal part of Nsp9 which spanned two discontinuous regions (Nsp9aa454-551 and Nsp9aa599-646). Taken together, these results suggest that TAT-Nb6 could be developed as a potential antiviral drug for the inhibition of PRRSV replication and controlling the PRRS disease.

    IMPORTANCE The pandemic outbreak of PRRS, which is caused by PRRSV, has greatly affected the swine industry. We still lack efficient vaccination and immense challenge to control its infection. An intracellularly expressed Nsp9-specific nanobody Nb6 has been shown to be able to inhibit PRRSV replication in MARC-145 cells. However, its application is limited because Nb6 cannot physically enter cells. Herein, we demonstrated that cell-penetrating peptide TAT could deliver Nb6 into cultured cells. In addition, TAT-Nb6 fusion protein could suppress the replication of various PRRSV strains in MARC-145 cells and PAMs. These findings may provide a new approach for the drug development to control PRRS.

  • The Alphavirus E2 Membrane-Proximal Domain Impacts Capsid Interaction and Glycoprotein Lattice Formation [Structure and Assembly]

  • Alphaviruses are small enveloped RNA viruses that bud from the host cell plasma membrane. Alphavirus particles have a highly organized structure, with a nucleocapsid core containing the RNA genome surrounded by the capsid protein, and a viral envelope containing 80 spikes, each a trimer of heterodimers of the E1 and E2 glycoproteins. The capsid protein and envelope proteins are both arranged in organized lattices that are linked via the interaction of the E2 cytoplasmic tail/endodomain with the capsid protein. We previously characterized the role of 2 highly conserved histidine residues, H348 and H352, located in an external, juxtamembrane region of the E2 protein termed the D-loop. Alanine substitutions of H348 and H352 inhibit virus growth by impairing late steps in the assembly/budding of virus particles at the plasma membrane. To investigate this budding defect, we selected for revertants of the E2-H348/352A double mutant. We identified eleven second-site revertants with improved virus growth and mutations in the capsid, E2 and E1 proteins. Multiple isolates contained the mutation E2-T402K in the E2 endodomain or E1-T317I in the E1 ectodomain. Both of these mutations were shown to partially restore H348/352A growth and virus assembly/budding, while neither rescued the decreased thermostability of H348/352A. Within the alphavirus particle, these mutations affect the E2-capsid interaction or the E1-mediated inter-trimer interactions at the 5-fold axis of symmetry. Together our results support a model in which the E2 D-loop promotes the formation of the glycoprotein lattice and its interactions with the internal capsid protein lattice.

    IMPORTANCE Alphaviruses include important human pathogens such as Chikungunya and the encephalitic alphaviruses. There are currently no licensed alphavirus vaccines or effective antiviral therapies, and more molecular information on virus particle structure and function are needed. Here we highlight the important role of the E2 juxtamembrane D-loop in mediating virus budding and particle production. Our results demonstrated that this E2 region affects both the formation of the external glycoprotein lattice and its interactions with the internal capsid protein shell.

  • Packaging of the influenza A virus genome is governed by a plastic network of RNA/protein interactions [Structure and Assembly]

  • The genome of influenza A virus is organized into eight ribonucleoproteins, each composed of a distinct RNA segment bound by the viral polymerase and oligomeric viral nucleoprotein. Packaging sequences unique to each RNA segment together with specific nucleoprotein amino acids are thought to ensure the precise incorporation of these eight ribonucleoproteins into single virus particles, yet the underlying interaction network remains largely unexplored. Here we show that the genome packaging mechanism of an H7N7 subtype influenza A virus widely tolerates the mutation of individual packaging sequences in three different RNA segments. However, combinations of these modified RNA segments cause distinct genome packaging defects, marked by the absence of specific RNA segment subsets from the viral particles. Furthermore, we find that combining a single mutated packaging sequence with sets of specific nucleoprotein amino acid substitutions greatly impairs the viral genome packaging process. Along with previous reports, our data propose that influenza A virus uses a redundant and plastic network of RNA-RNA and potentially RNA-nucleoprotein interactions to coordinately incorporate its segmented genome into virions.

    IMPORTANCE The genome of influenza A virus is organized into eight viral ribonucleoproteins (vRNPs), which provides evolutionary advantages but complicates genome packaging. Although it has been shown that RNA packaging sequences and specific amino acids in the viral nucleoprotein (NP), both components of each vRNP, ensure selective packaging of one copy of each vRNP per virus particle, the required RNA-RNA and RNA-NP interactions remain largely elusive. We identified that the genome packaging mechanism tolerates the mutation of certain individual RNA packaging sequences, while their combined mutation provokes distinct genome packaging defects. Moreover, we found that seven specific amino acid substitutions in NP impair the function of RNA packaging sequences, and that this defect is partially restored by another NP amino acid change. Collectively, our data indicate that packaging of the influenza A virus genome is controlled by a redundant and plastic network of RNA/protein interactions, which may facilitate natural reassortment processes.

  • Capture of a hyena-specific retroviral envelope gene with placental expression associated in evolution with the unique emergence among carnivorans of hemochorial placentation in Hyaenidae [Genetic Diversity and Evolution]

  • Capture of retroviral envelope genes from endogenous retroviruses has played a role in the evolution of mammals, with evidence for the involvement of these genes in the formation of the materno-fetal interface of the placenta. It has been shown that the diversity of captured genes is likely to be responsible for the diversity of placental structures, ranging from poorly invasive (epitheliochorial) to highly invasive (hemochorial) with an intermediate state (endotheliochorial) as found in carnivorans. The latter recapitulate part of this evolution, with the hyena being the sole carnivoran with a hemochorial placenta. Here we performed RNAsequencing on hyena placental transcripts and searched for endogenous retroviral envelope genes that have been captured specifically in the Hyaenidae clade and are not found in any other carnivoran. We identified an envelope gene that is expressed in the placenta at the level of the materno-fetal interface, as evidenced by in situ hybridization/immunohistochemistry. The gene entry is coincidental with the emergence of the Hyaenidae clade 30 Mya, being found at the same genomic locus in all 4 extant hyena species. Its coding sequence has further been maintained during all of Hyaenidae evolution. It is not found in any of the 30 other carnivorans nndash;both Felidae and Canidae- that we screened. This envelope protein does not disclose any fusogenic activity in ex vivo assays, at variance with the Syncytin-Car1 gene that is found in all carnivorans nndash;including hyena where it is still present, transcriptionally active in the placenta, and fusogenic. Altogether, the present results illustrate the permanent renewal of placenta-specific genes by retroviral captures, and de facto provide a candidate gene for the endotheliochorial to hemochorial transition of Hyaenidae among carnivorans.

    IMPORTANCE The placenta is the most diverse organ among mammals, due in part to stochastic capture of retroviral envelope genes. In carnivorans, capture of Syncytin-Car1 took place 80 Mya. It is fusogenic, expressed at the syncytialized placental materno-fetal interface and conserved among all carnivorans, consistent with their shared "endotheliochorial" placenta. Hyenas are a remarkable exception, with a highly invasive "hemochorial" placenta, as found in humans, where disruption of maternal blood vessels results in maternal blood bathing the syncytial materno-fetal interface. Here we identified a retroviral envelope gene capture and exaptation that took place about 30 Mya and is coincident with the emergence of the Hyaenidae, being conserved in all extant hyena species. It is expressed at the materno-fetal interface in addition to the shared syncytin-Car1 gene. This new env gene, not present in any other carnivoran, is a likely candidate to be responsible for the specific structure of the hyena placenta.

  • A human gain-of-function STING mutation causes immunodeficiency and gammaherpesvirus-induced pulmonary fibrosis in mice [Pathogenesis and Immunity]

  • We previously generated STING N153S knockin mice that have a human disease-associated gain-of-function mutation in STING. Patients with this mutation (STING N154S in human) develop STING-associated vasculopathy with onset in infancy (SAVI), a severe pediatric autoinflammatory disease characterized by pulmonary fibrosis. Since this mutation promotes up-regulation of antiviral type I interferon-stimulated genes (ISGs), we hypothesized that STING N153S knockin mice may develop more severe autoinflammatory disease in response to a virus challenge. To test this hypothesis, we infected heterozygous STING N153S mice with murine gammaherpesvirus 68 (HV68). STING N153S mice were highly vulnerable to infection and developed pulmonary fibrosis after infection. In addition to impairing CD8+ T cell responses and humoral immunity, STING N153S also promoted replication of HV68 in cultured macrophages. In further support of a combined innate and adaptive immunodeficiency, HV68 infection was more severe in Rag1nndash;/nndash; STING N153S mice than in Rag1nndash;/nndash; littermate mice, which completely lack adaptive immunity. Thus, a gain-of-function STING mutation creates a combined innate and adaptive immunodeficiency that leads to virus-induced pulmonary fibrosis.

    IMPORTANCE A variety of human rheumatologic disease-causing mutations have recently been identified. Some of these mutations are found in viral nucleic acid-sensing proteins, but whether viruses can influence the onset or progression of these human diseases is less well understood. One such autoinflammatory disease called STING-associated vasculopathy with onset in infancy (SAVI) affects children and leads to severe lung disease. We generated mice with a SAVI-associated STING mutation and infected them with HV68, a common DNA virus that is related to human Epstein-Barr virus. Mice with the human disease-causing STING mutation were more vulnerable to infection than wild-type littermate control animals. Furthermore, the STING mutant mice developed lung fibrosis similar to patients with SAVI. These findings reveal that a human STING mutation creates severe immunodeficiency, leading to virus-induced lung disease in mice.

  • A novel type of influenza A virus-derived defective interfering particle with nucleotide substitutions in its genome [Genetic Diversity and Evolution]

  • Defective interfering particles (DIPs) replicate at the expense of co-infecting, fully infectious homologous virus. Typically, they contain a highly deleted form of the viral genome. Utilizing single-cell analysis, we here report the discovery of a yet unknown DIP type, derived from influenza A viruses (IAV), termed OP7 virus. Instead of deletions, the genomic viral RNA (vRNA) of segment 7 (S7) carried 37 point mutations compared to the reference sequence, affecting promotor regions, encoded proteins and genome packaging signals. Co-infection experiments demonstrated strong interference of OP7 virus with IAV replication, manifested by a dramatic decrease in infectivity of released virions. Moreover, an over-proportional quantity of S7 in relation to other genome segments was observed, both intracellularly and in the released virus population. Concurrently, OP7 virions lacked a large fraction of other vRNA segments, which appears to constitute its defect in virus replication. OP7 virus might serve as a promising candidate for antiviral therapy. Furthermore, this novel form of DIP may also be present in other IAV preparations.

    IMPORTANCE Defective-interfering particles (DIPs) typically contain a highly deleted form of the viral genome, rendering them defective in virus replication. Yet, upon complementation through co-infection with fully infectious standard virus (STV), interference with the viral life cycle can be observed, leading to a suppressed STV replication and the release of mainly non-infectious DIPs. Interestingly, recent research indicates that DIPs may serve as an antiviral agent. We here report the discovery of a yet unknown type of influenza A virus-derived DIP (termed "OP7" virus) that contains numerous point mutations instead of large deletions in its genome. Furthermore, the underlying principles that render OP7 virions interfering and apparently defective seem to differ from conventional DIPs. In conclusion, we believe that OP7 virus might be a promising candidate for antiviral therapy. Moreover, it exerts strong effects, both on virus replication and on host cell response, and may have been overlooked in other IAV preparations.

  • Early Infection of Porcine Sapovirus Disrupts Tight Junction and Uses Occludin as a Co-receptor [Virus-Cell Interactions]

  • Sapovirus is a genus in the Caliciviridae family, and its members are common causative agents of severe, acute gastroenteritis in both humans and animals. Some caliciviruses are known to use either terminal sialic acids or histo-blood group antigens as attachment factors and/or cell surface proteins such as CD300lf, CD300ld, and junctional adhesion molecule 1 of tight junctions (TJs) as receptors. However, the role of TJs and their proteins in sapovirus entry has not been examined. In this study, we found that porcine sapovirus (PSaV) significantly decreased transepithelial electrical resistance and increased paracellular permeability early in infection of LLC-PK cells, suggesting that PSaV dissociates TJs of cells. This led to the interaction between PSaV particles and occludin, which travelled in a complex into late endosomes via Rab5- and Rab7-dependent trafficking. Inhibition of occludin using siRNA, specific antibody, or a dominant-negative mutant significantly blocked the entry of PSaV. Transient expression of occludin in non-permissible Chinese hamster ovary (CHO) cells conferred susceptibility to PSaV, but only for a limited time. Although claudin-1, another TJ protein, neither directly interacted, nor was internalized with PSaV particles, it facilitated PSaV entry and replication in the LLC-PK cells. We conclude that PSaV particles enter LLC-PK cells by binding to occludin as a co-receptor in PSaV-dissociated TJs. PSaV and occludin then form a complex that moves to late endosomes via Rab5- and Rab7-dependent trafficking. In addition, claudin-1 in the opened TJs by PSaV infection facilitates PSaV entry and infection as an entry factor.

    IMPORTANCE Sapoviruses (SaVs) cause severe acute gastroenteritis in humans and animals. Although they replicate in intestinal epithelial cells, which are tightly sealed by apical junctional complexes such as tight junctions (TJs), the mechanisms by which SaVs hijack TJs and their proteins for successful entry and infection remain largely unknown. Here, we demonstrate that porcine SaVs (PSaVs) induce early dissociation of TJs, allowing them to bind to the TJ protein, occludin, as a functional co-receptor. PSaVs then travel in a complex with occludin into late endosomes through Rab5- and Rab7-dependent trafficking. Claudin-1, another TJ protein, does not directly interact with PSaV, but facilitates the entry of PSaV into cells as an entry factor. This work contributes to our understanding of the entry of SaV and other caliciviruses into cells and may aid in the development of efficient and affordable drugs to treat SaV infections.

  • The host DHX9 DExH Box helicase is recruited to Chikungunya virus replication complexes for optimal genomic RNA translation [Virus-Cell Interactions]

  • Beyond their role in cellular RNA metabolism, DExD/H-box RNA helicases are hijacked by various RNA viruses in order to assist replication of the viral genome. Here, we identify the DExH-box RNA helicase 9 (DHX9) as a binding partner of Chikungunya virus (CHIKV) nsP3 mainly interacting with the C-terminal hypervariable domain. We show that during early CHIKV infection, DHX9 is recruited to the plasma membrane where it associates to replication complexes. At later stage of infection, DHX9 is however degraded through a proteasome-dependent mechanism. Using silencing experiments we demonstrate that while DHX9 negatively controls viral RNA synthesis, it is also required for optimal nsPs translation. Altogether this study identifies DHX9 as a novel cofactor for CHIKV replication in human cells that differently regulates the various steps of CHIKV life cycle and may therefore mediate a switch in RNA usage from translation to replication during the earliest steps of CHIKV replication.

    IMPORTANCE The re-emergence of chikungunya virus (CHIKV), an alphavirus that is transmitted to humans by Aedes mosquitoes, is a serious global health threat. In absence of effective antiviral drugs CHIKV infection has a significant impact on human health with chronic arthritis being one of the most serious complications. The molecular understanding of host-virus interactions is a prerequisite to the development of targeted therapeutics capable to interrupt viral replication and transmission. Here, we identify the host cell DHX9 DExH-Box helicase as an essential cofactor for early CHIKV genome translation. We demonstrate that CHIKV nsP3 protein acts as a key factor for DHX9 recruitment to replication complexes. We finally establish that DHX9 behaves as a switch that regulates the progression of the viral cycle from translation to genome replication. This study might therefore have a significant impact for the development of antiviral strategies.

  • Increased frequency of virus shedding by herpes simplex virus type 2-infected guinea pigs in the absence of CD4+ T lymphocytes. [Pathogenesis and Immunity]

  • Reactivation of HSV-2 results in infection of epithelial cells at the neuro-epithelial junction and shedding of virus at the epithelial surface. Virus shedding can occur in either the presence or absence of clinical disease and is usually of short duration although the shedding frequency varies among individuals. The basis for host control of virus shedding is not well understood although adaptive immune mechanisms are thought to play a central role. To determine the importance of CD4+ T cells in control of HSV-2 shedding, this subset of immune cells was depleted from HSV-2-infected guinea pigs by injection of an anti-CD4 monoclonal antibody (mAb). Guinea pigs were treated with the depleting mAb after establishment of a latent infection and vaginal swabs were taken daily to monitor shedding by qPCR. The cumulative number of HSV-2 shedding days and the mean number of days virus was shed was significantly increased in CD4-depleted compared to control-treated animals. However, there was no difference in the incidence of recurrent disease between the two treatment groups. Serum antibody levels and the number of HSV-specific antibody secreting cells in secondary lymphoid tissues were unaffected by depletion of CD4+ T cells, however, the frequency of functional HSV-specific, CD8+ IFN secreting cells was significantly decreased. Together, these results demonstrate an important role for CD4+ T lymphocytes in control of virus shedding that may be mediated in part by maintenance of HSV-specific CD8+ T cell populations. These results have important implications for development of therapeutic vaccines designed to control HSV-2 shedding.

    IMPORTANCE Sexual transmission of HSV-2 results from viral shedding following reactivation from latency. The immune cell populations and mechanisms that control HSV-2 shedding are not well understood. This study examined the role of CD4+ T cells in control of virus shedding using a guinea pig model of genital HSV-2 infection that recapitulates the shedding of virus experienced by humans. We found that the frequency of virus shedding episodes, but not the incidence of clinical disease was increased by depletion of CD4+ T cells. The HSV-specific antibody response was not diminished, but frequency of functional HSV-reactive CD8+ Tells was significantly diminished by CD4-depletion. These results confirm the role of cell-mediated immunity and highlight the importance of CD4+ T cells in controlling HSV shedding suggesting that therapeutic vaccines designed to reduce transmission by controlling HSV shedding should most likely include specific enhancement of HSV-specific CD4+ T cell responses.

  • Early T follicular helper cell responses and germinal center reactions are associated with viremia control in immunized Rhesus macaques [Pathogenesis and Immunity]

  • T follicular helper (TFH) cells are fundamental in germinal center (GC) maturation and selection of antigen-specific B cells within secondary lymphoid organs. GC-resident TFH cells have been fully characterized in human HIV infection. However, the role of GC TFH cells in GC B cell responses following various SIV vaccine regimens in Rhesus macaques (RMs) has not been fully investigated. We characterized GC TFH cells of RMs over the course of a mucosal/systemic vaccination regimen to elucidate GC formation and SIV humoral response generation. Animals were mucosally primed twice with replicating Ad5hr-SIV recombinants and systemically boosted with ALVAC/Env or DNAaamp;Env including SIV gp120 proteins. Lymph nodes were biopsied in macaque subgroups pre-vaccination and at days 3, 7, or 14 after the 2nd Ad5hr-SIV prime and the 2nd vector/Env boost. Evaluations of GC TFH and GC B cell dynamics including correlation analyses supported a significant role of early GC TFH cells in providing B cell help during initial phases of GC formation. GC TFH responses at day 3 post-mucosal priming were consistent with generation of Env-specific memory B cells in GCs and elicitation of prolonged Env-specific humoral immunity in the rectal mucosa. GC Env-specific memory B cell responses elicited early post-systemic boosting correlated significantly with decreased viremia post-infection. Our results highlight the importance of early GC TFH cell responses for robust GC maturation and generation of long-lasting SIV specific humoral responses at mucosal and systemic sites. Further investigation of GC TFH cell dynamics should facilitate development of an efficacious HIV vaccine.

    IMPORTANCE The modest HIV protection observed in the human RV144 vaccine trial associated antibody responses with vaccine efficacy. T follicular helper (TFH) cells are CD4+ T cells that select antibody secreting cells with high antigenic affinity in germinal centers (GCs) within secondary lymphoid organs. To evaluate the role of TFH cells in eliciting prolonged viral-specific humoral responses, we vaccinated Rhesus macaques with a combined mucosal prime/systemic boost regimen followed by repeated low-dose intrarectal challenges with SIV mimicking human exposure to HIV-1. Although the vaccine regimen did not prevent SIV infection, decreased viremia was observed in the immunized macaques. Importantly, vaccine-induced TFH responses elicited at day 3 post-immunization and robust GC maturation were strongly associated. Further, early TFH-dependent SIV-specific B cell responses were also correlated with decreased viremia. Our findings highlight the contribution of early vaccine-induced GC TFH responses to elicitation of SIV-specific humoral immunity and implicate their participation in SIV control.

  • Epidemiological Evidence for Lineage-specific Differences in the Risk of Inapparent Chikungunya Virus Infection [Genetic Diversity and Evolution]

  • In late 2013, chikungunya virus (CHIKV) was introduced into the Americas, leading to widespread epidemics. A large epidemic caused by the Asian chikungunya virus (CHIKV) lineage occurred in Managua, Nicaragua, in 2015. Literature reviews commonly state that the proportion of inapparent CHIKV infections ranges from 3 to 28%. This study estimates the symptomatic-to-asymptomatic ratio of CHIKV infections and identifies risk factors of infection. In October-November 2015, 60 symptomatic CHIKV-infected children were enrolled as index cases and prospectively followed, alongside 236 household contacts, in an index cluster study. Samples were collected upon enrollment and on days 14 or 35 and tested by rRT-PCR, IgM-ELISA, and Inhibition ELISA to detect pre- and post-enrollment CHIKV infections. Of 236 household contacts, 55 (23%) had experienced previous or very recent infections, 41 (17%) had active infections at enrollment, and 21 (9%) experienced incident infections. Vehicle ownership (multivariate-adjusted risk ratio (aRR): 1.58) increased the risk of CHIKV infection whereas gge;4 municipal trash collections/week (aRR: 0.38) and having an external water faucet (aRR: 0.52) protected against CHIKV infection. Among 63 active and incident infections, 31 (49%, 95% CI: 36%, 62%) were asymptomatic, yielding a symptomatic-to-asymptomatic ratio of 1:0.97 (95% CI: 1:0.56, 1:1.60). Although our estimate is outside the 3-28% range reported previously, Bayesian and simulation analyses, informed by a systematic literature search, suggested that the proportion of inapparent CHIKV infections is lineage-dependent and that more inapparent infections are associated with the Asian lineage than the ECSA lineage. Overall, these data substantially improve knowledge regarding chikungunya epidemics.

    Importance

    Chikungunya virus (CHIKV) is an understudied threat to human health. During the 2015 chikungunya epidemic in Managua, Nicaragua, we estimated the ratio of symptomatic-to-asymptomatic CHIKV infections, which is important for understanding transmission dynamics and public health impact. This index cluster study identified persons at risk of infection and followed them, enabling capture of asymptomatic infections. We estimated that 31 (49%) of 63 at-risk participants had asymptomatic CHIKV infections, which is significantly outside the 3-28% range reported in literature reviews. However, recent seroprevalence studies, including two large pediatric cohort studies in the same setting, had also found percentages of inapparent infections outside the 3-28% range. Bayesian and simulation analyses, informed by a systematic literature search, revealed that the percentage of inapparent infections in epidemic settings varies by the phylogenetic lineage of CHIKV. Our study quantifies and provides the first epidemiological evidence that chikungunya epidemic characteristics are strongly influenced by CHIKV lineage.

  • Oncogenic KSHV Upregulates Argininosuccinate Synthase 1, a Rate-Limiting Enzyme of the Citrulline-Nitric Oxide Cycle, to Activate the STAT3 Pathway and Promote Growth Transformation [Transformation and Oncogenesis]

  • Cancer cells are required to rewire existing metabolic pathways to support their abnormal proliferation. We have previously shown that, unlike glucose-addicted cancers, Kaposirrsquo;s sarcoma-associated herpesvirus (KSHV)-transformed cells depend on glutamine rather than glucose for energy production, and amino acid and nucleotide syntheses. High-level consumption of glutamine is tightly regulated and often coupled with the citrulline-nitric oxide (NO) cycle. We have found that KSHV infection accelerates the nitrogen efflux by upregulating the expression of argininosuccinate synthase 1 (ASS1), a key enzyme in the citrulline-NO cycle. KSHV utilizes multiple microRNAs to upregulate ASS1 expression. Depletion of either ASS1 or inducible nitric oxide synthase (iNOS) in KSHV-transformed cells suppresses growth proliferation, abolishes colony formation in soft agar, and decreases NO generation. Furthermore, by maintaining intracellular NO level, ASS1 expression facilitates KSHV-mediated activation of the STAT3 pathway, which is critical for virus-induced transformation. These results illustrate a novel mechanism by which an oncogenic virus hijacks a key metabolic pathway to promote growth transformation and reveal a potential novel therapeutic target for KSHV-induced malignancies.

    IMPORTANCE We have previously shown that Kaposirrsquo;s sarcoma-associated herpesvirus (KSHV)-transformed cells depend on glutamine rather than glucose for energy production, and amino acid and nucleotide syntheses. In this study, we have further examined how the KSHV-reprogramed metabolic pathways are regulated and discovered that KSHV hijacks the citrulline-nitric oxide (NO) cycle to promote growth proliferation and transformation. Multiple KSHV-encoded microRNAs upregulate argininosuccinate synthase 1 (ASS1), a key enzyme in the citrulline-NO cycle. ASS1 is required for KSHV-induced proliferation, colony formation in soft agar, and NO generation of KSHV-transformed cells, which also depends on inducible nitric oxide synthase. By maintaining intracellular NO level, ASS1 mediates KSHV activation of the STAT3 pathway, which is essential for KSHV-induced abnormal cell proliferation and transformation. These results illustrate a novel mechanism by which an oncogenic virus hijacks a key metabolic pathway to promote growth transformation and reveal a potential novel therapeutic target for KSHV-induced malignancies.

  • 2.8 A resolution cryo-EM structure of human parechovirus 3 in complex with Fab from a neutralizing antibody [Vaccines and Antiviral Agents]

  • Human parechovirus 3 (HPeV3) infection is associated with sepsis in neonates characterized by significant immune activation and subsequent tissue damage. Strategies to limit infection have been unsuccessful due to inadequate molecular diagnostic tools for early detection and lack of a vaccine or specific antiviral therapy. Towards the latter, we present a 2.8 AAring;-resolution structure of HPeV3 in complex with fragments from a neutralizing human monoclonal antibody AT12-015 using cryo-EM and image reconstruction. Modeling revealed that the epitope extends across neighboring asymmetric units with contributions from capsid proteins VP0, VP1, and VP3. Antibody decoration was found to block binding of HPeV3 to cultured cells. Additionally at high-resolution, it was possible to model a stretch of RNA inside the virion and from this identify the key features that drive and stabilize protein-RNA association during assembly.

    IMPORTANCE HPeV3 is receiving increasing attention as a prevalent cause of sepsis-like symptoms in neonates, which despite the severity of disease, there are no effective treatments available. Structural and molecular insights into virus neutralization are urgently needed, especially as clinical cases are on the rise. Towards this goal, we present the first structure of HPeV3 in complex with fragments from a neutralizing monoclonal antibody. At high-resolution it was possible to precisely define the epitope that when targeted, prevents virions from binding to cells. Such an atomic-level description is useful for understanding host-pathogen interaction, viral pathogenesis mechanisms, and for finding potential cures for infection and disease.

  • Defective viral genomes alter how Sendai virus interacts with cellular trafficking machinery leading to heterogeneity in the production of viral particles among infected cells [Virus-Cell Interactions]

  • Defective viral genomes (DVGs) generated during RNA virus replication determine infection outcome by triggering innate immunity, diminishing virulence, and, in many cases, facilitating the establishment of persistent infections. Despite the critical role of DVGs during virus-host interactions, the mechanisms regulating production and propagation of DVGs are poorly understood. Visualization of viral genomes using RNA fluorescent in situ hybridization revealed a striking difference in the intracellular localization of DVGs and full-length viral genomes during infections with the paramyxovirus Sendai. In cells enriched in full-length virus, viral genomes clustered in a perinuclear region and associated with cellular trafficking machinery, including microtubules and the GTPase Rab11a. However, in cells enriched in DVGs, defective genomes distributed diffusely throughout the cytoplasm and failed to interact with this cellular machinery. Consequently, cells enriched in full-length genomes produced both DVG and full-length genome-containing viral particles, while DVG-high cells poorly produced viral particles, yet strongly stimulated antiviral immunity. These findings reveal the selective production of both standard and DVG-containing particles by a subpopulation of cells during infection that can be differentiated by the intracellular localization of DVGs. This study highlights the importance of considering this functional heterogeneity in analyses of virus-host interactions during infection.

    Significance

    Defective viral genomes (DVGs) generated during Sendai virus infections accumulate in the cytoplasm of some infected cells and stimulate antiviral immunity and cell survival. DVGs are packaged and released as defective particles and have a significant impact on infection outcome. We show that the subpopulation of DVG-high cells poorly engage the virus packaging and budding machinery and do not effectively produce viral particles. In contrast, cells enriched in full-length genomes are the primary producers of both standard and defective viral particles during infection. This study demonstrates heterogeneity in the molecular intracellular interactions occurring within infected cells and highlights distinct functional roles for cells as either initiators of immunity or as producers and perpetuators of viral particles depending on their content and intracellular localization of viral genomes.

  • Existing host range mutations constrain further emergence of RNA viruses [Genetic Diversity and Evolution]

  • RNA viruses are capable of rapid host shifting, typically due to a point mutation that confers expanded host range. As additional point mutations are necessary for further expansions, epistasis among host range mutations can potentially affect the mutational neighborhood and frequency of niche expansion. We mapped the mutational neighborhood of host range expansion using three genotypes of the dsRNA bacteriophage phi6 (wildtype and two isogenic host range mutants) on the novel host Pseudomonas syringae pv. atrofaciens (PA). Sanger sequencing of fifty PA mutant clones for each genotype and population Illumina sequencing both revealed the same high frequency mutations allowing infection of PA. Wildtype phi6 had at least nine different ways of mutating to enter the novel host, eight of which are in p3 (host attachment protein gene), and 13/50 clones had unchanged p3 genes. However, the two isogenic mutants had dramatically restricted neighborhoods: only one or two mutations, all in p3. Deep sequencing revealed that wildtype clones without mutations in p3 likely had changes in p12 (morphogenic protein), a region that was not polymorphic for the two isogenic host range mutants. Sanger sequencing confirmed that 10/13 of the wildtype phi6 clones had nonsynonymous mutations in p12 and two others had point mutations in p9 and p5 nndash; none of these genes had previously been associated with host range expansion in phi6. We demonstrate, for the first time, epistatic constraint in an RNA virus due to host range mutations themselves, which has implications for models of serial host range expansion.

    Importance

    RNA viruses mutate rapidly and frequently expand their host ranges to infect novel hosts, leading to serial host shifts. Using an RNA bacteriophage model system (Pseudomonas phage phi6), we studied the impact of pre-existing host range mutations on another host range expansion. Results from both clonal Sanger and Illumina sequencing show extant host range mutations dramatically narrow the neighborhood of potential host range mutations compared to wildtype phi6. This research suggests that serial host shifting viruses may follow a small number of molecular paths to enter additional novel hosts. We also identified new genes involved in phi6 host range expansion, expanding our knowledge of this important model system in experimental evolution.

  • The Ectodomain of the Vaccinia Virus Glycoprotein A34 is Required for Cell Binding by Extracellular Virions and Contains a Large Region Capable of Interaction with the Glycoprotein B5 [Structure and Assembly]

  • An interaction between the orthopoxvirus glycoproteins A34 and B5 has been reported. The transmembrane and ectodomain of A34 is sufficient for interaction with B5, localization of B5 to the site of intracellular wrapping, and subsequent incorporation into the envelope of released extracellular virions. Several mutagenic approaches were undertaken to better define the B5 interaction domain on A34. A set of C-terminal truncations in A34 identified residues 1-80 as sufficient for interaction with B5. Additional truncations identified residues 80-130 of A34 as sufficient for interaction with B5. To better understand the function of this region, a set of recombinant viruses expressing an A34 with the full, partial, or no B5 interaction site (residues 1-130, 1-100, and 1-70, respectively) were constructed. All the recombinants expressing truncations of A34 incorporated B5 into extracellular virions but had a small plaque phenotype similar in size to a virus with the A34R gene deleted (vA34R). Further characterization indicated that the small plaque phenotype exhibited by these viruses is due to a combination of abrogated actin tail formation, reduced cell binding, and a defect in polyanion-induced non-fusogenic dissolution. Taken together, these results suggest that residues 80-130 of A34 are not necessary for the proper localization and incorporation of B5 into extracellular virions and furthermore, that the C-terminal residues of A34 are involved in cell binding and dissolution.

    IMPORTANCE

    Previous studies have shown that the vaccinia virus glycoproteins A34 and B5 interact, and in the absence of A34, B5 is mis-localized and not incorporated into extracellular virions. Here, using a transient transfection assay, residues 80-130 of the ectodomain of A34 were determined to be sufficient for interaction with B5. Recombinant viruses expressing an A34 with a full, partial, or no B5 interaction site were constructed and characterized. All of the A34 truncations interacted with B5 as predicted by the transient transfection studies but had a small plaque phenotype. Further analysis revealed that all of the recombinants incorporated detectable levels of B5 into released virions, but, were defective in cell binding and EV dissolution. This study is the first to directly demonstrate that A34 is involved in cell binding and implicate the ectodomain in this role.

  • RNA helicase A is an important host factor involved in dengue virus replication [Virus-Cell Interactions]

  • Dengue virus (DENV) utilizes host factors throughout its life cycle. In this study, we identified RNA helicase A (RHA), a member of the DEAD/H helicase family, as an important host factor of DENV. In response to DENV2 infection, nuclear RHA protein was partially redistributed into the cytoplasm. The siRNA-mediated knockdown of RHA significantly the amounts of infectious viral particles in various cells. The RHA knockdown reduced the multi-step viral growth of DENV2 and Japanese encephalitis virus, but not Zika virus. Further study showed that the absence of RHA resulted in a reduction of both viral RNA and protein levels, and the data obtained from the reporter replicon assay indicated that RHA does not directly promote viral protein synthesis. RHA binds to the DENV RNA, and associates with three nonstructural proteins including NS1, NS2B3, and NS4B. Further study showed that different domains of RHA mediate its interaction with these viral proteins. The expression of RHA or RHA-K417R mutant protein lacking ATPase/helicase activity in RHA-knockdown cells successfully restored DENV2 replication levels, suggesting that the helicase activity of RHA is dispensable for its proviral effect. Overall, our work reveals that RHA is an important factor of DENV and might serve as a target for antiviral agents.

    IMPORTANCE Dengue, caused by dengue virus, is the most rapidly spreading disease, and currently there are no treatments available. Host factors involved in the viral replication of dengue virus are potential antiviral therapeutic targets. Although RHA has been shown to promote the multiplication of several viruses such as HIV and adenovirus, its role in the flavivirus family, including dengue virus, Japanese encephalitis virus, and emerging Zika virus remains elusive. The current study revealed that RHA relocalized into the cytoplasm upon DENV infection, and associated with viral RNA and nonstructural proteins, implying that RHA was actively engaged in the viral life cycle. We further provide evidence that RHA promoted the viral yields of DENV2 independent of its helicase activity. These findings demonstrated that RHA is a new host factor required for DENV replication and might serve as a target for antiviral drugs.

  • Vectorial Release of Hepatitis E Virus in Polarized Human Hepatocytes [Virus-Cell Interactions]

  • Hepatitis E virus (HEV) is a common cause of acute viral hepatitis worldwide. Most HEV infections are asymptomatic but immunocompromised patients infected with HEV3, HEV4 or HEV7 may develop chronic infections. The HEV particles in stools are naked (nHEV) while those in the serum and culture supernatants (eHEV) are associated with lipids. Hepatocytes are polarized epithelial cells that have basolateral (to blood) and apical (to bile) exosomal pathways. We isolated a subclone, F2, from the human hepatocarcinoma cell line HepG2/C3A that grew as a polarized monolayer culture and had better HEV production than HepG2/C3A. F2 cells cultured on semi-permeable collagen inserts and infected basolaterally with nHEV3 released 94.6% of virus particles apically, those infected with eHEV3 released 96.8% apically, and eHEV1-infected cells released 99.3% apically. Transcytosis was not involved. Density gradient centrifugation and NP40 treatment showed that HEV particles released both apically and basolaterally were lipid-associated. The apically-released HEV3 and HEV1 particles were six- and nine-times more infectious than those released basolaterally, respectively. Confocal microscopy indicated that the ORF2 capsid protein colocalized apically with ORF3 virus protein, the apical marker DPP4, and the recycling endosome GTPase Rab27a. The amounts of soluble glycosylated ORF2 secreted apically and basolaterally were similar. These polarized hepatocyte data suggest that infectious HEV particles are mainly released to bile, while the small fraction released to blood could spread HEV throughout the host.

    Importance

    The hepatitis E virus (HEV) in stools is naked while those in culture supernatants and patientsrrsquo; blood are lipid-associated. Its life cycle in hepatocytes, polarized cells with basolateral side communicating with blood and apical side connected with bile, is incompletely understood. We have developed a polarized hepatocyte model, and used these cells to analyze the supernatants bathing the apical and basolateral sides and HEV subcellular distribution. HEV viral particles from both sides were lipid-associated and most infectious HEV particles left the cell via its apical side. Similar amounts of the soluble capsid protein ORF2 were secreted from both sides of the hepatocytes. This model mimicking physiological conditions should help clarify the HEV cell cycle in polarized hepatocytes.

  • Functional analysis and anti-virulent properties of a new depolymerase from a myovirus that infects Acinetobacter baumannii capsule K45 [Pathogenesis and Immunity]

  • Acinetobacter baumannii is an important pathogen causative of healthcare-associated infections and is able to rapidly develop resistance to all known antibiotics including colistin. As an alternative therapeutic agent, we have isolated a novel myovirus (vB_AbM_B9) which specifically infects and makes lysis from without in strains of the K45 and K30 capsule type, respectively. Phage B9 has a genome of 93,641 bp and encodes 167 predicted proteins, of which 29 were identified by mass spectrometry. This phage holds a capsule depolymerase (B9gp69) able to digest extracted exopolysaccharides of both K30 and K45 strains and that remains active in a wide range of pH values (5 to 9), ionic strengths (0 to 500 mM), and temperatures (20 to 80ddeg;C). B9gp69 demonstrated to be non-toxic in a cell line model of the human lung, and to make the K45 strain fully susceptible to serum killing in vitro. Contrary to the phage, no resistance development was observed by bacteria targeted with the B9gp69. Therefore, capsular depolymerases may represent attractive antimicrobial agents against A. baumannii infections.

    IMPORTANCE

    Currently, phage therapy has revived interest for controlling hard-to-treat bacterial infections. Acinetobacter baumannii is an emerging Gram-negative pathogen able to cause a variety of nosocomial infections. Additionally, this species is becoming more resistant to several classes of antibiotics. Herein, we describe the isolation of a novel lytic myophage B9 and its recombinant depolymerase. While the phage can be a promising alternative antibacterial agent, its success in the market will ultimately depend on new regulatory frameworks and general public acceptance. We therefore characterised the phage-encoded depolymerase which is a natural enzyme that can be more easily managed and used. To our knowledge, the therapeutic potential of phage depolymerase against A. baumannii is still unknown. We show for the first time that K45 capsule type is an important virulence factor of A. baumannii and that capsule removal via the recombinant depolymerase activity helps the host immune system to combat the bacterial infection.

  • The Autographa californica multiple nucleopolyhedrovirus ac51 gene is required for efficient nuclear egress of nucleocapsids and is essential for in vivo virulence [Structure and Assembly]

  • Alphabaculoviruses are lepidopteran-specific nucleopolyhedroviruses that replicate within the nucleus; however, the anterograde transport of the nucleocapsids of these viruses, which is an obligatory step for progeny virion production, is not well understood. In the present study, a unique Alphabaculovirus gene with unknown function, namely, the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac51 gene, was found to be required for efficient nuclear egress of AcMNPV nucleocapsids. Our results indicate that ac51 is a late gene, and Ac51 protein was detectable from 24 to 72 h postinfection using an antibody raised against Ac51. Ac51 is distributed in both the cytoplasm and nuclei of infected cells. Upon ac51 deletion, budded virion (BV) production by 96 h posttransfection was reduced by approximately 1,000-fold compared with that of wild-type AcMNPV. Neither viral DNA synthesis nor viral gene expression was affected. Ac51 was demonstrated to be a nucleocapsid protein of BVs, and ac51 deletion did not interrupt nucleocapsid assembly and ODV formation. However, BV production in the supernatants of transfected cells during a viral life cycle was substantially decreased when ac51 was deleted. Further analysis showed that compared with wild-type AcMNPV, ac51 deletion decreased nucleocapsid egress, while the numbers of nucleocapsids in the nuclei were comparable. Deletion of ac51 also eliminated the virulence of AcMNPV in vivo. Taken together, our results support the conclusion that ac51 plays an important role in the nuclear egress of nucleocapsids during BV formation and is essential for the in vivo virulence of AcMNPV.

    IMPORTANCE Little is known about the mechanism underlying the nuclear egress of baculovirus nucleocapsids. In particular, the mechanism by which nucleocapsids of alphabaculoviruses efficiently egress from the nucleus to produce high levels of BVs in cultured cells is not known. Some genes that are conserved in all baculoviruses have been identified as essential for not only the nuclear egress of nucleocapsids but also ODV formation. In this study, we show that ac51, a unique Alphabaculovirus gene, encodes a nucleocapsid protein of BVs and is required for high levels of BV production. Deletion of this gene impaired efficient nuclear egress of nucleocapsids but not nucleocapsid assembly or ODV formation, which is similar to the phenotype observed for ac141 or ac66 deletion. This result indicated that Ac51 is the third nucleocapsid protein that promotes the nuclear egress of nucleocapsids by a common pathway with Ac141 and Ac66.

  • Ubiquitination is essential for avibirnavirus replication by supporting VP1 Polymerase activity [Virus-Cell Interactions]

  • Ubiquitination is critical for several cellular physical processes. However, ubiquitin modification in virus replication is poorly understood. Therefore, the present study aimed to determine the presence and effect of ubiquitination on polymerase activity of viral protein 1 (VP1) of avibirnavirus. We reported that the replication of avibirnavirus is regulated by ubiquitination of its VP1 protein, the RNA-dependent RNA polymerase of infectious bursal disease virus (IBDV). In vivo detection revealed the ubiquitination of VP1 protein in IBDV infected target organs and different cells, but not in purified IBDV particles. Further analysis of ubiquitination confirms that VP1 is modified by K63-linked ubiquitin chain. Point mutation screening showed that the ubiquitination site of VP1 was at the K751 residue in the C-terminus. The K751 ubiquitination is independent of VP1rrsquo;s interaction with VP3 and eukaryotic initiation factor-4A II. Polymerase activity assays indicated that the K751 ubiquitination at the C-terminus of VP1 enhanced its polymerase activity. The K751 to R mutation of VP1 protein did not block the rescueing of IBDV, but decreased the replication ability of IBDV. Our data demonstrated that the ubiquitination of VP1 is crucial to regulate its polymerase activity and IBDV replication.

    IMPORTANCE

    Avibirnavirus protein VP1, the RNA-dependent RNA polymerase, is responsible for IBDV genome replication, gene expression and assembly. However, little is known about its chemical modification relating to its polymerase activity. In this study, we revealed the molecular mechanism of ubiquitin modification of VP1 via a K63-linked ubiquitin chain during infection. Lysine (K) residue 751 at the C-terminus of VP1 is the target site for ubiquitin and its ubiquitination is independent of VP1rrsquo;s interaction with VP3 and eukaryotic initiation factor-4A II. The K751 ubiquitination promotes the polymerase activity of VP1 and unubiquitinated VP1 mutant IBDV significantly impairs virus replication. We concluded that VP1 is the ubiquitin-modified protein and revealed the mechanism by which VP1 promotes avibirnavirus replication.

  • Two families of Env antibodies efficiently engage Fc-gamma receptors and eliminate HIV-1-infected cells [Pathogenesis and Immunity]

  • HIV-1 conceals epitopes of its envelope glycoproteins (Env) recognized by antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies. These antibodies (Abs), including anti-co-receptor binding site (CoRBS) and anti-cluster A, preferentially recognize Env in its "open" conformation. The binding of anti-CoRBS Abs has been shown to induce conformational changes that further opens Env allowing interaction of anti-cluster A antibodies. We explored the possibility that CoRBS Abs synergize with anti-cluster A Abs to engage Fc-gamma receptors to mediate ADCC. We found that binding of anti-CoRBS and anti-cluster A Abs to the same gp120 is required for interaction with soluble dimeric FcRIIIa in ELISA assays. We also show that Fc regions of both Abs are required to optimally engage FcRIIIa and mediate robust ADCC. Altogether, our results indicate that these two families of Abs act together in a sequential and synergistic fashion to promote FcRIIIa engagement and ADCC.

    IMPORTANCE The "open" CD4-bound conformation of HIV-1 envelope glycoproteins is the primary target of antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies present in HIV+ sera, such as anti-co-receptor binding site and anti-cluster A antibodies. Here we report that the binding of these two families of antibodies is required to engage FcRIIIa and mediate ADCC.

  • Tetherin inhibits Nipah virus but not Ebola virus replication in fruit bat cells [Virus-Cell Interactions]

  • Ebola virus (EBOV) and Nipah virus (NiV) infection of humans can cause fatal disease and constitutes a public health threat. In contrast, EBOV and NiV infection of fruit bats, the putative (EBOV) or proven (NiV) natural reservoir, is not associated with disease and it is currently unknown how these animals control the virus. The human interferon (IFN)-stimulated antiviral effector protein tetherin (CD317, BST-2) blocks release of EBOV- and NiV-like particles from cells and is counteracted by the EBOV glycoprotein (GP). In contrast, it is unknown whether fruit bat tetherin restricts virus infection and is susceptible to GP-driven antagonism. Here, we report the sequence of fruit bat tetherin and show that its expression is IFN-stimulated and associated with strong antiviral activity. Moreover, we demonstrate that EBOV-GP antagonizes tetherin orthologues of diverse species but fails to efficiently counteract fruit bat tetherin in virus-like particle (VLP) release assays. However, unexpectedly, tetherin was dispensable for robust IFN-mediated inhibition of EBOV spread in fruit bat cells. Thus, the VLP-based model system mimicking tetherin-mediated inhibition of EBOV release and its counteraction by GP seems not to adequately reflect all aspects of EBOV release from IFN-stimulated fruit bat cells, potentially due to differences in tetherin expression levels that could not be resolved by the present study. In contrast, tetherin expression was essential for IFN-dependent inhibition of NiV infection, demonstrating that IFN-induced fruit bat tetherin exerts antiviral activity and may critically contribute to control of NiV and potentially other highly virulent viruses in infected animals.

    IMPORTANCE Ebola virus and Nipah virus (EBOV, NiV) can cause fatal disease in humans. In contrast, infected fruit bats do not develop symptoms but can transmit the virus to humans. Why fruit bats but not humans control infection is largely unknown. Tetherin is an antiviral host cell protein and is counteracted by the EBOV glycoprotein in human cells. Here, employing model systems, we show that tetherin of fruit bats displays higher antiviral activity than human tetherin and is largely resistant against counteraction by the Ebola virus glycoprotein. Moreover, we demonstrate that induction of tetherin expression is critical for interferon-mediated inhibition of NiV but, for at present unknown reasons, not EBOV spread in fruit bat cells. Collectively, our findings identify tetherin as an antiviral effector of innate immune responses in fruit bats, which might allow these animals to control infection with NiV and potentially other viruses that cause severe disease in humans.

  • Recessive host range mutants and unsusceptible cells that inactivate virions without genome penetration: ecological and technical implications [Structure and Assembly]

  • Although microviruses do not possess a visible tail structure, one vertex rearranges after interacting with host lipopolysaccharides. Most examinations of host range, eclipse, and penetration were conducted before this "host-induced" unique vertex was discovered and before DNA sequencing became routine. Consequently, structure-function relationships dictating host range remain undefined. Biochemical and genetic analyses were conducted with two closely related microviruses, aalpha;3 and ST-1. Despite ~90% amino acid identity, the natural host of aalpha;3 is E. coli C; whereas ST-1 is a K12-specific phage. Virions attached and eclipsed to both native and unsusceptible hosts; however, they breached only the native hostrrsquo;s cell wall. This suggests that unsusceptible host-phage interactions promote off-pathway reactions that can inactivate viruses without penetration. This phenomenon may have broader ecological implications. To determine which structural proteins conferred host range specificity, chimeric virions were generated by individually interchanging the coat, spike, or DNA pilot proteins. Interchanging the coat protein switched host range. However, host range expansion could be conferred by single point mutations in the coat protein. The expansion phenotype was recessive: genetically mutant progeny from co-infected cells did not display the phenotype. Thus, mutant isolation required populations generated in low MOI environments: a phenomenon that may have impacted past host range studies in both prokaryotic and eukaryotic systems. The resulting genetic and structural data were consistent enough that host range expansion could be predicted, broadening the classical definition of antireceptors to include interfaces between protein complexes within the capsid.

    Importance

    To expand host range, viruses must interact with unsusceptible host cell surfaces, which could be detrimental. As observed in this study, virions were inactivated without genome penetration. This may be advantageous to potential new hosts; culling the viral population from which an expanded host range mutant could emerge. When identified, altered host range mutations were recessive. Accordingly, isolation required populations generated in low MOI environments. However, in laboratory settings, viral propagation includes high MOI conditions. Typically, infected cultures incubate until all cells produce progeny. Thus, coinfections dominate later replication cycles, masking recessive host range expansion phenotypes. This may have impacted similar studies with other viruses. Lastly, structural and genetic data could be used to predict site-directed mutant phenotypes, which may broaden the classic antireceptor definition to include interfaces between capsid complexes.

  • Conformational Differences Between Functional Human Immunodeficiency Virus (HIV-1) Envelope Glycoprotein Trimers and Stabilized Soluble Trimers [Structure and Assembly]

  • Binding to the receptor, CD4, triggers entry-related conformational changes in the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer ((gp120/gp41)3). Soluble versions of HIV-1 Env trimers (sgp140 SOSIP.664) stabilized by a gp120-gp41 disulfide bond and a change (I559P) in gp41 have been structurally characterized. Here we use crosslinking/mass spectrometry to evaluate the conformations of functional membrane Env and sgp140 SOSIP.664. Differences were detected in the gp120 trimer association domain and C-terminus and in the gp41 heptad repeat (HR1) region. Whereas the membrane Env trimer exposes the gp41 HR1 coiled coil only after CD4 binding, the sgp140 SOSIP.664 HR1 coiled coil was accessible to the gp41 HR2 peptide even in the absence of CD4. Our results delineate differences in both gp120 and gp41 subunits between functional membrane Env and the sgp140 SOSIP.664 trimer, and provide distance constraints that can assist validation of candidate structural models of the native HIV-1 Env trimer.

    IMPORTANCE The human immunodeficiency virus (HIV-1) envelope glycoprotein spikes mediate the entry of the virus into host cells and are a major target for vaccine-induced antibodies. Soluble forms of the envelope glycoproteins that are stable and easily produced have been characterized extensively and are being considered as vaccines. Here we present evidence that these stabilized soluble envelope glycoproteins differ in multiple respects from the natural HIV-1 envelope glycoproteins. By pinpointing these differences, our results can guide the improvement of envelope glycoprotein preparations to achieve greater similarity to the viral envelope glycoprotein spike, potentially increasing their effectiveness as a vaccine.

  • Cellular zinc finger protein 622 hinders human adenovirus lytic growth and limits binding of the viral pVII protein to virus DNA [Cellular Response to Infection]

  • Human adenovirus (HAdV) encodes a multifunctional DNA-binding protein pVII, which is involved in virus DNA packaging and extracellular immune signaling regulation. Although the pVII is an essential viral protein, its exact role in the virus life cycle and interplay with cellular proteins have remained to a large extent unclear. We have recently identified the cellular zinc finger protein 622 (ZNF622) as a potential pVII-interacting protein. In this study, we describe the functional consequences of the ZNF622-pVII interplay and the role of ZNF622 in the HAdV life cycle. ZNF622 protein expression increased and it accumulated similarly to the pVII protein in the nucleus of virus-infected cells. Lack of the ZNF622 protein specifically increased pVII binding to viral DNA in the infected cells and elevated the pVII protein levels in the purified virions. In addition, ZNF622 knock out cells showed an increased cell lysis and enhanced accumulation of the infectious virus particles. Protein interaction studies revealed that ZNF622 forms a trimeric complex with the pVII protein and the cellular histone chaperon protein nucleophosmin 1 (NPM1). The integrity of this complex is important since ZNF622 mutations and NPM1 deficiency changed pVII ability to bind viral DNA. Collectively, our results implicate that ZNF622 may act as a cellular anti-viral protein hindering lytic HAdV growth and limiting pVII protein binding to viral DNA.

    IMPORTANCE Human adenoviruses (HAdVs) are common human pathogens causing a wide range of acute infections. To counteract viral pathogenicity, cells encode a variety of anti-viral proteins and non-coding RNAs to block virus growth. In this study, we show that the cellular zinc finger protein 622 (ZNF622) interacts with an essential HAdV protein known as pVII. This mutual interaction limits pVII binding to viral DNA. Further, ZNF622 has a role in HAdV life cycle since the lack of ZNF622 correlates with increased lysis of the infected cells and accumulation of the infectious virions. Together, our study reveals a novel cellular anti-viral protein ZNF622, which may impede lytic HAdV growth.

  • Chikungunya virus fidelity variants exhibit differential attenuation and population diversity in cell culture and adult mice [Genetic Diversity and Evolution]

  • Chikungunya virus (CHIKV) is a re-emerging global health threat that produces debilitating arthritis in people. Like other RNA viruses with high mutation rates, CHIKV produces populations of genetically diverse genomes within a host. While several known CHIKV mutations influence disease severity in vertebrates and transmission by mosquitoes, the role of intrahost diversity in chikungunya arthritic disease is not studied. In this study, high and low fidelity CHIKV variants, previously characterized by altered in vitro population mutation frequencies, were used to evaluate how intrahost diversity influences clinical disease, CHIKV replication, and antibody neutralization in immunocompetent adult mice inoculated in the rear footpads. Both high and low fidelity mutations were hypothesized to attenuate CHIKV arthritic disease, replication, and neutralizing antibody levels compared to wildtype (WT) CHIKV. Unexpectedly, high fidelity mutants elicited more severe arthritic disease than WT despite comparable CHIKV replication, whereas a low fidelity mutant produces attenuated disease and replication. Serum antibody developed against both high and low fidelity CHIKV exhibited reduced neutralization of WT CHIKV. Using next-generation sequencing (NGS), the high fidelity mutations were demonstrated to be genetically stable, but produce more genetically diverse populations than WT CHIKV in mice. This enhanced diversification was subsequently reproduced after serial in vitro passage. The NGS results contrast with previously reported population diversities for fidelity variants, which focused mainly on part of the E1 gene, and highlight the need for direct measurements of mutation rates to clarify CHIKV fidelity phenotypes.

    IMPORTANCE Chikungunya virus (CHIKV) is a re-emerging global health threat that elicits debilitating arthritis in humans. There are currently no commercially available CHIKV vaccines. Like other RNA viruses, CHIKV has a high mutation rate and is capable of rapid intrahost diversification during an infection. In other RNA viruses, virus population diversity associates with disease progression; however, potential impacts of intrahost viral diversity on CHIKV arthritic disease have not been studied. Using previously characterized CHIKV fidelity variants, we addressed whether CHIKV population diversity influences the severity of arthritis and host antibody response in an arthritic mouse model. Our findings show that CHIKV populations with greater genetic diversity can cause more severe disease and stimulate antibody responses with reduced neutralization of low diversity virus populations in vitro. The discordant high fidelity phenotypes in this study highlight the complexity of inferring replication fidelity indirectly from population diversity.

  • Priming with a potent HIV-1 DNA vaccine frames the quality of immune responses prior to a poxvirus and protein boost [Vaccines and Antiviral Agents]

  • The use of heterologous immunization regimens and improved vector systems has led to increases in immunogenicity of HIV-1 vaccine candidates in non-human primates. In order to resolve interrelations between different delivery modalities, three different poxvirus boost regimens were compared. Three groups of rhesus macaques were each primed with the same DNA vaccine encoding for Gag, PolNef, and gp140. The groups were then boosted either with the vaccinia virus strain NYVAC or a variant with improved replication competence in human cells termed NYVAC-KC. The latter was either administered by scarification or intramuscularly. Finally, macaques were boosted with adjuvanted gp120 protein to enhance humoral responses. The regimen elicited very potent CD4+ and CD8+ T cell responses in a well-balanced manner, peaking two weeks after the boost. T cells were broadly reactive and polyfunctional. All animals exhibited antigen-specific humoral responses already after the poxvirus boost that further increased following protein administration. Polyclonal reactivity of IgG antibodies was highest against HIV-1 clade C Env-proteins with considerable cross-reactivity to other clades. Substantial effector functional activities (ADCC and ADCVI) were observed in sera obtained after the last protein boost. Notably, major differences between the groups were absent, indicating that the potent priming induced by the DNA vaccine initially framed the immune responses in such a way that the subsequent boosts with NYVAC and protein only led to an increase in the response magnitudes without skewing the quality. This study highlights the importance of selecting the best combination of vector systems in heterologous prime-boost vaccination regimens.

    Importance

    The evaluation of HIV vaccine efficacy trials indicates that protection would most likely correlate with a polyfunctional immune response involving several effector functions from all arms of the immune system. Heterologous prime-boost regimens have been shown to elicit vigorous T cell and antibody responses in non-human primates that, however, qualitatively and quantitatively differ depending on the respective vector systems used. The present study evaluated a DNA prime, poxvirus and protein boost regimen, and compared how two poxvirus vectors with varying degrees of replication capacity and two different delivery modalities nndash; conventional intramuscular delivery and percutaneous delivery by scarification nndash; impact several immune effectors. It was found that despite the different poxvirus boosts, the overall immune responses in the three groups were similar, suggesting the potent DNA priming as the major determining factor of immune responses. These findings emphasize the importance of selecting optimal priming agents in heterologous prime-boost vaccination settings.

  • Replication-competent NYVAC-KC yields improved immunogenicity to HIV-1 antigens in rhesus macaques, compared to non-replicating NYVAC [Vaccines and Antiviral Agents]

  • As part of the continuing effort to develop an effective HIV vaccine, we generated a poxviral vaccine vector (previously described) designed to improve on the results of the RV144 Phase III clinical trial. The construct, NYVAC-KC, is a replication-competent, attenuated recombinant of the vaccinia virus strain, NYVAC. NYVAC is a vector that has been used in many previous clinical studies but is replication deficient. Here we report a side-by-side comparison of replication-restricted NYVAC and replication-competent NYVAC-KC in a non-human primate study, which utilized a prime-boost regimen similar to that of RV144. NYVAC-C and NYVAC-C-KC express the HIV-1 antigens gp140 and Gag/Gag-Pol-Nef-derived VLPs from clade C and were used as the prime, with recombinant virus plus envelope protein as the boost. In nearly every T and B cell immune assay against HIV-1, including neutralization and antibody binding, NYVAC-C-KC induced a greater immune response than did NYVAC-C, indicating that replication competence in a poxvirus may improve upon the modestly successful regimen used in the RV144 clinical trial.

    Importance

    Though the RV144 Phase III clinical trial showed promise that an effective vaccine against HIV-1 is possible, a successful vaccine will require improvement over the vaccine candidate (ALVAC) used in the RV144 study. With that goal in mind, we have tested in non-human primates an attenuated, but replication-competent vector, NYVAC-KC, in direct comparison to its parental vector, NYVAC, which is replication-restricted in human cells, similar to the ALVAC vector used in RV144. We have utilized a prime-boost regimen for administration of the vaccine candidate that is similar to the one used in the RV144 study. The results of this study indicate that a replication-competent poxvirus vector may improve upon the effectiveness of the RV144 clinical trial vaccine candidate.

  • The Plant Non-canonical Antiviral Resistance Protein JAX1 Inhibits Potexviral Replication by Targeting the Viral RNA-dependent RNA Polymerase [Virus-Cell Interactions]

  • Understanding the innate immune mechanisms of plants is necessary for breeding of disease-resistant lines. Previously, we identified the antiviral resistance gene JAX1 from Arabidopsis thaliana, which inhibits infection by potexviruses. JAX1 encodes a unique jacalin-type lectin protein. In this study, we analyzed the molecular mechanisms of JAX1-mediated resistance. JAX1 restricted the multiplication of a potexviral replicon lacking movement-associated proteins, suggesting inhibition of viral replication. Therefore, we developed an in vitro potato virus X (PVX) translation/replication system using vacuole- and nucleus-free lysates from tobacco protoplasts, and revealed that JAX1 inhibits viral RNA synthesis but not translation of the viral RNA-dependent RNA polymerase (RdRp). JAX1 did not affect the replication of a resistance-breaking mutant of PVX. Blue native-polyacrylamide gel electrophoresis of fractions separated by sucrose gradient sedimentation showed that PVX RdRp constituted the high-molecular-weight complex that seems to be crucial for viral replication. JAX1 was detected in this complex of wild-type PVX replicon, but not in that of the resistance-breaking mutant. In addition, JAX1 interacted with the RdRp of the wild-type virus, but not with that of virus with a point mutation at the resistance-breaking residue. These results suggest that JAX1 targets RdRp to inhibit potexvirus replication.

    IMPORTANCE Resistance genes play a crucial role in plant antiviral innate immunity. The roles of conventional nucleotide binding-leucine rich repeat (NLR) proteins and the associated defense pathways have long been studied. In contrast, recently discovered resistance genes that do not encode NLR proteins (non-NLR resistance genes) have not been investigated extensively. Here we report that the non-NLR resistance factor JAX1, a unique jacalin-type lectin protein, inhibits potexviral de novo RNA synthesis by targeting the huge complex of viral replicase. This is unlike other known antiviral resistance mechanisms. Molecular elucidation of the target in lectin-type protein-mediated antiviral immunity will enhance our understanding of the plant non-NLR-mediated resistance system.

  • An HIV-1 broadly neutralizing antibody from a clade C infected pediatric elite neutralizer potently neutralizes the contemporaneous and autologous evolving viruses [Vaccines and Antiviral Agents]

  • Broadly neutralizing antibodies (bNAbs) have demonstrated protective effects against HIV-1 in primate studies and recent human clinical trials. Elite-neutralizers are potential candidates for isolation of HIV-1 bNAbs. The coexistence of bNAbs such as BG18 with neutralization susceptible autologous viruses in an HIV-1 infected adult elite controller has been suggested to control viremia. Disease progression is faster in HIV-1 infected children than adults. Plasma bNAbs with multiple epitope specificities are developed in HIV-1 chronically infected children with more potency and breadth than in adults. Therefore, we evaluated the specificity of plasma neutralizing antibodies of an antiretroviral naïve HIV-1 clade C chronically infected pediatric elite neutralizer AIIMS_330. The plasma antibodies showed broad and potent HIV-1 neutralizing activity with ggt;87% (29/33) breadth, median inhibitory dilution (ID50) value of 1246 and presence of N160 and N332-supersite dependent HIV-1 bNAbs. The sorting of BG505.SOSIP.664.C2 T332N gp140 HIV-1 antigen-specific single B cells of AIIMS_330 resulted in the isolation of an HIV-1 N332-supersite dependent bNAb AIIMS-P01. The AIIMS-P01 neutralized 67% of HIV-1 cross-clade viruses; exhibited substantial indels despite limited somatic hypermutations; interacted with native-like HIV-1 trimer as observed in negative stain electron microscopy and demonstrated high binding affinity. In addition, AIIMS-P01 neutralized the coexisting and evolving autologous viruses suggesting the coexistence of vulnerable autologous viruses and HIV-1 bNAbs in AIIMS_330 pediatric elite neutralizer. Such pediatric elite-neutralizers can serve as potential candidates for isolation of novel HIV-1 pediatric bNAbs and for understanding the coevolution of virus and host immune response.

    Importance

    More than 50% of the HIV-1 infections globally are caused by clade C viruses. Till date, there is no effective vaccine to prevent HIV-1 infection. Based on the structural information of the currently available HIV-1 bNAbs, attempts are underway to design immunogens that can elicit correlates of protection upon vaccination. Here we report the isolation and characterization of an HIV-1 N332-supersite dependent bNAb AIIMS-P01 from a clade C chronically infected pediatric elite neutralizer. The N332-supersite is an important epitope and is one of the current HIV-1 vaccine targets. AIIMS-P01 potently neutralized the contemporaneous and autologous evolving viruses and exhibits substantial indels despite low somatic hypermutations. Taken together with the information on infant bNAbs, further isolation and characterization of bNAbs contributing to the plasma breadth in HIV-1 chronically infected children may help to better understand their role in controlling HIV-1 infection.

  • A measles virus-based vaccine candidate mediates protection against Zika virus in an allogenic mouse pregnancy model [Vaccines and Antiviral Agents]

  • The impact of the Zika virus (ZIKV) epidemic highlights the need for vaccines that reduce or prevent infection and reliably prevent teratogenic complications. The live-attenuated measles virus (MV) vaccine strains are a promising vaccine platform since they induce robust humoral and cellular immune responses against additional antigens and have an excellent safety record. To explore its potential to protect against ZIKV, we compared a recombinant Schwarz strain MV that encodes ZIKV prM and soluble E proteins (MV-Zika-sE) with a prototypic alum-adjuvanted whole-inactivated ZIKV particle vaccine. Analysis of MV-Zika-sE-infected cells confirmed antigen expression, and the virus replicated with vaccine strain characteristics. Immunized IFNAR-/--CD46Ge mice developed E protein-specific and neutralizing antibodies, and ZIKV E-specific cellular immune responses were observed by IFN- ELISpot and in vitro T cell proliferation assays. To analyze protective efficacy, vaccinated female mice were challenged with ZIKV after allogenic mating. In MV-Zika-sE-vaccinated mice, weight gain was similar to uninfected mice, while no plasma viremia was detectable in the majority of the animals. In contrast, infected control animals gained less weight and experienced about 100-fold higher viremia over at least 3 days. Moreover, vaccination with MV-Zika-sE reduced ZIKV load in different organs, the placentas, and prevented infection of the fetus. Consequently, no fetal growth retardation, anemia, and death due to ZIKV infection were seen in MV-Zika-sE-vaccinated dams. In contrast, the inactivated ZIKV vaccine had little to no effect in our studies. Therefore, the MV-derived ZIKV vaccine is a promising candidate for further preclinical and clinical development.

    IMPORTANCE Zika virus (ZIKV) is a mosquito-borne flavivirus, which causes a variety of neurological complications, including congenital birth defects. Despite the urgent need, no ZIKV vaccine is licensed yet. Recombinant vaccine strain-derived measles viruses (MV) constitute a promising vector platform to induce immunity against foreign pathogens by expressing antigens from additional transcription units, while at the same time possessing a remarkable safety profile. This concept has already been validated against different pathogens including at least 3 other flaviviruses, and our data illustrate that vaccination with the MV expressing soluble ZIKV E protein significantly diminishes infection and prevents fetal loss or damage in an allogenic mouse pregnancy model. It can thus be regarded as a promising emergency vaccine candidate with the potential for inclusion in routine vaccination settings in endemic areas to prevent teratogenic effects of circulating ZIKV during pregnancy, comparable to standard rubella vaccination.

  • RNA sequencing of the in vivo human herpesvirus 6B transcriptome to identify targets for clinical assays distinguishing between latent and active infections [Genome Replication and Regulation of Viral Gene Expression]

  • Human herpesvirus 6B (HHV-6B) DNA is frequently detected in human samples. Diagnostic assays distinguishing HHV-6B reactivation from latency are limited. This has impaired strategies to diagnose and treat HHV-6B-associated diseases. We used RNA sequencing to characterize and compare the HHV-6B transcriptome in multiple sample types, including 1) whole blood from hematopoietic cell transplant (HCT) recipients with and without HHV-6B plasma viremia; 2) tumor tissue samples from subjects with large B cell lymphoma infected with HHV-6B; 3) lymphoblastoid cell lines (LCLs) from subjects with inherited chromosomally integrated HHV-6B or latent infection with HHV-6B; and 4) HHV-6B Z29 infected SupT1 CD4+ T cells. We demonstrated substantial overlap in the HHV-6B transcriptome observed in in vivo and in vitro samples, although there was variability in the breadth and quantity of gene expression across samples. The HHV-6B viral polymerase gene U38 was the only HHV-6B transcript detected in all RNA-seq data sets and was one of the most highly expressed genes. We developed a novel reverse transcription PCR assay targeting HHV-6B U38, which identified U38 messenger RNA in all tested whole blood samples from patients with concurrent HHV-6B viremia. No HHV-6B U38 transcripts were detected by RNA-seq or RT-qPCR in whole blood samples from subjects without HHV-6B plasma detection or from latently infected LCLs. A RT-qPCR assay for HHV-6B U38 may be useful to identify lytic HHV-6B infection in non-plasma samples and samples from individuals with inherited chromosomally integrated HHV-6B. This study also demonstrates the feasibility of transcriptomic analyses in HCT recipients.

    IMPORTANCE Human herpesvirus 6B (HHV-6B) is a DNA virus that infects most children within the first few years of life. After primary infection, HHV-6B persists as a chronic, latent infection in many cell types. Additionally, HHV-6B can integrate into germline chromosomes, resulting in individuals with viral DNA in every nucleated cell. Given that PCR to detect viral DNA is the mainstay for diagnosing HHV-6B infection, the characteristics of HHV-6B infection complicate efforts to distinguish between latent and active viral infection, particularly in immunocompromised patients who have frequent HHV-6B reactivation. In this study, we used RNA sequencing to characterize the HHV-6B gene expression profile in multiple sample types, and our findings identified evidence-based targets for diagnostic tests that distinguish between latent and active viral infection.

  • The Dietary Restriction-Like (drl-1) Gene, a Putative Serine/Threonine Kinase, is Essential for Orsay Virus Infection in Caenorhabditis elegans [Virus-Cell Interactions]

  • Orsay virus is the only known natural virus pathogen of Caenorhabditis elegans, and its discovery has enabled virus-host interactions studies in this model organism. Host genes required for viral infection remain understudied. We previously established a forward genetic screen based on a virus-inducible GFP transcriptional reporter to identify novel host factors essential for virus infection. Here we report the essential role in Orsay virus infection of the Dietary Restriction-Like (drl-1) gene, which encodes a serine/threonine kinase similar to the mammalian MEKK3 kinase. Ablation of drl-1 led to a ggt;10,000-fold reduction in Orsay virus RNA levels, which could be rescued by ectopic expression of DRL-1. DRL-1 was dispensable for Orsay replication from an endogenous transgene replicon, suggesting that DRL-1 affects a pre-replication stage of the Orsay life cycle. Thus, this study demonstrates the power of C. elegans as a model to identify novel virus-host interactions essential for virus infection.

    IMPORTANCE The recent discovery of Orsay virus, the only known natural virus of Caenorhabditis elegans, provides a unique opportunity to study virus-host interactions that mediate infection in a genetically tractable multicellular model organism. As viruses remain a global threat to human health, better insights into cellular components that enable virus infection and replication can ultimately lead to the development of new targets for antiviral therapeutics.

  • Infection of cell lines derived from diffuse large B-cell lymphoma (DLBCL) with the Epstein-Barr virus (EBV) alters the miRNA loading of the Ago2-complex [Transformation and Oncogenesis]

  • Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoid tumor which is occasionally Epstein-Barr virus (EBV)-positive and is further subtyped as activated B-cell (ABC) and germinal center B-cell (GCB) DLBCL, which has implications for prognosis and treatment.

    We performed Ago2-RNA immunoprecipitation followed by high throughput RNA sequencing (Ago2-RIP-Seq) to capture functionally active miRNAs in EBV-negative ABC-DLBCL and GC-DLBCL cell lines and their EBV-infected counterparts. In parallel, total miRNomes of these cells were sequenced to capture the cellular miRNA profile for comparison with the functionally active profile. Selected miRNAs with differential abundance were validated using RT-qPCR and Northern Blot. We found 6 miRNAs with differential abundance (2 upregulated and 4 downregulated miRNAs) between EBV-neg. and pos. ABC-DLBCL, and 12 miRNAs with differential abundance (3 upregulated and 9 downregulated miRNAs) between EBV-neg and -pos GC-DLBCL. Eight and twelve miRNAs were confirmed using RT-qPCR in ABC-DLBCL and GC-DLBCL, respectively. Selected miRNs were analyzed in additional type I/II vs. type III EBV latency DLBCL cell lines. Furthermore, up regulation of miR-221-3p and down regulation of let-7c-5p in ABC-DLBCL and up regulation of miR-363-3p and down regulation of 423-5p in GC-DLBCL was verified using RIP-Northern blot.

    Our comprehensive sequence analysis of the DLBCL miRNomes identified sets of deregulated miRNAs in the Ago2-RIP-seq. Our Ago2-IP-seq miRNomes profile could be considered as an important data set for detection of deregulated functionally active miRNAs in DLBCL and could possibly lead to identification of miRNAs as biomarkers for classification of DLBCL or even as targets for personalized targeted treatment.

    Importance: Diffuse large B-cell lymphoma (DLBCL) is a highly aggressive tumor of lymphoid origin which is occasionally Epstein-Barr virus (EBV)-positive. MicroRNAs are found in most multicellular organisms and even in viruses such as EBV. They regulate the synthesis of proteins by binding to their cognate messenger RNA (mRNA). MicroRNAs are tethered to their target mRNAs by "Argonaute" proteins. Here we compared the overall content of by differential loading of the Ago2-complex in comparison to the overall content of miRNAs in two DLBCL cell lines and their EBV-converted counterparts. In all cell lines, the Ago2-load was different from the overall expression of miRNAs. In addition, the loading of the Ago2-complex was changed upon infection with EBV. This indicates that the virus changes not only the overall content of miRNAs but also influences the expression of proteins by affecting the Ago-complexes.

  • The endosomal Toll-like receptors 7 and 9 cooperate in detection of MHV68 infection [Pathogenesis and Immunity]

  • Murine gammaherpesvirus 68 (MHV68) is an amenable small animal model for study of the human pathogens Epstein-Barr virus and Kaposirrsquo;s sarcoma-associated herpesvirus. Here, we have characterized the roles of the endosomal TLR escort protein UNC93B, endosomal TLR7, 9, and 13, and cell surface TLR2 in MHV68 detection. We found that the interferon aalpha; (IFNaalpha;) response of plasmacytoid dendritic cells (pDC) to MHV68 was reduced in Tlr9-/- cells compared to wildtype (WT), but not completely lost. Tlr7-/- pDC responded similarly to WT. However, we found that in Unc93b-/- pDC, as well as in Tlr7/Tlr9-/- double knockout pDC, the IFNaalpha; response to MHV68 was completely abolished. Thus, the only pattern recognition receptors contributing to the IFNaalpha; response to MHV68 in pDC are TLR7 and TLR9, but the contribution of TLR7 is masked by the presence of TLR9. To address the role of UNC93B and TLR for MHV68 infection in vivo, we infected mice with MHV68. Lytic replication of MHV68 after intravenous infection was enhanced in the lungs, spleen, and liver of UNC93B-deficient mice, in the spleen of TLR9-deficient mice, and in the liver and spleen of Tlr7/Tlr9-/- mice. The absence of TLR2 or TLR13 did not affect lytic viral titers. We then compared reactivation of MHV68 from latently infected WT, Unc93b-/-, Tlr7/Tlr9-/-, Tlr7-/-, and Tlr9-/- splenocytes. We observed enhanced reactivation and latent viral loads, particularly from Tlr7/Tlr9-/- splenocytes, compared to WT. Our data show that UNC93B-dependent TLR7 and TLR9 cooperate in and contribute to detection and control of MHV68 infection.

    IMPORTANCE STATEMENT The two human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), can cause aggressive forms of cancer. These herpesviruses are strictly host specific and therefore their murine homolog murid gammaherpesvirus 4 strain 68 (MHV68) is a widely used model to obtain in vivo insights into the interaction between these two gammaherpesviruses and their host. Like EBV and KSHV, MHV68 establishes lifelong latency in B cells. The innate immune system serves as one of the first lines of host defense, with pattern recognition receptors such as the Toll-like receptors playing a crucial role in mounting a potent antiviral immune response to various pathogens. Here we shed light on a yet unanticipated role of Toll-like receptor 7 in the recognition of MHV68 in a subset of immune cells called plasmacytoid dendritic cells, as well as the control of this virus in its host.

  • Enterovirus-A71 Infection Activates Human Immune Responses and Induces Pathological Changes in Humanized Mice [Pathogenesis and Immunity]

  • Since the discovery of enterovirus-A71 (EV-A71) half a century ago, it has caused large-scale outbreaks of hand-foot-and-mouth disease worldwide, in particular in the Asia-Pacific region, causing a great concern of public health and economic burden. Detailed mechanisms on the modulation of immune responses after EV-A71 infection have not been fully known, and the lack of appropriate models hinders the development of promising vaccines and drugs. In the present study, NOD-scid IL2R-/- (NSG) mice with human immune system (humanized mice) at the age of four-week-old were found to be susceptible to a human isolate of EV-A71 infection. After infection, humanized mice displayed limb weakness, which is similar to the clinical features found in some of the EV-A71-infected patients. Histopathological examination indicated the presence of vacuolation, gliosis or meningomyelitis in brain stem and spinal cord, which were accompanied by high viral loads detected in these organs. The numbers of activated human CD4+ and CD8+ T cells were upregulated after EV-A71 infection and EV-A71-specific human T cell responses were found. Furthermore, the secretion of several proinflammatory cytokines, such as human IFN-, IL-8 and IL-17A was elevated in the EV-A71-infected humanized mice. Taken together, our results suggested that the humanized mouse model permits insights into the human immune responses and the pathogenesis of EV-A71 infection, which may provide a platform for the evaluation of anti-EV-A71 drug candidates in the future.

    IMPORTANCE Despite causing self-limited hand-food-and-mouth disease in younger children, EV-A71 is consistently associated with severe forms of neurological complications and pulmonary edema. Nevertheless, only limited vaccines and drugs have been developed over the years, which is possibly due to a lack of models that can more accurately recapitulate human specificity, since human is the only natural host for wild type EV-A71 infection. Our humanized mouse model did not only mimic histological symptoms in patients, but also allows us to investigate the function of human immune system during the infection. It was found that human T cell responses were activated, accompanied by an increase in the production of proinflammatory cytokines in EV-A71-infected humanized mice, which might contribute to the exacerbation of disease pathogenesis. Collectively, this model allows us to delineate the modulation of human immune responses during EV-A71 infection and may provide a platform to evaluate anti-EV-A71 drug candidates in the future.

  • Elicitation of protective antibodies against 20 years of future H3N2 co-ciruculating influenza virus variants in ferrets preimmune to historical H3N2 influenza viruses. [Vaccines and Antiviral Agents]

  • The vast majority of people already have pre-existing immune responses to influenza viruses from one or more subtypes. However, almost all preclinical studies evaluate new influenza vaccine candidates in immunologically naïve animals. Recently, our group demonstrated that priming naive ferrets with broadly reactive H1 COBRA HA based vaccines boosted pre-existing antibodies induced by wild-type H1N1 virus infections. These H1 COBRA HA antigens induced antibodies with HAI activity against multiple antigenically different H1N1 viral variants. In this study, ferrets, preimmune to historical H3N2 viruses, were vaccinated with virus-like particle (VLP) vaccines expressing either an HA from a wild-type H3 influenza virus or a COBRA H3 HA antigen (T6, T7, T10, or T11). The elicited antisera had the ability to neutralize virus infection against a panel of viruses representing vaccine strains selected by the World Health Organization (WHO), or a set of viral variants that co-circulated during the same time period. Preimmune animals vaccinated with H3 COBRA T10 HA antigen elicited sera with higher HAI antibody titers than antisera elicited by VLP vaccines with wild-type HA VLPs in preimmune ferrets. However, while the T11 COBRA vaccine did not elicit HAI activity, the elicited antibodies did neutralize antigenically distinct H3N2 influenza viruses. Overall, H3 COBRA-based HA vaccines were able to neutralize both historical H3 and comtemporary, as well as future H3N2 viruses with higher titers than vaccines with wild-type H3 HA antigens. This is the first report demonstrating the effectiveness of a broadly reactive H3N3 vaccine in a preimmune ferret model.

    IMPORTANCE Following influenza virus exposure, the host generates neutralizing anti-hemagglutinin antibodies against that specific infecting influenza strain. These antibodies can also neutralize some, but not all, co-circulating strains. The goal of next generation influenza vaccines, such as HA head-based COBRA, is to stimulate broadly protective neutralizing antibodies against all strains circulating within a subtype, in particular those that persist over multiple influenza seasons, without requiring an update to the vaccine. To mimic the human condition, COBRA HA virus-like particle vaccines were tested in ferrets that were previously exposed to historical H3N2 influenza viruses. In this model, these vaccines elicited broadly protective antibodies that neutralized co-circulating H3N2 influenza viruses isolated over a 20-year period. This is the first study to show the effectiveness of H3N3 COBRA HA vaccines in a host with pre-existing immunity to influenza.

  • Infection and functional modulation of human monocytes and macrophages by varicella zoster virus. [Virus-Cell Interactions]

  • Varicella zoster virus (VZV) is associated with viraemia during primary infection that is presumed to stem from infection of circulating immune cells. Whilst VZV has been shown to be capable of infecting a number of different subsets of circulating immune cells such as T cells, dendritic cells and NK cells, less is known about the interaction between VZV and monocytes. Here, we demonstrate that blood derived human monocytes are permissive VZV replication in vitro. VZV infected monocytes exhibited each temporal class of VZV gene expression as evidenced by immunofluorescent staining. VZV virions were observed on the cell-surface and viral nucleocapsids were observed in the nucleus of VZV infected monocytes by scanning electron microscopy. In addition, VZV infected monocytes were able to transfer infectious virus to human fibroblasts. Infected monocytes displayed impaired dextran-mediated endocytosis and cell surface immunophenotyping revealed downregulation of CD14, HLA-DR, CD11b and the M-CSF receptor. Analysis of the impact of VZV infection on M-CSF stimulated monocyte to macrophage differentiation demonstrated loss of cell viability, indicating that VZV infected monocytes were unable to differentiate into viable macrophages. In contrast, macrophages differentiated from monocytes prior to exposure to VZV were highly permissive to infection. This study defines the permissiveness of these myeloid cell types to productive VZV infection and identifies functional impairment of VZV infected monocytes.

    IMPORTANCE Primary varicella infection results in widespread dissemination of the virus throughout the host. Viral transportation is known to be directly influenced by susceptible immune cells in the circulation. Moreover, infection of immune cells by VZV results in attenuation of the anti-viral mechanisms used to control infection and limit spread. Here, we provide evidence that human monocytes, which are highly abundant in the circulation, are permissive to productive VZV infection. Furthermore, monocyte derived macrophages were also highly permissive to VZV infection, although VZV infected monocytes are unable to differentiate into macrophages. Exploring the relationships between VZV and permissive immune cells such as human monocytes and macrophages elucidates novel immune evasion strategies and provides further insight into the control VZV has over the immune system.

  • HSV-1 ICP22 suppresses CD80 expression by murine dendritic cells [Cellular Response to Infection]

  • Herpes simplex virus type-1 (HSV-1) has the ability to delay its clearance from the eye during ocular infection. Here we show that ocular infection of mice with HSV-1 suppresses expression of the costimulatory molecule CD80, but not CD86, in the cornea. The presence of neutralizing anti-HSV-1 antibodies did not alleviate this suppression. At the cellular level, HSV-1 consistently downregulated the expression of CD80 by dendritic cells but not other antigen-presenting cells. Furthermore, flow cytometric analysis of HSV-1 infected corneal cells during a 7-day period reduced CD80 expression in dendritic cells (DCs) but not B cell, macrophages or monocytes. This suppression was associated with the presence of virus. Similar results were obtained using infected or transfected spleen cells or bone-marrow derive DCs. A combination of roscovitine treatment, transfection with immediate early genes (IE) and infection with a recombinant HSV-1 lacking ICP22 gene shown the importance of ICP22 in down-regulation of CD80 promoter but not CD86 promoter in vitro and in vivo. At the mechanistic level, we show that the HSV-1 immediate-early gene, ICP22 binds the CD80 promoter and that this interaction is required for HSV-1-mediated suppression of CD80 expression. Conversely, forced expression of CD80 by ocular infection of mice with a recombinant HSV-1 exacerbated corneal scarring in infected mice. Taken together these studies identify ICP22 mediated suppression of CD80 expression in dendritic cells as central to delayed clearance of the virus and limitation of the cytopathological response to primary infection in the eye.

    IMPORTANCE HSV-1-induced eye disease is a major public health problem. Eye disease is associated closely with immune responses to the virus and is exacerbated by delayed clearance of the primary infection. The immune system relies on antigen-presenting cells of the innate immune system to activate T cell response. We found that HSV-1 utilizes a robust and finely targeted mechanism of local immune evasion. It downregulates the expression of the costimulatory molecule CD80 but not CD86, on resident dendritic cells irrespective of the presence of anti-HSV-1 antibodies. The effect is mediated by direct binding of HSV-1 ICP22, the product of an immediate early gene of HSV-1, to the promoter of CD80. This immune evasion mechanism dampens the host immune response and thus reduces eye disease in ocularly infected mice. Therefore, ICP22 may be a novel inhibitor of CD80 that could be used to modulate immune response.

  • Cell-to-cell transmission is the main mechanism supporting bovine viral diarrhea virus spread in cell culture [Virus-Cell Interactions]

  • After initiation of an infective cycle, spread of virus infection can occur fundamentally in two different ways: (i) viral particles can be released into the external environment and diffuse through the extracellular space until they interact with a new host cell, or (ii) virions can remain associated with infected cells promoting the direct passage between infected and uninfected cells that is referred to as direct cell-to-cell transmission. Although evidence for cell-associated transmission has accumulated for many different viruses, the ability of members of the genus pestivirus to use this mode of transmission has not been reported. Here we used a novel recombinant virus expressing the envelope glycoprotein E2 fused to mCherry fluorescent protein to monitor the spreading of bovine viral diarrhea virus (BVDV, the type member of pestiviuses) infection. To demonstrate direct cell-to-cell transmission of BVDV we developed a cell co-culture system that allowed us to prove the direct transmission from infected to uninfected cells in the presence of neutralizing antibodies. This mode of transmission requires cell-cell contacts and clathrin-mediated receptor-dependent endocytosis. Notably, it overcomes antibody blocking of the BVDV receptor CD46 indicating that cell-to-cell transmission of the virus involves the engagement of co-receptors on the target cell.

    Importance. BVDV causes one of the economically most important viral infections for the cattle industry. The virus is able to cross the placenta and infect the fetus leading to the birth of persistently infected animals, which are reservoirs for the spread of BVDV. The occurrence of persistent infection has hampered the efficacy of vaccination because it requires eliciting levels of protection close to sterilizing immunity to prevent fetal infections. While vaccination prevents disease, BVDV can be detected when animals with neutralizing antibodies are challenged with the virus. Virus cell-to-cell transmission allows overcoming barriers imposed to free virus dissemination such as antibodies or epithelial barriers. Here, we show for the first time that BVDV exploits cell-cell contacts to propagate infection in a process that is resistant to antibody neutralization. Our results provide new insights into the mechanisms underlying the pathogenesis of BVDV infection and can aid the design of effective control strategies.

  • Selective Editing of HSV-1 Enables IFN Induction and Viral Replication That Destruct Malignant Cells [Virus-Cell Interactions]

  • Oncolytic herpes simplex virus 1 (HSV-1), devoid of the 134.5 gene, exerts antitumor activities. However, the oncolytic effect differs, ranging from pronounced to little responses. Although viral and host factors are involved, much remains to be deciphered. Here we report that engineered HSV-1 (N146), bearing amino acids 147-263 of 134.5, replicates competently in and lyses malignant cells refractory to the 134.5 null mutant. Upon infection, N146 precludes phosphorylation of translation initiation factor eIF2aalpha;, ensuring viral protein synthesis. On the other hand, N146 activates interferon regulatory factor 3 and IFN expression, known to prime immunity against virus and tumor. Nevertheless, N146 exhibits sustained replication even exposed to exogenous IFN-aalpha;. In a 4T1 tumor model, N146 markedly reduces tumor growth and metastasis formation. This coincides with viral replication or T cell infiltration in primary tumors. N146 is undetectable in normal tissues in vivo. Herein, targeted HSV-1 editing results in a unique anti-neoplastic agent that enables inflammation without major interference of viral growth within tumor cells.

    IMPORTANCE Oncolytic herpes simplex virus 1 is a promising agent for cancer immunotherapy. Due to a complex virus-host interaction, less is clear about what viral signature(s) constitutes a potent oncolytic backbone. Through molecular or genetic dissection, we show that selective editing of the 134.5 gene enables viral replication in malignant cells, activation of transcription factor IRF3 and subsequent induction of type I IFN. This translates into profoundly reduced primary tumor growth and metastasis burden in an aggressive breast carcinoma model in vivo. Our work reveals a distinct oncolytic platform that is amendable for further development.

  • The Deletion of Both Tyrosine-Based Endocytosis Signal and Endoplasmic Reticulum-Retrieval Signal in the Cytoplasmic Tail of Spike Protein Attenuates PEDV in Pigs [Pathogenesis and Immunity]

  • Porcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets. The PEDV spike (S) protein contains two intracellular sorting motifs Yxx (tyrosine-based motif YEVF or YEAF) and KVHVQ at the cytoplasmic tail, yet their functions have not been fully elucidated. Some Vero cell-adapted and/or attenuated PEDV variants contain ablations in these two motifs. We hypothesized that these motifs contribute to viral pathogenicity. By transiently expressing PEDV S proteins with mutations in the motifs, we confirmed that the motif KVHVQ is involved in retention of the S proteins in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). In addition, we identified that the Yxx motif triggers endocytosis of S proteins. These two motifs synergistically regulate the level of S expressed on the cell surface. To investigate their role in viral pathogenicity, we generated three recombinant PEDVs by introducing deletions or a mutation in the two motifs of the infectious clone of PEDV PC22A strain (icPC22A): 1) ic10aa (YxxEKVHVQ); 2) ic5aa (KVHVQ); and 3) icYA (Y1378A, to an inactivated motif AEVF). Infection of Vero cells with ic10aa resulted in larger syncytia and more virions with reduced number of S protein projections on the surface compared with icPC22A. Furthermore, we orally inoculated five groups of 5-day-old gnotobiotic piglets with the three mutants, icPC22A, or mock. The mutant ic10aa caused less severe diarrhea rate and significantly milder intestinal lesions than icPC22A, ic5aa and icYA. These data suggest that the deletion of both motifs can reduce the virulence of PEDV in piglets.

    IMPORTANCE Many coronaviruses (CoVs) possess conserved motifs Yxx and/or KxHxx/KKxx in the cytoplasmic tail of the S protein. The KxHxx/KKxx motif has been identified as the ER-retrieval signal, but the function of the Yxx motif in the intracellular sorting of CoV S proteins remains controversial. In this study, we showed that the Yxx of PEDV S protein is an endocytosis signal. Furthermore, using reverse genetics technology, we evaluated its role in PEDV pathogenicity in neonatal piglets. Our results explain one attenuation mechanism of Vero cell-adapted PEDV variants lacking functional Yxx and KVHVQ motifs. Knowledge from this study may aid in the design of efficacious live attenuated vaccines against PEDV, as well as other CoVs bearing the same motif in their S protein.

  • HIV-1 antisense protein of different clades induces autophagy and associates to the autophagy factor p62 [Virus-Cell Interactions]

  • The existence of the antisense transcript-encoded HIV-1 Antisense Protein (ASP) was recently reinforced by in silico analyses providing evidence for recent appearance of this gene in the viral genome. Our previous studies led to the detection of ASP in various cell lines by Western blot, flow cytometry and confocal microscopy analyses and reported that it induced autophagy, potentially through multimer formation. Herein, our goals were to assess autophagy induction by ASP from different clades, and to identify the implicated autophagy factors. We first demonstrated that ASP formed multimers, partly through its amino region and cysteine residues. Removal of this region was further associated with lower induction of autophagy, as assessed by autophagosome formation. ASP from different clades (A, B, C, D, and G) were next tested and were detected in monomeric and multimeric forms at various levels and all induced autophagy (clade A ASP being less efficient), as determined by LC3-II and p62 (SQSTM1) levels. Furthermore, CRISPR-based knock-out of ATG5, ATG7 and p62 genes led to increased ASP levels. Confocal microscopy analyses showed that ASP co-localized with p62 and LC3-II in autophagosome-like structures. Co-immunoprecipitation experiments further demonstrated that p62 associated with ASP through its PB1 domain. Interestingly, immunoprecipitation experiments supported that ASP is ubiquitinated and that ubiquitination was modulating its stability. We are thus suggesting that ASP induces autophagy through p62 interaction and that its abundance is controlled by autophagy, in which ubiquitin plays an important role. Understanding the mechanisms underlying ASP degradation is essential to better assess its function.

    IMPORTANCE In the present study, we provide the first evidence that a new HIV-1 protein termed ASP derived from different clades acts similarly in inducing autophagy, an important cellular process implicated in the degradation of excess or defective cellular material. We have gained further knowledge on the mechanism mediating the activation of autophagy. Our studies have important ramification in the understanding of viral replication and the pathogenesis associated with HIV-1 in infected individuals. Indeed, autophagy is implicated in antigen presentation during immune response and could thus be rendered inefficient in infected cells, such as dendritic cells. Furthermore, a possible link with HIV-1-associated Neurological Disorder (HAND) might also be a possible association with the capacity of ASP to induce autophagy. Our studies hence demonstrate the importance in conducting further studies on this protein, as it could represent a new interesting target for antiretroviral therapies and vaccine design.

  • Fatty acid synthase promotes the palmitoylation of Chikungunya virus nsP1 [Virus-Cell Interactions]

  • Chikungunya virus (CHIKV) is transmitted to people by mosquitoes, and CHIKV infection causes fever and joint pain. Fatty acid synthase (FASN) has been identified as a proviral factor for CHIKV. How FASN participates in CHIKV replication remains to be elucidated. In this study, we demonstrated that palmitic acid (PA) can restore the suppression of CHIKV replication by FASN inhibitors. The palmitoylation and plasma membrane localization of CHIKV nsP1 were reduced by FASN inhibitors. Triple mutation of Cys417, Cys418, and Cys419 in nsP1 blocked its palmitoylation and severely disrupted CHIKV replication. Furthermore, two zinc-finger D-H-H-C domain-containing palmitoyltransferases (ZDHHCs) including ZDHHC2 and ZDHHC19 promoted nsP1 palmitoylation and CHIKV replication. Our results not only identified the key enzymes for the palmitoylation of nsP1 but also provided mechanistic insights into the roles of FASN in CHIKV replication.

    Importance S-Palmitoylation is an important form of lipid posttranslational modification, which affects the function of proteins by regulating their transport, stability and localization. Previous studies have shown that FASN is critical for CHIKV replication; however, the mechanism for this function of FASN remains unknown. The key zinc-finger D-H-H-C domain-containing palmitoyltransferases involved in the palmitoylation of nsP1 are not clear. We demonstrated that FASN promoted CHIKV replication through nsP1 palmitoylation. ZDHHC2 and ZDHHC19 were identified as the major enzymes for nsP1 palmitoylation. Since nsP1 proteins are conserved in alphaviruses, our results highlight the mechanisms by which alphavirus nsP1 is palmitoylated.

  • FoxO1 Suppresses Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication and Controls Viral Latency [Virus-Cell Interactions]

  • Kaposirrsquo;s sarcoma-associated herpesvirus (KSHV) has latent and lytic replication phases, both of which contribute to the development of KSHV-induced malignancies. Among numerous factors identified to regulate KSHV life cycle, oxidative stress, caused by imbalanced clearing and production of reactive oxygen species (ROS), has been shown to robustly disrupt KSHV latency and induce viral lytic replication. In this study, we identify an important role of antioxidant defense factor forkhead box protein O1 (FoxO1) in KSHV lifecycle. Either chemical inhibition of the FoxO1 function or knockdown of FoxO1 expression led to the increase of intracellular ROS level that was subsequently sufficient to disrupt KSHV latency and induce viral lytic reactivation. On the other hand, treatment with N-acetyl-L-cysteine (NAC), an oxygen free radical scavenger, led to the reduction of FoxO1 inhibition-induced ROS level and ultimately the attenuation of KSHV lytic reactivation. These findings reveal that FoxO1 plays a critical role in keeping KSHV latency in check by maintaining intracellular redox balance.

    IMPORTANCE Kaposirrsquo;s sarcoma-associated herpesvirus (KSHV) is associated with several cancers including Kaposirrsquo;s sarcoma (KS). Both KSHV latent and lytic replication phases are important for the development of KS. Identification of factors regulating KSHV latent to lytic switch can provide insights into the pathogenesis of KSHV-induced malignancies. In this study, we have shown that antioxidant defense factor forkhead box protein O1 (FoxO1) maintains KSHV latency by suppressing viral lytic replication. Inhibition of FoxO1 disrupts KSHV latency and induces viral lytic replication by increasing intracellular ROS level. Significantly, treatment with an oxygen free radical scavenger N-acetyl-L-cysteine (NAC) attenuates FoxO1 inhibition-induced intracellular ROS level and KSHV lytic replication. Our works reveal a critical role of FoxO1 in suppressing KSHV lytic replication, which could be targeted for antiviral therapy.

  • Development of a primary human cell model for the study of human cytomegalovirus replication and spread within salivary epithelium [Virus-Cell Interactions]

  • Various aspects of human cytomegalovirus (HCMV) pathogenesis including its ability to replicate in specific cells and tissues and the mechanism(s) of horizontal transmission are not well understood, predominantly because of the strict species specificity exhibited by HCMV. Murine CMV (MCMV), which contains numerous gene segments highly similar to that of HCMV, has been useful for modeling some aspects of CMV pathogenesis, however, it remains essential to build relevant human cell-based systems to investigate how the HCMV counterparts function. The salivary gland epithelium is a site of persistence for both human and murine cytomegaloviruses and salivary secretions appear to play an important role in horizontal transmission. Therefore, it is important to understand how HCMV is replicating within the glandular epithelial cells so that it might be possible to therapeutically prevent transmission. In the current study, we describe the development of a salivary epithelial model derived from primary human "salispheres". Initial infection of these primary salivary cells with HCMV occurs in a manner similar to that reported for established epithelial lines, in that gH/gL/UL128/UL130/UL131A (Pentamer) positive strains can infect and replicate, while lab adapted Pentamer null strains do not. However, while HCMV does enter the lytic phase and produce virus in the salivary epithelial cells, it fails to exhibit robust spread throughout the culture and persists in a low percentage of salivary cells. The current study demonstrates the utility of these primary tissue derived cells for studying HCMV replication in salivary epithelial cells in vitro.

    IMPORTANCE Human cytomegalovirus (HCMV) infects the majority of the worldrrsquo;s population and although it typically establishes a quiescent infection with little to no disease in most individuals, the virus is responsible for a variety of devastating sequelae in immunocompromised adults and in developing fetuses. Therefore, identifying the viral properties essential for replication, spread and horizontal transmission are important areas of medical science. Our studies use novel human salivary gland-derived cellular models to investigate the molecular details by which HCMV replicates in salivary epithelial cells and provide insight into the mechanisms by which the virus persists in the salivary epithelium where it gains access to fluids centrally important for horizontal transmission.

  • Interferon-{alpha} Enhances NK Cell Function and the Suppressive Capacity of HIV-specific CD8+ T Cells [Pathogenesis and Immunity]

  • Current shock-and-kill strategies for the eradication of the HIV-1 reservoir have resulted in blips of viremia but not in a decrease in the size of the latent reservoir in patients on suppressive antiretroviral therapy (ART). This discrepancy could potentially be explained by an inability of the immune system to kill HIV-1-infected cells following the reversal of latency. Furthermore, some studies have suggested that certain latency-reversing agents (LRAs) may inhibit CD8+ T cell and NK cell responses. In this study, we tested the hypothesis that IFN-aalpha; could improve the function of NK cells from chronic progressors (CP) on ART. We show here that IFN-aalpha; treatment enhanced cytokine secretion, polyfunctionality, degranulation, and the cytotoxic potential of NK cells from healthy donors (HD) and CP. We also show that this cytokine enhanced the viral suppressive capacity of NK cells from HD and elite controllers or suppressors (ES). Furthermore, IFN-aalpha; enhanced global CP CD8+ T cell cytokine responses and the suppressive capacity of ES CD8+ T cells. Our data suggest that IFN-aalpha; treatment may potentially be used as an immunomodulatory agent in HIV-1 cure strategies.

    Importance: Data suggests that HIV+ individuals unable to control infection fail to do so due to impaired cytokine production and/cytotoxic effector cell function. Consequently, the success of cure agendas such as the shock-and-kill strategy will probably depend on enhancing patient effector cell function. In this regard, NK cells are of particular interest as they complement the function of CD8+ T cells. Here, we demonstrate the ability of short course IFN-aalpha; treatments to effectively enhance such effector functions in chronic progressor NK cells without inhibiting their general CD8+ T cell function. These results point to the possibility of exploring such short course IFN-aalpha; treatments for the enhancement of effector cell function in HIV+ patients in future cure strategies.

  • Sensitivity to Broadly Neutralizing Antibodies of Recently Transmitted HIV-1 Clade CRF02_AG Viruses with a Focus on Evolution over Time. [Vaccines and Antiviral Agents]

  • Broadly neutralizing antibodies (bnAbs) are promising agents for prevention and/or treatment of HIV-1 infection. However, the diversity among HIV-1 envelope (Env) glycoproteins impacts bnAbs potency and breadth. Neutralization data on CRF02_AG clade are scarce although it is highly prevalent in West Africa and Europe. We assessed the sensitivity to bnAbs of a panel of 33 early-transmitted CRF02_AG viruses over a 15-year period of the French epidemic (1997-2012). Env pseudotyped CRF02_AG viruses were best neutralized by the CD4 binding site (CD4bs)-directed bnAbs (VRC01, 3BNC117, NIH45-46G54W and N6) and the gp41 membrane proximal external region (MPER)-directed bnAb 10E8, in terms of both potency and breadth. We observed a higher resistance to bnAbs targeting the V1/V2-glycan region (PG9, PGT145) and the V3-glycan region (PGT121, 10-1074). Combinations were required to achieve a full coverage across this subtype. We observed an increased resistance to bnAbs targeting the CD4bs linked to the diversification of CRF02_AG Env over the course of the epidemic, a phenomenon which was previously described for subtypes B and C. These data on the sensitivity to bnAbs of CRF02_AG viruses, including only recently transmitted viruses, will inform future passive immunization studies. Considering the drift of the HIV-1 species towards higher resistance to neutralizing antibodies, it appears necessary to keep updating existing panels for evaluation of future vaccine and passive immunization studies.

    IMPORTANCE Major progress occurred during the last decade leading to isolation of human monoclonal antibodies, termed broadly neutralizing antibodies (bnAbs) due to their capacity to neutralize various strains of HIV-1. Several clinical trials are on the move in order to evaluate their efficacy in preventive or therapeutic strategies. However, no single bnAb is active against 100% of strains. It is important to gather data on the sensitivity to neutralizing antibodies of all genotypes, especially those more widespread in regions where the prevalence of HIV-1 infection is high. Here we assembled a large panel of clade CRF02_AG viruses, the most frequent genotype circulating in West Africa and the second most frequent in several European countries. We evaluated their sensitivity to bnAbs, including those the most advanced in clinical trials, and looked for the best combinations. In addition, we observed a trend toward increased resistance to bnAbs over the course of the epidemic.

  • Induction and suppression of NF-{kappa}B signalling by a DNA virus of Drosophila [Virus-Cell Interactions]

  • Interactions between the insect immune system and RNA viruses have been extensively studied in Drosophila, where RNA interference, NF-B and JAK-STAT pathways underlie antiviral immunity. In response to RNA interference, insect viruses have convergently evolved suppressors of this pathway that act by diverse mechanisms to permit viral replication. However, interactions between the insect immune system and DNA viruses have received less attention, primarily because few Drosophila-infecting DNA virus isolates are available. Here, we use a recently-isolated DNA virus of Drosophila melanogaster, Kallithea virus (family Nudiviridae), to probe known antiviral immune responses and virus evasion tactics in the context of DNA virus infection. We find that fly mutants for RNA interference and Immune deficiency (Imd), but not Toll, pathways are more susceptible to Kallithea virus infection. We identify the Kallithea virus-encoded protein gp83 as a potent inhibitor of Toll signalling, suggesting that Toll mediates antiviral defense against Kallithea virus infection, but that it is suppressed by the virus. We find that Kallithea virus gp83 inhibits Toll signalling through the regulation of NF-B transcription factors. Furthermore, we find that gp83 of the closely related Drosophila innubila nudivirus (DiNV) suppresses D. melanogaster Toll signalling, suggesting an evolutionary conserved function of Toll in defense against DNA viruses. Together, these results provide a broad description of known antiviral pathways in the context of DNA virus infection and identify the first Toll pathway inhibitor in a Drosophila virus, extending the known diversity of insect virus-encoded immune inhibitors.

    IMPORTANCE Co-evolution of multicellular organisms and their natural viruses may lead to an intricate relationship in which host survival requires effective immunity, and virus survival depends on evasion of such responses. Insect antiviral immunity, and reciprocal virus immune suppression tactics, have been well-studied in Drosophila melanogaster, primarily during RNA, but not DNA, virus infection. Therefore, we describe interactions between a recently-isolated Drosophila DNA virus (Kallithea virus - KV) and immune processes known to control RNA viruses, such as RNAi and Imd pathways. We find that KV suppresses the Toll pathway, and identify gp83 as a KV-encoded protein that underlies this suppression. This immunosuppressive ability is conserved in another nudivirus, suggesting the Toll pathway has conserved antiviral activity against DNA nudiviruses, which have evolved suppressors in response. Together, these results indicate that DNA viruses induce and suppress NF-B responses, and advance the application of KV as a model to study insect immunity.

  • Mutations in the spike protein of MERS-CoV transmitted in Korea increase resistance towards antibody-mediated neutralization [Virus-Cell Interactions]

  • The Middle East respiratory syndrome coronavirus (MERS-CoV) poses a threat to public health. The virus is endemic in the Middle East but can be transmitted to other countries by travel activity. The introduction of MERS-CoV into the Republic of Korea by an infected traveler resulted in a hospital outbreak of MERS that entailed 186 cases and 38 deaths. The MERS-CoV spike (S) protein binds to the cellular protein DPP4 via its receptor binding domain (RBD) and mediates viral entry into target cells. During the MERS outbreak in Korea emergence and spread of viral variants was observed that harbored mutations in the RBD, D510G and I529T. Counterintuitively, these mutations were found to reduce DPP4 binding and viral entry into target cells. Here, we investigated whether they also exerted pro-viral effects. We confirm that changes D510G and I529T reduce S protein binding to DPP4 but show that this reduction only translates into diminished viral entry when expression of DPP4 on target cells is low. Both mutations did not modulate S protein binding to sialic acids, S protein activation by host cell proteases and inhibition of S protein-driven entry by interferon-induced transmembrane proteins. In contrast, changes D510G and I529T increased resistance of S protein-driven entry to neutralization by monoclonal antibodies and serum from a convalescent MERS patient. These findings indicate that MERS-CoV variants with reduced neutralization sensitivity were transmitted during the Korean outbreak and that the responsible mutations were compatible with robust infection of cells expressing high levels of DPP4.

    IMPORTANCE MERS-CoV has pandemic potential and it is important to identify mutations in viral proteins that might augment viral spread. In the course of a large hospital outbreak of MERS in the Republic of Korea in 2015 the spread of a viral variant was observed that contained mutations in the viral spike protein. These mutations were found to reduce receptor binding and viral infectivity. However, it remained unclear whether they also exerted pro-viral effects. We demonstrate that these mutations reduce sensitivity to antibody-mediated neutralization and are compatible with robust infection of target cells expressing high amounts of the viral receptor DPP4.

  • Murine Cytomegalovirus glycoprotein O promotes epithelial cell infection in vivo [Pathogenesis and Immunity]

  • Cytomegaloviruses (CMVs) establish systemic infections across diverse cell types. Glycoproteins that alter tropism can potentially guide their spread. Glycoprotein O (gO) is a non-essential fusion complex component of both human (HCMV) and murine CMV (MCMV). We tested its contribution to MCMV spread from the respiratory tract. In vitro, MCMV lacking gO poorly infected fibroblasts and epithelial cells. Cell binding was intact but penetration was delayed. By contrast myeloid infection was preserved, and in the lungs, where myeloid and type 2 alveolar epithelial cells are the main viral targets, MCMV lacking gO showed a marked preference for myeloid infection. Its poor epithelial cell infection was associated with poor primary virus production and reduced virulence. Systemic spread, which proceeds via infected CD11c+ myeloid cells, was initially intact but then diminished, because less epithelial infection led ultimately to less myeloid infection. Thus, tight linkage between peripheral and systemic MCMV infections gave gO-dependent infection a central role in host colonization.

    IMPORTANCE Human cytomegalovirus is a leading cause of congenital disease. This reflects its capacity for systemic spread. A vaccine is needed, but the best viral targets are unclear. Attention has focussed on the virion membrane fusion complex. It has 2 forms, so we need to know what each contributes to host colonization. One includes the virion glycoprotein O. We used murine cytomegalovirus, which has equivalent fusion complexes, to determine the importance of glycoprotein O after mucosal infection. We show that it drives local virus replication in epithelial cells. It was not required to infect myeloid cells, which establish systemic infection, but poor local replication reduced systemic spread as a secondary effect. Therefore targeting glycoprotein O of human cytomegalovirus has the potential to reduce both local and systemic infections.

  • Reactivation of Simian Varicella Virus (SVV) in Rhesus Macaques After CD4 T Cell Depletion [Pathogenesis and Immunity]

  • Rhesus macaques intrabronchially inoculated with simian varicella virus (SVV), the counterpart of human varicella zoster virus (VZV), developed primary infection with viremia and rash, which resolved upon clearance of viremia followed by establishment of latency. To assess the role of CD4 T cell immunity in reactivation, monkeys were treated with a single 50 mg/kg dose of a humanized monoclonal anti-CD4 antibody; within 1 week, circulating CD4 T cells were reduced from 40-60% to 5-30% of the total T cell population and remained low for 2 months. Very low viremia was seen only in some of the treated monkeys. Zoster rash developed after 7 days in the monkey with the most extensive CD4 T cell depletion (5%) and in all other monkeys at 10-49 days post-treatment, with recurrent zoster in one treated monkey. SVV DNA was detected in the lung from 2 of 5 monkeys, in bronchial lymph nodes from 1 of the 5 monkeys, and in ganglia from at least two dermatomes in 3 of 5 monkeys. Immunofluorescence analysis of skin rash, lung, lymph node and ganglia revealed SVV ORF 63 protein in: sweat glands in skin; type II cells in lung alveoli, macrophages and dendritic cells in lymph nodes, and the neuronal cytoplasm of ganglia. Detection of SVV antigen in multiple tissues upon CD4 T cell depletion and virus reactivation suggests a critical role for CD4 T cell immunity in controlling varicella virus latency.

    IMPORTANCE Reactivation of latent VZV in humans can result in serious neurological complications. VZV-specific cell-mediated immunity is critical for the maintenance of latency. Similar to VZV in humans, SVV causes varicella in monkeys, establishes latency in ganglia, and reactivates to produce shingles. Herein, we show that depletion of CD4 T cells in rhesus macaques results in SVV reactivation, with virus antigens found in zoster rash and SVV DNA and antigens found in lungs, lymph nodes and ganglia. These results suggest the critical role of CD4 T cell immunity in controlling varicella virus latency.

  • Extensive Diversity of RNA Viruses in Australian Ticks [Genetic Diversity and Evolution]

  • Understanding the microbiome of ticks in Australia is of considerable interest given the ongoing debate over whether Lyme disease, and its causative agent the bacterium Borrelia burgdorferi sensu lato, are present in Australia. The diversity of bacteria infecting Australian ticks has been studied using both culture and metagenomics based techniques. However, little is known about the virome of Australian ticks, including whether this includes viruses with the potential to infect mammals. We used a meta-transcriptomics approach to reveal the diversity and evolution of viruses from Australian ticks collected from two locations on the central-east coast of Australia, including metropolitan Sydney. From this we identified 19 novel RNA viruses belonging to 12 families, as well as one previously described RNA virus. The majority of these viruses were related to arthropod-associated viruses suggesting that they do not utilize mammalian hosts. However, two novel viruses discovered in ticks feeding on bandicoot marsupials clustered closely within the mammalian-associated hepacivirus and pestivirus groups (family Flaviviridae). Another bandicoot tick yielded a novel coltivirus (family Reoviridae) nndash; a group of largely tick-associated viruses containing the known human pathogen Colorado tick fever virus and its relative Eyach virus. Importantly, our transcriptomic data provided no evidence for the presence of B. burgdorferi s.l. in any tick sample, providing further evidence against the presence of Lyme Disease in Australia. In sum, this study reveals that Australian ticks harbor a diverse virome, including some viruses that merit additional screening in the context of emerging infectious disease.

    IMPORTANCE Each year a growing number of individuals along the east coast of Australia experience debilitating disease following tick bites. As there is no evidence for the presence of the causative agent of Lyme disease, Borrelia Burgdorferi sensu lato, in Australian ticks, the etiological basis of this disease syndrome remains controversial. To characterize the viruses associated with Australian ticks, particularly those that might be associated with mammalian infection, we performed unbiased RNA sequencing on 146 ticks collected across two locations along the coast of New South Wales, Australia. This revealed 19 novel RNA viruses from a diverse set of families. Notably, three of these viruses clustered with known mammalian viruses, including a novel coltivirus that was related to the human pathogen Colorado tick fever virus.

  • Crustacean Genome Exploration Reveals the Evolutionary Origin of White Spot Syndrome Virus [Genetic Diversity and Evolution]

  • White spot syndrome virus (WSSV) is a crustacean-infecting, double-stranded DNA virus and is the most serious viral pathogen in the global shrimp industry. WSSV is the sole recognized member of the family Nimaviridae, and the lack of genomic data on other nimaviruses has obscured the evolutionary history of WSSV. Here, we investigated the evolutionary history of WSSV by characterizing WSSV relatives hidden in host genomic data. We surveyed 14 host crustacean genomes and identified five novel nimaviral genomes. Comparative genomic analysis of Nimaviridae identified 28 "core genes" that are ubiquitously conserved in Nimaviridae; unexpected conservation of 13 uncharacterized proteins highlighted yet unknown essential functions underlying the nimavirus replication cycle. The ancestral Nimaviridae gene set contained five baculoviral per os infectivity factor homologs and a sulfhydryl oxidase homolog, suggesting a shared phylogenetic origin of Nimaviridae and insect-associated double-stranded DNA viruses. Moreover, we show that novel gene acquisition and subsequent amplification reinforced the unique accessory gene repertoire of WSSV. Expansion of unique envelope protein and nonstructural virulence-associated genes may have been the key genomic event that made WSSV such a deadly pathogen.

    IMPORTANCE White spot syndrome virus (WSSV) is the deadliest viral pathogen threatening the global shrimp aquaculture. The evolutionary history of WSSV has remained a mystery, because few WSSV relatives, or nimaviruses, had been reported. Our aim was to trace the history of WSSV using the genomes of novel nimaviruses hidden in host genome data. We demonstrate that WSSV emerged from a diverse family of crustacean-infecting large DNA viruses. By comparing the genomes of WSSV and its relatives, we show that WSSV possesses an expanded set of unique host-viral interaction-related genes. This extensive gene gain may have been the key genomic event that made WSSV such a deadly pathogen. Moreover, conservation of insect-infecting virus protein homologs suggests a common phylogenetic origin of crustacean-infecting Nimaviridae and other insect-infecting DNA viruses. Our work redefines the previously poorly-characterized crustacean virus family and reveal the ancient genomic events that preordained the emergence of a devastating shrimp pathogen.

  • FMDV leader protease cleaves G3BP1 and G3BP2 and inhibits stress granule formation [Virus-Cell Interactions]

  • Like other viruses, the picornavirus foot-and-mouth disease virus (FMDV, genus aphthovirus), one of the most notorious pathogens in global livestock industry, needs to navigate antivirals host responses to establish an infection. There is substantial insight into how FMDV suppresses the type I IFN response, but it is largely unknown whether and how FMDV modulates the integrated stress response. Here, we show that the stress response is suppressed during FMDV infection. Using a chimeric recombinant encephalomyocarditis virus (EMCV), in which we functionally replaced the endogenous stress response antagonist by FMDV Lpro or 3Cpro, we demonstrate an essential role for Lpro in suppressing stress granule (SG) formation. Consistently, infection with a recombinant FMDV lacking Lpro resulted in SG formation. Additionally, we show that Lpro cleaves the known SG scaffold proteins G3BP1 and G3BP2, but not TIA-1. We demonstrate that the closely related equine rhinitis A virus (ERAV) Lpro also cleaves G3BP1 and G3BP2 and also suppresses SG formation, indicating that these abilities are conserved amongst aphthoviruses. Neither FMDV or ERAV Lpro interfered with phosphorylation of PKR or eIF2aalpha;, indicating that Lpro does not affect SG formation by inhibiting the PKR-triggered signaling cascade. Taken together, our data suggest that aphthoviruses actively target scaffolding proteins G3BP1 and G3BP2 and antagonize SG formation to modulate the integrated stress response.

    IMPORTANCE The picornavirus foot and mouth disease virus (FMDV) is a notorious animal pathogen that puts a major economic burden on global life stock industry. Outbreaks have significant consequences for animal health and product safety. Like many other viruses, FMDV must manipulate antiviral host responses to establish infection. Upon infection, viral dsRNA is detected which results in activation of the PKR-mediated stress response, leading to a stop in cellular and viral translation and the formation of stress granules (SG), which are thought to have antiviral properties. Here we show that FMDV can suppress SG formation via its leader protease (Lpro). Simultaneously we observed that Lpro can cleave the SG scaffolding proteins G3BP1 and G3BP2. Understanding the molecular mechanisms of FMDVrrsquo;s antiviral host response evasion strategies may help to develop countermeasures to control FMDV infections in the future.

  • Hepatitis B e antigen inhibits NF-{kappa}B activity by interrupting K63-linked ubiquitination of NEMO [Virus-Cell Interactions]

  • Viruses have adopted diverse strategies to suppress antiviral responses. Hepatitis B virus (HBV), a virus that is prevalent world-wide, manipulates the hostrrsquo;s innate immune system to evade scavenging. It is reported that the hepatitis B e antigen (HBeAg) can interfere with NF-B activity, which then leads to high viral loads, while HBV with the G1896A mutation remains infectious without the production of HBeAg but can induce more severe proinflammatory response and liver damage. The aim of current work was to study the molecular mechanism by which HBeAg suppresses IL-1bbeta;-stimulated NF-B activity, which leads to the suppression of the innate immune responses to HBV infection. Our study revealed that HBeAg could interact with NEMO, a regulatory subunit associated with IB kinase, which regulates the activation of NF-B. HBeAg suppressed the IL-1bbeta;-induced TRAF6-dependent K63-linked ubiquitination of NEMO, thereby downregulating NF-B activity and promoting virus replication. We further demonstrated the inhibitory effect of HBeAg on the NF-B signaling pathway using primary human hepatocytes, HBV-infected HepG2-NTCP cells and clinical liver samples. Our study reveals a molecular mechanism where HBeAg suppresses IL-1bbeta;-induced NF-B activation by decreasing the TRAF6-dependent K63-ubiquitination of NEMO, which may thereby enhance HBV replication and promote a persistent infection.

    IMPORTANCE The role of HBeAg in inflammatory responses during the infection of hepatitis B virus (HBV) is not fully understood, and several previous reports with regard to the NF-B pathway are controversial. In this report, we showed that HBeAg could suppress both TLR2 and IL-1bbeta;-induced activation of NF-B in cells and clinical samples and we further revealed novel molecular mechanisms. We found that HBeAg can associate with NEMO, the regulatory subunit for IB kinase (IKK) that controls the NF-B signaling pathway, and thereby inhibits TRAF6-mediated K63-linked ubiquitination of NEMO, resulting in down-regulation of NF-B activity and promotion of virus replication. In contrast, the HBeAg-negative HBV mutant can induce higher level of NF-B activity. These results are important for understanding the HBV-induced pathogenesis of chronic hepatitis and indicate that different clinical measures should be considered to treat HBeAg-positive and HBeAg-negative infections, our findings represent a conceptual advance in HBV-related suppression of NF-B signaling.

  • Components of the reovirus capsid differentially contribute to stability [Structure and Assembly]

  • The mammalian orthoreovirus (reovirus) outer capsid is composed of 200 mmu;1-3 heterohexamers and a maximum of 12 1 trimers. During cell entry, 3 is degraded by luminal or intracellular proteases to generate the infectious subviral particle (ISVP). When ISVP formation is prevented, reovirus fails to establish a productive infection, suggesting proteolytic priming is required for entry. ISVPs are then converted to ISVP*s, which is accompanied by mmu;1 rearrangements. The mmu;1 and 3 proteins confer resistance to inactivating agents; however, neither the impact on capsid properties nor the mechanism (or basis) of inactivation is fully understood. In this report, we utilized T1L/T3D M2 and T3D/T1L S4 to investigate the determinants of reovirus stability. Both reassortants encode mismatched subunits. When mmu;1-3 were derived from different strains, virions resembled wild type in structure and protease sensitivity. T1L/T3D M2 and T3D/T1L S4 ISVPs were less thermostable than wild type ISVPs. In contrast, virions were equally susceptible to heating. Virion associated mmu;1 adopted an ISVP*-like conformation concurrent with inactivation; 3 preserves infectivity by preventing mmu;1 rearrangements. Moreover, thermostability was enhanced by a hyperstable variant of mmu;1. Unlike the outer capsid, the inner capsid (core) was highly resistant to elevated temperatures. The dual layered architecture allowed for differential sensitivity to inactivating agents.

    IMPORTANCE Nonenveloped and enveloped viruses are exposed to the environment during transmission to a new host. Protein-protein and/or protein-lipid interactions stabilize the particle and protect the viral genome. Mammalian orthoreovirus (reovirus) is composed of two concentric, protein shells. The mmu;1 and 3 proteins form the outer capsid; contacts between neighboring subunits are thought to confer resistance to inactivating agents. We further investigated the determinants of reovirus stability. The outer capsid was disrupted concurrent with the loss of infectivity; virion associated mmu;1 rearranged into an altered conformation. Heat sensitivity was controlled by 3; however, particle integrity was enhanced by a single mmu;1 mutation. In contrast, the inner capsid (core) displayed superior resistance to heating. These findings reveal structural components that differentially contribute to reovirus stability.

  • Antibody responses towards the major antigenic sites of influenza B virus hemagglutinin in mice, ferrets and humans [Vaccines and Antiviral Agents]

  • The influenza B virus hemagglutinin contains four major antigenic sites (the 120 loop, the 150 loop, the 160 loop and the 190 helix) within the head domain. These immunodominant antigenic sites are the main targets of neutralizing antibodies, and are subject to antigenic drift. Yet, little is known about the specific antibody responses towards each site in terms of antibody prevalence and hemagglutination inhibition activity. Here, we used modified hemagglutinins of influenza B virus, which display only one or none of the major antigenic sites to measure antibody responses towards the classical as well as the non-canonical epitopes in mice, ferrets and humans. With our novel reagents, we found that both hemagglutination inhibition antibodies and total IgGs were mostly induced by the major antigenic sites. However, in human adults, we observed high hemagglutination inhibition antibody responses towards the non-canonical epitopes. By stratifying the human samples into age groups, the non-canonical antibody responses appeared to increase with age.

    IMPORTANCE This study dissects the specific antibody responses towards the major antigenic sites and the non-canonical epitopes of influenza B virus hemagglutinin in animals and humans using novel reagents. These findings will guide the design of the next generation influenza virus vaccines.

  • Absence of {beta}6 integrin reduces influenza disease severity in highly susceptible obese mice [Pathogenesis and Immunity]

  • Obese individuals are considered a high-risk group for developing severe influenza virus infection. While the exact mechanisms for increased disease severity remain under investigation, obese mouse models suggest that increased acute lung injury (ALI), potentially due to enhanced viral spread and decreased wound repair, are likely involved. We previously demonstrated that upregulation of the lung epithelial cell bbeta;6 integrin during influenza infection was involved in disease severity. Knocking out bbeta;6 (bbeta;6 KO) resulted in improved survival. Of interest, obese mice have increased lung bbeta;6 integrin levels at homeostasis. Thus, we hypothesized that the protective effect seen in bbeta;6 KO mice would extend to the highly susceptible obese mouse model. In the current study, we show that crossing bbeta;6 KO mice with genetically obese ob/ob mice (OBKO) resulted in reduced ALI and impaired viral spread, like their lean counterparts. Mechanistically, OBKO alveolar macrophages and epithelial cells had increased type I interferon (IFN) signaling potentially through upregulated type I IFN receptor expression, which was important for the enhanced protection during infection. Taken together, our results indicate that the absence of an epithelial integrin can beneficially alter the pulmonary microenvironment by increasing protective type I IFN responses even in a highly susceptible obese model. These studies increase our understanding of influenza pathogenesis in high-risk populations and may lead to the development of novel therapies.

    IMPORTANCE Obesity is a risk factor for developing severe influenza infection. However, the reasons for this are unknown. We found that the lungs of obese mice have increased expression of the epithelial integrin bbeta;6, a host factor associated with increased disease severity. Knocking out integrin bbeta;6 in obese mice favorably altered the pulmonary environment by increasing type I IFN interferon signaling resulting in decreased viral spread, reduced lung injury, and increased survival. This study furthers our understanding of influenza pathogenesis in the high-risk obese population and may potentially lead to the development of novel therapies for influenza infection.

  • Mutations in influenza A virus neuraminidase and hemagglutinin confer resistance against a broadly neutralizing hemagglutinin stem antibody [Vaccines and Antiviral Agents]

  • Influenza A virus (IAV), a major cause of human morbidity and mortality, continuously evolves in response to selective pressures. Stem-directed, broadly neutralizing antibodies (sBnAbs) targeting influenza hemagglutinin (HA) are a promising therapeutic strategy, but neutralization escape mutants can develop. We used an integrated approach combining viral passaging, deep sequencing, and protein structural analyses to define escape mutations and mechanisms of neutralization escape in vitro for the F10 sBnAb. IAV was propagated with escalating concentrations of F10 over serial passages in cultured cells to select for escape mutations. Viral sequence analysis revealed three mutations in HA and one in neuraminidase (NA). Introduction of these specific mutations into IAV through reverse genetics confirmed their roles in resistance to F10. Structural analyses revealed that the selected HA mutations (S123G, N460S, and N203V) are away from the F10 epitope but may indirectly impact influenza receptor binding, endosomal fusion, or budding. The NA mutation E329K, which was previously identified to be associated with antibody escape, affects the active site of NA, highlighting the importance of the balance between HA and NA function for viral survival. Thus, whole genome population sequencing enables the identification of viral resistance mutations responding to antibody-induced selective pressure.

    IMPORTANCE Influenza A virus is a public health threat for which currently available vaccines are not always effective. Broadly neutralizing antibodies that bind to the highly-conserved stem region of influenza hemagglutinin (HA) can neutralize many influenza strains. To understand how influenza virus can become resistant or "escape" such antibodies, we propagated influenza A virus in vitro with escalating concentrations of antibody and analyzed viral populations with whole genome sequencing. We identified HA mutations near and distal to the antibody binding epitope that conferred resistance to antibody neutralization. Additionally, we identified a neuraminidase (NA) mutation that allowed the virus to grow in the presence of high concentrations of the antibody. Virus carrying dual mutations in HA and NA also grew under high antibody concentrations. We show that NA mutations mediate the escape of neutralization by antibodies against HA, highlighting the importance of a balance between HA and NA for optimal virus function.

  • Mutation of influenza A virus PA-X decreases pathogenicity in chicken embryos and can increase the yield of reassortant candidate vaccine viruses [Pathogenesis and Immunity]

  • The PA-X protein of influenza A virus has roles in host cell shut-off and viral pathogenesis. While most strains are predicted to encode PA-X, strain-dependent variations in activity have been noted. We found that PA-X protein from A/PR/8/34 (PR8) strain had significantly lower repressive activity against cellular gene expression compared with PA-Xs from the avian strains A/turkey/England/50-92/91 (H5N1) (T/E) and A/chicken/Rostock/34 (H7N1). Loss of normal PA-X expression, either by mutation of the frameshift site or by truncating the X-ORF, had little effect on the infectious virus titre of PR8 or PR8 7:1 reassortants with T/E segment 3 grown in embryonated hensrrsquo; eggs. However, in both virus backgrounds, mutation of PA-X led to decreased embryo mortality and lower overall pathology; effects that were more pronounced in the PR8 strain than the T/E reassortant, despite the low shut-off activity of the PR8 PA-X. Purified PA-X mutant virus particles displayed an increased ratio of HA to NP and M1 compared to their WT counterparts, suggesting altered virion composition. When the PA-X gene was mutated in the background of poorly growing PR8 6:2 vaccine reassortant analogues containing the HA and NA segments from H1N1 2009 pandemic viruses or an avian H7N3 strain, HA yield increased up to 2-fold. This suggests that the PR8 PA-X protein may harbour a function unrelated to host cell shut-off and that disruption of the PA-X gene has the potential to improve the HA yield of vaccine viruses.

    IMPORTANCE Influenza A virus is a widespread pathogen that affects both man and a variety of animal species, causing regular epidemics and sporadic pandemics with major public health and economic consequences. A better understanding of virus biology is therefore important. The primary control measure is vaccination, which for humans, mostly relies on antigens produced in eggs from PR8-based viruses bearing the glycoprotein genes of interest. However, not all reassortants replicate well enough to supply sufficient virus antigen for demand. The significance of our research lies in identifying that mutation of the PA-X gene in the PR8 strain of virus can improve antigen yield, potentially by decreasing the pathogenicity of the virus in embryonated eggs.

  • Hepatitis C virus downregulates ubiquitin conjugating enzyme E2S expression to prevent proteasomal degradation of NS5A, leading to host cells more sensitive to DNA damage [Virus-Cell Interactions]

  • Hepatitis C virus (HCV) infection may cause chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV exploits cellular machineries to establish persistent infection. Herein we demonstrated that ubiquitin conjugating enzyme E2S (UBE2S), a member of ubiquitin conjugating enzyme family (E2s), was downregulated by ER stress caused by HCV in Huh7 cells. UBE2S interacted with domain I of HCV NS5A and degraded NS5A protein through Lys11-linked proteasome-dependent pathway. Overexpression of UBE2S suppressed viral propagation, while depletion of UBE2S expression increased viral infectivity. Enzymatically inactive UBE2S C95A mutant exerted no antiviral activity, suggesting that ubiquitin-conjugating enzymatic activity was required for the suppressive role of UBE2S. Chromatin ubiquitination plays a crucial role in the DNA damage response. We showed that the levels of UBE2S and Lys11 chains bound to the chromatin were markedly decreased in the context of HCV replication, rendering HCV-infected cells more sensitive to DNA damage. These data suggest that HCV counteracts antiviral activity of UBE2S to optimize viral propagation and may contribute to HCV-induced liver pathogenesis.

    IMPORTANCE Protein homeostasis is essential to normal cell function. HCV infection disturbs the protein homeostasis in the host cells. Therefore, host cells exert an anti-HCV activity in order to maintain normal cellular metabolism. We showed that UBE2S interacted with HCV NS5A and degraded NS5A protein through Lys11-linked proteasome-dependent pathway. However, HCV has evolved to overcome host antiviral activity. We demonstrated that UBE2S expression level was suppressed in HCV-infected cells. Since UBE2S is an ubiquitin conjugating enzyme and this enzyme activity is involved in DNA damage repair, HCV-infected cells are more sensitive to DNA damage and thus it may contribute to viral oncogenesis.

  • Determining the Mutation Bias of Favipiravir in Influenza Using Next-generation Sequencing [Vaccines and Antiviral Agents]

  • Favipiravir is a broad-spectrum antiviral drug that may be used to treat influenza. Previous research has identified that favipiravir likely acts as a mutagen but the precise mutation bias that favipiravir induces in influenza virus RNAs has not been described. Here, we use next-generation sequencing (NGS) with barcoding of individual RNA molecules to accurately and quantitatively detect favipiravir-induced mutations and to sample orders of magnitude more mutations than would be possible through Sanger sequencing. We demonstrate that favipiravir causes mutations and show that favipiravir primarily acts as a guanine analogue and secondarily as an adenine analogue resulting in the accumulation of transition mutations. We also use a standard NGS pipeline to show that the mutagenic effect of favipiravir can be measured by whole genome sequencing of virus.

    IMPORTANCE New antiviral drugs are needed as a first line of defence in the event of a novel influenza pandemic. Favipiravir is a broad-spectrum antiviral which is effective against influenza. The exact mechanism of how favipiravir works to inhibit influenza is still unclear. We used next-generation sequencing (NGS) to demonstrate that favipiravir causes mutations in influenza RNA. The greater depth of NGS sequence information over traditional sequencing methods allowed us to precisely determine the bias of particular mutations caused by favipiravir. NGS can also be used in a standard diagnostic pipeline to show that favipiravir is acting on the virus by revealing the mutation bias pattern typical to the drug. Our work will aid in testing whether viruses are resistant to favipiravir and may help demonstrate the effect of favipiravir on viruses in a clinical setting. This will be important if favipiravir is used during a future influenza pandemic.

  • Inhibition of Epstein-Barr Virus Replication in Human Papillomavirus-Immortalized Keratinocytes. [Genome Replication and Regulation of Viral Gene Expression]

  • Epstein-Barr virus (EBV) is implicated in the pathogenesis of human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OSCC). EBV-associated cancers harbor a latent EBV infection characterized by a lack of viral replication and the expression of viral oncogenes. Cellular changes promoted by HPV are comparable to those shown to facilitate EBV latency, though whether HPV-positive cells support a latent EBV infection has not been demonstrated. Using a model of direct EBV infection into HPV16-immortalized tonsillar cells grown in organotypic raft culture, we showed robust EBV replication in HPV-negative rafts but little to no replication in HPV-immortalized rafts. Reduced EBV replication was independent of immortalization as human telomerase-immortalized normal oral keratinocytes supported robust EBV replication. Furthermore, we observed reduced EBV lytic gene expression and increased expression of EBER1, a non-coding RNA highly expressed in latently-infected cells, in the presence of HPV. Human foreskin keratinocytes expressing HPV16 E6 and/or E7 showed that E7 was sufficient to reduce EBV replication. EBV replication is dependent upon epithelial differentiation and the differentiation-dependent expression of the transcription factor KLF4. While KLF4 levels were unaltered, expression of KLF4 transcriptional targets, including late differentiation markers, were reduced in HPV E6 and E7 rafts compared to parental rafts. However, the HPV E7-mediated block in EBV replication correlated with delayed expression of early differentiation markers. Overall, this study reveals a HPV16-mediated block in EBV replication through E7 which may facilitate EBV latency and long-term persistence in the tumor context.

    IMPORTANCE Using a model examining the establishment of EBV infection in HPV-immortalized tissues, we showed a HPV-induced interruption of the normal EBV life cycle reminiscent of a latent EBV infection. Our data support the notion that a persistent EBV epithelial infection depends upon pre-existing cellular alterations, and suggest the ability of HPV to promote such changes. More importantly these findings introduce a model for how EBV co-infection may influence HPV+ OSCC pathogenesis. Latently EBV-infected epithelial cells as well as other EBV-associated head-and-neck carcinomas exhibit oncogenic phenotypes commonly seen in HPV+ OSCC. Therefore, a HPV-induced shift in the EBV life cycle toward latency would not only facilitate EBV persistence but also provide additional viral oncogene expression, which can contribute to the rapid progression of HPV+ OSCC. These findings provide a step toward defining a role for EBV as a cofactor in HPV-positive oropharyngeal tumors.

  • Dolutegravir monotherapy of simian immunodeficiency virus-infected macaques selects for several patterns of resistance mutations with variable virological outcomes. [Vaccines and Antiviral Agents]

  • Drug resistance remains a major concern for HIV treatment. To date, very few resistance mutations have emerged in first-line combination therapy that includes the integrase strand transfer inhibitor (INSTI) dolutegravir (DTG). In vitro, DTG selects for several primary mutations that induce low-level DTG resistance; secondary mutations, while increasing the level of resistance, however further impair replication fitness, which raised the idea that DTG monotherapy may be feasible. The simian immunodeficiency virus (SIV) rhesus macaque model of HIV infection can be useful to explore this concept. Nine macaques were infected with virulent SIVmac251 and started on DTG monotherapy either during acute (n=2) or chronic infection (n=7). Within 4 weeks of treatment, all animals demonstrated a reduction in viremia of 0.8 to 3.5 log RNA copies/ml plasma. Continued treatment led to overall sustained benefits, but the outcome after 10-50 weeks of treatment was highly variable, and ranged from viral rebound to near pre-treatment levels, to sustained suppression, with viremia 0.5 to 5 log lower than expected based on pre-treatment viremia. A variety of mutations previously described to confer low-level resistance of HIV-1 to DTG or other INSTI were detected, sometimes followed by mutations believed to be compensatory. Some mutations, such as G118R, previously shown to severely impair replication capacity in vitro, were associated with more sustained virologic and immunologic benefits of continued DTG therapy, while other mutations such as E92Q and G140A/Q148K were associated with more variable outcomes. The observed variability of outcomes in macaques warrants avoidance of DTG monotherapy in HIV-infected people.

    IMPORTANCE A growing number of HIV drug combinations are effective in suppressing virus replication in HIV-infected persons. However, to reduce their cost and risk for toxicity, there is considerable interest in simplifying drug regimens. A major concern with single-drug regimens is the emergence of drug-resistant viral mutants. It has been speculated that DTG monotherapy may be a feasible option, because DTG may have a higher genetic barrier for the development of drug resistance than other commonly used antiretrovirals. To explore treatment initiation with DTG monotherapy, we started SIV-infected macaques on DTG either during acute or chronic infection. Although DTG initially reduced virus replication, continued treatment led to the emergence of a variety of viral mutations, previously described to confer low-level resistance of HIV-1 to DTG, and this was associated with variable clinical outcomes. This unpredictability of mutational pathways and outcomes warns against using DTG monotherapy as initial treatment for HIV-infected people.

  • Isolation and characterization of a distinct influenza A virus from Egyptian bats [Genetic Diversity and Evolution]

  • Recently, two genetically distinct influenza viruses were detected in bats in Guatemala and Peru. We conducted influenza A surveillance among four bat species in Egypt. Out of 1202 swabs, 105 were positive by RT-PCR. A virus was successfully isolated in eggs and propagated in MDCK cells in the presence of TPCK-treated trypsin. Genomic analysis revealed that the virus was phylogenetically distinct from all other influenza A viruses. Analysis of the HA gene suggested common ancestry with other H9 viruses and the virus showed low-level of cross-reactivity with sera raised against H9N2 viruses. Bats were seropositive for the isolated viruses. The virus replicated in the lungs of experimentally infected mice. While genetically distinct, this virus shares several avian influenza virus characteristics suggesting a more recent avian host origin.

    IMPORTANCE Through surveillance, we isolated and characterized an influenza A virus from Egyptian fruit bats. This virus had affinity to avian-like receptors but was also able to infect mice. Our findings indicate that bats may harbor a diversity of influenza A viruses. Such viruses may have the potential to cross the species barrier to infect other species including domestic birds and mammals and possibly humans.

  • Metagenomic sequencing of HIV-1 in the blood and female genital tract reveals little quasispecies diversity during acute infection [Genetic Diversity and Evolution]

  • Heterosexual transmission of human immunodeficiency virus-1 (HIV-1) is associated with a significant bottleneck in the viral quasispecies population, yet the timing of that bottleneck is poorly understood. We characterized HIV-1 diversity in the blood and female genital tract (FGT) within two weeks after detection of infection in three women enrolled in a unique prospective cohort in South Africa. We assembled full-length HIV-1 genomes from matched samples of cervicovaginal lavage (CVL) and plasma. Deep sequencing allowed us to identify intrahost single nucleotide variants (iSNVs) and characterize within-sample HIV-1 diversity.

    Our results demonstrated very little HIV-1 diversity in the FGT and plasma by the time of detectable viremia. Within each subject, the consensus HIV-1 sequences were identical in plasma and CVL. No iSNV was present at ggt;6% frequency. One subject had 77 low-frequency iSNVs across both CVL and plasma; another subject had 14 iSNVs in only CVL from the earliest time point; and the third subject had no iSNVs in CVL or plasma. Overall, the small amount of diversity that we detected was higher in the FGT than plasma and declined over the first two weeks after detectable viremia, compatible with a very early HIV-1 transmission bottleneck. To our knowledge, our study represents the earliest genomic analysis of HIV-1 in the FGT after transmission. Further, the use of metagenomic sequencing allowed us to characterize other organisms in the FGT, including commensal bacteria and sexually transmitted infections, highlighting the utility of this method to sequence both HIV-1 and its metagenomic environment.

    IMPORTANCE Due to error-prone replication, HIV-1 generates a diverse population of viruses within a chronically infected individual. When HIV-1 is transmitted to a new individual, one or a few viruses establish the new infection, leading to a genetic bottleneck in the virus population. Understanding the timing and nature of this bottleneck may provide insight into HIV-1 vaccine design and other preventative strategies. We examined the HIV-1 population in three women enrolled in a unique prospective cohort in South Africa, who were followed closely during the earliest stages of HIV-1 infection. We found very little HIV-1 diversity in the blood and female genital tract during the first two weeks after virus was detected in the bloodstream. These results are compatible with a very early HIV-1 population bottleneck, suggesting the need to study the HIV-1 population in the female genital tract before virus is detectable in the bloodstream.

  • Biogenesis of extracellular vesicles during herpes simplex virus type 1 infection: The role of the CD63 tetraspanin. [Virus-Cell Interactions]

  • Herpes simplex virus type-1 (HSV-1) infections afflict more than 80% of the population worldwide. The virus primarily infects mucoepithelial cells and establishes latent reservoirs in neurons in sensory ganglia. Frequent reactivation has been linked to severe diseases, especially in immunocompromised individuals. Earlier we reported that viral and host factors are packaged in extracellular vesicles (EVs) and delivered to uninfected cells where they activate antiviral responses and restrict virus infection. Here, we interrogated the effect of HSV-1 infection on EV biogenesis. We found that HSV-1 infection causes a decrease in the amount of intracellular CD63 protein with a concomitant increase in extracellular CD63. This observation correlates with our previous finding that infected cells release more CD63-positive EVs compared to uninfected cells. The stimulation of CD63 exocytosis requires virus replication. CD63 is a member of the tetraspanin family of proteins that traffics between the plasma membrane and endosomal compartments and has a role in sorting cargo into the EVs. Previously we reported that in cells depleted of CD63 HSV-1 virus yields increased, and here we provide data showing that in cells overexpressing CD63 HSV-1 virus yields decreased. Taken together, our data indicate that CD63 negatively impacts HSV-1 infection and that the CD63-positive EVs could control the dissemination of the virus in the host. Perhaps the EVs released by HSV-1 infected cells is a mechanism that controls virus dissemination.

    IMPORTANCE Intercellular communication, especially in neurons, largely relies on EVs and modulation of EVs is known to impact physiological processes. Here, we present that HSV-1 infection causes major alterations in the biogenesis of EVs, including an increase in their number and an increase in the CD63-positive population of EVs. These alterations result in an enrichment of the milieu of infection with EVs carrying "signatures" from infected cells. In addition to changes in the origin and type, EVs released by infected cells have differences in cargo as they carry viral and host factors determined by the virus. The tetraspanin CD63 negatively impacts the infection, as demonstrated by CD63-knockdown and overexpression assays. A proposed mechanism involves the activation of antiviral responses in cells receiving CD63-positive EVs released by infected cells. Overall, HSV-1 causes major alterations in EVs that could contribute to HSV-1 persistence and pathogenesis.

  • Low pH endocytic entry of the porcine alphaherpesvirus pseudorabies virus [Virus-Cell Interactions]

  • The alphaherpesvirus pseudorabies virus (PRV) is the causative agent of pseudorabies, a disease of great economic and welfare importance in swine. Other alphaherpesviruses, including herpes simplex virus (HSV), utilize low pH-mediated endocytosis to enter a subset of cell types. We investigated whether PRV used this entry pathway in multiple laboratory model cell lines. Inhibition of receptor-mediated endocytosis by treatment with hypertonic medium prevented PRV entry. PRV entry into several cell lines including porcine kidney (PK15) cells, and African green monkey kidney (Vero) cells was inhibited by noncytotoxic concentrations of the lysosomotropic agents ammonium chloride or monensin, which block the acidification of endosomes. Inactivation of virions by acid pretreatment is a hallmark of viruses that utilize a low pH-mediated entry pathway. Exposure of PRV virions to pH 5.0 in the absence of host cell membrane reduced entry into PK15 and Vero cells by greater than 80%. Together, these findings suggest that endocytosis and subsequent fusion with host membranes triggered by low endosomal pH is an important route of entry for PRV.

    IMPORTANCE PRV is a pathogen of great economic and animal welfare importance in many parts of the world. PRV causes neurologic, respiratory, and reproductive disorders often resulting in mortality of young and immunocompromised animals. Mortality, decreased production, and trade restrictions result in significant financial losses for the agricultural industry. Understanding the molecular mechanisms utilized by PRV to enter host cells is an important step in identifying novel strategies to prevent infection and spread. A thorough understanding of these mechanisms will contribute to a broader understanding of alphaherpesvirus entry. Here, we demonstrate PRV entry into multiple model cell lines via a low pH endocytosis pathway. Together, these results provide a framework for elucidating the early events of the PRV replicative cycle.

  • THE NS1 PROTEIN OF INFLUENZA A VIRUS PARTICIPATES IN NECROPTOSIS BY INTERACTING WITH MLKL AND INCREASING ITS OLIGOMERIZATION AND MEMBRANE TRANSLOCATION [Virus-Cell Interactions]

  • Elimination of infected cells by programmed cell death is a well-recognized host defense mechanism to control the spread of infection. In addition to apoptosis, necroptosis is also one of the mechanisms of cell death that can be activated by viral infection. Activation of necroptosis leads to the phosphorylation of mixed-lineage kinase domain-like protein (MLKL) by receptor interacting protein kinase (RIPK)-3 and results in MLKL oligomerization and membrane translocation, leading to membrane disruption and loss of cellular ion homeostasis. It has recently been reported that influenza A virus (IAV) infection induces necroptosis. However, the underlying mechanism of IAV-mediated necroptosis process, particularly the roles of IAV proteins in necroptosis, remains unexplored. Here, we report that IAV infection induces necroptosis in macrophages and epithelial cells. We demonstrate that the NS1 protein of IAV interacts with MLKL. The coiled-coil domain 2 of MLKL has a predominant role in mediating MLKL interaction with NS1. Interaction of NS1 with MLKL increases MLKL oligomerization and membrane translocation. Moreover, the MLKL-NS1 interaction enhances MLKL-mediated NLRP3 inflammasome activation, leading to increased IL-1bbeta; processing and secretion.

    IMPORTANCE Necroptosis is a programmed cell death that is inflammatory in nature owing to the release of danger-associated molecular patterns from the ruptured cell membrane. However, necroptosis also constitutes an important arm of host immune responses. Thus, a balanced inflammatory response determines the disease outcome. We report that the NS1 protein of IAV participates in necroptosis by interacting with MLKL, resulting in increased MLKL oligomerization and membrane translocation. These results reveal a novel function of the NS1 protein and the mechanism by which IAV induces necroptosis. Moreover, we show that this interaction enhances the NLRP3 inflammasome activation and IL-1bbeta; processing and secretion. The information may contribute to a better understanding of the role of necroptosis in IAV-induced inflammation.

  • Human norovirus epitope D plasticity allows escape from antibody immunity without loss of capacity for binding cellular ligands [Pathogenesis and Immunity]

  • Emergent strains of human norovirus seed pandemic waves of disease. These new strains have altered ligand binding and antigenicity characteristics. Study of viral variants isolated from immunosuppressed patients with long-term norovirus infection indicates that initial virus in vivo evolution occurs at the same antigenic sites as in pandemic strains. Here, cellular ligand binding and antigenicity of two co-circulating strains isolated from a patient with long-term norovirus infection were characterized. The isolated GII.4 viruses differed from previous strains and from each other at known blockade antibody epitopes. One strain had a unique sequence in epitope D, including loss of an insertion at residue 394, corresponding to decreased relative affinity for carbohydrate ligands. Replacement of 394 with alanine or restoration of the contemporary strain epitope D consensus sequence STT improved ligand binding relative affinity. However, monoclonal antibody blockade of binding potency was only gained for the consensus sequence, not by the alanine insertion. In depth study of unique changes in epitope D indicate that ligand binding, but not antibody blockade of ligand binding, is maintained despite sequence diversity, allowing escape from blockade antibodies without loss of capacity for binding cellular ligands.

    IMPORTANCE Human norovirus causes ~20% of all acute gastroenteritis and ~200,000 deaths per year, primarily in young children. Most epidemic and all pandemic waves of disease over the past 30 years have been caused by type GII.4 human norovirus strains. The capsid sequence of GII.4 strains is changing over time resulting in viruses with altered ligand and antibody binding characteristics. The carbohydrate binding pocket of these strains does not vary over time. Here, utilizing unique viral sequences, we study how residues in GII.4 epitope D balance the dual roles of variable antibody binding site and cellular ligand binding stabilization domain, demonstrating that amino acid changes in epitope D can result in loss of antibody binding without ablating ligand binding. This flexibility in epitope D likely contributes to GII.4 strain persistence by both allowing escape from antibody-mediated herd immunity and maintenance of cellular ligand binding and infectivity.

  • Functional mapping of regions involved in the negative imprinting of virion particles infectivity and in target cell protection by the Interferon-Induced Transmembrane Protein 3 (IFITM3) against HIV-1 [Cellular Response to Infection]

  • The interferon-induced transmembrane proteins (IFITMs) are a family of highly related antiviral factors that affect numerous viruses at two steps: in target cells by sequestering incoming viruses in endosomes and in producing cells by leading to the production of virions that package IFITMs and exhibit decreased infectivity. While most studies have focused on the former, little is known about the regulation of the negative imprinting of virion particles infectivity by IFITMs and about its relationship with target cell protection.

    Using a panel of IFITM3 mutants against HIV-1, we have explored these issues as well as others related to the biology of IFITM3, in particular virion packaging, stability, the relation to CD63/MVB, the modulation of cholesterol levels and the relationship between negative imprinting of virions and target cell protection.

    The results we have obtained exclude a role for cholesterol and indicate that CD63 accumulation does not directly relate to an antiviral behavior. We have defined regions that modulate the two antiviral properties of IFITM3, as well as novel domains that modulate the protein stability and that, in so doing, influence the extent of its packaging into virions. The results we have obtained however indicate that, even in the context of an IFITM-susceptible virus, IFITM3 packaging is not sufficient for the negative imprinting. Lastly, while most mutations concomitantly affect target cell protection and negative imprinting, a region in the CTD exhibits a differential behavior, potentially highlighting the regulatory role that this domain may play on the two antiviral activities of IFITM3.

    IMPORTANCE IFITM proteins have been associated to the sequestration of incoming virions in endosomes (target cell protection) and to the production of virion particles that incorporate IFITMs and exhibit decreased infectivity (negative imprinting of virion infectivity). How the latter is regulated and whether these two antiviral properties are related remain unknown.

    By examining the behavior of a large panel of IFITM3 mutants against HIV-1, we determine that IFITM3 mutants are essentially packaged into virions proportionally to their intracellular levels of expression. However, even in the context of an IFITM-susceptible virus, IFITM3 packaging is not sufficient for the antiviral effects. Most mutations were found to concomitantly affect both antiviral properties of IFITM3, but one CTD mutant exhibited a divergent behavior, possibly highlighting a novel regulatory role for this domain. These findings thus advance our comprehension of how this class of broad antiviral restriction factors acts.

  • N-Glycosylation of Seasonal Influenza Vaccine Hemagglutinins: Implication for potency testing and immune processing [Vaccines and Antiviral Agents]

  • Prior to each annual flu season, health authorities recommend three or four virus strains for inclusion in the annual influenza vaccine: a Type A:H1N1, a Type A:H3N2, and one or two Type B viruses. Antigenic differences between strains are found in the glycosylation patterns of the major influenza antigen, hemagglutinin (HA). Here we examine the glycosylation patterns of seven reference antigens containing HA used in influenza vaccine potency testing. These reagents are supplied by the Center for Biologics Evaluation and Research (CBER) or the National Institute for Biological Standards and Control (NIBSC) for use in vaccine testing. Those produced in hen egg, Madin Darby Canine Kidney (MDCK), and insect (Sf9) expression systems were examined. They are closely related or identical to antigens used in commercial vaccine. Reference antigens studied were used in the 2014-2015 influenza season and included A/California/07/2009 H1N1, A/Texas/50/2012 H3N2 and B/Massachusetts/02/2012. Released glycan and HA specific glycopeptide glycosylation patterns were examined. Also examined was the sensitivity of the Single Radial Immunodiffusion Assay (SRID) potency test to differences in HA antigen glycosylation. The SRID assay was not sensitive to any HA antigen glycosylation status from any cell system based on deglycosylation studies as applied using standard assay procedures. Mapping of glycosites with their occupying glycan to functional regions, including antigenic sites, lectin interaction regions and fusion domains was performed and has implications for immune processing, immune response and antigenic shielding. Differences in glycosylation patterns, as dictated by cell system used in expression, may impact on these functions.

    IMPORTANCE Here the glycosylation patterns of the 2014-2015 influenza vaccine season standard antigens A/California/07/2009 H1N1, A/Texas/50/2012 H3N2, and B/Massachusetts/02/2012 were revealed and sensitivity of the Single Radial Immunodiffusion Assay (SRID) potency test glycosylation was tested. Differences in hemagglutinin glycosylation site composition and heterogeneity seen in antigen produced in different cell substrates suggests differences in processing and downstream immune response. The SRID potency test used in vaccine release, is not sensitive to differences in glycosylation when applied under standard use conditions. This work reveals important differences in vaccine antigens and may point toward areas where improvements may be made concerning vaccine antigen preparation, immune processing and testing.

  • Murine leukemia virus glycosylated Gag reduces murine SERINC5 protein expression at steady-state levels via endosome/lysosome pathway to counteract the SERINC5 antiretroviral activity [Virus-Cell Interactions]

  • Glycosylated Gag (glycoGag) is an accessory protein expressed by most gamma-retroviruses including murine leukemia virus (MLV). MLV glycoGag not only enhances MLV replication and disease progression but also increases human immunodeficiency virus type 1 (HIV-1) infectivity as Nef does. Recently, SERINC5 (Ser5) was identified as the target for Nef, and the glycoGag Nef-like activity has been attributed to the Ser5 antagonism. Here, we investigated how glycoGag antagonizes Ser5 using MLV glycoMA and murine Ser5 proteins. We confirm previous observations that glycoMA re-localizes Ser5 from plasma membrane to perinuclear punctated compartments and the important role of its Y36XXL39 motif in this process. We find that glycoMA decreases Ser5 expression at steady-state levels, and identify two other glycoGag crucial residues P31 and R63 for the Ser5 downregulation. The glycoMA and Ser5 interaction is detected in live cells using a bimolecular fluorescence complementation (BiFC) assay. Ser5 is internalized via receptor-mediated endocytosis, and re-localized to Rab5+ early, Rab7+ late, and Rab11+ recycling endosomes by glycoMA. Although glycoMA is not polyubiquitinated, the Ser5 downregulation requires Ser5 polyubiquitination via the K48- and K63-linkage, resulting in Ser5 destruction in lysosomes. Although P31, Y36, L39, and R63 are not required for glycoMA interaction with Ser5, they are required for Ser5 re-localization to lysosomes for destruction. In addition, although murine Ser1, Ser2, and Ser3 exhibit very poor antiviral activity, they are also targeted by glycoMA for lysosomal destruction. We conclude that glycoGag has a broad activity to downregulate SERINC proteins via the cellular endosome/lysosome pathway, which promotes viral replication.

    IMPORTANCE MLV glycoGag not only enhances MLV replication but also increases HIV-1 infectivity similarly as Nef. Recent studies have discovered that both glycoGag and Nef antagonize a novel host restriction factor Ser5 and promote viral replication. Compared to Nef, the glycoGag antagonism of Ser5 is still poorly understood. MLV glycoGag is a transmembrane version of the structural Gag protein with an extra 88-amino-acid leader region that determines its activity. We now show that glycoGag interacts with Ser5 in live cells, and internalizes Ser5 via receptor-mediated endocytosis. Ser5 is polyubiquitinated and re-localized to endosomes and lysosomes for massive destruction. In addition to the previously identified tyrosine-based sorting signal, we find two more important residues for Ser5 re-localization and downregulation. We also find that the Ser5-sensitivity to glycoGag is conserved in SERINC family. Together, our findings highlight the important role of endosome/lysosome pathway in the enhancement of viral replication by viral proteins.

  • Group A Rotavirus VP1 Polymerase and VP2 Core Shell Proteins: Intergenotypic Sequence Variation and In vitro Functional Compatibility [Genome Replication and Regulation of Viral Gene Expression]

  • Group A rotaviruses (RVAs) are classified according to a nucleotide sequence-based system that assigns a genotype to each of the 11 double-stranded (ds) RNA genome segments. For the segment encoding the VP1 polymerase, 22 genotypes (R1-R22) are defined with an 83% nucleotide identity cut-off value. For the segment encoding the VP2 core shell protein, which is a functional VP1-binding partner, 20 genotypes (C1-C20) are defined with an 84% nucleotide identity cut-off value. However, the extent to which the VP1 and VP2 proteins encoded by these genotypes differ in their sequences or interactions has not been described. Here, we sought to (i) delineate the relationships and sites of variation for VP1 and VP2 proteins belonging to the known RVA genotypes and (ii) correlate intergenotypic sequence diversity with functional VP1-VP2 interaction(s) during dsRNA synthesis. Using bioinformatic approaches, we revealed which VP1 and VP2 genotypes encode divergent proteins and identified the positional locations of amino acid changes in the context of known structural domains/subdomains. We then employed an in vitro dsRNA synthesis assay to test whether genotype R1, R2, R4, and R7 VP1 polymerases could be enzymatically-activated by genotype C1, C2, C4, C5, and C7 VP2 core shell proteins. Genotype combinations that were incompatible informed the rational design and in vitro testing of chimeric mutant VP1 and VP2 proteins. The results of this study connect VP1 and VP2 nucleotide-level diversity to protein-level diversity for the first time, and they provide new insights into regions/residues critical for VP1-VP2 interaction(s) during viral genome replication.

    IMPORTANCE Group A rotaviruses (RVAs) are widespread in nature, infecting numerous mammalian and avian hosts and causing severe gastroenteritis in human children. RVAs are classified using a system that assigns a genotype to each viral gene according to its nucleotide sequence. To date, 22 genotypes have been described for the gene encoding the viral polymerase (VP1) and 20 genotypes for the gene encoding the core shell protein (VP2). Here, we analyzed if/how the VP1 and VP2 proteins encoded by the known RVA genotypes differ from each other in their sequences. We also used a biochemical approach to test whether the intergenotypic sequence differences influenced how VP1 and VP2 functionally engage each other to mediate RNA synthesis in a test tube. This work is important because it increases our understanding of RVA protein-level diversity and raises new ideas about VP1-VP2 binding interface(s) that are important for viral replication.

  • A genome-wide Epstein-Barr virus polyadenylation map and its antisense RNA to EBNA [Genome Replication and Regulation of Viral Gene Expression]

  • Epstein-Barr virus (EBV) is a ubiquitous human pathogen associated with Burkitt lymphoma and nasopharyngeal carcinoma. Although the EBV genome encodes more than a hundred genes, a full transcription map with EBV polyadenylation profiles remains unknown. To elucidate the 3rrsquo; ends of all EBV transcripts genome-wide we performed the first comprehensive analysis of viral polyadenylation sites (pA sites) using our published PA-seq technology. We identified that EBV utilizes a total of 62 pA sites in JSC-1, 60 in Raji, and 53 in Akata cells for the expression of EBV genes from both plus and minus DNA strands; 42 of these pA sites are commonly used in all three cell lines. The majority of identified pA sites were mapped to the intergenic regions downstream of previously annotated EBV open reading frames (ORFs) and viral promoters. The pA sites lacking association with any known EBV genes were also identified, mostly to the minus DNA strand within the EBNA locus, a major locus responsible for maintenance of viral latency and cell transformation. The expression of these novel antisense transcripts to EBNA were verified by 3rrsquo;RACE and Northern blot analyses in several EBV+ cell lines. In contrast to EBNA RNA expressed during latency, expression of EBNA-antisense transcripts, which is restricted in latent cells, can be significantly induced by viral lytic infection, suggesting potential regulation of viral gene expression by EBNA-antisense transcription during lytic EBV infection. Our data provide the first evidence that EBV has an unrecognized mechanism that regulates EBV reactivation from latency.

    IMPORTANCE Epstein-Barr virus represents an important human pathogen with an etiological role in the development of several cancers. By elucidation of a genome-wide polyadenylation landscape of EBV in JSC-1, Raji, and Akata cells, we have re-defined the EBV transcriptome and mapped individual Pol II transcripts of viral genes to each one of the mapped pA sites at single nucleotide resolution, as well as the depth of expression. By unveiling a new class of viral lytic RNA transcripts antisense to latent EBNAs, we provide a novel mechanism of how EBV might control the expression of viral lytic genes and lytic infection. Thus, this report takes another step closer to understanding EBV gene structure and expression and paves a new path for antiviral approaches.

  • Structural basis for human norovirus capsid binding to bile acids [Structure and Assembly]

  • A recently developed human norovirus cell culture system revealed that the presence of bile enhanced or was an essential requirement for the growth of certain genotypes. Before this discovery, histo-blood group antigens (HBGAs) were the only well-studied co-factor known for human noroviruses and there was evidence that several genotypes poorly bound HBGAs. Therefore, the purpose of this study was to investigate how human norovirus capsid interact with bile acids. We found that bile acids had low micromolar affinities to GII.1, GII.10, and GII.19 capsids, but did not bind GI.1, GII.3, GII.4, or GII.17. We showed that bile acid bound at a partially conserved pocket on the norovirus capsid-protruding (P) domain using X-ray crystallography. An amino acid sequence alignment and structural analysis delivered an explanation of selective bile acid binding. Intriguingly, we discovered that binding of the bile acid was the critical step to stabilize several P domain loops that optimally placed an essential amino acid side chain (Asp375) to bind HBGAs in an otherwise HBGA non-binder (GII.1). Furthermore, bile acid enhanced HBGA binding for a known HBGA binder (GII.10). Altogether, these new data suggests that bile acid functions as a loop stabilizing regulator and enhancer of HBGA binding for certain norovirus genotypes.

    IMPORTANCE Given that human norovirus virions likely interact with bile acid during a natural infection and our evidence that an HBGA non-binder (GII.1) can be converted to an HBGA binder after bile acid binding is of major significance. Our data provides direct evidence that like HBGAs, bile acid interaction on the capsid is an important co-factor for certain genotypes. However, more unanswered questions seem to arise from these new discoveries. For example, is there an association between the bile acid requirement and prevalence of certain genotypes? That is, the GII.1 and GII.10 (bile acid binders) genotypes rarely caused outbreaks, whereas the GII.4 and GII.17 genotypes (bile acid non-binders) were responsible for large epidemics. Therefore, it seems plausible that certain genotypes require bile acids, whereas others have modified their bile acid requirements on the capsid.

  • Minute Virus of Canines NP1 protein interacts with the cellular factor CPSF6 to regulate viral alternative RNA processing [Genome Replication and Regulation of Viral Gene Expression]

  • The NP1 protein of minute virus of canines (MVC) governs production of the viral capsid proteins via its role in pre-mRNA processing. NP1 suppresses polyadenylation and cleavage at its internal site (pA)p to allow accumulation of RNAs that extend into the capsid gene, and it enhances splicing of the upstream adjacent 3rd intron, which is necessary to properly enter the capsid protein open reading frame. We find the (pA)p region to be complex. It contains redundant classical cis-acting signals necessary for the cleavage and polyadenylation reaction and splicing of the adjacent upstream 3rd intron, as well as regions outside of the classical motifs that were necessary for responding to NP1. NP1, but not processing mutants of NP1, bound to MVC RNA directly. The cellular RNA processing factor CPSF6 interacted with NP1 in transfected cells and participated with NP1 to modulate its effects. These experiments further characterize the role of NP1 in parvovirus gene expression.

    IMPORTANCE The Parvovirinae are small non-enveloped icosahedral viruses that are important pathogens in many animal species including humans. Unlike other parvoviruses, the bocavirus genus controls expression of its capsid proteins via alternative RNA processing, by both suppressing polyadenylation at an internal site, (pA)p, and facilitating splicing of an upstream adjacent intron. This regulation is mediated by a small genus-specific protein, NP1. Understanding the cis-acting targets of NP1, as well as the cellular factors with which it interacts, is necessary to more clearly understand this unique mode of parvovirus gene expression.

  • Encapsidation of viral RNA in Picornavirales: studies on cowpea mosaic virus demonstrate dependence on viral replication [Structure and Assembly]

  • To elucidate linkage between replication and encapsidation in Picornavirales, we have taken advantage of the bipartite nature of the plant-infecting member of the order, cowpea mosaic virus (CPMV), to decouple the two processes. RNA-free virus-like particles (eVLPs) can be generated by transiently co-expressing the RNA-2-encoded coat protein precursor (VP60) with the RNA-1-encoded 24K protease, in the absence of the replication machinery (Saunders et al., Virology 393, 329nndash;337, 2009). We have made use of the ability to produce assembled capsids of CPMV in the absence of replication to examine the putative linkage between RNA replication and packaging in the Picornavirales. We have created a series of mutant RNA-1 and RNA-2 molecules and have assessed the effect of the mutations on both the replication and packaging of the viral RNAs. We demonstrate that mutations that affect replication have a concomitant impact on encapsidation, and that RNA-1 -mediated replication is required for encapsidation of both RNA-1 and RNA-2. This close coupling between replication and encapsidation provides a means for the specific packaging of viral RNAs. Moreover, we demonstrate that this feature of CPMV can be used to specifically encapsidate custom RNA by placing a sequence of choice between the RNA-2 sequences required for replication.

    IMPORTANCE The mechanism whereby members of the order Picornavirales specifically package their genomic RNAs is poorly understood. Research with monopartite members of the order, such as poliovirus, have indicated that packaging is linked to replication, though the presence of "packaging signals" along the length of the viral RNA has also been suggested. Thanks to the bipartite nature of the CPMV genome which allows the manipulation of RNA-1 without modifying RNA-2, we show here that this specificity is due to a functional linking between the two processes of viral replication and encapsidation. This has important implications for our understanding of the fundamental molecular biology of Picornavirales,and opens the door to novel research and therapeutic applications in the field of custom RNA packaging and delivery technologies.

  • Isolation of neutralizing monoclonal antibodies to human astrovirus and characterization of virus variants that escape neutralization [Structure and Assembly]

  • Human astroviruses (HAstVs) cause severe diarrhea and represent an important health problem in children under two years of age. Despite their medical importance, the study of these pathogens has been neglected. To better understand the astrovirus antigenic structure and the basis of protective immunity, in this work we produced a panel of neutralizing monoclonal antibodies (Nt-MAbs) to HAstV serotypes 1, 2, and 8, and identified the mutations that allow the viruses to escape neutralization. We first tested the capacity of the recombinant HAstV capsid core and spike domains to elicit Nt-Ab. Hyperimmunization of animals with the two domains showed that although both induced a potent immune response, only the spike was able to elicit antibodies with neutralizing activity. Based on this finding, we used a mixture of the recombinant spike domains belonging to the three HAstV serotypes to immunize mice. Five Nt-MAbs were isolated and characterized; all of them were serotype-specific, two were directed to HAstV-1, one to HAstV-2, and two to HAstV-8. These antibodies were used to select single and double neutralization-escape variant viruses, and determination of the amino acid changes that allow the viruses to escape neutralization permitted us to define the existence of four potentially independent neutralization epitopes on the HAstV capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies, and tools to explore the possibility to develop a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry.

    Relevance Human astroviruses (HAstVs) are common etiological agents of acute gastroenteritis in children, the elderly, and immunocompromised patients; some virus strains have also been associated with neurological disease. Despite their medical importance, the study of these pathogens has advanced at a slow pace. In this work, we produced neutralizing antibodies to the virus and mapped the epitopes they recognize on the virus capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies, as well as tools to explore the development of a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry.

  • Plasticity of amino acid residue 145 near the receptor binding site of H3 swine influenza A viruses and its impact on receptor binding and antibody recognition. [Genetic Diversity and Evolution]

  • The hemagglutinin (HA), a glycoprotein on the surface of influenza A virus (IAV), initiates the virus life cycle by binding to terminal sialic acid (SA) residues on host cells. The HA gradually accumulates amino acid (aa) substitutions that allow IAV to escape immunity through a mechanism known as antigenic drift. We recently confirmed that a small set of aa residues are largely responsible for driving antigenic drift in swine-origin H3 IAV. All identified residues are located adjacent to the HA receptor binding site (RBS), suggesting that substitutions associated with antigenic drift may also influence receptor binding. Among those substitutions, residue 145 was shown to be a major determinant of antigenic evolution. To determine whether there are functional constraints to substitutions near the RBS and their impact on receptor binding and antigenic properties, we carried out site-directed mutagenesis experiments at the single aa level. We generated a panel of viruses carrying substitutions at residue 145 representing all 20 amino acids. Despite limited amino acid usage in nature, most substitutions at residue 145 were well tolerated without major impact on virus replication in vitro. All substitutions retained receptor binding specificity, but frequently led to decreased receptor binding. Glycan microarray analysis showed that substitutions at residue 145 modulate binding to a broad range of glycans. Furthermore, antigenic characterization identified specific substitutions at residue 145 that altered antibody recognition. This work provides a better understanding of the functional effects of aa substitutions near the RBS and the interplay between receptor binding and antigenic drift.

    IMPORTANCE The complex and continuous antigenic evolution of IAVs remains a major hurdle for vaccine selection and effective vaccination. On the virusrrsquo; hemagglutinin (HA) of the H3N2 IAVs, the aa substitution N145K causes significant antigenic changes. We show that aa 145 displays remarkable amino acid plasticity in vitro tolerating multiple aa substitutions, many of which have not yet been observed in nature. Mutant viruses carrying substitutions at residue 145 showed no major impairment on virus replication in the presence of lower receptor binding avidity. However, their antigenic characterization confirmed the impact of the 145K substitution in antibody immunodominance. We provide a better understanding of the functional effects of aa substitutions implicated in antigenic drift and its consequences on receptor binding and antigenicity. The mutation analyses presented in this report represent a significant dataset to aid and test computational approachesrrsquo; ability to predict binding of glycans and in antigenic cartography analyses.

  • Released tryptophanyl-tRNA synthetase stimulates innate immune responses against viral infection [Cellular Response to Infection]

  • Tryptophanyl-tRNA synthetase (WRS) is one of the aminoacyl-tRNA synthetases (ARSs) that possesses non-canonical functions. Full-length WRS is released during bacterial infection and primes the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex to elicit innate immune responses. However, the role of WRS in viral infection remains unknown. Here, we show that full-length WRS is secreted by immune cells in the early phase of viral infection and functions as an antiviral cytokine. Treatment of cells with recombinant WRS protein promotes the production of inflammatory cytokines and type 1 interferons (IFNs) and curtails virus replication in THP-1 and Raw264.7 cells, but not in TLR4-/- or MD2-/- bone marrow-derived macrophages (BMDMs). Intravenous and intranasal administration of recombinant WRS protein induces an innate immune response and blocks viral replication in vivo. These findings suggest that secreted full-length WRS has a non-canonical role in inducing innate immune responses to viral infection as well as to bacterial infection.

    IMPORTANCE ARSs are essential enzymes in translation that link specific amino acids to their cognate tRNAs. In higher eukaryotes, some ARSs possess additional, non-canonical functions in the regulation of cell metabolism. Here, we report a novel non-canonical function of WRS in antiviral defense. WRS is rapidly secreted in response to viral infection and primes the innate immune response by inducing the secretion of pro-inflammatory cytokines and type 1 IFNs, resulting in the inhibition of virus replication both in vitro and in vivo. Thus, we consider WRS to be a member of the antiviral innate immune response. The results of this study enhance our understanding of host defense systems and provide additional information on the non-canonical functions of ARSs.

  • A microRNA derived from adenovirus virus-associated RNAII promotes virus infection via post-transcriptional gene silencing [Virus-Cell Interactions]

  • The adenovirus (Ad) serotype 5 genome encodes two non-coding small RNAs (virus-associated RNAs: VA-RNAI and II), which are approximately 160nt-long RNAs transcribed by RNA polymerase III. It is well-known that VA-RNAI supports Ad infection via the inhibition of double-stranded RNA-dependent protein kinase (PKR), which recognizes double-stranded RNA and acts as an antiviral system. Recent studies revealed that VA-RNAs are processed into VA-RNA-derived microRNAs (miRNAs) (mivaRNAI, II); however, we and another group recently demonstrated that mivaRNAI does not promote Ad replication. On the other hand, the roles of VA-RNAII and VA-RNAII-derived miRNA (mivaRNAII) in Ad replication have remained to be clarified. In this study, we demonstrate mivaRNAII-mediated promotion of Ad replication. Transfection with chemically synthesized 3'-mivaRNAII-138, one of the most abundant mivaRNAII, significantly enhanced Ad replication, while the other species of mivaRNAII did not. We identified 8 putative target genes of 3'-mivaRNAII-138 by microarray analysis and in silico analysis. Among the 8 candidates, knockdown of the cullin4A (CUL4A) gene, which encodes a component of the ubiquitin ligase complex, most significantly enhanced Ad replication. CUL4A expression was significantly suppressed by 3'-mivaRNAII-138 via post-transcriptional gene silencing, indicating that CUL4A is a target gene of 3'-mivaRNAII-138 and mivaRNAII functions as a viral miRNA promoting Ad infection. It has been reported that CUL4A is involved in degradation of c-Jun, which acts as a transcription factor in the Jun-N-terminal kinase (JNK) signaling cascade. Treatment with JNK inhibitors dramatically suppressed Ad replication, suggesting that mivaRNAII-mediated down-regulation of CUL4A enhanced JNK signaling, and thereby promoted Ad infection.

    IMPORTANCE Several types of viruses encode viral miRNAs which regulate host and/or viral gene expression via post-transcriptional gene silencing, leading to efficient viral infection. Adenovirus (Ad) expresses miRNAs derived from VA-RNAs (mivaRNAI, II); however, recent studies have revealed that processing of VA-RNAI into mivaRNAI inhibits Ad replication. Conversely, we demonstrated here that mivaRNAII significantly promotes Ad replication, and that mivaRNAII-mediated suppression of CUL4A expression via post-transcriptional gene silencing induces accumulation of c-Jun, leading to promotion of Ad infection. These results exhibited the significance of VA-RNAII for supporting Ad infection through a mechanism complementary to that of VA-RNAI. These observations could provide important clues toward a new perspective on host-virus interaction. Moreover, Ad is widely used as a basic framework for viral vectors and oncolytic viruses. Our findings will help to regulate Ad infection, and will promote the development of novel Ad vectors and oncolytic Ad.

  • BCL6 Inhibitor-mediated Downregulation of pSAMHD1 and T Cell Activation Are Associated with Decreased HIV Infection and Reactivation [Vaccines and Antiviral Agents]

  • Clearance of HIV-infected germinal center (GC) CD4+ follicular helper T cells (Tfh) after combination antiretroviral therapy (ART) is essential to an HIV cure. Blocking B cell lymphoma 6 (BCL6, the master transcription factor for Tfh cells) represses HIV infection of tonsillar CD4+ Tfh ex vivo, reduces GC formation and limits immune activation in vivo. We assessed the anti-HIV activity of a novel BCL6 inhibitor, FX1, in Tfh/non-Tfh CD4+ T cells, and its impact on T cell activation and SAMHD1 phosphorylation (Thr592). FX1 repressed HIV-1 infection of peripheral CD4+ T cells, tonsillar Tfh/non-Tfh CD4+ T cells (pllt;0.05), total elongated-and multi-spliced HIV-1 RNA production during the first round of viral life cycle (pllt;0.01). Using purified circulating CD4+ T cells from uninfected donors, we demonstrate that FX1 treatment resulted in down-regulation pSAMHD1 expression (pllt;0.05) and T cell activation (HLA-DR, CD25, Ki67, pllt;0.05) ex vivo corresponding with inhibition of HIV-1 and HIV-2 replication. Ex vivo HIV-1 reactivation using purified peripheral CD4+ T cells from HIV-infected ART-suppressed donors was also blocked by FX1 treatment (pllt;0.01). Our results indicate that BCL6 function contributes to Tfh/non-Tfh CD4+ T cell activation and cellular susceptibility to HIV infection. BCL6 inhibition represents a novel therapeutic strategy to potentiate HIV suppression in Tfh/non-Tfh CD4+ T cells without reactivation of latent virus.

    IMPORTANCE The expansion and accumulation of HIV-infected BCL6+ Tfh CD4+ T cells are thought to contribute to the persistence of viral reservoirs in infected subjects undergoing ART. Two mechanisms have been raised for the preferential retention of HIV within Tfh CD4+ T cells: (a) antiretroviral drugs have limited tissue distribution, resulting in insufficient tissue concentration and lower efficacy in controlling HIV replication in lymphoid tissues; (b) cytotoxic CD8+ T cells within lymphoid tissues express low levels of chemokine receptor (CXCR5) thus limiting their ability to enter the GCs to control/eliminate HIV-infected Tfh cells. Our results indicate that the BCL6 inhibitor FX1 can not only repress HIV infection of tonsillar Tfh ex vivo, but also suppress HIV infection and reactivation in primary, non-Tfh CD4+ T cells. Our study provides a rationale for targeting BCL6 protein to extend ART-mediated reduction of persistent HIV and/or support strategies towards HIV remission beyond ART cessation.

  • The Impact of Replication Stress in Human Papillomavirus Pathogenesis [Gem]

  • Inactivation of critical cell cycle checkpoints by the HPV oncoprotein E7 results in replication stress (RS) that leads to genomic instability in premalignant lesions. Intriguingly, RS tolerance is achieved through several mechanisms, allowing HPV to exploit the cellular RS response for viral replication and to facilitate viral persistence in the presence of DNA damage. As such, inhibitors of the RS response pathway may provide a novel approach to target HPV-associated lesions and cancers.

  • Eurasian avian-like swine influenza A viruses escape human MxA restriction by distinct mutations in their nucleoprotein [Genetic Diversity and Evolution]

  • To cross the human species barrier, influenza A viruses (IAV) of avian origin have to overcome the interferon-induced host restriction factor MxA by acquiring distinct mutations in their nucleoprotein (NP). We recently demonstrated that North American classical swine IAV are able to escape MxA restriction partially. Here, we investigated whether the Eurasian avian-like swine IAV lineage currently circulating in European swine would likewise evade restriction by human MxA. We found that the NP of the isolate A/swine/Belzig/2/2001 (Belzig) exerts increased MxA escape similar in extent to human IAV NPs. Mutational analysis revealed that the MxA escape mutations in Belzig-NP differ from the known MxA resistance cluster of the North American classical swine lineage and human-derived IAV NPs. A mouse-adapted avian IAV of the H7N7 subtype encoding Belzig-NP showed significantly enhanced viral growth in both MxA-expressing cells and MxA-transgenic mice compared to control viruses lacking the MxA escape mutations. Similarly, growth of recombinant Belzig virus was only marginally affected in MxA-expressing cells and MxA-transgenic mice compared to Belzig mutant viruses lacking MxA escape mutations in NP. Phylogenetic analysis of the Eurasian avian-like swine IAV revealed that the NP amino acids required for MxA escape were acquired successively and were maintained after their introduction. Our results suggest that circulation of IAV in the swine population can result in the selection of NP variants with a high degree of MxA resistance, thereby increasing the zoonotic potential of these viruses.

    SIGNIFICANCE The human MxA protein efficiently blocks replication of IAV from non-human species. In rare cases, however, these IAV overcome the species barrier and become pandemic. All known pandemic viruses have acquired and maintained MxA escape mutations in the viral NP and are thus not efficiently controlled by MxA. Intriguingly, partial MxA resistance can also be acquired in other hosts that express antivirally active Mx proteins such as swine. To perform a risk assessment of IAV circulating in the European swine population, we analyzed the degree of MxA resistance of Eurasian avian-like swine IAV. Our data demonstrate that these viruses carry yet undescribed Mx resistance mutations in NP that mediate efficient escape from human MxA. We conclude that Eurasian avian-like swine IAV possess a substantial zoonotic potential.

  • DUSP10 Negatively Regulates the Inflammatory Response to Rhinovirus Through IL-1{beta} Signalling. [Cellular Response to Infection]

  • Rhinoviral infection is a common trigger of the excessive inflammation observed during exacerbations of asthma and chronic obstructive pulmonary disease. Rhinovirus (RV) recognition by pattern recognition receptors activates the MAPK pathways, common inducers of inflammatory gene production. A family of dual-specificity phosphatases (DUSPs) can regulate MAPK function, but their roles in rhinoviral infection are not known. We hypothesised that DUSPs would negatively regulate the inflammatory response to RV infection. Our results revealed that p38 and JNK MAPKs play key roles in the inflammatory response of epithelial cells to RV infection. Three DUSPs previously shown to have roles in innate immunity, 1, 4 and 10, were expressed in primary bronchial epithelial cells, one of which, DUSP10, was down regulated by RV infection. Small interfering-RNA knock down of DUSP10 identified a role for the protein in negatively regulating inflammatory cytokine production in response to IL-1bbeta; alone and in combination with RV, without any effect on RV replication. This study identifies DUSP10 as an important regulator of airway inflammation in respiratory viral infection.

    Importance Rhinoviruses are one of the causes of the common cold. In patients with asthma or chronic obstructive pulmonary disease, viral infections, including rhinovirus, are the commonest cause of exacerbations. Novel therapeutics to limit viral inflammation are clearly required. The work presented here identifies DUSP10 as an important protein involved in limiting the inflammatory response in the airway without affecting immune control of the virus.

  • Acute infection and subsequent subclinical reactivation of herpes simplex virus-2 after vaginal inoculation of rhesus macaques [Pathogenesis and Immunity]

  • Herpes simplex virus-2 (HSV-2) is a common sexually transmitted infection with a highly variable clinical course. Many infections quickly become subclinical, with episodes of spontaneous virus reactivation. To study the host/HSV-2 interactions an animal model of subclinical HSV-2 infection is needed. In an effort to develop a relevant model, rhesus macaques (RM) were inoculated intravaginally with two or three HSV-2 strains (186, 333 and/or G); total dose 1x107 pfu HSV-2 per animal. Infectious HSV-2 and HSV-2 DNA was consistently shed in vaginal swabs for the first 7-14 days after each inoculation. Proteins associated with wound healing, innate immunity and inflammation were significantly increased in cervical secretions immediately after HSV-2 inoculation. Histologic evidence of acute herpesvirus pathology including: acantholysis in the squamous epithelium, ballooning degeneration of, and intranuclear inclusion bodies, in epithelial cells, with HSV antigen in mucosal epithelial cells and keratinocytes. Further, an intense inflammatory infiltrate was found in the cervix and vulva. Evidence of latent infection and reactivation was demonstrated by the detection of spontaneous HSV-2 shedding post-acute inoculation (102-103 DNA copies/swab) in 80% of RM. Further, HSV-2 DNA was detected in ganglia in most necropsied animals. HSV-2-specifc T-cell responses were detected in all animals albeit antibodies to HSV-2 were detected in only 30% of the animals. Thus, HSV-2 infection of RM recapitulates many of the key features of subclinical HSV-2 infection in women, but seems to be more limited, as virus shedding was undetectable more than 40 days after the last virus inoculation.

    Importance Herpes simplex virus-2 (HSV-2) infects nearly 500 million persons globally with an estimated 21 million incident cases each year, making it one of the most common sexually transmitted infections (STIs). HSV-2 is associated with increased human immunodeficiency virus-1 (HIV-1) acquisition and this risk does not decline with the use of anti-herpes drugs. As initial acquisition of both HIV and HSV-2 infections is subclinical, study of the initial molecular interactions of the two agents require an animal model. We found that HSV-2 can infect RMs after vaginal inoculation, establish latency in the nervous system and spontaneously reactivate which mimics some of the key features of HSV-2 infection in women. Studying HSV-2 infected RM may provide an animal model to develop strategies to prevent HSV-2 acquisition and reactivation.

  • Dynamics of Virus-Specific Memory B Cells and Plasmablasts following CNS Viral Infection [Cellular Response to Infection]

  • Humoral responses within the central nervous system (CNS) are common to many neurotropic viral infections, with antibody (Ab) secreting cells (ASC) contributing to local protection. However, a role for virus-specific memory B cells (Bmem) within the CNS is poorly explored due to lack of robust phenotypic or functional identification in mice. This study takes advantage of mice expressing tamoxifen-inducible Cre recombinase (Cre-ERT2) under the Aicda promoter crossed with Rosa26-loxP-tdTomato reporter mice (AIDCre-Rosa26tdTomato) to monitor B cells having undergone activation-induced cytidine deaminase (AID)-mediated somatic hypermutation (SHM) following neurotropic coronavirus infection. AID detection via tdTomato expression allowed tracking of virus-specific ASC and Bmem in priming and effector sites throughout infection. In draining lymph nodes tdTomato+ ASC were most prevalent prior to germinal center (GC) formation, but total tdTomato+ B cells only peaked with robust GC formation at day 14 p.i. Moreover, their proportion of Bmem dominated over ASC throughout infection. In the CNS tdTomato+ cells started emerging at day 14 p.i. While they initially comprised mainly Bmem, the proportions of ASC and Bmem became similar as tdTomato+ B cells increased throughout viral persistence. Delayed tamoxifen treatment demonstrated ongoing CNS recruitment of tdTomato+ B cells, mainly ASC, primed late during GC reactions. Overall, the data support that virus-induced B cells exhibiting SHM require peripheral GC formation to emerge in the CNS. Ongoing GC reactions and regional signals further regulate dynamics within the CNS, with preferential maintenance of tdTomato+ B cells in spinal cords relative to brains during viral persistence.

    Importance The prevalence and role of Ag-specific Bmem in the CNS is largely undefined during viral encephalomyelitis. Lack of reliable markers identifying murine Bmem has made it difficult to assess their contribution to local anti-viral protection via antigen presentation or conversion to ASC. Using reporter mice infected with neurotropic coronavirus to track virus-specific Bmem and ASC, this report demonstrates that both subsets only emerge in the CNS following peripheral GC formation and subsequently prevail. While early GC reactions supported preferential Bmem accumulation in the CNS, late GC reactions favored ASC accumulation, although Bmem outnumbered ASC in draining lymph nodes throughout infection. Importantly, virus-specific B cells undergoing sustained GC selection were continually recruited to the persistently-infected CNS. Elucidating the factors governing temporal events within GCs, as well as regional CNS cues during viral persistence, will aid intervention to modulate CNS humoral responses in the context of infection and associated autoimmune pathologies.

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

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

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

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