Virus Imaging

Select by virus name
About Images
Art Gallery
Covers Gallery
ICTV 8th Color Plates
PS10 Screen Saver   

Virus Structure Tutorials

Triangulation Number
Topography Maps 3D


Virology Links

In the News

- News -
- Video -
- Blogs -
 * Virology Highlights
- Flu & H1N1 - (CDC|WHO)

Journal Contents

Science
Nature
Nature Structural & Molecular Biology

Structure & Assembly (J.Virol)
Journal of Virology
J. General Virology
Retrovirology
Virology
Virology Journal
Virus Genes
Viruses

Educational Resouces


Video Lectures  NEW 
TextBook  NEW 
Educational Links
Educational Kids

Legacy

Archived Web Papers

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]

  • Functional Analysis and Antivirulence 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 health care-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_AbaM_B9) which specifically infects and makes lysis from without in strains of the K45 and K30 capsule types, 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 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 was demonstrated to be nontoxic 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 case with 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. Here 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 characterized 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 the 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.

  • 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 HEV genotype 3 (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 (oriented toward the blood) and apical (oriented toward the 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 cells. F2 cells cultured on semipermeable 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 NP-40 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 open reading frame 2 (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 into bile, while the small fraction released into blood could spread HEV throughout the host.

    IMPORTANCE Hepatitis E virus (HEV) in stools is naked, while that in culture supernatants and patientsrrsquo; blood is lipid associated. Its life cycle in hepatocytes, polarized cells with a basolateral side communicating with blood and an apical side connected with bile, is incompletely understood. We have developed a polarized hepatocyte model and used the cells to analyze the supernatants bathing the apical and basolateral sides and HEV subcellular distribution. HEV particles from both sides were lipid associated, and most infectious HEV particles left the cell via its apical side. Similar amounts of the open reading frame 2 (ORF2) soluble capsid protein were secreted from both sides of the hepatocytes. This model mimicking physiological conditions should help clarify the HEV cell cycle in polarized hepatocytes.

  • Cellular RNA Helicase DHX9 Interacts with the Essential Epstein-Barr Virus (EBV) Protein SM and Restricts EBV Lytic Replication [Virus-Cell Interactions]

  • Epstein-Barr virus (EBV) SM protein is an RNA-binding protein that has multiple posttranscriptional gene regulatory functions essential for EBV lytic replication. In this study, we identified an interaction between SM and DHX9, a DExH-box helicase family member, by mass spectrometry and coimmunoprecipitation. DHX9 participates in many cellular pathways involving RNA, including transcription, processing, transport, and translation. DHX9 enhances virus production or infectivity of a wide variety of DNA and RNA viruses. Surprisingly, an increase in EBV late gene expression and virion production occurred upon knockdown of DHX9. To further characterize the SM-DHX9 interaction, we performed immunofluorescence microscopy of EBV-infected cells and found that DHX9 partially colocalized with SM in nuclear foci during EBV lytic replication. However, the positive effect of DHX9 depletion on EBV lytic gene expression was not confined to SM-dependent genes, indicating that the antiviral effect of DHX9 was not mediated through its effects on SM. DHX9 enhanced activation of innate antiviral pathways comprised of several interferon-stimulated genes that are active against EBV. SM inhibited the transcription-activating function of DHX9, which acts through cAMP response elements (CREs), suggesting that SM may also act to counteract DHX9rrsquo;s antiviral functions during lytic replication.

    IMPORTANCE This study identifies an interaction between Epstein-Barr virus (EBV) SM protein and cellular helicase DHX9, exploring the roles that this interaction plays in viral infection and host defenses. Whereas most previous studies established DHX9 as a proviral factor, we demonstrate that DHX9 may act as an inhibitor of EBV virion production. DHX9 enhanced innate antiviral pathways active against EBV and was needed for maximal expression of several interferon-induced genes. We show that SM binds to and colocalizes DHX9 and may counteract the antiviral function of DHX9. These data indicate that DHX9 possesses antiviral activity and that SM may suppress the antiviral functions of DHX9 through this association. Our study presents a novel host-pathogen interaction between EBV and the host cell.

  • 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 short interfering RNA-mediated knockdown of RHA significantly reduced the amounts of infectious viral particles in various cells. The RHA knockdown reduced the multistep 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 bound to the DENV RNA and associated with three nonstructural proteins, including NS1, NS2B3, and NS4B. Further study showed that different domains of RHA mediated 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 a 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.

  • 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 Glycoprotein B5 [Structure and Assembly]

  • An interaction between the orthopoxvirus glycoproteins A34 and B5 has been reported. The transmembrane and ectodomain of A34 are 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 to 80 as sufficient for interaction with B5. Additional truncations identified residues 80 to 130 of A34 as sufficient for interaction with B5. To better understand the function of this region, a set of recombinant viruses expressing A34 with the full, partial, or no B5 interaction site (residues 1 to 130, 1 to 100, and 1 to 70, respectively) was constructed. All the recombinants expressing truncations of A34 incorporated B5 into extracellular virions but had a small-plaque phenotype similar to that of 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 nonfusogenic dissolution. Taken together, these results suggest that residues 80 to 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 mislocalized and not incorporated into extracellular virions. Here, using a transient-transfection assay, residues 80 to 130 of the ectodomain of A34 were determined to be sufficient for interaction with B5. Recombinant viruses expressing 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 extracellular virion (EV) dissolution. This study is the first to directly demonstrate that A34 is involved in cell binding and implicate the ectodomain in this role.

  • 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 double-stranded RNA (dsRNA) bacteriophage 6 (wild type and two isogenic host range mutants) on the novel host Pseudomonas syringae pv. atrofaciens. Both Sanger sequencing of 50 P. syringae pv. atrofaciens mutant clones for each genotype and population Illumina sequencing revealed the same high-frequency mutations allowing infection of P. syringae pv. atrofaciens. Wild-type 6 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 wild-type 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 wild-type 6 clones had nonsynonymous mutations in p12, and 2 others had point mutations in p9 and p5. None of these genes had previously been associated with host range expansion in 6. 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 6), we studied the impact of preexisting host range mutations on another host range expansion. Results from both clonal Sanger and Illumina sequencing show that extant host range mutations dramatically narrow the neighborhood of potential host range mutations compared to that of wild-type 6. 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 6 host range expansion, expanding our knowledge of this important model system in experimental evolution.

  • 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 superinfection, 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 a 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 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 A few individuals can control HIV infection without the need for antiretroviral treatment and are referred to as HIV controllers. We have studied HIV controllers who suddenly lose 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 naive 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 it may offer an important tool to anticipate viral rebound in individuals in clinical studies that include combination antiretroviral therapy (cART) treatment interruptions and which, if not treated quickly, could pose a significant risk to the trial participants.

  • 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 target human immunodeficiency virus type 1 (HIV-1) gp120 and block CD4-induced conformational changes required for viral entry. It is now in phase III clinical trials and is expected to be approved by the U.S. Food and Drug Administration (FDA) in the near future. Although fostemsavir is very potent against HIV in vitro and in vivo, a number of resistant mutants have already been 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 retained 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 (CD4bs)-targeting bNAbs in neutralizing HIV-1 strains at low concentrations. This synergistic effect was enhanced against virus harboring mutations conferring resistance to BMS-626529. The mechanistic basis of the observed synergy is likely enhanced inhibition of CD4 binding to the 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 against HIV 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, the attachment inhibitors, 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.

  • Spatiotemporal Differences in Presentation of CD8 T Cell Epitopes during Hepatitis B Virus Infection [Virus-Cell Interactions]

  • Distinct populations of hepatocytes infected with hepatitis B virus (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 levels of activation of HBV-specific CD8+ T cells. Using antibodies specific to two distinct HLA-A*02:01/HBV epitope complexes of HBV nucleocapsid and envelope proteins, we mapped their topological distributions in liver biopsy specimens of two anti-hepatitis B e antigen-positive (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 the presence of gamma interferon (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 efficacies.

    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 is lacking. In this work, analysis of CHB patient liver parenchyma and in vitro HBV infection models shows a nonuniform distribution of HBV CD8+ T cell epitopes that is influenced by the 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.

  • 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 in 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-naive 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, a median inhibitory dilution (ID50) value of 1,246, 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 the 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. To 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 under way 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 exhibited 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 provide a better understanding of their role in controlling HIV-1 infection.

  • 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-reactive 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) was evident for an attached substrate but not for unattached small-molecule cleavage of sialic acid. This finding 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(ab')2 fragments that occupied reduced area without loss of avidity or disrupted HA/NA interactions showed 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 protection mediated by HA stalk-reactive antibodies.

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

  • 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 their critical role during virus-host interactions, the mechanisms regulating the 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 virus. 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 infected cells 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.

    IMPORTANCE 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 engages 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 interactions occurring within infected cells and highlights distinct functional roles for cells as either initiators of immunity or producers and perpetuators of viral particles depending on their content of viral genomes and their intracellular localization.

  • A 2.8-Angstrom-Resolution Cryo-Electron Microscopy 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 characterized by significant immune activation and subsequent tissue damage in neonates. Strategies to limit infection have been unsuccessful due to inadequate molecular diagnostic tools for early detection and the lack of a vaccine or specific antiviral therapy. Toward 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-electron microscopy (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 Human parechovirus 3 (HPeV3) is receiving increasing attention as a prevalent cause of sepsis-like symptoms in neonates, for 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. Toward 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 interactions and viral pathogenesis mechanisms and for finding potential cures for infection and disease.

  • Oncogenic Kaposis Sarcoma-Associated Herpesvirus 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 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 levels, 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 levels, 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.

  • 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 ratio of symptomatic to asymptomatic CHIKV infections and identifies risk factors of infection. In October to November 2015, 60 symptomatic CHIKV-infected children were enrolled as index cases and prospectively monitored, alongside 236 household contacts, in an index cluster study. Samples were collected upon enrollment and on day 14 or 35 and tested by real-time reverse transcription-PCR (rRT-PCR), IgM capture enzyme-linked immunosorbent assays (IgM-ELISAs), and inhibition ELISAs to detect pre- and postenrollment 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 (multivariable-adjusted risk ratio [aRR], 1.58) increased the risk of CHIKV infection, whereas gge;4 municipal trash collections/week (aRR, 0.38) and having externally piped water (aRR, 0.52) protected against CHIKV infection. Among 63 active and incident infections, 31 (49% [95% confidence interval {CI}, 36%, 62%]) were asymptomatic, yielding a ratio of symptomatic to asymptomatic infections of 1:0.97 (95% CI, 1:0.56, 1:1.60). Although our estimate is outside the 3% to 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 East/Central/South African (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 the public health impact of CHIKV. This index cluster study identified and monitored persons at risk of infection, 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% to 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% to 28% range. Bayesian and simulation analyses, informed by a systematic literature search, revealed that the percentage of inapparent infections in epidemic settings varies by CHIKV phylogenetic lineage. Our study quantifies and provides the first epidemiological evidence that chikungunya epidemic characteristics are strongly influenced by CHIKV lineage.

  • 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, and 355 and found that NAb responses induced by the 241-glycan-bearing BG505 trimers were frequently redirected to the newly opened sites. The strongest evidence for redirection of the NAb response to neoepitopes, 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 underoccupied, we found some evidence for redirection of the NAb response to a neoepitope 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.

  • Directed Nucleosome Sliding during the Formation of the Simian Virus 40 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 mapped the locations of nucleosomes containing acetylated or methylated lysines in the histone tails of H3 and H4 present in the chromatin from 48-h-postinfection 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 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 cycles, since chromatin structure plays a major role in the regulation of the life cycle. In order to determine the role of SV40 chromatin structure late in infection, we mapped the locations of nucleosomes and their histone tail modifications in SV40 minichromosomes and in the SV40 chromatin found in virions using chromatin immunoprecipitation-DNA sequencing (ChIP-Seq). We have identified a novel viral transcriptional control mechanism in which a nucleosome found in the regulatory region of the 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.

  • Verdinexor (KPT-335), a Selective Inhibitor of Nuclear Export, Reduces Respiratory Syncytial Virus Replication In Vitro [Vaccines and Antiviral Agents]

  • Respiratory syncytial virus (RSV) is a leading cause of hospitalization of infants and young children, causing considerable respiratory disease and repeat infections that may lead to chronic respiratory conditions such as asthma, wheezing, and bronchitis. RSV causes ~34 million new episodes of lower respiratory tract illness (LRTI) in children younger than 5 years of age, with ggt;3 million hospitalizations due to severe RSV-associated LRTI. The standard of care is limited to symptomatic relief as there are no approved vaccines and few effective antiviral drugs; thus, a safe and efficacious RSV therapeutic is needed. Therapeutic targeting of host proteins hijacked by RSV to facilitate replication is a promising antiviral strategy as targeting the host reduces the likelihood of developing drug resistance. The nuclear export of the RSV M protein, mediated by the nuclear export protein exportin 1 (XPO1), is crucial for RSV assembly and budding. Inhibition of RSV M protein export by leptomycin B correlated with reduced RSV replication in vitro. In this study, we evaluated the anti-RSV efficacy of Verdinexor (KPT-335), a small molecule designed to reversibly inhibit XPO1-mediated nuclear export. KPT-335 inhibited XPO1-mediated transport and reduced RSV replication in vitro. KPT-335 was effective against RSV A and B strains and reduced viral replication following prophylactic or therapeutic administration. Inhibition of RSV replication by KPT-335 was due to a combined effect of reduced XPO1 expression, disruption of the nuclear export of RSV M protein, and inactivation of the NF-B signaling pathway.

    IMPORTANCE RSV is an important cause of LRTI in infants and young children for which there are no suitable antiviral drugs offered. We evaluated the efficacy of KPT-335 as an anti-RSV drug and show that KPT-335 inhibits XPO1-mediated nuclear export, leading to nuclear accumulation of RSV M protein and reduction in RSV levels. KPT-335 treatment also resulted in inhibition of proinflammatory pathways, which has important implications for its effectiveness in vivo.

  • 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 immunodeficiency virus (HIV) infection. However, the role of GC TFH cells in GC B cell responses following various simian immunodeficiency virus (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 adenovirus type 5 host range mutant (Ad5hr)-SIV recombinants and systemically boosted with ALVAC-SIVM766Gag/Pro/gp120-TM and SIVM766aamp;CG7V gD-gp120 proteins formulated in alum hydroxide (ALVAC/Env) or DNA encoding SIVenv/SIVGag/rhesus interleukin 12 (IL-12) plus SIVM766aamp;CG7V gD-gp120 proteins formulated in alum phosphate (DNAaamp;Env). Lymph nodes were biopsied in macaque subgroups prevaccination and at day 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 for 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 postinfection. 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 virus-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 postimmunization 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.

  • Analysis of a Subacute Sclerosing Panencephalitis Genotype B3 Virus from the 2009-2010 South African Measles Epidemic Shows That 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 analyzed 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, MIBE F did not. Both F proteins had decreased thermal stability compared to that of 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 analyzed 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 hyperfusogenic MeV F proteins is associated with infection of the brain. We also demonstrate that hyperfusogenic 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.

  • Increased Frequency of Virus Shedding by Herpes Simplex Virus 2-Infected Guinea Pigs in the Absence of CD4+ T Lymphocytes [Pathogenesis and Immunity]

  • Reactivation of herpes simplex virus 2 (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 quantitative PCR. The cumulative number of HSV-2 shedding days and the mean number of days virus was shed were 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+ gamma interferon-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+ T cells 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 include specific enhancement of HSV-specific CD4+ T cell responses.

  • 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 been shown to regulate different biological responses, such as angiogenesis, coagulation, and fibrinolysis. Here, we found that HRG almost completely abrogated the infection of Ghost cells, Jurkat cells, CD4+ T cells, and macrophages by HIV-1 at a low pH (range, 6.5 to 5.5) but not at a neutral pH. HRG was shown to interact with the heparan sulfate expressed by target cells, inhibiting an early postbinding step associated with 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 defense 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 2 (HSV-2), respectively, suggesting that HRG might display 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 to be 1 in every 1,000 episodes of sexual intercourse, reflecting the 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 2, was also markedly inhibited by HRG at low pH values, suggesting that extracellular acidosis enables HRG to display broad antiviral activity.

  • 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 with replication complexes. At a 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 mature nonstructural protein 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 reemergence of chikungunya virus (CHIKV), an alphavirus that is transmitted to humans by Aedes mosquitoes, is a serious global health threat. In the 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. Finally, we 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 on the development of antiviral strategies.

  • Early Porcine Sapovirus Infection Disrupts Tight Junctions and Uses Occludin as a Coreceptor [Virus-Cell Interactions]

  • The genus Sapovirus belongs to the family Caliciviridae, 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 roles of TJs and their proteins in sapovirus entry have 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 traveled in a complex into late endosomes via Rab5- and Rab7-dependent trafficking. Inhibition of occludin using small interfering RNA (siRNA), a specific antibody, or a dominant-negative mutant significantly blocked the entry of PSaV. Transient expression of occludin in nonpermissive 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 coreceptor 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 TJs opened 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 coreceptor. 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.

  • Development of Stable Rotavirus Reporter Expression Systems [Gene Delivery]

  • Engineered recombinant viruses expressing reporter genes have been developed for real-time monitoring of replication and for mass screening of antiviral inhibitors. Recently, we reported using a reverse genetics system to develop the first recombinant reporter rotaviruses (RVs) that expressed NanoLuc (NLuc) luciferase. Here, we describe a strategy for developing stable reporter RVs expressing luciferase and green or red fluorescent proteins. The reporter genes were inserted into the open reading frame of NSP1 and expressed as a fusion with an NSP1 peptide consisting of amino acids 1 to 27. The stability of foreign genes within the reporter RV strains harboring a shorter chimeric NSP1-reporter gene was greater than that of those in the original reporter RV strain, independent of the transgene inserted. The improved reporter RV was used to screen for neutralizing monoclonal antibodies (MAbs). Sequence analysis of escape mutants from one MAb clone (clone 29) identified an amino acid substitution (arginine to glycine) at position 441 in the VP4 protein, which resides within neutralizing epitope 5-1 in the VP5* fragment. Furthermore, to express a native reporter protein lacking NSP1 amino acids 1 to 27, the 5'- and 3'-terminal region sequences were modified to restore the predicted secondary RNA structure of the NSP1-reporter chimeric gene. These data demonstrate the utility of reporter RVs for live monitoring of RV infections and also suggest further applications (e.g., RV vaccine vectors, which can induce mucosal immunity against intestinal pathogens).

    IMPORTANCE Development of reporter RVs has been hampered by the lack of comprehensive reverse genetics systems. Recently, we developed a plasmid-based reverse genetics system that enables generation of reporter RVs expressing NLuc luciferase. The prototype reporter RV had some disadvantages (i.e., the transgene was unstable and was expressed as a fusion protein with a partial NSP1 peptide); however, the improved reporter RV overcomes these problems through modification of the untranslated region of the reporter-NSP1 chimeric gene. This strategy for generating stable reporter RVs could be expanded to diverse transgenes and be used to develop RV transduction vectors. Also, the data improve our understanding of the importance of 5'- and 3'-terminal sequences in terms of genome replication, assembly, and packaging.

  • 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 coinfecting, fully infectious homologous virus. Typically, they contain a highly deleted form of the viral genome. Utilizing single-cell analysis, here we report the discovery of a yet-unknown DIP type, derived from influenza A viruses (IAVs), 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 promoter regions, encoded proteins, and genome packaging signals. Coinfection experiments demonstrated strong interference of OP7 virus with IAV replication, manifested by a dramatic decrease in the infectivity of released virions. Moreover, an overproportional 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 coinfection with fully infectious standard virus (STV), interference with the viral life cycle can be observed, leading to suppressed STV replication and the release of mainly noninfectious DIPs. Interestingly, recent research indicates that DIPs may serve as an antiviral agent. Here we 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 those of 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 the host cell response, and may have been overlooked in other IAV preparations.

  • A Human Gain-of-Function STING Mutation Causes Immunodeficiency and Gammaherpesvirus-Induced Pulmonary Fibrosis in Mice [Pathogenesis and Immunity]

  • We previously generated STING N153S knock-in mice that have a human disease-associated gain-of-function mutation in STING. Patients with this mutation (STING N154S in humans) develop STING-associated vasculopathy with onset in infancy (SAVI), a severe pediatric autoinflammatory disease characterized by pulmonary fibrosis. Since this mutation promotes the upregulation of antiviral type I interferon-stimulated genes (ISGs), we hypothesized that STING N153S knock-in 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 the 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 that of patients with SAVI. These findings reveal that a human STING mutation creates severe immunodeficiency, leading to virus-induced lung disease in mice.

  • 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 maternofetal 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. In this study, we performed RNA sequencing 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 maternofetal interface, as evidenced by in situ hybridization/immunohistochemistry. The gene entry is coincidental with the emergence of the Hyaenidae clade 30 million years ago (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 carnivoransmmdash;both Felidae and Canidaemmdash;that we screened. This envelope protein does not disclose any fusogenic activity in ex vivo assays, at variance with the syncytin-Car1 gene, which is found in all carnivorans, including the hyena, in which it is still present, transcriptionally active in the placenta, and fusogenic. Together, the present results illustrate the permanent renewal of placenta-specific genes by retroviral capture 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 maternofetal 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 maternofetal interface. In this study, 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 maternofetal 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.

  • Antibody-Mediated Protective Mechanisms Induced by a Trivalent Parainfluenza Virus-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 broad protection against the three ebolaviruses. While GP consensus-based antigens failed to elicit neutralizing antibodies, polyvalent vaccine 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 sera from guinea pigs receiving the monovalent vaccines, sera from guinea pigs receiving the trivalent vaccine bound and neutralized EBOV and SUDV at equivalent levels and BDBV at only a slightly reduced level. Peptide microarrays revealed a preponderance of binding to amino acids 389 to 403, 397 to 415, and 477 to 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 infection survivors demonstrated that the immune sera block the binding of antibodies specific for the GP glycan cap, the GP1-GP2 interface, the mucin-like domain, and the membrane-proximal external region. Thus, administration of a cocktail of three ebolavirus vaccines induces a desirable broad antibody response, without skewing of the response toward preferential recognition of a single virus.

    IMPORTANCE The symptoms of the disease caused by the ebolaviruses Ebola, Bundibugyo, and Sudan are similar, and their areas of endemicity overlap. However, because of the limited antigenic relatedness of the ebolavirus glycoprotein (GP) used in all candidate vaccines against these viruses, they protect only against homologous and not against heterologous ebolaviruses. Therefore, a broadly specific pan-ebolavirus vaccine is required, and this 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, an in-depth analysis of the antibody responses to administration of a cocktail of human parainfluenza virus 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.

  • Packaging of the Influenza Virus Genome Is Governed by a Plastic Network of RNA- and Nucleoprotein-Mediated 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, and yet the underlying interaction network remains largely unexplored. We show here 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); this 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.

  • 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 two 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 are positioned to affect the E2-capsid interaction or the E1-mediated intertrimer 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 is 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.

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

  • A large proportion of the world population harbors herpes simplex virus 1 (HSV-1), a major cause of infectious corneal blindness. HSV-specific CD8+ T cells protect from herpesvirus 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 nonprotected symptomatic (SYMP) individuals, with a history of numerous episodes of recurrent herpetic disease, using the high-throughput digital NanoString nCounter system and flow cytometry. Interestingly, our results demonstrated that 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 multifunctional HSV-specific effector memory CD62Llow CD44high CD8+ TEM cells were detected in ASYMP individuals compared to more of monofunctional central memory CD62Lhigh CD44high CD8+ 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 vaccines.

    IMPORTANCE A staggering number of the world population harbors herpes simplex virus 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 multifunctional HSV-specific effector memory CD8+ TEM cells were detected in ASYMP individuals. In contrast, nonprotected SYMP individuals had more 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.

  • 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 the 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 the cell cycle at different phases. We observed that all compounds inducing cell cycle arrest in G2/M phase strongly enhanced the replication of VSV-M51 in cells with functional antiviral signaling. G2/M arrest strongly inhibited type I and type III interferon (IFN) production as well as expression of IFN-stimulated genes in response to exogenously added IFN. Moreover, G2/M arrest enhanced the replication of Sendai virus (a paramyxovirus), which is also highly sensitive to the type I IFN response but did not stimulate the 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 could represent an "Achillesrrsquo; 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 the cell cycle affects replication of VSV and VSV-M51. We show that G2/M cell cycle arrest strongly enhances the 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 "Achillesrrsquo; 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.

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

  • Porcine reproductive and respiratory syndrome (PRRS) is of great concern to the swine industry due to pandemic outbreaks of the disease, current ineffective vaccinations, and a 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 protein entry into cells. TAT-Nb6 was expressed as an inclusion body in Escherichia coli, and indirect enzyme-linked immunosorbent assays 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 porcine alveolar macrophages (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 and spanned two discontinuous regions (Nsp9aa454nndash;551 and Nsp9aa599nndash;646). Taken together, these results suggest that TAT-Nb6 can be developed as an antiviral drug for the inhibition of PRRSV replication and controlling PRRS disease.

    IMPORTANCE The pandemic outbreak of PRRS, which is caused by PRRSV, has greatly affected the swine industry. We still lack an efficient vaccine, and it is an 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. Here, we demonstrated that the 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 drug development to control PRRS.

  • HDAC6 Restricts Influenza A Virus by Deacetylation of the RNA Polymerase PA Subunit [Virus-Cell Interactions]

  • The life cycle of influenza A virus (IAV) is modulated by various cellular host factors. Although previous studies indicated that IAV infection is controlled by HDAC6, the deacetylase involved in the regulation of PA remained unknown. Here, we demonstrate that HDAC6 acts as a negative regulator of IAV infection by destabilizing PA. HDAC6 binds to and deacetylates PA, thereby promoting the proteasomal degradation of PA. Based on mass spectrometric analysis, Lys(664) of PA can be deacetylated by HDAC6, and the 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 an HDAC6 deacetylase dead mutant (HDAC6-DM; H216A, H611A). Finally, we also demonstrate that overexpression of HDAC6 suppresses IAV RNA polymerase activity, but HDAC6-DM does not. Taken together, our findings provide initial evidence that HDAC6 plays a negative role in IAV RNA polymerase activity by deacetylating PA and thus restricts IAV RNA transcription and replication.

    IMPORTANCE Influenza A virus (IAV) continues to threaten global public health due to drug resistance and the emergence of frequently mutated strains. Thus, it is critical to find new strategies to control IAV infection. Here, we discover one host protein, HDAC6, that can inhibit viral RNA polymerase activity by 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 of aalpha;-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 at various periods of viral infection may illuminate novel strategies for developing antiviral drugs.

  • Mapping the pH Sensors Critical for Host Cell Entry by a Complex Nonenveloped Virus [Virus-Cell Interactions]

  • Bluetongue virus (BTV), in the family Reoviridae, is an insect-borne, double-capsid virus causing hemorrhagic 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 a lethal effect, indicating that the location of histidine in VP5 is critical to inducing changes in VP5 conformation that facilitates membrane penetration. Further, we show that the VP5 anchoring domain alone recapitulates sensing of low pH. Our data reveal a novel, multiconformational process that overcomes entry barriers faced by this multicapsid 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 multilayered 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 reveal a novel mechanism of cell entry for a nonenveloped virus and highlight mechanisms which may also be used by other viruses.

  • The Herpesviridae Conserved Multifunctional Infected-Cell Protein 27 (ICP27) Is Important but Not Required for Replication and Oncogenicity of Mareks Disease Alphaherpesvirus [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, in which 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 those of 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 showing that 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. We studied the Herpesviridae conserved ICP27 regulatory protein in cell culture and during MDV infection in chickens. We determined that 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.

  • Hepatitis C Virus Escape Studies of Human Antibody AR3A Reveal 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 an elevated lifetime 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 that can reduce AR3A binding to expressed H77 envelope were introduced into chimeric cell culture-infectious HCV recombinants (HCVcc) H77(core-NS2)/JFH1. Substitutions G523A, G530A, and D535A greatly reduced fitness, and S424A, P525A, and N540A, although 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 the 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 coreceptor dependency. Common substitutions at positions 431 and 442 did not confer high-level resistance in other genotype 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 on 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 succeeded in inducing only 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 coreceptor dependency, both in an HVR1-dependent fashion. Thus, we identify novel AR3A-specific resistance substitutions and the role of HVR1 in protecting HCV from AR3-targeting antibodies. These viral escape mechanisms should be taken into consideration in future HCV vaccine development.

  • Efficient Inhibition of Avian and Seasonal Influenza A Viruses by a Virus-Specific Dicer-Substrate Small Interfering RNA 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 virus 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 25- to 27-nucleotide-long siRNA molecules are processed by endogenous Dicer into 21-nucleotide siRNAs and are thus designated 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 pretransfection 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 100 different antiviral RNA molecules targeting the most conserved regions of the 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.

  • Inhibition of DNA-Sensing Pathway by Marek's 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 the beta interferon (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.

  • Correction for Cadena-Nava et al., "Self-Assembly of Viral Capsid Protein and RNA Molecules of Different Sizes: Requirement for a Specific High Protein/RNA Mass Ratio" [Author Correction]

  • Human Papillomavirus 16 E2 Regulates Keratinocyte Gene Expression Relevant to Cancer and the Viral Life Cycle [Cellular Response to Infection]

  • Human papillomaviruses (HPVs) 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 the 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 telomerase reverse transcriptase (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. Transcriptome sequencing (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. Small interfering RNA (siRNA) targeting of E2 reversed the 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 the 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 the regulation of the host genome in cancers. Patients with HPV16-positive HNC with a loss of E2 expression exhibited a worse clinical outcome, and we discuss how this could, at least partially, be related to the loss of E2 host gene regulation.

    IMPORTANCE Human papillomavirus 16 (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 the 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.

  • 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 organized by J. Matthijnssens, J. S. L. 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 double-stranded RNA (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-electron microscopic (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 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.

  • Genetic Compatibility of Reassortants between Avian H5N1 and H9N2 Influenza Viruses with Higher Pathogenicity in Mammals [Pathogenesis and Immunity]

  • The cocirculation 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. The 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 cocirculation 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.

  • Autophagy Promotes Replication of Influenza A Virus In Vitro [Virus-Cell Interactions]

  • Influenza A virus (IAV) infection could induce autophagosome accumulation. However, the impact of the autophagy machinery on IAV infection remains controversial. Here, we showed that induction of cellular autophagy by starvation or rapamycin treatment increases progeny virus production, while disruption of autophagy using a small interfering RNA (siRNA) and pharmacological inhibitor reduces progeny virus production. Further studies revealed that alteration of autophagy significantly affects the early stages of the virus life cycle or viral RNA synthesis. Importantly, we demonstrated that overexpression of both the IAV M2 and NP proteins alone leads to the lipidation of LC3 to LC3-II and a redistribution of LC3 from the cytosol to punctate vesicles indicative of authentic autophagosomes. Intriguingly, both M2 and NP colocalize and interact with LC3 puncta during M2 or NP transfection alone and IAV infection, leading to an increase in viral ribonucleoprotein (vRNP) export and infectious viral particle formation, which indicates that the IAV-host autophagy interaction plays a critical role in regulating IAV replication. We showed that NP and M2 induce the AKT-mTOR-dependent autophagy pathway and an increase in HSP90AA1 expression. Finally, our studies provided evidence that IAV replication needs an autophagy pathway to enhance viral RNA synthesis via the interaction of PB2 and HSP90AA1 by modulating HSP90AA1 expression and the AKT-mTOR signaling pathway in host cells. Collectively, our studies uncover a new mechanism that NP- and M2-mediated autophagy functions in different stages of virus replication in the pathogenicity of influenza A virus.

    IMPORTANCE Autophagy impacts the replication cycle of many viruses. However, the role of the autophagy machinery in IAV replication remains unclear. Therefore, we explored the detailed mechanisms utilized by IAV to promote its replication. We demonstrated that IAV NP- and M2-mediated autophagy promotes IAV replication by regulating the AKT-mTOR signaling pathway and HSP90AA1 expression. The interaction of PB2 and HSP90AA1 results in the increase of viral RNA synthesis first; subsequently the binding of NP to LC3 favors vRNP export, and later the interaction of M2 and LC3 leads to an increase in the production of infectious viral particles, thus accelerating viral progeny production. These findings improve our understanding of IAV pathogenicity in host cells.

  • The Dengue Virus Nonstructural 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 nonstructural proteins. Nonstructural 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 and is responsible for the cholesterol traffic inside the cell. In this work, we demonstrate that in mosquito cells, but not in vertebrate cells, NS1 associates with 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 CAV-1 or FKBP52 with short interfering RNAs or the inhibition of CyA by cyclosporine resulted in significant decrease in NS1 secretion, again without affecting virion release. Colocalization, coimmunoprecipitation, and proximity ligation assays indicated that NS1 colocalizes and interacts with all 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 follows the same secretory pathway as that 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, which are in charge of cholesterol transport inside the cell. Results obtained with ZIKV-infected mosquito cells suggest that 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.

  • 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 the 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 piglets were 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 similar progressive increases in viral loads 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. The levels of proinflammatory cytokines and chemokines, including interleukin 1 beta (IL-1bbeta;), IL-6, IL-23aalpha;, gamma interferon (IFN-), tumor necrosis factor alpha (TNF-aalpha;), and chemokine ligand 5 (CCL5), were significantly upregulated in both groups of inoculated piglets. Eight-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 the 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 similar increases in viral loads 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.

  • 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 (i) EV-A71 strain type and RNA sequence diversity impacts recombination frequency in a predictable manner that mimics the observations found in nature; (ii) recombination is primarily a replicative process mediated by the RNA-dependent RNA polymerase; (iii) a mutation shown to reduce recombination in PV (L420A) similarly reduces EV-A71 recombination, suggesting conservation in mechanism(s); and (iv) sequencing of intraserotypic recombinant genomes indicates that template switching occurs 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 RNA-dependent RNA polymerase (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, which is indicative of conservation in mechanism.

  • Novel Mutations in nsP2 Abolish Chikungunya Virus-Induced Transcriptional Shutoff and Make the 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, by using 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 interferon (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 also is 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.

  • 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 closest evolutionary 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 30 kDa 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 natural killer cells or T cell recognition, the evasion of antibody-mediated effects does not seem to be absolutely required for infection or reinfection. 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 nonhuman primates greatly broadened the understanding of mechanisms by which CMVs evade or reprogram T cell and natural killer 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 nonhuman primate models.

  • Articles of Significant Interest in This Issue [Spotlight]

  • Return To Top of the Page

    Advanced Publication Articles

    Return To Top of the Page

    JVI Accepts: Articles Published Ahead of Print

  • Cytoplasm and Beyond: The Dynamic Innate Immune Sensing of Influenza A Virus by RIG-I [Gem]

  • Innate immune sensing of influenza A virus (IAV) requires RIG-I, a fundamental cytoplasmic RNA sensor. How RIG-I's cytoplasmic localization reconciles with the nuclear replication nature of IAV is poorly understood. Recent findings provide advanced insights into the spatiotemporal RIG-I sensing of IAV and highlight the contribution of various RNA ligands to RIG-I activation. Understanding a compartment-specific RIG-I sensing paradigm would facilitate the identification of the full spectrum of physiological RIG-I ligands produced during IAV infection.

  • Genome characterization, prevalence and transmission mode of a novel picornavirus associated with the Threespine Stickleback fish (Gasterosteus aculeatus) [Structure and Assembly]

  • The complete genome sequence of an RNA virus was assembled from RNA sequencing of virus particles purified from threespine stickleback intestine tissue samples. This new virus is most closely related to the Eel Picornavirus and can be assigned to the genus Potamipivirus in the family Picornaviridae. Its unique genetic properties are enough to establish a new species, dubbed the Threespine Stickleback Picornavirus (TSPV). Due to their broad geographic distribution throughout the northern hemisphere and parallel adaptation to freshwater, threespine sticklebacks have become a model in evolutionary ecology. Further analysis using diagnostic PCRs revealed that TSPV is highly prevalent in both anadromous and freshwater populations of threespine sticklebacks, infects almost all fish tissues and is transmitted vertically to offspring obtained from in vitro fertilization in laboratory settings. Finally, TSPV was found in Sequence Read Archives of transcriptome of G. aculeatus further demonstrating its wide distribution and unsought prevalence in samples. It is thus necessary to test the impact of TSPV on the biology of threespine sticklebacks as this widespread virus could interfere with the behavioral, physiological, or immunological studies that employ this fish as model system.

    Abstract Importance

    The threespine stickleback species complex is an important model system in ecological and evolutionary studies because of the large number of isolated divergent populations that are experimentally tractable. For similar reasons, its co-evolution with the cestode parasite Schistocephalus solidus, its interaction with gut microbes, and the evolution of its immune system are of growing interest. Herein we describe the discovery of an RNA virus that infects both freshwater and anadromous populations of sticklebacks. We show that the virus is transmitted vertically in laboratory settings and found it in Sequence Read Archives suggesting that experiments using sticklebacks were conducted in presence of the virus. This discovery can serve as a reminder that the presence of viruses in wild-caught animals is possible, even when animals appear healthy. Regarding threespine sticklebacks, the impact of TSPV on the fish biology should be investigated further to ensure that it does not interfere with experimental results.

  • Adaptation of an R5 Simian-human Immunodeficiency Virus Encoding an HIV Clade A Envelope With or Without Ablation of Adaptive Host Immunity: Differential Selection of Viral Mutants [Pathogenesis and Immunity]

  • Simian-human immunodeficiency virus (SHIV) infection in rhesus macaques (RMs) resembles human immunodeficiency virus type 1 (HIV-1) infection in humans and serves as a tool to evaluate candidate AIDS vaccines. HIV-1 clade A (HIV-A) predominates in parts of Africa. We constructed an R5 clade A SHIV (SHIV-A; strain SHIV-KNH1144) carrying env from a Kenyan HIV-A. SHIV-A underwent rapid serial passage through six RMs. To allow unbridled replication without adaptive immunity, we simultaneously ablated CD8+ and B cells with cytotoxic monoclonal antibodies in the next RM, resulting in extremely high viremia and CD4+ T-cell loss. Infected blood was then transferred into two non-immune-depleted RMs, where progeny SHIV-A showed increased replicative capacity and caused AIDS. We reisolated SHIV-KNH1144p4, which was replication-competent in peripheral blood mononuclear cells (PBMC) of all RMs tested. Next-generation sequencing of early and late-passage SHIV-A strains identified mutations that arose due to "fitness" virus optimization in the former and mutations exhibiting signatures typical for adaptive host immunity in the latter. "Fitness" mutations are best described as mutations that allow for better fit of the HIV-A Env with SIV-derived virion building blocks or host proteins and mutations in non-coding regions that accelerate virus replication, all of which result in the outgrowth of virus variants in the absence of adaptive T-cell and antibody-mediated host immunity.

    IMPORTANCE In this study, we constructed a simian-human immunodeficiency virus carrying an R5 Kenyan HIV-1 clade A env (SHIV-A). To bypass host immunity, SHIV-A was rapidly passaged in naïve macaques or animals depleted of both CD8+ and B cells. Next-generation sequencing identified different mutations that resulted from optimization of viral replicative fitness in the absence of adaptive immunity or due to pressure of the latter.

  • The US11 gene of Herpes Simplex Virus 1 promotes neuroinvasion and periocular replication following corneal infection [Pathogenesis and Immunity]

  • Herpes simplex virus type 1 (HSV-1) cycles between phases of latency in sensory neurons and replication in mucosal sites. HSV-1 encodes two key proteins that antagonize the shutdown of host translation; US11 through preventing PKR activation, and ICP34.5 through mediating eIF2aalpha; dephosphorylation. While the profound attenuation of ICP34.5 deletion mutants has been repeatedly demonstrated, a role for US11 in HSV-1 pathogenesis remains unclear. We therefore generated an HSV-1 strain 17 US11 null virus and examined its properties in vitro and in vivo. In U373 glioblastoma cells, US11 cooperated with ICP34.5 to prevent eIF2aalpha; phosphorylation late in infection. This effect, however, was muted in human corneal epithelial cells (HCLEs), which did not accumulate phosphorylated eIF2aalpha; unless both US11 and ICP34.5 were absent. Low levels of phosphorylated eIF2aalpha; correlated with continued protein synthesis and with the ability of virus lacking US11 to overcome antiviral immunity in HCLE and U373 cells. Neurovirulence following intracerebral inoculation of mice was not affected by the deletion of US11. In contrast, the time to endpoint criteria following corneal infection was greater for US11-null compared to wild-type virus. Replication in trigeminal ganglia and periocular tissue was promoted by US11, as was periocular disease. The establishment of latency and the frequency of virus reactivation from trigeminal ganglia was unaffected by US11 deletion, although emergence of the US11-null virus occurred with slowed kinetics. Considered together, the data indicate that US11 facilitates the countering of anti-viral response of infected cells and promotes the efficient emergence of virus following reactivation.

    IMPORTANCE

    Alphaherpesviruses are ubiquitous DNA viruses including the human pathogens herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), and are significant causes of ulcerative mucosal sores, infectious blindness, encephalitis, and devastating neonatal disease. Successful primary infection and persistent co-existence with host immune defenses is dependent on the ability of these viruses to counter the anti-viral response. HSV-1 and 2 and other primate viruses within the Simplexvirus genus encode US11, an immune antagonist that promotes virus production by preventing shutdown of protein translation. Here we investigated the impact of US11 deletion on HSV-1 growth in vitro and pathogenesis in vivo. This work supports a role for US11 in pathogenesis and emergence from latency, elucidating immunomodulation by this medically important cohort of viruses.

  • Prime boost immunisations with DNA, MVA and protein-based vaccines elicit robust HIV-1, Tier 2 neutralizing antibodies against the CAP256 superinfecting virus. [Vaccines and Antiviral Agents]

  • A vaccine regimen that elicits broadly neutralizing antibodies (bNAbs) is a major goal in HIV-1 vaccine research. Here we assessed the immunogenicity of the CAP256SU envelope protein delivered by modified vaccinia virus Ankara (M) and DNA (D) vaccines in different prime/boost combinations followed by a soluble protein (P) boost. The envelope protein (Env) contained a flexible, glycine linker and I559P mutation. Trimer-specific bNAbs PGT145, PG16 and CAP256 VRC26_08 efficiently bound to the membrane-bound CAP256 envelope expressed on the surface of cells transfected or infected with the DNA and MVA vaccines respectively. The vaccines were tested in two different vaccination regimens in rabbits (MMPPP and DDMMPP). Both regimens elicited autologous Tier 2 neutralizing antibodies (NAbs) and high titre binding antibodies to the matching CAP256 Env and CAP256 V1V2 loop scaffold. The immunogenicity of DNA and MVA vaccines expressing membrane-bound Env alone was compared to that of Env stabilised in a more native-like conformation on the surface of Gag VLPs. The inclusion of Gag in the DNA and MVA vaccines resulted in earlier development of Tier 2 NAbs for both vaccination regimens. In addition, a higher proportion of the rabbits primed with DNA and MVA vaccines that included Gag developed Tier 2 NAbs as compared to those primed expressing Env alone. Previously, these DNA and MVA vaccines expressing subtype C mosaic HIV-1 Gag were shown to elicit strong T cell responses in mice. Here we show that when the CAP256 SU envelope protein is included, these vaccines elicit autologous Tier 2 NAbs.

    Importance

    A vaccine is urgently needed to combat HIV-1, particularly in sub-Saharan Africa which remains disproportionately affected by the pandemic and accounts for the majority of new infections and AIDS-related deaths. In this study two different vaccination regimens were compared. Rabbits that received two DNA primes followed by two MVA and two protein inoculations developed better immune responses to those that received two MVA and three protein inoculations. In addition, DNA and MVA vaccines that expressed mosaic Gag VLPS presenting a stabilised Env antigen elicited better responses than Env alone, which supports the inclusion of Gag VLPs in an HIV-1 vaccine.

  • Molecular basis of a protective/neutralizing monoclonal antibody targeting envelope proteins of both tick-borne encephalitis virus and louping ill virus [Vaccines and Antiviral Agents]

  • Tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) are members of the tick-borne flaviviruses (TBFVs) in the family Flaviviridae, which cause encephalomeningitis and encephalitis in humans and other animals. Although vaccines against TBEV and LIV are available, infection rates are rising due to the low vaccination coverage. To date, no specific therapeutics have been licensed. Several neutralizing monoclonal antibodies (MAbs) show promising effectiveness in the control of TBFVs, but the underlying molecular mechanisms are yet to be characterized. Here, we determined the crystal structures of LIV envelope protein (E) and report the comparative structural analysis of a TBFV broadly neutralizing murine MAb (MAb 4.2) in complex with either LIV or TBEV E proteins. The structures reveal that MAb 4.2 binds to the lateral ridge of Domain III (EDIII) of LIV-E or TBEV-E, an epitope also reported for other potently neutralizing MAbs against mosquito-borne flaviviruses (MBFVs), but adopts a unique binding orientation. Further structural analysis suggested that MAb 4.2 may neutralize flavivirus infection by preventing the structural rearrangement required for membrane fusion during virus entry. These findings extend our understanding of the vulnerability of TBFVs and other flaviviruses (including MBFVs) and provide an avenue for antibody-based TBFVs antiviral development.

    Importance

    Understanding the mechanism of antibody neutralization/protection against a virus is crucial for antiviral counter-measures development. Tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) are tick-borne flaviviruses (TBFVs) in the family Flaviviridae. They cause encephalomeningitis and encephalitis in humans and other animals. Although vaccines for both viruses are available, infection rates are rising due to the low vaccination coverage. In this study, we solved the crystal structures of LIV envelope protein (E) and a broadly-neutralizing/protective TBFV MAb, MAb 4.2, in complex with E from either TBEV or LIV. Key structural features shared by TBFV E proteins were analyzed. Structures of E-antibody complexes show that MAb 4.2 targets the lateral ridge of both TBEV and LIV E proteins, a vulnerable site in flaviviruses for other potent neutralizing MAbs. Thus, this site represents a promising target for TBFV antiviral development. Further, these structures provide important information for understanding TBFV antigenicity.

  • The I{kappa}B kinases restrict human cytomegalovirus infection [Virus-Cell Interactions]

  • Human Cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes disease in immunosuppressed populations. HCMV has a complex relationship with innate immune signaling pathways. Specifically, HCMV has been found to block some aspects of inflammatory signaling while benefiting from others. Through analysis of knockout cell lines targeting the NFB regulatory kinases IKKaalpha; and IKKbbeta;, we find that the IKK kinases are host restriction factors that contribute to cytokine-mediated resistance to viral infection, limit the initiation of HCMV infection, and attenuate viral cell-to-cell spread. The HCMV UL26 protein is a viral immune modulator important for HCMV infection that has been shown to inhibit host cell NFB signaling, yet it has remained unclear how UL26-mediated NFB modulation contributes to infection. Here, we find that UL26's modulation of NFB signaling is separable from its contribution to high-titer viral replication. However, we find that IKKbbeta; is required for the induction of cytokine expression associated with UL26 infection. Collectively, our data indicate that the IKK kinases restrict infection, but that HCMV targets their signaling to modulate the cellular inflammatory environment.

    Importance

    Innate immune signaling is a critical defense against virus infection, and represents a central host-virus interaction that frequently determines the outcome of infection. NFB signaling is an essential component of innate immunity that is extensively modulated by HCMV, a significant cause of morbidity in neonates and the immunosuppressed. However, the roles that various facets of NFB signaling play during HCMV infection have remained elusive. We find that the two major regulatory kinases in this pathway, IKKaalpha; and IKKbbeta;, limit the initiation of infection, viral replication, and cell-to-cell spread. In addition, our results indicate that these kinases contribute differently to the host cell response to infection in the absence of a virally encoded NFB inhibitor, UL26. Given its importance to viral infection, elucidating the contributions of various NFB constituents to infection is an essential first step towards the possibility of targeting this pathway therapeutically.

  • Comparison of HSV-1 strains circulating in Finland demonstrates the uncoupling of whole-genome relatedness and phenotypic outcomes of viral infection [Genetic Diversity and Evolution]

  • A majority of adults in Finland are seropositive carriers of herpes simplex viruses (HSV). Infection occurs at epithelial or mucosal surfaces, after which virions enter innervating nerve endings, eventually establishing lifelong infection in neurons of the sensory or autonomic nervous system. Recent data have highlighted the genetic diversity of HSV-1 strains, and demonstrated apparent geographic patterns in strain similarity. Though multiple HSV-1 genomes have been sequenced from Europe to date, there is a lack of sequenced genomes from Nordic countries. Finland's history includes at least two major waves of human migration, suggesting the potential for diverse viruses to persist in the population. Here we used HSV-1 clinical isolates from Finland to test the relationship between viral phylogeny, genetic variation, and phenotypic characteristics. We found that Finnish HSV-1 isolates separated into two distinct phylogenetic groups, potentially reflecting historical waves of human (and viral) migration into Finland. Each HSV-1 isolate harbored a distinct set of phenotypes in cell culture, including differences in virus production, extracellular virus release, and cell-type-specific fitness. Importantly, the phylogenetic clusters were not predictive of any detectable pattern in phenotypic differences, demonstrating that whole-genome relatedness is not a proxy for overall viral phenotype. Instead, we highlight specific gene-level differences that may contribute to observed phenotypic differences, and we note that strains from different phylogenetic groups contain the same genetic variations.

    Importance

    Herpes simplex viruses (HSV) infect a majority of adults. Recent data have highlighted the genetic diversity of HSV-1 strains, and demonstrated apparent genomic relatedness between strains from the same geographic region. We use HSV-1 clinical isolates from Finland to test the relationship between viral genomic and geographic relationships, differences in specific genes, and characteristics of viral infection. We found that viral isolates from Finland separated into two distinct groups of genomic and geographic relatedness, potentially reflecting historical patterns of human and viral migration into Finland. These Finland HSV-1 isolates had distinct infection characteristics in multiple cell types tested, which were specific to each isolate and did not group according to genomic and geographic relatedness. This demonstrates that HSV-1 strain differences in specific characteristics of infection are set by a combination of host cell type and specific viral gene-level differences.

  • Turnip mosaic virus is a second example of a virus using transmission activation for plant-to-plant propagation by aphids [Virus-Cell Interactions]

  • Cauliflower mosaic virus (CaMV, family Caulimoviridae) responds to the presence of aphid vectors on infected plants by forming specific transmission morphs. This phenomenon, coined transmission activation (TA), controls plant-to-plant propagation of CaMV. A fundamental question is whether other viruses rely on TA. Here, we demonstrate that transmission of the unrelated Turnip mosaic virus (TuMV, family Potyviridae) is activated by the reactive oxygen species H2O2 and inhibited by the calcium channel blocker LaCl3. H2O2-triggered TA manifested itself by the induction of intermolecular cysteine bonds between viral HC-Pro molecules and by formation of viral transmission complexes, composed of TuMV particles and HC-Pro that mediates vector-binding. Consistently, LaCl3 inhibited intermolecular HC-Pro cysteine bonds and HC-Pro interaction with viral particles. These results show that TuMV is a second virus using TA for transmission, but using an entirely different mechanism than CaMV. We propose that TuMV TA requires ROS and calcium signaling and that it is operated by a redox switch.

    Importance

    Transmission activation, i.e. a viral response to the presence of vectors on infected hosts that regulates virus acquisition and thus transmission, is an only recently described phenomenon. It implies that viruses contribute actively to their transmission, something that has been shown before for many other pathogens but not for viruses. However, transmission activation has been described so far for only one virus, and it was unknown whether other viruses rely also on transmission activation. Here we present evidence that a second virus uses transmission activation, suggesting that it is a general transmission strategy.

  • Manipulation of aphid behavior by a persistent plant virus [Cellular Response to Infection]

  • Plants are frequently infected with cytoplasmic RNA viruses that persist for many generations through nearly 100% vertical transmission without producing any symptoms. Movement between plant cells and horizontal transmission has not been observed in these viruses; instead they are distributed to all host cells through host cell division. Jalapenntilde;o peppers (Capsicum annuum) are all infected with Pepper cryptic virus 1 (PCV 1, family Partitiviridae). We compared the effect of odor cues from PCV 1 infected (J+) and virus free (J-) Jalapenntilde;o pepper on the aphid Myzus periscae, a common vector of acute plant viruses. Pairwise preference experiments showed a stark contrast to insect-plant interactions in acute virus infections: virus infected plants deterred aphids. The acute plant virus Cucumber mosaic virus (CMV) manipulates its host's volatile emission to attract aphid vectors and facilitate its transmission. We inoculated J+ and J- with CMV. Volatiles of J+ and J- CMV infected plants were more attractive to aphids than J+ and J- mock inoculated plants. However, in pairwise preference between J+ CMV- and J- CMV-infected plants, aphids preferred the J- CMV volatile blend. Aphid reproduction on J+ and J- plants was measured as an indicator for the effect of PCV 1 on host quality for aphids. Aphid reproduction on J+ plants was more than two fold lower than on J- plants.

    IMPORTANCE

    This study demonstrates that a plant persistent virus can manipulate aphid behavior. This manipulation is in stark contrast with previously described effects of acute viruses on their host to facilitate their transmission. This study demonstrates a positive relationship between Pepper cryptic virus 1 and Jalapenntilde;o pepper plants by protecting the plants from the vector of acute viruses, and reducing aphid herbivory. This work reveals an important implication of persistent plant viruses for pest and pathogen management in agriculture.

  • Effect of Previous Respiratory Syncytial Virus Infection on Murine Immune Responses to F and G Protein Containing Virus-like Particles [Vaccines and Antiviral Agents]

  • Most individuals are infected with respiratory syncytial virus (RSV) by age two, but infection does not result in long-term protective immunity to subsequent infections. Previous RSV infection may, however, impact responses to an RSV vaccine. The goal of these studies was to explore the effect of previous RSV infection on murine antibody responses to RSV F and G protein containing virus-like particles (VLP), comparing responses to those resulting from VLP immunization of RSV naïve animals. These studies showed that after RSV infection, immunization with a single dose of VLPs containing a conformation stabilized pre-fusion F protein stimulated high titers of neutralizing antibodies (NA) while an immunization with post-F containing VLPs or a second RSV infection only weakly stimulated NA even though total anti-F protein IgG antibody levels in both VLP immunized animals were similar. Furthermore, single Pre-F or Post-F VLP immunization of RSV previously infected (primed) animals resulted in total anti-F antibody titers that were 10 to 12-fold higher than titers after a VLP prime and boost of RSV naïve animals or after two consecutive RSV infections. The avidities of serum antibodies as well as numbers of splenic B cells and bone marrow cells after different immunization protocols were also assessed. The combined results show that RSV infection can quite effectively prime animals for the production of protective antibodies that can be efficiently activated by a Pre-F VLP boost but not by a Post-F VLP boost or a second RSV infection.

    Importance

    In contrast to most virus infections, humans may experience repeated RSV infections caused by the same virus serotype indicating that immune memory responses to RSV are defective. However, the effects of any residual but non-protective immunity on responses to RSV vaccines are not clear. This study demonstrates that a VLP vaccine candidate containing a stabilized pre-fusion F protein can robustly stimulate protective immunity in animals previously infected with RSV while a second RSV infection or a post-fusion F containing VLP cannot. This result shows that a properly constructed immunogen can be an effective vaccine in animals previously infected with RSV. The results also suggest that the defect in RSV memory may not be in the induction of that memory but rather in its activation by a subsequent RSV infection.

  • HIV-1 subtype C with PYxE insertion has enhanced binding of Gag-p6 to host cell protein ALIX and increased replication fitness [Pathogenesis and Immunity]

  • Human immunodeficiency virus type 1 subtype C (HIV-1C) has a natural deletion of a YPxL motif in its Gag-p6 late domain. This domain mediates the binding of Gag to host cell protein ALIX and subsequently facilitates viral budding. In a subset of HIV-1C infected individuals, the tetrapeptide insertion PYxE has been identified at the deleted YPxL motif site. Here, we report the consequences of PYxE insertion on the interaction with ALIX and the relevance regarding replication fitness and drug sensitivity. In our three HIV-1C cohorts, PYKE and PYQE were most prevalent among PYxE variants. Through in silico predictions and in vitro experiments, we showed that HIV-1C Gag has an increased binding to ALIX when PYxE motif is present. To go more into the clinical relevance of the PYxE insertion, we obtained patient-derived gag-pol sequences from HIV-1CPYxEi viruses and inserted them in a reference HIV-1. Viral growth was increased, and the sensitivity to protease inhibitor (PI) lopinavir (LPV) and nucleoside reverse transcriptase inhibitor tenofovir alafenamide (TAF) was decreased for some of the HIV-1C PYxE variants compared to wild-type variants. Our data suggest that PYxE insertion in Gag restores the ability of Gag to bind ALIX and correlates with enhanced viral fitness in the absence or presence of LPV and TAF. The high prevalence and increased replication fitness of the HIV-1C virus with PYxE insertion could indicate the clinical importance of these viral variants.

    Importance

    Genomic differences within HIV-1 subtypes is associated with a varying degree of viral spread, disease progression, and clinical outcome. Viral budding is essential in the HIV-1 life cycle and mainly mediated through the interaction of Gag with host proteins. Two motifs within Gag-p6 mediate binding of host cell proteins and facilitate budding. HIV-1 subtype C (HIV-1C) has a natural deletion of one of these two motifs resulting in an inability to bind to host cell protein ALIX. Previously, we have identified a tetrapeptide (PYxE) insertion at this deleted motif site in a subset of HIV-1C patients. Here, we report the incidence of PYxE insertions in three different HIV-1C cohorts, and the insertion restores the binding of Gag to ALIX. It also increases viral growth even in the presence of antiretroviral drugs lopinavir and tenofovir alafenamide. Hence, PYxE insertion in HIV-1C might be biologically relevant for viruses and clinically significant among patients.

  • Dissecting the cell entry pathway of baculovirus by single particle tracking and quantitative electron microscopic analysis [Virus-Cell Interactions]

  • The budded virus of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infects insect cells through mainly clathrin-mediated endocytosis. However, the cell entry pathway of AcMNPV remains unclear. In this study, by using population-based analysis of single-virus tracking and electron microscopy, we investigated the internalization, fusion behavior and endocytic trafficking of AcMNPV. AcMNPV internalization into host insect cells was facilitated by actin polymerization and dynamin. After incorporation into early endosomes, AcMNPV envelope fused with the membranes of early endosome, allowing for nucleocapsid release into the cytoplasm. Microtubules were implicated in the bidirectional and long-range transport of virus-containing endosomes. In addition, microtubule depolymerization reduced the motility of virus-bearing early endosomes, impairing the progression of infection beyond enlarged early endosomes. These findings demonstrated that AcMNPV internalization was facilitated by actin polymerization in a dynamin dependent manner, and nucleocapsid release occurred in early endosomes in a microtubule dependent manner. This study provides mechanistic and kinetic insights into AcMNPV infection and enhance our understanding of the infection pathway of baculoviruses.

    IMPORTANCE

    Baculoviruses are used widely as environmentally benign pesticides, protein expression systems and potential mammalian gene delivery vectors. Despite the significant application value, little is known about the cell entry and endocytic trafficking pathways of baculoviruses. In this study, we demonstrate that the alphabaculovirus AcMNPV exhibited actin- and microtubule-dependent transport for nucleocapsid release predominantly from within early endosomes. In contrast to AcMNPV transduction in mammalian cells, its infection in host insect cells is facilitated by actin polymerization for internalization and microtubules for endocytic trafficking within early endosomes, implying that AcMNPV exhibits cell type specificity in the requirement of the cytoskeleton network. In addition, experimental depolymerization of microtubule impaired the progression of infection beyond enlarged early endosomes. This is the first study that dissects the cell entry pathway of baculoviruses in host cells at the single particle level, which advances our understanding of the early steps of baculovirus entry.

  • Regulation of HCV infection by Cellular Retinoic Acid Binding Proteins through the modulation of lipid droplet abundance [Virus-Cell Interactions]

  • Retinoid (vitamin A) is an essential diet constituent that governs a broad range of biological processes. Its biologically active metabolite, all-trans retinoic acid (ATRA), exhibits a potent antiviral property by enhancing both innate and adaptive antiviral immunity against a variety of viral pathogens such as, but not limited to, HIV, RSV, HSV, and measles. Even though the hepatocyte is highly enriched with retinoid and its metabolite, ATRA, it supports the establishment of efficient hepatitis C virus (HCV) replication. Here, we demonstrate the hepatocyte-specific cell-intrinsic mechanism by which ATRA exerts either proviral or antiviral effect, depending on how it engages cellular retinoic acid binding proteins (CRABPs). We found that the engagement of CRABP1 by ATRA potently supported viral infection by promoting the accumulation of lipid droplets (LDs), which robustly enhanced the formation of a replication complex on LD-associated ER membrane. In contrast, ATRA binding to CRABP2 potently inhibited HCV via suppression of LD accumulation. However, this antiviral effect of CRABP2 was abrogated due to the functional and quantitative predominance of CRABP1 in the hepatocytes. In summary, our study demonstrated that CRABPs serve as an on-off switch that modulates the efficiency of HCV lifecycle and elucidated how HCV evades the antiviral properties of ATRA via the exploitation of CRABP1 functionality.

    IMPORTANCE ATRA, a biologically active metabolite of vitamin A, exerts pleiotropic biological effects including the activation of both innate and adaptive immunity, thereby serving as a potent antimicrobial compound against numerous viral pathogens. Despite the enrichment of hepatocytes with vitamin A, HCV still establishes an efficient viral lifecycle. Here, we discovered that the hepatocellular response to ATRA creates either a proviral or an antiviral environment depending on its engagement with CRABP1 or 2 respectively. CRABP1 supports the robust replication of HCV while CRABP2 potently inhibited the efficiency of viral replication. Our biochemical, genetic, and microscopic analyses reveal that the pro- and antiviral effects of CRABPs are mediated by modulation of the LD abundance where HCV establishes the platform for viral replication and assembly on the LD-associated ER membrane. This study uncovered a cell-intrinsic mechanism by which HCV exploits the proviral function of CRABP1 to establish an efficient viral lifecycle.

  • Comparison of the whole genome sequence of an Oka varicella vaccine from China with other Oka vaccine strains reveals sites putatively critical for vaccine efficacy [Vaccines and Antiviral Agents]

  • Varicella-Zoster Virus (VZV) infection results in varicella mostly in children. Reactivation of the virus causes Herpes Zoster (HZ) mostly in adults. A live attenuated vaccine (vOka-Biken hereafter) was originally derived from the parental strain pOka. Several live attenuated vaccines based on the Oka strain are currently available worldwide. In China, varicella vaccines have been licensed by 4 manufacturers. In this study, we analyze the Whole Genome Sequence (WGS) of vOka-BK produced by Changchun BCHT Biotechnology also known as Baike. vOka-BK WGS was compared against the genomic sequence of 4 other Oka strains: pOka, vOka-Biken, vOka-Varilrix from GlaxoSmithKline (GSK) and vOka-Varivax from Merck aamp; Co.. A previous study identified 137 Single Nucleotide Polymorphisms (SNPs) shared by all vOkas. The current analysis used this data as a reference to compare with vOka-BK WGS and focused on 54 SNPs located in the unique regions of the genome. Twenty-eight non-synonymous substitutions were identified, ORF62 and ORF55 featuring the most amino acid changes with 9 and 3 respectively. Among the 54 SNPs, 10 had a different mutation profile in vOka-BK compared to the other 3 vaccines. A comparison with the clade 3 strain Ellen, known to be attenuated, identified 3 shared amino acid changes, *130R in ORF0, R958G and S628G in ORF62. This analysis provides the first comparison of a Chinese varicella vaccine to the other vaccines available worldwide and identifies sites potentially critical for VZV vaccine efficacy.

    IMPORTANCE Varicella, also known as chickenpox, is a highly contagious disease, caused by Varicella Zoster Virus (VZV). Varicella is a common childhood disease that can be prevented by a live attenuated vaccine. The first available vaccine was derived from the parental Oka strain in Japan in 1974. Several live attenuated vaccines based on the Oka strain are currently available worldwide. Among the 4 vaccines produced in China, the vaccine manufactured by Changchun BCHT Biotechnology also known as Baike, has been reported to be very efficacious. Comparative genomic analysis of the Baike vaccine with other Oka vaccine strains identified sites that might be involved in vaccine efficacy as well as important for the biology of the virus.

  • Infectious herpes simplex virus in the brainstem is correlated with reactivation in the trigeminal ganglia [Pathogenesis and Immunity]

  • Herpes simplex virus (HSV) establishes latency in neurons of the peripheral and central nervous systems (CNS). Evidence is mounting that HSV latency and reactivation in the nervous system has the potential to promote neurodegenerative processes. Understanding how this occurs is an important human health goal. In the mouse model, in vivo viral reactivation in the peripheral nervous system, triggered by hyperthermic stress, has been well-characterized with respect to frequency and cell type. However, characterization of in vivo reactivation in the CNS is extremely limited. Further, it remains unclear whether virus reactivated in the peripheral nervous system is transported to the CNS in an infectious form, how often this occurs and what parameters underlie the efficiency and outcomes of this process. In this study, reactivation was quantified in the trigeminal ganglia (TG) and the brainstem from the same latently infected animal using direct assays of equivalent sensitivity. Reactivation was detected more frequently in the TG than brainstem and in all but one case, the amount of virus recovered was greater in the TG than that detected in the brainstem. Viral protein positive neurons were observed in the TG, but a cellular source for reactivation in the brainstem was not identified, despite serially sectioning and examining the entire tissue (0/6 brainstems). These findings suggest that infectious virus detected in the brainstem is primarily the result of transport of reactivated virus from the TG into the brainstem.

    IMPORTANCE Latent herpes simplex virus (HSV) DNA has been detected in the central nervous system (CNS) of humans post-mortem and infection with HSV has been correlated with the development of neurodegenerative diseases. However, whether HSV can directly reactivate in the CNS, and/or infectious virus can be transported to the CNS following reactivation in peripheral ganglia, has been unclear. In this study, infectious virus was recovered from both the trigeminal ganglia and the brainstem of latently infected mice following a reactivation stimulus, but a higher frequency of reactivation and increased titers of infectious virus were recovered from the trigeminal ganglia. Viral proteins were detected in neurons of the trigeminal ganglia, but a cellular source of infectious virus could not be identified in the brainstem. These results suggest that infectious virus is transported from the ganglia to the CNS following reactivation, but do not exclude the potential for direct reactivation in the CNS.

  • Generation of infectious recombinant human rotaviruses from just 11 cloned cDNAs encoding the rotavirus genome [Genome Replication and Regulation of Viral Gene Expression]

  • The generation of recombinant group A rotaviruses (RVAs) entirely from cloned cDNAs has been described only for a single animal RVA strain, simian SA11-L2. We recently developed an optimized RVA reverse genetics system based on only RVA cDNAs (11-plasmid system), in which the concentration of cDNA plasmids containing the NSP2 and NSP5 genes is 3- or 5-fold increased in relation to that of the other plasmids. Based on this approach, we generated a recombinant human RVA (HuRVA)-based monoreassortant virus containing the VP4 gene of the simian SA11-L2 virus using the 11-plasmid system. In addition to this monoreassortant virus, authentic HuRVA (strain KU) could also be generated with the 11-plasmid system with some modifications. Our results demonstrate that the 11-plasmid system involving just RVA cDNAs can be used for the generation of recombinant HuRVA and recombinant HuRVA-based reassortant viruses.

    IMPORTANCE HuRVA is a leading pathogen causing severe diarrhea in young children worldwide. In this paper, we describe the generation of recombinant HuRVA (strain KU) from only 11 cloned cDNAs encoding the HuRVA genome by reverse genetics. The growth properties of the recombinant HuRVA were similar to those of the parental RVA, providing a powerful tool for better understanding of HuRVA replication and pathogenesis. Furthermore, the ability to manipulate the genome of HuRVAs 'to order' will be useful for next-generation vaccine production for this medically important virus and for engineering of clinical vectors expressing any foreign genes.

  • Medusavirus, a novel large DNA virus discovered from hot spring water [Genetic Diversity and Evolution]

  • Recent discoveries of new large DNA viruses reveal high diversity in their morphologies, genetic repertoires, and replication strategies. Here, we report the novel features of Medusavirus, a large DNA virus newly isolated from hot spring water in Japan. Medusavirus with a diameter of 260 nm shows a T=277 icosahedral capsid with unique spherical-headed spikes on its surface. It has a 381 kb genome encoding 461 putative proteins, 86 of which have their closest homologs in Acanthamoeba castellanii, whereas 279 (61%) are ORFans. The virus lacking the genes of DNA topoisomerase II and RNA polymerase showed that the DNA replication takes place in the host nucleus while the progeny virions are assembled in the cytoplasm. Furthermore, Medusavirus encoded all of five types of histones (H1, H2A, H2B, H3, and H4) and one DNA polymerase, which are phylogenetically placed at the root of the eukaryotic clades. By contrast, the host amoeba encoded many Medusavirus homologs including the major capsid protein. These facts strongly suggested that amoeba is indeed the most promising natural host of Medusavirus, and lateral gene transfers have taken place repeatedly and bidirectionally between the virus and its host since the early stage of their co-evolution. Medusavirus reflects the traces of direct evolutionary interactions between the virus and eukaryotic hosts, which may be caused by sharing the DNA replication compartment and evolutionarily long lasting viral-host relationships. Based on its unique morphological characteristics and phylogenomic relationships with other known large DNA viruses, we propose that Medusavirus forms a new family Medusaviridae.

    IMPORTANCE We have isolated a new NCLDV virus from hot spring water in Japan, named Medusavirus. This new NCLDV is phylogenetically placed at the root of the eukaryotic clades based on the phylogenies of several key genes including DNA polymerase, and surprisingly encodes the full set of histone homologs. Furthermore, its laboratory host, Acanthamoeba castellanii, encodes many Medusavirus homologs in its genome including the major capsid protein, suggesting that the amoeba is the genuine natural host of this new virus from ancient times, and lateral gene transfers have occurred between the virus and amoeba repeatedly. These results suggest that Medusavirus is a unique NCLDV preserving ancient footprints of evolutionary interactions with its hosts, thus providing clues to elucidate the evolution of NCLDVs, eukaryotes, and viral-host interaction. Based on the dissimilarities with other known NCLDVs, we propose that Medusavirus forms a new viral family Medusaviridae.

  • The pseudoknots region of the 5' untranslated region is a determinant of viral tropism and virulence of foot-and-mouth disease virus [Pathogenesis and Immunity]

  • Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease. It is well-characterized by the genetic instability and different antigenic properties. The nonstructural protein, 3A, is a primary determinant of viral tropism and virulence of Cathay topotype FMDVs. However, several other determinants are also speculated to be involved in the viral tropism and virulence. Deletion of 43 nucleotides (nt) in the pseudoknots (PKs) region of the 5' untranslated region (UTR) has been found to coexist with the identified 3A deletion in Cathay topotype FMDV genomes. In this study, we isolated an O/ME-SA/PanAsia lineage FMDV strain O/GD/CHA/2015 that included an 86-nt deletion in the PKs region and showed a porcinophilic phenotype. To investigate the potential role of PKs region in viral pathogenicity, we generated a recombinant FMDV strain with an incomplete PKs region and compared its virulence and pathogenesis with the intact FMDV strain in swine and bovine. Deletion of the 86-nt in the PKs had no major effects on the pathogenicity of the virus in swine, but significantly attenuated its ability to infect bovine cells and cattle, indicating that the PKs region is a newly discovered determinant of viral tropism and virulence. The role of the 43-nt deletion existing in the Cathay topotype FMDV was also investigated by evaluating the infection properties of genetically engineered viruses. Consistently, the 43-nt deletion in the PKs region significantly decreased the pathogenicity of the virus in bovines. Overall, our findings suggest that the PKs region deletion occurred naturally in FMDV genome, and that the PKs region is highly associated with viral host range and functions as a novel determinant for FMDV pathogenesis.

    IMPORTANCE This study demonstrates that the deletion in PKs region occurred naturally in FMDV genome. The isolated O/ME-SA/PanAsia lineage FMDV with an 86-nt deletion in PKs region showed a pig-adapted characteristic that could only cause clinical signs in swine but not bovines. Compared with the wildtype FMDV strain which possessed full infection capacity in both swine and bovines, deletion of the 86-nt in the PKs region resulted in the deficiency of the recombinant virus to cause disease in bovines. Deletion of the previously reported 43-nt in the PKs region also led to significantly decreased pathogenicity of FMDV in bovines. This study indicates that the PKs region is a novel determinant of viral tropism and virulence of FMDV.

  • Coronavirus endoribonuclease activity in porcine epidemic diarrhea virus suppresses type I and type III interferon responses [Pathogenesis and Immunity]

  • Identifying viral antagonists of innate immunity and determining if they contribute to pathogenesis is critical for developing effective strategies to control emerging viruses. Previously, we reported that an endoribonuclease (EndoU) encoded by murine coronavirus plays a pivotal role in evasion of host innate immune defenses in macrophages. Here, we asked if the EndoU activity of porcine epidemic diarrhea coronavirus (PEDV), which causes acute diarrhea in swine, plays a role in antagonizing the innate response in porcine epithelial cells and macrophages, the sites of viral replication. We constructed an infectious clone of PEDV-Colorado strain (icPEDV-wt) and an EndoU-mutant PEDV (icPEDV-EnUmt) by changing the codon for a catalytic histidine residue of EndoU to alanine (His226Ala). We found that both icPEDV-wt and icPEDV-EnUmt propagated efficiently in interferon (IFN) deficient Vero cells. In contrast, the propagation of icPEDV-EnUmt was impaired in porcine epithelial cells (LLC-PK1), where we detected an early and robust transcriptional activation of type I and type III IFNs. Infection of piglets with the parental Colorado strain, icPEDV-wt or icPEDV-EnUmt revealed that all viruses replicated in the gut and induced diarrhea, however there was reduced viral shedding and mortality in the icPEDV-EnUmt-infected animals. These results demonstrate that the EndoU activity is not required for PEDV replication in immortalized, IFN-deficient Vero cells, but is important for suppressing IFN response in epithelial cells and macrophages, which facilitates replication, shedding and pathogenesis in vivo. We conclude that PEDV EndoU activity is a key virulence factor that suppresses both type I and type III IFN responses.

    Importance Coronaviruses (CoVs) can emerge from an animal reservoir into a naïve host species to cause pandemic respiratory or gastrointestinal diseases with significant mortality in humans or domestic animals. Porcine epidemic diarrhea virus (PEDV), an alpha-CoV, infects gut epithelial cells and macrophages, inducing diarrhea resulting in high mortality in piglets. How PEDV suppresses the innate immune response was unknown. We found that mutating a viral endoribonuclease, EndoU, results in a virus that activates both the type I interferon-aalpha;/bbeta; response and the type III interferon- response in macrophages and epithelial cells. This activation of interferon limited viral replication in epithelial cell cultures and was associated with reduced virus shedding and mortality in piglets. This study reveals a role for EndoU activity as a virulence factor in PEDV infection and provides an approach for generating live-attenuated vaccine candidates for emerging coronaviruses.

  • Punctuated evolution of myxoma virus: rapid and disjunct evolution of a recent viral lineage in Australia [Genetic Diversity and Evolution]

  • Myxoma virus (MYXV) has been evolving in a novel host species nndash; European rabbits nndash; in Australia since 1950. Previous studies of viruses sampled from 1950 to 1999 revealed a remarkably clock-like evolutionary process across all Australian lineages of MYXV. Through an analysis of 49 newly generated MYXV genome sequences isolated in Australia between 2008 and 2017 we show that MYXV evolution in Australia can be characterized by three lineages, one of which exhibited a greatly elevated rate of evolutionary change and a dramatic break-down of temporal structure. Phylogenetic analysis revealed that this apparently punctuated evolutionary event occurred between 1996 and 2012. The branch leading to the rapidly evolving lineage contained a relatively high number of non-synonymous substitutions, and viruses in this lineage reversed a mutation found in the progenitor standard laboratory strain (SLS) and all previous sequences that disrupts the reading frame of the M005L/R gene. Analysis of genes encoding proteins involved in DNA synthesis or RNA transcription did not reveal any mutations likely to cause rapid evolution. Although there was some evidence for recombination across the MYXV phylogeny, this was not associated with the increase in evolutionary rate. The period from 1996 to 2012 saw significant declines in wild rabbit numbers, due to the introduction of rabbit hemorrhagic disease and prolonged drought in south-eastern Australia, followed by the partial recovery of populations. It is therefore possible that a rapidly changing environment for virus transmission changed the selection pressures faced by MYXV, altering the course and pace of virus evolution.

    IMPORTANCE The co-evolution of myxoma virus (MYXV) and European rabbits in Australia is one of the most important natural llsquo;experimentsrrsquo; in evolutionary biology, providing insights into virus adaptation to new hosts and the evolution of virulence. Previous studies of MYXV evolution have also shown that the virus evolves both relatively rapidly and in a strongly clock-like manner. Using newly acquired MYXV genome sequences from Australia we show that the virus has experienced a dramatic change in evolutionary behavior over the last 20 years, with a break-down in clock-like structure, the appearance of a rapidly evolving virus lineage, and the accumulation of multiple non-synonymous and indel mutations. We suggest that this punctuated evolutionary event may reflect a change in selection pressures as rabbit numbers declined following the introduction of rabbit hemorrhagic disease virus and drought in the geographic regions inhabited by rabbits.

  • Hepatitis Delta Antigen Regulates mRNA and Antigenome RNA Levels during Hepatitis Delta Virus Replication [Genome Replication and Regulation of Viral Gene Expression]

  • Hepatitis delta virus (HDV) is a satellite of hepatitis B virus that increases the severity of acute and chronic liver disease. HDV produces three processed RNAs that accumulate in infected cells: the circular genome, the circular antigenome, which serves as a replication intermediate, and lesser amounts of the mRNA, which encodes the sole viral protein, hepatitis delta antigen (HDAg). The HDV genome and antigenome RNAs form ribonucleoprotein complexes with HDAg. Although HDAg is required for HDV replication, it is not known how the relative amounts of HDAg and HDV RNA affect replication, nor whether HDAg synthesis is regulated by the virus. Using a novel transfection system in which HDV replication is initiated using in vitro-synthesized circular HDV RNAs, HDV replication was found to depend strongly on the relative amounts of HDV RNA and HDAg. HDV controls these relative amounts via differential effects of HDAg on the production of HDV mRNA and antigenome RNA, both of which are synthesized from the genome RNA template. mRNA synthesis is favored at low HDAg levels but becomes saturated at high HDAg concentrations. Antigenome RNA accumulation increases linearly with HDAg and dominates at high HDAg levels. These results provide a conceptual model for how HDV antigenome RNA production and mRNA transcription are controlled from the earliest stage of infection onwards and also demonstrate that, in this control, HDV behaves similarly to other negative strand RNA viruses, even though there is no genetic similarity between them.

    IMPORTANCE Hepatitis delta virus (HDV) is a satellite of hepatitis B virus that increases the severity of liver disease; approximately 15 million people are chronically infected worldwide. There are no licensed therapies available. HDV is not related to any known virus and few details regarding its replication cycle are known. One key question is whether and how HDV regulates the relative amounts of viral RNA and protein in infected cells. Such regulation might be important because the HDV RNA and protein form complexes that are essential for HDV replication and the proper stoichiometry of these complexes could be critical for their function. Our results show that the relative amounts of HDV RNA and protein in cells are indeed important for HDV replication and that the virus does control them. These observations indicate that further study of these regulatory mechanisms is required to better understand replication of this serious human pathogen.

  • The DNase Activity of KSHV SOX Protein Serves an Important Role in Viral Genome Processing During Lytic Replication [Genome Replication and Regulation of Viral Gene Expression]

  • The KSHV alkaline exonuclease SOX, encoded by ORF37, is a bifunctional, early-lytic phase protein that possesses an alkaline 5'- to 3'- DNase activity and promotes host shutoff at the mRNA level during productive lytic infection. While the SOX protein is well characterized for drastically impairing cellular gene expression, little is known about the impact of its DNase activity on KSHV genome, life cycle and biology of KSHV infections. Here, we introduced a previously described DNase-inactivating Glu129His/Q129H mutation into ORF37 gene of the viral genome to generate ORF37-Q129H recombinant virus (Q129H mutant) and investigated the effects of loss/inactivation of DNase activity on viral genome replication, cleavage and packaging. We, for the first time, provide experimental evidences that the DNase activity of SOX protein does not affect the viral latent/lytic DNA synthesis, but is required for cleavage and processing of the KSHV genome during lytic replication. Interestingly, the Q129H mutation severely impaired intranuclear processing of progeny virions compared to the wild-type ORF37, as assessed by pulse-field/Gardella gel electrophoresis, electron microscopy, and SMARD (Single Molecule Analysis of Replicating DNA) assays. Complementation with ORF37-wt or BGLF5 (KSHV's homolog of Epstein-Barr Virus) in 293L/Q129H cells restored the viral genome encapsidation defects. Together, these results indicated that ORF37's proposed DNase activity is essential for viral genome processing and encapsidation, hence, can be targeted for designing antiviral agents to block KSHV virion production.

    IMPORTANCE: Kaposi's sarcoma-associated herpesvirus is the causative agent of multiple malignancies predominantly in immunocompromised individuals including HIV/AIDS patients. Reduced incidence of KS in HIV/AIDS patients receiving anti-herpetic drugs to block lytic replication confirms the role of lytic DNA replication and gene products in KSHV-mediated tumorigenesis. Herpesvirus lytic replication results in the production of complex, concatemeric DNA, which is cleaved into unit-length viral DNA for packaging into the infectious virions. The conserved herpesviral alkaline exonucleases play an important role in viral genome cleavage and packaging. Here, by using the previously described Q129H mutant virus that selectively lacks DNase activity but retains host shutoff activity, we provide experimental evidences confirming that the DNase function of KSHV SOX protein is essential for viral genome processing, packaging and capsid maturation into the cytoplasm during lytic replication in infected cells. This led to the identification of ORF37's DNase activity as a potential target for antiviral therapeutics.

  • Zika virus infection in Tupaia belangeri causes dermatological manifestations and confers protection against secondary infection [Pathogenesis and Immunity]

  • Animal models of Zika virus (ZIKV) infection have recently been established in mice, guinea pigs, and nonhuman primates. Tree shrews (Tupaia belangeri) are an emerging experimental animal in biomedical applications, but their susceptibility to ZIKV infection has not been explored. In the present study, we showed that subcutaneous inoculation of ZIKV led to rapid viremia and viral secretion in saliva, as well as to typical dermatological manifestations characterized by massive diffuse skin rash on the trunk. Global transcriptomic sequencing of peripheral blood mononuclear cells isolated from ZIKV-infected animals revealed systematic gene expression changes related to the inflammatory response and dermatological manifestations. Importantly, ZIKV infection readily triggered the production of high-titer neutralizing antibodies, thus preventing secondary homologous infection, in tree shrews. However, neonatal tree shrews succumbed to ZIKV challenge upon intracerebral infection. The tree shrew model described here recapitulates the most common dermatological manifestations observed in ZIKV-infected patients and may greatly facilitate the elucidation of ZIKV pathogenesis and the development of novel vaccines and therapeutics.

    IMPORTANCE

    The reemergence of Zika virus (ZIKV) has caused a global public health crisis since 2016, and there are currently no vaccines or antiviral drugs to prevent or treat ZIKV infection. However, considerable advances have been made in understanding the biology and pathogenesis of ZIKV infection. In particular, various animal models have been successfully established to mimic ZIKV infection and its associated neurological diseases and to evaluate potential countermeasures. However, the clinical symptoms in these mouse and nonhuman primate models are different from the common clinical manifestations seen in human ZIKV patients; in particular, dermatological manifestations are rarely recapitulated in these animal models. Here, we developed a new animal model of ZIKV infection in tree shrews, a rat-sized, primate-related mammal. In vitro and in vivo characterization of ZIKV infection in tree shrews established a direct link between ZIKV infection and the immune responses and dermatological manifestations. The tree shrew model described here, as well as other available animal models, provides a valuable platform to study ZIKV pathogenesis and to evaluate vaccines and therapeutics.

  • Measles virus bearing MIBE-derived fusion protein is pathogenic after infection via the respiratory route [Virus-Cell Interactions]

  • A clinical isolate of measles virus (MeV) bearing a single amino acid alteration in the viral fusion protein (F; L454W) was previously identified in two patients with lethal sequelae of MeV central nervous system (CNS) infection. The mutation dysregulated the viral fusion machinery so that the mutated F protein mediated cell fusion in the absence of known MeV cellular receptors. While this virus could feasibly have arisen via intra-host evolution of the wildtype (wt) virus, it was recently shown that the same mutation emerged under the selective pressure of small molecule antiviral treatment. Under these conditions, a potentially neuropathogenic variant emerged outside the CNS. While CNS adaptation of MeV was thought to generate viruses that are less fit for inter-host spread, we show that two animal models can be readily infected with CNS-adapted MeV via the respiratory route. Despite bearing a fusion protein that is less stable at 37ddeg;C than the wt MeV F, this virus infects and replicates in cotton rat lung tissue more efficiently than the wt virus, and is lethal in a suckling mouse model of MeV encephalitis even with a lower inoculum. Thus, either during lethal MeV CNS infection or during antiviral treatment in vitro, neuropathogenic MeV can emerge, can infect new hosts via the respiratory route, and is more pathogenic (at least in these animal models) than wt MeV.

    Importance: Measles (MeV) infection can be severe in immunocompromised individuals and lead to complications including measles inclusion body encephalitis (MIBE). In some cases, MeV persistence and subacute sclerosing panencephalitis (SSPE) occur even in the face of an intact immune response. While relatively rare complications of MeV infection, MIBE and SSPE are lethal. This work addresses the hypothesis that despite a dysregulated viral fusion complex, CNS adapted measles virus can spread outside the CNS within an infected host.

  • Blocking HIV-1 infection by chromosomal integrative expression of human CD4 on the surface of Lactobacillus helveticus R0052 [Vaccines and Antiviral Agents]

  • Lactobacillus bacteria are potential delivery vehicles for biopharmaceutical molecules because they are well-recognized as safe microorganisms that naturally inhabit the human body. The goal of this study is to employ these lactobacilli to combat human immunodeficiency virus (HIV-1) infection and transmission. By using a chromosomal integration method, we engineered the Lactobacillus helveticus R0052 strain to display human CD4, the HIV-1 receptor, on the cell surface. Since human CD4 can bind to any infectious HIV-1 particles, the engineered lactobacilli can potentially capture HIV-1 of different sub-types and prevent infection. Our data demonstrate that the CD4-carrying bacteria are able to adsorb HIV-1 particles and reduce infection significantly in vitro, and also block intrarectal HIV-1 infection in a humanized mouse model in preliminary tests in vivo. Our results support the potential of this approach to decrease the efficiency of HIV-1 sexual transmission.

    IMPORTANCE In the absence of an effective vaccine, alternative approaches to block HIV-1 infection and transmission with commensal bacteria expressing antiviral proteins are being considered. This report provides a proof-of-concept by using Lactobacillus stably expressing the HIV-1 receptor, CD4, to capture and neutralize HIV-1 in vitro and in a humanized mouse model. The stable expression of antiviral proteins like CD4 following genomic integration of the corresponding genes into this Lactobacillus strain may contribute to the prevention of HIV-1 sexual transmission.

  • Human papillomavirus E6/E7 and lncRNA TMPOP2 mutually upregulated gene expression in cervical cancer cells [Genome Replication and Regulation of Viral Gene Expression]

  • TMPOP2 was previously suggested to be an oncogenic long noncoding RNA which is excessively expressed in cervical cancer cells and inhibits E-cadherin gene expression by recruiting transcription repressor EZH2 to gene promoter. So far, the function and regulation of TMPOP2 in cervical cancer remains largely unknown. Herein, we found that TMPOP2 expression was correlated with human papillomavirus HPV16/18 E6 and E7 in cervical cancer cell CaSki and HeLa. Tumor suppressor p53, which is targeted for degradation by HPV16/18, was demonstrated to associate with two p53-response elements in the TMPOP2 promoter to repress the transcription of TMPOP2 gene. Reciprocally, ectopic expression of TMPOP2 was demonstrated to sequester tumor repressor miRNAs miR-375 and miR-139 which target HPV16/18 E6/E7 mRNA and resulted in an upregulation of HPV16/18 E6/E7 genes. Thereby, HPV16/18 E6/E7 and the lncRNA TMPOP2 form a positive feedback loop to mutually derepress gene expression in cervical cancer cells. Moreover, results of RNA sequencing and cell cycle analysis showed that knockdown of TMPOP2 impaired the expression of cell cycle genes, induced cell cycle arrest and inhibited HeLa cell proliferation. Together, our results indicate that TMPOP2 and HPV16/18 E6/E7 mutually strengthen their expression in cervical cancer cells to enhance tumorigenic activities.

    IMPORTANCE: Type 16 and type 18 HPVs are the main causative agents of cervical cancer. Viral proteins HPV16/18 E6 and E7 are constitutively expressed in cancer cells to maintain oncogenic phenotypes. Accumulating evidences suggest that HPVs are correlated with the deregulation of lncRNAs in cervical cancer although the mechanism was unexplored in most cases. TMPOP2 is a newly identified lncRNA excessively expressed in cervical cancer. However, the mechanism for the upregulation of TMPOP2 in cervical cancer cells remains largely unknown and its relationship with HPVs is still elusive. The significance of our research is in revealing the mutual upregulation of HPV16/18 E6/E7 and TMPOP2 with the molecular mechanisms explored. This study will expand our understandings to the oncogenic activities of human papillomaviruses and lncRNAs.

  • Conserved gammaherpesvirus protein kinase selectively promotes irrelevant B cell responses. [Pathogenesis and Immunity]

  • Gammaherpesviruses are ubiquitous pathogens that are associated with B cell lymphomas. In the early stages of chronic infection these viruses infect naïve B cells and subsequently usurp the B cell differentiation process through the germinal center response to ensure latent infection of long-lived memory B cells. A unique feature of early gammaherpesvirus chronic infection is a robust differentiation of irrelevant, virus-nonspecific B cells with reactivities against self and other species' antigens. In contrast, protective, virus-specific humoral responses do not reach peak levels until a much later time. While several host factors are known to either promote or selectively restrict gammaherpesvirus-driven germinal center response, viral mechanisms that contribute to the irrelevant B cell response have not been defined. In this report we show that expression and the enzymatic activity of the gammaherpesvirus-encoded conserved protein kinase selectively facilitates the irrelevant, but not virus-specific B cell responses. Further, we show that lack of IL-1 receptor attenuates gammaherpesvirus-driven B cell differentiation and viral reactivation. Because germinal center B cells are thought to be the target of malignant transformation during gammaherpesvirus-driven lymphomagenesis, identification of host and viral factors that promote germinal center responses during gammaherpesvirus infection may offer an insight into the mechanism of gammaherpesvirus pathogenesis.

    IMPORTANCE Gammaherpesviruses are ubiquitous cancer-associated pathogens that usurp the B cell differentiation process to establish life-long latent infection in memory B cells. A unique feature of early gammaherpesvirus infection is the robust increase in differentiation of B cells that are not specific for viral antigens and instead encode antibodies that react with self and other species' antigens. Viral mechanisms that are involved in driving such irrelevant B cell differentiation are not known. Here we show that gammaherpesvirus-encoded conserved protein kinase and host IL-1 signaling promote irrelevant B cell responses and gammaherpesvirus-driven germinal center response, with the latter thought to be the target of viral transformation.

  • Early human B cell response to Ebola virus in four U.S. survivors of infection [Pathogenesis and Immunity]

  • The human B cell response to natural filovirus infections early after recovery is poorly understood. Previous serologic studies suggest that some Ebola virus survivors exhibit delayed antibody responses with low magnitude and quality. Here, we sought to study the population of individual memory B cells induced early in convalescence. We isolated monoclonal antibodies (mAbs) from memory B cells from four survivors treated for Ebola virus disease (EVD) one or three-months after discharge from hospital. At the early time points post-recovery, the frequency of Ebola-specific B cells was low and dominated by clones that were cross-reactive with both Ebola glycoprotein (GP) and with the secreted form (sGP). Of 25 mAbs isolated from four donors, only one exhibited neutralization activity. This neutralizing mAb, designated mAb EBOV237, recognizes an epitope in the glycan cap of the surface glycoprotein. In vivo murine lethal challenge studies showed that EBOV237 conferred protection when given prophylactically at a level similar to that of the ZMapp component mAb 13C6. The results suggest that the human B cell response to EVD one to three months post-discharge is characterized by a paucity of broad or potent neutralizing clones. However, the neutralizing epitope in the glycan cap recognized by EBOV237 may play a role in the early human antibody response to EVD and should be considered in rational design strategies for new Ebola virus vaccine candidates.

    IMPORTANCE The pathogenesis of Ebola virus disease (EVD) in humans is complex, and the mechanisms contributing to immunity are poorly understood. In particular, it appears that the quality and magnitude of the human B cell response early after recovery from EVD may be reduced compared to most viral infections. Here we isolated human monoclonal antibodies from B cells of four survivors of EVD at one or three months after hospital discharge. Ebola-specific memory B cells early in convalescence were low in frequency, and the antibodies they encoded demonstrated poor neutralizing potencies. One neutralizing antibody that protected mice from lethal infection, EBOV237, was identified in the panel of 25 human antibodies isolated. Recognition of the glycan cap epitope recognized by EBOV237 suggests this antigenic site should be considered in vaccine design and treatment strategies for EVD.

  • Broad hemagglutinin-specific memory B cell expansion by seasonal influenza virus infection reflects early-life imprinting and adaptation to the infecting virus [Pathogenesis and Immunity]

  • Memory B cells (MBCs) are key determinants of the B cell response to influenza virus infection and vaccination, but the effect of different forms of influenza antigen exposure on MBC populations has received little attention. We analyzed peripheral blood mononuclear cells and plasma collected following human H3N2 influenza infection to investigate the relationship between hemagglutinin-specific antibody production and changes in the size and character of hemagglutinin-reactive MBC populations. Infection produced increased concentrations of plasma IgG reactive to the H3 head of the infecting virus, to the conserved stalk, and to a broad chronological range of H3s consistent with original antigenic sin responses. H3-reactive IgG MBC expansion after infection included reactivity to head and stalk domains. Notably, expansion of H3 head-reactive MBC populations was particularly broad and reflected original antigenic sin patterns of IgG production. Findings also suggest that early-life H3N2 infection "imprints" for strong H3 stalk-specific MBC expansion. Despite the breadth of MBC expansion, the MBC response included an increase in affinity for the H3 head of the infecting virus. Overall, our findings indicate that H3-reactive MBC expansion following H3N2 infection is consistent with maintenance of response patterns established early in life, but nevertheless includes MBC adaptation to the infecting virus.

    IMPORTANCE Rapid and vigorous virus-specific antibody responses to influenza virus infection and vaccination result from activation of preexisting virus-specific memory B cells (MBCs). Understanding the effects of different forms of influenza virus exposure on MBC populations is therefore an important guide to the development of effective immunization strategies. We demonstrate that exposure to the influenza hemagglutinin via natural infection enhances broad protection through expansion of hemagglutinin-reactive MBC populations that recognize head and stalk regions of the molecule. Notably, we show that hemagglutinin-reactive MBC expansion reflects imprinting by early-life infection and that this might apply to stalk-reactive, as well as to head-reactive, MBCs. Our findings provide experimental support for the role of MBCs in maintaining imprinting effects and suggest a mechanism by which imprinting might confer heterosubtypic protection against avian influenza viruses. It will be important to compare our findings to the situation after influenza vaccination.

  • Baculovirus IE2 interacts with viral DNA through Daxx to generate an organized nuclear body structure for gene activation in Vero cells [Virus-Cell Interactions]

  • Upon virus infection of a cell, the uncoated DNA is usually blocked by the host intrinsic immune system inside the nucleus. Although it is crucial for the virus to counteract the host intrinsic immune system and access its genome, little is known about how viruses can knock down host restriction and identify their blocked genomes for later viral gene activation and replication. We found that upon baculovirus transduction into Vero E6 cells, the invading viral DNA is trapped by the cellular death domain-associated protein (Daxx) and histone H3.3 in the nucleus, resulting in gene inactivation. IE2, a baculovirus transactivator, targets host Daxx through IE2 SUMO-interacting motifs (SIMs) to indirectly access viral DNA, and forms unique nuclear body structures, we term clathrate cage-like apparatus (CCLA), at the early transduction stage. At the later transduction stage, CCLAs gradually enlarge, and IE2 continues to closely interact with viral DNA but no longer associates with Daxx at this later transduction stage. The association with Daxx is essential for IE2 CCLA formation, and the enlarged CCLAs are capable of transactivating viral but not chromosomal DNA of the Vero E6 cells. Our study reveals that baculovirus IE2 counteracts the cellular intrinsic immune system by specifically targeting Daxx and H3.3 to associate with viral DNA indirectly and efficiently. IE2 then utilizes this association with viral DNA to establish a unique CCLA cellular nanomachinery, which is visible under light microscopy, as an enclosed environment for proper viral gene expression.

    IMPORTANCE The major breakthrough of this work is that viral protein IE2 localizes and transactivates its own viral DNA through a most unlikely route, i.e., host proteins Daxx and H3.3 that are designed to efficiently restrict viral DNA from expression. By interacting with these host intrinsic immune factors, IE2 can thus target the viral DNA, and then form a unique spherical nuclear body, which we name the CCLA, to enclose the viral DNA and necessary factors to assist in high-level transactivation. Our study represents one of the most complete investigations of nuclear body formation. In addition, so far only RNA or protein molecules were reported as potential nucleators for initiating nuclear body formation, our study may represent the first example showing that DNA can be a nucleator for a new class of nuclear body formation.

  • Differential Antibody-Based Immune Response Against Isolated GP1 Receptor-Binding Domains from Lassa and Junin Viruses [Pathogenesis and Immunity]

  • There are two predominant subgroups in the Arenaviridae family of viruses, the Old-World and the New-World viruses that use distinct cellular receptors for entry. While New-World viruses typically elicit good neutralizing antibody responses, the Old-World viruses generally evade such responses. Antibody based immune responses are directed against the glycoprotein spike complexes that decorate the viruses. A thick coat of glycans reduces the accessibility of antibodies to the surface of spike complexes from Old-World viruses but other mechanisms may further hamper the development of efficient humoral responses. Specifically, it was suggested that the GP1 receptor-binding module of the Old-World Lassa virus might help evading humoral response. Here we investigate the immunogenicity of the GP1 domain from Lassa virus and compare it to GP1 domain from the New-World Juniiacute;n virus. We found striking differences in the ability of antibodies that were developed against these immunogens to target the same GP1 receptor-binding domains in the context of the native spike complexes. Whereas GP1 from Juniiacute;n virus elicited productive neutralizing responses, GP1 from Lassa virus elicited only non-productive responses. These differences can be rationalized by conformational changes that GP1 from Lassa virus but not from Juniiacute;n virus, undergoes after dissociating from the trimeric spike complex. Hence shedding of GP1 in the case of Lassa virus can indeed serve as a mechanism to subvert the humoral immune response. Moreover, the realization of using a recombinant protein for eliciting productive response against the New-World Juniiacute;n virus may suggests a novel and safe way to design future vaccines.

    IMPORTANCE Some viruses that belong to the Arenaviridae family like Lassa and Juniiacute;n viruses are notorious human pathogens, which may lead to fatal outcomes when they infect people. It is thus important to develop means to combat these viruses. For developing effective vaccines, it is vital to understand the basic mechanisms that these viruses utilize in order to evade or overcome host immune responses. It was previously noted that the GP1 receptor-binding domain from Lassa virus is shedded and accumulates in the sera of infected individuals. This raised the possibility that Lassa GP1 may function as an immunological decoy. Here we demonstrate that mice develop non-productive immune responses against GP1 from Lassa virus, which is in contrast to effective neutralizing responses that GP1 from Juniiacute;n virus elicits. Thus, GP1 from Lassa virus is indeed an immunological decoy and GP1 from Juniiacute;n virus may serve as a constituent of a future vaccine.

  • Engineering Responses to Amino Acid Substitutions in the VP0 and VP3 Coding Regions of PanAsia-1 Strains of Foot-and-Mouth Disease Virus Serotype O [Virus-Cell Interactions]

  • The presence of sequence divergence through adaptive mutations in the major capsid protein VP1, and also in VP0 (VP4 and VP2) and VP3 of foot-and-mouth disease virus (FMDV) is relevant to a broad range of viral characteristics. To explore the potential role of isolate-specific residues in the VP0 and VP3 coding regions of PanAsia-1 strains in genetic and phenotypic properties of FMDV, a series of recombinant full-length genomic clones were constructed, by using a Cathay topotype infectious cDNA as the original backbone. The deleterious and compensatory effects of individual amino acid substitutions at positions 4008, 3060, and in several different domains of VP2 illustrated that the chain-based spatial interaction patterns of VP1nndash;3 as well as between the internal VP4 and the three external capsid proteins of FMDV might contribute to the assembly of eventually viable viruses. The Y2079H site-directed mutants dramatically induced a decrease in plaque size on BHK-21 cells and viral pathogenicity in suckling mice. Remarkably, the 2079H-encoding viruses displayed a moderate increase in acid sensitivity correlated with NH4Cl resistance, as compared to the Y2079-encoding viruses. Interestingly, none of all the 16 recued viruses were able to infect heparan sulfate-expressing CHO-K1 cells. However, viral infection in BHK-21 cells was facilitated by utilizing non-integrin-dependent, heparin-sensitive receptor(s), and replacements of four uncharged amino acids at position 3174 in VP3 of FMDV had no apparent influence on heparin affinity. These results provide particular insights into the correlation of evolutionary biology with genetic diversity in adapting populations of FMDV.

    IMPORTANCE The sequence variation within the capsid proteins occurs frequently in the infection of susceptible tissue cultures, reflecting the high levels of genetic diversity of FMDV. A systematic study for the functional significance of isolate-specific residues in VP0 and VP3 of FMDV PanAsia-1 strains suggested that the interaction of amino acid side chains between the N-terminus of VP4 and several potential domains of VP1nndash;3 had cascading effects on the viability and developmental characteristics of progeny viruses. Y2079H in VP0 of the indicated FMDVs could affect plaque size and pathogenicity as well as acid sensitivity correlated with NH4Cl resistance, whereas no inevitable correlation in viral plaque and acid-sensitive phenotypes. The heparin affinity of non-integrin-dependent FMDVs implied the difference in structure of heparan sulfate proteoglycans on the surface of different cell lines. These results may contribute to understanding the distinct phenotypic properties of FMDV in vitro and in vivo.

  • An epigenetic journey: Epstein-Barr virus transcribes chromatinized and subsequently unchromatinized templates during its lytic cycle [Minireviews]

  • The Epstein-Barr virus lytic phase, like that of all herpesviruses, proceeds via an orderly cascade that integrates DNA replication and gene expression. EBV early genes are expressed independent of viral DNA amplification, and several early gene products facilitate DNA amplification. On the other hand, EBV late genes are defined by their dependence upon viral DNA replication for expression. Recently, a set of orthologous genes found in bbeta; and -herpesviruses have been determined to encode a viral pre-initiation complex (vPIC) that mediates late gene expression. EBV vPIC requires an origin of lytic replication in cis, implying that vPIC mediates transcription from newly replicated DNA. Consistent with this implication, EBV late gene mRNAs localize to replication factories. Notably, these factories exclude canonical histones. In this review we compare and contrast the mechanisms and epigenetics of EBV early versus late gene expression. We summarize recent findings, propose a model explaining the dependence of EBV late gene expression on lytic DNA amplification, and suggest some directions for future study.

  • GADD45{gamma} activated early in the course of HSV-1 infection suppresses the activation of a network of innate immunity genes [Cellular Response to Infection]

  • The stress response genes encoding GADD45, and to a lesser extent GADD45bbeta; are activated early in infection with HSV-1. Cells depleted of GADD45 by transfection of shRNA or in which the gene had been knocked out (GADD45) yield significantly less virus than untreated infected cells. Consistent with lower virus yields the GADD45 cells, both uninfected or infected with HSV-1 exhibit significantly higher levels of transcripts encoding a cluster of a network of innate immunity genes that include those encoding IFI16, IFIT1, MDA5 and RIG-I. Members of this cluster of genes have been reported by this laboratory to be activated concurrent with significantly reduced virus yields in cell depleted of LGP2 or HDAC4. We conclude the following: (i) Innate immunity to HSV-1 is normally repressed in unstressed cells. The repression appears to be determined by two mechanisms. The first, illustrated here is by activation by HSV-1 infection of the gene encoding GADD45. The second mechanism requires constitutively active expression of LGP2 and HDAC4.

    IMPORTANCE Previous studies from our lab reported that knockout of some innate immunity genes is associated with increases in the expression of overlapping networks of genes and significant loss of ability to support the replication of HSV-1; knockout of other genes is associated with decreases in the expression of overlapping networks of genes and no effect on viral replication. In this report we have documented evidence that depletion of GADD45 reduced virus yields concurrently with significant upregulation of expression of a cluster of innate immunity genes comprising IFI16, IFIT1, MDA5 and RIG-I. The current report differs from the preceding study in an important aspect: the preceding study found no evidence to support the hypothesis that HSV-1 maintained adequate levels of LGP2 or HDAC4 to block upregulation of the cluster of innate immunity genes. We show that HSV-1 causes upregulation of the GADD45 gene to prevent the upregulation of innate immunity genes.

  • Arbidol and other Low Molecular Weight Drugs That Inhibit Lassa and Ebola Viruses [Vaccines and Antiviral Agents]

  • Antiviral therapies that impede virus entry are attractive because they act on the first phase of the infectious cycle. Drugs that target pathways common to multiple viruses are particularly desirable when laboratory-based viral identification may be challenging, e.g. in an outbreak setting. We are interested in identifying drugs that block both Ebola virus (EBOV) and Lassa virus (LASV), two unrelated but highly pathogenic hemorrhagic fever viruses that have caused outbreaks in similar regions in Africa and share features of virus entry: use of cell surface attachment factors, macropinocytosis, endosomal receptors and low pH to trigger fusion in late endosomes. Towards this goal, we directly compared the potency of eight drugs known to block EBOV entry with their potency as inhibitors of LASV entry. Five drugs (amodiaquine, apilimod, arbidol, niclosamide, and zoniporide) showed roughly equivalent inhibition of LASV and EBOV glycoprotein (GP)-bearing pseudoviruses; three (clomiphene, sertraline and toremifene) were more potent against EBOV. We then focused on arbidol, which is licensed abroad as an anti-influenza drug and exhibits activity against a diverse array of clinically relevant viruses. We found that arbidol inhibits infection by authentic LASV, inhibits LASV GP-mediated cell-cell fusion and virus-cell fusion and, reminiscent of its activity on influenza hemagglutinin, stabilizes LASV GP to low pH exposure. Our findings suggest that arbidol inhibits LASV fusion, which may partly involve blocking conformational changes in LASV GP. We discuss our findings in terms of the potential to develop a drug cocktail that could inhibit both LASV and EBOV.

    IMPORTANCE Lassa and Ebola viruses continue to cause severe outbreaks in humans, yet there are only limited therapeutic options to treat the deadly hemorrhagic fever diseases they cause. Because of overlapping geographic occurrences and similarities in mode of entry into cells, we seek a practical drug or drug cocktail that could be used to treat infections by both viruses. Towards this goal, we directly compared eight drugs, approved or in clinical testing, for their ability to block entry mediated by the glycoproteins of both viruses. We identified five drugs with approximately equal potency against both. Among these we investigated the modes of action of arbidol, a drug licensed abroad to treat influenza infections. We found, as shown for influenza, that arbidol blocks fusion mediated by the Lassa virus glycoprotein. Our findings encourage the development of a combination of approved drugs to treat both Lassa and Ebola virus diseases.

  • Assessing the protective potential of H1N1 influenza virus hemagglutinin head and stalk antibodies in humans [Pathogenesis and Immunity]

  • Seasonal influenza viruses are a major cause of human disease worldwide. Most neutralizing antibodies (Abs) elicited by influenza viruses target the head domain of the hemagglutinin (HA) protein. Anti-HA head Abs can be highly potent, but they have limited breadth since the HA head is variable. There is great interest in developing new universal immunization strategies that elicit broadly neutralizing Abs against conserved regions of HA, such as the stalk domain. Although HA stalk Abs can provide protection in animal models, it is unknown if they are present at sufficient levels in humans to provide protection against naturally-acquired influenza virus infections. Here, we quantified H1N1 HA head and stalk-specific Abs in 179 adults hospitalized during the 2015-2016 influenza virus season. We found that HA head Abs, as measured by hemagglutinin-inhibition (HAI) assays, were associated with protection against naturally-acquired H1N1 infection. HA stalk-specific serum total IgG titers were also associated with protection, but this association was attenuated and not statistically significant after adjustment for HA head-specific Ab titers. We found slightly higher titers of HA stalk-specific IgG1 and IgA Abs in sera from uninfected participants compared to sera from infected participants; however, we found no difference in sera in vitro antibody dependent cellular cytotoxicity activity. In passive transfer experiments, sera from participants with high HAI activity efficiently protected mice, while sera with low HAI activity protected mice to a lower extent. Our data suggest that HA head Abs are more efficient at protecting against H1N1 infection compared to HA stalk Abs.

    IMPORTANCE Abs targeting the HA head of influenza viruses are often associated with protection from influenza virus infections. These Abs typically have limited breadth since mutations frequently arise in HA head epitopes. New vaccines targeting the more conserved HA stalk domain are being developed. Abs that target the HA stalk are protective in animal models, but it is unknown if these Abs exist at protective levels in humans. Here, we completed experiments to determine if Abs against the HA head and stalk were associated with protection from naturally-acquired human influenza virus infections during the 2015-2016 influenza season.

  • Expression of the pseudorabies virus gB glycoprotein triggers NK cell cytotoxicity and increases binding of the activating NK cell receptor PILRbeta [Pathogenesis and Immunity]

  • Natural killer (NK) cells are members of the innate immunity and are key players in the defense against viral infected and malignant cells. NK cells are particularly important in the innate defense against herpesviruses, including alphaherpesviruses. Aggravated and life-threatening alphaherpesvirus-induced disease has been reported in patients with NK cell deficiencies. NK cells are regulated by a diversity of activating and inhibitory cell surface receptors that recognize specific ligands on the plasma membrane of viral infected or malignant target cells. Although alphaherpesviruses have developed several evasion strategies against NK cell-mediated attack, alphaherpesvirus-infected cells are still readily recognized and killed by NK cells. However, the (viral) factors that trigger NK cell activation against alphaherpesvirus-infected cells are largely unknown.

    In this study, we show that expression of the gB glycoprotein of the alphaherpesvirus pseudorabies virus (PRV) triggers NK cell-mediated cytotoxicity, both in PRV-infected and in gB-transfected cells. In addition, we report that, like their human and murine counterpart, porcine NK cells express the activating receptor paired immunoglobulin-like type 2 receptor bbeta; (PILRbbeta;) and show that gB expression triggers increased binding of recombinant porcine PILRbbeta; to the surface of PRV-infected cells and gB-transfected cells.

    Importance Natural killer (NK) cells display a prominent cytolytic activity against virus-infected cells and are indispensable in the innate antiviral response, particularly against herpesviruses. Despite their importance in the control of alphaherpesvirus infections, relatively little is known about the mechanisms that trigger NK cell cytotoxicity against alphaherpesvirus-infected cells. Here, using the porcine alphaherpesvirus pseudorabies virus (PRV), we report that the conserved alphaherpesvirus glycoprotein gB triggers NK cell-mediated cytotoxicity, both in virus-infected and gB-transfected cells. In addition, we report that gB expression results in increased cell surface binding of porcine paired immunoglobulin-like type 2 receptor bbeta; (PILRbbeta;), an activating NK cell receptor. The interaction between PILRbbeta; and viral gB may possibly have consequences that stretch beyond the interaction with NK cells, including virus entry in host cells. The identification of gB as an NK cell-activating viral protein may be of importance in the construction of future vaccines and therapeutics requiring optimized interactions of alphaherpesviruses with NK cells.

  • Rare Detection of Antiviral Functions of Polyclonal IgA Isolated from Plasma and Breast Milk Compartments in HIV-1 Chronically Infected Women [Pathogenesis and Immunity]

  • The humoral response to invading mucosal pathogens comprises multiple antibody isotypes, derived from systemic and mucosal compartments. To understand the contribution of each antibody isotype/source to the mucosal humoral response, parallel investigation of the specificities and functions of antibodies within and across isotypes and compartments is required. The role of IgA against HIV-1 is complex, with studies supporting a protective role as well as a role for serum IgA in blocking effector functions. Thus, we explored the fine specificity and function of IgA in both plasma and mucosal secretions important to infant HIV-1 infection: breast milk. IgA and IgG were isolated from milk and plasma from 20 HIV-1 infected lactating Malawian women. HIV-1 binding specificities, neutralization potency, inhibition of virus-epithelial cell binding, and antibody-mediated phagocytosis were measured. Fine specificity mapping showed IgA and IgG responses to multiple HIV-1 Env epitopes, including conformational V1/V2 and linear V2, V3, and C5. Env IgA was heterogeneous between the milk and systemic compartments (Env IgA = 0.00- 0.63, p=0.0046- 1.00). Furthermore, IgA and IgG appeared compartmentalized, as there was a lack of correlation between the specificities of Env-specific IgA and IgG (in milk, = -0.07-0.26, p=0.35-0.83). IgA and IgG also differed in function: while neutralization and phagocytosis were consistently mediated by milk and plasma IgG, they were rarely detected in IgA from both milk and plasma. Understanding the ontogeny of the divergent IgG and IgA antigen specificity repertoires, and their effects on antibody function, will inform vaccination approaches targeted toward mucosal pathogens.

    IMPORTANCE Antibodies within the mucosa are part of the first line of defense against mucosal pathogens. Evaluating mucosal antibody isotypes, specificities and antiviral functions in relationship to the systemic antibody profile can provide insights on whether the antibody response is coordinated in response to mucosal pathogens. In a natural immunity cohort of HIV-infected lactating women, we mapped the fine specificity and function of IgA in breast milk and plasma and compared these with the autologous IgG responses. Antigen specificity and function differed between IgG and IgA, with antiviral functions (neutralization and phagocytosis) predominantly mediated by the IgG fraction in both milk and plasma. Furthermore, the specificity of milk IgA differed from systemic IgA. Our data suggest that milk IgA and systemic IgA should be separately examined as potential correlates of risk. Preventive vaccines may need to employ different strategies to elicit functional antiviral immunity by both antibody isotypes in the mucosa.

  • Transmission of A Novel Genotype Hepatitis E Virus from Bactrian Camels to Cynomolgus Macaques [Pathogenesis and Immunity]

  • Hepatitis E virus (HEV) is zoonotic and a major cause of acute viral hepatitis worldwide. Recently, we identified a novel HEV genotype 8 (HEV8) in Bactrian camels in Xinjiang, China. However, the epidemiology, pathogenicity and zoonotic potential of HEV8 are unclear. Here, we present the prevalence of HEV8 in China, and investigate its pathogenicity and cross-species transmission in cynomolgus macaques. Fresh fecal and milk samples of Bactrian camels collected from four provinces/regions in China were screened for HEV RNA by RT-PCR. An HEV8-positive sample was used to inoculate two cynomolgus macaques to examine the potential for cross-species infection. The pathogenicity of HEV8 was analyzed by testing HEV markers and liver function during the study period, and histopathology of liver biopsies at 3, 13 and 25 weeks post-inoculation. Extra-hepatic replication was tested by using RT-qPCR and immunofluorescence assay. The overall prevalence of HEV8 RNA in Chinese Bactrian camels was 1.4% (4/295) and positive samples were found in three different provinces/regions in China. Histopathology confirmed acute and chronic HEV8 infection respectively in the two monkeys. Multiple tissues were positive for HEV RNA and ORF2 proteins. Renal pathology was observed in the monkey with chronic hepatitis. Whole genome sequencing showed only 1-3 mutations in the HEV8 in the fecal samples of the two monkeys compared to that of the camel. HEV8 is circulating in multiple regions in China. Infection of two monkeys with HEV8 induced chronic and systemic infections demonstrating the high potential zoonotic risk of HEV8.

    IMPORTANCE It is estimated that one third of the world population have been exposed to HEV. In developed countries and China, zoonotic HEV strains are responsible for almost all acute and chronic HEV infection cases. It is always of immediate interest to investigate the zoonotic potential of novel HEV strains. In 2016, we discovered a novel HEV genotype in Bactrian camels, HEV8. But the epidemiology, zoonotic potential and pathogenicity of the virus were unknown. In the present study, we demonstrated that HEV8 was circulating in multiple regions in China and was capable of infecting cynomolgus macaques, a surrogate of human, posing high risk of zoonosis. Chronic hepatitis, systemic infection and renal pathology were observed. Collectively, these date indicate that HEV8 exhibits high potential of zoonotic transmission. Considering the importance of Bactrian camels as livestock animals, risk groups, such as camelid meat and milk consumers, should be screened for HEV8 infection.

  • JC Polyomavirus Entry by Clathrin-Mediated Endocytosis is Driven by {beta}-arrestin [Virus-Cell Interactions]

  • JC polyomavirus (JCPyV) establishes a persistent, lifelong, asymptomatic infection within the kidney of the majority of the human population. Under conditions of severe immunosuppression or immune modulation, JCPyV can reactivate in the central nervous system (CNS) and cause progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease. Initiation of infection is mediated through viral attachment to aalpha;2,6-sialic acid containing lactoseries tetrasaccharide c (LSTc) on the surface of host cells. JCPyV internalization is dependent on serotonin 5-hydroxytryptamine (5-HT)2 receptors, and entry is thought to occur by clathrin-mediated endocytosis (CME). However, the JCPyV entry process and the cellular factors involved in viral internalization remain poorly understood. Treatment of cells with small molecule chemical inhibitors and RNA interference of 5-HT2R endocytic machinery, including bbeta;-arrestin, clathrin, AP2, and dynamin, significantly reduced JCPyV infection. However, infectivity of polyomavirus simian virus 40 (SV40) was not affected by CME-specific treatments. Inhibition of clathrin or bbeta;-arrestin specifically reduced JCPyV internalization but did not affect viral attachment. Furthermore, mutagenesis of a bbeta;-arrestin binding domain (Ala-Ser-Lys) within the intracellular C-terminus of 5-HT2AR, severely diminished internalization and infection, suggesting that bbeta;-arrestin interactions with 5-HT2AR are critical for JCPyV infection and entry. These conclusions illuminate key host factors that regulate clathrin-mediated endocytosis of JCPyV, which is necessary for viral internalization and productive infection.

    IMPORTANCE Viruses usurp cellular factors to invade host cells. Activation and utilization of these proteins upon initiation of viral infection is therefore required for productive infection and resultant viral disease. The majority of healthy individuals are asymptomatically infected by JC polyomavirus (JCPyV), but if the host immune system is compromised, JCPyV can cause progressive multifocal leukoencephalopathy (PML), a rare, fatal demyelinating disease. Individuals infected with HIV or taking prolonged immunomodulatory therapies have a heightened risk for developing PML. The cellular proteins and pathways utilized by JCPyV to mediate viral entry are poorly understood. Our findings further characterize how JCPyV utilizes the clathrin-mediated endocytosis pathway to invade host cells. We have identified specific components of this pathway that are necessary for the viral entry process and infection. Collectively, the conclusions increase our understanding of JCPyV infection and pathogenesis and may contribute to the future development of novel therapeutic strategies for PML.

  • Characterization of intact proviruses in blood and lymph node from HIV-infected individuals undergoing analytical treatment interruption [Genetic Diversity and Evolution]

  • The role of lymphoid tissue as a potential source of HIV-1 rebound following interruption of antiretroviral therapy is uncertain. To address this issue, we compared the latent viruses obtained from CD4+ T cells in peripheral blood and lymph nodes to viruses emerging during treatment interruption. Latent viruses were characterized by sequencing near full-length (NFL) proviral DNA, and env from viral outgrowth cultures (VOAs). 5 HIV-1 infected individuals on antiretroviral therapy (ART) were studied, 4 of whom participated in a clinical trial of a TLR9 agonist that included an analytical treatment interruption. We found that 98% of intact or replication competent clonal sequences overlapped between blood and lymph node. In contrast, there was no overlap between 205 latent reservoir and 125 rebound sequences in the 4 individuals who underwent treatment interruption. However, rebound viruses could be accounted for by recombination. The data suggests that CD4+ T cells carrying latent viruses circulate between blood and lymphoid tissues in individuals on ART and support the idea that recombination may play a role in the emergence of rebound viremia.

    IMPORTANCE HIV-1 persists as a latent infection in CD4+ T cells that can be found in lymphoid tissues in infected individuals during ART. However, the importance of this tissue reservoir and its contribution to viral rebound upon ART interruption is not clear. In this study, we sought to compare latent HIV-1 from blood and lymph node CD4+ T cells from 5 HIV-1 infected individuals. Further, we analyzed the contribution of lymph node viruses to viral rebound. We observed that the frequency of intact proviruses was the same in blood and lymph node. Moreover, expanded clones of T cells bearing identical proviruses were found in blood and lymph node. These latent reservoir sequences did not appear to be the direct origin of rebound virus. Instead, latent proviruses were found to contribute to the rebound compartment by recombination.

  • The molecular basis for antigenic drift of human A/H2N2 influenza viruses [Genetic Diversity and Evolution]

  • Influenza A/H2N2 viruses caused a pandemic in 1957 and continued to circulate in humans until 1968. The antigenic evolution of A/H2N2 viruses over time and the amino acid substitutions responsible for this antigenic evolution are not known. Here, the antigenic diversity of a representative set of human A/H2N2 viruses isolated from 1957 until 1968 was characterized. Antigenic change of influenza A/H2N2 viruses during the 12 years that this virus circulated was modest. Two amino acid substitutions, T128D and N139K, located in the head domain of the H2 hemagglutinin molecule were identified as important determinants of antigenic change during A/H2N2 virus evolution. The rate of A/H2N2 virus antigenic evolution during the twelve-year period after introduction in humans was half of that of A/H3N2 viruses, despite similar rates of genetic change.

    IMPORTANCE While influenza A viruses of subtype H2N2 were at the origin of the Asian influenza pandemic, little is known about the antigenic changes that occurred during the twelve years of circulation in humans, the role of preexisting immunity and evolutionary rates of the virus. In this study, the antigenic map derived from hemagglutination inhibition titers of cell-cultured virus isolates and ferret post-infection sera displayed a directional evolution of viruses away from earlier isolates. Furthermore, individual mutations in close proximity to the receptor-binding site of the HA molecule determined the antigenic reactivity confirming that individual amino acid substitutions in A/H2N2 viruses can confer major antigenic changes. This study adds to our understanding of virus evolution with respect to antigenic variability, rates of virus evolution, and potential escape mutants of A/H2N2.

  • Identification of NK cell subpopulations that differentiate HIV-infected subject cohorts with diverse level of virus control [Pathogenesis and Immunity]

  • HIV infection is controlled immunologically in a small subset of infected individuals without antiretroviral therapy (ART), though the mechanism of control is unclear. CD8+ T cells are a critical component of HIV control in many immunologic controllers. NK cells are also believed to have a role in controlling HIV infection, though their role is less well-characterized. We used mass cytometry to simultaneously measure expression of 24 surface markers on peripheral NK cells from HIV-infected subjects with varying degrees of HIV natural control; we then used machine learning to identify NK cell subpopulations that differentiate HIV controllers from non-controllers. Using CITRUS (Cluster identification, characterization, and regression), we identified 3 NK cell subpopulations that differentiated subjects with chronic HIV viremia (Viremic Non-Controllers, VNC) from individuals with undetectable HIV viremia without ART (Elite Controllers, EC). In a parallel approach, we identified 11 NK cell subpopulations that differentiated HIV-infected subject groups using k-means clustering after dimensionality reduction by t-neighbor Stochastic Neighbor Embedding (tSNE) or Linear Discriminant Analysis (LDA). Of these additional 11 subpopulations, the frequencies of 5 correlated with HIV DNA levels; importantly, significance was retained in 2 subpopulations when only including cohorts without detectable viremia. By comparing the surface marker expression patterns of all identified subpopulations, we revealed that the CD11b+CD57-CD161+Siglec-7+ subpopulation of CD56dimCD16+ NK cells are more abundant in EC and HIV-negative controls compared to VNC, and the frequency of these cells correlated with HIV DNA levels. We hypothesize that this population may have a role in immunologic control of HIV infection.

    Importance:

    HIV infection results in the establishment of a stable reservoir of latently infected cells; ART is usually required to keep viral replication in control and disease progression at bay, though a small subset of HIV-infected subjects can control HIV infection without ART through immunological mechanisms. In this study, we sought to identify subpopulations of NK cells that may be involved in the natural immunological control of HIV infection. We used mass cytometry to measure surface marker expression on peripheral NK cells. Using two distinct semi-supervised machine learning approaches, we identified a CD11b+CD57-CD161+Siglec-7+ subpopulation of CD56dimCD16+ NK cells that differentiates HIV controllers from non-controllers. These cells can be sorted out for future functional studies to assess their potential role in the immunologic control of HIV infection.

  • S-like phase CDKs stabilize the Epstein-Barr virus BDLF4 protein to temporally control late gene transcription [Genome Replication and Regulation of Viral Gene Expression]

  • Temporally controlled gene expression is necessary for the propagation of herpesviruses. To achieve this, herpesviruses encode several transcriptional regulators. In Epstein-Barr virus, BcRF1 associates with five viral proteins (BDLF4, BGLF3, BFRF2, BVLF1 and BDLF3.5) to form the viral late (L) gene regulatory complex, which is called the viral Pre-Initiation Complex (vPIC), on TATT-containing promoters. However, regulation of the vPIC has been largely unexplored. Here, we performed two screens using a kinase inhibitor library, and identified a series of CDK inhibitors that downregulated the expression of L genes without any impact on viral DNA replication through de-stabilization of BDLF4 protein. Knockdown of CDK2 by shRNA and proteasome inhibitor treatment showed that phosphorylation of BDLF4 protein prevented ubiquitin-mediated degradation. Moreover, we demonstrated that cyclin A- and E-associated CDK2 complexes phosphorylated BDLF4 in vitro and identified several serine/threonine phosphorylation sites in BDLF4. Phospho-inactive and -mimic mutants revealed that phosphorylation at threonine 91 plays a role in stabilizing BDLF4. Therefore, our findings indicate that S-like phase CDKs mediate regulation of L gene expression through stabilization of the BDLF4 protein, which makes the temporal L gene expression system more robust.

    IMPORTANCE Late (L) genes represent more than one-third of the herpes virus genome, suggesting that many of these genes are indispensable for the life cycle of the virus. With the exception of BCRF1, BDLF2 and BDLF3, Epstein-Barr virus L genes are transcribed by viral regulators, which are known as the viral Pre-Initiation Complex (vPIC) and the host RNA polymerase II complex. Because the vPIC is conserved in beta- and gamma-herpesviruses, studying the control of viral L gene expression by the vPIC contributes to the development of drugs that specifically inhibit these processes in beta- and gamma-herpesvirus infections/diseases. In this study, we demonstrated that CDK inhibitors induced de-stabilization of the vPIC component BDLF4, leading to a reduction in L gene expression and subsequently progeny production. Our findings suggest that CDK inhibitors may be a therapeutic option against beta- and gamma-herpesviruses in combination with existing inhibitors of herpesvirus lytic replication such as ganciclovir.

  • An emerging issue in oncogenic virology: the role of beta HPV types in development of cutaneous squamous cell carcinoma [Gem]

  • Evidence suggests that beta HPVs, together with ultraviolet radiation, contribute to the development of cutaneous squamous cell carcinoma. Beta HPVs appear to be not the main drivers of carcinogenesis, but rather facilitators of the accumulation of ultraviolet-induced DNA mutations. Beta HPVs are promoters of skin carcinogenesis, although they are dispensable for the maintenance of the malignant phenotype. Therefore, beta HPV represents a target for skin cancer prevention, especially in high-risk populations.

  • Ceramide suppresses influenza A virus replication in vitro [Virus-Cell Interactions]

  • Annual influenza outbreaks are associated with significant morbidity and mortality worldwide despite the availability of seasonal vaccines. Influenza pathogenesis depends on the manipulation of host cell signaling to promote virus replication. Ceramide is a sphingosine-derived lipid that regulates diverse cellular processes. Studies highlighted the differential role of ceramide de novo biosynthesis on the propagation of various viruses. Whether ceramide plays, a role in influenza virus replication is not known. In this study, we assessed the potential interplay between the influenza A (IAV) and ceramide biosynthesis pathways.

    Accumulation of ceramide in human lung epithelial cells infected with influenza A/H1N1 virus strains was evaluated using thin layer chromatography and/or confocal microscopy. Virus replication was assessed upon the regulation of the de novo ceramide biosynthesis pathway. A significant increase in ceramide accumulation was observed in cells infected with IAV in a dose- and time-dependent manner. Inoculating the cells with UV-inactivated IAV or treating them with sialidase prior to virus inoculation did not result in ceramide accumulation in the cells suggesting that the induction of ceramide required an active virus replication. Inhibiting de novo ceramide significantly decreased ceramide accumulation and enhanced virus replication. The addition of exogenous C6-ceramide prior to infection mediated an increase in cellular ceramide levels and significantly attenuated IAV replication and reduced viral titers (1log10 pfu/ml unit) Therefore, our data demonstrate that ceramide accumulation through de novo biosynthesis pathway plays a protective and antiviral role against IAV infection. These findings propose new avenues for development of antiviral molecules and strategies.

    Importance: Understanding the effect of sphingolipid metabolism on viral pathogenesis provide important insights into the development of therapeutic strategies against microbial infections. In this study, we demonstrate a critical role of ceramide during influenza A virus infection. We demonstrate that ceramide produced through de novo biosynthesis possess an antiviral role. These observations unlock new opportunities for the development of novel antiviral therapies against influenza.

  • Validating Enterovirus D68-2Apro as an Antiviral Drug Target and the Discovery of Telaprevir as a Potent D68-2Apro Inhibitor [Vaccines and Antiviral Agents]

  • Enterovirus D68 (EV-D68) is a viral pathogen that leads to severe respiratory illness and has been linked with the development of acute flaccid myelitis (AFM) in children. No vaccines or antivirals are currently available for EV-D68 infection, and treatment options for hospitalized patients are limited to supportive care. Here, we report the expression of the EV-D68 2A protease (2Apro) and characterization of its enzymatic activity. Furthermore, we discovered telaprevir, an FDA-approved drug used for the treatment of Hepatitis C virus infections, as a potent antiviral against EV-D68 by targeting the 2Apro enzyme. Using FRET-based substrate cleavage assay, we showed that the purified EV-D68 2Apro has proteolytic activity selective against a peptide sequence corresponding to the viral VP1-2A polyprotein junction. Telaprevir inhibits EV-D68 2Apro through a nearly irreversible, biphasic binding mechanism. In cell culture, telaprevir showed submicromolar to low micromolar potency against several recently circulating neurotropic strains of EV-D68 in different human cell lines. To further confirm the antiviral drug target, serial viral passage experiments were performed to select for resistance against telaprevir. An N84T mutation near the active site of 2Apro was identified in resistant viruses, and this mutation reduced the potency of telaprevir in both the enzymatic and cellular antiviral assays. Collectively, we report for the first time the in vitro enzymatic activity of EV-D68 2Apro and the identification of telaprevir as a potent EV-D68 2Apro inhibitor. These findings implicate EV-D68 2Apro as an antiviral drug target and highlight the repurposing potential of telaprevir to treat EV-D68 infection.

    IMPORTANCE: A 2014 EV-D68 outbreak in the United States has been linked to the development of acute flaccid myelitis in children. Unfortunately, no treatment options against EV-D68 are currently available, and the development of effective therapeutics is urgently needed. Here, we characterize and validate a new EV-D68 drug target, the 2Apro, and identify telaprevirmmdash;an FDA approved drug used to treat hepatitis C virus (HCV) infectionsmmdash;as a potent antiviral with a novel mechanism towards 2Apro. 2Apro functions as a viral protease that cleaves a peptide sequence corresponding to the VP1-2A polyprotein junction. Binding of telaprevir potently inhibits its enzymatic activity, and using drug resistance selection, we show that the potent antiviral activity of telaprevir was due to 2Apro inhibition. This is the first inhibitor to selectively target the 2Apro from EV-D68 and can be used as a starting point for the development of selective EV-D68 therapeutics.

  • MECHANISM OF TETHERIN INHIBITION OF ALPHAVIRUS RELEASE [Virus-Cell Interactions]

  • Tetherin is an interferon-inducible, antiviral host factor that broadly restricts enveloped virus release by tethering budded viral particles to the plasma membrane. In response, many viruses have evolved tetherin antagonists. The human tetherin gene can express two isoforms, long and short, due to alternative translation initiation sites in the N-terminal cytoplasmic tail. The long isoform (L-tetherin) contains 12 extra amino acids in its N-terminus, including a dual tyrosine motif (YDYCRV) that is an internalization signal for clathrin-mediated endocytosis and a determinant of NF-B activation. Tetherin restricts alphaviruses, which are highly organized enveloped RNA viruses that bud from the plasma membrane. L-tetherin is more efficient than S-tetherin in inhibiting alphavirus release in 293 cells. Here, we demonstrated that alphaviruses do not encode an antagonist for either tetherin isoform. Instead, the isoform specificity reflected a requirement for tetherin endocytosis. The YxY motif in L-tetherin was necessary for alphavirus restriction in 293 cells, but was not required for rhabdovirus restriction. L-tetherin's inhibition of alphavirus release correlated with its internalization but did not involve NF-B activation. In contrast, in U-2 OS cells the YxY motif and the L-tetherin N-terminal domain were not required for either robust tetherin internalization or alphavirus inhibition. Tetherin forms that were negative for restriction accumulated at the cell surface of infected cells, while the levels of tetherin forms that restrict were decreased. Together, our results suggest that tetherin-mediated virus clearance plays an important role in the restriction of alphavirus release, and that cell type-specific cofactors may promote tetherin endocytosis.

    IMPORTANCE The mechanisms of tetherin's antiviral activities and viral tetherin antagonism have been studied in detail for a number of different viruses. Although viral countermeasures against tetherin can differ significantly, overall tetherin's antiviral activity correlates with physical tethering of virus particles to prevent their release. While tetherin can mediate virus endocytic uptake and clearance, this has not been observed to be required for restriction. Here we show that efficient tetherin inhibition of alphavirus release requires efficient tetherin endocytosis. Our data suggest that this endocytic uptake can be mediated by tetherin itself or by a tetherin cofactor that promotes uptake of an endocytosis-deficient variant of tetherin.

  • Effective Suppression of HIV-1 Replication by Cytotoxic T Lymphocytes Specific for Pol Epitopes in Conserved Mosaic Vaccine Immunogens [Pathogenesis and Immunity]

  • Cytotoxic T lymphocytes (CTLs) with strong abilities to suppress HIV-1 replication and recognize circulating HIV-1 could be key for both HIV-1 cure and prophylaxis. We recently designed conserved mosaic T-cell vaccine immunogens tHIVconsvX composed of 6 Gag and Pol regions. Since the tHIVconsvX vaccine targets conserved regions common to most global HIV-1 variants and employs a bi-valent mosaic design, it is expected that it could be universal if the vaccine works. Although we recently demonstrated that CTLs specific for 5 Gag epitopes in the vaccine immunogens had strong ability to suppress HIV-1 replication in vitro and in vivo, it remains unknown whether or not the Pol region-specific CTLs are equally efficient. Here, we studied CTLs specific for Pol epitopes in the immunogens in treatment-naïve Japanese patients infected with HIV-1 clade B. Overall, we mapped 20 reported and 5 novel Pol conserved epitopes in tHIVconsvX. Responses to 6 Pol epitopes were significantly associated with good clinical outcome, suggesting that CTLs specific for these 6 Pol epitopes had a strong ability to suppress HIV-1 replication in HIV-1-infected individuals. In vitro T-cell analyses further confirmed that the Pol-specific CTLs could effectively suppress HIV-1 replication. The present study thus demonstrated that the Pol regions of the vaccine contained protective epitopes. T-cell responses to the previous 5 Gag and present 6 Pol protective epitopes together also showed a strong correlation with better clinical outcome. These findings support the testing of the conserved mosaic vaccine in HIV-1 cure and prevention in humans.

    IMPORTANCE It is likely necessary for an effective AIDS vaccine to elicit CD8+ T cells with the ability to recognize circulating HIV-1 and suppress their replication. We recently developed novel bivalent-mosaic T-cell vaccine immunogens composed of conserved regions of the Gag and Pol proteins match to at least 80% globally circulating HIV-1 isolates. Nevertheless, it remains to be proven if vaccine with these immunogens can elicit T cells with the ability to suppress HIV-1 replication. It is well known that Gag-specific T cells can suppress HIV-1 replication more effectively than T cells specific for epitopes in other proteins. We recently identified 5 protective Gag epitopes in the vaccine immunogens. We here identified T cells specific for 6 Pol epitopes present in the immunogens with strong abilities to suppress HIV-1 in vivo and in vitro. This study further encourages clinical testing of the conserved mosaic T-cell vaccine in HIV-1 prevention and cure.

  • Expression of MDM2 in macrophages promotes the early post-entry steps of HIV-1 infection through inhibition of p53 [Virus-Cell Interactions]

  • The molecular basis for HIV-1 susceptibility in primary human monocyte-derived macrophages (MDMs) was previously evaluated by comparing the transcriptome of infected and bystander populations. Careful analysis of the data suggested that the ubiquitin ligase MDM2 acted as a positive regulator of HIV-1 replication in MDMs. In this study, MDM2 silencing through transcript-specific small interfering RNAs in MDMs induced a reduction in HIV-1 reverse transcription and integration along with an increase in the expression of p53-induced genes, including CDKN1A. Experiments with Nutlin-3, a pharmacological inhibitor of MDM2 p53-binding activity, showed a similar effect on HIV-1 infection, thus suggesting that the observed restriction in HIV-1 production results from the release/activation of p53 and not the absence of MDM2 per se. Knockdown and inhibition of MDM2 also both correlate with a decrease in the Thr592-phosphorylated inactive form of SAMHD1. The expression level of MDM2 and the p53 activation status are therefore important factors in the overall susceptibility of macrophages to HIV-1 infection, bringing a new understanding of signaling events controlling the process of virus replication in this cell type.

    IMPORTANCE Macrophages, with their long lifespan in vivo and their resistance to HIV-1-mediated cytopathic effect, might serve as viral reservoirs, contributing to the virus persistence in an infected individual. Identification of host factors that increase the overall susceptibility of macrophages to HIV-1 might provide new therapeutic targets for the efficient control of viral replication in these cells and limit the formation of reservoirs in exposed individuals. In this study, we demonstrate the importance of p53 regulation by MDM2, which creates a cellular environment more favorable to the early steps of HIV-1 replication. Moreover, we show that p53 stabilization reduces virus infection in human macrophages, highlighting the important role of p53 in antiviral immunity.

  • CD4-dependent Modulation of HIV-1 Entry by LY6E [Virus-Cell Interactions]

  • LY6E is a GPI-anchored, interferon-inducible protein that has been shown to modulate viral infection in a cell type-dependent manner. Our recent work showed that LY6E promotes HIV-1 infection in some high CD4-expressing cells, including human peripheral blood mononuclear cell (PBMCs) and SupT1 cell line. In this work, we provide evidence that LY6E inhibits HIV-1 entry and spread in low CD4-expressing Jurkat cells and human monocyte-derived macrophages (MDMs) through a downregulation of the viral receptor CD4. We found that knockdown of LY6E in Jurkat cells and MDMs increases HIV-1 infection yet overexpression of LY6E in Jurkat cells inhibits HIV-1 entry and replication. LY6E was found to be co-localized with CD4 on the plasma membrane of Jurkat cells and MDMs, and enhances CD4 internalization. We artificially manipulated CD4 level in Jurkat and SupT1 cells, and found that overexpression of CD4 in Jurkat cells overcomes the inhibitory effect of LY6E; conversely, blocking the function of CD4 in SupT1 with a neutralizing antibody eliminates the enhancement of LY6E on HIV-1 entry. The CD4-dependent inhibitory phenotype of LY6E in low CD4-expressing human MDMs can be recapitulated for a panel of transmitted founder viruses and lab-adapted HIV-1 strains. Given that HIV-1 can target low CD4-expressing cells during the acute infection yet replicates efficiently in high CD4-expressing T cells at the late stage of diseases, our observation that LY6E differentially modulates HIV-1 replication in a CD4-dependent manner have implications for understanding the complex roles of IFN-induced proteins in AIDS pathogenesis.

    IMPORTANCE The role of IFN-induced genes (ISGs) in viral infection remains incompletely understood. While most ISGs are antiviral, some ISGs have been shown to promote viral infection, including HIV-1. We previously showed that IFN-inducible LY6E protein promotes HIV-1 infection in human PMBCs and high CD4-expresing SupT1 cells. Here we found that LY6E inhibits HIV-1 entry and replication in low CD4-expressing MDMs and Jurkat cells. Mechanistically, we demonstrated that LY6E downregulates the cell surface receptor CD4, thus impairing the virus binding to target cells. This is in contrast to the situation of high CD4 cells, where LY6E predominantly promotes viral membrane fusion. The opposing role of IFN-inducible LY6E in modulating HIV-1 infection highlights the complex roles of ISGs in viral infection and viral pathogenesis.

  • Pathogenesis, host innate immune response and aerosol transmission of Influenza D virus in cattle [Pathogenesis and Immunity]

  • The recently discovered influenza D virus (IDV) of the Orthomyxoviridae family has been detected in swine and ruminants with a worldwide distribution. Cattle are considered to be the primary host and reservoir and previous studies suggested a tropism of IDV for the upper respiratory tract and a putative role in the Bovine Respiratory Disease complex. This study aimed to characterize the pathogenicity of IDV in naive calves, as well as the ability of this virus to transmit by air. Eight naive calves were infected by aerosol with a recent French isolate, D/bovine/France/5920/2014. Results show that IDV replicates not only in the upper but also the lower respiratory tracts (LRT), inducing moderate bronchopneumonia with restricted lesions of interstitial pneumonia. Inoculation was followed by IDV-specific IgG1 production as early as 10 days post challenge, and likely both Th1 and Th2 responses. Study of the innate immune response in the LRT of IDV infected calves indicated the overexpression of pathogen recognition receptors and of chemokines CCL2, CCL3 and CCL4, but without overexpression of genes involved in the type I interferon pathway. Finally, virological examination of three aerosol-sentinel animals, housed 3 meters apart from inoculated calves, and IDV detection in air samples collected in different areas showed that IDV can be airborne transmitted and infect naïve contact calves on short distances. This study suggests that IDV is a respiratory virus with moderate pathogenicity and probably a high level of transmission. It consequently can be considered as predisposing or co-factor of respiratory disease.

    IMPORTANCE Influenza D virus (IDV), a new Genus of the Orthomyxoviridae family, has a broad geographical distribution and can infect several animal species. Cattle are so far considered as the primary host for IDV, but the pathogenicity and the prevalence of this virus is still unclear. We demonstrated that under experimental conditions (in a controlled environment and in the absence of co-infecting pathogens), IDV is able to cause mild to moderate disease and targets both the upper and lower respiratory tracts. The virus can transmit by direct as well as aerosol contacts. While this study evidenced overexpression of pathogen recognition receptors and chemokines in the lower respiratory tract, IDV-specific IgG1 production as early as 10 days post challenge, and likely both Th1 and Th2 responses, further studies are warranted to better understand the immune responses triggered by IDV and its role as part of the Bovine Respiratory Disease complex.

  • HoxA10 Facilitates SHP-1-catalized Dephosphorylation of p38 MAPK/STAT3 to Repress Hepatitis B Virus Replication by a Feedback Regulatory Mechanism [Cellular Response to Infection]

  • Hepatitis B virus (HBV) infection is the leading cause of chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). This study reveals a distinct mechanism underlying the regulation of HBV replication. HBV activates the homeobox A10 (HoxA10) in human hepatocytes, leukocytes, peripheral blood mononuclear cells (PBMCs), HepG2-NTCP cells, leukocytes isolated from CHB patients, and HBV-associated HCC tissues. HoxA10 in turn represses HBV replication in human hepatocytes, HepG2-NTCP cells, and BABL/c mice. Interestingly, we show that during early HBV infection, p38 MAPK and STAT3 were activated to facilitate HBV replication, however, during late HBV infection, HoxA10 was induced to attenuate HBV replication. Detailed studies reveal that HoxA10 binds to p38 MAPK and recruits the SH2 containing protein tyrosine phosphatase 1 (SHP-1) to facilitate SHP-1 in catalyzing dephosphorylation of p38 MAPK/STAT3, and thereby attenuates p38 MAPK/STAT3 activation and HBV replication. Furthermore, HoxA10 binds to HBV EnhI/X promoter and competes with STAT3 in the binding of the promoter, and thereby repress HBV transcription. Taken together, HoxA10 attenuates HBV replication through repressing the p38 MAPK/STAT3 pathway by two approaches: HoxA10 interacts with p38 MAPK and recruits SHP-1 to repress HBV replication; HoxA10 binds to EnhI/X promoter and competes with STAT3 to attenuate HBV transcription. Thus, the function of HoxA10 is similar to the action of interferon (IFN) in term of inhibition of HBV infection; however, the mechanism of HoxA10-mediated repression of HBV replication is different from the mechanism underlying IFN-induced inhibition of HBV infection.

    IMPORTANCE Two billion people have been infected with HBV worldwide, about 240 million of infected patients developed into chronic hepatitis B (CHB), and 650,000 die each year from liver cirrhosis (LC) or hepatocellular carcinoma (HCC). This work elucidates a mechanism underlying the control of HBV replication. HBV infection activates HoxA10, a regulator of cell differentiation and cancer progression, in human cells and patients with CHB and HCC. HoxA10 subsequently inhibits HBV replication in human tissue culture cells and mice. Additionally, HoxA10 interacts with p38MAPK to repress the activation of p38MAPK and STAT3, and recruits and facilitates SHP-1 to catalyze dephosphorylation of p38MAPK and STAT3. Moreover, HoxA10 competes with STAT3 in the binding of HBV X promoter to repress HBV transcription. Thus, this work reveals a negative regulatory mechanism underlying the control of HBV replication and would provide new insights into the development of potential agents to control of HBV infection.

  • Potent Anti-Hepatitis C (HCV) T Cell Immune Responses Induced in Mice Vaccinated with DNA-launched RNA Replicons and MVA-HCV [Vaccines and Antiviral Agents]

  • Hepatitis C is a liver disease caused by the Hepatitis C virus (HCV) affecting 71 million people worldwide with no licensed vaccines that prevent infection. Here, we have generated four novel alphavirus-based DNA-launched self-amplifying RNA replicon (DREP) vaccines expressing either structural Core-E1-E2 or nonstructural p7-NS2-NS3 HCV proteins of genotype 1a placed under the control of an alphavirus promoter, with or without an alphaviral translational enhancer (grouped as DREP-HCV and DREP-e-HCV, respectively). DREP vectors are known to induce cross-priming and further stimulation of immune responses through apoptosis, and here we demonstrate that they efficiently trigger apoptosis-related proteins in transfected cells. Mice immunized with the DREP vaccines as a prime followed by a heterologous boost with a recombinant modified vaccinia virus Ankara (MVA) vector expressing the nearly full-length genome of HCV (MVA-HCV), induced potent and long-lasting HCV-specific CD4+ and CD8+ T cell immune responses that were significantly stronger compared to a homologous MVA-HCV prime/boost immunization, being the DREP-e-HCV/MVA-HCV combination the most immunogenic regimen. HCV-specific CD4+ and CD8+ T cell responses were highly polyfunctional, of effector memory phenotype and mainly directed against E1-E2 and NS2-NS3, respectively. Additionally, DREP/MVA-HCV immunization regimens induced higher antibody levels against HCV E2 protein than homologous MVA-HCV immunization. Collectively, these results provided an immunization protocol against HCV inducing high levels of HCV-specific T cell responses, as well as humoral responses. These findings reinforce the combined use of DREP-based vectors and MVA-HCV as promising prophylactic and therapeutic vaccines against HCV.

    IMPORTANCE

    HCV represents a global health problem as more than 71 million people are chronically infected worldwide. Direct-acting antiviral agents can cure HCV infection in most patients, but due to their high cost and the emergence of resistant mutants they do not represent a feasible and affordable strategy to eradicate the virus. Therefore, a vaccine is an urgent goal that requires efforts in understanding the correlates of protection for HCV clearance. Here, we describe for the first time the generation of novel vaccines against HCV based on alphavirus DNA replicons expressing HCV antigens. We demonstrate that potent T cell immune responses, as well as humoral immune responses against HCV can be achieved in mice by using a combined heterologous prime/boost immunization protocol consisting in the administration of alphavirus replicon DNA vectors as a prime followed by a boost with a recombinant modified vaccinia virus Ankara vector expressing HCV antigens.

  • Mini-chromosome Maintenance Proteins Cooperate with LANA During the G1/S Phase of the Cell Cycle to Support Viral DNA Replication [Genome Replication and Regulation of Viral Gene Expression]

  • Latency Associated Nuclear Antigen (LANA) is essential for maintaining the viral genome by regulating replication and segregation of the viral episomes. The virus maintains 50-100 episomal copies during latency and replicates in synchrony with the cellular DNA of the infected cells. Since virus lacks its own replication machinery, it utilizes the cellular proteins for replication and maintenance and LANA is shown to make many of these proteins available for replication by directly recruiting them to the viral origin of replication within the terminal repeat (TR) region. Our studies identified members of the Minichromosome maintenance (MCM) complex as potential LANA interacting proteins. Here, we show that LANA specifically interacts with the components of MCM complex, primarily during the G1/S phase of the cell cycle. MCM3 and 4 of the MCM complex specifically bound to the amino-terminal domain, while MCM6 bound to both, the amino and carboxyl terminal domains of LANA. MCM binding region in the N-terminal domain mapped to the chromatin binding domain (CBD). LANA with point mutations in the carboxyl terminal domain identified MCM6 binding domain and over expression of that domain (1100-1150aa) abolished TR replication. Introduction of peptide encompassing 1104-1123aa region of LANA reduced MCM6 association with LANA and TR replication. Moreover, a recombinant KSHV (BAC16 1100-1150) with deleted 1100-1150aa domain of LANA showed reduced replication and persistence of viral genome copies as compared to the wild type BAC16. Additionally, the role of MCMs on viral replication was confirmed by depleting MCMs and assaying transient and long-term maintenance of the viral episomes. The recruitment of MCMs to the replication origins through LANA was demonstrated through chromatin immunoprecipitation and isolation of proteins on nascent replicated DNA (iPOND). These data clearly show the role of MCMs in latent DNA replication and potential for targeting C-terminal domain of LANA for blocking viral persistence.

    Significance: LANA-mediated latent DNA replication is essential for an efficient maintenance of KSHV episomes in the host. During latency, virus relies on the host cellular machinery for replication, which occurs in synchrony with the cellular DNA. LANA interacts with the components of multiple cellular pathways including cellular replication machinery and recruits them to the viral origin for DNA replication. In this study, we characterize the interactions between LANA and Minichromosome maintenance (MCMs) proteins, a member of the cellular replication complex. We demonstrated a cell-cycle dependent interaction between LANA and MCMs and determined their importance on viral genome replication and maintenance through biochemical assays. In addition, we mapped a 50-amino acid region in LANA, which was capable of abrogating the association of MCM6 with LANA and blocking DNA replication. We also detected LANA along with MCM at the replication forks using a novel approach, isolation of proteins on nascent DNA (iPOND).

  • Cryo-EM structures of novel viruses from mud crab Scylla Paramamosain with multiple infections [Structure and Assembly]

  • Viruses associated with sleeping disease (SD) in crabs cause great economic losses to aquaculture, and no effective measures are available for their prevention. Herein, to help develop novel antiviral strategies, single-particle cryo-electron microscopy was applied to investigate viruses associated with SD. The results not only revealed the structure of mud crab dicistrovirus (MCDV), but also identified a novel mud crab tombus-like virus (MCTV) not previously detected using molecular biology methods. The structure of MCDV at 3.5 AAring; resolution reveals three major capsid proteins (VP1-3) organized into a pseudo-T3 icosahedral capsid, and affirms the existence of VP4. Unusually, MCDV VP3 contains a long C-terminal region and forms a novel protrusion that has not been observed in other dicistrovirus. Our results also reveal that MCDV can release its genome via conformation changes of the protrusions when viral mixtures are heated. The structure of MCTV at 3.3 AAring; resolution reveals a T=3 icosahedral capsid with common features of both tombusviruses and nodaviruses. Furthermore, MCTV has a novel hydrophobic tunnel beneath the 5-fold vertex and 30 dimeric protrusions composed of the P-domains of the capsid protein at the 2-fold axes that are exposed on the virion surface. The structural features of MCTV are consistent with a novel type of virus.

    Importance:

    Pathogen identification is vital for unknown infectious outbreaks, especially for dual or multiple infections. Sleeping disease (SD) in crabs causes great economic losses to aquaculture worldwide. Herein, we report the discovery and identification of a novel virus in mud crabs with multiple infections that was not previously detected by molecular, immune, or traditional electron microscopy (EM) methods. High resolution structures of pathogenic viruses are essential for a molecular understanding and developing new disease prevention methods. The 3D structure of the mud crab tombus-like virus (MCTV) and mud crab dicistrovirus (MCDV) determined herein could assist the development of antiviral inhibitors. The identification of a novel virus in multiple infections previously missed using other methods demonstrates the usefulness of this strategy for investigating multiple infectious outbreaks, even in humans and other animals.

  • Acetylation of E2 by P300 mediates topoisomerase entry at the papillomavirus replicon [Virus-Cell Interactions]

  • Human papillomavirus (HPV) E2 proteins are integral for the transcription of viral genes and for the replication and maintenance of viral genomes in host cells. E2 recruits the viral DNA helicase E1 to the origin. A lysine (K111) highly conserved among almost all PV E2 proteins is a target for P300 (EP300) acetylation and is critical for viral DNA replication (1, 2). Since the viral genome exists as a covalently closed circle of double stranded DNA, topoisomerase 1 (Topo1) is thought to be required for progression of the replication forks. Due to the specific effect of K111 mutations on DNA unwinding (2), we demonstrate that the E2 protein targets Topo1 to the viral origin, and this depends on acetylation of K111. The effect was corroborated by functional replication assays, in which higher levels of P300 but not its homolog CBP caused enhanced replication with wild-type E2 but not the acetylation defective K111 arginine mutant. These data reveal a novel role for lysine acetylation during viral DNA replication by regulating topoisomerase recruitment to the replication origin.

    IMPORTANCE

    Human papillomaviruses affect an estimated 75% of the sexually active adult population in the US and 5.5 million new cases emerging every year. More than 200 HPV genotypes have been identified; a subset is linked the development of cancers from these epithelial infections. Specific antiviral medical treatments for infected individuals are not available. This project examines the mechanisms that control viral genome replication and may allow the development of novel therapeutics.

  • Sirtuin 6 Attenuates Kaposi's Sarcoma-associated herpesvirus (KSHV) Reactivation via Suppressing the Ori-Lyt Activity and Expression of RTA [Virus-Cell Interactions]

  • Kaposi's sarcomanndash;associated herpesvirus (KSHV, also called human herpesvirus 8, HHV-8), upon being reactivated, causes serious diseases in immunocompromised individuals. Its reactivation has not been fully understood, especially how the cellular regulating mechanisms play roles in KSHV gene expression and viral DNA replication. In searching for the cellular factors that regulate KSHV gene expression, we found that several histone deacytelases (HDACs) and sirtuins (SIRTs), including HDAC 2, 7, 8 and 11, and SIRT 4 and 6, repress KSHV ori-Lyt promoter activity. Interestingly, the nuclear protein, SIRT6 presents the greatest inhibitory effect on ori-Lyt promoter activity. A more detailed investigation reveals that SIRT6 exerted repressive effects on multiple promoter of KSHV. As a consequence of inhibiting the KSHV promoters, SIRT6 not only represses viral protein production, but also inhibits viral DNA replication as investigated in a KSHV-contained cell line, SLK-iBAC-gfpK52. Depletion of the SIRT6 protein using the siRNA cannot directly reactivate KSHV from the SLK-iBAC-gfpK52, but makes it more effective in reactivating KSHV by a low amount of the reactivator (Doxycyclin) and enhances the viral DNA replication in KSHV infection system. We performed DNA chromatin immunoprecipitation (ChIP) assays for SIRT6 in SLK-iBAC-gfpK52 cell line to determine whether SIRT6 interacts with KSHV genome in order to play the regulatory effects. Our results suggest that SIRT6 interacts with KSHV ori-Lyt and ORF50 promoters. Furthermore, the SIRT6-KSHV DNA interaction is significantly negated by reactivation. Therefore, we identified a cellular regulator, SIRT6 that represses KSHV replication through interacting with KSHV DNA and inhibiting viral gene expression.

    IMPORTANCE Kaposi's sarcomanndash;associated herpesvirus (KSHV) is a pathogen causing cancer in the immune-deficiency population. The reactivation of KSHV from latency is important for it to be carcinogenic. Our finding that SIRT6 plays inhibitory effects on KSHV reactivation through interaction with viral genome and suppressing viral gene expression is important because it might lead to a strategy of interfering with KSHV reactivation. Overexpression of SIRT6 represses several KSHV promoters' activities leading to a reduced gene expression and DNA replication of KSHV in a KSHV BAC-containing cell line. Depletion of SIRT6 favors reactivation of KSHV from SLK-iBACV-gfpK52. More importantly, we reveal that SIRT6 interacts with KSHV DNA. Whether the interaction of SIRT6 with KSHV DNA occurs at a global level will be further studied in the future.

  • Reduction of Kaposi's sarcoma-associated herpesvirus latency using CRISPR-Cas9 to edit the latency-associated nuclear antigen gene [Vaccines and Antiviral Agents]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), an AIDS defining cancer in HIV-1 infected individuals or immune suppressed transplant patients. The prevalence for both KSHV and KS are highest in sub-Saharan Africa where HIV-1 infection is also epidemic. There is no effective treatment for advanced KS; therefore, the survival rate is low. Similar to other herpesviruses, KSHV's ability to establish latent infection in the host presents a major challenge to KS treatment or prevention. Strategies to reduce KSHV episomal persistence in latently infected cells might lead to approaches to prevent KS development. The CRISPR-Cas9 system is a gene editing technique that has been used to specifically manipulate the HIV-1 genome, but also EBV which like KSHV belongs to the -herpesvirus family. Among KSHV gene products, the latency-associated nuclear antigen (LANA) is absolutely required in the maintenance, replication and segregation of KSHV episomes during mitosis, which makes LANA an ideal target for CRISPR-Cas9 editing. In this study, we designed a replication-incompetent adenovirus type 5 to deliver LANA-specific Cas9 system (Ad-CC9-LANA) into various KSHV latent target cells. We showed that KSHV latently infected epithelial and endothelial cells transduced with Ad-CC9-LANA underwent significant reductions in the KSHV episome burden, LANA RNA and protein expression over time, but this effect is less profound in BC3 cells due to the low infection efficiency of adenovirus type 5 for B cells. The use of adenovirus vector might confer potential in-vivo applications of LANA-specific Cas9 against KSHV infection and KS.

    IMPORTANCE The ability for Kaposi's sarcoma-associated herpesvirus (KSHV), the causative agent of Kaposi's sarcoma (KS), to establish and maintain latency has been a major challenge to clearing infection and preventing KS development. This is the first study to demonstrate the feasibility of using a KSHV LANA-targeted CRISPR-Cas9 and adenoviral delivery system to disrupt KSHV latency in infected epithelial and endothelial cell lines. Our system significantly reduced the KSHV episomal burden over time. Given the safety record of adenovirus as vaccine or delivery vectors, this approach to limit KSHV latency may also represent a viable strategy against other tumorigenic viruses and may have potentially benefits developing countries where the viral cancer burden is high.

  • The non-structural NS1 protein of influenza viruses modulates TP53 splicing through the host factor CPSF4 [Virus-Cell Interactions]

  • Influenza A viruses (IAV) are known to modulate and "hijack" several cellular host mechanisms, including gene splicing and RNA maturation machineries. These modulations alter host cellular responses and enable an optimal expression of viral products throughout infection. The interplay between the host protein p53 and IAV, in particular through the viral nonstructural protein NS1, has been shown to be supportive for IAV replication. However, it remains unknown whether alternatively spliced isoforms of p53, known to modulate p53 transcriptional activity, are affected by IAV infection and contribute in IAV replication. Using a TP53 minigene, which mimics intron 9 alternative splicing, we have shown here that the NS1 protein of IAV changes the expression pattern of p53 isoforms. Our results demonstrated that CPSF4 (cellular protein cleavage and polyadenylation specificity factor 4) independently and the interaction between NS1 and CPSF4 modulate the alternative splicing of TP53 transcripts which may result in the differential activation of p53 responsive genes. Finally, we reported that CPSF4 and most likely beta and gamma spliced p53 isoforms affect both viral replication and IAV-associated type I interferon secretion. Altogether, our data showed that cellular p53 and CPSF4 factors, both interacting with viral NS1, have a crucial role during IAV replication that allows IAV to interact and alter the expression of alternatively spliced p53 isoforms in order to regulate the cellular innate response, especially via type I interferon secretion, and perform efficient viral replication.

    Importance

    Influenza A viruses (IAV) constitute a major public health issue, causing illness and death in high-risk populations during seasonal epidemics or pandemics. IAV are known to modulate cellular pathways to promote their replication and avoid immune restriction via the targeting of several cellular proteins. One of these proteins, p53, is a master regulator involved in a large panel of biological processes, including cell cycle arrest, apoptosis or senescence. This "cellular gatekeeper" is also involved in the control of viral infections, and viruses have developed a wide diversity of mechanisms to modulate/hijack p53 functions to achieve an optimal replication in their hosts. Our group and others have previously shown that p53 activity was finely modulated by different multi-level mechanisms during IAV infection. Here, we characterized IAV non-structural protein NS1 and the cellular factor CPSF4 as major partners involved in the IAV-induced modulation of the TP53 alternative splicing that was associated with a strong modulation of p53 activity and notably the p53-mediated antiviral response.

  • The Polar Region of HIV-1 Envelope Protein Determines Viral Fusion and Infectivity by Stabilizing gp120-gp41 Association [Virus-Cell Interactions]

  • HIV-1 enters cells through binding between viral envelope glycoprotein (Env) and cellular receptors to initiate virus and cell fusion. HIV-1 Env precursor (gp160) is cleaved into two units non-covalently bound to form a trimer on virions, including a surface unit (gp120) and a transmembrane unit (gp41) responsible for virus binding and membrane fusion, respectively. The polar region (PR) at the N-terminus of gp41 comprises 17 residues, including 7 polar amino acids. Previous studies suggested that the PR contributes to HIV-1 membrane fusion and infectivity; however, the precise role of the PR in Env-mediated viral entry and the underlying mechanisms remain unknown. Here, we show that the PR is critical for HIV-1 fusion and infectivity by stabilizing Env trimers. Through analyzing the PR sequences of 57,645 HIV-1 isolates, we performed targeted mutagenesis and functional studies of three highly conserved polar residues in the PR (S532P, T534A and T536A), which have not been characterized previously. We found that single or combined mutations of these three residues abolished or significantly decreased HIV-1 infectivity without affecting viral production. These PR mutations abolished or significantly reduced HIV-1 fusion with target cells and also Env-mediated cell-cell fusion. Three PR mutations containing S532P substantially reduced gp120 and gp41 association, Env trimer stability, and increased gp120 shedding. Furthermore, S532A mutation significantly reduced HIV-1 infectivity and fusogenicity, but not Env expression and cleavage. Our findings suggest that the PR of gp41, particularly the key residue S532, is structurally essential for maintaining HIV-1 Env trimer, viral fusogenicity and infectivity.

    IMPORTANCE Although extensive studies of the transmembrane unit (gp41) of HIV-1 Env have led to a fusion inhibitor clinically used to block viral entry, the functions of different domains of gp41 in HIV-1 fusion and infectivity are not fully elucidated. The polar region (PR) of gp41 has been proposed to participate in HIV-1 membrane fusion in biochemical analyses, but its role in viral entry and infectivity remain unclear. In our effort to characterize three nucleotide mutations of an HIV-1 RNA element that partially overlap with the PR coding sequence, we identified a novel function of the PR that determines viral fusion and infectivity. We further demonstrated the structural and functional impact of six PR mutations on HIV-1 Env stability, viral fusion and infectivity. Our findings reveal the previously unappreciated function of the PR and the underlying mechanisms, highlighting the important role of the PR in regulating HIV-1 fusion and infectivity.

  • Equine herpesvirus 1 bridles T-lymphocytes to reach its target organs [Virus-Cell Interactions]

  • Equine herpesvirus 1 (EHV1) replicates in the respiratory epithelium and disseminates through the body via a cell-associated viremia in leukocytes, despite the presence of neutralizing antibodies. llsquo;Hijackedrrsquo; leukocytes, previously identified as monocytic cells and T-lymphocytes, transmit EHV1 to endothelial cells of the endometrium or central nervous system, causing reproductive (abortigenic variants) or neurological disorders (neurological variants). Here, we questioned the potential route of EHV1 infection of T-lymphocytes, and how EHV1 misuses T-lymphocytes as a vehicle to reach the endothelium of the target organs, in the absence or presence of the immune surveillance. Viral replication was evaluated in activated/quiescent primary T-lymphocytes, and demonstrated increased infection of activated versus quiescent, CD4+ versus CD8+, and blood- versus lymph nodal-derived T-cells. Moreover, primarily infected respiratory epithelial cells and circulating monocytic cells efficiently transferred virions to T-lymphocytes in the presence of neutralizing antibodies. Albeit the early expression of all classes of viral proteins, the expression of viral glycoproteins on the T-cell surface was restricted. In addition, the release of viral progeny was hampered, resulting in the accumulation of viral nucleocapsids in the T-cell nucleus. During contact of infected T-lymphocytes with endothelial cells, (a) late viral protein(s) orchestrate(s) T-cell polarization and synapse formation, followed by anterograde dynein-mediated transport and transfer of viral progeny to the engaged cell. This represents a sophisticated, but efficient immune evasion strategy to allow transfer of progeny virus from T-lymphocytes towards adjacent target-cells. These results demonstrate that T-lymphocytes are susceptible for EHV1 infection and that cell-cell contact transmits infectious virus to and from T-lymphocytes.

    Importance

    Equine herpesvirus 1 (EHV1) is an ancestral alphaherpesvirus related to herpes simplex virus 1, causing respiratory, reproductive and neurological disorders in Equidae. EHV1 is indisputably a master in exploiting leukocytes to reach its target organs, accordingly evading the host-immunity. However, the role of T-lymphocytes in cell-associated viremia remains poorly understood. Here, we show that activated T-lymphocytes efficiently become infected and support viral replication, despite the presence of the protective immunity. We demonstrated a restricted expression of viral proteins on the cell-surface of infected T-cells, preventing immune-recognition. In addition, we indicated a hampered progeny release, resulting in the accumulation of nucleocapsids in the T-cell nucleus. Upon engagement with target endothelium, late viral proteins orchestrate a viral synapse formation, and viral transfer to the contact cell. Our findings have significant implications to understand the EHV1 pathogenesis, which is essential to develop innovative therapies, to prevent the devastating clinical symptoms.

  • The Carboxyl Terminus of Tegument Protein pUL21 Contributes to Pseudorabies Virus Neuroinvasion [Pathogenesis and Immunity]

  • Following its entry into cells, pseudorabies virus (PRV) utilizes microtubules to deliver its nucleocapsid to the nucleus. Previous studies have shown that PRV VP1/2 is an effector of dynein-mediated capsid transport. However, the mechanism of PRV recruiting microtubule motor proteins for successful neuroinvasion and neurovirulence is not well understood. Here, we provide evidence that PRV pUL21 is an inner tegument protein. We tested its interaction with the cytoplasmic light chains using a bimolecular fluorescence complementation (BiFC) assay and observed that PRV pUL21 interacts with Roadblock-1. This interaction was confirmed by co-immunoprecipitation (co-IP) assays. We also determined the efficiency of retrograde and anterograde axonal transport of PRV strains in explanted neurons using a microfluidic chamber system and investigated pUL21 contribution to PRV neuroinvasion in vivo. Further data showed that the carboxyl terminus of pUL21 is essential for its interaction with Roadblock-1 and this domain contributes to PRV retrograde axonal transport in vitro and in vivo. Our findings suggest that the carboxyl terminus of pUL21 contributes to PRV neuroinvasion.

    Importance

    Herpesviruses are a group of DNA viruses that infect both humans and animals. Alpha herpesviruses are distinguished by their ability to establish latent infection in peripheral neurons. After entering neurons, the herpesvirus capsid interacts with cellular motor proteins and undergoes retrograde transport on axon microtubules. This elaborate process is vital to the herpesvirus lifecycle, but the underlying mechanism remains poorly understood. Here, we determined that pUL21 is an inner tegument protein of pseudorabies virus (PRV) and that it interacts with the cytoplasmic dynein light chain Roadblock-1. We also observed that pUL21 promotes retrograde transport of PRV in neuronal cells. Furthermore, our findings confirm that pUL21 contributes to PRV neuroinvasion in vivo. Importantly, the carboxyl terminus of pUL21 is responsible for interaction with Roadblock-1 and this domain contributes to PRV neuroinvasion. This study offers fresh insights into alpha herpesvirus neuroinvasion and the interaction between virus and host during PRV infection.

  • Effects of alterations to the CX3C motif and secreted form of human respiratory syncytial virus (RSV) G protein on immune responses to a parainfluenza virus vector expressing the RSV G protein [Vaccines and Antiviral Agents]

  • Human respiratory syncytial virus (RSV) is a major pediatric respiratory pathogen. The attachment (G) and fusion (F) glycoproteins are major neutralization and protective antigens. RSV G is expressed as membrane-anchored (mG) and secreted (sG) forms, both containing a central fractalkine-like CX3C motif. The CX3C motif and sG are thought to interfere with host immune responses and have been suggested to be omitted from a vaccine. We used a chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) vector to express RSV wild-type (wt) G and modified forms, including sG alone, mG alone, mutants with ablated CX3C, and G with enhanced packaging into vector virions. In hamsters, these viruses replicated to similar titers. When assayed with a complement-enhanced neutralization assay in Vero cells, sG did not reduce the serum RSV- or PIV3-neutralizing antibody (NAb) responses, whereas ablating CX3C drastically reduced the RSV NAb response. Protective efficacy against RSV challenge was not reduced by sG, but was strongly dependent on the CX3C motif. In ciliated human airway epithelial (HAE) cells, NAbs induced by wt G, but not by wt F, completely blocked RSV infection in the absence of added complement. This activity was dependent on the integrity of the CX3C motif. In hamsters, the rB/HPIV3 expressing wt G conferred better protection against RSV challenge than that expressing wt F. Codon-optimization of the wt G further increased its immunogenicity and protective efficacy. This study showed that ablation of the CX3C motif or sG in an RSV vaccine, as has been suggested previously, would be ill-advised.

    Importance

    Human respiratory syncytial virus (RSV) is the leading viral cause of severe pediatric respiratory illness. An RSV vaccine is not yet available. The RSV attachment protein G is an important protective and neutralization antigen. G contains a conserved fractalkine-like CX3C motif and is expressed in membrane-bound (mG) and secreted (sG) forms. sG and the CX3C motif are thought to interfere with host immune responses, but this remains poorly characterized. Here, we used an attenuated chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) vector to express various modified forms of RSV G, and demonstrated that strong antibody and protective responses could be induced by G alone, and that this was highly dependent on the integrity of the CX3C motif. There was no evidence that sG or the CX3C motif impaired immune responses against RSV G or the rB/HPIV3 vector. rB/HPIV3 expressing wt RSV G provides a bivalent vaccine against RSV and HPIV3.

  • Residues on AAV Capsid Lumen Dictate Interactions and Compatibility With the Assembly-activating Protein [Structure and Assembly]

  • The adeno-associated virus (AAV) serves as a broadly used vector system for in vivo gene delivery. The process of AAV capsid assembly remains poorly understood. The viral co-factor assembly-activating protein (AAP) is required for maximum AAV production and has multiple roles in capsid assembly: trafficking of the structural proteins (VP) to the nuclear site of assembly, promoting stability of VP against multiple degradation pathways, and facilitating stable interactions between VP monomers. The N-terminal 60 amino acids of AAP (AAPN) are essential for these functions. Presumably, AAP must physically interact with VP to execute its multiple functions, but the molecular nature of the AAP-VP interaction is not well understood. Here, we query structurally related AAVs in how they functionally engage AAP from AAV serotype 2 (AAP2) toward virion assembly. These studies led to the identification of key residues on the lumenal capsid surface important for AAP-VP and for VP-VP interactions. Replacing a cluster of glutamic acid residues with a glutamine-rich motif on the conserved VP beta barrel structure of variants incompatible with AAP2 creates a gain-of-function mutant compatible with AAP2. Conversely, mutating positively charged residues within AAP2's hydrophobic region and conserved core domains within AAPN creates a gain-of-function AAP2 mutant that rescues assembly of the incompatible variant. Our results suggest a model for capsid assembly where surface charge/neutrality dictates an interaction between AAPN and the lumenal VP surface to nucleate capsid assembly.

    IMPORTANCE

    Efforts to engineer the AAV capsid to gain desirable properties for gene therapy (e.g. tropism, reduced immunogenicity, higher potency) require that capsid modifications do not affect particle assembly. The relationship between VP and the cofactor that facilitates its assembly, AAP, is central to both assembly preservation and vector production. Understanding the requirements for this compatibility can inform manufacturing strategies to maximize production and reduce costs. Additionally, library-based approaches that simultaneously examine a large number of capsid variants would benefit from a universally functional AAP, which could hedge against overlooking variants with potentially valuable phenotypes but that were lost during vector library production due to incompatibility with the cognate AAP. Studying interactions between AAV's structural and nonstructural components enhances our fundamental knowledge of capsid assembly mechanisms and the protein-protein interactions required for productive assembly of the icosahedral capsid.

  • Biological and molecular characterization of Chenopodium quinoa mitovirus 1 reveals a distinct sRNA response compared to cytoplasmic RNA viruses. [Cellular Response to Infection]

  • Indirect evidence of mitochondrial viruses in plants comes from discovery of genomic fragments integrated into the nuclear and mitochondrial DNA of a number of plant species. Here, we report the existence of replicating mitochondrial virus in plants: from RNAseq data of infected Chenopodium quinoa, a plant species commonly used as a test plant in virus host-range experiments, among other virus contigs, we could assemble a 2.7 Kb contig that had highest similarity to mitoviruses found in plant genomes. Northern blot analyses confirmed the existence of plus and minus strand RNA corresponding to the mitovirus genome. No DNA corresponding to the genomic RNA was detected, excluding the endogenization of such virus. We have tested a number of C. quinoa accessions, and the virus was present in a number of commercial varieties, but absent from a large collection of Bolivian and Peruvian accessions. The virus could not be transmitted mechanically or by grafting, but it is transmitted vertically through seeds at a 100% rate. Small RNA analysis of a C. quinoa line carrying the mitovirus and infected by alfalfa mosaic virus showed that the typical anti-viral silencing response active against cytoplasmic viruses (21-22 nt vsRNA peaks) is not active against CqMV1, since in this specific case the highest accumulating vsRNA length is 16, which is the same as that corresponding to RNA from mitochondrial genes. This is evidence of a distinct viral RNA degradation mechanism active inside mitochondria that could possibly have also an anti-viral effect.

    IMPORTANCE

    This paper reports the first biological characterization of a bona fide plant mitovirus in an important crop, Chenopodium quinoa, providing data supporting that mitoviruses have the typical features of cryptic (persistent) plant viruses. We for the first time demonstrate that plant mitoviruses are associated with mitochondria in plants. In contrast with fungal mitoviruses, plant mitoviruses are not substantially affected by the anti-viral silencing pathway, and the most abundant mitovirus small RNA length is 16 nt.

  • Adenosine Deaminase Acting on RNA 1 Associates with Orf Virus OV20.0 and Enhances Viral Replication [Virus-Cell Interactions]

  • Orf virus (ORFV) infects sheep and goats and is also an important zoonotic pathogen. The viral protein OV20.0 has been shown to suppress innate immunity by targeting the double-stranded RNA (dsRNA)-activated protein kinase (PKR) by multiple mechanisms. These mechanisms include a direct interaction with PKR and binding with two PKR activators, dsRNA and the cellular PKR activator (PACT), which ultimately leads to the inhibition of PKR activation. In the present study, we identified a novel association between OV20.0 and adenosine deaminase acting on RNA 1 (ADAR1). OV20.0 bound directly to the dsRNA binding domains (RBDs) of ADAR1 in the absence of dsRNA. Additionally, OV20.0 preferentially interacted with RBD1 of ADAR1, which was essential for its dsRNA binding ability and for the homodimerization that is critical for intact adenosine (A)-to-inosine (I) editing activity. Finally, the association with OV20.0 suppressed the A-to-I editing ability of ADAR1, while ADAR1 played a proviral role during ORFV infection by inhibiting PKR phosphorylation. These observations revealed a new strategy used by OV20.0 to evade antiviral responses via PKR.

    Importance

    Viruses evolve specific strategies to counteract host innate immunity. ORFV, an important zoonotic pathogen, encodes OV20.0 to suppress PKR activation via multiple mechanisms, including interactions with PKR and two PKR activators. In this study, we demonstrated that OV20.0 interacts with ADAR1, a cellular enzyme responsible for converting adenosine (A) to inosine (I) in RNA. The RNA binding domains, but not the catalytic domain, of ADAR1 are required for this interaction. The OV20.0-ADAR1 association affects the functions of both proteins; OV20.0 suppressed the A-to-I editing of ADAR1, while ADAR1 elevated OV20.0 expression. The proviral role of ADAR1 is likely due to the inhibition of PKR phosphorylation. As RNA editing by ADAR1 contributes to the stability of the genetic code and the structure of RNA, these observations suggest that in addition to serving as a PKR inhibitor, OV20.0 might modulate ADAR1-dependent gene expression to combat antiviral responses or achieve efficient viral infection.

  • Clade C HIV-1 envelope vaccination regimens differ in their ability to elicit antibodies with moderate neutralization breadth against genetically diverse tier 2 HIV-1 envelope variants [Vaccines and Antiviral Agents]

  • The goals of pre-clinical HIV vaccine studies in nonhuman primates are to develop and test different approaches for their ability to generate protective immunity. Here, we compared the impact of 7 different vaccine modalities all expressing the HIV-1 1086.C clade C envelope (Env) on (i) the magnitude and durability of antigen-specific serum antibody responses, and (ii) autologous and heterologous neutralizing antibody capacity. These vaccination regimens included immunization with different combinations of DNA, modified vaccinia Ankara (MVA), soluble gp140 protein, and different adjuvants. Serum samples collected from 130 immunized monkeys at two key time points were analyzed using the TZM-bl assay: 2 weeks after the final immunization (week 40/41) and the day of challenge (week 58). Key initial findings were that inclusion of a gp140 protein boost had a significant impact on the magnitude and durability of Env-specific IgG antibodies, and addition of 3M-052 adjuvant was associated with better neutralizing activity against the SHIV1157ipd3N4 challenge virus and a heterologous HIV-1 CRF_01 Env CNE8. We measured neutralization against a panel of twelve tier 2 Envs using a newly described computational tool to quantify serum neutralization potency by factoring in the pre-determined neutralization tier of each reference Env. This analysis revealed modest neutralization breadth, with DNA/MVA immunization followed by gp140 protein boosts in 3M-052 adjuvant producing the best scores. This study highlights that protein-containing regimens provide a solid foundation for the further development of novel adjuvants and inclusion of trimeric Env immunogens that could eventually elicit a higher level of neutralizing antibody breadth.

    IMPORTANCE Despite much progress, we still do not have a clear understanding of how to elicit a protective neutralizing antibody response against HIV-1 through vaccination. There have been great strides in the development of envelope immunogens that mimic the virus particle, but less is known about how different vaccination modalities and adjuvants contribute to shaping the antibody response. We compared seven different vaccines that were administered to rhesus macaques and delivered the same envelope protein through various modalities and with different adjuvants. The results demonstrate that some vaccine components are better than others at eliciting neutralizing antibodies with breadth.

  • Porcine circovirus type 2 induces ORF3-independent mitochondrial apoptosis via PERK activation and elevation of cytosolic calcium [Pathogenesis and Immunity]

  • Our previous studies have demonstrated that porcine circovirus type 2 (PCV2) triggers unfolded protein response (UPR) in PK-15 cells by activating the PERK/eIF2aalpha; pathway of endoplasmic reticulum stress (ER stress) that, in turn, facilitates viral replication. PCV2 is found to cause oxidative stress and up-regulation of cytoplasmic Ca2+. The virus is reported to employ its ORF3 to induce apoptosis. We wonder if and how PCV2-induced UPR would lead to apoptosis independent of ORF3. Using an ORF3-deficient PCV2 mutant (ORF3), apoptotic responses in infected PK-15 and PAM cells were still apparent, though lower than its parental PCV2 strain. We hypothesized that apoptosis induced by ORF3 might result from UPR. We found that ORF3-induced apoptosis was significantly reduced when the infected cells were treated with a selective PERK blocker GSK2606414 (GSK) or a general ER stress attenuator 4-phenylbutyrate (4-PBA). Such treatments also ameliorated elevation of cytoplasmic Ca2+ and reactive oxygen species (ROS) in PK-15 and PAM cells, two predisposing factors of apoptosis via disruption of the ER-mitochondrial units. Treatment of ORF3-infected cells with GSK and 4-PBA also decreased mitochondrial Ca2+ load and increased mitochondrial membrane potential (MMP). By transient expression of the structural protein capsid (Cap) in combination with PERK silencing, we found that Cap induced collapse of MMP and mitochondrial apoptosis could result from UPR and elevation of Ca2+ and ROS that were inhibitable by down-regulation of PERK. We propose that PCV2-driven ER stress is Cap-dependent and could lead to mitochondrial apoptotic response independent of ORF3 via perturbation of intracellular Ca2+ homeostasis and accumulation of ROS.

    IMPORTANCE Porcine circovirus type 2 (PCV2) encoded protein ORF3, a putative protein having pro-apoptotic activity. Our early studies have shown that PCV2 infection triggers ER stress via selective activation of the PERK pathway, a branch of ER stress pathways, in permissive cells for enhanced replication and its infection increased level of cytosolic Ca2+ and ROS. Here we clearly show that PCV2 infection or capsid protein (Cap) expression induces ORF3-independent apoptosis via increased cytosolic and mitochondrial Ca2+ and cellular ROS as a result of activation of PERK pathway.

  • Epigenetic modification is regulated by the interaction of influenza A virus nonstructural protein-1 with the de novo DNA methyltransferase DNMT3B and subsequent transport to the cytoplasm for K48-linked polyubiquitination [Virus-Cell Interactions]

  • The influenza virus NS1 protein is a non-structural protein that plays a major role in antagonizing host interferon responses during infection. However, a clear role for NS1 in epigenetic modification has not been established. In this study, NS1 regulates the expression of some key regulators of JAK-STAT signaling by inhibiting the DNA methylation of their promoters. Furthermore, DNA methyltransferase 3B (DNMT3B) is responsible for this process. Upon investigating the mechanisms underlying this event, NS1 interacts with DNMT3B, but not DNMT3A, leading to the dissociation of DNMT3B from the promoters of the corresponding genes. In addition, the interaction between NS1 and DNMT3B changes the localization of DNMT3B from the nucleus to the cytosol, resulting in K48-linked ubiquitination and degradation of DNMT3B in the cytosol. Conclusion: NS1 interacts with DNMT3B and changes its localization to mediate K48-linked polyubiquitination, subsequently contributing to the modulation of the expression of JAK-STAT signaling suppressors.

    Importance The NS1 protein of influenza A virus (IAV) is a multifunctional protein that counters cellular antiviral activities and is a virulence factor. However, the involvement of NS1 protein in DNA methylation during IAV infection has not been established. Here, we reveal that the NS1 protein binds the cellular DNMT3B DNA methyltransferase, thereby inhibiting the methylation of the promoters of genes encoding suppressors of JAK-STAT signaling. As a result, these suppressor genes are induced, and JAK-STAT signaling is inhibited. Further, we demonstrate that the NS1 protein transports DNMT3B to the cytoplasm for ubiquitination and degradation. Thus, we identify the NS1 protein as a potential trigger of the epigenetic deregulation of JAK-STAT signaling suppressors and illustrate a novel mechanism underlying the regulation of host immunity during IAV infection.

  • Involvement of a non-structural protein in poliovirus capsid assembly. [Genetic Diversity and Evolution]

  • Virus capsid proteins must perform a number of roles. These include the ability to selfnndash;assemble and to maintain stability under challenging environmental conditions, while retaining the conformational flexibility necessary to uncoat and deliver the viral genome into a host cell. Fulfilling these roles could place conflicting constraints on the innate abilities encoded within the protein sequences. In a previous study, we identified a number of mutations within the capsid coding sequence of poliovirus (PV) that were established in the population during selection for greater thermostability by sequential treatment at progressively higher temperatures. Two mutations in the VP1 protein acquired at an early stage were maintained throughout this selection procedure. One of these mutations prevented virion assembly when introduced into a wild type (wt) infectious clone. Here, we show by sequencing beyond the capsid coding region of the heat selected virions, that two mutations had arisen within the coding region for the 2A protease. Both mutations were maintained throughout the selection process. Introduction of these mutations into a wt infectious clone by site-directed mutagenesis considerably reduced replication. However, they permitted a low level of assembly of infectious virions containing the otherwise lethal mutation in VP1. The 2Apro mutations were further shown to slow the kinetics of viral polyprotein processing and we suggest that this delay improves the correct folding of the mutant capsid precursor protein to permit virion assembly.

    IMPORTANCE RNA viruses including poliovirus evolve rapidly due to the error-prone nature of the polymerase enzymes involved in genome replication. Fixation of advantageous mutations may require the acquisition of complementary mutations which can act in concert to achieve a favourable phenotype. This study highlights a compensatory role of a non-structural regulatory protein, 2Apro, for an otherwise lethal mutation of the structural VP1 protein to facilitate increased thermal resistance. Studying how viruses respond to selection pressures is important for understanding mechanisms which underpin emergence of resistance and could be applied to the future development of antiviral agents and vaccines.

  • Antigenicity and Immunogenicity of Differentially Glycosylated HCV E2 Envelope Proteins Expressed in Mammalian and Insect Cells [Vaccines and Antiviral Agents]

  • Development of a prophylactic vaccine for hepatitis C virus (HCV) remains a global health challenge. Cumulative evidence supports the importance of antibodies targeting the HCV E2 envelope glycoprotein to facilitate viral clearance. However, a significant challenge for a B cell-based vaccine is focusing the immune response on conserved E2 epitopes capable of eliciting neutralizing antibodies not associated with viral escape. We hypothesized that glycosylation might influence the antigenicity and immunogenicity of E2. Accordingly, we performed head-to-head molecular, antigenic and immunogenic comparisons of soluble E2 (sE2) produced in (i) mammalian (HEK293) cells, which confer mostly complex and high mannose type glycans; and (ii) insect (Sf9) cells, which impart mainly paucimannose type glycans. Mass spectrometry demonstrated that all 11 predicted N-glycosylation sites were utilized in both HEK293- and Sf9-derived sE2, but that N-glycans in insect sE2 were on average smaller and less complex. Both proteins bound CD81 and were recognized by conformation-dependent antibodies. Mouse immunogenicity studies revealed that similar polyclonal antibody responses were generated against antigenic domains Anndash;E of E2. Although neutralizing antibody titers showed that Sf9-derived sE2 induced moderately stronger responses than HEK293-derived sE2 against the homologous HCV H77c isolate, the two proteins elicited comparable neutralization titers against heterologous isolates. Given that global alteration of HCV E2 glycosylation by expression in different hosts did not appreciably affect antigenicity or overall immunogenicity, a more productive approach to increasing the antibody response to neutralizing epitopes may be complete deletion, rather than just modification, of specific N-glycans proximal to these epitopes.

    IMPORTANCE Development of a vaccine for hepatitis C virus (HCV) remains a global health challenge. A major challenge for vaccine development is focusing the immune response on conserved regions of the HCV envelope protein, E2, capable of eliciting neutralizing antibodies. Modification of E2 by glycosylation might influence the immunogenicity of E2. Accordingly, we performed molecular and immunogenic comparisons of E2 produced in mammalian and insect cells. Mass spectrometry demonstrated that the predicted glycosylation sites were utilized in both mammalian and insect cell E2, although the glycan types in insect cell E2 were smaller and less complex. Mouse immunogenicity studies revealed similar polyclonal antibody responses. However, insect cell E2 induced stronger neutralizing antibody responses against the homologous isolate used in the vaccine, albeit the two proteins elicited comparable neutralization titers against heterologous isolates. A more productive approach for vaccine development may be complete deletion of specific glycans in the E2 protein.

  • RNA-Binding Motif Protein 24 (RBM24) Is Involved in pgRNA Packaging by Mediating Interaction between HBV Polymerase and the Epsilon Element [Virus-Cell Interactions]

  • Encapsidation of pregenome RNA (pgRNA) is a curial step in HBV replication. Binding by viral polymerase (Pol) to the epsilon stem loop () on the 5rrsquo; terminal region (TR) of pgRNA is required for pgRNA packaging. However, the detailed mechanism is not well understood. RNA-binding motif protein 24 (RBM24) inhibits core translation by binding to the 5rrsquo; TR of pgRNA. Here, we demonstrate that RBM24 is also involved in pgRNA packaging. RBM24 directly binds to the lower bulge of via RNA recognition submotifs (RNPs). RBM24 also interacts with Pol in an RNA-independent manner. The alanine-rich domain (ARD) of RBM24 and the reverse transcriptase domain (RT) of Pol are essential for binding between RBM24 and Pol. In addition, overexpression of RBM24 increases Pol- interaction, whereas RBM24 knockdown decreases interaction. RBM24 was able to rescue binding between and mutant Pol lacking -binding activity, further showing that RBM24 mediates interaction between Pol and by forming a Pol-RBM24- complex. Finally, RBM24 significantly promotes the packaging efficiency of pgRNA. In conclusion, RBM24 mediates Pol- interaction and formation of a Pol-RBM24- complex, which inhibits translation of pgRNA and results in pgRNA packing into capsids/virions for reverse transcription and DNA synthesis.

    IMPORTANCE Hepatitis B virus (HBV) is a ubiquitous human pathogen, and HBV infection is a major global health burden. Chronic HBV infection is associated with the development of liver diseases including fulminant hepatitis, hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. A currently approved vaccine can prevent HBV infection, and medications are able to reduce viral load and prevent liver disease progression. However, current treatments rarely achieve a cure for chronic infection. Thus, it is important to gain insight into the mechanisms of HBV replication. In this study, we found that the host factor RBM24 is involved in pregenomic RNA (pgRNA) packaging and regulates HBV replication. These findings highlight a potential target for antiviral therapeutics of HBV infection.

  • KSHV LANA adjacent regions with distinct functions in episome segregation or maintenance [Virus-Cell Interactions]

  • Kaposi's sarcoma herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is an 1162 amino acid protein that mediates episome persistence of viral genomes. LANA binds KSHV terminal repeat (TR) sequence through its carboxy-terminal domain to mediate DNA replication. LANA simultaneously binds mitotic chromosomes and TR DNA to segregate virus genomes to daughter cell nuclei. Amino-terminal LANA attaches to chromosomes by binding histones H2A/H2B, and carboxy-terminal LANA contributes to mitotic chromosome binding. Although amino- and carboxy-terminal LANA are essential for episome persistence, they are not sufficient since deletion of all internal LANA sequence renders LANA highly deficient for episome maintenance. Internal LANA sequence upstream of the internal repeat elements contributes to episome segregation and persistence. Here, we investigate this region with a panel of LANA deletion mutants. Mutants retained the ability to associate with mitotic chromosomes and bind TR DNA. In contrast to prior results, deletion of most of this sequence did not reduce LANA's ability to mediate DNA replication. Deletions of upstream sequence within this region compromised segregation of TR DNA to daughter cells, as assessed by retention of GFP expression from a replication deficient TR plasmid. However, deletion of this upstream sequence did not reduce episome maintenance. In contrast, deletions that include an 80 amino acid sequence immediately downstream resulted in highly deficient episome persistence. LANA deleted for this downstream sequence maintained the ability to replicate and segregate TR DNA, suggesting a unique role for these residues. Therefore, this work identifies adjacent LANA regions with distinct roles in episome segregation and persistence.

    IMPORTANCE KSHV LANA mediates episomal persistence of viral genomes. LANA binds KSHV terminal repeat (TR) sequence to mediate DNA replication and tethers KSHV DNA to mitotic chromosomes to segregate genomes to daughter cell nuclei. Here, we investigate LANA sequence upstream of the internal repeat elements that contributes to episome segregation and persistence. Mutants with deletions within this sequence maintained the ability to bind mitotic chromosomes or bind and replicate TR DNA. Deletion of upstream sequence within this region reduced segregation of TR DNA to daughter cells, but not episome maintenance. In contrast, mutants deleted for 80 amino acids immediately downstream were highly deficient for episome persistence, yet maintained the ability to replicate and segregate TR DNA, the two principle components of episome persistence, suggesting another role for these residues. In summary, this work identifies adjacent LANA sequence with distinct roles in episome segregation and persistence.

  • US3 kinase-mediated phosphorylation of tegument protein VP8 plays a critical role in the cellular localization of VP8 and its effect on the lipid metabolism of bovine herpesvirus-1-infected cells [Virus-Cell Interactions]

  • Bovine herpesvirus-1 (BoHV-1) infects bovine species, causing respiratory infections, genital disorders and abortions. VP8 is the most abundant tegument protein of BoHV-1 and critical for virus replication in cattle. In this study the cellular transport of VP8 in BoHV-1-infected cells and its ability to alter the cellular lipid metabolism was investigated. A viral kinase, US3, was found to be involved in regulating these processes. In the early stages of infection VP8 was localized in the nucleus. Subsequently, presumably after completion of its role in the nucleus, VP8 was translocated to the cytoplasm. When US3 was deleted or the essential US3-phosphorylation site of VP8 was mutated in BoHV-1, the majority of VP8 was localized in the nucleus of infected cells. This suggests that phosphorylation by US3 may be critical for cytoplasmic localization of VP8. Eventually, the cytoplasmic VP8 was accumulated in the cis-Golgi apparatus, but not in the trans-Golgi network, implying that VP8 was not involved in virion transport towards and budding from the cell membrane. VP8 caused lipid droplet (LD) formation in the nucleus of transfected cells and increased cellular cholesterol levels. Lipid droplets were not found in the nucleus of BoHV-1-infected cells, when VP8 is cytoplasmic in the presence of US3. However, when US3 was deleted or phosphorylation residues in VP8 were mutated, nuclear VP8 and LDs appeared in BoHV-1-infected cells. The total cholesterol level was increased in BoHV-1-infected cells, but not in UL47-BoHV-1-infected cells further supporting a role for VP8 in altering the cellular lipid metabolism during infection.

    IMPORTANCE Nuclear localization signals (NLSs) and nuclear export signals (NESs) are important elements directing VP8 to the desired locations in the BoHV-1-infected cell. In this study, a critical regulator was identified that switches the nuclear and cytoplasmic localization of VP8 in BoHV-1 infected cells. BoHV-1 used viral kinase US3 to regulate the cellular localization of VP8. Early during BoHV-1 infection VP8 was localized in the nucleus, where it performs various functions; once US3 was expressed, phosphorylated VP8 was cytoplasmic and ultimately accumulated in the cis-Golgi apparatus, presumably to be incorporated into virions. The Golgi localization of VP8 was only observed in virus-infected cells and not in US3 co-transfected cells, suggesting that this is mediated by other viral factors. Interestingly, VP8 was shown to cause increased cholesterol levels, which is a novel function for VP8 and a potential strategy to supply lipid for viral replication.

  • Rhesus Macaque Rhadinovirus Encodes a Viral Interferon Regulatory Factor to Disrupt Promyelocytic Leukemia Nuclear Bodies and Antagonize Type I Interferon Signaling. [Virus-Cell Interactions]

  • Interferon (IFN) production and the subsequent induction of IFN stimulated genes (ISGs) are highly effective innate strategies utilized by cells to protect against invading pathogens, including viruses. Critical components involved in this innate process are the Promyelocytic Leukemia Nuclear Bodies (PML-NBs), which are sub-nuclear structures required for the development of a robust IFN response. As such, PML-NBs serve as an important hurdle for viruses to overcome to successfully establish an infection. Both Kaposi's sarcoma-associated Herpesvirus (KSHV) and the closely related rhesus macaque rhadinovirus (RRV) are unique for encoding viral homologues to IFN regulatory factors (termed vIRFs) that can manipulate the host immune response by multiple mechanisms. All four KSHV vIRFs inhibit the induction of IFN while vIRF1 and vIRF2 can inhibit ISG induction downstream of the IFN receptor. Less is known about the RRV vIRFs. RRV vIRF R6 can inhibit the induction of IFN by IRF3, however, it is not known whether any RRV vIRFs inhibit ISG induction following IFN receptor signaling. In our current study we demonstrate that the RRV vIRF R12 aids viral replication in the presence of the type I IFN response. This is achieved in part through the disruption of PML-NBs and the inhibition of robust ISG transcription.

    IMPORTANCE KSHV and RRV encode a unique set of homologs to cellular IFN regulatory factors, termed vIRFs, which are hypothesized to help these viruses evade the innate immune response and establish infections in their respective hosts. Our work elucidates the role of one RRV vIRF, R12, and demonstrates that RRV can dampen the type I IFN response downstream of IFN signaling which would be important for establishing a successful infection in vivo.

  • A mini-genome study of Hazara Nairovirus genomic promoters [Genome Replication and Regulation of Viral Gene Expression]

  • Hazara Nairovirus (HAZV) is a tri-segmented RNA virus most closely related to Crimean-Congo hemorrhagic fever virus, in the order Bunyavirales. The terminal roughly 20 nt of their genome ends are highly complementary, similar to those of other segmented negative-strand RNA viruses (sNSV), and act as promoters for RNA synthesis. These promoters contain two elements; the very termini of both strands (promoter element 1 (PE1)) are conserved and virus-specific, and are found bound to separate sites on the polymerase surface in crystal structures of promoter-polymerase complexes. The following sequences (PE2) are segment-specific with the potential to form dsRNA, and this latter aspect is also important for promoter activity. Nairovirus genome promoters differ from those of peribunya and arenaviruses in that they contain a short ss-region between the two regions of complementarity. Using a HAZV mini-genome system, we find the single-stranded nature of this region, as well as the potential of the following sequence to form dsRNA, are essential for reporter gene expression. Most unexpectedly, the sequence of the PE2 dsRNA appears to be equally important for promoter activity. These differences in sNSV PE2 promoter elements are discussed in the light of our current understanding of the initiation of RNA synthesis.

    IMPORTANCE A mini-genome system for HAZV, closely related to CCHFV, was used to study its genome replication. HAZV genome ends, like those of other sNSV such as peribunya- and arenaviruses, are highly complementary and serve as promoters for genome synthesis. These promoters are composed of two elements; the very termini of both 3rrsquo; and 5rrsquo; strands that are initially bound to separate sites on the polymerase surface in a sequence-specific fashion, and the following sequences with the potential to anneal, but whose sequence is not important. Nairovirus promoters differ from the other sNSV cited in that they contain a short ssRNA region between the two elements. The ss nature of this region is an essential element of this promoter, whereas its sequence is unimportant. The sequence of the following complementary region is unexpectedly also important, a possible rare example of sequence-specific dsRNA recognition.

  • Establishment of A Novel Humanized Mouse Model To Investigate In Vivo Activation and Depletion of Patient-Derived HIV Latent Reservoirs [Pathogenesis and Immunity]

  • Curing HIV infection has been thwarted by the persistent reservoir of latently-infected CD4+ T cells, which reinitiate systemic infection after antiretroviral therapy (ART) interruption. To evaluate reservoir-depletion strategies, we developed a novel pre-clinical in vivo model consisting of immunodeficient mice intrasplenically injected with peripheral blood mononuclear cells (PBMC) from long-term ART-suppressed HIV-infected donors. In the absence of ART, these mice developed rebound viremia which, two weeks after PBMC injection, was 1,000-fold higher (mean=9,229,281 HIV copies/ml) in mice injected intrasplenically than in mice injected intraperitoneally (mean=6,838 HIV copies/ml) or intravenously (mean=591 HIV copies/ml). One week after intrasplenic PBMC injection, in situ hybridization of the spleen demonstrated extensive disseminated HIV infection, likely initiated from in vivo reactivated primary latently infected cells. Time-to-viremia was delayed significantly by treatment with a broadly neutralizing antibody, 10-1074, as compared to treatment with 10-1074-FcRnull, suggesting that 10-1074 mobilized Fc-mediated effector mechanisms to deplete the replication-competent reservoir. This was supported by phylogenetic analysis of Env sequences from viral-outgrowth cultures and untreated, 10-1074-treated or 10-1074-FcRnull-treated mice. The predominant sequence cluster detected in viral-outgrowth cultures and untreated mouse plasma was significantly reduced in plasma of 10-1074-treated mice, while two new clusters emerged which were not detected in viral-outgrowth cultures or plasma from untreated mice. These new clusters lacked mutations associated with 10-1074 resistance. Taken together, these data indicated that 10-1074 treatment depletes the reservoir of latently infected cells harboring replication competent HIV. Furthermore, this mouse model represents a new in vivo approach for the preclinical evaluation of new HIV-cure strategies.

    IMPORTANCE Sustained remission of HIV infection is prevented by a persistent reservoir of latently infected cells capable of reinitiating systemic infection and viremia. To evaluate strategies to reactivate and deplete this reservoir, we developed and characterized a new humanized mouse model consisting of highly immunodeficient mice intrasplenically injected with peripheral blood mononuclear cells from long-term ART-suppressed HIV-infected donors. Reactivation and dissemination of HIV infection was visualized in the mouse spleens in parallel with the onset of viremia. The applicability of this model for evaluating reservoir depletion treatments was demonstrated by establishing, through delayed time-to-viremia and phylogenetic analysis of plasma virus, that treatment of these humanized mice with a broadly neutralizing antibody, 10-1074, depleted the patient-derived population of latently infected cells. This mouse model represents a new in vivo approach for the preclinical evaluation of new HIV-cure strategies.

  • Host range and population survey of Spodoptera frugiperda rhabdovirus [Genetic Diversity and Evolution]

  • The Sf9 and Sf21 cell lines derived from ovarian tissues of the wide host range phytophagous Lepidopteran Spodoptera frugiperda are widely used for research and commercial-scale production of recombinant proteins. These cell lines are chronically infected with a rhabdovirus (Sf-RV) that does not cause any overt cytopathic effects. We demonstrate that wild populations of S. frugiperda in the eastern United States and Caribbean are infected with genetically diverse strains of Sf-RV, and that this virus is also capable of infecting cells of S. exigua, Heliothis subflexa and Bombyx mori. Feeding studies demonstrated the ability of S. frugiperda larvae to deposit Sf-RV onto human-consumed vegetables during feeding. Although no evidence for replication in two species of plant cells was detected, subcellular localization studies demonstrated that the Sf-RV nucleocapsid was targeted to plasmodesmata, while two forms of the accessory protein could be differentiated on the basis of their ability to localize to nuclei. Collectively, this study suggests that environmental exposure of humans to Sf-RV is likely to be commonplace and frequent, but its inability to replicate in plant or human cells suggests that there is no substantial risk to human health.

    IMPORTANCE Insect-derived cell lines are widely used commercially for the production of vaccines and protein-based pharmaceuticals. After decades safe and beneficial use, it was a surprise to the biotechnology industry to discover an endemic rhabdovirus in Sf9 cells. This discovery was made possible only due to substantial advancements in DNA sequencing technologies. Given public health concerns associated with many rhabdovirus species, several initiatives were undertaken to establish that Sf-RV does not pose a threat to humans. Such actions include the generation of cell lines that have been cleared of Sf-RV. Given that Sf9 is derived from a moth whose larva feed on human-edible foods, we explored the prevalence of Sf-RV in its wild and lab-grown populations, as well as its ability to be deposited on food items during feeding. Collectively, our data suggest that there is no overt risk from exposure to Sf-RV.

  • Infectious Entry of Merkel Cell Polyomavirus [Virus-Cell Interactions]

  • Merkel Cell Polyomavirus (MCPyV) is a small, non-enveloped tumor virus associated with an aggressive form of skin cancer, the Merkel cell carcinoma (MCC). MCPyV infections are highly prevalent in the human population with MCPyV virions being continuously shed from human skin. However, the precise host cell tropism(s) of MCPyV remains unclear: MCPyV is able to replicate within a subset of dermal fibroblasts, but MCPyV DNA has also been detected in a variety of other tissues. However, MCPyV appears different from other polyomaviruses as it requires sulfated polysaccharides such as heparan sulfates and/or chondroitin sulfates for initial attachment. Like other polyomaviruses, MCPyV engages sialic acid as a (co-)receptor. To explore the infectious entry process of MCPyV, we analyzed the cell biological determinants of MCPyV entry into A549 cells, a highly transducible lung carcinoma cell line, in comparison to well-studied simian virus 40 and a number of other viruses. Our results indicate that MCPyV enters cells via caveolar/lipid raft-mediated endocytosis but not macropinocytosis, clathrin-mediated endocytosis or glycosphingolipid-enriched carriers. The viruses internalized in small endocytic pits that led the virus to endosomes and from there to the endoplasmic reticulum (ER). Similar to other polyomaviruses, trafficking required microtubular transport, acidification of endosomes, and a functional redox environment. To our surprise, the virus was found to acquire a membrane envelope within endosomes, a phenomenon not reported for other viruses. Only minor amounts of viruses reached the ER, while the majority was retained in endosomal compartments suggesting that endosome-to-ER trafficking is a bottleneck during infectious entry.

    IMPORTANCE MCPyV is the first polyomavirus directly implicated in the development of an aggressive human cancer, the Merkel Cell Carcinoma (MCC). Although MCPyV is constantly shed from healthy skin, MCC incidence increases among aging and immunocompromised individuals. To date, the events connecting initial MCPyV infection and subsequent transformation still remain elusive. MCPyV differs from other known polyomaviruses concerning its cell tropism, entry receptor requirements, and infection kinetics. In this study, we examined the cellular requirements for endocytic entry as well as the subcellular localization of incoming virus particles. A thorough understanding of the determinants of the infectious entry pathway and the specific biological niche will benefit prevention of virus-derived cancers such as MCC.

  • Unique transcriptional architecture in airway epithelial cells and macrophages shapes distinct responses following influenza virus infection ex vivo. [Cellular Response to Infection]

  • Airway epithelial cells and macrophages differ markedly in their responses to influenza A virus (IAV) infection. To investigate transcriptional responses underlying these differences, purified subsets of type II airway epithelial cells (ATII) and alveolar macrophages (AM) recovered from the lungs of mock- or IAV-infected mice at 9 hours post-infection were subjected to RNA sequencing. This time point was chosen to allow for characterization of cell types first infected with virus inoculum, prior to multicycle virus replication and the infiltration of inflammatory cells into the airways. In the absence of infection, AM predominantly expressed genes related to immunity whereas ATII expressed genes consistent with their physiological roles in the lung. Following IAV infection, AM almost exclusively activated cell-intrinsic antiviral pathways that were dependent on interferon regulatory factor (IRF)3/7 and/or type I interferon (IFN) signaling. In contrast, IAV-infected ATII activated a broader range of physiological responses, including cell-intrinsic antiviral pathways, which were both independent and dependent on IRF3/7 and/or type I IFN. These data suggest that transcriptional profiles hardwired during development are a major determinant underlying the different responses of ATII and AM to IAV infection.

    IMPORTANCE Airway epithelial cells (AEC) and airway macrophages (AM) represent major targets of influenza A virus (IAV) infection in the lung, yet the two cell types respond very differently to IAV infection. We have used RNA sequencing to define the host transcriptional responses in each cell type under steady-state conditions, as well as following IAV infection. To do this, different cell subsets isolated from the lungs of mock- and IAV-infected mice were subjected to RNA sequencing. Under steady-state conditions, AM and AEC express distinct transcriptional activity, consistent with distinct physiological roles in the airways. Not surprisingly, these also exhibited major differences in transcriptional responses following IAV infection. These studies shed light on how the different transcriptional architecture of airway cells from two different lineages drive transcriptional responses to IAV infection.

  • The human CD4+ T cell response against mumps virus targets a broadly recognized nucleoprotein epitope [Cellular Response to Infection]

  • Mumps outbreaks among vaccinated young adults stress the need for a better understanding of the mumps virus (MuV)-induced immunity. Antibody responses to MuV are well-characterized, but studies on T cell responses are limited. We recently isolated a MuV-specific CD4+ T cell clone by stimulating PBMCs of a mumps case with the viral nucleoprotein (MuV-N). In this study, we further explored the identity and relevance of the epitope recognized by the CD4+ T cell clone, and ex vivo by T cells in a cohort of mumps cases. Using a 2D-matrix peptide pool of 15-mers peptides covering the complete MuV-N, the epitope recognized by the T cell clone was identified to be MuV-N110-124 GTYRLIPNARANLTA, present in a well-conserved region of the viral protein. Upon peptide-specific stimulation, the T cell clone expressed the activation marker CD137, produced IFN-, TNF and IL-10 in a HLA-DR4 restricted manner. Moreover, the CD4+ T cells exerted a cytotoxic phenotype and specifically killed cells presenting MuV-N110-124. Furthermore, the identified peptide is widely applicable to the general population as it is predicted to bind various common HLA-DR molecules, and epitope-specific CD4+ T cells displaying cytotoxic/Th1-type properties were found in all tested mumps cases expressing different HLA-DR alleles.

    This first broadly recognized human MuV-specific CD4+ T cell epitope could provide a useful tool to detect and evaluate virus-specific T cell responses upon MuV infection or following vaccination.

    IMPORTANCE Recent outbreaks of mumps amongst vaccinated young adults have been reported worldwide. Humoral responses against mumps virus (MuV) are well characterized although no correlate of protection has been elucidated, stressing the need to better understand cellular MuV-specific immunity. In this study, we identified the first MuV T cell epitope, which is derived from the viral nucleoprotein (MuV-N) and was recognized by a cytotoxic/Th1 CD4+ T cell clone that was isolated from a mumps case. Moreover, the epitope was predicted to bind a broad variety of common HLA-DRB1 alleles, which was confirmed by the epitope-specific cytotoxic/Th1 CD4+ T cell responses observed in multiple mumps cases with various HLA-DRB1 genotypes. The identified epitope is completely conserved amongst various mumps strains. These findings qualify this promiscuous MuV T cell epitope as a useful tool for further in depth exploration of MuV-specific T cell immunity after natural mumps virus infection or induced by vaccination.

  • Natural inhibitor of human cytomegalovirus in human seminal plasma [Virus-Cell Interactions]

  • Human cytomegalovirus (HCMV) is the most frequent viral cause of congenital infections that can lead to severe birth defects. Although HCMV is frequently detected in semen and thus potentially sexually transmitted, the role of semen for HCMV transmission is largely unclear. Here, we describe that human seminal plasma (SP; the cell-free supernatant of semen) inhibits HCMV infection. The inhibition of HCMV infection was dose-dependent and effective for different cell types, virus strains and semen donors. This inhibitory effect was specific for HCMV, as herpes simplex virus 2 (HSV-2) and human immunodeficiency virus 1 (HIV-1) infections were enhanced by SP. Mechanistically, SP inhibited infection by interfering with the attachment of virions to cells most likely via an interaction with the trimeric glycoprotein complex of gH/gL/gO. Together, our findings suggest that semen contains a factor that potentially limits sexual transmission of HCMV.

    IMPORTANCE The role of semen in sexual transmission of the human cytomegalovirus (HCMV) is currently unclear. This is surprising as HCMV is frequently detected in this body fluid and infection is of high danger for neonates and pregnant women. Here, we found that seminal plasma (SP) dose-dependently inhibited HCMV infection. The infection inhibition was specific for HCMV, as other viruses such as human immunodeficiency virus 1 (HIV-1) and herpes simplex virus 2 (HSV-2) were not inhibited by SP. SP must contain a soluble, heat resistant factor that limits attachment of HCMV particles to cells probably by interaction with the trimeric glycoprotein complex of gH/gL/gO. This novel virus-host interaction could possibly limit transmission of HCMV via semen during sexual intercourse.

  • Acute respiratory infection in human dipeptidyl peptidase 4-transgenic mice infected with Middle East respiratory syndrome coronavirus [Pathogenesis and Immunity]

  • Middle East respiratory syndrome coronavirus (MERS-CoV) infection can manifest as a mild illness, acute respiratory distress, organ failure, or death. Several animal models have been established to study disease pathogenesis and to develop vaccines and therapeutic agents. Here, we developed transgenic (Tg) mice on a C57BL/6 background; these mice expressed human CD26/dipeptidyl peptidase 4 (hDPP4), a functional receptor for MERS-CoV, under the control of an endogenous hDPP4 promoter. We then characterized this mouse model of MERS-CoV. The expression profile of hDPP4 in these mice was almost equivalent to that in human tissues, including kidney and lung; however, hDPP4 was overexpressed in murine CD3-positive cells within peripheral blood and lymphoid tissues. Intranasal inoculation of young and adult Tg mice with MERS-CoV led to infection of the lower respiratory tract and pathological evidence of acute multifocal interstitial pneumonia within 7 days, with only transient loss of body weight. However, the immunopathology in young and adult Tg mice was different. On Day 5 or 7 post-inoculation, lungs of adult Tg mice contained higher levels of pro-inflammatory cytokines and chemokines associated with migration of macrophages. These results suggest that the immunopathology of MERS infection in the Tg mouse is age-dependent. The mouse model described herein will increase our understanding of disease pathogenesis and host mediators that protect against MERS-CoV infection.

    IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) infections are endemic in the Middle East and a threat to public health worldwide. Rodents are not susceptible to the virus because they do not express functional receptors; therefore, we generated a new animal model of MERS-CoV infection based on transgenic mice expressing human (h)DPP4. The pattern of hDPP4 expression in this model was similar to that in human tissues (except lymphoid tissue). In addition, MERS-CoV was limited to the respiratory tract. Here, we focused on host factors involved in immunopathology in MERS-CoV infection and clarified differences in antiviral immune responses between young and adult transgenic mice. This new small animal model could contribute to more in-depth study of the pathology of MERS-CoV infection and aid development of suitable treatments.

  • TMPRSS2 contributes to virus spread and immunopathology in the airways of murine models after coronavirus infection [Pathogenesis and Immunity]

  • Transmembrane serine protease TMPRSS2 activates the spike protein of highly pathogenic human coronaviruses such as severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV). In vitro, activation induces virus-cell membrane fusion at the cell surface. However, the roles of TMPRSS2 during coronavirus infection in vivo are unclear. Here, we used animal models of SARS-CoV and MERS-CoV infection to investigate the role of TMPRSS2. Th-1-prone C57BL/6 mice and TMPRSS2-knockout (KO) mice were used for SARS-CoV infection, and transgenic mice expressing the human MERS-CoV receptor, hDPP4-Tg mice, and TMPRSS2-KO hDPP4-Tg mice were used for MERS-CoV infection. After experimental infection, TMPRSS2-deficient mouse strains showed reduced body weight loss and viral kinetics in the lungs. Lack of TMPRSS2 affected the primary sites of infection and virus spread within the airway, accompanied by less severe immunopathology. However, TMPRSS2-KO mice showed weakened inflammatory chemokine and/or cytokine responses to intranasal stimulation with poly (I:C), a Toll-like receptor 3 agonist. In conclusion, TMPRSS2 plays a crucial role in viral spread within the airway of murine models infected by SARS-CoV and MERS-CoV and in the resulting immunopathology.

    IMPORTANCE Broad-spectrum antiviral drugs against highly pathogenic coronaviruses and other emerging viruses are desirable to enable a rapid response to pandemic threats. Transmembrane protease serine type2 (TMPRSS2), a protease belonging to the type II transmembrane serine protease family, cleaves the coronavirus spike protein, making it a potential therapeutic target for coronavirus infections. Here, we examined the role of TMPRSS2 using animal models of SARS-CoV and MERS-CoV infection. The results suggest that lack of TMPRSS2 in the airways reduces the severity of lung pathology after infection by SARS-CoV and MERS-CoV. Taken together, the results will facilitate development of novel targets for coronavirus therapy.

  • Viral replicative capacity, antigen availability via hematogenous spread and high TFH:TFR ratios drive induction of potent neutralizing antibody responses [Vaccines and Antiviral Agents]

  • Live viral vaccines elicit protective, long-lived humoral immunity but the underlying mechanisms through which this occurs are not fully elucidated. Generation of affinity matured, long-lived protective antibody responses involve close interactions between T follicular helper (TFH) cells, germinal center (GC) B cells, and T follicular regulatory (TFR) cells. We postulated that escalating concentrations of antigens from replicating viruses or live vaccines, spread through the hematogenous route, is essential for the induction and maintenance of long-lived protective antibody responses. Using replicating and poorly- or non-replicating orthopox and influenza A viruses we show that the magnitude of TFH cell, GC B cell and neutralizing antibody responses is directly related to virus replicative capacity. Further, we have identified that both lymphoid and circulating TFH:TFR cell ratios during the peak GC response can be used as an early predictor of protective, long-lived antibody response induction. Finally, administration of poorly or non-replicating viruses to allow hematogenous spread generates significantly stronger TFH:TFR ratios and robust TFH, GC B cell and neutralizing antibody responses.

    Importance: Neutralizing antibody response is the best-known correlate of long-term protective immunity for most of the currently licensed clinically effective viral vaccines. However, the host immune and viral factors that are critical for the induction of robust and durable antiviral humoral immune responses are not well understood. Our study provides insight into the dynamics of key cellular mediators of germinal center reaction during live virus infections and the influence of viral replicative capacity on the magnitude of antiviral antibody response and effector function. The significance of our study lies in two key findings. First, systemic spread of even poorly- or non-replicating viruses to mimic the spread of antigens from replicating viruses due to escalating antigen concentration is fundamental to induction of durable antibody responses. Second, the TFH:TFR ratio may be used as an early predictor of protective antiviral humoral immune responses long before memory responses are generated.

  • West Nile virus-inclusive single-cell RNA sequencing reveals heterogeneity in the type I interferon response within single cells [Cellular Response to Infection]

  • West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus of global importance. Neuroinvasive WNV infection results in encephalitis and can lead to prolonged neurological impairment or death. Type I interferon (IFN-I) is crucial for promoting antiviral defenses through the induction of antiviral effectors, which function to restrict viral replication and spread. However, our understanding of the antiviral response to WNV infection is mostly derived from analysis of bulk cell populations. It is becoming increasingly apparent that substantial heterogeneity in cellular processes exists among individual cells, even within a seemingly homogenous cell population. Here, we present WNV-inclusive single-cell RNA sequencing (scRNA-seq), an approach to examine the transcriptional variation and viral RNA burden across single cells. We observed that only a few cells within the bulk population displayed robust transcription of IFN-bbeta; mRNA, and this did not appear to depend on viral RNA abundance within the same cell. Furthermore, we observed considerable transcriptional heterogeneity in the IFN-I response, with genes displaying high unimodal and bimodal expression patterns. Broadly, IFN-stimulated genes negatively correlated with viral RNA abundance, corresponding with a precipitous decline in expression in cells with high viral RNA levels. Altogether, we demonstrated the feasibility and utility of WNV-inclusive scRNA-seq as a high-throughput technique for single-cell transcriptomics and WNV RNA detection. This approach can be implemented in other models to provide insights into the cellular features of protective immunity and identify novel therapeutic targets.

    IMPORTANCE West Nile virus (WNV) is a clinically relevant pathogen responsible for recurrent epidemics of neuroinvasive disease. Type I interferon is essential for promoting an antiviral response against WNV infection; however, it is unclear how heterogeneity in the antiviral response at the single-cell level impacts viral control. Specifically, conventional approaches lack the ability to distinguish differences across cells with varying viral abundance. The significance of our research is to demonstrate a new technique for studying WNV infection at the single-cell level. We discovered extensive variation in antiviral gene expression and viral abundance across cells. This protocol can be applied to primary cells or in vivo models to better understand the underlying cellular heterogeneity following WNV infection for the development of targeted therapeutic strategies.

  • Identification of clotrimazole-derivatives as specific inhibitors of Arenavirus fusion [Virus-Cell Interactions]

  • Arenaviruses are a large family of emerging enveloped negative strand RNA viruses that include several causative agents of viral hemorrhagic fevers. For cell entry, human pathogenic arenaviruses use different cellular receptors and endocytic pathways that converge at the level of acidified late endosomes, where the viral envelope glycoprotein mediates membrane fusion. Inhibitors of arenavirus entry hold promise for therapeutic antiviral intervention and the identification of "druggable" targets is of high priority. Using a recombinant vesicular stomatitis virus pseudotype platform, we identified the clotrimazole-derivative TRAM-34, a highly selective antagonist of the calcium-activated potassium channel KCa3.1, as a specific entry inhibitor for arenaviruses. TRAM-34 specifically blocked entry of most arenaviruses, including hemorrhagic fever viruses, but not Lassa virus and other enveloped viruses. Anti-arenaviral activity was likewise observed with the parental compound clotrimazole and the derivative senicapoc, whereas structurally unrelated KCa3.1 inhibitors showed no antiviral effect. Deletion of KCa3.1 by CRISPR/Cas9 technology did not affect the anti-arenaviral effect of TRAM-34, indicating that the observed antiviral effect of clotrimazoles was independent of the known pharmacological target. The drug affected neither virus-cell attachment, nor endocytosis, suggesting an effect on later entry steps. Employing a quantitative cell-cell fusion assay that bypasses endocytosis, we demonstrate that TRAM-34 specifically inhibits arenavirus-mediated membrane fusion. In sum, we uncover a novel anti-arenaviral action of clotrimazoles that currently undergo in vivo evaluation in the context of other human diseases. Their favorable in vivo toxicity profiles and stability opens the possibility to repurpose clotrimazole-derivatives for therapeutic intervention against human pathogenic arenaviruses.

    IMPORTANCE Emerging human pathogenic arenaviruses are causative agents of severe hemorrhagic fevers with high mortality and represent serious public health problems. The current lack of a licensed vaccine and the limited treatment options makes the development of novel anti-arenaviral therapeutics an urgent need. Using a recombinant pseudotype platform, we uncovered that clotimazole drugs, in particular TRAM-34, specifically inhibit cell entry of a range of arenaviruses, including important emerging human pathogens, with the exception of Lassa virus. The antiviral effect was independent of the known pharmacological drug target and involved inhibition of the unusual membrane fusion mechanism of arenaviruses. TRAM-34 and its derivatives currently undergo evaluation against a number of human diseases and show favorable toxicity profiles and high stability in vivo. Our study provides the basis for further evaluation of clotrimazole-derivatives as antiviral drug candidates. Their advanced stage of drug development will facilitate re-purposing for therapeutic intervention against human pathogenic arenaviruses.

  • USP15 participates in HCV propagation through the regulation of viral RNA translation and lipid droplet formation [Virus-Cell Interactions]

  • Hepatitis C virus (HCV) utilizes cellular factors for an efficient propagation. Ubiquitin is covalently conjugated to the substrate to alter its stability or to modulate signal transduction. In this study, we examined the importance of ubiquitination for HCV propagation. We found that inhibition of de-ubiquitinating enzymes (DUBs) or overexpression of non-specific DUBs impaired HCV replication, suggesting that ubiquitination regulates HCV replication. To identify specific DUBs involved in HCV propagation, we set up an RNAi screening against DUBs and successfully identified ubiquitin-specific protease 15 (USP15) as a novel host factor for HCV propagation. Our studies showed that USP15 is involved in translation of HCV RNA and production of infectious HCV particles. In addition, deficiency of USP15 in human hepatic cell lines (Huh7 and Hep3B/miR122 cells) but not in a non-hepatic cell line (293T cells) impaired HCV propagation, suggesting that USP15 participates in HCV propagation through the regulation of hepatocyte-specific functions. Moreover, we showed that loss of USP15 had no effect on innate immune responses in vitro and in vivo. We also found that USP15-deficient Huh7 cells showed reductions in the amounts of lipid droplets (LDs), and addition of palmitic acids restored the production of infectious HCV particles. Taken together, these data suggest that USP15 participates in HCV propagation by regulating the translation of HCV RNA and formation of LDs.

    IMPORTANCE Although ubiquitination has been shown to play important roles in the HCV life cycle, the roles of de-ubiquitinating enzymes (DUBs), which cleave ubiquitin chains from their substrates, in HCV propagation have not been investigated. Here, we identified USP15 as a DUB regulating HCV propagation. USP15 showed no interaction with viral proteins and no participation in innate immune responses. Deficiency of USP15 in Huh7 cells resulted in suppression of the translation of HCV RNA and reduction in the amounts of lipid droplets, and addition of fatty acids partially restored the production of infectious HCV particles. These data suggest that USP15 participates in HCV propagation in hepatic cells through the regulation of viral RNA translation and lipid metabolism.

  • Broadly cross-reactive, non-neutralizing antibodies against the influenza B virus hemagglutinin demonstrate effector function dependent protection against lethal viral challenge in mice [Vaccines and Antiviral Agents]

  • Protection from influenza virus infection is canonically associated with antibodies that neutralize the virus by blocking the interaction between the viral hemagglutinin and host cell receptors. However, protection can also be conferred by other mechanisms including antibody-mediated effector functions. Here we report the characterization of 22 broadly cross-reactive, non-neutralizing antibodies specific for influenza B virus hemagglutinin. The majority of these antibodies recognized influenza B viruses isolated over the period of 73 years and bind the conserved stalk domain of the hemagglutinin. A proportion of the characterized antibodies protected mice from both morbidity and mortality after challenge with a lethal dose of influenza B virus. Activity in an antibody-dependent cell mediated cytotoxicity reporter assay correlated strongly with protection, suggesting that Fc-dependent effector function determines protective efficacy. The information regarding mechanism of action and epitope location stemming from our characterization of these antibodies will inform the design of urgently needed vaccines that could induce broad protection against influenza B viruses.

    IMPORTANCE While broadly protective antibodies against the influenza A virus hemagglutinin have been well studied, very limited information is available for similar antibodies that recognize influenza B viruses. Similarly, the development of a universal or broadly protective influenza B virus vaccine lags behind the development of such a vaccine for influenza A virus. More information about epitope location and mechanism of action of broadly protective influenza B virus antibodies is required to inform vaccine development. In addition, protective antibodies could be a useful tool to treat or prevent influenza B virus infection in pediatric cohorts or in a therapeutic setting in immunocompromised individuals in conjugation with existing treatment avenues.

  • Chloroviruses lure hosts through long-distance chemical signaling [Virus-Cell Interactions]

  • Chloroviruses exist in aquatic systems around the planet where they infect certain eukaryotic green algae that are mutualistic endosymbionts in a variety of protists and metazoans. Natural chlorovirus populations are seasonally dynamic but the precise temporal changes in these populations and the mechanisms that underlie them have, heretofore, been unclear. We recently reported the novel concept that predator/prey-mediated virus activation regulates chlorovirus population dynamics, and in the current manuscript demonstrate virus packaged chemotactic modulation of prey behavior.

    IMPORTANCE Viruses have not previously been reported to act as chemotactic/chemo-attractive agents. Rather, viruses as extracellular entities are generally viewed as non-metabolically active spore-like agents that await further infection events upon collisions with appropriate host cells. That a virus might actively contribute to its fate via chemotaxis and change the behavior of an organism independent of infection is unprecedented.

  • Geminiviral V2 Protein Suppresses Transcriptional Gene Silencing through Interaction with AGO4 [Pathogenesis and Immunity]

  • In plants, RNA-directed DNA methylation (RdDM)-mediated transcriptional gene silencing (TGS) is a natural antiviral defense against geminiviruses. Several geminiviral proteins have been shown to target the enzymes related to methylcycle or histone modification; however, it remains largely unknown whether and by which mechanism geminiviruses directly inhibit RdDM-mediated TGS. In this study, we show that Cotton Leaf Curl Multan virus (CLCuMuV) V2 directly interacts with NbAGO4 and the L76S mutation in V2 (V2L76S) abolishes such interaction. We further showed that V2, but not V2L76S can suppresses RdDM and TGS. Silencing of NbAGO4 inhibits TGS, reduces viral methylation level and enhances CLCuMuV DNA accumulation. In contrast, the V2L76S substitution mutant attenuates CLCuMuV infection and enhances viral methylation level. These findings reveal that CLCuMuV V2 contributes to viral infection by interaction with NbAGO4 to suppress RdDM-mediated TGS in plants.

    IMPORTANCE In plants, RNA-directed DNA methylation (RdDM) pathway is a natural antiviral defense mechanism against geminiviruses. However, how geminiviruses counter RdDM-mediated defense is largely unknown. Our findings reveal that Cotton Leaf Curl Multan virus V2 contributes to viral infection by interaction with NbAGO4 to suppress RNA-directed DNA methylation-mediated transcriptional gene silencing in plants. Our work provides the first evidence that a geminiviral protein is able to directly target core RdDM component to counter RdDM-mediated TGS antiviral defense in plants, which extends our current understanding of viral counter- host antiviral defense.

  • Examination and reconstruction of three ancient endogenous parvovirus capsid protein gene remnants found in rodent genomes [Structure and Assembly]

  • Parvovirus-derived endogenous viral elements (EVEs) have been found in the genomes of many different animal species, resulting from integration events that may have occurred from more than 50 million years ago to much more recently. Here, we further investigate the properties of autonomous parvovirus EVEs and describe their relationships to contemporary viruses. While we did not find any intact capsid protein open reading frames in the integrated viral sequences, we examined three EVEs that could be repaired to form full-length sequences with relatively few changes. These sequences were found in the genomes of Rattus norvegicusggt; (brown rat), Mus spretus (Algerian mouse), and Apodemus sylvaticus (wood mouse). The R. norvegicus sequence was not present in the genomes of the closely related species R. rattus, R. tanezumi, R. exulans, and R. everetti, indicating that it was less than 2 million years old, and the M. spretus and A. sylvaticus sequences were not found in the published genomes of other mouse species, also indicating relatively recent insertions. The M. spretus VP2 sequence assembled into capsids, which had high thermal stability, bound the sialic acid N-acetyl neuraminic acid, and entered murine L cells. The 3.89AAring; structure of the M. spretus VLPs, determined using cryo-electron microscopy, showed similarities to rodent and porcine parvovirus capsids. The repaired VP2 sequences from R. norvegicus and A. sylvaticus did not assemble as first prepared, but chimeras combining capsid surface loops from R. norvegicus with canine parvovirus assembled, allowing some of that capsidrrsquo;s structures and functions to be examined.

    IMPORTANCE. Parvovirus EVEs incorporated into the genomes of different animals represent remnants of the DNA sequences ancient viruses that infected the ancestors of those animals millions of years ago, but we know little about their properties or how they differ from currently circulating parvoviruses. By expressing the capsid proteins of different parvovirus EVEs that were found integrated into the genomes of three different rodents, we can examine their structures and functions. A VP2 (major capsid protein) EVE sequence from a mouse genome assembled into capsids that had a similar structure and biophysical properties to extant parvoviruses, and also bound sialic acids and entered rodent cells. Chimeras formed from combinations of canine parvovirus and portions of the parvovirus sequences from the brown rat genome allowed us to examine the structures and functions of the surface loops of that EVE capsid.

  • Why Are CD8 T Cell Epitopes of Human Influenza A Virus Conserved? [Vaccines and Antiviral Agents]

  • The high-degree of conservation of CD8 T cell epitopes of influenza A virus (IAV) may allow for the development of T cell-inducing vaccines that provide protection across different strains and subtypes. This conservation is not fully explained by functional constraint, since additional mutation(s) can compensate the replicative fitness loss of IAV escape-variants. Here, we propose three additional mechanisms that contribute to the conservation of CD8 T cell epitopes of IAV. First, influenza-specific CD8 T cells may protect predominantly against severe pathology rather than infection and may have only a modest effect on transmission. Second, polymorphism of human MHC-I gene restricts the advantage of an escape-variant to only a small fraction of human population, who carry the relevant MHC-I alleles. Finally, infection with CD8 T cell escape-variants may result in compensatory increase in the responses to other epitopes of IAV. We use a combination of population genetics and epidemiological models to examine how the interplay between these mechanisms affects the rate of invasion of IAV escape-variants. We conclude that for a wide range of biologically reasonable parameters the invasion of an escape-variant virus will be slow with a timescale of a decade or more. The results suggest T cell-inducing vaccines may not engender the rapid evolution of IAV. Finally, we identify key parameters whose measurement will allow for more accurate quantification of the long-term effectiveness and impact of universal T cell-inducing influenza vaccines.

    Importance. Universal influenza vaccines against the conserved epitopes of influenza A virus have been proposed to minimize the burden of seasonal outbreaks and prepare for the pandemics. However, it is not clear how rapidly T cell-inducing vaccines will select for viruses that escape these T cell responses. Our mathematical models explore the factors that contribute to the conservation of CD8 T cell epitopes and how rapidly the virus will evolve in response to T cell-inducing vaccines. We identify the key biological parameters to be measured and questions that need to be addressed in future studies.

  • Horizontal transfer of a retrotransposon from the rice planthopper to the genome of an insect DNA virus [Genetic Diversity and Evolution]

  • Horizontal transfer of genetic materials between virus and host have been frequently identified. Three rice planthoppers, Laodelphax striatellus, Nilaparvata lugens, and Sogatella furcifera are agriculturally important insects because they are destructive rice pests and also the vector of a number of phytopathogenic viruses. In this study, we discovered that a small region (~300 nt) of the genome of invertebrate iridescent virus 6 (IIV-6, genus Iridovirus, family Iridoviridae), a giant DNA virus that infects invertebrates but is not known to infect planthoppers, is highly homologous to the sequences present in high copy numbers in these three planthopper genomes. These sequences are related to the short interspersed nuclear elements (SINEs), a class of non-LTR retrotransposon (retroposon), suggesting a horizontal transfer event of a transposable element from the rice planthopper genome to IIV-6 genome. In addition, a number planthopper transcripts mapped to these rice planthopper SINE-like sequences (RPSlSs) were identified and appear to be transcriptionally regulated along the different developmental stages of planthoppers. Small RNAs derived from these RPSlSs are predominantly 26-28 nt long, which is the typical characteristic of PIWI-interacting RNAs. Phylogenetic analysis suggests that IIV6 might acquire a SINE-like retrotransposon from S. furcifera after the separation of the three rice planthoppers. This study provides further example of the horizontal transfer of an insect transposon to virus and suggests the association of rice planthoppers with iridoviruses in the past or present.

    IMPORTANCE This study provides an example of the horizontal transfer event from a rice planthopper genome to an invertebrate iridescent virus 6 (IIV-6, a iridovirus) genome. A small region of IIV-6 (~300 nt) genome is highly homologous to the sequences presented in high copy numbers of three rice planthopper genomes that are related to the short interspersed nuclear elements (SINEs), a class of non-LTR retrotransposon (retroposon). The expression of these planthopper SINE-like sequences was confirmed and corresponding Piwi-interacting RNA-like small RNAs were identified and comprehensively characterized. Phylogenetic analysis suggests that the giant invertebrate iridovirus IIV-6 might obtain this SINE related sequence from Sogatella furcifera through an horizontal transfer event in the past. To the best of our knowledge, this is the first report of horizontal transfer event between a planthopper and a giant DNA virus, and also the first evidence for the eukaryotic origin of a genetic material in iridoviruses.

  • Investigation of host range of and host defense against a mitochondrially replicating mitovirus. [Cellular Response to Infection]

  • Mitoviruses (genus Mitovirus, family Narnaviridae) are mitochondrially replicating viruses that have the simplest positive-sense RNA genomes of 2.3 to 3.6 kb with a single ORF encoding an RNA-dependent RNA polymerase. Cryphonectria mitovirus 1 (CpMV1) from US strain NB631 of the chestnut blight fungus, Cryphonectria parasitica, was the first virus identified as a mitochondrially-replicating virus. Despite subsequent discovery of many other mitoviruses from diverse fungi, no great advances in understanding mitovirus biology have emerged, partly because of the lack of inoculation methods. Here we developed a protoplast fusion-based protocol for horizontal transmission of CpMV1 that entailed fusion of recipient and donor protoplasts, hyphal anastomosis, and single conidium isolation. This method allowed expansion of the host range to many other C. parasitica strains. Species within and outside the family Cryphonectriacea, C. radicalis and Valsa spermatoma, also supported the replication of CpMV1 at a level comparable to that in the natural host. No stable maintenance of CpMV1 was observed in Helminthosporium victoriae. Polymerase chain reaction-based haplotyping of virus-infected fungal strains confirmed the recipient mitochondrial genetic background. Phenotypic comparison between CpMV1-free and -infected isogenic strains revealed no overt effects of the virus. Taking advantage of the infectivity to the standard strain of C. parasitica EP155, accumulation levels were compared among antiviral RNA silencing-proficient and deficient strains in the EP155 background. Comparable accumulation levels were observed among these strains, suggesting the avoidance of antiviral RNA silencing by CpMV1, consistent with its mitochondrial replication. Collectively, this study provides a foundation to further explore the biology of mitoviruses.

    IMPORTANCE Capsidless mitoviruses, ubiquitously detected in filamentous fungi, have the simplest RNA genomes of 2.3 to 3.6 kb encoding only RNA-dependent RNA polymerase. Despite their simple genomes, detailed biological characterization of mitoviruses has been hampered by their mitochondrial location within the cell, posing challenges to their experimental introduction and study. Here we developed a protoplast fusion-based protocol for horizontal transfer of the prototype mitovirus, Cryphonectria mitovirus 1 (CpMV1) isolated from strain NB631 of the chestnut blight fungus (Cryphonectria parasitica), a model filamentous fungus for studying virus/host interactions. The host range of CpMV1 has been expanded to many different strains of C. parasitica, different fungal species within and outside the Cryphonectriaceae. Comparison of CpMV1 accumulation among various RNA silencing-deficient and competent strains showed clearly the virus to be unaffected by RNA silencing. This study provides a solid foundation for further exploration of mitovirus/host interactions.

  • GII.4 NOROVIRUS PROTEASE SHOWS pH-SENSITIVE PROTEOLYSIS WITH A UNIQUE ARG-HIS PAIRING IN THE CATALYTIC SITE [Structure and Assembly]

  • Human noroviruses (NoV) are the main cause of epidemic and sporadic gastroenteritis. Phylogenetically noroviruses are divided into seven genogroups with each divided into multiple genotypes. NoVs belonging to genogroup II and genotype 4 (GII.4) are globally most prevalent. Genetic diversity among the NoVs and the periodic emergence of novel strains present a challenge for the development of vaccines and antivirals to treat the NoV infection. NoV protease is essential for viral replication and an attractive target for the development of antivirals. The available structure of GI.1 protease provided a basis for the design of inhibitors targeting the active site of the protease. These inhibitors, though potent against the GI proteases, poorly inhibit the GII proteases for which structural information is lacking. To elucidate the structural basis for this difference in the inhibitor efficiency, we determined the crystal structure of a GII.4 protease. The structure revealed significant changes in the S2 substrate-binding pocket, making it noticeably smaller, and in the active site with the catalytic triad residues showing conformational changes. Further, a conserved arginine is found inserted into the active site interacting with the catalytic histidine and restricting substrate/inhibitor access to the S2 pocket. This interaction alters the relationships between the catalytic residues and may allow for a pH-dependent regulation of protease activity. The changes we observed in the GII.4 protease structure may explain the reduced potency of the GI-specific inhibitors against the GII protease and therefore must be taken into account when designing broadly cross-reactive antivirals against NoVs.

    IMPORTANCE Human noroviruses (NoV) cause sporadic and epidemic gastroenteritis worldwide. They are divided into seven genogroups (GInndash;GVII), with each genogroup further divided into several genotypes. Human NoVs belonging to genogroup II and genotype 4 (GII.4) are the most prevalent. Currently, there are no vaccines or antiviral drugs available for NoV infection. The protease encoded by the NoV because of its essential role in replication is considered a valuable target. NoV protease structures have only been determined for the GI genogroup. We show here that the structure of GII.4 protease exhibits several significant changes from GI proteases including a unique pairing of an arginine with the catalytic histidine that makes the proteolytic activity of GII.4 protease pH-sensitive. A comparative analysis of NoV protease structures may provide a rational framework for structure-based drug design of broadly cross-reactive inhibitors targeting NoVs.

  • Porcine circovirus 2 uses a multitude of weak binding sites to interact with heparan sulfate, and the interactions do not follow the symmetry of the capsid [Virus-Cell Interactions]

  • Porcine circovirus 2 is the smallest pathogenic virus capable of autonomous replication within its host. Infections result in immunosuppression and subsequent death of the host, and are initiated via the attachment of the PCV2 icosahedral capsid to heparan sulfate and chondroitin sulfate B glycosaminoglycans on the cell surface. However, the underlying mechanism of structural recognition remains to be explored. Using heparin, a routinely used analog of heparan sulfate, we demonstrate that increasing lengths of heparin exhibit greater affinity towards PCV2. Our competition assays indicate that dextra sulfate (8kDa) has higher affinity than heparin (12kDa), chondroitin sulfate B (41kDa) hyaluronic acid (1.6MDa), and dextran (6kDa) for PCV2. This suggests that polymers high in sulfate content are capable of competing with the PCV2-heparan sulfate interaction, and thus have the potential to inhibit PCV2 infection. Finally, we visualize the interaction between heparin and the PCV2 capsid using cryo-electron microscopy single particle analysis, symmetry expansion, and focused classification. The image reconstructions provide the first example of an asymmetric distribution of heparin on the surface of an icosahedral virus capsid. We demonstrate that each of the 60 capsid subunits that generate the T=1 capsid can bind heparin via one of five binding sites. However, not all of the binding sites are occupied by heparin and only one- to two-thirds of the binding sites are occupied. The binding sites are defined by arginine, lysine, and polar amino acids. Mutating the arginine, lysine, and polar amino acids to alanine diminishes the binding capacity of PCV2 to heparin.

    Importance: It has been demonstrated that porcine circovirus 2 (PCV2) attaches to cells via heparan sulfate (HS) and chondroitin sulfate B (CSB) glycosaminoglycans; however, the underlying structural mechanism describing the HS/CSB recognition by PCV2 remains to be explored. We use cryo-electron microscopy with single particle analysis, symmetry expansion, and focused classification to visualize the interaction between the PCV2 capsid and heparin, an analog of heparan sulfate, to better than 3.6AAring; resolution. We observe that the interaction between the PCV2 and heparin does not adhere to the icosahedral symmetry of the capsid. To the best of our knowledge, this is the first example where the interaction between heparin and an icosahedral capsid does not follow the symmetry elements of the capsid. Our findings also suggest that anionic polymers such as dextran sulfate may act to inhibit PCV2 infection.

  • Lund Human Mesencephalic (LUHMES) Neuronal Cell Line Supports HSV-1 Latency in vitro [Virus-Cell Interactions]

  • Lund human mesencephalic (LUHMES) cells are human embryonic neuronal precursor cells that can be maintained as proliferating cells due to the expression of a tetracycline-regulatable (Tet-Off) v-myc transgene. They can be differentiated to post-mitotic neurons by the addition of tetracycline, GDNF and dibutyryl cAMP. We demonstrate that these cells can be infected with HSV-1 at a MOI of 3 with the majority of cells surviving. By 6 days post infection, there is a loss of lytic gene transcription and an increase in numbers of neurons that express the latency associated transcripts (LATs). Importantly, the virus can then be reactivated by the addition of a phosphoinositide 3-kinase inhibitor, which has previously been shown to reactivate HSV-1 in rat neuron cultures. While rodent primary culture neuron systems have been described, these are limited by their lack of scalability, as it is difficult to obtain more than 500,000 neurons to employ for a given experiment. Several recent papers have described a human DRG neuron culture model and human iPSC neuron culture models that are scalable, but they require that the presence of antiviral suppression be maintained following HSV-1 infection. The human LUHMES cell model of HSV-1 infection described here may be especially useful for studying HSV-1 latency and reactivation on account of its scalability, its amenability to maintenance of latency without the continual use of antiviral inhibitors, and its latent gene expression profile which mirrors many properties observed in vivo, importantly the heterogeneity of cells expressing the LATs.

    IMPORTANCE Herpes Simplex Virus (HSV) is responsible for significant morbidity in humans due to its ability to cause oral and genital lesions, ocular disease, and encephalitis. While antivirals can attenuate the severity and frequency of disease, there is no vaccine or cure. Understanding the molecular details of HSV latency and reactivation is key to the development of new therapies. One of the difficulties in studying HSV latency has been the need to rely on establishment of latent infections in animal models. While rodent primary neuron culture models have shown promise, they yield relatively small numbers of latently infected neurons for biochemical and molecular analyses. Here we present the use of a human CNS-derived conditionally proliferating cell line that can be differentiated into mature neurons and latently infected with HSV-1. This model shows promise as a scalable tool to study molecular and biochemical aspects of HSV-1 latency and reactivation in human neurons.

  • LXR alpha Restricts Gammaherpesvirus Reactivation from Latently-Infected Peritoneal Cells. [Pathogenesis and Immunity]

  • Gammaherpesviruses are ubiquitous viruses that establish lifelong infections. Importantly, these viruses are associated with numerous cancers and lymphoproliferative diseases. While risk factors for developing gammaherpesvirus-driven cancers are poorly understood, it is clear that elevated viral reactivation from latency often precedes oncogenesis. Here we demonstrate that the Liver X Receptor alpha isoform (LXRaalpha;) restricted gammaherpesvirus reactivation in an anatomic site-specific manner. We have previously demonstrated that deficiency of both LXR isoforms (aalpha; and bbeta;) leads to increase in the fatty acid and cholesterol synthesis in primary macrophage cultures, with a corresponding increase in gammaherpesvirus replication. Interestingly, expression of fatty acid synthesis genes was not derepressed in LXRaalpha; deficient hosts, indicating that the antiviral effects of LXRaalpha; are independent of lipogenesis. Additionally, the critical host defenses against gammaherpesvirus reactivation, virus-specific CD8+ T cells and interferon (IFN) signaling, remained intact in the absence of LXRaalpha;. Remarkably, using an MHV68 reporter virus, we discovered that LXRaalpha; expression dictates the cellular tropism of MHV68 in the peritoneal cavity. Specifically, LXRaalpha;-/- mice exhibit reduced latency within the peritoneal B cell compartment, and elevated latency within F4/80+ cells. Thus LXRaalpha; restricts gammaherpesvirus reactivation through a novel mechanism, which is independent of the known CD8+ T cell-based antiviral responses or changes in lipid synthesis, and likely involves changes in the tropism of MHV68 in the peritoneal cavity.

    IMPORTANCE Liver X receptors (LXRs) are nuclear receptors that mediate cholesterol and fatty acid homeostasis. Importantly, as ligand activated transcription factors, LXRs represent potential targets for the treatment of hypercholesterolemia and atherosclerosis. Here we demonstrate that LXRaalpha;, one of the two LXR isoforms restricts reactivation of latent gammaherpesvirus from peritoneal cells. As gammaherpesviruses are ubiquitous oncogenic agents, LXRs may represent a targetable host factor for the treatment of poorly controlled gammaherpesvirus infection and associated lymphomagenesis.

  • Inhibition of Ongoing Influenza A Virus Replication Reveals Different Mechanisms of RIG-I Activation [Pathogenesis and Immunity]

  • The pattern-recognition receptors provide essential nonself immune surveillance within distinct cellular compartments. The retinoic acid-inducible gene I (RIG-I) is one of the primary cytosolic RNA sensors with an emerging role in the nucleus. It is involved in the spatiotemporal sensing of influenza A virus (IAV) replication, leading to the induction of type I interferons (IFN). Nonetheless, the physiological viral ligands activating RIG-I during IAV infection remain underexplored. Other than full-length viral genomes, cellular constraints that impede ongoing viral replication likely potentiate an erroneous viral polymerase generating aberrant viral RNA species with RIG-I activating potential. Here we interrogate the origins of RIG-I activating viral RNA under two such constraints. Using chemical inhibitors that inhibit continuous viral protein synthesis, we identify the incoming but not de novo synthesized viral defective interfering (DI) genomes contributing to RIG-I activation. In comparison, deprivation of viral nucleoprotein (NP), the key RNA chain elongation factor for the viral polymerase, leads to the production of aberrant viral RNA species activating RIG-I; however, their nature is likely to be distinct from DI RNA. Moreover, RIG-I activation in response to NP deprivation is not adversely affected by expression of the nuclear export protein (NEP), which diminishes the generation of a major subset of aberrant viral RNA but facilitates the accumulation of small viral RNA (svRNA). Overall, our results indicate the existence of fundamentally different mechanisms of RIG-I activation under cellular constraints that impede ongoing IAV replication.

    IMPORTANCE The induction of an IFN response by IAV is mainly mediated by the RNA sensor RIG-I. The physiological RIG-I ligands produced during IAV infection are not fully elucidated. Cellular constraints leading to the inhibition of ongoing viral replication likely potentiate an erroneous viral polymerase producing aberrant viral RNA species activating RIG-I. Here we demonstrate that RIG-I activation during chemical inhibition of continuous viral protein synthesis is attributable to the incoming DI genomes. Erroneous viral replication driven by NP deprivation promotes the generation of RIG-I activating aberrant viral RNA, but their nature is likely to be distinct from DI RNA. Our results thus reveal distinct mechanisms of RIG-I activation by IAV under cellular constraints impeding ongoing viral replication. A better understanding of RIG-I sensing of IAV infection provides insight into the development of novel interventions to combat influenza infection.

  • The glucocorticoid receptor (GR) stimulates Herpes Simplex Virus 1 productive infection, in part because the infected cell protein 0 (ICP0) promoter is cooperatively transactivated by the GR and Kruüppel-like transcription factor 15 [Virus-Cell Interactions]

  • Following acute infection, herpes simplex virus 1 (HSV-1) establishes life-long latency in neurons. Physical, emotional, and chemical stress are linked to increasing the incidence of reactivation from latency, but the mechanism of action is not well understood. In general, stress increases corticosteroid levels leading to activation of the glucocorticoid receptor (GR), a pioneer transcription factor. Consequently, we hypothesized that stress-mediated activation of the GR can stimulate productive infection and viral gene expression. New studies demonstrated that the GR-specific antagonist (CORT-108297) significantly reduced HSV-1 productive infection in mouse neuroblastoma cells (Neuro-2A). Additional studies demonstrated that the activated GR and Krüppel-like transcription factor 15 (KLF15) cooperatively transactivated the infected cell protein 0 (ICP0) promoter, a crucial viral regulatory protein. Interestingly, the synthetic corticosteroid dexamethasone and GR or KLF15 alone had little effect on ICP0 promoter activity in transfected Neuro-2A or Vero cells. Chromatin immunoprecipitation (ChIP) studies revealed the GR and KLF15 occupied ICP0 promoter sequences important for trans-activation at 2 and 4 hours after infection: however, binding was not readily detected at 8 or 16 hours after infection. Similar results were obtained in cells transfected with the full-length ICP0 promoter. ICP0 promoter sequences lack a consensus "whole" GR response element (GRE), but contain putative 1/2 GREs that were important for dexamethasone induced promoter activity. The activated GR stimulates expression of, and interacts with KLF15; consequently, these data suggest KLF15 and the GR form a feed-forward loop that can activate viral gene expression and productive infection following stressful stimuli.

    IMPORTANCE The ability of herpes simplex virus 1 (HSV-1) to periodically reactivate from latency results in virus transmission and recurrent disease. The incidence of reactivation from latency is increased by chronic or acute stress. Stress increases corticosteroid levels, which bind and activate the glucocorticoid receptor (GR). Since GR activation is an immediate early response to stress, we tested whether the GR influences productive infection and the promoter that drives infected cell protein 0 (ICP0) expression. Pretreatment of cells with the GR-specific antagonist (CORT-108297) significantly reduced virus replication. Although the GR had little effect on ICP0 promoter activity alone, the Krüppel-like transcription factor 15 (KLF15) cooperated with the GR to stimulate promoter activity in transfected cells. In transfected or infected cells, the GR and KLF15 occupied ICP0 sequences important for transactivation. Collectively, these studies provide insight into how stress can directly stimulate productive infection and viral gene expression.

  • Baculovirus per os Infectivity Factor Complex: Components and Assembly [Structure and Assembly]

  • Baculovirus entry into insect midgut cells is dependent on a multiprotein complex of per os infectivity factors (PIFs) on the envelope of occlusion derived virions (ODVs). The structure and assembly of the PIF complex is largely unknown. To reveal the complete members of the complex, a combination of blue native polyacrylamide gel electrophoresis, liquid chromatography-tandem mass spectrometry, and Western blots were conducted on three different baculoviruses. The results showed that the PIF complex is ~500-kDa in size consisting of nine PIFs including a newly discovered member (PIF9). To decipher the assembly process, each pif was knockout from the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome individually using synthetic baculovirus technology and the impact on PIF complex formation was investigated. Deletion of pif8 resulted in the formation of a ~400 kDa subcomplex. Deletion of pif0, 4, 6, 7, or 9 resulted in a subcomplex of ~230 kDa, but deletion of pif1, 2 or 3 abolished formation of any complex. Taken together, a core-complex of ~230-kDa consisting of PIF1, 2 and 3 was identified. This revised the previous knowledge that the core-complex was about 170 kDa and contained PIF1 to 4. Analysis of the PIF complex in cellular fractions suggested that it is assembled in the cytoplasm before being transported to the nucleus and subsequent incorporation into the envelope of ODV. Only the full complex, and not the subcomplexes, is resistant to proteolytic attack, indicating the essentiality of correct complex assembly for oral infection.

    IMPORTANCE Entry of baculovirus in host insects is mediated by a per os infectivity factor (PIF) complex on the envelope of occlusion-derived virus (ODV). Knowledge of the composition and structure of the PIF complex is fundamental to understanding its mode of action. By using multiple approaches, we provide here a complete list of proteins (nine) in the PIF complex. Different from previous knowledge in the field, the core-complex is revised to ~230 kDa in size and consisting of PIF1-3 but not PIF4. Interestingly, our results suggest that the PIF complex is formed in the cytoplasm prior to its transport to the nucleus and subsequent incorporation into ODV. Only the full complex is resistant to proteolytic degradation in insect midgut implying the critical role of the entire complex. These findings provide the baseline for future studies on the ODV entry mechanism mediated by the multiprotein complex.

  • Structural Basis of Nanobodies Targeting the Prototype Norovirus [Structure and Assembly]

  • Human norovirus infections are a major disease burden. In this study, we analyzed three new norovirus-specific Nanobodies that interacted with the prototype human norovirus (i.e., GI.1). We showed that the Nanobodies bound on the side (Nano-7 and Nano-62) and top (Nano-94) of the capsid-protruding (P) domain using X-ray crystallography. Nano-7 and Nano-62 bound at a similar region on the P domain, but the orientation of these two Nanobodies clashed with the shell (S) domain and neighboring P domains on intact particles. This finding suggested that the P domains on the particles should shift in order for Nano-7 and Nano-62 to bind to intact particles. Interestingly, both Nano-7 and Nano-94 were capable of blocking norovirus virus-like particles (VLPs) from binding to histo-blood group antigens (HBGAs), which are important co-factors for norovirus infection. Previously, we showed that the GI.1 HBGA pocket could be blocked with the soluble human milk oligosaccharide 2-fucosyllactose (2rrsquo;FL). In this current study, we showed that a combined treatment of Nano-7 or Nano-94 with 2rrsquo;FL enhanced the blocking potential with an additive (Nano-7) or synergistic effect (Nano-94). We also found that GII Nanobodies with 2rrsquo;FL also enhanced the inhibition. The Nanobody inhibition likely occurred by different mechanisms, including particle aggregation or particle disassembly, whereas the 2rrsquo;FL blocked the HBGA binding site. Overall, these new data showed that the positive effect of the addition of 2rrsquo;FL was not limited to a single mode of action of Nanobodies or to a single norovirus genogroup.

    IMPORTANCE The discovery of vulnerable regions on norovirus particles is instrumental in the development of effective inhibitors, particularly for GI noroviruses that are genetically diverse. Analysis of these GI.1-specific Nanobodies has shown that similar to GII norovirus particles, the GI particles have vulnerable regions. The only known co-factor region, the HBGA binding pocket, represents the main target for inhibition. With a combination treatment, i.e., addition of Nano-7 or Nano-94 with 2rrsquo;FL, the effect of inhibition was increased. Therefore combination drug treatments might offer a better approach to combat norovirus infections, especially since the GI genotypes are highly diverse, are continually changing the capsid landscape, and few conserved epitopes have so far been identified.

  • HIV subtype and Nef-mediated immune evasion function correlate with viral reservoir size in early-treated individuals [Pathogenesis and Immunity]

  • The HIV accessory protein Nef modulates key immune evasion and pathogenic functions and its encoding gene region exhibits high sequence diversity. Given the recent identification of early HIV-specific adaptive immune responses as novel correlates of HIV reservoir size, we hypothesized that viral factors that facilitate evasion of such responses - namely, Nef genetic and functional diversity - might also influence reservoir establishment and/or persistence. We isolated baseline plasma HIV RNA-derived nef clones from 30 acute/early-infected individuals who participated in a clinical trial of early cART (llt;6 months following infection) and assessed each Nef clone's ability to downregulate CD4 and HLA class I in vitro. We then explored the relationships between baseline clinical, immunologic and virologic characteristics, and HIV reservoir size measured 48 weeks following initiation of suppressive cART (where reservoir size was quantified in terms of proviral DNA loads as well as levels of replication-competent HIV in CD4+ T-cells). Maximal within-host Nef-mediated downregulation of HLA, but not CD4, correlated positively with post-cART proviral DNA levels (Spearman's R=0.61; p=0.0004) and replication-competent reservoir sizes (Spearman's R=0.36; p=0.056) in univariable analyses. Furthermore, Nef-mediated HLA downregulation function was retained in final multivariable models adjusting for established clinical and immunologic correlates of reservoir size. Finally, HIV subtype B infected persons (N=25) harbored significantly larger viral reservoirs compared to non-B infected persons (2 CRF01_AE and 3 subtype G infections). Our results highlight a potentially important role of viral factors - in particular HIV subtype and accessory protein function - in modulating viral reservoir establishment and persistence.

    IMPORTANCE While combination antiretroviral therapies (cART) have transformed HIV into a chronic manageable condition, they do not act upon the latent HIV reservoir and are therefore not curative. As HIV cure or remission should be more readily achievable in individuals with smaller HIV reservoirs, achieving a deeper understanding of clinical, immunologic and virologic determinants of reservoir size is critical to eradication efforts. We performed a post-hoc analysis of 30 participants of a clinical trial of early cART who had previously been assessed in detail for their clinical, immunologic and reservoir size characteristics. We observed that HIV subtype and autologous Nef-mediated HLA downregulation function correlated with viral reservoir size measured approximately one year post-cART initiation. Our findings highlight virologic characteristics - both genetic and functional - as possible novel determinants of HIV reservoir establishment and persistence.

  • In vitro and in vivo characterization of novel neuraminidase substitutions in influenza A(H1N1)pdm09 identified using Laninamivir-mediated in vitro selection [Vaccines and Antiviral Agents]

  • Neuraminidase inhibitors (NAIs) are widely used antiviral drugs for the treatment of humans with influenza virus infections. There have been widespread reports of NAI resistance among seasonal A(H1N1) viruses, and most have been identified in oseltamivir-exposed patients or those treated with other NAIs. Thus, monitoring and identifying NA markers conferring resistance to NAIsmmdash;particularly newly introduced treatmentsmmdash;is critical to manage viral infections. Therefore, we screened and identified substitutions conferring resistance to laninamivir by enriching random mutations in the NA gene of the 2009 pandemic influenza [A(H1N1)pdm09] followed by deep sequencing of the laninamivir-selected variants. After the generation of single mutants possessing each identified mutation, two A(H1N1)pdm09 recombinants possessing novel NA gene substitutions (ie, D199E and P458T) were shown to confer resistance to more than one NAI. Of note, mutants possessing P458Tmmdash;which is located outside of the catalytic or framework residue of the NA active sitemmdash;exhibited highly reduced inhibition by all four approved NAIs. Using MDCK cells, we observed that the in vitro viral replication of the two recombinants was lower compared with WT. Additionally, in infected mice, decreased mortality and/or mean lung viral titers were observed in mutants compared with WT. Reverse mutations to WT were observed in the lung homogenate samples from D199E-infected mice after 3 serial passages. Overall, the novel NA substitutions identified which could possibly emerge in influenza A(H1N1)pdm09 during laninamivir therapy and have altered NAI susceptibility, but the compromised in vitro/in vivo viral fitness may limit viral spreading.

    IMPORTANCE With the widespread emergence of NAI-resistant influenza virus strains, continuous monitoring of mutations that confer antiviral resistance is needed. Laninamivir is the most recently approved NAIs in several countries; little data exists related to the in vitro selection of viral mutations conferring resistance to laninamivir. Thus, we screened and identified substitutions conferring resistance to laninamivir by random mutagenesis in the NA gene of the 2009 pandemic influenza [A(H1N1)pdm09] strain followed by deep sequencing of the laninamivir-selected variants. We found several novel substitutions in NA (D199E and P458T) in an A(H1N1)pdm09 background which demonstrated resistance to NAIs and which have an impact on viral fitness. Our study highlights the importance of continued surveillance for potential antiviral-resistant variants and the development of alternative therapeutics.

  • High permissiveness for genetic exchanges between enteroviruses of species A, including enterovirus 71, favours evolution through intertypic recombination in Madagascar [Genetic Diversity and Evolution]

  • Human enteroviruses of species A (EV-A) are the leading cause of hand-foot-and-mouth disease (HFMD). EV-A71 is frequently implicated in HFMD outbreaks and can also cause severe neurological manifestations. We investigated the molecular epidemiological processes at work and the contribution of genetic recombination to the evolutionary history of EV-A in Madagascar, focusing on the recently described EV-A71 genogroup F in particular. Twenty-three EV-A isolates, mostly collected in 2011 from healthy children living in various districts of Madagascar, were characterised by whole-genome sequencing. Eight different types were identified, highlighting the local circulation and diversity of EV-A. Comparative genome analysis revealed evidence of frequent recent intra- and intertypic genetic exchanges between the non-capsid sequences of Madagascan EV-A isolates. The three EV-A71 isolates had different evolutionary histories in terms of recombination, with one isolate displaying a mosaic genome resulting from recent genetic exchanges with Madagascan coxsackieviruses A7 and possibly -A5 and -A10 or common ancestors. The engineering and characterisation of recombinants generated from progenitors belonging to different EV-A types or EV-A71 genogroups with distantly related non-structural sequences indicated a high level of permissiveness for intertypic genetic exchange in EV-A. This permissiveness suggests that the primary viral functions associated with the non-structural sequences have been highly conserved, through the diversification and evolution of the EV-A species. No outbreak of disease due to EV-A has yet been reported in Madagascar, but the diversity, circulation and evolution of these viruses justify surveillance of EV-A circulation and HFMD cases, to prevent possible outbreaks due to emerging strains.

    IMPORTANCE Human enteroviruses of species A (EV-A), including EV-A71, are the leading cause of hand-foot-and-mouth disease (HFMD), and may also cause severe neurological manifestations. We investigated the circulation and molecular evolution of EV-A in Madagascar, focusing particularly on the recently described EV-A71 genogroup F. Eight different types, mostly collected in 2011, were identified, highlighting the local circulation and diversity of EV-A. Comparative genome analysis revealed evidence of frequent genetic exchanges between the different types of isolates. The three EV-A71 isolates had different evolutionary histories in terms of recombination. The engineering and characterisation of recombinants involving progenitors belonging to different EV-A types indicated a high degree of permissiveness for genetic exchange in EV-A. No outbreak of disease due to EV-A has yet been reported in Madagascar, but the diversity, circulation and evolution of these viruses justify the surveillance of EV-A circulation, to prevent possible HFMD outbreaks due to emerging strains.

  • Peroxiredoxin 1, a novel HBx-interacting protein, interacts with Exosc5 and negatively regulates HBV propagation through degradation of HBV RNA. [Virus-Cell Interactions]

  • Hepatitis B virus (HBV) infection is a major risk factor for the development of chronic liver diseases, including cirrhosis and hepatocellular carcinoma (HCC). A growing body of evidence suggests that HBV X protein (HBx) plays a crucial role in viral replication and HCC development. Here we identified peroxiredoxin 1 (Prdx1), a cellular hydrogen peroxide scavenger, as a novel HBx-interacting protein. Co-immunoprecipitation analysis coupled with site-directed mutagenesis revealed that the region from amino acid 17 to 20 of the HBx, particularly HBx Cys17, is responsible for the interaction with Prdx1. Knockdown of Prdx1 by siRNA significantly increased the levels of intracellular HBV RNA, HBV antigens and extracellular HBV DNA, whereas knockdown of Prdx1 did not increase the activities of HBV core, enhancer I (Enh1)/X, preS1 and preS2/S promoters. Kinetic analysis of HBV RNA showed that knockdown of Prdx1 inhibited HBV RNA decay, suggesting that Prdx1 reduces HBV RNA levels posttranscriptionally. The RNA co-immunoprecipitation assay revealed that Prdx1 interacted with HBV RNA. The exosome component 5 (Exosc5), a member of the RNA exosome complexes, was co-immunoprecipitated with Prdx1, suggesting its role in regulation of HBV RNA stability. Taken together, these results suggest that Prdx1 and Exosc5 play crucial roles in host defense mechanisms against HBV infection.

    IMPORTANCE HBV infection is a major global health problem. HBx plays important roles in HBV replication and viral carcinogenesis through its interaction with host factors. In this study, we identified Prdx1 as a novel HBx-binding protein. We provide evidence suggesting that Prdx1 promotes HBV RNA decay through interaction with HBV RNA and Exosc5, leading to down-regulation of HBV RNA. These results suggest that Prdx1 negatively regulates HBV propagation. Our findings may shed new light on the roles of Prdx1 and Exosc5 in host defense mechanisms in HBV infection.

  • The virulence of different Vaccinia virus strains is directly proportional to their ability in down modulating specific cell-mediated immune compartments in vivo. [Pathogenesis and Immunity]

  • Vaccinia virus is a notorious virus for a number of scientific reasons; however, most of its notoriety comes from the fact it was used as vaccine against smallpox, being ultimately responsible for the eradication of that disease. Nonetheless, many different Vaccinia virusrrsquo; strains have been obtained over the years, some suitable to be used as vaccines whereas others are virulent and unsuitable for this purpose. Interestingly, different vaccinia strains elicit different immune responses in vivo, and this is a direct result of the genomic differences amongst strains. In order to evaluate the net result of virus-encoded immune evasion strategies by vaccinia viruses, we compared anti-viral immune responses in mice intranasally infected by the highly attenuated and non-replicative MVA strain; the attenuated and replicative Lister strain; or the virulent WR strain. Overall, cell responses elicited upon WR infections are down modulated when compared to MVA and Lister infections, especially in determined cell compartments such as macrophages/monocytes and T-CD4+. CD4+ T-cells are not only diminished in WR-infected mice, but they are also less activated, as evaluated by the expression of co-stimulatory molecules such as CD25, CD212 and CD28, and by the production of cytokines including TNFaalpha;, IFN , IL-4 and IL-10. On the other hand, MVA-infections are able to induce strong T-cell responses in mice, whereas Lister infections consistently induced intermediary responses between WR and MVA. Together, our results support a model in which the virulence of a VACV strain is proportional to their potential to down modulate the hostrrsquo;s immune responses.

    IMPORTANCE: Vaccinia virus was used as vaccine against smallpox and was instrumental in the successful eradication of that disease. Although smallpox vaccination is no longer in place in the overall population, the use of Vaccinia virus in the development of viral vector-based vaccines has become popular. Nonetheless, different vaccinia strains are known and induce different immune responses. To look into that, we compared immune responses triggered by mice infections with the non-replicative MVA strain; the attenuated Lister strain; or the virulent WR strain. We observed that the WR strain was capable of down modulating mice cell responses whereas the highly attenuated MVA strain induced high levels of cell-mediated immunity. Infections by the intermediately attenuated Lister strain induced cell responses that were intermediary between WR and MVA. We propose that the virulence of a Vaccinia virus strain is directly proportional to its ability in down modulating specific compartments of antiviral cell responses.

  • Endogenous viral elements are widespread in arthropod genomes and commonly give rise to piRNAs [Genetic Diversity and Evolution]

  • Arthropod genomes contain sequences derived from integrations of DNA and non-retroviral RNA viruses. These sequences, known as endogenous viral elements (EVEs), have been acquired over the course of evolution and have been proposed to serve as a record of past viral infections. Recent evidence indicates that EVEs can function as templates for the biogenesis of PIWI-interacting RNAs (piRNAs) in some mosquito species and cell lines, raising the possibility that EVEs may serve as a source of immunological memory in these organisms. However, whether piRNAs are derived from EVEs or serve an antiviral function in other arthropod species is unknown. Here we used publically available genome assemblies and small RNA sequencing datasets to characterize the repertoire and function of EVEs across 48 arthropod genomes. We found that EVEs are widespread in arthropod genomes and primarily correspond to unclassified ssRNA viruses and viruses belonging to the Rhabdoviridae and Parvoviridae families. Additionally, EVEs were enriched in piRNA clusters in a majority of species and we found that production of primary piRNAs from EVEs is common, particularly for EVEs located within piRNA clusters. While the abundance of EVEs within arthropod genomes and the frequency with which EVEs give rise to primary piRNAs generally supports the hypothesis that EVEs contribute to an antiviral response via the piRNA pathway, limited nucleotide identity between currently described viruses and EVEs identified here likely limits the extent to which this process plays a role during infection with known viruses in the arthropod species analyzed.

    IMPORTANCE Our results greatly expand knowledge of EVE abundance, diversity, and function in an exceptionally wide range of arthropod species. We found that while previous findings in mosquitoes regarding the potential of EVEs to serve as sources of immunological memory via the piRNA pathway may be generalized to other arthropod species, speculation regarding the antiviral function of EVE-derived piRNAs should take into context the fact that EVEs are, in the vast majority of cases, not similar enough to currently described viruses at the nucleotide level to serve as sources of antiviral piRNAs against them.

  • M segment-based minigenomes and virus-like particle assays as an approach to assess the potential of tick-borne Phlebovirus genome reassortment. [Genome Replication and Regulation of Viral Gene Expression]

  • Bunyaviruses have a tripartite negative-sense RNA genome. Due to the segmented nature of these viruses, if two closely related viruses co-infect the same host or vector cell, it is possible that RNA segments from either of the two parental viruses are incorporated into progeny virions to give reassortant viruses. Little is known about the ability of tick-borne phleboviruses to reassort. The present study describes the development of minigenome assays for the tick-borne viruses Uukuniemi phlebovirus (UUKV) and Heartland phlebovirus (HRTV). We used these minigenome assays in conjunction with the existing minigenome system of SFTS phlebovirus (SFTSV) to assess the ability of viral N and L proteins to recognize, transcribe and replicate the M segment-based minigenome of a heterologous virus. The highest minigenome activity was detected with the M segment-based minigenome of cognate viruses. However, our findings indicate that several combinations utilizing N and L proteins of heterologous viruses resulted in M segment minigenome activity. This suggests that the M segment untranslated regions (UTRs) are recognised as a functional promoter of transcription and replication by the N and L proteins of related viruses. Further, virus-like particle assays demonstrated that HRTV glycoproteins can package UUKV and SFTSV S and L segment-based minigenomes. Taken together, these results suggest that co-infection of these viruses could lead to the generation of viable reassortant progeny. Thus, the tools developed herein could aid in understanding the role of genome reassortment in the evolution of these emerging pathogens under an experimental setting.

    IMPORTANCE In recent years, there has been a large expansion in the number of tick-borne viruses emerging that are assigned to the Phlebovirus genus. Bunyaviruses have a tripartite segmented genome and infection of the same host cell by two closely related bunyaviruses can in theory result in eight potential progeny viruses, with different genome segment combinations. We used genome analogues expressing reporter genes to assess the ability of phlebovirus nucleocapsid protein and RNA-dependent RNA polymerase to recognize the untranslated region of a genome segment of related phleboviruses, and virus-like particle assays to assess whether viral glycoproteins can package genome analogues of related phleboviruses. Our results provide strong evidence that these emerging pathogens could reassort their genomes if they were to meet in nature in an infected host or vector. This reassortment process can result in viruses with new pathogenic properties.

  • RESISTANCE TO SECOND-GENERATION HIV-1 MATURATION INHIBITORS [Vaccines and Antiviral Agents]

  • A betulinic acid-based compound, bevirimat (BVM), inhibits HIV-1 maturation by blocking a late step in protease-mediated Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. Previous studies showed that mutations conferring resistance to BVM cluster around the CA-SP1 cleavage site. Single amino acid polymorphisms in the SP1 region of Gag and the C-terminus of CA reduced HIV-1 susceptibility to BVM, leading to the discontinuation of BVMrrsquo;s clinical development. We recently reported a series of "second-generation" BVM analogs that display markedly improved potency and breadth of activity relative to the parent molecule. Here, we demonstrate that viral clones bearing BVM-resistance mutations near the C-terminus of CA are potently inhibited by second-generation BVM analogs. We performed de novo selection experiments to identify mutations that confer resistance to these novel compounds. Selection experiments with subtype B HIV-1 identified an Ala-to-Val mutation at SP1 residue 1 and a Pro-to-Ala mutation at CA residue 157 within the major homology region (MHR). In selection experiments with subtype C HIV-1, we identified mutations at CA residue 230 (CA-V230M), and SP1 residues 1 (SP1-A1V), 5 (SP1-S5N), and 10 (SP1-G10R). The positions at which resistance mutations arose are highly conserved across multiple subtypes of HIV-1. We demonstrate that the mutations confer modest to high-level maturation inhibitor resistance. In most cases, resistance was not associated with a detectable increase in the kinetics of CA-SP1 processing. These results identify mutations that confer resistance to second-generation maturation inhibitors, and provide novel insights into the mechanism of resistance.

    IMPORTANCE HIV-1 maturation inhibitors are a class of small-molecule compounds that block a late step in the viral protease-mediated processing of the Gag polyprotein precursor, the viral protein responsible for the formation of virus particles. The first-in-class HIV-1 maturation inhibitor, bevirimat, was highly effective in blocking HIV-1 replication but its activity was compromised by naturally occurring sequence polymorphisms within Gag. Recently developed bevirimat analogs, referred to as "second-generation" maturation inhibitors, overcome this issue. To understand more about how these second-generation compounds block HIV-1 maturation, here we selected for HIV-1 mutants that are resistant to these compounds. Selections were performed in the context of two different subtypes of HIV-1. We identified a small set of mutations at highly conserved positions within the capsid and spacer peptide 1 domains of Gag that confer resistance. Identification and analysis of these maturation inhibitor-resistant mutants provides insights into the mechanisms of resistance to these compounds.

  • Flexibility in vitro of amino acid 226 in the receptor-binding site of an H9 subtype influenza A virus and its effect in vivo on virus replication, tropism, and transmission. [Virus-Cell Interactions]

  • Influenza A viruses remain a significant public health threat causing more than 300,000 hospitalizations in the United states during 2015-2016 season alone. While only few IAVs of avian origin have been associated with human infections, the ability of these viruses to cause zoonotic infections further increases the public health risk of influenza. Of these, H9N2 viruses in Asia are of particular importance as they have contributed internal gene segments to other emerging zoonotic IAVs. Notably, recent H9N2 viruses have acquired molecular markers that allow for a transition from "avian-like" to "human-like" terminal sialic acid (SA) receptor recognition via a single amino acid change at position 226 (H3 numbering), from glutamine (Q226) to leucine (L226), within the HA receptor-binding site (RBS). We sought to determine the plasticity of amino acid 226 and the biological effects of alternative amino acids on variant viruses. We created a library of viruses with the potential of having any of the 20 amino acids at position 226 on a prototypic H9 HA subtype IAV. We isolated H9 viruses that carried naturally occurring amino acids, variants found in other subtypes and variants not found in any subtype at position 226. Fitness studies in quails revealed that some natural amino acids conferred an in vivo replication advantage. This study shows the flexibility of position 226 of the HA of H9 influenza viruses and the resulting effect of single amino acid changes on the phenotype of variants in vivo and in vitro.

    Importance

    A single amino acid change at position 226 in the hemagglutinin (HA) from glutamine (Q) to leucine (L) has been shown to play a key role in receptor specificity switching in various influenza HA subtypes, including H9. We tested the flexibility of amino acid usage and determine the effects of such changes. The results reveal that amino acids other than L226 and Q226 are well tolerated and that some amino acids allow for the recognition of both avian and human influenza receptors in the absence of other changes. Our results can inform better avian influenza surveillance efforts as well as contribute to rational vaccine design and improve structural molecular dynamics algorithms.

  • Interferon-induced Transmembrane Protein 1 restricts replication of virus that enter cells via the plasma membrane. [Virus-Cell Interactions]

  • The acute anti-viral response is mediated by a family of interferon stimulated genes (ISG), providing cell-intrinsic immunity. Mutations in genes encoding these proteins are often associated with increased susceptibility to viral infections. One family of ISGs with anti-viral function are the interferon-inducible transmembrane proteins (IFITM) of which IFITM3 has been studied extensively. By contrast, IFITM1 has not been studied in detail. Since IFITM1 can localise to the plasma membrane, we investigated its function with a range of enveloped viruses thought to infect cells by fusion with the plasma membrane. Overexpression of IFITM1 prevented infection by a number of Paramyxoviridae and Pneumoviridae, including Respiratory Syncytial Virus (RSV), mumps virus and human metapneumovirus (HMPV). IFITM1 also restricted infection with an enveloped DNA virus that can enter via the plasma membrane, herpes simplex virus 1 (HSV-1). To test the importance of plasma membrane localisation for IFITM1 function, we identified blocks of amino acids in the conserved intracellular loop (CIL) domain that altered the subcellular localisation of the protein and reduced anti-viral activity. Screening published datasets, twelve rare non-synonymous SNPs were identified in human IFITM1, some of which are in the CIL domain. Using an Ifitm1-/- knock-out mouse we show that RSV infection was more severe, thereby extending the range of viruses restricted in vivo by IFITM proteins and suggesting overall that IFITM1 is broadly anti-viral and this anti-viral function is associated with cell surface localisation.

    IMPORTANCE Host susceptibility to viral infection is multifactorial, but early control of viruses not previously encountered is predominantly mediated by the interferon stimulated gene (ISG) family. There are upwards of 300 of these genes, the majority of which do not have a clearly defined function or mechanism of action. The cellular location of these proteins may have an important effect on their function. One ISG located at the plasma membrane is Interferon inducible transmembrane protein 1 (IFITM1). Here we demonstrate that IFITM1 can restrict a range of viruses that enter via the plasma membrane. Mutant IFITM1 proteins that were unable to localise to the plasma membrane did not restrict viral infection. We also observed for the first time that IFITM1 plays a role in vivo, Ifitm1-/- knock-out mice were more susceptible to viral lung infection. This data contributes to our understanding of how ISG prevent viral infections.

  • Comparative analysis of gammaherpesvirus circRNA repertoires: conserved and unique viral circRNAs. [Genetic Diversity and Evolution]

  • Recent studies have identified circular RNAs (circRNAs) expressed from the Epstein Barr virus (EBV) and Kaposirrsquo;s sarcoma herpesvirus (KSHV) human DNA tumor viruses. To gain initial insights into the potential relevance of EBV circRNAs in virus biology and disease, we assessed the circRNAome of the interspecies homologue, rhesus macaque lymphocryptovirus (rLCV) in a naturally occurring lymphoma from a simian immunodeficiency (SIV) virus infected rhesus macaque. This analysis revealed rLCV orthologs of the latency-associated EBV circular RNAs, circRPMS1_E4_E3a and circEBNA_U. Also identified in two samples displaying unusually high lytic gene expression was a novel rLCV circRNA that contains both conserved and rLCV-specific RPMS1 exons and whose backsplice junctions flank an rLCV lytic origin of replication (OriLyt). Analysis of a lytic infection model for the murid herpesvirus 68 (MHV68) rhadinovirus identified a cluster of circRNAs near an MHV68 lytic origin of replication with the most abundant of these, circM11_ORF69 spanning the OriLyt. Lastly, analysis of KSHV latency and reactivation models revealed the latency associated circRNA originating from the vIRF4 gene as the predominant viral circRNA. Together, this study broadens our appreciation for circRNA repertoires in the lymphocryptovirus and rhadinovirus genera of gammaherpesviruses and provides evolutionary support for viral circRNA functions in latency and viral replication.

    IMPORTANCE

    Infection with oncogenic gammaherpesviruses leads to long-term viral persistence through a dynamic interplay between the virus and the host immune system. Critical for remodeling of the host cell environment after the immune responses are viral non-coding RNAs that modulate host signaling pathways without attracting adaptive immune recognition. Despite the importance of non-coding RNAs in persistent infection, the circRNA class of non-coding RNAs has only recently been identified in gammaherpesviruses. Accordingly, their roles in virus infection and associated oncogenesis are unknown. Here we report evolutionary conservation of EBV encoded circRNAs by assessing the circRNAome in rLCV infected lymphomas from an SIV infected rhesus macaque and we report latent and lytic circRNAs from KSHV and MHV68. These experiments demonstrate utilization of the circular RNA class of RNAs across 4 members of the gammaherpesvirus subfamily and they identify orthologs and potential homoplastic circRNAs, implying conserved circRNA functions in virus biology and associated malignancies.

  • Glycoprotein K8.1A of Kaposi's sarcoma-associated herpesvirus is a critical B cell tropism determinant, independent of its heparan sulfate binding activity [Virus-Cell Interactions]

  • B lymphocytes are the major cellular reservoir in individuals infected with Kaposirrsquo;s sarcoma-associated herpesvirus (KSHV), and the virus is etiologically linked to two B cell lymphoproliferative disorders. We previously described the MC116 human B cell line as a KSHV-susceptible model to overcome the paradoxical refractoriness of B cell lines to experimental KSHV infection. Here, using monoclonal antibody inhibition and a deletion mutant virus, we demonstrate that the KSHV virion glycoprotein K8.1A is critical for infection of MC116 as well as tonsillar B cells; by contrast, we confirm previous reports on the dispensability of this glycoprotein for infection of primary endothelial cells and other commonly studied non-B cell targets. Surprisingly, we found that the role of K8.1A in B cell infection is independent of its only known biochemical activity of binding to surface heparan sulfate, suggesting the possible involvement of an additional molecular interaction(s). Our finding that K8.1A is a critical determinant for KSHV B cell tropism parallels the importance of proteins encoded by positionally homologous genes for cell tropism of other gammaherpesviruses.

    Importance: Elucidating the molecular mechanisms by which KSHV infects B lymphocytes is critical for understanding how this virus establishes lifelong persistence in infected people, in whom it can cause life-threatening B cell lymphoproliferative disease. Here we show that K8.1A, a KSHV-encoded glycoprotein on the surface of the virus particles, is critical for infection of B cells. This finding stands in marked contrast to previous studies with non-B lymphoid cell types, for which K8.1A is known to be dispensable. We also show that the required function of K8.1A in B cell infection does not involve its binding to cell surface heparan sulfate, the only known biochemical activity of this glycoprotein. The discovery of this critical role of K8.1A in KSHV B cell tropism opens promising new directions toward unraveling the complex mechanisms underlying infection and disease caused by this viral human pathogen.

  • A Recombinant Rabies Virus Expressing the Marburg Virus Glycoprotein is Dependent Upon ADCC for Protection Against Marburg Virus Disease in a Murine Model [Vaccines and Antiviral Agents]

  • Marburg virus (MARV) is a filovirus related to Ebola virus (EBOV) associated with human hemorrhagic disease. Outbreaks are sporadic and severe with a reported case mortality rate upward of 88%. There is currently no antiviral or vaccine available. Given the sporadic nature of outbreaks, vaccines provide the best approach for long-term control of MARV in endemic regions. We have developed an inactivated rabies virus-vectored MARV vaccine (FILORAB3) to protect against Marburg virus disease. Immunogenicity studies in our lab have shown that a Th1-biased seroconversion to both RABV and MARV glycoproteins is beneficial for protection in a preclinical murine model. As such, we adjuvanted FILORAB3 with GLA-SE, a TLR-4 agonist. Across two different BALB/c mouse challenge models, we achieved 92% protection against murine-adapted Marburg virus (ma-MARV). Although our vaccine elicited strong MARV GP antibodies, it did not strongly induce neutralizing antibodies. Through both in vitro and in vivo approaches, we elucidated a critical role for NK cell-dependent antibody-mediated cellular cytotoxicity (ADCC) in vaccine-induced protection. Overall, these findings demonstrated that FILORAB3 is a promising vaccine candidate for Marburg virus disease.

    IMPORTANCE

    Marburg virus (MARV) is a virus similar to Ebola virus and also causes a hemorrhagic disease, which is highly lethal. In contrast to EBOV, only a few vaccines are developed against MARV and researcher do not understand what kind of immune responses are required to protect from MARV. Here we show that antibodies directed against MARV after application of our vaccine protect in an animal system but fail to neutralize the Virus in widely used virus neutralization assay against MARV. This newly discovered activity needs to be more considered when analyzing MARV vaccines or infections.

  • Parvulin 14 and parvulin 17 bind to HBx and cccDNA and upregulate HBV replication from cccDNA to virion in a HBx-dependent manner [Virus-Cell Interactions]

  • The parvulin 14 (Par14) and parvulin 17 (Par17) proteins, which are both encoded by PIN4 gene, play roles in protein folding, chromatin remodeling, DNA binding, ribosome biogenesis, and cell cycle progression. However, the effects of Par14 and Par17 on viral replication have never been explored. In this study, we found that, in the presence of HBx, either Par14 or Par17 could upregulate hepatitis B virus (HBV) replication, whereas in the absence of HBx, neither Par14 nor Par17 had any effect on replication. Overexpression of Par14/Par17 markedly increased formation of covalently closed circular DNA (cccDNA), synthesis of HBV RNA and DNA, and virion secretion. Conversely, PIN4 knockdown significantly decreased HBV replication in HBV-transfected and -infected cells. Co-immunoprecipitation revealed that Par14/Par17 engaged in direct physical interactions with HBx in the cytoplasm, nucleus, and mitochondria, possibly mediated through substrate-binding residues on Par14/Par17 (E46/D74 and E71/D99, respectively) and conserved 19R20P-28R29P motifs on HBx. Furthermore, these interactions enhanced HBx stability, promoted HBx translocation to the nuclear and mitochondrial fractions, and increased HBV replication. Chromatin immunoprecipitation assays revealed that, in the presence of HBx, Par14/Par17 were efficiently recruited to cccDNA and promoted transcriptional activation via specific DNA-binding residues (S19/44). By contrast, in the absence of HBx, Par14/Par17 bound cccDNA only at the basal level and did not promote transcriptional activation. Taken together, our results demonstrate that Par14 and Par17 upregulate HBV RNA transcription and DNA synthesis, thereby increasing the HBV cccDNA level, through formation of the cccDNAnndash;Par14/17nndash;HBx complex.

    IMPORTANCE OF THIS STUDY HBx protein plays an essential regulatory role in HBV replication. We found that substrate-binding residues on the human parvulin peptidylprolyl cis/trans isomerase proteins Par14 and Par17 bound to conserved argininenndash;proline (RP) motifs on HBx in the cytoplasm, nucleus, and mitochondria. The HBxnndash;Par14/Par17 interaction stabilized HBx, promoted its translocation to the nucleus and mitochondria, and stimulated multiple steps of HBV replication, including cccDNA formation, HBV RNA and DNA synthesis, and virion secretion. In addition, in the presence of HBx, Par14 and Par17 proteins bound to cccDNA and promoted its transcriptional activation. Our results suggest that inhibition or knockdown of Par14 and 17 may represent a novel therapeutic option against HBV infection.

  • PF74 Inhibits HIV-1 Integration by Altering The Composition of the Preintegration Complex [Virus-Cell Interactions]

  • The HIV-1 capsid protein (CA) facilitates reverse transcription and nuclear entry of the virus. However, CArrsquo;s role in post-nuclear entry steps remains speculative. We describe a direct link between CA and integration by employing the capsid inhibitor PF74 as a probe coupled with the biochemical analysis of HIV-1 preintegration complexes (PICs) isolated from acutely infected cells. At a low micromolar concentration, PF74 potently inhibited HIV-1 infection without affecting reverse transcription. Surprisingly, PF74 markedly reduced proviral integration owing to inhibition of nuclear entry and/or integration. However, a two-fold reduction in nuclear entry by PF74 did not quantitatively correlate with the level of antiviral activity. Titration of PF74 against the integrase inhibitor raltegravir showed an additive antiviral effect that is dependent on a block at the post-nuclear entry step. PF74rrsquo;s inhibitory effect was not due to the formation of defective viral DNA ends or a delay in integration, suggesting that the compound inhibits PIC-associated integration activity. Unexpectedly, PICs recovered from cells infected in the presence of PF74 exhibited elevated integration activity. PF74rrsquo;s effect on PIC activity is CA-specific as the compound did not increase the integration activity of PICs of a PF74-resistant HIV-1 CA mutant. Sucrose gradient based fractionation studies revealed that PICs assembled in the presence of PF74 contained lower levels of CA, suggesting a negative association between CA and PIC-associated integration activity. Finally, addition of a CA-specific antibody or PF74 inhibited PIC-associated integration activity. Collectively, our results demonstrate that PF74rrsquo;s targeting of PIC-associated CA results in impaired HIV-1 integration.

    IMPORTANCE Antiretroviral therapy (ART) that uses various combinations of small molecule inhibitors has been highly effective in controlling HIV. However, the drugs used in the ART regimen are expensive, cause side effects, and face viral resistance. The HIV-1 CA plays critical roles in the virus life cycle and is an attractive therapeutic target. While currently there is no CA-based therapy, highly potent CA-specific inhibitors are being developed as a new class of antivirals. Efforts to develop a CA-targeted therapy can be aided through a clear understanding of the role of CA in HIV-1 infection. CA is well-established to co-ordinate reverse transcription and nuclear entry of the virus. However the role of CA in post-nuclear entry steps of HIV-1 infection is poorly understood. We show that a CA-specific drug PF74 inhibits HIV-1 integration revealing a novel role of this multifunctional viral protein in a post-nuclear entry step of HIV-1 infection.

  • Positive selection at key residues in the HIV Envelope distinguishes broad and strain-specific plasma neutralizing antibodies [Pathogenesis and Immunity]

  • The development of HIV broadly neutralizing antibodies (bNAbs) has previously been shown to be associated with viral evolution and high levels of genetic diversity in the HIV envelope (Env) glycoprotein. However, few studies have examined Env evolution in those who fail to develop breadth, to assess whether bNAbs result from distinct evolutionary pathways. We compared Env evolution in eight HIV-1 infected participants who developed bNAbs to six donors with similar viral loads who did not develop bNAbs over three years of infection. We focused on Env V1V2 and C3V4 as these are major targets for both strain-specific nAbs and bNAbs. Overall evolutionary rates (ranging from 9.92 x 10nndash;3 to 4.1 x 10nndash;2 substitutions/site/year) and viral diversity (from 1.1% to 6.5%) across Env, and within targeted epitopes, did not distinguish bNAb donors from non-bNAb donors. However, bNAb participants had more positively selected residues within epitopes than those without bNAbs, and several of these were common among bNAb donors. Comparison of the kinetics of strain-specific nAbs and bNAbs indicated that selection pressure at these residues increased with the onset of breadth. These data suggest that highly targeted viral evolution rather than overall envelope diversity, is associated with neutralization breadth. The association of shared, positively selected sites with the onset of breadth highlights the importance of diversity at specific positions in these epitopes for bNAb development, with implications for the development of sequential and cocktail immunization strategies.

    Importance

    Millions of people are still being infected with HIV decades since the first recognition of this virus. Currently, no vaccine is able to elicit bNAbs that will prevent infection by global HIV strains. Several studies have implicated HIV Env diversity in the development of breadth. However, Env evolution in individuals who fail to develop breadth despite mounting potent, strain-specific neutralizing responses has not been well-defined. Using longitudinal neutralization, epitope mapping and sequence data from 14 participants, we found that overall measures of viral diversity were similar in all donors. However, the number of positively selected sites within Env epitopes was higher in bNAb participants than strain-specific donors. We further identifed common sites that were positively selected as bNAbs developed. These data indicate that while viral diversity is required for breadth, this should be highly targeted to specific residues to shape the elicitation of bNAbs by vaccination.

  • Identification and characterization of a poliovirus capsid mutant with enhanced thermal stability [Genetic Diversity and Evolution]

  • Enteric viruses, including poliovirus, are spread by the fecal-oral route. In order to persist and transmit to a new host, enteric virus particles must remain stable once they are in the environment. Environmental stressors such as heat and disinfectants can inactivate virus particles and prevent viral transmission. It has been previously demonstrated that bacteria or bacterial surface glycans can enhance poliovirus virion stability and limit inactivation from heat or bleach. While investigating the mechanisms underlying bacterial-enhanced virion thermal stability, we identified and characterized a poliovirus mutant with increased resistance to heat inactivation. This poliovirus mutant, M132V, harbors a single amino acid change in the VP1 capsid-coding that is sufficient to confer heat resistance, but not bleach resistance. Although the M132V virus was stable in the absence of bacteria or feces at most temperatures, M132V virus was stabilized by feces at very high temperatures. M132V PV had reduced specific infectivity and RNA uncoating compared with WT PV, but viral yields in HeLa cells were similar. In orally-inoculated mice, M132V had a slight fitness cost since fecal titers were lower and 12.5% of fecal viruses reverted to WT. Overall, this work sheds light on factors that influence virion stability and fitness.

    IMPORTANCE

    Viruses spread by the fecal-oral route need to maintain viability in the environment to ensure transmission. Previous work indicated that bacteria and bacterial surface polysaccharides can stabilize viral particles and enhance transmission. To explore factors that influence viral particle stability, we isolated a mutant poliovirus that is heat resistant. This mutant virus does not require feces for stability at most temperatures, but can be stabilized by feces at very high temperatures. Even though the mutant virus is heat resistant, it is susceptible to inactivation by treatment with bleach. This work provides insight into how viral particles maintain infectivity in the environment.

  • Functional and physical interaction between the Arf activator GBF1 and hepatitis C virus NS3 protein [Virus-Cell Interactions]

  • GBF1 has emerged as a host factor required for the genome replication of RNA viruses of different families. During the hepatitis C virus (HCV) life cycle, GBF1 performs a critical function at the onset of genome replication, but is dispensable when the replication is established. To better understand how GBF1 regulates HCV infection, we have looked for interactions between GBF1 and HCV proteins. NS3 was found to interact with GBF1 in yeast two-hybrid, in co-immunoprecipitation and in proximity ligation assays, and to interfere with GBF1 function and alter GBF1 intracellular localization in cells expressing NS3. The interaction was mapped to the Sec7 domain of GBF1 and the protease domain of NS3. A reverse yeast two-hybrid screen to identify mutations altering NS3-GBF1 interaction yielded an NS3 mutant (N77D, Con1 strain) that is non-replicative despite conserved protease activity, and does not interact with GBF1. The mutated residue is exposed at the surface of NS3, suggesting it could be part of the domain of NS3 that interacts with GBF1. The corresponding mutation in strain JFH-1 strain (S77D) produces a similar phenotype. Our results provide evidence for an interaction between NS3 and GBF1 and suggest that an alteration of this interaction is detrimental to HCV genome replication.

    IMPORTANCE Single-stranded, positive-sense RNA viruses rely to a significant extent on host factors to achieve the replication of their genome. GBF1 is such a cellular protein that is required for the replication of several RNA viruses, but its mechanism of action during viral infections is not yet defined. In this study, we investigated potential interactions that GBF1 might engage in with proteins of hepatitis C virus (HCV), a GBF1-dependent virus. We found that GBF1 interacts with NS3, a non-structural protein involved in HCV genome replication, and our results suggest that this interaction is important for GBF1 function during HCV replication. Interestingly, GBF1 interaction with HCV appears different from its interaction with enteroviruses, another group of GBF1-dependent RNA viruses, in keeping with the fact that HCV and enteroviruses use different functions of GBF1.

  • Solution structure, self-assembly and membrane interactions of the matrix protein from Newcastle disease virus at neutral and acidic pH [Structure and Assembly]

  • Newcastle disease virus (NDV) is an enveloped paramyxovirus. The matrix protein of the virus (M-NDV) has an innate propensity to produce virus-like particles being budded from the plasma membrane of the expressing cell without recruiting other viral proteins. The virus predominantly infects the host cell via fusion with the host plasma membrane or, alternatively, can use receptor-mediated endocytic pathways. The question arises: what are the mechanisms supporting such diversity, especially concerning the assembling and membrane binding properties of the virus protein scaffold both in neutral and acidic pH conditions? Here we suggest a novel method of the M-NDV isolation in physiological ionic strength and employ a combination of small-angle X-ray scattering, atomic force microscopy with complimentary structural techniques and membrane interaction measurements to characterize the solution behavior/structure of the protein as well as its binding to lipid membranes at pH 4.0 and pH 7.0. We demonstrate that the minimal structural unit of the protein in solution is a dimer that spontaneously assembles in a neutral milieu into hollow helical oligomers by repeating the protein tetramers. Acidic pH conditions decrease the protein oligomerization state to the individual dimers, tetramers and octamers without changing the density of the protein layer and lipid membrane affinity, thus indicating that the endocytic pathway is a possible facilitator of the NDV entry into a host cell through an enhanced scaffold disintegration.

    Importance

    The matrix protein of the Newcastle disease virus (NDV) is one of the most abundant viral proteins that regulates the formation of progeny virions. NDV is an avian pathogen that impacts the economics of bird-husbandry due to its resulting morbidity and high mortality rates. Moreover, it belongs to Avulavirus subfamily of Paramyxoviridae family of Mononegavirales that include dangerous representatives such as respiratory syncytial virus, human parainfluenza virus, and measles virus. Here we investigate the solution structure and membrane binding properties of this protein both in acidic and at neutral pH to distinguish between possible virus entry pathways and propose a mechanism of assembly of the viral matrix scaffold. This work is fundamental for understanding the mechanisms of viral entry as well as to inform subsequent proposals that the virus maybe act as an adequate template for future drug or vaccine delivery.

  • Tetraspanins: Architects of viral entry and exit platforms [Gem]

  • Host factors render cells susceptible to viral infection. One family of susceptibility factors, the tetraspanin proteins, facilitate enveloped virus entry by promoting virus-cell membrane fusion. They also facilitate viral egress from infected cells. In this article, we discuss recent insights into how tetraspanins assemble viral entry and exit platforms on cell membranes, and we speculate that tetraspanins contribute to non-viral membrane fusions by similar mechanisms.

  • Koala and wombat gammaherpesviruses encode the first known viral nucleoside triphosphate diphosphohydrolase (NTPDase) homologs and are phylogenetically divergent from all known gammaherpesviruses. [Genetic Diversity and Evolution]

  • There is a large taxonomic gap in our understanding of mammalian herpesvirus genetics and evolution corresponding to those herpesviruses that infect marsupials, which diverged from eutherian mammals approximately 150 million years ago (mya). We compare the genomes of two marsupial gammaherpesviruses; Phascolarctid gammaherpesvirus 1 (PhaHV1) and Vombatid gammaherpesvirus 1 (VoHV1), which infect koalas (Phascolartos cinereus) and wombats (Vombatus ursinus), respectively. The core viral genomes were approximately 117 kbp and 110 kbp in length, respectively, sharing 69% nucleotide sequence pair-wise identity. Phylogenetic analyses showed that PhaHV1 and VoHV1 formed a separate branch and may indicate a new gammaherpesvirus genus. The genomes contained 60 predicted ORFs homologous to those in eutherian herpesviruses, and 20 ORFs not yet found in any other herpesvirus. Seven of these ORFs were shared by the two viruses, indicating that they were probably acquired pre-speciation, approximately 30-40 mya. One of these shared genes encodes a putative nucleoside triphosphate diphosphohydrolase (NTPDase). NTPDases are usually found in mammals and higher order eukaryotes, with a very small number found in bacteria. This is the first time an NTPDase has been identified in any viral genome. Interrogation of public transcriptomic datasets from two koalas identified PhaHV1-specific transcripts in multiple host tissues, including transcripts for the novel NTPDase. PhaHV1 ATPase activity was also demonstrated in vitro, suggesting that the encoded NTPDase is functional during viral infection. In mammals, NTPDases are important in downregulation of the inflammatory and immune responses, but the role of the PhaHV1 NTPDase during viral infection remains to be determined.

    IMPORTANCE: The genome sequences of the koala and wombat gammaherpesviruses show that the viruses form a distinct branch, indicative of a novel genus within the Gammaherpesvirinae. Their genomes contain several new ORFs, including ORFs encoding an ST6Gal that is phylogenetically closest to poxvirus and insect homologs, and the first reported viral NTPDase. NTPDases are ubiquitously expressed in mammals, and are also present in several parasitic, fungal and bacterial pathogens. In mammals, these cell-surface localized NTPDases play essential roles in thromboregulation, inflammation and immune suppression. In this study, we demonstrate that the virus-encoded NTPDase is enzymatically active and is transcribed during natural infection of the host. Understanding how these enzymes benefit viruses can help to inform how they may cause disease or evade host immune defenses.

  • Evolution of the Envelope Glycoprotein of HIV-1 Clade B towards Higher Infectious Properties over the Course of the Epidemic [Virus-Cell Interactions]

  • We showed previously that during the HIV/AIDS epidemic the envelope glycoprotein (Env) of HIV-1, and in particular the gp120 subunit, has evolved towards an increasing resistance to neutralizing antibodies at a population level. Here, we considered whether the antigenic evolution of the HIV-1 Env has been associated with modifications of its functional properties, focusing on cell-entry efficacy and interactions with the receptor and co-receptors. We tested the infectivity of a panel of Env-pseudotyped viruses derived from patients infected by subtype B viruses at three periods of the epidemic (1987-1991, 1996-2000, 2006-2010). Pseudotyped viruses harboring Env from patients infected during the most recent period were approximately ten fold more infectious in cell culture than those from patients infected at the beginning of the epidemic. This was associated with faster viral entry kinetics: contemporary viruses entered target cells approximately twice as fast as historical viruses. Contemporary viruses were also twice as resistant as historical viruses to the fusion inhibitor enfuvirtide. Resistance to enfuvirtide correlated with a resistance to CCR5 antagonists, suggesting that contemporary viruses expanded their CCR5 usage efficiency. Viruses were equally captured by DC-SIGN but after binding to DC-SIGN, contemporary viruses infected target cells more efficiently than historical viruses. Thus, we report evidence that the infectious properties of the envelope glycoprotein of HIV-1 increased during the course of the epidemic. It is plausible that these changes have affected viral fitness during the transmission process and might have contributed to an increasing virulence of HIV-1.

    IMPORTANCE

    Following primary infection by HIV-1, neutralizing antibodies (NAbs) exert a selective pressure on the HIV-1 envelope glycoprotein (Env) driving the evolution of the viral population. Previous studies suggested that, as a consequence, Env has evolved at the HIV species level since the start of the epidemic so as to display greater resistance to NAbs. Here, we investigated whether the antigenic evolution of the HIV-1 Env has been associated with modifications of its functional properties, focusing on cell-entry efficacy and interactions with the receptor and co-receptors. Our data provide evidence that infectious properties of the HIV-1 Env has increased during the course of the epidemic. These changes may have contributing to increasing virulence of HIV-1 and an optimization of transmission between individuals.

  • Human Norovirus Neutralized by a Monoclonal Antibody Targeting the HBGA Pocket [Structure and Assembly]

  • Temporal changes in the GII.4 human norovirus capsid sequences occasionally result in the emergence of genetic variants capable of causing new epidemics. The GII.4 persistence is believed to be associated with the recognition of numerous histo-blood group antigen (HBGA) types and antigenic drift. We found that one of the earliest known GII.4 isolate (1974) and a more recent epidemic GII.4 variant (2012) had varied norovirus-specific monoclonal antibody (MAb) reactivities, yet similar HBGA binding profiles. To better understand the binding interaction of one MAb (10E9) that had varied reactivities with these GII.4 variants, we determined the X-ray crystal structure of the NSW-2012 GII.4 P domain 10E9 Fab complex. We showed that the 10E9 Fab interacted with conserved and variable residues, which could be associated with antigenic drift. Interestingly, the 10E9 Fab binding pocket partially overlapped the HBGA pocket and had direct competition for conserved HBGA binding residues (i.e., Arg345 and Tyr444). Indeed, the 10E9 MAb blocked norovirus VLPs from binding to several sources of HBGAs. Moreover, the 10E9 antibody completely abolished virus replication in the human norovirus intestinal enteroid cell culture system. Our new findings provide first direct evidence that competition for GII.4 HBGA binding residues and steric obstruction could lead to norovirus neutralization. On the other hand, the 10E9 MAb recognized residues flanking the HBGA pocket, which are often substituted as the virus evolves. This mechanism of antigenic drift likely influences herd immunity and impedes the possibility of acquiring broadly reactive HBGA-blocking antibodies.

    IMPORTANCE The emergence of new epidemic GII.4 variants is thought to be associated with changes in antigenicity and HBGA binding capacity. Here, we show that HBGA binding profiles remain unchanged between 1974 and 2012 GII.4 variants, whereas these variants showed varying levels of reactivities against a panel of GII.4 MAbs. We identified a MAb that bound at the HBGA pocket and blocked norovirus VLPs from binding to HBGAs and neutralized norovirus virions in the cell culture system. Raised against GII.4 2006 strain this MAb was unreactive to GII.4 1987 isolate, but was able to neutralize newer 2012 strain, which has important implications for vaccine design. Altogether, these new findings suggested that the amino acid variations surrounding HBGA pocket lead to temporal changes in antigenicity without affecting the ability of GII.4 variants to bind HBGAs, which are known co-factors for infection.

  • Influenza infection enhances antibody-mediated NK cell functions via Type I interferon dependent pathways [Pathogenesis and Immunity]

  • NK cells are an important component in the control of influenza infection, acting to both clear virus-infected cells and release antiviral cytokines. Engagement of CD16 on NK cells by antibody-coated influenza-infected cells results in antibody-dependent cellular cytotoxicity (ADCC). Increasing the potency of antibody-mediated NK cell activity could ultimately lead to improved control of influenza infection. To understand if NK cells can be functionally enhanced following exposure to influenza virus-infected cells, we co-cultured human PBMCs with influenza-infected human alveolar epithelial (A549) cells and evaluated the capacity of NK cells to mediate antibody-dependent functions. Pre-incubation of PBMCs with influenza-infected cells markedly enhanced the ability of NK cells to respond to immune complexes containing HA and anti-HA antibodies or transformed allogenic cells in the presence or absence of a therapeutic monoclonal antibody. Cytokine multiplex, RNA sequencing, supernatant transfer, trans-well and cytokine blocking/supplementation experiments showed that type I interferons released from PBMCs were primarily responsible for the influenza-induced enhancement of antibody-mediated NK cell functions. Importantly, the influenza-mediated increase in antibody-dependent NK cell functionality was mimicked by the type I interferon agonist poly(I:C). We conclude that type I interferon secretion induced by influenza virus infection enhances the capacity of NK cells to mediate ADCC, and this pathway could be manipulated to alter the potency of anti-influenza therapies and vaccines.

    Significance: Protection from severe influenza may be assisted by antibodies that engage NK cells to kill infected cells through ADCC. Studies have primarily focused on antibodies that have ADCC activity, rather than the capacity of NK cells to become activated and mediate ADCC during an influenza infection. We found that type I interferon released in response to influenza infection primes NK cells to become highly reactive to anti-influenza ADCC antibodies. Enhancing the capacity of NK cells to mediate ADCC could assist in controlling influenza virus infections.

  • Hantavirus RdRp requires a host cell factor for cap snatching [Virus-Cell Interactions]

  • The hantavirus RNA dependent RNA polymerase (RdRp) snatches 5rrsquo; capped mRNA fragments from the host cell transcripts and uses them as primers to initiation transcription and replication of the viral genome in the cytoplasm of infected cells. Hantavirus nucleocapsid protein (N protein) binds to the 5rrsquo; caps of host cell mRNA and protects them from the attack of cellular decapping machinery. N protein rescues long capped mRNA fragments in cellular P bodies that are later processed by an unknown mechanism to generate 10-14 nucleotide long capped RNA primers with a 3rrsquo; "G" residue. Hantavirus RdRp has an N-terminal endonuclease domain and a C-terminal uncharacterized domain that harbors a binding site for the N protein. The purified endonuclease domain of RdRp nonspecifically degraded RNA in vitro, puzzling how such nonspecific endonuclease activity generates primers of appropriate length and specificity during cap snatching. We fused the N-terminal endonuclease domain with the C-terminal uncharacterized domain of the RdRp. The resulting NC mutant with the assistance of N protein generated capped primers of appropriate length and specificity from a test mRNA in cells. Bacterially expressed and purified NC mutant and N protein required further incubation with the lysates of human umbilical vein endothelial cells (HUVEC) for the specific endonuleolytic cleavage of a test mRNA to generate capped primers of appropriate length and defined 3rrsquo; terminus in vitro. Our results suggest that an unknown host cell factor likely facilitates the interaction between N protein and NC mutant, that brings the N protein bound capped RNA fragments in close proximity to the endonuclease domain of the RdRp for the specific cleavage at a precise length from the 5rrsquo; cap. These studies have provided critical insights in the cap snatching mechanism of cytoplasmic viruses, and have revealed potential new targets for their therapeutic intervention.

    Importance: Humans acquire hantavirus infection by the inhalation of aerosolized excreta of infected rodent hosts. Hantavirus infections cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) with the mortality rates of 15% and 50 %, respectively (1). Annually 150000-200000 cases of hantavirus infections are reported worldwide for which there is no treatment at present. Cap snatching is the early event in the initiation of virus replication in the infected hosts. Interruption in cap snatching will inhibit virus replication and will likely improve the prognosis of the hantavirus disease. Our studies provide mechanistic insight in the cap-snatching mechanism and demonstrate the requirement of a host cell factor for successful cap snatching. Identification of this host cell factor will reveal a novel therapeutic target for combating this viral illness.

  • Contrasting roles of the PD-1 signaling pathway in dendritic cell-mediated induction and regulation of HIV-1-specific effector T cell functions [Cellular Response to Infection]

  • Eliciting highly functional CD8+ cytotoxic T lymphocyte (CTL) responses against a broad range of epitopes will likely be required for immunotherapeutic control of HIV-1 infection. However, the combination of CTL exhaustion and the ability of HIV-1 to rapidly establish CTL escape variants represent major hurdles towards this goal. Our previous work highlighted the use of monocyte derived, mature, high IL-12-producing type-1 polarized dendritic cells (MDC1) to selectively induce more potent effector CTLs derived from naïve, rather than memory, CD8+ T cell precursors isolated from HIV-1 positive participants in the Multicenter AIDS Cohort Study. In this study, we report that these highly stimulatory antigen presenting cells also express enhanced levels of the co-inhibitory molecule programmed cell death ligand 1 (PD-L1), the ligand for PD-1, which is further upregulated upon subsequent stimulation with the CD4+ T helper cell-derived factor CD40L. Interestingly, blocking the PD-1 signaling pathway during DC1 induction of HIV-1-specific CTL responses inhibited the priming, activation, and differentiation of naïve CD8+ T cells into T-box transcription factor high (Tbethi) and eomesodermin (Eomes)+ effector T cells. In contrast, PD-1 blockade enhanced the overall magnitude of memory HIV-specific CTL responses and reversed the exhausted memory phenotype from a T-betlow/Eomes+ to a Tbethi/Eomes+ phenotype. These results indicate that the PD-L1/PD-1 signaling pathway has a previously unappreciated dual role in the induction and regulation of HIV-1-specific CTL immunity, which is greatly determined by the context and differentiation stage of the responsive CD8+ T cells.

    IMPORTANCE Targeting the PD-1/PD-L1 immune checkpoint axis with signaling inhibitors has proven to be a powerful immunotherapeutic strategy to enhance the functional quality and survival of existing antigen-specific effector T cells. However, our study brings to attention that the context and timing of PD-1 signaling in T cells greatly impacts the outcome of the effector response. In particular, we show that PD-1 activation plays a positive role during the DC-mediated initiation stage of the primary T cell response, while it serves as an inhibitory mechanism during the effector phase of the response. Therefore, caution should be taken in the design of therapies that include targeting of the PD-1/PD-L1 signaling pathway in order to avoid potential negative impacts on the induction of de novo T cell responses.

  • Insulin-like Growth Factor 2 Receptor Expression is Promoted by Human Herpesvirus 8-Encoded Interleukin-6 and Contributes to Viral Latency and Productive Replication [Virus-Cell Interactions]

  • Human herpesvirus 8 (HHV-8) viral interleukin-6 (vIL-6) localizes largely to the endoplasmic reticulum (ER) and here associates functionally with both the gp130 signal transducer and the novel ER membrane protein vitamin K epoxide reductase complex subunit 1 variant-2 (VKORC1v2). The latter interaction contributes to the viability of latently infected primary effusion lymphoma (PEL) cells and to HHV-8 productive replication, in part via promotion of ER-associated degradation (ERAD) of nascent pro-cathepsin D (pCatD) and consequent suppression of lysosome-localized pro-apoptotic mature CatD. Here we report that VKORC1v2 associates with insulin-like growth factor 2 receptor (IGF2R), also known as cation-independent mannose-6-phosphate receptor, which is involved in trafficking of mannose-6-phosphate-conjugated glycoproteins to lysosomes. VKORC1v2 effected reduced IGF2R expression in a manner dependent on VKORC1v2-IGF2R interaction, while vIL-6, which could inhibit VKORC1v2-IGF2R interaction, effected increased expression of IGF2R. These effects were independent of changes in IGF2R mRNA levels, indicating likely posttranslational mechanisms. In kinetic analyses involving labeling of either newly-synthesized or pre-existing IGF2R, vIL-6 promoted accumulation of the former while having no detectable effect on the latter. Furthermore, vIL-6 led to decreased K48-linked ubiquitination of IGF2R and suppression of ERAD proteins effected increased IGF2R expression and loss of IGF2R regulation by vIL-6. Depletion-based experiments identified IGF2R as a promoter of PEL cell viability and virus yields from lytically reactivated cultures. Our findings identify ER-transiting nascent IGF2R as an interaction partner of VKORC1v2 and target of vIL-6 regulation and IGF2R as a positive contributor to HHV-8 biology, thereby extending understanding of the mechanisms of VKORC1v2-associated vIL-6 function.

    IMPORTANCE HHV-8 vIL-6 promotes productive replication in the context of reactivated lytic replication in primary effusion lymphoma (PEL) and endothelial cells and sustains latently infected PEL cell viability. Viral IL-6 is also considered to contribute significantly to HHV-8-associated pathogenesis, as vIL-6 can promote cell proliferation, cell survival, and angiogenesis that are characteristic of HHV-8-associated Kaposirrsquo;s sarcoma, PEL and multicentric Castlemanrrsquo;s disease (MCD), in addition to pro-inflammatory activities observed in MCD-like "Kaposirrsquo;s sarcoma-associated herpesvirus-induced cytokine syndrome". We show in the present study that vIL-6 can promote productive replication and latent PEL cell viability through upregulation of the mannose-6-phosphate- and peptide hormone-interacting receptor IGF2R, which is a positive factor in HHV-8 biology via these activities. VKORC1v2-enhanced ER-associated degradation of IGF2R and vIL-6 promotion of IGF2R expression through prevention of its interaction with VKORC1v2 and consequent rescue from degradation represent newly recognized activities of VKOCR1v2 and vIL-6.

  • Bluetongue virus VP6 and genomic RNA interaction is essential for genome packaging [Structure and Assembly]

  • The genomes of the Reoviridae, including the animal pathogen Bluetongue virus (BTV), are multi-segmented double-stranded (ds) RNA. During replication, single-stranded (ss) positive-sense RNA segments are packaged into the assembling virus capsid, triggering genomic dsRNA synthesis. However, exactly how this packaging event occurs is not clear. A minor capsid protein VP6, unique for the orbiviruses, has been proposed to be involved in the RNA packaging process. In this study, we sought to characterize the RNA binding activity of VP6 and its functional relevance. A novel proteomic approach was utilized to map the ss/dsRNA binding sites of a purified recombinant protein and the genomic dsRNA binding sites of the capsid-associated VP6. The data revealed each VP6 has multiple distinct RNA binding regions and only one region is shared between recombinant and capsid-associated VP6. A combination of targeted mutagenesis and reverse genetics identified the RNA-binding region that is essential for virus replication. Using an in vitro RNA-binding competition assay, a unique cell-free assembly assay and an in vivo single cycle replication assay, it was possible to identify a motif within the shared binding region that binds BTV ssRNA preferentially consistent with specific RNA recruitment during capsid assembly. These data highlight the critical roles this unique protein plays in orbivirus genome packaging and replication.

    IMPORTANCE Genome packaging is a critical stage during virus replication. For virus with segmented genome, the genome segments need to be correctly packaged into a newly formed capsid. However, the detailed mechanism of this packaging is unclear. Here we focus on VP6, a minor viral protein of Bluetongue virus, which is critical for genome packaging. We use multiple approaches including a robust RNA-protein finger-printing assay, which map the ssRNA binding sites of recombinant VP6 and the genomic dsRNA binding sites of the capsid-associated VP6. Together with virological and biochemical methods, within VP6, we for the first time identify the viral RNA packaging motif of a segmented dsRNA virus.

  • Genome-wide identification of direct RTA targets reveals key host factors for KSHV lytic reactivation [Virus-Cell Interactions]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus, which maintains the persistent infection of the host by intermittently reactivating from latently infected cells to produce viral progenies. While it is established that the viral transcription factor RTA is required for the induction of lytic viral genes for KSHV lytic reactivation, it is still unknown to what extent RTA alters the host transcriptome to promote KSHV lytic cycle and viral pathogenesis. To address this question, we performed a comprehensive time course transcriptome analysis during KSHV reactivation in B-cell lymphoma cells and determined RTA-binding sites on both the viral and host genomes, which resulted in the identification of the core RTA-induced host genes (core RIGs). We found that the majority of RTA-binding sites at core RIGs contained the canonical RBP-J-binding DNA motif. Subsequently, we demonstrated the vital role of the Notch signaling transcription factor RBP-J for RTA-driven rapid host gene induction, which is consistent with RBP-J being essential for KSHV lytic reactivation. Importantly, many of the core RIGs encode plasma membrane proteins and key regulators of signaling pathways and cell death, however, their contribution to the lytic cycle is largely unknown. We show that the cell cycle and chromatin regulator Geminin and the plasma membrane protein Gamma-glutamyltransferase 6, two of the core RIGs, are required for efficient KSHV reactivation and virus production. Our results indicate that host genes that RTA rapidly and directly induces can be pivotal for driving the KSHV lytic cycle.

    IMPORTANCE The lytic cycle of KSHV is involved not only in the dissemination of the virus but also viral oncogenesis in which the effect of RTA on the host transcriptome is still unclear. Using genomics approaches, we identified a core set of host genes, which are rapidly and directly induced by RTA in the early phase of KSHV lytic reactivation. We found that RTA does not need viral co-factors, but requires its host co-factor RBP-J for inducing many of its core RIGs. Importantly, we show a critical role for two of the core RIGs in the efficient lytic reactivation and replication, highlighting their significance in KSHV lytic cycle. We propose that the unbiased identification of RTA-induced host genes can uncover potential therapeutic targets for inhibiting KSHV replication and viral pathogenesis.

  • New isolates of pandoraviruses: contribution to the study of replication cycle steps [Virus-Cell Interactions]

  • Giant viruses are complex members of the virosphere, exhibiting outstanding structural and genomic features. Among these viruses, the pandoraviruses are one of the most intriguing members, exhibiting giant particles and genomes presenting up to 2.5 Mb, with many genes having no known function. In this work, we analyzed, by virological and microscopical methods, the replication cycle steps of three new pandoraviruses isolated from samples collected in different regions of Brazil. Our data indicate that all analyzed pandoravirus isolates can deeply modify the Acanthamoeba cytoplasmic environment, recruiting mitochondria and membranes into and around the electron-lucent viral factories. We also observed that the viral factories start forming before the complete degradation of the cellular nucleus. Variable patterns of pandoravirus particle morphogenesis were observed, and the assembly of the particles seemed to be started either by the apex or by its opposite side. Based on the counting of viral particles during the infection time course, we observed that pandoravirus particles could be exocytosed after their morphogenesis in a process that involves intense recruitment of membranes that wrap the just-formed particles. The treatment of infected cells with brefeldin affects particles exocytosis in two of the three analyzed strains, indicating biological variability among isolates. Despite such particle exocytosis, the lysis of host cells also contributes to viral release. This work reinforces and reveals important steps in the replication cycle of pandoraviruses.

    IMPORTANCE The emerging Pandoraviridae family is composed of some of the most complex viruses known to date. Only a few pandoravirus isolates have been described until now, and many aspects of their life cycle remain to be elucidated. A comprehensive description of the replication cycle is pivotal to a better understanding of the virus biology. In this study, we describe new pandoraviruses and use different methods to better characterize the replication cycle steps of this new group of viruses. Our results provide new information about the diversity and biology of these giant viruses.

  • Global proteomic profiling of Salmonella infection by a giant phage [Structure and Assembly]

  • The 240-kb Salmonella phage SPN3US genome encodes 264 gene products, many of which are functionally uncharacterized. We have previously used mass spectrometry to define the proteomes of wild-type and mutant forms of the SPN3US virion. In this study we sought to determine if this technique was suitable for the characterization of the SPN3US proteome during liquid infection. Mass spectrometry of SPN3US-infected cells identified 232 SPN3US and 1994 Salmonella proteins. SPN3US proteins with related functions, such as proteins with roles in DNA replication, transcription and virion formation, were coordinately expressed in a temporal manner. Mass spectral counts showed the four most abundant SPN3US proteins to be the major capsid, two head ejection proteins, and the functionally unassigned protein, gp22. This high abundance of gp22 in infected bacteria contrasted with its absence from mature virions, suggesting it might be the scaffold protein, an essential head morphogenesis protein yet to be identified in giant phages. We identified homologs to SPN3US gp22 in 45 related giant phages, including KZ, whose counterpart is also abundant in infected bacteria but absent in the virion. We determined the KZ counterpart to be cleaved in vitro by its prohead protease, an event that has been observed to promote head maturation of some other phages. Our findings are consistent with a scaffold protein assignment for SPN3US gp22, although direct evidence is required for its confirmation. These studies demonstrate the power of mass spectral analyses for facilitating the acquisition of new knowledge into the molecular events of viral infection.

    IMPORTANCE "Giant" phages with genomes llt;200 kb are being isolated in increasing numbers from a range of environments. With hosts such as Salmonella enterica, Pseudomonas aeruginosa and Erwinia amylovora, these phages are of interest for phage therapy of multi-drug resistant pathogens. However, our understanding of how these complex phages interact with their hosts is impeded by the proportion (~80%) of their gene products that are functionally uncharacterized. To develop the repertoire of techniques for analysis of phages, we analyzed a liquid infection of Salmonella phage SPN3US (240 kb genome) using third generation mass spectrometry. We observed the temporal production of phage proteins whose genes collectively represent 96% of the SPN3US genome. These findings demonstrate the sensitivity of mass spectrometry for global proteomic profiling of virus-infected cells and the identification of a candidate for a major head morphogenesis protein will facilitate further studies into giant phage head assembly.

  • Co-administration of CH31 broadly neutralizing antibody does not affect development of vaccine-induced anti-HIV-1 envelope antibody responses in infant Rhesus macaques [Vaccines and Antiviral Agents]

  • Prevention of mother to child transmission (MTCT) is an indispensable component of the combat against the global AIDS epidemic. A combination of passive broadly neutralizing antibody (bnAb) infusion and active vaccination promises to provide protection of infants against MTCT from birth through the breastfeeding period, and could prime the immune system for life-long immunity. In this study, we investigate the impact of a single infusion of CD4 binding site (CD4bs) bnAb administered at birth on de novo antibody responses elicited by concurrent active HIV envelope vaccination. Four groups of infant macaques received active immunizations with subunit Env protein or MVA-vectored Env and subunit Env protein, with or without a single intravenous co-administration of CH31 bnAb at birth. Vaccinated animals were monitored to evaluate binding and functional antibody responses elicited by the active vaccinations. Despite achieving plasma concentrations that were able to neutralize tier 2 viruses, co-administration of CH31 did not have a large impact on the kinetics, magnitude, specificity, or avidity of vaccine-elicited binding or functional antibody responses including epitope-specificity, the development of CD4bs antibodies, neutralization, binding to infected cells, or ADCC. We conclude that infusion of CD4bs bnAb CH31 at birth does not interfere with de novo antibody responses to active vaccination, and that a combination of passive bnAb infusion and active HIV-1 Env vaccination is a viable strategy for immediate and prolonged protection against MTCT.

    IMPORTANCE Our study is the first to evaluate the impact of passive infusion of a broadly neutralizing antibody in newborns on the de novo development of antibody responses following active vaccinations in infancy. We demonstrated the safety and the feasibility of bnAb administration to achieve biologically relevant levels of the antibody and showed that the passive infusion did not impair the de novo antibody production following HIV-1 Env vaccination. Our study paved the way for further investigations of the combination strategy of passive + active immunization, to provide protection of infants born to HIV-1 positive mothers over the entire period of risk for mother to child transmission.

  • Constraints of viral RNA synthesis on codon usage of negative strand RNA virus [Structure and Assembly]

  • Negative Strand RNA viruses (NSV) include some of the most pathogenic human viruses known. NSVs completely rely on the host cell for protein translation, but their codon usage bias is often different than that of the host. This discrepancy may have originated from the unique mechanism of NSV viral RNA synthesis in that the genomic RNA sequestered in the nucleocapsid serves as the template. The stability of the genomic RNA in the nucleocapsid appears to regulate its accessibility to the viral RNA polymerase, thus placing constraints on codon usage to balance viral RNA synthesis. By in situ analyses of vesicular stomatitis virus RNA synthesis, specific activities of viral RNA synthesis were correlated with the genomic RNA sequence. It was found that by simply altering the sequence and not the amino acid it encoded, a significant reduction, up to ~ 750 fold reduction, in viral RNA transcripts occurred. Through a subsequent sequence analysis and Thermal Shift assays, it was found that the purine/pyrimidine content modulates the overall stability of the polymerase complex, resulting in altering the activity of viral RNA synthesis. The codon usage is therefore constrained by the obligation of the NSV genome to viral RNA synthesis.

    Significance Statement: Negative strand RNA viruses (NSVs) include most pathogenic viruses known. New methods to monitor their evolutionary trends are urgently needed for development of antivirals and vaccines. The protein translation machinery of the host cell is currently recognized as a main genomic regulator of RNA virus evolution, which works especially well for positive strand RNA viruses. However, this approach fails for NSVs because it does not consider the unique mechanism of their viral RNA synthesis. For NSVs, the viral RNA dependent RNA polymerase (vRdRp) must gain access to the genome sequestered in the nucleocapsid. Our work suggests a paradigm shift that the interactions between the RNA genome and the nucleocapsid protein regulate the activity of vRdRp, which selects the codon usage.

  • ROTAVIRUS INFECTION ALTERS SPLICING OF THE STRESS-REGULATED TRANSCRIPTION FACTOR XBP1. [Virus-Cell Interactions]

  • XBP1 is a stress-regulated transcription factor also involved in mammalian host defenses and innate immune response. Our investigation of XBP1 RNA splicing during rotavirus infection revealed that an additional XBP1 RNA (XBP1es) that corresponded to exon-skipping in the XBP1 pre RNA was induced depending on the rotavirus strain used. We showed that the translation product of XBP1es (XBP1es) has trans-activation properties similar to those of XBP1 on ER stress response element (ERSE) containing promoters. Using mono-reassortant between ES+ ("skipping") and ES- ("nonskipping") strains of rotavirus, we showed that gene 7 encoding the viral translation enhancer NSP3 was involved in this phenomenon and that exon-skipping paralleled the nuclear relocalization of cytoplasmic PABP. We further showed, using recombinant rotaviruses carrying chimeric gene 7, that the ES+ phenotype was linked to the eIF4G-binding domain of NSP3. Because the XBP1 transcription factor is involved in stress and immunological responses, our results suggest an alternative way to activate XBP1 upon viral infection or nuclear localization of PABP.

    IMPORTANCE Rotavirus is one of the most important pathogens causing severe gastroenteritis in young children worldwide. Here we show that infection with several rotavirus strains induces an alternative splicing of the RNA encoding the stressed-induced transcription factor XBP1. The genetic determinant of XBP1 splicing is the viral RNA- translation-enhancer NSP3. XBP1 being involved in cellular stress and immune responses and the XBP1 protein made from the alternatively spliced RNA being an active transcription factor, our observations raise the question of alternative splicing being a cellular response to rotavirus infection.

  • Evolution of Hepatitis B Virus Receptor NTCP Reveals Differential Pathogenicity and Species-Specificities of Hepadnaviruses in Primates, Rodents and Bats [Genetic Diversity and Evolution]

  • Human hepatitis B virus (HBV) is a global health problem, affecting more than 250 million people worldwide. HBV-like viruses, named orthohepadnaviruses, also naturally infect non-human primates, rodents and bats, but their pathogenicity and evolutionary history are unclear. Here, we determined the evolutionary history of HBV receptors, NTCP and GPC5, over millions of years of primate, rodent and bat evolution. We use this as a proxy to understand the pathogenicity of orthohepadnaviruses in mammalian hosts and to determine the implications for species-specificity. We found that NTCP, but not GPC5, has evolved under positive selection in primates (27 species), rodents (18 species) and bats (21 species), although at distinct residues. Notably, the positively selected codons map to the HBV-binding sites in primate NTCP, suggesting past genetic "arms-races" with pathogenic orthohepadnaviruses. In rodents, the positively selected codons fall outside and within the presumed HBV-binding sites, which may contribute to the restricted circulation of rodent orthohepadnaviruses. In contrast, the presumed HBV-binding motifs in bat NTCP are conserved, and none of the positively selected codons map to this region. This suggests that orthohepadnaviruses may bind to different surfaces in bat NTCP. Alternatively, the patterns may reflect adaptive changes associated to metabolism rather than pathogens. Overall, our findings further point to NTCP as a naturally-occurring genetic barrier for cross-species transmissions in primates, which may contribute for the narrow host range of HBV. In contrast, this constraint seems less important in bats, which may correspond with greater orthohepadnavirus circulation and diversity.

    IMPORTANCE Chronic infection with hepatitis B virus (HBV) is a major cause of liver disease and cancer in humans. Mammalian HBV-like viruses are also found in non-human primates, rodents and bats. As most viruses, HBV requires a successful interaction with a host receptor for replication. Cellular receptors are thus key determinants of host susceptibility, as well as specificity. One hallmark of pathogenic virus-host relationships is the reciprocal evolution of host receptor and viral envelope proteins, as a result of their antagonistic interaction over time. The dynamics of these so-called "evolutionary arms-races" can leave signatures of adaptive selection, which in turn reveal the evolutionary history of the virus-host interaction, as well as the viral pathogenicity and the genetic determinants of species-specificity. Here, we show how HBV-like viruses have shaped the evolutionary history of their mammalian host receptor, as a result of their ancient pathogenicity, and decipher the genetic determinants of cross-species transmissions.

  • Porcine hemagglutinating encephalomyelitis virus activation of the integrin {alpha}5{beta}1-FAK-Cofilin pathway causes cytoskeletal rearrangement to promote its invasion of N2a cells [Virus-Cell Interactions]

  • Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurotropic virus that causes diffuse neuronal infection with neurological damage and high mortality. Virus-induced cytoskeletal dynamics are thought to be closely related to this type of nerve damage. Currently, the regulation pattern of the actin cytoskeleton and its molecular mechanism remain unclear when PHEV enters the host cells. Here, we demonstrate that entry of PHEV into N2a cells induces a biphasic remodeling of the actin cytoskeleton and a dynamic change in cofilin activity. Viral entry is affected by the disruption of actin kinetics or alteration of cofilin activity. PHEV binds to integrin aalpha;5bbeta;1 and then initiates the integrin aalpha;5bbeta;1-FAK signaling pathway, leading to virus-induced early cofilin phosphorylation and F-actin polymerization. Additionally, Ras-related C3 botulinum toxin substrate 1 (Rac1), cell division cycle 42 (Cdc42) and downstream regulatory genes p21-activated protein kinases (PAKs) are recruited as downstream mediators of PHEV-induced dynamic changes of the cofilin activity pathway. In conclusion, we demonstrate that PHEV utilizes the integrin aalpha;5bbeta;1-FAK-Rac1/Cdc42-PAK-LIMK-Cofilin pathway to cause an actin cytoskeletal rearrangement to promote its own invasion, providing theoretical support for the development of PHEV pathogenic mechanisms and new antiviral targets.

    IMPORTANCE Porcine hemagglutinating encephalomyelitis virus (PHEV), a member of the Coronaviridae family, is a typical neurotropic virus that primarily affects the nervous system of piglets to produce typical neurological symptoms. However, the mechanism of nerve damage caused by the virus is not fully elucidated. Actin is an important component of the cytoskeleton of eukaryotic cells and serves as the first obstacle to the entry of pathogens into host cells. Additionally, the morphological structure and function of nerve cells depend on the dynamic regulation of the actin skeleton. Therefore, exploring the mechanism of neuronal injury induced by PHEV from the perspective of the actin cytoskeleton not only helps elucidate the pathogenesis of PHEV but also provides a theoretical basis for the search for new antiviral targets. This is the first report to define a mechanistic link between alterations in signaling from cytoskeleton pathways and the mechanism of PHEV invading nerve cells.

  • HIV Infection Functionally Impairs Mycobacterium tuberculosis-specific CD4 and CD8 T-cell responses [Cellular Response to Infection]

  • HIV infection is the major risk factor predisposing for Mycobacterium tuberculosis (Mtb) progression from latent tuberculosis infection (LTBI) to tuberculosis disease (TB). Since long-term treated aviremic HIV-infected individuals remained at higher risk of developing TB as compared to HIV-uninfected individuals, we hypothesized that progression from LTBI to pulmonary TB (PTB) might not only be due to CD4 T-cell depletion but also to Mtb-specific CD4 T-cell functional impairment. To test this hypothesis, Mtb-specific T-cell frequencies and cytokine profiles were investigated in untreated Tanzanian individuals suffering from LTBI (n=20) or PTB (n=67) and compared to those of untreated Mtb/HIV co-infected individuals suffering from LTBI (n=15) or PTB (n=10). We showed that HIV infection significantly reduced the proportion of Th2 (IL-4/IL-5/IL-13) producing Mtb-specific CD4 T cells and IL-2 producing Mtb-specific CD4 and CD8 T cells in both individuals with LTBI or PTB (Pllt;0.05). Interestingly, the loss of IL-2 production was associated with a significant increase of PD-1 expression on Mtb-specific CD4 and CD8 T cells (Pllt;0.05), while the loss of Th2 cytokine production was associated with a significant reduction of Gata-3 expression in memory CD4 T cells (Pllt;0.05). Finally, we showed that the serum levels of IL-1aalpha;, IL-6, CRP, IL-23 and IP-10 were significantly reduced in Mtb/HIV co-infected individuals with PTB as compared to HIV negative individuals with PTB (Pllt;0.05), suggesting that HIV infection significantly suppresses Mtb-induced systemic pro-inflammatory cytokine responses. Taken together, this study suggests that in addition to deplete Mtb-specific CD4 T cells, HIV infection significantly impairs functionally favorable Mtb-specific CD4 T-cell responses in Tanzanian individuals with LTBI or PTB.

    IMPORTANCE Mycobacterium tuberculosis (Mtb) and Human Immunodeficiency Virus (HIV) infections are co-endemic in several regions of the world and Mtb/HIV co-infected individuals are more susceptible to progress to tuberculosis disease. We therefore hypothesized that HIV infection would potentially impair Mtb-specific protective immunity in individuals suffering from latent tuberculosis infection (LTBI) or active pulmonary tuberculosis (PTB). In this study, we demonstrated that Mtb/HIV co-infected individuals have less circulating Mtb-specific CD4 T cells and those that remained were functionally impaired in both LTBI and PTB settings. In addition, we showed that HIV infection significantly interferes with Mtb-induced systemic pro-inflammatory cytokine/chemokine responses. Taken together, these data suggest that HIV infection impairs functionally favorable Mtb-specific immunity.

  • Triticum mosaic virus IRES relies on a picornavirus-like YX-AUG motif to designate the preferred translation initiation site and to likely target the 18S rRNA. [Genome Replication and Regulation of Viral Gene Expression]

  • Several viruses encode an internal ribosome entry site (IRES) at the 5rrsquo; end of their RNA, which, unlike most cellular mRNAs, initiates translation in the absence of a 5rrsquo; m7GpppG cap. Here we report a uniquely regulated translation enhancer found in the 739-nt long sequence of Triticum mosaic virus leader sequence that distinguishes the preferred initiation site from a plethora of IRES-encoded AUG triplets. Through deletion mutations of the TriMV 5rrsquo; untranslated region (UTR), we show that the TriMV 5rrsquo; UTR encodes a cis-acting picornaviral Y16-X11-AUG like-motif (with a 16-nt polypyrimidine CU-tract (Y16), at a precise, 11-nt distance (X11) from the preferred 13th AUG. Phylogenetic analyses indicate that this motif is conserved among potyviral leader sequences with multiple AUGs. Consistent with a broadly conserved mechanism, the motif could be functionally replaced with known picornavirus YX-AUG motifs predicted to function as target sites for 18S rRNA recruitment by direct base pairing. Accordingly, mutations that disrupted overall complementarity to the 18S rRNA markedly reduced TriMV IRES activity, as did the delivery of anti-sense oligonucleotides designed to block YX-AUG accessibility. To our knowledge, this is the first report of a plant viral IRES YX-AUG motif, and suggests a conserved mechanism regulates translation for multiple economically-important plant and animal positive single-stranded RNA viruses.

    IMPORTANCE Uncapped viral RNAs often rely on their 5rrsquo; leader sequences to initiate translation, and the Triticum mosaic virus (TriMV) devotes an astonishing 7% of its genome to directing ribosomes to the correct AUG. Here we uncover a novel mechanism by which a TriMV cis-regulatory element controls cap-independent translation. The upstream region of the functional AUG contains a 16-nt polypyrimidine tract located 11-nt from the initiation site. Based on functional redundancy with similar motifs derived from human picornaviruses, the motif is likely to operate by directing ribosome targeting through base pairing with 18S rRNA. Our results provide the first report of a broad-spectrum mechanism regulating translation initiation for both plant and animal-hosted picornaviruses.

  • Porcine Intestinal Enteroids: a New Model for Studying Enteric Coronavirus PEDV Infection and the Host Innate Response [Cellular Response to Infection]

  • Porcine epidemic diarrhea virus (PEDV), a member of the group of alphacoronaviruses, is the pathogen of a highly contagious gastrointestinal swine disease. The elucidation of the events associated with the intestinal epithelial response to PEDV infection has been limited by the absence of good in vitro porcine intestinal models that recapitulate the multicellular complexity of the gastrointestinal tract. Here, we generated swine enteroids from the intestinal crypt stem cells of the duodenum, jejunum, or ileum, and found that the generated enteroids are able to satisfactorily recapitulate the complicated intestinal epithelium in vivo and are susceptible to infection by PEDV. PEDV infected multiple types of cells including enterocytes, stem cells, and goblet cells, and exhibited segmental infection discrepancies compared with ileal enteroids and colonoids, and this finding was verified in vivo. Moreover, the clinical isolate PEDV-JMS propagated better in ileal enteroids than the cell-adapted PEDV CV777, and PEDV infection suppressed IFN production early during the infection course. IFN-lambda elicited a potent antiviral response and inhibited PEDV in enteroids more efficiently than IFN-aalpha;. Therefore, swine enteroids provide a novel in vitro model for exploring the pathogenesis of PEDV and for the in vitro study of the interplay between a host and a variety of swine enteric viruses.

    IMPORTANCE PEDV is a highly contagious enteric coronavirus that causes significant economic losses, and the lack of a good in vitro model system is a major roadblock to an in-depth understanding of PEDV pathogenesis. Here, we generated a porcine intestinal enteroid model for PEDV infection. Utilizing porcine intestinal enteroids, we demonstrated that PEDV infects multiple lineages of the intestinal epithelium and preferably infects ileal enteroids over colonoids and that enteroids prefer to respond to IFN-lambda 1 over IFN-aalpha;. These events recapitulate the events that occur in vivo. This study constitutes the first use of a primary intestinal enteroid model to investigate the susceptibility of porcine enteroids to PEDV and to determine the antiviral response following infection. Our study provides important insights into the events associated with PEDV infection of the porcine intestine and provides a valuable in vitro model for studying not only PEDV but also other swine enteric viruses.

  • Strawberry mottle virus (family Secoviridae, order Picornavirales) encodes a novel glutamic protease to process the RNA2 polyprotein at two cleavage sites [Genome Replication and Regulation of Viral Gene Expression]

  • Strawberry mottle virus (SMoV) belongs to the family Secoviridae (order Picornavirales) and has a bipartite genome with each RNA encoding one polyprotein. All characterized secovirids encode a single protease related to the picornavirus 3C-protease. The SMoV 3C-like protease was previously shown to cut the RNA2 polyprotein (P2) at a single site between the predicted movement protein and coat protein (CP) domains. However, the SMoV P2 polyprotein includes an extended C-terminal region with a coding capacity of up to 70 kDa downstream of the presumed CP domain, an unusual characteristic for this family. In this study, we identified a novel cleavage event at a PAFP sequence immediately downstream of the CP domain. Following deletion of the PAFP sequence, the polyprotein was processed at or near a related PKFP sequence 40 kDa further downstream, defining two protein domains in the C-terminal region of the P2 polyprotein. Both processing events were dependent on a novel protease domain located between the two cleavage sites. Mutagenesis of amino acids that are conserved amongst isolates of SMoV and of the related Black raspberry necrosis virus did not identify essential cysteine, serine or histidine residues, suggesting that the RNA2-encoded SMoV protease is not related to serine or cysteine proteases of other picorna-like viruses. Rather, two highly conserved glutamic acid residues spaced by 82 residues were found to be strictly required for the protease activity. We conclude that processing of SMoV polyproteins requires two viral proteases, the RNA1-encoded 3C-like protease and a novel glutamic protease encoded by RNA2.

    IMPORTANCE Many viruses encode proteases to release mature proteins and intermediate polyproteins from viral polyproteins. Polyprotein processing allows regulating the accumulation and activity of viral proteins. Many viral proteases also cleave host factors to facilitate virus infection. Thus, viral proteases are key virulence factors. To date, viruses with a positive-strand RNA genome are only known to encode cysteine or serine proteases, most of which are related to the cellular papain, trypsin or chymotrypsin proteases. Here, we characterize the first glutamic protease encoded by a plant virus or by a positive-strand RNA virus. The novel glutamic protease is unique to a few members of the family Secoviridae suggesting that it is a recent acquisition in the evolution of this family. The protease does not resemble known cellular proteases. Rather, it is predicted to share structural similarities with a family of fungal and bacterial glutamic proteases that adopt a lectin fold.

  • A coreceptor-mimetic peptide enhances the potency of V3-glycan antibodies [Vaccines and Antiviral Agents]

  • Broadly neutralizing antibodies (bNAbs) target five major epitopes on the HIV-1 envelope glycoprotein (Env). The most potent bNAbs have median IC50 values in the nanomolar range and the broadest bNAbs neutralize up to 98% of HIV-1 strains. The engineered HIV-1 entry inhibitor eCD4-Ig has greater breadth than and similar potency to bNAbs. eCD4-Ig is markedly more potent than CD4-Ig due to its C-terminal coreceptor-mimetic peptide. Here we investigated whether the coreceptor-mimetic peptide mim6 improved the potency of bNAbs with different epitopes. We observed that when mim6 was appended to the C-terminus of the heavy chains of bNAbs, this sulfopeptide improved the potency of all classes of bNAbs against HIV-1 isolates that are sensitive to neutralization by the sulfopeptide alone. However, mim6 did not significantly enhance neutralization of other isolates when appended to most classes of bNAbs, with one exception. Specifically, mim6 improved the potency of bNAbs of the V3-glycan class, including PGT121, PGT122, PGT128, and 10-1074, by an average of 2-fold for all HIV-1 isolates assayed. Despite this difference, 10-1074 does not induce exposure of the coreceptor-binding site, and addition of mim6 to 10-1074 did not promote shedding of the gp120 subunit of Env. Mixtures of 10-1074 and an Fc domain fused to mim6 neutralized less efficiently than a 10-1074/mim6 fusion, indicating that mim6 enhances the avidity of this fusion. Our data show that mim6 can consistently improve the potency of V3-glycan antibodies, and suggest that these antibodies bind in an orientation that facilitates mim6 association with Env.

    IMPORTANCE HIV-1 requires both the cellular receptor CD4 and a tyrosine-sulfated coreceptor to infect its target cells. CD4-Ig is a fusion of the HIV-1-binding domains of CD4 with an antibody Fc domain. Previous studies have demonstrated that the potency of CD4-Ig is markedly increased by appending a coreceptor-mimetic sulfopeptide to its C-terminus. We investigated whether this coreceptor-mimetic peptide improves the potency of broadly neutralizing antibodies (bNAbs) targeting five major epitopes on the HIV-1 envelope glycoprotein (Env). We observed that inclusion of the sulfopeptide dramatically improved the potency of all bNAb classes against isolates with more open Env structures, typically those that utilize the coreceptor CXCR4. In contrast, the sulfopeptide improved only V3-glycan antibodies when neutralizing primary isolates, on average by two-fold. These studies improve the potency of one class of bNAbs, show that coreceptor-mimetic sulfopeptides enhance neutralization through distinct mechanisms, and provide insight for the design of novel multi-specific entry inhibitors.

  • Novel MVA Vector Expressing Anti-apoptotic Gene B13R Delays Apoptosis and Enhances Humoral Responses [Vaccines and Antiviral Agents]

  • Modified vaccinia Ankara (MVA), an attenuated poxvirus, has been developed as a potential vaccine vector for use against cancer and multiple infectious diseases including HIV. MVA is highly immunogenic and elicits strong cellular and humoral responses in pre-clinical models and humans. However, there is potential to further enhance the immunogenicity of MVA as MVA-infected cells undergo rapid apoptosis leading to faster clearance of recombinant antigens and potentially blunting a greater response. Here, we generated MVA-B13R by replacing the fragmented 181R/182R genes of MVA with a functional anti-apoptotic gene B13R and confirmed its anti-apoptotic function against chemically induced apoptosis in vitro. In addition, MVA-B13R showed a significant delay in induction of apoptosis in muscle cells derived from mice and humans as well as pDCs and CD141+ DCs from rhesus macaques compared to MVA infected cells. MVA-B13R expressing SIV Gag, Pol, and HIV envelope (SHIV) produced higher levels of envelope in the supernatants compared to MVA/SHIV infected DF-1 cells in vitro. Immunization of BALB/c mice showed induction of higher Envelope-specific antibody secreting cells, IgG antibody titers, memory B cells, and better persistence of antibody titers by MVA-B13R/SHIV compared to MVA/SHIV. Gene set enrichment analysis of draining lymph nodes cells from day 1 after immunization showed a negative enrichment for interferon responses in MVA-B13R/SHIV mice compared to MVA/SHIV mice. Taken together, these results demonstrate that restoring B13R functionality in MVA significantly delays MVA-induced apoptosis in muscle and antigen-presenting cells in vitro and augments vaccine-induced humoral immunity in mice.

    IMPORTANCE MVA is an attractive viral vector for vaccine development due to its safety and immunogenicity in multiple species and humans even under conditions of immunodeficiency. Here, to further improve the immunogenicity of MVA, we developed a novel vector MVA-B13R by replacing the fragmented anti-apoptotic genes 181R/182R with a functional version derived from vaccinia virus, B13R. Our results show that MVA-B13R significantly delays apoptosis in antigen-presenting cells and muscle cells in vitro and augments vaccine-induced humoral immunity in mice leading to the development of a novel vector for vaccine development against infectious diseases and cancer.

  • The potato virus X TGBp2 protein plays dual functional roles in viral replication and movement [Genome Replication and Regulation of Viral Gene Expression]

  • Plant viruses usually encode one or more movement proteins (MP) to accomplish their intercellular movement. A group of positive-strand RNA plant viruses requires three viral proteins (TGBp1, TGBp2, and TGBp3) that are encoded by an evolutionarily conserved genetic module of three partially overlapping open reading frames (ORFs), termed the triple gene block (TGB). However, how these three viral movement proteins function cooperatively in viral intercellular movement is still elusive. Using a novel in vivo double-stranded RNA (dsRNA) labeling system, we showed that the dsRNAs generated by potato virus X (PVX) RNA-dependent RNA polymerase (RdRp) are colocalized with viral RdRp, which are further tightly covered by "chainmail"-like TGBp2 aggregates and localizes alongside TGBp3 aggregates. We also discovered that TGBp2 interacts with the C-terminal domain of PVX RdRp, and this interaction is required for the localization of TGBp3 and itself to the RdRp/dsRNA bodies. Moreover, we reveal that the central and C-terminal hydrophilic domains of TGBp2 are required to interact with viral RdRp. Finally, we demonstrate that knock-out of the entire TGBp2 or the domain involved in interacting with viral RdRp attenuates both PVX replication and movement. Collectively, these findings suggest that TGBp2 plays dual functional roles in PVX replication and intercellular movement.

    IMPORTANCE Many plant viruses encode three partially overlapping open reading frames (ORFs), termed the triple gene block (TGB), for intercellular movement. However, how these three proteins coordinate their functions remains obscure. In the present study, we provided multiple lines of evidence supporting PVX TGBp2 functions as the molecular adaptor bridging the interaction between the RdRp/dsRNA body and TGBp3 by forming "chainmail"-like structures in the RdRp/dsRNA body, which can also enhance viral replication. Taken together, our results provide new insights into the replication and movement of PVX and possibly also other TGB-encoding plant viruses.

  • Vaccine-induced T-cell responses do not predict the rate of acquisition after repeated intrarectal SIVmac239 challenges in Mamu-B*08+ rhesus macaques [Vaccines and Antiviral Agents]

  • Approximately 50% of rhesus macaques (RMs) expressing the major histocompatibility complex class I (MHC-I) allele Mamu-B*08 spontaneously control chronic phase viremia after infection with the pathogenic simian immunodeficiency virus (SIV)mac239 clone. CD8+ T-cell responses in these animals are focused on immunodominant Mamu-B*08-restricted SIV epitopes in Vif and Nef, and prophylactic vaccination with these epitopes increases the incidence of elite control in SIVmac239-infected Mamu-B*08+ RMs. Here we evaluated if robust vaccine-elicited CD8+ T-cell responses against Vif and Nef can prevent systemic infection in Mamu-B*08+ RMs following mucosal SIV challenges. Ten Mamu-B*08+ RMs were vaccinated with a heterologous prime/boost/boost regimen encoding Vif and Nef, while six sham-vaccinated MHC-I-matched RMs served as the controls for this experiment. Vaccine-induced CD8+ T-cells against Mamu-B*08-restricted SIV epitopes reached high frequencies in blood but were present at lower levels in lymph node and gut biopsies. Following repeated intrarectal challenges with SIVmac239, all control RMs became infected by the sixth SIV exposure. By comparison, four vaccinees were still uninfected after six challenges and three of them remained aviremic after 3-4 additional challenges. The rate of SIV acquisition in vaccinees was numerically lower (albeit not statistically significant) than that of controls. However, peak viremia was significantly reduced in infected vaccinees compared to control animals. We found no T-cell markers that distinguished vaccinees that acquired SIV infection versus those that did not. Additional studies will be needed to validate these findings and determine if cellular immunity can be harnessed to prevent the establishment of productive immunodeficiency virus infection.

    IMPORTANCE It is generally accepted that the antiviral effects of vaccine-induced classical CD8+ T-cell responses against human immunodeficiency virus (HIV) are limited to partial reductions in viremia after the establishment of productive infection. Here we show that rhesus macaques (RMs) vaccinated with Vif and Nef acquired simian immunodeficiency virus (SIV) infection at a slower (albeit not statistically significant) rate than control RMs following repeated intrarectal challenges with a pathogenic SIV clone. All animals in the present experiment expressed the elite control-associated major histocompatibility complex class-I (MHC-I) molecule Mamu-B*08 that binds immunodominant epitopes in Vif and Nef. Though preliminary, these results provide tantalizing evidence that the protective efficacy of vaccine-elicited CD8+ T-cells may be greater than previously thought. Future studies should examine if vaccine-induced cellular immunity can prevent systemic viral replication in RMs that do not express MHC-I alleles associated with elite control of SIV infection.

  • Splicing-Dependent Subcellular Targeting of Borna Disease Virus Nucleoprotein Isoforms [Genome Replication and Regulation of Viral Gene Expression]

  • Targeting of viral proteins to specific subcellular compartments is a fundamental step for viruses to achieve successful replication in infected cells. Borna disease virus-1 (BoDV), a non-segmented, negative-strand RNA virus, uniquely replicates and persists in the cell nucleus. Here, it is demonstrated that BoDV nucleoprotein (N) transcripts undergo mRNA splicing to generate truncated isoforms. In combination with alternative usage of translation initiation sites, the N gene potentially expresses at least six different isoforms, which exhibit diverse intracellular localizations, including the nucleoplasm, cytoplasm and endoplasmic reticulum (ER), as well as intranuclear viral replication sites. Interestingly, the ER-targeting signal peptide in the N is exposed by removing the intron by mRNA splicing. Furthermore, the spliced isoforms inhibit viral polymerase activity. Consistently, recombinant BoDVs lacking the N-splicing signals acquire the ability to replicate faster than wild-type virus in cultured cells, suggesting that N isoforms created by mRNA splicing negatively regulate BoDV replication. These results not only provided the mechanism of how mRNA splicing generates viral proteins that have distinct function but also a novel strategy for replication control of RNA viruses using the isoforms with different subcellular localization.

    IMPORTANCE Borna disease virus (BoDV) is a highly neurotropic RNA virus that belongs to the genus orthobornavirus. A zoonotic orthobornavirus that is genetically related to BoDV has recently been identified in squirrels, thus increasing the importance of understanding the replication and pathogenesis of orthobornaviruses. BoDV replicates in the nucleus and uses alternative mRNA splicing to express viral proteins. However, it is unknown whether the virus uses splicing to create protein isoforms with different functions. The present study demonstrated that the nucleoprotein transcript undergoes splicing and produces four new isoforms in coordination with alternative usage of translation initiation codons. The spliced isoforms showed a distinct intracellular localization, including in the endoplasmic reticulum, and recombinant viruses lacking the splicing signals replicated more efficiently than wild-type. The results provided not only a new regulation of BoDV replication but also insights into how RNA viruses produce protein isoforms from small sized genomes.

  • The Epstein-Barr virus induces the expression of the LPAM-1 integrin in B-cells in vitro and in vivo [Virus-Cell Interactions]

  • The Epstein-Barr virus (EBV) infects the oropharynx but surprisingly frequently induces B-cell proliferations in the gut of immunosuppressed individuals. We found that EBV infection in vitro induces the expression of the LPAM-1 integrin on tonsillar B cells and increases it on peripheral blood cells. Similarly, LPAM-1 was induced in the tonsils of patients undergoing primary infectious mononucleosis. EBV-induced LPAM-1 bound to the MAdCAM-1 addressin that allows B-cell homing to the gastrointestinal mucosa-associated lymphoid tissue (GALT). Thus, we hypothesized that EBV-induced LPAM-1 could induce relocation of infected B-cells from the tonsil to the GALT. In situ hybridization with an EBER-specific probe revealed the frequent presence of EBV-infected cells in the pericolic lymph nodes of healthy individuals. Relocation of infected B-cells into the GALT would expand the EBV reservoir, possibly protects it from T-cells primed in the oropharynx and explain why EBV induces lymphoid tumors in the gut.

    IMPORTANCE The Epstein-Barr virus (EBV) causes tumors in multiple organs, in particular the oro- and nasopharyngeal area, but also in the digestive system. This virus enters the body in the oropharynx and establishes a chronic infection in this area. The observation that the virus causes tumors in the digestive system implies that the infected cells can move to this organ. We found that EBV infection induces the expression of integrin beta 7 (ITGB7), an integrin that associates with integrin alpha 4 to form the LPAM-1 dimer. LPAM-1 is key for homing of B cells to the gastrointestinal tract, suggesting that induction of this molecule is the mechanism through which EBV-infected cells to enter this organ. In favor of this hypothesis, we could also detect EBV-infected cells in the lymph nodes adjacent to the colon and in the appendix.

  • A highly attenuated vesicular stomatitis virus-based vaccine platform controls HBV replication in mouse models of hepatitis B [Vaccines and Antiviral Agents]

  • Therapeutic vaccines may be an important component of a treatment regimen for curing chronic hepatitis B virus (HBV) infection. We previously demonstrated that recombinant wild-type vesicular stomatitis virus (VSV) expressing the HBV middle surface glycoprotein (MHBs) elicits functional immune responses in mouse models of HBV replication. However, VSV has some undesirable pathogenic properties, and the use of this platform in humans requires further viral attenuation. We therefore generated a highly attenuated VSV that expresses MHBs and contains two attenuating mutations. This vector was evaluated for immunogenicity, pathogenesis, and anti-HBV function in mice. Compared to wild-type VSV, the highly attenuated virus displayed markedly reduced pathogenesis but induced similar MHBs-specific CD8+ T cell and antibody responses. The CD8+ T cell responses elicited by this vector in naive mice prevented HBV replication in animals that were later challenged by hydrodynamic injection or transduction with adeno-associated virus encoding the HBV genome (AAV-HBV). In mice in which persistent HBV replication was first established by AAV-HBV transduction, subsequent immunization with the attenuated VSV induced MHBs-specific CD8+ T cell responses that corresponded with a reduction in serum and liver HBV antigens and nucleic acids. HBV control was associated with an increase in intrahepatic HBV-specific CD8+ T cells and a transient elevation in serum alanine aminotransferase activity. The ability of VSV to induce a robust multi-specific T cell response that controls HBV replication combined with the improved safety profile of the highly attenuated vector suggests that this platform offers a new approach for HBV therapeutic vaccination.

    IMPORTANCE A curative treatment for chronic hepatitis B must eliminate the virus from the liver, but current antiviral therapies typically fail to do so. Immune-mediated resolution of infection occurs in a small fraction of chronic HBV patients, which suggests the potential efficacy of therapeutic strategies that boost the patientrrsquo;s own immune response to the virus. We modified a safe form of VSV to express an immunogenic HBV protein, and evaluated the efficacy of this vector in the prevention and treatment of HBV infection in mouse models. Our results show that this vector elicits HBV-specific immune responses that prevent the establishment of HBV infection and reduce viral proteins in the serum and viral DNA/RNA in the liver of mice with persistent HBV replication. These findings suggest that highly attenuated and safe virus-based vaccine platforms have the potential to be utilized for the development of an effective therapeutic vaccine against chronic HBV.

  • Equine-origin immunoglobulin fragments protects nonhuman primates from Ebola virus disease [Vaccines and Antiviral Agents]

  • Ebola virus (EBOV) infections result in aggressive hemorrhagic fever in humans with fatality rates reaching 90%, with no licensed, specific therapeutics to treat ill patients. Advances over the past 5 years have firmly established monoclonal antibody (mAb)-based products as the most promising therapeutics for treating EBOV infections, but production is costly, quantities are limited, and thus mAbs are not the best candidates for mass use in the case of an epidemic. To address this need, we generated EBOV-specific polyclonal immunoglobulin fragments F(abrrsquo;)2 from horses hyperimmunized with an EBOV vaccine. The F(abrrsquo;)2 was found to potently neutralize West and Central African EBOV in vitro. Treatment of nonhuman primates (NHPs) with seven doses of 100mg/kg F(abrrsquo;)2 beginning at 3 or 5 days post-infection (dpi) resulted in 100% survival. Notably, NHPs that initiated treatment at 5 dpi were already highly viremic with observable signs of EBOV disease, demonstrating that F(abrrsquo;)2 was still effective as a therapeutic even in symptomatic patients. These results show that F(abrrsquo;)2 should be accelerated for clinical testing in preparation of future EBOV outbreaks and epidemics.

    IMPORTANCE Ebola is one of the deadliest viruses to humans. It has been over 40 years since Ebola was first reported, but no cure is available. Research breakthroughs over the last 5 years have shown that monoclonal antibodies (mAbs) constitute an effective therapy for Ebola. However, mAbs are expensive, difficult to produce in large amounts and therefore may only play a limited role during an epidemic. A cheaper alternative is required, especially since Ebola is endemic in several third-world countries with limited medical resources. Here, we used a standard protocol to produce large amounts of antisera fragments (F(abrrsquo;)2) from horses vaccinated with an Ebola vaccine, and tested its protectiveness in monkeys. We showed that F(abrrsquo;)2 was effective in 100% of monkeys even after these animals were visibly ill with Ebola. Thus, F(abrrsquo;)2 could be a very good option for large-scale treatments of patients and should be advanced to clinical testing.

  • Role of sphingomyelin in alphaherpesvirus entry [Virus-Cell Interactions]

  • Bovine herpesvirus 1 (BoHV-1) is an alphaherpesvirus that causes disease in cattle populations worldwide. Sphingomyelin (SM) is the most abundant sphingolipid in the mammalian cell membrane, where it preferentially associates with cholesterol to form lipid raft domains. SM is a substrate for the lysosome-resident enzyme acid sphingomyelinase, which plays a role in cell membrane repair following injury. Treatment of cells with noncytotoxic concentrations of Staphylococcus aureus-derived sphingomyelinase successfully reduced cell surface-exposed sphingomyelin, but did not significantly inhibit BoHV-1 entry and infection as measured by beta-galactosidase reporter assay. Interestingly, entry of the porcine alphaherpesvirus pseudorabies virus (PRV) was inhibited by sphingomyelin-depletion of cells. Treatment of BoHV-1 particles with sphingomyelinase inhibited viral entry activity, suggesting that viral SM plays a role in BoHV-1 entry, while cellular SM does not. Treatment of cells with noncytotoxic concentrations of the functional inhibitors of host acid sphingomyelinase, imipramine and amitriptyline, which induce degradation of the cellular enzyme, did not significantly inhibit BoHV-1 entry. In contrast, inhibition of cellular acid sphingomyelinase inhibited PRV entry. Entry of the human alphaherpesvirus herpes simplex virus 1 (HSV-1) was independent of both host SM and acid sphingomyelinase, in a manner similar to BoHV-1. Together, the results suggest that among the alphaherpesviruses, there is variability in entry requirements for cellular sphingomyelin and acid sphingomyelinase activity.

    IMPORTANCE BoHV-1 is a ubiquitous pathogen affecting cattle populations worldwide. Infection can result in complicated, polymicrobial infections due to the immunosuppressive properties of the virus. Available vaccines limit disease severity and spread, but do not prevent infection. The financial and animal welfare ramifications of BoHV-1 are significant. In order to develop more effective prevention and treatment regimens, a more complete understanding of the initial steps in viral infection is necessary. We recently identified a low pH endocytosis pathway for BoHV-1. Here we examine the role of cellular factors responsible for membrane integrity and repair in alphaherpesviral entry. This study allows comparisons of the BoHV-1 entry pathway with those of other alphaherpesviruses (PRV and HSV-1). Lastly, this is the first report of sphingomyelin and lysosomal sphingomyelinase playing a role in the entry of a herpesvirus. The results may lead to development of more effective prevention and treatment regimens.

  • Iterons homologous to helper geminivirus are essential for efficient replication of betasatellite [Genome Replication and Regulation of Viral Gene Expression]

  • Betasatellites associated with geminiviruses can be replicated promiscuously by distinct geminiviruses but exhibit a preference for cognate helper viruses. However, the cis elements responsible for betasatellite origin recognition have not been characterized. In this study, we identified an iteron-like repeated sequence motif, 5'-GAGGACC-3', in a tobacco curly shoot betasatellite (TbCSB) associated with tobacco curly shoot virus (TbCSV). Competitive DNA binding assays revealed that two core repeats 5'-GGACC-3' are required for specific binding to TbCSV Rep, TbCSB iteron mutants accumulated to greatly reduced levels and lost the cognate helper-mediated replication preference. Interestingly, TbCSV also contains identical repeated sequences that are essential for specific Rep binding and in vivo replication. To gain an insight into the mechanism by which TbCSB has acquired the cognate iterons, we performed a SELEX assay to identify the high-affinity Rep binding ligands from a large pool of randomized sequences. Analysis of SELEX winners showed that all of the sequences contained at least one core iteron-like motif, suggesting that TbCSB has evolved to contain cognate iterons for high-affinity Rep binding. Further analyses of various betasatellite sequences revealed a region upstream of the satellite conserved region replete with iterative sequence motifs, including species-specific repeats and a general repeat 5'-GGTAAAT-3'. Remarkably, the species-specific repeats in many betasatellites are homologous to their respective cognate helper begomoviruses, whereas the general repeat is widespread in most of betasatellite molecules analyzed. These data collectively suggest that many betasatellites have evolved to acquire homologous iteron-like sequences for cognate helper virus-mediated efficient replication.

    IMPORTANCE Geminivirus-encoded replication initiator protein (Rep) binds to repeated sequence elements (also known as iterons) in the origin of replication that serve as essential cis elements for specific viral replication. Betasatellites associated with begomoviruses can be replicated by cognate or noncognate helper viruses, but the cis elements responsible for betasatellite origin recognition have not been characterized. Using a betasatellite (TbCSB) associated with tobacco curly shoot virus (TbCSV) as a model, we identify two tandem repeats (iterons) in the Rep-binding motif (RBM) as required for specific Rep binding and efficient replication, and show that identical iteron sequences present in the TbCSV are also necessary for Rep binding and replication of helper virus. Extensive analysis of begomovirus/betasatellite sequences show that many betasatellites contain iteron-like elements homologous to their respective cognate helper begomoviruses. Our data suggest that many betasatellites have evolved to acquire homologous iteron-like sequences for cognate helper virus-mediated efficient replication.

  • A Single Adaptive Mutation in Sodium Taurocholate Cotransporting Polypeptide Induced by Hepadnaviruses Determines Virus Species Specificity [Genetic Diversity and Evolution]

  • Hepatitis B virus (HBV) and its hepadnavirus relatives infect a wide range of vertebrates from fish to human. Hepadnaviruses and their hosts have a long history of acquiring adaptive mutations. However, there are no reports providing direct molecular evidence for such a coevolutionary "arms race" between hepadnaviruses and their hosts. Here, we present evidence suggesting the adaptive evolution of the sodium taurocholate cotransporting polypeptide (NTCP), an HBV receptor, has been influenced by virus infection. Evolutionary analysis of the NTCP-encoding genes from 20 mammals showed that most NTCP residues are highly conserved among species, exhibiting evolution under negative selection (dN/dS llt;1); this observation implies that the evolution of NTCP is restricted by maintaining its original protein function. However, 0.7% of NTCP amino acid (aa) residues exhibit rapid evolution under positive selection (dN/dS llt;1). Notably, a substitution at aa 158, a positively selected residue, converting the human NTCP to a monkey-type sequence abrogated the capacity to support HBV infection; conversely, a substitution at this residue converting the monkey Ntcp to the human sequence was sufficient to confer HBV susceptibility. Together, these observations suggested a close association of the aa 158 positive selection with the pressure by virus infection. Moreover, the aa 158 sequence determined attachment of the HBV envelope protein to host cell, demonstrating the mechanism whereby HBV infection would create positive selection at this NTCP residue. In summary, we provide the first evidence in agreement with the function of hepadnavirus as a driver for inducing adaptive mutation in host receptor.

    IMPORTANCE Hepatitis B virus (HBV) and its hepadnavirus relatives infect a wide range of vertebrates, with a long infectious history (hundreds of millions of years). Such a long history generally allows adaptive mutations in hosts to escape from infection, while simultaneously allowing adaptive mutations in viruses to overcome host barriers. However, there is no published molecular evidence for such a coevolutionary "arms race" between hepadnaviruses and hosts. In the present study, we performed coevolutionary phylogenetic analysis between hepadnaviruses and the sodium taurocholate cotransporting polypeptide (NTCP), an HBV receptor, combined with virological experimental assays for investigating the biological significance of NTCP sequence variation. Our data provide the first molecular evidences supporting that HBV-related hepadnaviruses drive adaptive evolution in the NTCP sequence, including a mechanistic explanation of how NTCP mutations determine host viral susceptibility. Our novel insights enhance our understanding of how hepadnaviruses evolved with their hosts, permitting the acquisition of strong species-specificity.

  • Cross-presentation of skin-targeted rAAV2/1 transgene induces potent resident memory CD8+ T cell responses. [Vaccines and Antiviral Agents]

  • A key aspect to consider for vaccinal protection is the induction of a local line of defense consisting of non-recirculating tissue-resident memory T cell (TRM), in parallel to the generation of systemic memory CD8+ T cell responses. The potential to induce TRM has now been demonstrated for a number of pathogens and viral vectors. This potential, however, has never been tested for recombinant adeno-associated (rAAV) vectors, which are weakly inflammatory and poor transducer of dendritic cells. Using a model rAAV2/1-based vaccine, we report herein that a single intradermal immunization with rAAV2/1 vectors in mice induces fully functional TRM at the local site of immunization. The optimal differentiation of rAAV-induced transgene-specific skin TRM was dependent on local transgene expression and additional CD4+ T cell help. Transgene expression in dendritic cells, however, appeared dispensable for the priming of transgene-specific skin-TRM, suggesting that this process solely depends on the cross-presentation of transgene products. Overall, this study provides needed information to properly assess rAAV vectors as T cell inducing vaccine carriers.

    IMPORTANCE rAAV display numerous characteristics that could make them extremely attractive as vaccine carriers, including an excellent safety profile in humans and great flexibility regarding serotypes and choice of target tissue. Studies addressing rAAV ability to induce protective T cell responses, however, are scarce. Notably, the potential to induce tissue-resident memory T cell response has never been described for rAAV vectors, strongly limiting further interest for their use as vaccine carriers. Using a model rAAV2/1 vaccine delivered to the skin, our study demonstrates that rAAV vectors can induce bona-fide skin-resident TRM and provides additional clues regarding the cellular mechanisms underlying this process. These results will help widen the field of rAAV applications.

  • Identification of the receptor used by the ecotropic mouse GLN endogenous retrovirus [Virus-Cell Interactions]

  • Approximately 10% of the mouse genome is composed of endogenous retroviruses belonging to different families. In contrast to the situation in the human genome, several of these families correspond to recent, still infectious elements capable of encoding complete viral particles. The mouse endogenous retrovirus GLN is one of these active families. We previously identified one fully functional provirus from the sequenced genome of the C57BL/6 mouse strain. The GLN envelope protein gives the infectious viral particles an ecotropic host range, and we had demonstrated that the receptor was neither CAT1 nor SMIT1, the two previously identified receptors for mouse ecotropic retroviral envelope proteins. In this study, we have identified SLC19A1, the reduced folate carrier, as the cellular protein used as a receptor by the GLN retrovirus. The ecotropic tropism exhibited by this envelope is due to the presence or absence of a N-linked glycosylation site in the first extracellular loop as well as the specific amino acid sequence of the extracellular domains of the receptor. Like all the other retroviral envelope proteins from the gammaretrovirus genus whose receptors have been identified, the GLN envelope protein uses a member of the solute carrier superfamily as a receptor.

    IMPORTANCE Endogenous retroviruses are genomic traces of past infections present in all vertebrates. Most of these elements degenerate over time and become nonfunctional, but the mouse genome still contains several families with full infection abilities. The GLN retrovirus is one of them and its members encode particles that are only able to infect mouse cells. Here, we identified the cellular protein used as a receptor by GLN for cell entry. It is SLC19A1, the reduced folate carrier. We show that GLN infection is limited to mouse cells due to both a mutation in the mouse gene preventing glycosylation of SLC19A1, and also to other residues conserved within the rat, but not in the hamster and human proteins. Like all other gammaretroviruses whose receptors have been identified, GLN uses a member of the solute carrier superfamily for cell entry, highlighting the role of these proteins for retroviral infection in mammals.

  • 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.

  • 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.

  • 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.

    Return To Top of the Page