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

Table of Contents for this page:

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

    JVI Current Issue

  • Editorial Board [Masthead]

  • Characterization of Novel Reoviruses Wad Medani Virus (Orbivirus) and Kundal Virus (Coltivirus) Collected from Hyalomma anatolicum Ticks in India during Surveillance for Crimean Congo Hemorrhagic Fever [Genetic Diversity and Evolution]

  • In 2011, ticks were collected from livestock following an outbreak of Crimean Congo hemorrhagic fever (CCHF) in Gujarat state, India. CCHF-negative Hyalomma anatolicum tick pools were passaged for virus isolation, and two virus isolates were obtained, designated Karyana virus (KARYV) and Kundal virus (KUNDV), respectively. Traditional reverse transcription-PCR (RT-PCR) identification of known viruses was unsuccessful, but a next-generation sequencing (NGS) approach identified KARYV and KUNDV as viruses in the Reoviridae family, Orbivirus and Coltivirus genera, respectively. Viral genomes were de novo assembled, yielding 10 complete segments of KARYV and 12 nearly complete segments of KUNDV. The VP1 gene of KARYV shared a most recent common ancestor with Wad Medani virus (WMV), strain Ar495, and based on nucleotide identity we demonstrate that it is a novel WMV strain. The VP1 segment of KUNDV shares a common ancestor with Colorado tick fever virus, Eyach virus, Tai Forest reovirus, and Tarumizu tick virus from the Coltivirus genus. Based on VP1, VP6, VP7, and VP12 nucleotide and amino acid identities, KUNDV is proposed to be a new species of Coltivirus. Electron microscopy supported the classification of KARYV and KUNDV as reoviruses and identified replication morphology consistent with other orbi- and coltiviruses. The identification of novel tick-borne viruses carried by the CCHF vector is an important step in the characterization of their potential role in human and animal pathogenesis.

    IMPORTANCE Ticks and mosquitoes, as well Culicoides, can transmit viruses in the Reoviridae family. With the help of next-generation sequencing (NGS), previously unreported reoviruses such as equine encephalosis virus, Wad Medani virus (WMV), Kammavanpettai virus (KVPTV), and, with this report, KARYV and KUNDV have been discovered and characterized in India. The isolation of KUNDV and KARYV from Hyalomma anatolicum, which is a known vector for zoonotic pathogens, such as Crimean Congo hemorrhagic fever virus, Babesia, Theileria, and Anaplasma species, identifies arboviruses with the potential to transmit to humans. Characterization of KUNDV and KARYV isolated from Hyalomma ticks is critical for the development of specific serological and molecular assays that can be used to determine the association of these viruses with disease in humans and livestock.

  • Equine Influenza Virus in Asia: Phylogeographic Pattern and Molecular Features Reveal Circulation of an Autochthonous Lineage [Genetic Diversity and Evolution]

  • Equine influenza virus (EIV) causes severe acute respiratory disease in horses. Currently, the strains belonging to the H3N8 subtype are divided into two clades, Florida clade 1 (FC1) and Florida clade 2 (FC2), which emerged in 2002. Both FC1 and FC2 clades were reported in Asian and Middle East countries in the last decade. In this study, we described the evolution, epidemiology, and molecular characteristic of the EIV lineages, with focus on those detected in Asia from 2007 to 2017. The full genome phylogeny showed that FC1 and FC2 constituted separate and divergent lineages, without evidence of reassortment between the clades. While FC1 evolved as a single lineage, FC2 showed a divergent event around 2004 giving rise to two well-supported and coexisting sublineages, European and Asian. Furthermore, two different spread patterns of EIV in Asian countries were identified. The FC1 outbreaks were caused by independent introductions of EIV from the Americas, with the Asian isolates genetically similar to the contemporary American lineages. On the other hand, the FC2 strains detected in Asian mainland countries conformed to an autochthonous monophyletic group with a common ancestor dated in 2006 and showed evidence of an endemic circulation in a local host. Characteristic aminoacidic signature patterns were detected in all viral proteins in both Asian-FC1 and FC2 populations. Several changes were located at the top of the HA1 protein, inside or near antigenic sites. Further studies are needed to assess the potential impact of these antigenic changes in vaccination programs.

    IMPORTANCE The complex and continuous antigenic evolution of equine influenza viruses (EIVs) remains a major hurdle for vaccine development and the design of effective immunization programs. The present study provides a comprehensive analysis showing the EIV evolutionary dynamics, including the spread and circulation within the Asian continent and its relationship to global EIV populations over a 10-year period. Moreover, we provide a better understanding of EIV molecular evolution in Asian countries and its consequences on the antigenicity. The study underscores the association between the global horse movement and the circulation of EIV in this region. Understanding EIV evolution is imperative in order to mitigate the risk of outbreaks affecting the horse industry and to help with the selection of the viral strains to be included in the formulation of future vaccines.

  • Phosphorylation of Human Papillomavirus Type 16 L2 Contributes to Efficient Virus Infectious Entry [Virus-Cell Interactions]

  • The human papillomavirus (HPV) capsid comprises two viral proteins, L1 and L2, with the L2 component being essential to ensure efficient endocytic transport of incoming viral genomes. Several studies have previously reported that L1 and L2 are posttranslationally modified, but it is uncertain whether these modifications affect HPV infectious entry. Using a proteomic screen, we identified a highly conserved phospho-acceptor site on the HPV-16 and bovine papillomavirus 1 (BPV-1) L2 proteins. The phospho-modification of L2 and its presence in HPV pseudovirions (PsVs) were confirmed using anti-phospho-L2-specific antibodies. Mutation of the phospho-acceptor sites of both HPV-16 and BPV-1 L2 resulted in the production of infectious virus particles, with no differences in efficiencies of packaging the reporter DNA. However, these mutated PsVs showed marked defects in infectious entry. Further analysis revealed a defect in uncoating, characterized by a delay in the exposure of a conformational epitope on L1 that indicates capsid uncoating. This uncoating defect was accompanied by a delay in the proteolysis of both L1 and L2 in mutated HPV-16 PsVs. Taken together, these studies indicate that phosphorylation of L2 during virus assembly plays an important role in optimal uncoating of virions during infection, suggesting that phosphorylation of the viral capsid proteins contributes to infectious entry.

    IMPORTANCE The papillomavirus L2 capsid protein plays an essential role in infectious entry, where it directs the successful trafficking of incoming viral genomes to the nucleus. However, nothing is known about how potential posttranslational modifications may affect different aspects of capsid assembly or infectious entry. In this study, we report the first phospho-specific modification of the BPV-1 and HPV-16 L2 capsid proteins. The phospho-acceptor site is very highly conserved across multiple papillomavirus types, indicating a highly conserved function within the L2 protein and the viral capsid. We show that this modification plays an essential role in infectious entry, where it modulates susceptibility of the incoming virus to capsid disassembly. These studies therefore define a completely new means of regulating the papillomavirus L2 proteins, a regulation that optimizes endocytic processing and subsequent completion of the infectious entry pathway.

  • Deep Mutational Scan of the Highly Conserved Influenza A Virus M1 Matrix Protein Reveals Substantial Intrinsic Mutational Tolerance [Genetic Diversity and Evolution]

  • Influenza A virus matrix protein M1 is involved in multiple stages of the viral infectious cycle. Despite its functional importance, our present understanding of this essential viral protein is limited. The roles of a small subset of specific amino acids have been reported, but a more comprehensive understanding of the relationship between M1 sequence, structure, and virus fitness remains elusive. In this study, we used deep mutational scanning to measure the effect of every amino acid substitution in M1 on viral replication in cell culture. The map of amino acid mutational tolerance we have generated allows us to identify sites that are functionally constrained in cell culture as well as sites that are less constrained. Several sites that exhibit low tolerance to mutation have been found to be critical for M1 function and production of viable virions. Surprisingly, significant portions of the M1 sequence, especially in the C-terminal domain, whose structure is undetermined, were found to be highly tolerant of amino acid variation, despite having extremely low levels of sequence diversity among natural influenza virus strains. This unexpected discrepancy indicates that not all sites in M1 that exhibit high sequence conservation in nature are under strong constraint during selection for viral replication in cell culture.

    IMPORTANCE The M1 matrix protein is critical for many stages of the influenza virus infection cycle. Currently, we have an incomplete understanding of this highly conserved proteinrrsquo;s function and structure. Key regions of M1, particularly in the C terminus of the protein, remain poorly characterized. In this study, we used deep mutational scanning to determine the extent of M1rrsquo;s tolerance to mutation. Surprisingly, nearly two-thirds of the M1 sequence exhibits a high tolerance for substitutions, contrary to the extremely low sequence diversity observed across naturally occurring M1 isolates. Sites with low mutational tolerance were also identified, suggesting that they likely play critical functional roles and are under selective pressure. These results reveal the intrinsic mutational tolerance throughout M1 and shape future inquiries probing the functions of this essential influenza A virus protein.

  • First Crystal Structure of a Nonstructural Hepatitis E Viral Protein Identifies a Putative Novel Zinc-Binding Protein [Structure and Assembly]

  • Hepatitis E virus (HEV) is a 7.2-kb positive-sense, single-stranded RNA virus containing three partially overlapping reading frames, ORF1 to ORF3. All nonstructural proteins required for viral replication are encoded by ORF1 and are transcribed as a single transcript. Computational analysis of the complete ORF1 polyprotein identified a previously uncharacterized region of predicted secondary structure bordered by two disordered regions coinciding partially with a region predicted as a putative cysteine protease. Following successful cloning, expression, and purification of this region, the crystal structure of the identified protein was determined and identified to have considerable structural homology to a fatty acid binding domain. Further analysis of the structure revealed a metal binding site, shown unambiguously to specifically bind zinc via a nonclassical, potentially catalytic zinc-binding motif. Based on the structural homology of the HEV protein with known structures, along with the presence of a catalytic zinc-binding motif, it is possible that the identified protein corresponds to the HEV protease, which could require activation or repression through the binding of a fatty acid. This represents a significant step forward in the characterization and the understanding of the molecular mechanisms of the HEV genome. We present analysis for the first time of this identified nonstructural protein, expanding the knowledge and understanding of the complex mechanisms of HEV biology.

    IMPORTANCE Hepatitis E virus (HEV) is an emerging virus found predominately in developing countries; it causes an estimated 20 million infections, which result in approximately 57,000 deaths a year. Although it is known that the nonstructural proteins of HEV ORF1 are expressed as a single transcript, there is debate as to whether ORF1 functions as a single polyprotein or if it is processed into separate domains via a viral or endogenous cellular protease. Here we present the first structural and biophysical characterization of an HEV nonstructural protein using a construct that has partially overlapping boundaries with the predicted putative cysteine protease.

  • Fundamental Contribution and Host Range Determination of ANP32A and ANP32B in Influenza A Virus Polymerase Activity [Virus-Cell Interactions]

  • The polymerase of the influenza virus is part of the key machinery necessary for viral replication. However, the avian influenza virus polymerase is restricted in mammalian cells. The cellular protein ANP32A has been recently found to interact with viral polymerase and to influence both polymerase activity and interspecies restriction. We report here that either human ANP32A or ANP32B is indispensable for human influenza A virus RNA replication. The contribution of huANP32B is equal to that of huANP32A, and together they play a fundamental role in the activity of human influenza A virus polymerase, while neither human ANP32A nor ANP32B supports the activity of avian viral polymerase. Interestingly, we found that avian ANP32B was naturally inactive, leaving avian ANP32A alone to support viral replication. Two amino acid mutations at sites 129 to 130 in chicken ANP32B lead to the loss of support of viral replication and weak interaction with the viral polymerase complex, and these amino acids are also crucial in the maintenance of viral polymerase activity in other ANP32 proteins. Our findings strongly support ANP32A and ANP32B as key factors for both virus replication and adaptation.

    IMPORTANCE The key host factors involved in the influenza A viral polymerase activity and RNA replication remain largely unknown. We provide evidence here that ANP32A and ANP32B from different species are powerful factors in the maintenance of viral polymerase activity. Human ANP32A and ANP32B contribute equally to support human influenza viral RNA replication. However, unlike avian ANP32A, the avian ANP32B is evolutionarily nonfunctional in supporting viral replication because of a mutation at sites 129 and 130. These sites play an important role in ANP32A/ANP32B and viral polymerase interaction and therefore determine viral replication, suggesting a novel interface as a potential target for the development of anti-influenza strategies.

  • Subclinical Cytomegalovirus DNA Is Associated with CD4 T Cell Activation and Impaired CD8 T Cell CD107a Expression in People Living with HIV despite Early Antiretroviral Therapy [Virus-Cell Interactions]

  • Most people living with HIV (PLWH) are coinfected with cytomegalovirus (CMV). Subclinical CMV replication is associated with immune dysfunction and with increased HIV DNA in antiretroviral therapy (ART)-naive and -suppressed PLWH. To identify immunological mechanisms by which CMV could favor HIV persistence, we analyzed 181 peripheral blood mononuclear cell (PBMC) samples from 64 PLWH starting ART during early HIV infection with subsequent virologic suppression up to 58 months. In each sample, we measured levels of CMV and Epstein-Barr virus (EBV) DNA by droplet digital PCR (ddPCR). We also measured expression of immunological markers for activation (HLA-DR+ CD38+), cycling (Ki-67+), degranulation (CD107a+), and the immune checkpoint protein PD-1 on CD4+ and CD8+ T cell memory subsets. Significant differences in percentages of lymphocyte markers by CMV/EBV shedding were identified using generalized linear mixed-effects models. Overall, CMV DNA was detected at 60/181 time points. At the time of ART initiation, the presence of detectable CMV DNA was associated with increased CD4+ T cell activation and CD107a expression and with increased CD8+ T cellular cycling and reduced CD107a expression on CD8+ T cells. While some effects disappeared during ART, greater CD4+ T cell activation and reduced CD107a expression on CD8+ T cells persisted when CMV was present (P llt; 0.01). In contrast, EBV was not associated with any immunological differences. Among the covariates, peak HIV RNA and CD4/CD8 ratio had the most significant effect on the immune system. In conclusion, our study identified immune differences in PLWH with detectable CMV starting early ART, which may represent an additional hurdle for HIV cure efforts.

    IMPORTANCE Chronic viral infections such as with HIV and CMV last a lifetime and can continually antagonize the immune system. Both viruses are associated with higher expression of inflammation markers, and recent evidence suggests that CMV may complicate efforts to deplete HIV reservoirs. Our group and others have shown that CMV shedding is associated with a larger HIV reservoir. Subclinical CMV replication could favor HIV persistence via bystander effects on our immune system. In this study, we collected longitudinal PBMC samples from people starting ART and measured immune changes associated with detectable CMV. We found that when CMV was detectable, CD4+ T cell activation was higher and CD8+ T cell degranulation was lower. Both results may contribute to the slower decay of the size of the reservoir during CMV replication, since activated CD4+ T cells are more vulnerable to HIV infection, while the loss of CD8+ T cell degranulation may impede the proper killing of infected cells.

  • Hepatitis B Virus Precore Protein p22 Inhibits Alpha Interferon Signaling by Blocking STAT Nuclear Translocation [Virus-Cell Interactions]

  • Antagonism of host immune defenses against hepatitis B virus (HBV) infection by the viral proteins is speculated to cause HBV persistence and the development of chronic hepatitis. The circulating hepatitis B e antigen (HBeAg, p17) is known to manipulate host immune responses to assist in the establishment of persistent viral infection, and HBeAg-positive (HBeAg+) patients respond less effectively to IFN-aalpha; therapy than do HBeAg-negative (HBeAgnndash;) patients in clinical practice. However, the function(s) of the intracellular form of HBeAg, previously reported as the precore protein intermediate (p22) without the N-terminal signal peptide, remains elusive. Here, we report that the cytosolic p22 protein, but not the secreted HBeAg, significantly reduces interferon-stimulated response element (ISRE) activity and the expression of interferon-stimulated genes (ISGs) upon alpha interferon (IFN-aalpha;) stimulation in cell cultures. In line with this, HBeAg+ patients exhibit weaker induction of ISGs in their livers than do HBeAgnndash; patients upon IFN-aalpha; therapy. Mechanistically, while p22 does not alter the total STAT1 or pSTAT1 levels in cells treated with IFN-aalpha;, it blocks the nuclear translocation of pSTAT1 by interacting with the nuclear transport factor karyopherin aalpha;1 through its C-terminal arginine-rich domain. In summary, our study suggests that HBV precore protein, specifically the p22 form, impedes JAK-STAT signaling to help the virus evade the host innate immune response and, thus, causes resistance to IFN therapy.

    IMPORTANCE Chronic hepatitis B virus (HBV) infection continues to be a major global health concern, and patients who fail to mount an efficient immune response to clear the virus will develop a life-long chronic infection that can progress to chronic active hepatitis, cirrhosis, and primary hepatocellular carcinoma. There is no definite cure for chronic hepatitis B, and alpha interferon (IFN-aalpha;) is the only available immunomodulatory drug, to which only a minority of chronic patients are responsive, with hepatitis B e antigen (HBeAg)-negative patients responding better than HBeAg-positive patients. We herein report that the intracellular HBeAg, also known as precore or p22, inhibits the antiviral signaling of IFN-aalpha;, which sheds light on the enigmatic function of precore protein in shaping HBV chronicity and provides a perspective toward areas that need to be further studied to make the current therapy better until a cure is achieved.

  • Characterizing a Murine Model for Astrovirus Using Viral Isolates from Persistently Infected Immunocompromised Mice [Pathogenesis and Immunity]

  • Human astroviruses are single-stranded RNA enteric viruses that cause a spectrum of disease ranging from asymptomatic infection to systemic extragastrointestinal spread; however, they are among the least-characterized enteric viruses, and there is a lack of a well-characterized small animal model. Finding that immunocompromised mice were resistant to human astrovirus infection via multiple routes of inoculation, our studies aimed to determine whether murine astrovirus (MuAstV) could be used to model human astrovirus disease. We experimentally infected wild-type mice with MuAstV isolated from immunocompromised mice and found that the virus was detected throughout the gastrointestinal tract, including the stomach, but was not associated with diarrhea. The virus was also detected in the lung. Although virus levels were higher in recently weaned mice, the levels were similar in male and female adult mice. Using two distinct viruses isolated from different immunocompromised mouse strains, we observed virus strain-specific differences in the duration of infection (3 versus 10 weeks) in wild-type mice, indicating that the within-host immune pressure from donor mice shaped the virus kinetics in immunocompetent recipient hosts. Both virus strains elicited minimal pathology and a lack of sustained immunity. In summary, MuAstV represents a useful model for studying asymptomatic human infection and gaining insight into the astrovirus pathogenesis and immunity.

    IMPORTANCE Astroviruses are widespread in both birds and mammals; however, little is known about the pathogenesis and the immune response to the virus due to the lack of a well-characterized small-animal model. Here we describe two distinct strains of murine astrovirus that cause infections in immunocompetent mice that mirror aspects of asymptomatic human infections, including minimal pathology and short-lived immunity. However, we noted that the duration of infection differed greatly between the strains, highlighting an important facet of these viruses that was not previously appreciated. The ubiquitous nature and diversity of murine astroviruses coupled with the continuous likelihood of reinfection raise the possibility of viral interference with other mouse models of disease.

  • RNA Sequencing Analyses of Gene Expression during Epstein-Barr Virus Infection of Primary B Lymphocytes [Virus-Cell Interactions]

  • Epstein-Barr virus (EBV) infection of human primary resting B lymphocytes (RBLs) leads to the establishment of lymphoblastoid cell lines (LCLs) that can grow indefinitely in vitro. EBV transforms RBLs through the expression of viral latency genes, and these genes alter host transcription programs. To globally measure the transcriptome changes during EBV transformation, primary human resting B lymphocytes (RBLs) were infected with B95.8 EBV for 0, 2, 4, 7, 14, 21, and 28 days, and poly(A) plus RNAs were analyzed by transcriptome sequencing (RNA-seq). Analyses of variance (ANOVAs) found 3,669 protein-coding genes that were differentially expressed (false-discovery rate [FDR] llt; 0.01). Ninety-four percent of LCL genes that are essential for LCL growth and survival were differentially expressed. Pathway analyses identified a significant enrichment of pathways involved in cell proliferation, DNA repair, metabolism, and antiviral responses. RNA-seq also identified long noncoding RNAs (lncRNAs) differentially expressed during EBV infection. Clustered regularly interspaced short palindromic repeat (CRISPR) interference (CRISPRi) and CRISPR activation (CRISPRa) found that CYTOR and NORAD lncRNAs were important for LCL growth. During EBV infection, type III EBV latency genes were expressed rapidly after infection. Immediately after LCL establishment, EBV lytic genes were also expressed in LCLs, and ~4% of the LCLs express gp350. Chromatin immune precipitation followed by deep sequencing (ChIP-seq) and POLR2A chromatin interaction analysis followed by paired-end tag sequencing (ChIA-PET) data linked EBV enhancers to 90% of EBV-regulated genes. Many genes were linked to enhancers occupied by multiple EBNAs or NF-B subunits. Incorporating these assays, we generated a comprehensive EBV regulome in LCLs.

    IMPORTANCE Epstein-Barr virus (EBV) immortalization of resting B lymphocytes (RBLs) is a useful model system to study EBV oncogenesis. By incorporating transcriptome sequencing (RNA-seq), chromatin immune precipitation followed by deep sequencing (ChIP-seq), chromatin interaction analysis followed by paired-end tag sequencing (ChIA-PET), and genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen, we identified key pathways that EBV usurps to enable B cell growth and transformation. Multiple layers of regulation could be achieved by cooperations between multiple EBV transcription factors binding to the same enhancers. EBV manipulated the expression of most cell genes essential for lymphoblastoid cell line (LCL) growth and survival. In addition to proteins, long noncoding RNAs (lncRNAs) regulated by EBV also contributed to LCL growth and survival. The data presented in this paper not only allowed us to further define the molecular pathogenesis of EBV but also serve as a useful resource to the EBV research community.

  • Oral Vaccination with Replication-Competent Adenovirus in Mice Reveals Dissemination of the Viral Vaccine beyond the Gastrointestinal Tract [Vaccines and Antiviral Agents]

  • Since the 1970s, replication-competent human adenoviruses 4 and 7 have been used as oral vaccines to protect U.S. soldiers against the severe respiratory diseases caused by these viruses. These vaccines are thought to establish a digestive tract infection conferring protection against respiratory challenge through antibodies. The success of these vaccines makes replication-competent adenoviruses attractive candidates for use as oral vaccine vectors. However, the inability of human adenoviruses to replicate efficiently in laboratory animals has hampered the study of such vectors. Here, we used mouse adenovirus type 1 (MAV-1) in mice to study oral replication-competent adenovirus-based vaccines. We show that MAV-1 oral administration provides protection that recapitulates the protection against homologous respiratory challenge observed with adenovirus 4 and 7 vaccines. Moreover, live oral MAV-1 vaccine better protected against a respiratory challenge than inactivated vaccines. This protection was linked not only with the presence of MAV-1-specific antibodies but also with a better recruitment of effector CD8 T cells. However, unexpectedly, we found that such oral replication-competent vaccine systemically spread all over the body. Our results therefore support the use of MAV-1 to study replication-competent oral adenovirus-based vaccines but also highlight the fact that those vaccines can disseminate widely in the body.

    IMPORTANCE Replication-competent adenoviruses appear to be promising vectors for the development of oral vaccines in humans. However, the study and development of these vaccines suffer from the lack of any reliable animal model. In this study, mouse adenovirus type 1 was used to develop a small-animal model for oral replication-competent adenovirus vaccines. While this model reproduced in mice what is observed with human adenovirus oral vaccines, it also highlighted that oral immunization with such a replication-competent vaccine is associated with the systemic spread of the virus. This study is therefore of major importance for the future development of such vaccine platforms and their use in large human populations.

  • Mechanisms of B-Cell Oncogenesis Induced by Epstein-Barr Virus [Gem]

  • Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus which asymptomatically infects the majority of the world population. Under immunocompromised conditions, EBV can trigger human cancers of epithelial and lymphoid origin. The oncogenic potential of EBV is demonstrated by in vitro infection and transformation of quiescent B cells into lymphoblastoid cell lines (LCLs). These cell lines, along with primary infection using genetically engineered viral particles coupled with recent technological advancements, have elucidated the underlying mechanisms of EBV-induced B-cell lymphomagenesis.

  • Reduced Folate Carrier: an Entry Receptor for a Novel Feline Leukemia Virus Variant [Virus-Cell Interactions]

  • Feline leukemia virus (FeLV) is horizontally transmitted among cats and causes a variety of hematopoietic disorders. Five subgroups of FeLV, A to D and T, each with distinct receptor usages, have been described. Recently, we identified a new FeLV Env (TG35-2) gene from a pseudotyped virus that does not belong to any known subgroup. FeLV-A is the primary virus from which other subgroups have emerged via mutation or recombination of the subgroup A env gene. Retrovirus entry into cells is mediated by the interaction of envelope protein (Env) with specific cell surface receptors. Here, phenotypic screening of a human/hamster radiation hybrid panel identified SLC19A1, a feline reduced folate carrier (RFC) and potential receptor for TG35-2-phenotypic virus. RFC is a multipass transmembrane protein. Feline and human RFC cDNAs conferred susceptibility to TG35-2-pseudotyped virus when introduced into nonpermissive cells but did not render these cells permissive to other FeLV subgroups or feline endogenous retrovirus. Moreover, human cells with genomic deletion of RFC were nonpermissive for TG35-2-pseudotyped virus infection, but the introduction of feline and human cDNAs rendered them permissive. Mutation analysis of FeLV Env demonstrated that amino acid substitutions within variable region A altered the specificity of the Env-receptor interaction. We isolated and reconstructed the full-length infectious TG35-2-phenotypic provirus from a naturally FeLV-infected cat, from which the FeLV Env (TG35-2) gene was previously isolated, and compared the replication of the virus in hematopoietic cell lines with that of FeLV-A 61E by measuring the viral RNA copy numbers. These results provide a tool for further investigation of FeLV infectious disease.

    IMPORTANCE Feline leukemia virus (FeLV) is a member of the genus Gammaretrovirus, which causes malignant diseases in cats. The most prevalent FeLV among cats is FeLV subgroup A (FeLV-A), and specific binding of FeLV-A Env to its viral receptor, thiamine transporter feTHTR1, is the first step of infection. In infected cats, novel variants of FeLV with altered receptor specificity for viral entry have emerged by mutation or recombination of the env gene. A novel FeLV variant arose from a subtle mutation of FeLV-A Env, which altered the specific interaction of the virus with its receptor. RFC, a folate transporter, is a potential receptor for the novel FeLV variant. The perturbation of specific retrovirus-receptor interactions under selective pressure by the host results in the emergence of novel viruses.

  • Interplay between the Epigenetic Enzyme Lysine (K)-Specific Demethylase 2B and Epstein-Barr Virus Infection [Virus-Cell Interactions]

  • The histone modifier lysine (K)-specific demethylase 2B (KDM2B) plays a role in the differentiation of hematopoietic cells, and its expression appears to be deregulated in certain cancers of hematological and lymphoid origins. We have previously found that the KDM2B gene is differentially methylated in cell lines derived from Epstein-Barr virus (EBV)-associated endemic Burkitt lymphoma (eBL) compared with that in EBV-negative sporadic Burkitt lymphoma-derived cells. However, whether KDM2B plays a role in eBL development has not been previously investigated. Oncogenic viruses have been shown to hijack the host cell epigenome to complete their life cycle and to promote the transformation process by perturbing cell chromatin organization. Here, we investigated whether EBV alters KDM2B levels to enable its life cycle and promote B-cell transformation. We show that infection of B cells with EBV leads to downregulation of KDM2B levels. We also show that LMP1, one of the main EBV transforming proteins, induces increased DNMT1 recruitment to the KDM2B gene and augments its methylation. By altering KDM2B levels and performing chromatin immunoprecipitation in EBV-infected B cells, we show that KDM2B is recruited to the EBV gene promoters and inhibits their expression. Furthermore, forced KDM2B expression in immortalized B cells led to altered mRNA levels of some differentiation-related genes. Our data show that EBV deregulates KDM2B levels through an epigenetic mechanism and provide evidence for a role of KDM2B in regulating virus and host cell gene expression, warranting further investigations to assess the role of KDM2B in the process of EBV-mediated lymphomagenesis.

    IMPORTANCE In Africa, Epstein-Barr virus infection is associated with endemic Burkitt lymphoma, a pediatric cancer. The molecular events leading to its development are poorly understood compared with those leading to sporadic Burkitt lymphoma. In a previous study, by analyzing the DNA methylation changes in endemic compared with sporadic Burkitt lymphoma cell lines, we identified several differential methylated genomic positions in the proximity of genes with a potential role in cancer, and among them was the KDM2B gene. KDM2B encodes a histone H3 demethylase already shown to be involved in some hematological disorders. However, whether KDM2B plays a role in the development of Epstein-Barr virus-mediated lymphoma has not been investigated before. In this study, we show that Epstein-Barr virus deregulates KDM2B expression and describe the underlying mechanisms. We also reveal a role of the demethylase in controlling viral and B-cell gene expression, thus highlighting a novel interaction between the virus and the cellular epigenome.

  • Aerosol Transmission of Gull-Origin Iceland Subtype H10N7 Influenza A Virus in Ferrets [Pathogenesis and Immunity]

  • Subtype H10 influenza A viruses (IAVs) have been recovered from domestic poultry and various aquatic bird species, and sporadic transmission of these IAVs from avian species to mammals (i.e., human, seal, and mink) are well documented. In 2015, we isolated four H10N7 viruses from gulls in Iceland. Genomic analyses showed four gene segments in the viruses were genetically associated with H10 IAVs that caused influenza outbreaks and deaths among European seals in 2014. Antigenic characterization suggested minimal antigenic variation among these H10N7 isolates and other archived H10 viruses recovered from human, seal, mink, and various avian species in Asia, Europe, and North America. Glycan binding preference analyses suggested that, similar to other avian-origin H10 IAVs, these gull-origin H10N7 IAVs bound to both avian-like alpha 2,3-linked sialic acids and human-like alpha 2,6-linked sialic acids. However, when the gull-origin viruses were compared with another Eurasian avian-origin H10N8 IAV, which caused human infections, the gull-origin virus showed significantly higher binding affinity to human-like glycan receptors. Results from a ferret experiment demonstrated that a gull-origin H10N7 IAV replicated well in turbinate, trachea, and lung, but replication was most efficient in turbinate and trachea. This gull-origin H10N7 virus can be transmitted between ferrets through the direct contact and aerosol routes, without prior adaptation. Gulls share their habitat with other birds and mammals and have frequent contact with humans; therefore, gull-origin H10N7 IAVs could pose a risk to public health. Surveillance and monitoring of these IAVs at the wild bird-human interface should be continued.

    IMPORTANCE Subtype H10 avian influenza A viruses (IAVs) have caused sporadic human infections and enzootic outbreaks among seals. In the fall of 2015, H10N7 viruses were recovered from gulls in Iceland, and genomic analyses showed that the viruses were genetically related with IAVs that caused outbreaks among seals in Europe a year earlier. These gull-origin viruses showed high binding affinity to human-like glycan receptors. Transmission studies in ferrets demonstrated that the gull-origin IAV could infect ferrets, and that the virus could be transmitted between ferrets through direct contact and aerosol droplets. This study demonstrated that avian H10 IAV can infect mammals and be transmitted among them without adaptation. Thus, avian H10 IAV is a candidate for influenza pandemic preparedness and should be monitored in wildlife and at the animal-human interface.

  • Replication of Marek's Disease Virus Is Dependent on Synthesis of De Novo Fatty Acid and Prostaglandin E2 [Virus-Cell Interactions]

  • Marekrrsquo;s disease virus (MDV) causes deadly lymphoma and induces an imbalance of the lipid metabolism in infected chickens. Here, we discovered that MDV activates the fatty acid synthesis (FAS) pathway in primary chicken embryo fibroblasts (CEFs). In addition, MDV-infected cells contained high levels of fatty acids and showed increased numbers of lipid droplets (LDs). Chemical inhibitors of the FAS pathway (TOFA and C75) reduced MDV titers by approximately 30-fold. Addition of the downstream metabolites, including malonyl-coenzyme A and palmitic acid, completely restored the inhibitory effects of the FAS inhibitors. Furthermore, we could demonstrate that MDV infection activates the COX-2/prostaglandin E2 (PGE2) pathway, as evident by increased levels of arachidonic acid, COX-2 expression, and PGE2 synthesis. Inhibition of the COX-2/PGE2 pathway by chemical inhibitors or knockdown of COX2 using short hairpin RNA reduced MDV titers, suggesting that COX-2 promotes virus replication. Exogenous PGE2 completely restored the inhibition of the COX-2/PGE2 pathway in MDV replication. Unexpectedly, exogenous PGE2 also partially rescued the inhibitory effects of FAS inhibitors on MDV replication, suggesting that there is a link between these two pathways in MDV infection. Taken together, our data demonstrate that the FAS and COX-2/PGE2 pathways play an important role in the replication of this deadly pathogen.

    IMPORTANCE Disturbances of the lipid metabolism in chickens infected with MDV contribute to the pathogenesis of disease. However, the role of lipid metabolism in MDV replication remained unknown. Here, we demonstrate that MDV infection activates FAS and induces LD formation. Moreover, our results demonstrate that MDV replication is highly dependent on the FAS pathway and the downstream metabolites. Finally, our results reveal that MDV also activates the COX-2/PGE2 pathway, which supports MDV replication by activating PGE2/EP2 and PGE2/EP4 signaling pathways.

  • Complex Membrane Remodeling during Virion Assembly of the 30,000-Year-Old Mollivirus Sibericum [Virus-Cell Interactions]

  • Cellular membranes ensure functional compartmentalization by dynamic fusion-fission remodeling and are often targeted by viruses during entry, replication, assembly, and egress. Nucleocytoplasmic large DNA viruses (NCLDVs) can recruit host-derived open membrane precursors to form their inner viral membrane. Using complementary three-dimensional (3D)-electron microscopy techniques, including focused-ion beam scanning electron microscopy and electron tomography, we show that the giant Mollivirus sibericum utilizes the same strategy but also displays unique features. Indeed, assembly is specifically triggered by an open cisterna with a flat pole in its center and open curling ends that grow by recruitment of vesicles never reported for NCLDVs. These vesicles, abundant in the viral factory (VF), are initially closed but open once in close proximity to the open curling ends of the growing viral membrane. The flat pole appears to play a central role during the entire virus assembly process. While additional capsid layers are assembled from it, it also shapes the growing cisterna into immature crescent-like virions and is located opposite to the membrane elongation and closure sites, thereby providing virions with a polarity. In the VF, DNA-associated filaments are abundant, and DNA is packed within virions prior to particle closure. Altogether, our results highlight the complexity of the interaction between giant viruses and their host. Mollivirus assembly relies on the general strategy of vesicle recruitment, opening, and shaping by capsid layers similar to all NCLDVs studied until now. However, the specific features of its assembly suggest that the molecular mechanisms for cellular membrane remodeling and persistence are unique.

    IMPORTANCE Since the first giant virus Mimivirus was identified, other giant representatives are isolated regularly around the world and appear to be unique in several aspects. They belong to at least four viral families, and the ways they interact with their hosts remain poorly understood. We focused on Mollivirus sibericum, the sole representative of "Molliviridae," which was isolated from a 30,000-year-old permafrost sample and exhibits spherical virions of complex composition. In particular, we show that (i) assembly is initiated by a unique structure containing a flat pole positioned at the center of an open cisterna, (ii) core packing involves another cisterna-like element seemingly pushing core proteins into particles being assembled, and (iii) specific filamentous structures contain the viral genome before packaging. Altogether, our findings increase our understanding of how complex giant viruses interact with their host and provide the foundation for future studies to elucidate the molecular mechanisms of Mollivirus assembly.

  • Seeking Closure: How Do Herpesviruses Recruit the Cellular ESCRT Apparatus? [Minireviews]

  • The Herpesviridae are structurally complex DNA viruses whose capsids undergo primary envelopment at the inner nuclear membrane and secondary envelopment at organelles in the cytoplasm. In both locations, there is evidence that envelope formation and scission involve the participation of multiple viral proteins and also the cellular ESCRT apparatus. It nevertheless appears that the best-understood viral strategies for ESCRT recruitment, those adopted by the retroviruses and many other families of enveloped RNA viruses, are not utilized by the Herpesviridae, at least during envelopment in the cytoplasm. Thus, although a large number of herpesvirus proteins have been assigned roles in envelopment, there is a dearth of candidates for the acquisition of the ESCRT complex and the control of envelope scission. This review summarizes our current understanding of ESCRT association by enveloped viruses, examines what is known of herpesvirus ESCRT utilization in the nucleus and cytoplasm, and identifies candidate cellular and viral proteins that could link enveloping herpesviruses to cellular ESCRT components.

  • Aedes aegypti NeSt1 Protein Enhances Zika Virus Pathogenesis by Activating Neutrophils [Pathogenesis and Immunity]

  • Saliva from the mosquito vector of flaviviruses is capable of changing the local immune environment, leading to an increase in flavivirus-susceptible cells at the infected bite site. In addition, an antibody response to specific salivary gland (SG) components changes the pathogenesis of flaviviruses in human populations. To investigate whether antigenic SG proteins are capable of enhancing infection with Zika virus (ZIKV), a reemerging flavivirus primarily transmitted by the Aedes aegypti mosquito, we screened for antigenic SG proteins using a yeast display library and demonstrate that a previously undescribed SG protein we term neutrophil stimulating factor 1 (NeSt1) activates primary mouse neutrophils ex vivo. Passive immunization against NeSt1 decreases pro-interleukin-1bbeta; and CXCL2 expression, prevents macrophages from infiltrating the bite site, protects susceptible IFNARnndash;/nndash; IFNGRnndash;/nndash; (AG129) mice from early ZIKV replication, and ameliorates virus-induced pathogenesis. These findings indicate that NeSt1 stimulates neutrophils at the mosquito bite site to change the immune microenvironment, allowing a higher level of early viral replication and enhancing ZIKV pathogenesis.

    IMPORTANCE When a Zika virus-infected mosquito bites a person, mosquito saliva is injected into the skin along with the virus. Molecules in this saliva can make virus infection more severe by changing the immune system to make the skin a better place for the virus to replicate. We identified a molecule that activates immune cells, called neutrophils, to recruit other immune cells, called macrophages, that the virus can infect. We named this molecule neutrophil-stimulating factor 1 (NeSt1). When we used antibodies to block NeSt1 in mice and then allowed Zika virus-infected mosquitoes to feed on these mice, they survived much better than mice that do not have antibodies against NeSt1. These findings give us more information about how mosquito saliva enhances virus infection, and it is possible that a vaccine against NeSt1 might protect people against severe Zika virus infection.

  • Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication [Virus-Cell Interactions]

  • Here, we show that the cellular DNA replication protein and ATR substrate SMARCAL1 is recruited to viral replication centers early during adenovirus infection and is then targeted in an E1B-55K/E4orf6- and cullin RING ligase-dependent manner for proteasomal degradation. In this regard, we have determined that SMARCAL1 is phosphorylated at S123, S129, and S173 early during infection in an ATR- and CDK-dependent manner, and that pharmacological inhibition of ATR and CDK activities attenuates SMARCAL1 degradation. SMARCAL1 recruitment to viral replication centers was shown to be largely dependent upon SMARCAL1 association with the RPA complex, while Ad-induced SMARCAL1 phosphorylation also contributed to SMARCAL1 recruitment to viral replication centers, albeit to a limited extent. SMARCAL1 was found associated with E1B-55K in adenovirus E1-transformed cells. Consistent with its ability to target SMARCAL1, we determined that E1B-55K modulates cellular DNA replication. As such, E1B-55K expression initially enhances cellular DNA replication fork speed but ultimately leads to increased replication fork stalling and the attenuation of cellular DNA replication. Therefore, we propose that adenovirus targets SMARCAL1 for degradation during infection to inhibit cellular DNA replication and promote viral replication.

    IMPORTANCE Viruses have evolved to inhibit cellular DNA damage response pathways that possess antiviral activities and utilize DNA damage response pathways that possess proviral activities. Adenovirus has evolved, primarily, to inhibit DNA damage response pathways by engaging with the ubiquitin-proteasome system and promoting the degradation of key cellular proteins. Adenovirus differentially regulates ATR DNA damage response signaling pathways during infection. The cellular adenovirus E1B-55K binding protein E1B-AP5 participates in ATR signaling pathways activated during infection, while adenovirus 12 E4orf6 negates Chk1 activation by promoting the proteasome-dependent degradation of the ATR activator TOPBP1. The studies detailed here indicate that adenovirus utilizes ATR kinase and CDKs during infection to promote the degradation of SMARCAL1 to attenuate normal cellular DNA replication. These studies further our understanding of the relationship between adenovirus and DNA damage and cell cycle signaling pathways during infection and establish new roles for E1B-55K in the modulation of cellular DNA replication.

  • The CCCTC Binding Factor, CTRL2, Modulates Heterochromatin Deposition and the Establishment of Herpes Simplex Virus 1 Latency In Vivo [Genome Replication and Regulation of Viral Gene Expression]

  • The cellular insulator protein CTCF plays a role in herpes simplex virus 1 (HSV-1) latency through the establishment and regulation of chromatin boundaries. We previously found that the CTRL2 regulatory element downstream from the latency-associated transcript (LAT) enhancer was bound by CTCF during latency and underwent CTCF eviction at early times postreactivation in mice latently infected with 17syn+ virus. We also showed that CTRL2 was a functional enhancer-blocking insulator in both epithelial and neuronal cell lines. We hypothesized that CTRL2 played a direct role in silencing lytic gene expression during the establishment of HSV-1 latency. To test this hypothesis, we used a recombinant virus with a 135-bp deletion spanning only the core CTRL2 insulator domain (CTRL2) in the 17syn+ background. Deletion of CTRL2 resulted in restricted viral replication in epithelial cells but not neuronal cells. Following ocular infection, mouse survival decreased in the CTRL2-infected cohort, and we found a significant decrease in the number of viral genomes in mouse trigeminal ganglia (TG) infected with CTRL2, indicating that the CTRL2 insulator was required for the efficient establishment of latency. Immediate early (IE) gene expression significantly increased in the number of ganglia infected with CTRL2 by 31 days postinfection relative to the level with 17syn+ infection, indicating that deletion of the CTRL2 insulator disrupted the organization of chromatin domains during HSV-1 latency. Finally, chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) analyses of TG from CTRL2-infected mice confirmed that the distribution of the repressive H3K27me3 (histone H3 trimethylated at K27) mark on the CTRL2 recombinant genomes was altered compared to that of the wild type, indicating that the CTRL2 site modulates the repression of IE genes during latency.

    IMPORTANCE It is becoming increasingly clear that chromatin insulators play a key role in the transcriptional control of DNA viruses. The gammaherpesviruses Epstein-Barr virus (EBV) and Kaposirrsquo;s sarcoma-associated herpesvirus (KSHV) utilize chromatin insulators to order protein recruitment and dictate the formation of three-dimensional DNA loops that spatially control transcription and latency. The contribution of chromatin insulators in alphaherpesvirus transcriptional control is less well understood. The work presented here begins to bridge that gap in knowledge by showing how one insulator site in HSV-1 modulates lytic gene transcription and heterochromatin deposition as the HSV-1 genome establishes latency.

  • Antagonizing the Glucocorticoid Receptor Impairs Explant-Induced Reactivation in Mice Latently Infected with Herpes Simplex Virus 1 [Virus-Cell Interactions]

  • Herpes simplex virus 1 (HSV-1) establishes lifelong latent infections in neurons. Reactivation from latency can lead to serious recurrent disease, including stromal keratitis, corneal scarring, blindness, and encephalitis. Although numerous studies link stress to an increase in the incidence of reactivation from latency and recurrent disease, the mechanism of action is not well understood. We hypothesized that stress, via corticosteroid-mediated activation of the glucocorticoid receptor (GR), stimulates viral gene expression and productive infection during reactivation from latency. Consequently, we tested whether GR activation by the synthetic corticosteroid dexamethasone influenced virus shedding during reactivation from latency using trigeminal ganglion (TG) explants from Swiss Webster mice latently infected with HSV-1, strain McKrae. TG explants from the latently infected mice shed significantly higher levels of virus when treated with dexamethasone. Conversely, virus shedding from TG explants was significantly impaired when they were incubated with medium containing a GR-specific antagonist (CORT-108297) or stripped fetal bovine serum, which lacks nuclear hormones and other growth factors. TG explants from latently infected, but not uninfected, TG contained significantly more GR-positive neurons following explant when treated with dexamethasone. Strikingly, VP16 protein expression was detected in TG neurons at 8 hours after explant whereas infected-cell protein 0 (ICP0) and ICP4 protein expression was not readily detected until 16 hours after explant. Expression of all three viral regulatory proteins was stimulated by dexamethasone. These studies indicated corticosteroid-mediated GR activation increased the number of TG neurons expressing viral regulatory proteins, which enhanced virus shedding during explant-induced reactivation from latency.

    IMPORTANCE Herpes simplex virus 1 (HSV-1) establishes lifelong latent infections in neurons within trigeminal ganglia (TG); periodically, reactivation from latency occurs, leading to virus transmission and recurrent disease. Chronic or acute stress increases the frequency of reactivation from latency; how this occurs is not well understood. Here, we demonstrate that the synthetic corticosteroid dexamethasone stimulated explant-induced reactivation from latency. Conversely, a glucocorticoid receptor (GR) antagonist significantly impaired reactivation from latency, indicating that GR activation stimulated explant-induced reactivation. The viral regulatory protein VP16 was readily detected in TG neurons prior to infected-cell protein 0 (ICP0) and ICP4 during explant-induced reactivation. Dexamethasone induced expression of all three viral regulatory proteins following TG explant. Whereas the immunosuppressive properties of corticosteroids would facilitate viral spread during reactivation from latency, these studies indicate GR activation increases the number of TG neurons that express viral regulatory proteins during early stages of explant-induced reactivation.

  • Correction for Sun et al., "Risk Assessment of Fifth-Wave H7N9 Influenza A Viruses in Mammalian Models" [Author Correction]

  • Roles of Type 1, 2, and 3 Innate Lymphoid Cells in Herpes Simplex Virus 1 Infection In Vitro and In Vivo [Pathogenesis and Immunity]

  • Innate lymphoid cells (ILCs) play important roles in host defense and inflammation. They are classified into three distinct groups based on their cytokine and chemokine secretion patterns and transcriptome profiles. Here, we show that ILCs isolated from mice can be infected with herpes simplex virus 1 (HSV-1) but that subsequent replication of the virus is compromised. After infection, type 2 ILCs expressed significantly higher levels of granulocyte colony-stimulating factor (G-CSF), interleukin 1aalpha; (IL-1aalpha;), IL-6, IL-9, RANTES, tumor necrosis factor alpha (TNF-aalpha;), CXCL1, CXCL2, CXCL10, CCL3, and CCL4 than infected type 1 or type 3 ILCs. Transcriptome-sequencing (RNA-seq) analysis of the ILCs 24 h after HSV-1 infection revealed that 77 herpesvirus genes were detected in the infected type 3 ILCs, whereas only 11 herpesvirus genes were detected in infected type 1 ILCs and 27 in infected type 2 ILCs. Compared with uninfected cells, significant upregulation of over 4,000 genes was seen in the HSV-1-infected type 3 ILCs, whereas 414 were upregulated in the infected type 1 ILCs and 128 in the infected type 2 ILCs. In contrast, in all three cell types, only a limited number of genes were significantly downregulated. Type 1, type 2, and type 3 ILC-deficient mice were used to gain insights into the effects of the ILCs on the outcome of ocular HSV-1 infection. No significant differences were found on comparison with similarly infected wild-type mice or on comparison of the three strains of deficient mice in terms of virus replication in the eyes, levels of corneal scarring, latency-reactivation in the trigeminal ganglia, or T-cell exhaustion. Although there were no significant differences in the survival rates of infected ILC-deficient mice and wild-type mice, there was significantly reduced survival of the infected type 1 or type 3 ILC-deficient mice compared with type 2 ILC-deficient mice. Adoptive transfer of wild-type T cells did not alter survival or any other parameters tested in the infected mice. Our results indicate that type 1, 2, and 3 ILCs respond differently to HSV-1 infection in vitro and that the absence of type 1 or type 3, but not type 2, ILCs affects the survival of ocularly infected mice.

    IMPORTANCE In this study, we investigated for the first time what roles, if any, innate lymphoid cells (ILCs) play in HSV-1 infection. Analysis of isolated ILCs in vitro revealed that all three subtypes could be infected with HSV-1 but that they were resistant to replication. The expression profiles of HSV-1-induced cytokines/chemokines and cellular and viral genes differed among the infected type 1, 2, and 3 ILCs in vitro. While ILCs play no role or a redundant role in the outcomes of latency-reactivation in infected mice, absence of type 1 and type 3, but not type 2, ILCs affects the survival of infected mice.

  • Phosphoproteome Analysis of Cells Infected with Adapted and Nonadapted Influenza A Virus Reveals Novel Pro- and Antiviral Signaling Networks [Cellular Response to Infection]

  • Influenza A viruses (IAVs) quickly adapt to new environments and are well known to cross species barriers. To reveal a molecular basis for these phenomena, we compared the Ser/Thr and Tyr phosphoproteomes of murine lung epithelial cells early and late after infection with mouse-adapted SC35M virus or its nonadapted SC35 counterpart. With this analysis we identified a large set of upregulated Ser/Thr phosphorylations common to both viral genotypes, while Tyr phosphorylations showed little overlap. Most of the proteins undergoing massive changes of phosphorylation in response to both viruses regulate chromatin structure, RNA metabolism, and cell adhesion, including a focal adhesion kinase (FAK)-regulated network mediating the regulation of actin dynamics. IAV also affected phosphorylation of activation loops of 37 protein kinases, including FAK and several phosphatases, many of which were not previously implicated in influenza virus infection. Inhibition of FAK proved its contribution to IAV infection. Novel phosphorylation sites were found on IAV-encoded proteins, and the functional analysis of selected phosphorylation sites showed that they either support (NA Ser178) or inhibit (PB1 Thr223) virus propagation. Together, these data allow novel insights into IAV-triggered regulatory phosphorylation circuits and signaling networks.

    IMPORTANCE Infection with IAVs leads to the induction of complex signaling cascades, which apparently serve two opposing functions. On the one hand, the virus highjacks cellular signaling cascades in order to support its propagation; on the other hand, the host cell triggers antiviral signaling networks. Here we focused on IAV-triggered phosphorylation events in a systematic fashion by deep sequencing of the phosphoproteomes. This study revealed a plethora of newly phosphorylated proteins. We also identified 37 protein kinases and a range of phosphatases that are activated or inactivated following IAV infection. Moreover, we identified new phosphorylation sites on IAV-encoded proteins. Some of these phosphorylations support the enzymatic function of viral components, while other phosphorylations are inhibitory, as exemplified by PB1 Thr223 modification. Our global characterization of IAV-triggered patterns of phospho-proteins provides a rich resource to further understand host responses to infection at the level of phosphorylation-dependent signaling networks.

  • Human Astrovirus MLB Replication In Vitro: Persistence in Extraintestinal Cell Lines [Virus-Cell Interactions]

  • MLB astroviruses were identified 10 years ago in feces from children with gastroenteritis of unknown etiology and have been unexpectedly detected in severe cases of meningitis/encephalitis, febrile illness of unknown etiology, and respiratory syndromes. The aim of this study was to establish a cell culture system supporting MLB astrovirus replication. We used two clinical strains to infect several cell lines, an MLB1 strain from a gastroenteritis case, and an MLB2 strain associated with a neurologic infection. Efforts to propagate the viruses in the Caco-2 cell line were unsuccessful. In contrast, we identified two human nonintestinal cell lines, Huh-7 and A549, permissive for both genotypes. After serial passages in the Huh-7.5 cell line, the adapted strains were able to establish persistent infections in the Huh-7.5, Huh-7AI, and A549 cell lines, with high viral loads (up to 10 log10 genome copies/ml) detected by quantitative reverse transcription-PCR (RT-qPCR) in the culture supernatant. Immunofluorescence assays demonstrated infection in about 10% of cells in persistently infected cultures. Electron microscopy revealed particles of 32 to 33 nm in diameter after negative staining of cell supernatants and capsid arrays in ultrathin sections with a particularly high production in Huh-7.5 cells. Interferon (IFN) expression by infected cells and effect of exogenous IFN varied depending on the type of infection and the cell line. The availability of a cell culture system to propagate MLB astroviruses represents a key step to better understand their replicative cycle, as well as a source of viruses to conduct a wide variety of basic virologic studies.

    IMPORTANCE MLB astroviruses are emerging viruses infecting humans. More studies are required to determine their exact epidemiology, but several reports have already identified them as the cause of unexpected clinical diseases, including severe neurologic diseases. Our study provides the first description of a cell culture system for the propagation of MLB astroviruses, enabling the study of their replicative cycle. Moreover, we demonstrated the unknown capacity of MLB astrovirus to establish persistent infections in cell culture. Whether these persistent infections are also established in vivo remains unknown, but the clinical consequences would be of high interest if persistence was confirmed in vivo. Finally, our analysis of IFN expression provides some trails to understand the mechanism by which MLB astroviruses can cause persistent infections in the assayed cultures.

  • Senecavirus-Specific Recombination Assays Reveal the Intimate Link between Polymerase Fidelity and RNA Recombination [Genetic Diversity and Evolution]

  • Senecavirus A (SVA) is a reemerging virus, and recent evidence has emphasized the importance of SVA recombination in vivo on virus evolution. In this study, we report the development of an infectious cDNA clone for the SVA/HLJ/CHA/2016 strain. We used this strain to develop a reporter virus expressing enhanced green fluorescent protein (eGFP), which we then used to screen for a recombination-deficient SVA by an eGFP retention assay. Sequencing of the virus that retained the eGFP following passage allowed us to identify the nonsynonymous mutations (S460L alone and I212V-S460L in combination) in the RNA-dependent RNA polymerase (RdRp) region of the genome. We developed a Senecavirus-specific cell culture-based recombination assay, which we used to elucidate the role of RdRp in SVA recombination. Our results demonstrate that these two polymerase variants (S460L and I212/S460L) have reduced recombination capacity. These results indicate that the RdRp plays a central role in SVA replicative recombination. Notably, our results showed that the two recombination-deficient variants have higher replication fidelity than the wild type (WT) and display decreased ribavirin sensitivity compared to the WT. In addition, these two mutants exhibited significantly increased fitness in vitro compared to the WT. These results demonstrate that recombination and mutation rates are intimately linked. Our results have important implications for understanding the crucial role of the RdRp in virus recombination and fitness, especially in the molecular mechanisms of SVA evolution and pathogenicity.

    IMPORTANCE Recent evidence has emphasized the importance of SVA recombination on virus evolution in vivo. We describe the first assays to study Senecavirus A recombination. The results show that the RNA-dependent RNA polymerase plays a crucial role in recombination and that recombination can impact the fitness of SVA in cell culture. Further, SVA polymerase fidelity is closely related to recombination efficiency. The results provide key insights into the role of recombination in positive-strand RNA viruses.

  • Nipah and Hendra Virus Glycoproteins Induce Comparable Homologous but Distinct Heterologous Fusion Phenotypes [Virus-Cell Interactions]

  • Nipah and Hendra viruses (NiV and HeV) exhibit high lethality in humans and are biosafety level 4 (BSL-4) paramyxoviruses in the growing genus Henipavirus. The attachment (G) and fusion (F) envelope glycoproteins are both required for viral entry into cells and for cell-cell fusion, which is pathognomonic of henipaviral infections. Here, we compared the fusogenic capacities between homologous and heterologous pairs of NiV and HeV glycoproteins. Importantly, to accurately measure their fusogenic capacities, as these depend on glycoprotein cell surface expression (CSE) levels, we inserted identical extracellular tags to both fusion (FLAG tags) or both attachment (hemagglutinin [HA] tags) glycoproteins. Importantly, these tags were placed in extracellular sites where they did not affect glycoprotein expression or function. NiV and HeV glycoproteins induced comparable levels of homologous HEK293T cell-cell fusion. Surprisingly, however, while the heterologous NiV F/HeV G (NF/HG) combination yielded a hypofusogenic phenotype, the heterologous HeV F/NiV G (HF/NG) combination yielded a hyperfusogenic phenotype. Pseudotyped viral entry levels primarily corroborated the fusogenic phenotypes of the glycoprotein pairs analyzed. Furthermore, we constructed G and F chimeras that allowed us to map the overall regions in G and F that contributed to these hyperfusogenic or hypofusogenic phenotypes. Importantly, the fusogenic phenotypes of the glycoprotein combinations negatively correlated with the avidities of F-G interactions, supporting the F/G dissociation model of henipavirus-induced membrane fusion, even in the context of heterologous glycoprotein pairs.

    IMPORTANCE The NiV and HeV henipaviruses are BSL-4 pathogens transmitted from bats. NiV and HeV often lead to human death and animal diseases. The formation of multinucleated cells (syncytia) is a hallmark of henipaviral infections and is caused by fusion of cells coordinated by interactions of the viral attachment (G) and fusion (F) glycoproteins. We found via various assays that viral entry and syncytium formation depend on the viral origin of the glycoproteins, with HeV F and NiV G promoting higher membrane fusion levels than their counterparts. This is important knowledge, since both viruses use the same bat vector species and potential coinfections of these or subsequent hosts may alter the outcome of disease.

  • Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal Degradation of VP1 and Attenuating Its Antagonistic Role in the Beta Interferon Signaling Pathway [Virus-Cell Interactions]

  • DnaJ heat shock protein family (Hsp40) member A3 (DNAJA3) plays an important role in viral infections. However, the role of DNAJA3 in replication of foot-and-mouth-disease virus (FMDV) remains unknown. In this study, DNAJA3, a novel binding partner of VP1, was identified using yeast two-hybrid screening. The DNAJA3-VP1 interaction was further confirmed by coimmunoprecipitation and colocalization in FMDV-infected cells. The J domain of DNAJA3 (amino acids 1 to 168) and the lysine at position 208 (K208) of VP1 were shown to be critical for the DNAJA3-VP1 interaction. Overexpression of DNAJA3 dramatically dampened FMDV replication, whereas loss of function of DNAJA3 elicited opposing effects against FMDV replication. Mechanistical study demonstrated that K208 of VP1 was critical for reducing virus titer caused by DNAJA3 using K208A mutant virus. DNAJA3 induced lysosomal degradation of VP1 by interacting with LC3 to enhance the activation of lysosomal pathway. Meanwhile, we discovered that VP1 suppressed the beta interferon (IFN-bbeta;) signaling pathway by inhibiting the phosphorylation, dimerization, and nuclear translocation of IRF3. This inhibitory effect was considerably boosted in DNAJA3-knockout cells. In contrast, overexpression of DNAJA3 markedly attenuated VP1-mediated suppression on the IFN-bbeta; signaling pathway. Poly(Issdot;C)-induced phosphorylation of IRF3 was also decreased in DNAJA3-knockout cells compared to that in the DNAJA3-WT cells. In conclusion, our study described a novel role for DNAJA3 in the hostrrsquo;s antiviral response by inducing the lysosomal degradation of VP1 and attenuating the VP1-induced suppressive effect on the IFN-bbeta; signaling pathway.

    IMPORTANCE This study pioneeringly determined the antiviral role of DNAJA3 in FMDV. DNAJA3 was found to interact with FMDV VP1 and trigger its degradation via the lysosomal pathway. In addition, this study is also the first to clarify the mechanism by which VP1 suppressed IFN-bbeta; signaling pathway by inhibiting the phosphorylation, dimerization, and nuclear translocation of IRF3. Moreover, DNAJA3 significantly abrogated VP1-induced inhibitive effect on the IFN-bbeta; signaling pathway. These data suggested that DNAJA3 plays an important antiviral role against FMDV by both degrading VP1 and restoring of IFN-bbeta; signaling pathway.

  • Type I Interferon Receptor Signaling Drives Selective Permissiveness of Astrocytes and Microglia to Measles Virus during Brain Infection [Pathogenesis and Immunity]

  • Fatal neurological syndromes can occur after measles virus (MeV) infection of the brain. The mechanisms controlling MeV spread within the central nervous system (CNS) remain poorly understood. We analyzed the role of type I interferon (IFN-I) receptor (IFNAR) signaling in the control of MeV infection in a murine model of brain infection. Using organotypic brain cultures (OBC) from wild-type and IFNAR-knockout (IFNARKO) transgenic mice ubiquitously expressing the human SLAM (CD150) receptor, the heterogeneity of the permissiveness of different CNS cell types to MeV infection was characterized. In the absence of IFNAR signaling, MeV propagated significantly better in explant slices. In OBC from IFNAR-competent mice, while astrocytes and microglia were infected on the day of explant preparation, they became refractory to infection with time, in contrast to neurons and oligodendrocytes, which remained permissive to infection. This selective loss of permissiveness to MeV infection was not observed in IFNARKO mouse OBC. Accordingly, the development of astrogliosis related to the OBC procedure was exacerbated in the presence of IFNAR signaling. In the hippocampus, this astrogliosis was characterized by a change in the astrocyte phenotype and by an increase of IFN-I transcripts. A proteome analysis showed the upregulation of 84 out of 111 secreted proteins. In the absence of IFNAR, only 27 secreted proteins were upregulated, and none of these were associated with antiviral activities. Our results highlight the essential role of the IFN-I response in astrogliosis and in the permissiveness of astrocytes and microglia that could control MeV propagation throughout the CNS.

    IMPORTANCE Measles virus (MeV) can infect the central nervous system (CNS), with dramatic consequences. The mechanisms controlling MeV invasion of the CNS remain ill-defined since most previous data were obtained from postmortem analysis. Here, we highlight for the first time the crucial role of the type I interferon (IFN-I) response not only in the control of CNS invasion but also in the early permissiveness of glial cells to measles virus infection.

  • Discovery of Small-Molecule Inhibitors Targeting the E3 Ubiquitin Ligase Activity of the Herpes Simplex Virus 1 ICP0 Protein Using an In Vitro High-Throughput Screening Assay [Virus-Cell Interactions]

  • Herpes simplex virus 1 (HSV-1) has infected more than 80% of the population. Reactivation of the virus causes diseases ranging in severity from benign cold sores to fatal encephalitis. Current treatments involve viral DNA replication inhibitors, but the emergence of drug-resistant mutants is observed frequently, highlighting the need for novel antiviral therapies. Infected cell protein 0 (ICP0) of HSV-1 is encoded by an immediate early gene and plays a fundamental role during infection, because it enables viral gene expression and blocks antiviral responses. One mechanism by which ICP0 functions is through an E3 ubiquitin ligase activity that induces the degradation of targeted proteins. A ICP0 virus or mutants with deficiencies in E3 ligase activity cannot counteract beta interferon (IFN-bbeta;)-induced restriction of viral infection, are highly immunogenic, are avirulent, and fail to spread. Thus, small molecules interfering with essential and conserved ICP0 functions are expected to compromise HSV-1 infection. We have developed a high-throughput screening assay, based on the autoubiquitination properties of ICP0, to identify small-molecule inhibitors of ICP0 E3 ubiquitin ligase activity. Through a pilot screening procedure, we identified nine compounds that displayed dose-dependent inhibitory effects on ICP0 but not on Mdm2, a control E3 ubiquitin ligase. Following validation, one compound displayed ICP0-dependent inhibition of HSV-1 infection. This compound appeared to bind ICP0 in a cellular thermal shift assay, it blocked ICP0 self-elimination, and it blocked wild-type but not ICP0-null virus gene expression. This scaffold displays specificity and could be used to develop optimized ICP0 E3 ligase inhibitors.

    IMPORTANCE Since acyclovir and its derivatives were launched for herpesviruses control almost four decades ago, the search for novel antivirals has waned. However, as human life expectancy has increased, so has the number of immunocompromised individuals who receive prolonged treatment for HSV recurrences. This has led to an increase in unresponsive patients due to acquired viral drug resistance. Thus, novel treatments need to be explored. Here we explored the HSV-1 ICP0 E3 ligase as a potential antiviral target because (i) ICP0 is expressed before virus replication, (ii) it is essential for infection in vivo, (iii) it is required for efficient reactivation of the virus from latency, (iv) inhibition of its E3 ligase activity would sustain host immune responses, and (v) it is shared by other herpesviruses. We report a compound that inhibits HSV-1 infection in an ICP0-dependent manner by inhibiting ICP0 E3 ligase activity.

  • Comparative Analysis of African and Asian Lineage-Derived Zika Virus Strains Reveals Differences in Activation of and Sensitivity to Antiviral Innate Immunity [Virus-Cell Interactions]

  • In recent years, Asian lineage Zika virus (ZIKV) strains emerged to cause pandemic outbreaks associated with a high rate of congenital ZIKV syndrome (CZVS). The reasons for the enhanced spread and severe disease caused by newly emerging strains are not fully understood. Here we compared viral sequences, viral replication, and innate immune signaling induction of three different ZIKV strains derived from African and Asian lineages and West Nile virus, another flavivirus. We found pronounced differences in activation of innate immune signaling and inhibition of viral replication across ZIKV strains. The newly emerged Asian ZIKV strain Brazil Fortaleza 2015, which is associated with a higher rate of neurodevelopmental disorders like microcephaly, induced much weaker and delayed innate immune signaling in infected cells. However, superinfection studies to assess control of innate immune signaling induced by Sendai virus argue against an active block of IRF3 activation by the Brazilian strain of ZIKV and rather suggest an evasion of detection by host cell pattern recognition receptors. Compared to the Asian strain FSS13025 isolated in Cambodia, both ZIKV Uganda MR766 and ZIKV Brazil Fortaleza appear less sensitive to the interferon-induced antiviral response. ZIKV infection studies of cells lacking the different RIG-I-like receptors identified RIG-I as the major cytosolic pattern recognition receptor for detection of ZIKV.

    IMPORTANCE Zika Virus (ZIKV), discovered in 1947, is divided into African and Asian lineages. Pandemic outbreaks caused by currently emerging Asian lineage strains are accompanied by high rates of neurological disorders and exemplify the global health burden associated with this virus. Here we compared virological and innate immunological aspects of two ZIKV strains from the Asian lineage, an emerging Brazilian strain and a less-pathogenic Cambodian strain, and the prototypic African lineage ZIKV strain from Uganda. Compared to the replication of other ZIKV strains, the replication of ZIKV Brazil was less sensitive to the antiviral actions of interferon (IFN), while infection with this strain induced weaker and delayed innate immune responses in vitro. Our data suggest that ZIKV Brazil directs a passive strategy of innate immune evasion that is reminiscent of a stealth virus. Such strain-specific properties likely contribute to differential pathogenesis and should be taken into consideration when choosing virus strains for future molecular studies.

  • Articles of Significant Interest in This Issue [Spotlight]

  • Comparisons of Human Immunodeficiency Virus Type 1 Envelope Variants in Blood and Genital Fluids near the Time of Male-to-Female Transmission [Pathogenesis and Immunity]

  • To better understand the transmission of human immunodeficiency virus type 1 (HIV-1), the genetic characteristics of blood and genital viruses from males were compared to those of the imputed founding virus population in their female partners. Initially serodiscordant heterosexual African couples with sequence-confirmed male-to-female HIV-1 transmission and blood and genital specimens collected near the time of transmission were studied. Single viral templates from blood plasma and genital tract RNA and DNA were sequenced across HIV-1 env gp160. Eight of 29 couples examined yielded viral sequences from both tissues. Analysis of these couplesrrsquo; sequences demonstrated, with one exception, that the womenrrsquo;s founding viral populations arose from a single viral variant and were CCR5 tropic, even though CXCR4 variants were detected within four males. The median genetic distance of the imputed most recent common ancestor of the womenrrsquo;s founder viruses showed that they were closer to the semen viruses than to the blood viruses of their transmitting male partner, but this finding was biased by detection of a greater number of viral clades in the blood. Using multiple assays, the blood and genital viruses were consistently found to be compartmentalized in only two of eight men. No distinct amino acid signatures in the menrrsquo;s viruses were found to link to the womenrrsquo;s founders, nor did the womenrrsquo;s env sequences have shorter variable loops or fewer N-linked glycosylation sites. The lack of selective factors, except for coreceptor tropism, is consistent with othersrrsquo; findings in male-to-female and high-risk transmissions. The infrequent compartmentalization between the transmittersrrsquo; blood and semen viruses suggests that cell-free blood virus likely includes HIV-1 sequences representative of those of viruses in semen.

    IMPORTANCE Mucosal transmissions account for the majority of HIV-1 infections. Identification of the viral characteristics associated with transmission would facilitate vaccine design. This study of HIV strains from transmitting males and their seroconverting female partners found that the malesrrsquo; genital tract viruses were rarely distinct from the blood variants. The imputed founder viruses in women were genetically similar to both the blood and genital tract variants of their male partners, indicating a lack of evidence for genital tract-specific lineages. These findings suggest that targeting vaccine responses to variants found in blood are likely to also protect from genital tract variants.

  • Asparagine Is a Critical Limiting Metabolite for Vaccinia Virus Protein Synthesis during Glutamine Deprivation [Virus-Cell Interactions]

  • Viruses actively interact with host metabolism because viral replication relies on host cells to provide nutrients and energy. Vaccinia virus (VACV; the prototype poxvirus) prefers glutamine to glucose for efficient replication to the extent that VACV replication is hindered in glutamine-free medium. Remarkably, our data show that VACV replication can be fully rescued from glutamine depletion by asparagine supplementation. By global metabolic profiling, as well as genetic and chemical manipulation of the asparagine supply, we provide evidence demonstrating that the production of asparagine, which exclusively requires glutamine for biosynthesis, accounts for VACVrrsquo;s preference of glutamine to glucose rather than glutaminerrsquo;s superiority over glucose in feeding the tricarboxylic acid (TCA) cycle. Furthermore, we show that sufficient asparagine supply is required for efficient VACV protein synthesis. Our study highlights that the asparagine supply, the regulation of which has been evolutionarily tailored in mammalian cells, presents a critical barrier to VACV replication due to a high asparagine content of viral proteins and a rapid demand of viral protein synthesis. The identification of asparagine availability as a critical limiting factor for efficient VACV replication suggests a new direction of antiviral strategy development.

    IMPORTANCE Viruses rely on their infected host cells to provide nutrients and energy for replication. Vaccinia virus, the prototypic member of the poxviruses, which comprise many significant human and animal pathogens, prefers glutamine to glucose for efficient replication. Here, we show that the preference is not because glutamine is superior to glucose as the carbon source to fuel the tricarboxylic acid cycle for vaccinia virus replication. Rather interestingly, the preference is because the asparagine supply for efficient viral protein synthesis becomes limited in the absence of glutamine, which is necessary for asparagine biosynthesis. We provide further genetic and chemical evidence to demonstrate that asparagine availability plays a critical role in efficient vaccinia virus replication. This discovery identifies a weakness of vaccinia virus and suggests a possible direction to intervene in poxvirus infection.

  • A Genome-Wide Haploid Genetic Screen Identifies Heparan Sulfate-Associated Genes and the Macropinocytosis Modulator TMED10 as Factors Supporting Vaccinia Virus Infection [Virus-Cell Interactions]

  • Vaccinia virus is a promising viral vaccine and gene delivery candidate and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to modified vaccinia virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2, and TMED10 (TMP21/p23/p24) as important host factors for vaccinia virus infection. The critical roles of EXT1 in heparan sulfate synthesis and vaccinia virus infection were confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, presumably by regulating the cell surface expression of the TAM receptor Axl.

    IMPORTANCE Poxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells and are used as a vaccine vector against tuberculosis, influenza, and coronaviruses. Poxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knockout library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, facilitates virus uptake by modulating the cellular response to phosphatidylserine.

  • Carnivore Parvovirus Ecology in the Serengeti Ecosystem: Vaccine Strains Circulating and New Host Species Identified [Genetic Diversity and Evolution]

  • Carnivore parvoviruses infect wild and domestic carnivores, and cross-species transmission is believed to occur. However, viral dynamics are not well understood, nor are the consequences for wild carnivore populations of the introduction of new strains into wild ecosystems. To clarify the ecology of these viruses in a multihost system such as the Serengeti ecosystem and identify potential threats for wildlife conservation, we analyzed, through real-time PCR, 152 samples belonging to 14 wild carnivore species and 62 samples from healthy domestic dogs. We detected parvovirus DNA in several wildlife tissues. Of the wild carnivore and domestic dog samples tested, 13% and 43%, respectively, were positive for carnivore parvovirus infection, but little evidence of transmission between the wild and domestic carnivores was detected. Instead, we describe two different epidemiological scenarios with separate routes of transmission: first, an endemic feline parvovirus (FPV) route of transmission maintained by wild carnivores inside the Serengeti National Park (SNP) and, second, a canine parvovirus (CPV) route of transmission among domestic dogs living around the periphery of the SNP. Twelve FPV sequences were characterized; new host-virus associations involving wild dogs, jackals, and hyenas were discovered; and our results suggest that mutations in the fragment of the vp2 gene were not required for infection of different carnivore species. In domestic dogs, 6 sequences belonged to the CPV-2a strain, while 11 belonged to the CPV-2 vaccine-derived strain. This is the first description of a vaccine-derived parvovirus strain being transmitted naturally.

    IMPORTANCE Carnivore parvoviruses are widespread among wild and domestic carnivores, which are vulnerable to severe disease under certain circumstances. This study furthers the understanding of carnivore parvovirus epidemiology, suggesting that feline parvoviruses are endemic in wild carnivores in the Serengeti National Park (SNP), with new host species identified, and that canine parvoviruses are present in the dog population living around the SNP. Little evidence of transmission of canine parvoviruses into wild carnivore species was found; however, the detection of vaccine-derived virus (described here for the first time to be circulating naturally in domestic dogs) highlights the importance of performing epidemiological research in the region.

  • Regulation of Human Cytomegalovirus Secondary Envelopment by a C-Terminal Tetralysine Motif in pUL71 [Structure and Assembly]

  • Human cytomegalovirus (HCMV) secondary envelopment requires the viral tegument protein pUL71. The lack of pUL71 results in a complex ultrastructural phenotype with increased numbers of viral capsids undergoing envelopment at the cytoplasmic virus assembly complex. Here, we report a role of the pUL71 C terminus in secondary envelopment. Mutant viruses expressing C-terminally truncated pUL71 (TB71del327-361 and TB71del348-351) exhibited an impaired secondary envelopment in transmission electron microscopy (TEM) studies. Further mutational analyses of the C terminus revealed a tetralysine motif whose mutation (TB71mutK348-351A) resulted in an envelopment defect that was undistinguishable from the defect caused by truncation of the pUL71 C terminus. Interestingly, not all morphological alterations that define the ultrastructural phenotype of a TB71stop virus were found in cells infected with the C-terminally mutated viruses. This suggests that pUL71 provides additional functions that modulate HCMV morphogenesis and are harbored elsewhere in pUL71. This is also reflected by an intermediate growth defect of the C-terminally mutated viruses compared to the growth of the TB71stop virus. Electron tomography and three-dimensional visualization of different stages of secondary envelopment in TB71mutK348-351A-infected cells showed unambiguously the formation of a bud neck. Furthermore, we provide evidence for progressive tegument formation linked to advancing grades of capsid envelopment, suggesting that tegumentation and envelopment are intertwined processes. Altogether, we identified the importance of the pUL71 C terminus and, specifically, of a positively charged tetralysine motif for HCMV secondary envelopment.

    IMPORTANCE Human cytomegalovirus (HCMV) is an important human pathogen that causes severe symptoms, especially in immunocompromised hosts. Furthermore, congenital HCMV infection is the leading viral cause of severe birth defects. Development of antiviral drugs to prevent the production of infectious virus progeny is challenging due to a complex and multistep virion morphogenesis. The mechanism of secondary envelopment is still not fully understood; nevertheless, it represents a potential target for antiviral drugs. Our identification of the role of a positively charged motif in the pUL71 C terminus for efficient HCMV secondary envelopment underlines the importance of pUL71 and, especially, its C terminus for this process. It furthermore shows how cell-associated spread and virion release depend on secondary envelopment. Ultrastructural analyses of different stages of envelopment contribute to a better understanding of the mechanisms underlying the process of secondary envelopment. This may bring us closer to the development of novel concepts to treat HCMV infections.

  • Enterovirus A71 Containing Codon-Deoptimized VP1 and High-Fidelity Polymerase as Next-Generation Vaccine Candidate [Vaccines and Antiviral Agents]

  • Enterovirus A71 (EV-A71) is a major pathogen that causes hand-foot-and-mouth disease (HFMD), which occasionally results in severe neurological complications. In this study, we developed four EV-A71 (rgEV-A71) strains by reverse genetics procedures as possible vaccine candidates. The four rgEV-A71 viruses contained various codon-deoptimized VP1 capsid proteins (VP1-CD) and showed replication rates and antigenicity similar to that of the wild-type virus, while a fifth virus, rg4643C4VP-CD, was unable to form plaques but was still able to be examined by median tissue culture infectious dose (TCID50) titers, which were similar to those of the others, indicating the effect of CD on plaque formation. However, the genome stability showed that there were some mutations which appeared during just one passage of the VP1-CD viruses. Thus, we further constructed VP1-CD rgEV-A71 containing high-fidelity determinants in 3D polymerase (CD-HF), and the number of mutations in CD-HF rgEV-A71 was shown to have decreased. The CD-HF viruses showed less virulence than the parental strain in a mouse infection model. After 14 days postimmunization, antibody titers had increased in mice infected with CD-HF viruses. The mouse antisera showed similar neutralizing antibody titers against various CD-HF viruses and different genotypes of EV-A71. The study demonstrates the proof of concept that VP1 codon deoptimization combined with high-fidelity 3D polymerase decreased EV-A71 mutations and virulence in mice but retained their antigenicity, indicating it is a good candidate for next-generation EV-A71 vaccine development.

    IMPORTANCE EV-A71 can cause severe neurological diseases with fatality in infants and young children, but there are still no effective drugs to date. Here, we developed a novel vaccine strategy with the combination of CD and HF substitutions to generate the genetically stable reverse genetics virus. We found that CD combined with HF polymerase decreased the virulence but maintained the antigenicity of the virus. This work demonstrated the simultaneous introduction of CD genome sequences and HF substitutions as a potential new strategy to develop attenuated vaccine seed virus. Our work provides insight into the development of a low-virulence candidate vaccine virus through a series of genetic editing of virus sequences while maintaining its antigenicity and genome stability, which will provide an additional strategy for next-generation vaccine development of EV-A71.

  • Phenotypic and Functional Differences between Human Herpesvirus 6- and Human Cytomegalovirus-Specific T Cells [Pathogenesis and Immunity]

  • Human herpesvirus 6 (HHV-6) infects ggt;90% of the population and establishes a latent infection with asymptomatic episodes of reactivation. However, HHV-6 reactivation is associated with morbidity and sometimes mortality in immunocompromised patients. To date, control of the virus in healthy virus carriers and the failure to control it in patients with disease remain poorly understood. In particular, knowledge of HHV-6-specific T-cell responses is limited. Here, we characterized HHV-6A- and HHV-6B-specific CD4+ and CD8+ T-cell responses from peripheral blood mononuclear cells (PBMCs) of healthy donors. We studied the phenotype of effector HHV-6-specific T cells ex vivo, as well as of induced specific suppressive regulatory CD4+ T cells in vitro poststimulation, in comparison to human cytomegalovirus (HCMV) responses. Compared to that for HCMV, we show that ex vivo T-cell reactivity in peripheral blood is detectable but at very low frequency, both for HHV-6A and -6B viruses. Interestingly, the phenotype of the specific T cells also differs between the viruses. HHV-6A- and HHV-6B-specific CD4+ T lymphocytes are less differentiated than HCMV-specific T cells. Furthermore, we show a higher frequency of HHV-6-specific suppressive regulatory T cells (eTregs) than HCMV-specific eTregs in coinfected individuals. Despite the strong similarity of HHV-6 and HCMV from a virologic point of view, we observed immunological differences, particularly in relation to the frequency and phenotype of effector/memory and regulatory virus-specific T cells. This suggests that different immune factors are solicited in the control of HHV-6 infection than in that of HCMV infection.

    IMPORTANCE T cells are central to an effective defense against persistent viral infections that can be related to human cytomegalovirus (HCMV) or human herpesvirus 6 (HHV-6). However, knowledge of HHV-6-specific T-cell responses is limited. In order to deepen our knowledge of T-cell responses to HHV-6, we characterized HHV-6A- and HHV-6B-specific CD4+ and CD8+ T-cell responses directly ex vivo from healthy coinfected blood donors. Despite the strong similarity of HHV-6 and HCMV from a virologic point of view, we observed immunological differences, particularly in relation to the frequency and phenotype of effector/memory and regulatory virus-specific T cells. This suggests that different immune factors are solicited in the control of HHV-6 infection than in that of HCMV infection. Our findings may encourage immunomonitoring of patients with viral replication episodes to follow the emergence of effector versus regulatory T cells.

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    JVI Accepts: Articles Published Ahead of Print

  • Rational design of Zika virus subunit vaccine with enhanced efficacy [Vaccines and Antiviral Agents]

  • Zika virus (ZIKV) infection in pregnant women can lead to fetal deaths and malformations. We have previously reported that ZIKV envelope protein domain III (EDIII) is a subunit vaccine candidate with cross-neutralization activity; however, like many other subunit vaccines, its efficacy is limited. To improve the efficacy of this subunit vaccine, we identified a non-neutralizing epitope on ZIKV EDIII surrounding residue 375, which is buried in the full-length envelope protein but becomes exposed in recombinant EDIII. We then shielded this epitope with an engineered glycan probe. Compared to the wild-type EDIII, the mutant EDIII induced significantly stronger neutralizing antibodies in three mouse strains, and also demonstrated significantly improved efficacy by fully protecting mice, particularly pregnant mice and their fetuses, against high-dose lethal ZIKV challenge. Moreover, the mutant EDIII immune sera significantly enhanced the passive protective efficacy by fully protecting mice against lethal ZIKV challenge; this passive protection was positively associated with neutralizing antibody titers. We further showed that the enhanced efficacy of the mutant EDIII was due to the shielding of the immunodominant non-neutralizing epitope surrounding residue 375, which led to immune refocusing on the neutralizing epitopes. Taken together, this study reveals that an intrinsic limitation of subunit vaccines is their artificially exposed immunodominant non-neutralizing epitopes, which can be overcome through glycan shielding. Additionally, the mutant ZIKV protein generated in this study is a promising subunit vaccine candidate with high efficacy in preventing ZIKV infections in mice.

    Significance

    Viral subunit vaccines generally suffer low efficacy. Here we revealed an intrinsic limitation of subunit vaccine designs - artificially exposed surfaces of subunit vaccines contain epitopes unfavorable for vaccine efficacy. More specifically, we identified an epitope on the Zika virus (ZIKV) envelope protein domain III (EDIII) that is buried in the full-length envelope protein, but becomes exposed in the recombinant EDIII. We further shielded this epitope with a glycan, and the resulting mutant EDIII vaccine demonstrated significantly enhanced efficacy over the wild-type EDIII vaccine in protecting animal models from ZIKV infections. Therefore, the intrinsic limitation of subunit vaccines can be overcome through shielding these artificially exposed unfavorable epitopes. The engineered EDIII vaccine generated in this study is a promising vaccine candidate that can be further developed to battle ZIKV infections.

  • Dense bodies of a gH/gL/UL128-131 pentamer repaired Towne strain of human cytomegalovirus induce an enhanced neutralizing antibody response [Vaccines and Antiviral Agents]

  • The development of a vaccine against human cytomegalovirus infection (HCMV) is a high-priority medical goal. The viral pentameric protein complex consisting of gH/gL/UL128-131A (PC) is considered to be an important vaccine component. Its relevance to the induction of a protective antibody response is, however, still a matter of debate. We addressed this issue by using subviral dense bodies (DBs) of HCMV. DBs are exceptionally immunogenic. Laboratory HCMV strain DBs harbour important neutralizing antibody targets like the glycoproteins B, H, L, M, and N, but they are devoid of the PC. To be able to directly compare the impact of the PC on the levels of neutralizing antibody (NT-abs) responses, a PC-positive variant of the HCMV laboratory strain Towne was established by BAC-mutagenesis (Towne-UL130rep). This strain synthesized PC-positive DBs upon infection of fibroblasts. These DBs were used in side-by-side immunizations with PC-negative Towne DBs. Mouse and rabbit sera were tested to address the impact of the PC on DB-immunogenicity. The neutralizing antibody response to PC-positive DBs was superior, as tested on fibroblasts, epithelial cells and endothelial cells and for both animal species used. The experiments revealed the potential of the PC to enhance the antibody response against HCMV. Of particular interest was that PC-positive DBs induced an antibody response that blocked the infection of fibroblasts by a PC-positive viral strain more efficiently, compared to sera following immunizations with PC-negative particles.

    IMPORTANCE Infections with the human cytomegalovirus (HCMV) may cause severe and even life-threatening disease manifestations in newborns and immunosuppressed individuals. Several strategies for the development of a vaccine against this virus are currently pursued. A critical question in this respect refers to the antigenic composition of a successful vaccine. Using a subviral particle vaccine candidate, we show here that one protein complex of HCMV, termed the pentameric complex (PC) enhances the neutralizing antibody response against viral infection of different cell types. We further show for the first time that this not only relates to the infection of epithelial- or endothelial cells. The presence of the PC in the particles also enhanced the neutralizing antibody response against infection of fibroblasts by HCMV. Together these findings argue in favor of including the PC in strategies for HCMV vaccine development.

  • Tissue-specific gene expression during productive HPV16 infection of cervical, foreskin and tonsil epithelium [Cellular Response to Infection]

  • Epidemiological data confirms a much higher incidence of high risk human papillomavirus HPV16 mediated carcinogenesis of the cervical epithelium, compared to other target sites. In order to elucidate tissue specific responses to virus infection, we compared gene expression changes induced by the productive HPV16 infection of cervical, foreskin and tonsil organotypic rafts. These rafts closely mimic persistent HPV16 infection, much before carcinogenesis sets in. The total number of gene expression changes varied considerably across the tissue types, with only 32 genes being regulated in common. Among them, we confirmed the kelch-like family protein KLHL35 and Laminin-5 complex to be upregulated and downregulated respectively, in all the three tissues. HPV16 infection induces upregulation of genes involved in cell cycle control, cell division, mitosis, DNA replication and DNA damage repair, in all the three tissues, indicative of a hyper-proliferative environment. In the cervical and tonsil epithelium, we observe significant downregulation of genes involved in epidermis development, keratinocyte differentiation and extracellular matrix organization. On the other hand, in the HPV16 foreskin tissue, several genes involved in interferon mediated innate immunity; cytokine signaling and cellular defenses were downregulated. Furthermore, pathway analysis and experimental validations identified important cellular pathways like STAT1 and TGFbbeta; to be differentially regulated among the three tissue types. The differential modulation of important cellular pathways like TGFbbeta;1 and STAT1 can explain the sensitivity of the tissues for HPV cancer progression.

    IMPORTANCE

    Although the high risk human papillomavirus HPV16 infects anogenital and oropharyngeal sites, the cervical epithelium has a unique vulnerability to progression of cancer. Host responses during persistent infection and pre-neoplastic stages can shape the outcome of cancer progression, in a tissue dependent manner. Our study for the first time reports differential regulation of critical cellular functions and signaling pathway, during productive HPV16 infection of the cervical, foreskin and tonsil tissue. While the virus induces hyperproliferation in the infected cells it downregulates epithelial differentiation, epidermal development and innate immune responses, according to the tissue type. Modulation of these biological functions can determine virus fitness, pathogenesis and illuminate key cellular mechanisms the virus employs to establish persistence and finally initiate disease progression.

  • Japanese encephalitis virus induces apoptosis and encephalitis by activating the PERK pathway [Virus-Cell Interactions]

  • Accumulated evidence demonstrates that Japanese encephalitis virus (JEV) infection triggers endoplasmic reticulum (ER) stress and neuron apoptosis. ER stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) has been reported to induce apoptosis under acute or prolonged ER stress. However, the precise role of PERK in JEV-induced apoptosis and encephalitis remains unknown. Here, we reported that JEV infection activates the PERK-ATF4-CHOP apoptosis pathway both in vitro and in vivo. PERK activation also promotes the formation of stress granule, which in turn represses JEV-induced apoptosis. However, PERK inhibitor reduces apoptosis, indicating that JEV-activated PERK predominantly induces apoptosis via the PERK-ATF4-CHOP apoptosis pathway. Among JEV proteins that have been reported to induce ER stress, only JEV NS4B can induce PERK activation. PERK has been reported to form an active molecule by dimerization. The coimmunoprecipitation assay shows that NS4B interacts with PERK. Moreover, glycerol gradient centrifugation shows that NS4B induces PERK dimerization. Both the LIG-FHA and LIG-WD40 domains within NS4B are required to induce PERK dimerization, suggesting that JEV NS4B pulls two PERK molecules together by simultaneously interacting with them via different motifs. PERK deactivation reduces brain cell damage and encephalitis during JEV infection. Furthermore, expression of JEV NS4B is sufficient to induce encephalitis via PERK in mice, indicating that JEV activates PERK primarily via its NS4B to cause encephalitis. Taken together, our findings provide a novel insight into JEV-caused encephalitis.

    Importance

    Japanese encephalitis virus (JEV) infection triggers endoplasmic reticulum (ER) stress and neuron apoptosis. ER stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) has been reported to induce apoptosis under acute or prolonged ER stress. However, whether the PERK pathway of ER stress response plays important roles in JEV-induced apoptosis and encephalitis remains unknown. Here, we found that JEV infection activates ER stress sensor PERK in neuronal cells and mouse brains. PERK activation induces apoptosis via the PERK-ATF4-CHOP apoptosis pathway upon JEV infection. Among the JEV proteins including prM, E, NS1, NS2A, NS2B and NS4B, only NS4B activates PERK. Moreover, activated PERK participates in apoptosis and encephalitis induced by JEV and NS4B. These findings provide a novel therapeutic approach for JEV-caused encephalitis.

  • The KSHV ORF34 protein Interacts and Stabilizes HIF-2{alpha} via Binding to the HIF-2{alpha} bHLH and PAS Domains [Virus-Cell Interactions]

  • Hypoxia and hypoxia inducible factors (HIFs) play important roles in the Kaposi's sarcoma-associated herpesvirus (KSHV) life cycle. KSHV is the causative agents of Kaposi's sarcoma (KS) and other AIDS related malignancies. Kaposi's sarcoma is a highly vascular tumor, which preferentially develops in the lower extremities of the body where blood vessels are often poorly oxygenated. The main cellular responses to hypoxia are mediated mainly by two isoforms of HIF, HIF-1aalpha; and HIF-2aalpha;. Both HIF-1aalpha; and HIF-2aalpha; have common as well as distinct functions, although they are similar in structure and function. Previously, we showed that the KSHV ORF34 protein binds HIF-1aalpha; and facilitates its degradation through the ubiquitin-proteasome pathway causing negative regulation of HIF-1aalpha;-dependent genes (Haque and Kousoulas, J Virol. 87:2164-73, 2013). Herein, we show that the ORF34 gene is involved in the regulation of KSHV lytic gene expression, since deletion of the ORF34 resulted in reduced immediate early and early lytic gene expression and blocked late gene expression. Coimmunoprecipitation experiments revealed that the ORF34 protein physically interacted with HIF-2aalpha; in transfected, as well as in KSHV-infected cells. Utilization of ORF34 truncations revealed that three distinct domains bind HIF-2aalpha; and that both bHLH and PAS domains of HIF-2aalpha; interacted with ORF34. Unlike HIF-1aalpha;, dose dependent co-expression of ORF34 stabilized the HIF-2aalpha; protein ensuring HIF-2aalpha; dependent transcriptional activity. The ORF34 protein enhanced HIF-2aalpha; ubiquitination at the bHLH and PAS domains. The results show that the KSHV ORF34 protein is involved in the KSHV life cycle by regulating the expression of HIF-1aalpha; aamp; HIF-2aalpha; proteins.

    Importance

    The Hypoxia inducible factors-1aalpha; (HIF-1aalpha;) and -2aalpha; (HIF-2aalpha;) are transcription factors which play important roles in the Kaposi's sarcoma-associated herpesvirus (KSHV) latent and lytic gene replication. Herein, we show that the ORF34 gene is involved in the regulation of KSHV lytic gene expression, since deletion of the ORF34 resulted in reduced immediate early and early lytic gene expression and blocked late gene expression. In addition, we demonstrate that the KSHV ORF34 protein binds and stabilizes HIF-2aalpha;, in contrast to its role in binding HIF-1aalpha; and causing its degradation via the proteasome pathway. Thus, the KSHV ORF34 protein plays a regulatory role in the KSHV life cycle by regulating HIF-1aalpha; and HIF-2aalpha; expression.

  • HIV diversity and genetic compartmentalization in blood and testes during suppressive antiretroviral therapy [Genetic Diversity and Evolution]

  • HIV's ability to persist during suppressive antiretroviral therapy is the main barrier to cure. Immune privileged tissues such as the testes may constitute distinctive sites of HIV persistence, but this has been challenging to study in humans. We analyzed proviral burden and genetics in blood and testes of 10 individuals on suppressive therapy who underwent elective gender-affirming surgery. HIV DNA levels in matched blood and testes were quantified by qPCR and subgenomic proviral sequences (nef region) were characterized from single templates. HIV diversity, compartmentalization and immune escape burden were assessed using genetic and phylogenetic approaches. Diverse proviruses were recovered from blood (396 sequences; 354 nef-intact) and testes (326 sequences; 309 nef-intact) of all participants. Notably, the frequency of identical HIV sequences varied markedly between and within individuals. Nevertheless, proviral loads, within-host unique HIV sequence diversity, and immune escape burden correlated positively between blood and testes. When all intact nef sequences were evaluated, 60% of participants exhibited significant blood-testes genetic compartmentalization, but none did so when restricting to unique sequences per site, suggesting that compartmentalization, when present, is attributable to clonal expansion of HIV-infected cells. Our observations confirm the testes as a site of HIV persistence and suggest that individuals with larger and more diverse blood reservoirs will have larger and more diverse testes reservoirs. Furthermore, while the testes microenvironment may not be sufficiently unique to facilitate the seeding of unique viral populations therein, differential clonal expansion dynamics may be at play, which may complicate HIV eradication.

    Importance

    Two key questions in HIV reservoir biology are whether immune privileged tissues, such as the testes, harbor distinctive proviral populations during suppressive therapy, and if so by what mechanism. While our results indicated that blood-testes HIV genetic compartmentalization was reasonably common (60%), it was always attributable to differential frequencies of identical HIV sequences between sites. No blood/tissue dataset retained evidence of compartmentalization when only unique HIV sequences per site were considered; moreover, HIV immune escape mutation burdens were highly concordant between sites. We conclude that the principal mechanism by which blood and testes reservoirs differ is not via seeding of divergent HIV sequences therein, but rather due to differential clonal expansion of latently-infected cells. Thus, while viral diversity and escape-related barriers to HIV eradication are of broadly similar magnitude across blood and testes, clonal expansion represents a challenge. Results support individualized analysis of within-host reservoir diversity to inform curative approaches.

  • Replication-defective West Nile virus with NS1 deletion as a new vaccine platform for flavivirus [Vaccines and Antiviral Agents]

  • We previously produced a replication-defective West Nile virus lacking NS1 (WNV-NS1) that could propagate at low levels (105 IU/ml) in a 293T cell line expressing wild-type (WT) NS1. This finding indicates the potential of developing WNV-NS1 as a non-infectious vaccine. To explore this idea, we developed an NS1-expressing Vero cell line (VeroNS1) that significantly improved the yield of WNV-NS1 (108 IU/ml). We evaluated the safety and efficacy of WNV-NS1 in mice. WNV-NS1 appeared to be safe as no replicative virus on naïve Vero cells after continuously culturing of WNV-NS1 on VeroNS1 cells for fifteen rounds. WNV-NS1 was non-infectious in mice, even when IFNAR-/- mice were administrated with a high dose of WNV-NS1. A single dose of WNV-NS1 vaccination protected mice from a highly lethal challenge of WT WNV. The antibody response against WNV correlated well with the protection of vaccinated mice. Our study demonstrates the potential of the NS1 trans complementation system as a new platform for flavivirus vaccine development.

    IMPORTANCE

    Many flaviviruses are significant human pathogens that frequently cause outbreaks and epidemics around the world. Development of novel vaccine platforms against these pathogens is a public health priority. Using West Nile virus (WNV) as a model, we developed a new vaccine platform for flaviviruses. WNV containing a NS1 deletion (WNV-NS1) could be efficiently trans complemented in Vero cells that constitutively express WT NS1 protein. A single-dose immunization of WNV-NS1 elicited robust immune response in mice. The immunized animals were fully protected against pathogenic WNV infection. No adverse effects related to the WNV-NS1 vaccination were observed. The results have demonstrated the potential of the NS1 complementation system as an alternative platform for flaviviruses vaccine development, especially for highly pathogenic flaviviruses.

  • Marek's disease virus-encoded miR-155 ortholog critical for the induction of lymphomas is not essential for the proliferation of transformed cell lines [Transformation and Oncogenesis]

  • MicroRNAs (miRNAs) are small non-coding RNAs with profound regulatory roles in many areas of biology, including cancer. MicroRNA 155 (miR-155), one of the extensively studied multifunctional miRNAs, is important in several human malignancies such as diffuse large B cell lymphoma and chronic lymphocytic leukemia. Moreover, miR-155 orthologs KSHV-miR-K12-11 and MDV-miR-M4, encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) and Marek's disease virus (MDV) respectively, are also involved in oncogenesis. In MDV-induced T-cell lymphomas and lymphoblastoid cell lines derived from them, MDV-miR-M4 is highly expressed. Using excellent disease models of infection in natural avian hosts, we showed previously that MDV-miR-M4 is critical for the induction of T-cell lymphomas as mutant viruses with precise deletions were significantly compromised in their oncogenicity. However, these studies did not elucidate whether continued expression of MDV-miR-M4 is essential for maintaining the transformed phenotype of tumor cells. Here using an in situ CRISPR/Cas9 editing approach, we deleted MDV-miR-M4 from the MDV-induced lymphoma-derived lymphoblastoid cell line MDCC-HP8. Precise deletion of MDV-miR-M4 was confirmed by PCR, sequencing, quantitative RT-PCR and functional analysis. Continued proliferation of the MDV-miR-M4-deleted cell lines demonstrated that MDV-miR-M4 expression is non-essential for maintaining the transformed phenotype, despite its initial critical role in the induction of lymphomas. Ability to examine the direct role of oncogenic miRNAs in situ in tumour cell lines is valuable in delineating distinct determinants and pathways associated with the induction or maintenance of transformation in cancer cells and will also contribute significantly to gain further insights into the biology of oncogenic herpesviruses.

    Importance

    Marek's disease virus (MDV) is an alphaherpesvirus associated with Marek's disease, a highly contagious neoplastic disease of chickens. MD serves as an excellent model for studying virus-induced T-cell lymphomas in the natural chicken hosts. Among the limited set of genes associated with MD oncogenicity, MDV-miR-M4, a highly expressed viral ortholog of the oncogenic miR-155, has received extensive attention due to its direct role in the induction of lymphomas. Using a targeted CRISPR-Cas9-based gene editing approach in MDV-transformed lymphoblastoid cell lines, we show that MDV-miR-M4, despite its critical role in the induction of tumours, is not essential for maintaining the transformed phenotype and continuous proliferation. As far as we know, this is the first study where precise editing of an oncogenic miRNA has been carried out in situ in MD lymphoma-derived cell lines to demonstrate that it is not essential in maintaining the transformed phenotype.

  • Effect of geographic isolation on the nasal virome of indigenous children [Genetic Diversity and Evolution]

  • The influence of living in small remote villages on the diversity of viruses in the nasal mucosa was investigated in three Colombian villages with widely different levels of geographic isolation. Metagenomics was used to characterize viral nucleic acids in nasal swabs from 63 apparently healthy young children. Sequences from human virus members of the families Anelloviridae, Papillomaviridae, Picornaviridae, Herpesviridae, Polyomaviridae, Adenoviridae, and Paramyxoviridae were detected in a decreasing fraction of children. The number of papillomavirus infections detected was greater among Hispanic-speaking children most exposed to outside contacts while anellovirus infections were more common in the isolated indigenous villages. The diversity of the other human viruses detected did not differ between villages. Closely related variants of rhinoviruses A or B were identified in 2 to 4 children from each village reflecting ongoing transmission clusters. Genomes of viruses not currently known to infect humans, including in the family Parvoviridae, Partitiviridae, Dicistroviridae, and Iflaviridae and circular Rep expressing ssDNA genomes (CRESS-DNA) were also detected in nasal swabs possibly reflecting environmental contamination from insect, fungal, or unknown sources. Despite the high level of geographic and cultural isolation, the overall diversity of human viruses in the nasal passages of children was not reduced in highly isolated indigenous villages indicating ongoing exposure to globally circulating viruses.

    Importance

    Extreme geographic and cultural isolation can still be found in some indigenous South American villages. Such isolation may be expected to limit the introduction of otherwise common and widely distributed viruses. Very small population size may also result in rapid local viral extinction due to lack of sero-negative subjects to maintain transmission chains of rapidly cleared viruses. We compared the viruses in the nasal passage of young children in three villages with increasing level of geographic isolation. We find that isolation did not reduce the overall diversity of viral infections. Multiple infections with nearly identical rhinoviruses could be detected within each villages likely reflecting recent viral introductions and transmission clusters amongst epidemiologically linked members of these very small communities. We conclude that despite their geographic isolation remote indigenous villages show evidence of ongoing exposures to globally circulating viruses.

  • REVISITING THE CORRELATE OF REDUCED HIV INFECTION RISK IN THE RV144 VACCINE TRIAL [Commentary]

  • The RV144 vaccine trial is the only clinical study to have shown a modest but statistically significant decrease in HIV infection risk. RV144 and the subsequent studies identifying the level of V1V2-specific antibodies as a correlate of reduced infection risk are still controversial despite many papers supporting and expanding the initial study. We address these controversies and summarize active and passive immunization experiments in non-human primates that support the initial finding.

  • Extracellular albumin and endosomal ions prime enterovirus particles for uncoating that can be prevented by fatty acid saturation [Virus-Cell Interactions]

  • There is limited information about the molecular triggers leading to the uncoating of enteroviruses in physiological conditions. Using real-time spectroscopy and sucrose gradients with radioactively-labeled virus we show at 37 ddeg;C, formation of a low amount of albumin-triggered, metastable, uncoating intermediate of echovirus 1 without receptor engagement. This conversion was blocked by saturating the albumin with fatty acids. High potassium but low sodium and calcium concentrations, mimicking the endosomal environment, also induced the formation of a metastable uncoating intermediate of echovirus 1. Together, these factors boosted the formation of the uncoating intermediate and infectivity of this intermediate was retained, as judged by end-point titration. Cryo-electron microscopy reconstruction of the virions treated with albumin and high potassium, low sodium and low calcium concentrations resulted in a 3.6 AAring; resolution model revealing a fenestrated capsid showing 4 % expansion and loss of the pocket factor, similarly to altered (A-) particles described for other enteroviruses. The dimer interface between VP2 molecules was opened, the VP1 N-termini disordered and most likely externalised. The RNA was clearly visible, anchored to the capsid. The results presented here suggest that extracellular albumin, partially saturated with fatty acids, likely leads to the formation of the infectious uncoating intermediate prior to the engagement with the cellular receptor. In addition, changes in mono- and divalent cations, likely occurring in endosomes, promote capsid opening and genome release.

    Importance

    There is limited information about uncoating of enteroviruses in physiological conditions. Here, we focused on physiologically relevant factors that likely contribute to opening of echovirus 1 and other B-group enteroviruses. By combining biochemical and structural data, we show, that before entering cells, extracellular albumin is capable of priming the virus into a metastable, yet infectious intermediate state. The ionic changes that are suggested to occur in endosomes, can further contribute to uncoating and promote genome release, once the viral particle is endocytosed. Importantly, we provide a detailed high-resolution structure of a virion after treatment with albumin and a preset ion composition, showing pocket factor release, capsid expansion and fenestration, and the clearly visible genome still anchored to the capsid. This study provides valuable information about the physiological factors that contribute to the opening of B-group enteroviruses.

  • Retrograde regulation by the viral protein kinase epigenetically sustains the EBV latency-to-lytic switch to augment virus production [Virus-Cell Interactions]

  • Herpesviruses are ubiquitous and infection by some, like Epstein-Barr virus (EBV), is nearly universal. To persist, EBV must periodically switch from a latent to a replicative/lytic phase. This productive phase is responsible for most herpesvirus-associated diseases. EBV encodes a latency-to-lytic switch protein which upon activation sets off a vectorially-constrained cascade of gene expression that results in production of infectious virus. While triggering expression of the switch protein ZEBRA is essential to lytic cycle entry, sustaining its expression is equally important to avoid premature termination of the lytic cascade. We report that the viral protein kinase (vPK), encoded by a gene that is kinetically downstream of the lytic switch, sustains expression of ZEBRA, amplifies the lytic cascade increasing virus production, and importantly, prevents abortive lytic cycle. We find that vPK, through a non-canonical site phosphorylation, activates the cellular PI3-kinase-related kinase ATM to cause phosphorylation of the heterochromatin enforcer KAP1/TRIM28 even in the absence of EBV genomes or other EBV proteins. Phosphorylation of KAP1 renders it unable to restrain ZEBRA, thereby further derepressing and sustaining its expression to culminate in virus production. This partnership with a host kinase and a transcriptional corepressor enables retrograde regulation by vPK of ZEBRA nndash; an observation that is counter to the unidirectional regulation of gene expression reminiscent of most DNA viruses.

    Importance

    Herpesviruses infect nearly all humans and persist quiescently for the life of the host. These viruses intermittently activate into the lytic phase to produce infectious virus, thereby causing disease. To ensure that lytic activation is not prematurely terminated, expression of the virally-encoded lytic switch protein needs to be sustained. In studying Epstein-Barr virus, one of the most prevalent human herpesviruses that also causes cancer, we have discovered that a viral kinase activated by the viral lytic switch protein partners with a cellular kinase to deactivate a silencer of the lytic switch protein, thereby providing a positive feedback loop to ensure successful completion of the viral productive phase. Our findings highlight key nodes of interaction between the host and virus that could be exploited to treat lytic phase-associated diseases by terminating the lytic phase or kill cancer cells harboring herpesviruses by accelerating the completion of the lytic cascade.

  • Analysis of paramyxovirus transcription and replication by high-throughput sequencing [Genome Replication and Regulation of Viral Gene Expression]

  • We have developed a high-throughput sequencing (HTS) workflow for investigating paramyxovirus transcription and replication. We show that sequencing of oligo-dT selected polyadenylated mRNAs, without considering the orientation of the RNAs from which they had been generated, cannot accurately be used to analyse the abundance of viral mRNAs because genomic RNA co-purifies with the viral mRNAs. The best method is directional sequencing of infected cell RNA that has physically been depletion of ribosomal and mitochondrial RNA followed by bioinformatic steps to differentiate data originating from genomes from viral mRNAs and antigenomes. This approach has the advantage that the abundance of viral mRNA (and antigenomes) and genomes can be analysed and quantified from the same data. We investigated the kinetics of viral transcription and replication during infection of A549 cells with parainfluenza virus type 2 (PIV2), PIV3, PIV5 or mumps virus, and determined the abundance of individual viral mRNAs and readthrough mRNAs. We found that the mRNA abundance gradients differed significantly between all four viruses, but that for each virus the pattern remained relatively stable throughout infection. We suggest that rapid degradation of nonpolyadenylated mRNAs may be primarily responsible for the shape of the mRNA abundance gradient in parainfluenza virus 3, whereas a combination of this factor and disengagement of RNA polymerase at intergenic sequences, particularly those at the NP:P and P:M gene boundaries, may be responsible in the other viruses.

    IMPORTANCE High throughput sequencing (HTS) of virus infected cells can be used to study in great detail the patterns of virus transcription and replication. For paramyxoviruses, and by analogy for all other negative strand RNA viruses, we show that directional sequencing must be used to distinguish between genomic RNA and mRNA/antigenomic RNA because significant amounts of genomic RNA co-purify with polyA-selected mRNA. We found that the best method is directional sequencing of total cell RNA, after the physical removal of ribosomal RNA (and mitochondrial RNA), because quantitative information on the abundance of both genomic RNA and mRNA/antigenomes can be simultaneous derived. Using this approach, we reveal new details of the kinetics of virus transcription and replication for parainfluenza virus (PIV) type 2, PIV3, PIV5 and mumps virus, as well as on the relative abundance of the individual viral mRNAs.

  • PRRSV activates lipophagy to facilitate viral replication through down-regulation of NDRG1 expression [Cellular Response to Infection]

  • Autophagy maintains cellular homeostasis by degrading organelles, proteins, and lipids in lysosomes. Autophagy is involved in the innate and adaptive immune responses to a variety of pathogens. Some viruses can hijack host autophagy to enhance their replication. However, the role of autophagy in Porcine reproductive and respiratory syndrome virus (PRRSV) infection is unclear. Here, we show that N-Myc downstream regulated gene 1 (NDRG1) deficiency induced autophagy, which facilitated PRRSV replication by regulating lipid metabolism. NDRG1 mRNA is expressed ubiquitously in most porcine tissues and most strongly in white adipose tissue. PRRSV infection downregulated the expression of NDRG1 mRNA and protein, while NDRG1 deficiency contributed to PRRSV RNA replication and progeny virus assembly. NDRG1 deficiency reduced intracellular lipid droplets (LDs) number, but the expression levels of key genes in lipogenesis and lipolysis were not altered. Our results also show that NDRG1 deficiency promoted autophagy and increased the subsequent yields of hydrolyzed free fatty acids (FFAs). The reduced LD numbers, increased FFA levels, and enhanced PRRSV replication were abrogated in the presence of an autophagy inhibitor. Overall, our findings suggest that NDRG1 plays a negative role in PRRSV replication by suppressing autophagy and LD degradation.

    Importance

    Porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped single-positive-stranded RNA virus, causes acute respiratory distress in piglets and reproductive failure in sows. It has led to tremendous economic losses in the swine industry worldwide since it was first documented in the late 1980s. Vaccination is currently the major strategy used to control the disease. However, conventional vaccines and other strategies do not provide satisfactory or sustainable prevention. Therefore, safe and effective strategies to control PRRSV are urgently required. The significance of our research is that we demonstrate a previously unreported relationship between PRRSV, NDRG1, and lipophagy in the context of viral infection. Furthermore, our data point to a new role for NDRG1 in autophagy and lipid metabolism. Thus, NDRG1 and lipophagy will have significant implications for understanding PRRSV pathogenesis for developing new therapeutics.

  • Disassembly of the TRIM23-TBK1 Complex by the Us11 Protein of Herpes Simplex Virus 1 Impairs Autophagy [Virus-Cell Interactions]

  • The Us11 protein encoded by herpes simplex virus 1 (HSV-1) functions to impair autophagy; however, the molecular mechanisms of this inhibition remain to be fully established. Here, we report that the Us11 protein targets tripartite motif protein 23 (TRIM23), which is a key regulator of autophagy-mediated antiviral defense mediated by TANK-binding kinase 1 (TBK1). In virus-infected cells, the Us11 protein drastically reduces the formation of autophagosomes mediated by TRIM23 or TBK1. This autophagy-inhibitory effect is attributable to the binding of the Us11 protein to the ARF domain in TRIM23. Furthermore, such interaction spatially excludes TBK1 from the TRIM23 complex that also contains heat shock protein 90 (Hsp90). When stably expressed alone in host cells, the Us11 protein recapitulates the observed phenotypes seen in cells infected with the US11 expressing or wild type virus. Consistently, expression of the Us11 protein promotes HSV-1 growth while, on the other hand, expression of TRIM23 restricts HSV-1 replication in the absence of US11. Together, these results suggest that disruption of the TRIM23-TBK1 complex by the Us11 protein inhibits autophagy-mediated restriction of HSV-1 infection.

    IMPORTANCE

    Autophagy is an evolutionarily conserved process that restricts certain intracellular pathogens, including HSV-1. Although HSV-1 is well-known to inhibit autophagy, little is known about the precise molecular mechanisms of autophagy inhibition. We demonstrate that the Us11 protein of HSV-1 spatially disrupts the TRIM23-TBK1 complex, which subsequently suppresses autophagy and autophagy-mediated virus restriction. Thus, expression of the Us11 protein facilitates HSV-1 replication. These data unveil new insight into viral escape from autophagy-mediated host restriction mechanisms.

  • Allosteric regulation of HIV-1 capsid structure for Gag assembly, virion production, and viral infectivity by a disordered interdomain linker [Structure and Assembly]

  • Retroviral Gag-capsid (CA) interdomain linker is an unstructured peptide segment connecting structured N-terminal and C-terminal domains. Although the region is reported to play roles in virion morphogenesis and infectivity, underlying molecular mechanisms remain unexplored. To address this issue, we determined biological and molecular phenotypes of HIV-1 CA linker mutants by experimental and in silico approaches. Among the nine linker mutants tested, eight exhibited attenuation of viral particle production to varying extents mostly in parallel with a reduction in viral infectivity. Sucrose density gradient, confocal microscopy, and live-cell protein interaction analyses indicated that the defect is accompanied by attenuation of Gag-Gag interactions following Gag plasma membrane targeting in the cells. In silico analyses denoted distinct distributions of interaction-prone hydrophobic patches between immature and mature CA proteins. Molecular dynamics simulations predicted that the linker mutations can allosterically alter structural fluctuations including the interaction surfaces apart from the mutation sites in both the immature and mature CA proteins. These results suggest that the HIV-1 CA interdomain linker is a cis-modulator of the CA interaction surfaces to optimize efficiency of Gag assembly, virion production, and viral infectivity.

    Importance

    HIV-1 particle production and infection are highly ordered processes. Viral Gag proteins play a central role in the assembly and disassembly of viral molecules. Of these, capsid protein (CA) is a major contributor to the Gag-Gag interactions. CA consists of two structured domains, i.e., N-terminal (NTD) and C-terminal (CTD) domains, connected by an unstructured domain named interdomain linker. While multiple regions in the NTD and CTD domains are reported to play roles in virion morphogenesis and infectivity, the roles of the linker region in Gag assembly and virus particle formation remain elusive. In this report, we show by biological and molecular analyses that the linker region functions as an intramolecular modulator to tune Gag assembly, virion production, and viral infectivity. Our study thus illustrates a hitherto unrecognized mechanism, an allosteric regulation of CA structure by the disordered protein element, for HIV-1 replication.

  • Influenza virus with increased pH of HA activation has improved replication in cell culture but at the cost of infectivity in human airway epithelium [Virus-Cell Interactions]

  • Pandemic H1N1 (pH1N1) influenza virus emerged from swine in 2009 with adequate capability to infect and transmit between people. In subsequent years it has circulated as a seasonal virus and evolved further human-adapting mutations. Mutations in the haemagglutinin (HA) stalk that increase pH stability have been associated with human adaptation and airborne transmission of pH1N1 virus. Yet, our understanding of how pH stability impacts virus/host interactions is incomplete. Here, using recombinant viruses with point mutations that alter the pH stability of pH1N1 HA, we found distinct effects on virus phenotypes in different experimental models. Increased pH sensitivity enabled virus to uncoat in endosomes more efficiently, manifesting as increased replication rate in typical continuous cell cultures under single-cycle conditions. A more acid labile HA also conferred a small reduction in sensitivity to antiviral therapeutics that act at the pH-sensitive HA fusion step. Conversely, in primary human airway epithelium cultured at air-liquid interface, increased pH sensitivity attenuated multicycle viral replication, by compromising virus survival in the extracellular microenvironment. In a mouse model of influenza pathogenicity, there was an optimum HA activation pH and viruses with either more or less pH stable HA were less virulent. Opposing pressures inside and outside the host cell that determine pH stability may influence zoonotic potential. The distinct effects that changes in pH stability exert on viral phenotypes underscore the importance of using the most appropriate systems for assessing virus titre and fitness, which has implications for vaccine manufacture, antiviral drug development and pandemic risk assessment.

    Importance

    The pH stability of the haemagglutinin surface protein varies between different influenza strains and subtypes and can affect the virus' ability to replicate and transmit. Here, we demonstrate a delicate balance the virus strikes within and without the target cell. We show that a pH-stable haemagglutinin enables a human influenza virus to replicate more effectively in human airway cells and mouse lungs by facilitating virus survival in the extracellular environment of the upper respiratory tract. Conversely after entering target cells, being more pH-stable confers relative disadvantage, resulting in less efficient delivery of the viral genome to the host cell nucleus. Since the balance we describe will be affected differently in different host environments, it may restrict virus' ability to cross species. In addition, our findings imply that different influenza viruses may show variation in how well they are controlled by antiviral strategies targeting pH-dependent steps in the virus replication cycle.

  • Novel small molecule targeting the hemagglutinin stalk of influenza viruses [Vaccines and Antiviral Agents]

  • Combating influenza is one of the perennial global public health issues to be managed. Antiviral drugs are useful for the treatment of influenza in the absence of an appropriate vaccine. However, the appearance of resistant strains necessitates a constant search for new drugs. In this study, we investigated novel anti-influenza drug candidates using in vitro and in vivo assays. We identified anti-influenza hit compounds using a high-throughput screening method with a green fluorescent protein-tagged recombinant influenza virus. Through subsequent analyses of their cytotoxicity and pharmacokinetic properties, one candidate (IY7640) was selected for further evaluation. In a replication kinetics analysis, IY7640 showed greater inhibitory effects during the early phase of viral infection than the viral neuraminidase-inhibitor oseltamivir. In addition, we observed that hemagglutinin (HA)-mediated membrane fusion was inhibited by the IY7640 treatment, indicating that the HA stalk region, which is highly conserved across various (sub)types of influenza viruses, may be the molecular target of IY7640. In an escape mutant analysis in cells, amino acid mutations were identified at the HA stalk region of the 2009 pandemic H1N1 (pH1N1) virus. Even though the in vivo efficacy of IY7640 did not reach to complete protection in a lethal challenge study in mice, these results suggest that IY7640 has potential to be developed as a new type of anti-influenza drug.

    IMPORTANCE

    Anti-influenza drugs with broad-spectrum efficacy against antigenically diverse influenza viruses can be highly useful when no vaccines are available. To develop new anti-influenza drugs, we screened a number of small molecules and identified a strong candidate, IY7640. When added at the time of or after influenza virus infection, IY7640 was observed to successfully inhibit or reduce viral replication in cells. We subsequently discovered that IY7640 targets the stalk region of the influenza HA protein, which exhibits a relatively high degree of amino acid sequence conservation across various (sub)types of influenza viruses. Furthermore, IY7640 was observed to block HA-mediated membrane fusion of H1N1, H3N2, and influenza B viruses in cells. Although it appears less effective against strains other than H1N1 subtype viruses in a challenge study in mice, we suggest that the small molecule IY7640 has potential to be optimized as a new anti-influenza drug.

  • SIV infection modulates CD94 (KLRD1)+ NK cells in rhesus macaques [Pathogenesis and Immunity]

  • Recently others and we have shown that NK cells exhibit memory-like recall responses against CMV and HIV/SIV infections. Although the mechanism(s) have not been fully delineated, several groups have shown that the activating receptor NKG2C is elevated on NK cells in the context of rhesus CMV (rhCMV) or human CMV (hCMV) infections. CD94, which heterodimerizes with NKG2C is also linked to adaptive NK cell responses. Because non-human primates (NHP) play a crucial role in modeling HIV (SIV) infections, it is crucial to be able to assess and characterize the NKG2 family in NHP. Unfortunately it is not possible to detect CD94 using commercially available antibodies in NHP. Our work, a first for NHP, has focused on developing RNA-flow cytometry using mRNA transcripts as proxies distinguishing NKG2C from NKG2A. We have expanded the application of this technology and here we show the first characterization of CD94 (KLRD1)+ NK cells in NHP using multiparametric RNA-Flow cytometry. Peripheral blood mononuclear cells (PBMCs) from naïve and matched acutely (n=4) or chronically (n=12) SIV-infected rhesus macaques were analyzed by flow cytometry using commercially available antibodies, determining expression of transcripts for NKG2A, NKG2C and CD94 (KLRC1, KLRC2 and KLRD1 respectively) on NK cells using RNA-flow cytometry. Our data show that KLRC1pplusmn;KLRC2+KLRD1+ NK cells decrease following chronic, but not acute, infection with SIV. This approach will allow us to investigate the kinetics of infection and NK memory formation, and will further improve our understanding of basic NK cell biology, especially in the context of SIV infection.

    Importance:

    Non-human primates play a crucial role in approximating human biology and many diseases that are difficult, if not impossible, to achieve in other animal models, notably HIV. Current advances in adaptive NK cell research positions us to address fundamental deficiencies in our fight against infection and disease at the earliest moments following infection, or substantially earlier in disease progression. Here we show that we can identify specific NK cell subpopulations that are modulated following chronic, but not acute, SIV infection. The ability to identify these subsets more precisely will inform therapeutic and vaccine strategies targeting an optimized NK cell response.

  • Human hemoglobin subunit beta functions as a pleiotropic regulator of the RIG-I/MDA5-mediated antiviral innate immune responses [Cellular Response to Infection]

  • Hemoglobin is an important oxygen-carrying protein and plays crucial roles in establishing host resistance against pathogens and regulating innate immune responses. The hemoglobin subunit beta (HB) is an essential component of hemoglobin, and we have previously demonstrated that the antiviral role of the porcine HB (pHB) is mediated by promoting the type I interferon pathways. Thus, considering the high homology between human HB (hHB) and pHB, we hypothesized that hHB also play an important role in the antiviral innate immunity. In this study, we characterized hHB as a regulatory factor for the replication of RNA viruses by differentially regulating the RIG-I- and MDA5-mediated antiviral signaling pathways. Furthermore, we showed that hHB directly inhibited the MDA5-mediated signaling through reducing the MDA5-dsRNA affinity. Additionally, hHB required hHB-induced reactive oxygen species to promote the RIG-I-mediated signaling through enhancing the K63-linked RIG-I ubiquitination. Taken together, our findings suggest that hHB is a pleiotropic regulator of the RIG-I/MDA5-mediated antiviral responses and further highlight the importance of intercellular microenvironment including redox state in regulating the antiviral innate immune responses.

    Importance

    Hemoglobin, the most important oxygen-carrying protein, is involved in the regulation of innate immune responses. We have previously reported that the hemoglobin subunit beta (HB) of porcine exerts an antiviral ability through regulating the type I interferon production. However, the antiviral activities and the underlying mechanisms of HBs originated from other animals have been poorly understood. Here, we identified human HB (hHB) as a pleiotropic regulator of the replication of RNA viruses through regulating the RIG-I/MDA5-mediated signaling pathways. hHB enhances the RIG-I-mediated antiviral responses through promoting the RIG-I ubiquitination depending on the hHB-induced reactive oxygen species (ROS), while it blocks the MDA5-mediated antiviral signaling through suppressing the MDA5-dsRNA interaction. Our results contribute to understand the crucial roles of hHB in the regulation of the RIG-I/MDA5-mediated signaling pathways. We also provide a novel facet to the correlation of the intercellular redox state with the regulation of antiviral innate immunity.

  • A novel phenotype links HIV-1 capsid stability to cGAS-mediated DNA sensing [Virus-Cell Interactions]

  • HIV-1 capsid executes essential functions that are regulated by capsid stability and host factors. In contrast to increasing knowledge on functional roles of capsid-interacting host proteins during post-entry steps, less is known about capsid stability and its impact on intracellular events. Here, using the antiviral compound PF74 as a probe for capsid function, we uncover a novel phenotype of capsid stability that has a profound effect on innate sensing of viral DNA by the DNA sensor cGAS. A single mutation, R143A, in the capsid protein conferred resistance to high concentrations of PF74, without affecting capsid binding to PF74. A cell-free assay showed that the R143A mutant partially counteracted the capsid-destabilizing activity of PF74, pointing to capsid stabilization as a resistance mechanism for R143A. In monocytic THP-1 cells, the R143A virus, but not the wild-type virus, suppressed cGAS-dependent innate immune activation. These results suggest that capsid stabilization improves the shielding of viral DNA from innate sensing. We found that a naturally occurring transmitted founder (T/F) variant shares the same properties as the R143A mutant with respect to PF74 resistance and DNA sensing. Imaging assays revealed delayed uncoating kinetics of this T/F variant and the R143A mutant. All these phenotypes of this T/F variant were controlled by a genetic polymorphism located at the trimeric interface between capsid hexamers, thus linking these capsid-dependent properties. Overall, this work functionally connects capsid stability to innate sensing of viral DNA and reveals naturally occurring phenotypic variation in HIV-1 capsid stability.

    Importance

    The HIV-1 capsid, which is made from individual viral capsid proteins (CA), is a target for a number of antiviral compounds including the small-molecule inhibitor PF74. In the present study, we utilized PF74 to identify a transmitted/founder (T/F) strain that shows increased capsid stability. Interestingly, PF74-resistant variants prevented cGAS-dependent innate immune activation in a condition where the other T/F strains induced type I interferon. These observations thus reveal a new CA-specific phenotype that couples capsid stability to viral DNA recognition by cytosolic DNA sensors.

  • CD28 costimulation is required for development of herpetic stromal keratitis but does not prevent establishment of latency [Pathogenesis and Immunity]

  • Corneal infection with HSV-1 leads to infection of trigeminal ganglia (TG) typically followed by establishment of latency in the infected neurons. When latency is disrupted the virus reactivates and migrates back to the cornea where it re-stimulates the immune response leading to lesions in a disease called herpetic stromal keratitis (HSK). HSK requires T cell activation as in the absence of T cells there is no disease. We decided to determine if CD28 costimulation of T cells was required in HSK. Results indicated that mice (B6-CD28-/- and BALB-CD28-/- failed to develop recurrent HSK while their wild-type counterparts did. In order to better understand the dynamics of TG infection in these mice, we evaluated the amount of virus in infected TG and the number of individual neurons harboring latent virus. Results indicate that CD28-/- mice possessed significantly increased genome levels in their TG, but much fewer LAT+ cells as compared to wild-type mice from Day 7 to Day 30, but that after Day 30 these differences became non-significant. We next evaluated total and antigen-specific CD8+ T cells in TG. Results indicated that there were significantly fewer CD8 T cells in TG from Day 10 to Day 25, but that after that differences were not significant. Taken together, these data suggest that CD28 costimulation is required for HSK, but that while initial infection of TG is greater in CD28-/- mice this begins to normalize with time and that this normalization is concurrent with the delayed development of antigen specific CD8+ T cells.

    IMPORTANCE

    We study the pathogenesis of herpes simplex-mediated corneal disease. T cells play a critical role in both disease and in the maintenance of latency in neurons. Consequently, the focus of this report was to evaluate the role that T cell costimulation plays in both corneal disease and in controlling the ability of the virus to maintain a stable infection of the ganglia that innervates the cornea. We demonstrate that in the absence of costimulation with CD28, corneal disease does not take place. However, this costimulation does not prevent the ability to CD8+ T cells to develop and thus control latent infection of neurons. We conclude from these studies that CD28 costimulation is required for corneal destructive immune responses, but that CD8+ T cells will develop over time and help to maintain latency.

  • Contemporary circulating enterovirus D68 strains infect and undergo retrograde axonal transport in spinal motor neurons independent of sialic acid [Virus-Cell Interactions]

  • Enterovirus D68 (EV-D68) is an emerging virus that has been identified as a cause of recent outbreaks of acute flaccid myelitis (AFM), a poliomyelitis-like spinal cord syndrome that can result in permanent paralysis and disability. In experimental mouse models, EV-D68 spreads to, infects, and kills spinal motor neurons following various routes of inoculation. The topography of virus-induced motor neuron loss correlates with the pattern of paralysis. The mechanism(s) by which EV-D68 spreads to target motor neurons remains unclear. We sought to determine the capacity of EV-D68 to spread by the neuronal route and to determine the role of known EV-D68 receptors, sialic acid and intracellular adhesion molecule 5 (ICAM-5), in neuronal infection. To do this, we utilized a microfluidic chamber culture system in which human iPSC motor neuron cell bodies and axons can be compartmentalized for independent experimental manipulation. We found that EV-D68 can infect motor neurons via their distal axons and spread by retrograde axonal transport to the neuronal cell bodies. Virus was not released from the axons via anterograde axonal transport after infection of the cell bodies. Prototypic strains of EV-D68 depended on sialic acid for axonal infection and transport, while contemporary circulating strains isolated during the 2014 EV-D68 outbreak did not. The pattern of infection did not correspond with ICAM-5 distribution and expression in either human tissue, the mouse model, or the iPSC motor neurons.

    IMPORTANCE Enterovirus D68 (EV-D68) infections are on the rise worldwide. Since 2014, the United States has experienced biennial spikes in EV-D68-associated acute flaccid myelitis (AFM) that have left hundreds of children paralyzed. Much remains to be learned about the pathogenesis of EV-D68 in the central nervous system (CNS). Herein we investigate the mechanisms of EV-D68 CNS invasion through neuronal pathways. A better understanding of EV-D68 infection in experimental models may allow for better prevention and treatment strategies of EV-D68 CNS disease.

  • Variation in intra-individual lentiviral evolution rates: A systematic review of human, non-human primate and felid species. [Genetic Diversity and Evolution]

  • Lentiviral replication mediated by reverse transcriptase is considered to be highly error prone, leading to a high intra-individual evolution rate that promotes evasion of neutralization and persistent infection. Understanding lentiviral intra-individual evolutionary dynamics on a comparative basis can therefore inform research strategies to aid in studies of pathogenesis, vaccine design, and therapeutic intervention. We conducted a systematic review of intra-individual evolution rates for three species groups of lentiviruses- feline immunodeficiency virus (FIV), simian immunodeficiency virus (SIV), and human immunodeficiency virus (HIV). Overall, intra-individual rate estimates differed by virus, but not by host, gene or viral strain. Lentiviral infections in spillover (nonadapted hosts) approximated infections in primary (adapted) hosts. Our review consistently documents that FIV evolution rates within individuals are significantly slower than rates recorded for HIV and SIV. FIV intra-individual evolution rates were noted to be equivalent to FIV inter-individual rates. These findings document inherent differences in evolution of FIV relative to primate lentiviruses which may signal intrinsic difference of reverse transcriptase between these viral species or different host-viral interactions. Analyses of lentiviral evolutionary selection pressures at individual versus population level is valuable for understanding transmission dynamics, emergence of virulent and avirulent strains, and provides novel insight for approaches to interrupt lentiviral infections.

    Importance

    To the best of our knowledge, this is the first study that compares intra-individual evolution rates for FIV, SIV and HIV following systematic review of the literature. Our findings have important implications for informing research strategies in the field of intra-individual virus dynamics for lentiviruses. We observed that FIV evolves more slowly than HIV and SIV at the intra-individual level and found that mutation rates may differ by gene sequence length, but not by host, gene, strain, experimental settings (relative to natural) or spillover host infections (relative to primary).

  • TAF family proteins and MEF2C are essential for Epstein-Barr virus MYC super-enhancer activity [Transformation and Oncogenesis]

  • Super-enhancers (SEs) are clusters of enhancers marked by extraordinary high and broad chromatin immune-precipitation followed by deep sequencing (ChIP-seq) signals for H3K27ac or other transcription factors (TFs). SEs play pivotal roles in development and oncogenesis. Epstein-Barr virus (EBV) super-enhancers (ESEs) are co-occupied by all essential EBV oncogenes and EBV-activated NF-kB subunits. Perturbation of ESEs stops lymphoblastoid cell line (LCL) growth. To further characterize ESEs and identify proteins critical for ESEs function, MYC ESEs were cloned upstream of a GFP reporter. Reporters driven by MYC ESEs 525kb and 428kb upstream of MYC (525ESE and 428ESE) had very high activities in LCLs but not in EBV-negative BJAB cells. EBNA2 activated MYC ESE driven Luciferase reporters. CRISPRi targeting 525ESE significantly decreased MYC expression. Genome-wide CRISPR screens identified factors essential for ESE activity. TAF family proteins including TAF8, TAF11, and TAF3 were essential for the activity of the integrated 525ESE driven reporter in LCLs. TAF8 and TAF11 knockout significantly decreased 525ESE activity and MYC transcription. MEF2C was also identified to be essential for 525ESE activity. Depletion of MEF2C decreased 525ESE reporter activity, MYC expression, and LCL growth. MEF2C cDNA resistant to CRIPSR cutting rescued MEF2C knockout, restored 525ESE reporter activity and MYC expression. MEF2C depletion decreased IRF4, EBNA2, and SPI1 binding to 525ESE in LCLs. MEF2C depletion also affected the expression of other ESE target genes including ETS1 and BCL2. These data indicated that in addition to EBNA2, TAF family members and MEF2C are essential for ESE activity, MYC expression, and LCL growth.

    Importance

    SEs play critical roles in cancer development. Since SEs assemble much bigger protein complexes on enhancers than typical enhancers (TEs), they are more sensitive than TEs to perturbations. Understanding the protein composition of SEs that are linked to key oncogenes may identify novel therapeutic targets. A genome-wide CRISPR screen specifically identified proteins essential for MYC ESE activity but not SV40 enhancer. These proteins were not only essential for the reporter activity[underln],[/underln] but were also important for MYC expression and LCL growth. Targeting these proteins may lead to new therapies for EBV-associated cancers.

  • Pentosan polysulfate demonstrates anti-HTLV-1 activities in vitro and in vivo [Vaccines and Antiviral Agents]

  • HTLV-1 infection causes T-cell leukemia and inflammatory diseases, most notably including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The underlying mechanism for the pathogenesis of HAM/TSP remains unclear. According to a recent clinical trial, a humanized antibody that targets CCR4+ cells ameliorates inflammation by reducing the number of infected cells in the central nervous system; this result suggests that transmigration of HTLV-1 infected cells plays a crucial role in HAM/TSP. Partly due to the blood-brain barrier, current treatments for HAM/TSP are mostly palliative. Pentosan polysulfate (PPS), a semisynthetic glycosaminoglycan, has recently been used to treat HAM/TSP and was found to alleviate the symptoms. In this study, we investigate the effect of PPS on HTLV-1 infected cells and provide evidence for its efficacy in HAM/TSP. PPS is cytotoxic to certain HTLV-1 infected cells and significantly suppresses HTLV-1 virion production. PPS also efficiently inhibits HTLV-1 cell-cell transmission in T cells. In addition, PPS blocks HTLV-1 infection of primary endothelial cells (HUVEC) and suppresses the subsequent induction of proinflammatory cytokine expression. Furthermore, PPS was found to inhibit the adhesion and transmigration of HTLV-1 infected cells. We also confirmed the anti-HTLV-1 effect of PPS in vivo using two mouse models. PPS blocked HTLV-1 infection in a PBMC humanized mouse model, huPBMC NSG mice. PPS was also found to suppress the development of dermatitis and lung damage in HBZ-transgenic (HBZ-Tg) mice, an HTLV-1 transgenic mouse model that develops systemic inflammation.

    Importance

    HTLV-1 is the first human retrovirus to have been identified and is endemic in certain areas worldwide. HTLV-1 infection leads to the development of an inflammatory disease called HAM/TSP, a myelopathy characterized by slowly progressive spastic paraparesis. There have been no effective therapeutics available for HAM/TSP, but recently a semisynthetic glycosaminoglycan named pentosan polysulfate (PPS) has been found to alleviate the symptoms of HAM/TSP. Here we conduct a comprehensive study on the effect of PPS both in vitro and in vivo. PPS demonstrates anti-HTLV-1 potential in infected cell lines, as shown by its suppressive effects on HTLV-1 replication and transmission and on transmigration of infected T cells. Moreover, results obtained from two HTLV-1 mouse models demonstrate that PPS inhibits HTLV-1 infection and inflammation development in vivo. Our work offers insights into the treatment of HAM/TSP by PPS and also suggests its possible use for treating other HTLV-1 induced inflammatory diseases.

  • Programmed -2/-1 Ribosomal Frameshifting in Simarteriviruses: An Evolutionarily Conserved Mechanism [Genome Replication and Regulation of Viral Gene Expression]

  • The -2/-1 programmed ribosomal frameshifting (-2/-1 PRF) mechanism in porcine reproductive and respiratory syndrome virus (PRRSV) leads to the translation of two additional viral proteins, nsp2TF and nsp2N. This -2/-1 PRF mechanism is transactivated by a viral protein nsp1bbeta; and cellular poly(rC) binding proteins (PCBPs). Critical elements for -2/-1 PRF, including a slippery sequence and a downstream C-rich motif, were also identified in eleven simarteriviruses. However, the slippery sequences (XXXUCUCU instead of XXXUUUUU) in seven simarteriviruses can only facilitate -2 PRF to generate nsp2TF. The nsp1bbeta; of simian hemorrhagic fever virus (SHFV) was identified as a key factor that transactivates both -2 and -1 PRF, and universally conserved Tyr111 and Arg114 in nsp1bbeta; are essential for this activity. In vitro translation experiments demonstrated the involvement of PCBPs in simarterivirus -2/-1 PRF. Using SHFV reverse genetics, we confirmed critical roles of nsp1bbeta;, slippery sequence and C-rich motif in -2/-1 PRF in SHFV-infected cells. Attenuated virus growth ability was observed in SHFV mutants with impaired expression of nsp2TF and nsp2N. Comparative genomic sequence analysis showed that key elements of -2/-1 PRF are highly conserved in all known arteriviruses, except equine arteritis virus (EAV) and wobbly possum disease virus (WPDV). Furthermore, -2/-1 PRF with SHFV PRF signal RNA can be stimulated by heterotypic nsp1bbeta;s of all tested non-EAV arteriviruses. Taken together, these data suggest that -2/-1 PRF is an evolutionarily conserved mechanism employed in non-EAV/WPDV arteriviruses for the expression of additional viral proteins that are important for viral replication.

    IMPORTANCE

    Simarteriviruses are a group of arteriviruses infecting nonhuman primates, and a number of new species have been established in recent years. Although these arteriviruses are widely distributed among African nonhuman primates of different species and some of them caused lethal hemorrhagic fever disease, this group of viruses has been under-characterized. Since wild nonhuman primates are historically important sources or reservoirs of human pathogens, there is concern that simarteriviruses may be "preemergent" zoonotic pathogens. Thus, molecular characterization of simarteriviruses is becoming a priority in arterivirology. In this study, we demonstrated that an evolutionarily conserved ribosomal frameshifting mechanism is used by simarteriviruses and other distantly related arteriviruses for the expression of additional viral proteins. This mechanism is unprecedented in eukaryotic systems. Given the crucial role of ribosome function in all living systems, the potential impact of the in-depth characterization of this novel mechanism reaches beyond the field of virology.

  • Peste des petits ruminants virus nucleocapsid protein inhibits interferon-{beta} production by interacting with IRF3 to block its activation [Virus-Cell Interactions]

  • Peste des petits ruminants virus (PPRV) is the etiological agent of Peste des petits ruminants, causing acute immunosuppression in its natural hosts. However, the molecular mechanisms by which PPRV antagonizes the host immune responses have not been fully characterized. In particular, how PPRV suppresses the activation of the host RIG-I-like receptors (RLRs) pathway has yet to be clarified. In this study, we demonstrated that PPRV infection significantly suppressed the RLRs pathway activation and type I interferon (IFN) production, and identified PPRV N protein as an extremely important antagonistic viral factor that suppressed IFN-bbeta; and IFN-stimulated genes (ISGs) expression. A detailed analysis showed that PPRV N protein inhibited type I IFN production by targeting interferon regulatory factor 3 (IRF3), a key molecule in RLRs pathway required for type I IFN induction. PPRV N protein interacted with IRF3 (but not with other components of RLRs pathway, including MDA5, RIG-I, VISA, TBK1, and MITA) and abrogated the phosphorylation of IRF3. As expected, PPRV N protein also considerably impaired the nuclear translocation of IRF3. The TBK1nndash;IRF3 interaction was significantly involved in IRF3 phosphorylation, and we showed that PPRV N protein inhibited the association between TBK1 and IRF3, which in turn inhibited IRF3 phosphorylation. Amino acid 106nndash;210 region of PPRV N protein was determined to be essential for suppressing the nuclear translocation of IRF3 and IFN-bbeta; production; and the 140-400 region of IRF3 was identified as the crucial region for the Nnndash;IRF3 interaction. Together, our findings demonstrated a new mechanism evolved by PPRV to inhibit type I IFN production and provide structural insights into the immunosuppression caused by PPRV.

    IMPORTANCE

    Peste des petits ruminants is a highly contagious animal disease affecting small ruminants, which threatens both small livestock and endangered susceptible wildlife populations in many countries. The causative agent, Peste des petits ruminants virus (PPRV), often causes acute immunosuppression in its natural hosts during infection. Here, for the first time, we demonstrated that N protein, the most abundant protein of PPRV, played an extremely important role in suppression of IRF3 function and type I IFN production by interfering with the formation of the TBK1-IRF3 complex. This study explored a novel antagonistic mechanism of PPRV.

  • Novel Function of Bluetongue Virus NS3 Protein in Regulation of the MAPK/ERK Signaling Pathway [Pathogenesis and Immunity]

  • Bluetongue virus (BTV) is an arbovirus transmitted by blood-feeding midges to a wide range of wild and domestic ruminants. In this report, we showed that BTV, through its non-structural protein NS3 (BTV-NS3), is able to activate the MAPK/ERK pathway as assessed by phosphorylation levels of ERK1/2 and the translation initiation factor eIF4E. By combining immunoprecipitation of BTV-NS3 and mass spectrometry analysis from both BTV-infected and NS3-transfected cells, we identified the serine/threonine-protein kinase B-Raf (BRAF), a crucial player of the MAPK/ERK pathway, as a new cellular interactor of BTV-NS3. BRAF silencing led to a significant decrease of the MAPK/ERK activation by BTV supporting a model where BTV-NS3 interacts with BRAF to activate this signaling cascade. This positive regulation acts independently of the role of BTV-NS3 in counteracting the induction of the IFN-aalpha;/bbeta; response. Furthermore, the intrinsic ability of BTV-NS3 to bind BRAF and activate the MAPK/ERK pathway is conserved throughout multiple serotypes/strains but appears to be specific to BTV compared to other members of Orbivirus genus. Inhibition of MAPK/ERK pathway with U0126 reduced viral titers, suggesting that BTV manipulates this pathway for its own replication. Altogether, our data provide molecular mechanisms that unravel new essential functions of NS3 during BTV infection.

    IMPORTANCE Bluetongue Virus (BTV) is responsible of the arthropod-borne disease Bluetongue (BT) transmitted to ruminants by blood-feeding midges. In this report, we found that BTV, through its non-structural protein NS3 (BTV-NS3), interacts with BRAF, a key component of the MAPK/ERK pathway. In response to growth factors, this pathway promotes cell survival and increases protein translation. We showed that BTV-NS3 enhances the MAPK/ERK pathway and this activation is BRAF-dependent. Treatment of MAPK/ERK pathway with the pharmacologic inhibitor U0126 impairs viral replication, suggesting that BTV manipulates this pathway for its own benefit. Our results illustrate, at the molecular level, how a single virulence factor has evolved to target a cellular function to increase its viral replication.

  • Ocular Glands become Infected Secondary to Infectious Keratitis and Play a Role in Corneal Resistance to Infection [Pathogenesis and Immunity]

  • Ocular glands play a critical role in eye health through the excretion of factors directly onto the ocular surface. The cornea is a normally transparent tissue necessary for visual acuity located in the anterior segment of the eye. Corneal damage can occur during microbial infection of the cornea resulting in potentially permanent visual deficits. The involvement of ocular glands during corneal infection has been only briefly described. We hypothesized that ocular glands contribute to resistance as an arm of the eye-associated lymphoid tissue and may also be susceptible to infection secondary to microbial keratitis. Utilizing a mouse model of herpes simplex virus type-1 (HSV-1) keratitis, we found that infection of corneas resulted in subsequent infection of ocular glands, including harderian glands (HGs) and extraorbital glands. Similarly, infection of corneas with Pseudomonas aeruginosa resulted in secondary infection of ocular glands. A robust immune response, characterized by increased numbers of immune cells and inflammatory mediators, occurred within ocular glands following HSV-1 keratitis. Removal of HGs altered corneal resistance to HSV-1 as measured by increased viral load, decreased corneal edema, and decreased inflammatory cell infiltration. These novel findings suggest that ocular glands are involved in microbial keratitis through their susceptibility to secondary infection and contribution to corneal resistance.

    IMPORTANCE Microbial keratitis accounts for up to 700,000 clinical visits annually in the United States. The involvement of ocular glands during microbial keratitis is not readily appreciated and treatment options do not address the consequences of ocular gland dysfunction. The current study shows that ocular glands are susceptible to direct infection by common ocular pathogens, including HSV-1 and Pseudomonas aeruginosa, subsequent to microbial keratitis. Additionally, ocular glands contribute soluble factors that play a role in corneal resistance to HSV-1 and alter viral load, corneal edema, and immune cell infiltration. Further studies are needed to elucidate the mechanisms by which this occurs.

  • Hepatitis B Virus X Protein Function Requires Zinc Binding [Virus-Cell Interactions]

  • The host structural maintenance of chromosomes 5/6 complex (Smc5/6) suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the cellular DDB1-containing E3 ubiquitin ligase to target Smc5/6 for degradation. However, the details of how HBx modulates the interaction between DDB1 and Smc5/6 remain to be determined. In this study, we performed biophysical analyses of recombinant HBx and functional analysis of HBx mutants in HBV-infected primary human hepatocytes (PHH) to identify key regions and residues that are required for HBx function. We determined that recombinant HBx is soluble and exhibits stoichiometric zinc binding when expressed in the presence of DDB1. Mass spectrometry-based hydrogen-deuterium exchange and cysteine-specific chemical footprinting of the HBx:DDB1 complex identified several HBx cysteine residues (located between amino acids 61-137) that are likely involved in zinc binding. These cysteine residues did not form disulphide bonds in HBx expressed in human cells. In line with the biophysical data, functional analysis demonstrated that HBx amino acids 45-140 are required for Smc6 degradation and HBV transcription in PHH. Furthermore, site-directed mutagenesis determined that C61, C69, C137 and H139 are necessary for HBx function, although are likely not essential for DDB1 binding. This CCCH motif is highly conserved in HBV as well as in the X protein from various mammalian hepadnaviruses. Collectively, our data indicate that the essential HBx cysteine and histidine residues form a zinc-binding motif that is required for HBx function.

    IMPORTANCE The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses HBV transcription. HBV counters this restriction by expressing HBV X protein (HBx), which redirects a host ubiquitin ligase to target Smc5/6 for degradation. Despite this recent advance in understanding HBx function, the key regions and residues of HBx required for Smc5/6 degradation have not been determined. In the current study, we performed biochemical, biophysical and cell-based analyses of HBx. By doing so, we mapped the minimal functional region of HBx and identified a highly conserved CCCH motif in HBx that is likely responsible for coordinating zinc and is essential for HBx function. We also developed a method to produce soluble recombinant HBx protein that likely adopts a physiologically relevant conformation. Collectively, this study provides new insights into the HBx structure-function relationship and suggests a new approach for structural studies of this enigmatic viral regulatory protein.

  • Spatiotemporal Analysis of Hepatitis B Virus X Protein in Primary Human Hepatocytes [Virus-Cell Interactions]

  • The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the host DDB1 E3 ubiquitin ligase to target Smc5/6 for degradation. HBx is an attractive therapeutic target for the treatment of chronic hepatitis B (CHB), but it is challenging to study this important viral protein in the context of natural infection due to the lack of a highly specific and sensitive HBx antibody. In this study, we developed a novel monoclonal antibody that enables detection of HBx protein in HBV-infected primary human hepatocytes (PHH) by Western blot and immunofluorescence. Confocal imaging studies with this antibody demonstrated that HBx is predominantly located in the nucleus of HBV-infected PHH, where it exhibits a diffuse staining pattern. In contrast, a DDB1-binding deficient HBx mutant was detected in both the cytoplasm and nucleus, suggesting that the DDB1 interaction plays an important role in the nuclear localization of HBx. Our study also revealed that HBx is expressed early after infection and has a short half-life (~3 hours) in HBV-infected PHH. In addition, we found that treatment with siRNAs that target DDB1 or HBx mRNA decreased HBx protein levels and led to the reappearance of Smc6 in the nuclei of HBV-infected PHH. Collectively, these studies provide the first spatiotemporal analysis of HBx in a natural infection system and also suggest that HBV transcriptional silencing by Smc5/6 can be restored by therapeutic targeting of HBx.

    IMPORTANCE Hepatitis B virus X protein (HBx) is a promising drug target since it promotes the degradation of the host structural maintenance of chromosomes 5/6 complex (Smc5/6) that inhibits HBV transcription. To date, it has not been possible to study HBx in physiologically relevant cell culture systems due to the lack of a highly specific and selective HBx antibody. In this study, we developed a novel monoclonal HBx antibody and performed a spatiotemporal analysis of HBx in a natural infection system. This revealed that HBx localizes to the nucleus of infected cells, is expressed shortly after infection and has a short half-life. In addition, we demonstrated that inhibiting HBx expression or function promotes the reappearance of Smc6 in the nucleus of infected cells. These data provide new insights into HBx and underscores its potential as a novel target for the treatment of chronic HBV infection.

  • Comprehensive characterization of the HERV-K(HML-6): overview of their structure, phylogeny and contribution to the human genome [Genetic Diversity and Evolution]

  • The 8% human genome is composed of Human Endogenous Retroviruses (HERVs), remnants of ancestral germline infections by exogenous retroviruses, which have been vertically transmitted as Mendelian characters. The HML-6 group, a member of the class II Betaretrovirus-like, includes several proviral loci with an increased transcriptional activity in cancer, and at least two elements that are known for retaining an intact open reading frame (ORF) and for encoding small proteins such as ERVK3-1, that is expressed in various healthy tissues, and HERV-K-MEL, a small Env peptide expressed in samples of cutaneous and ocular melanoma, but not in normal tissues.

    Importance: We reported the distribution and genetic composition of 66 HML-6 elements. We analyzed the phylogeny of the HML-6 sequences and identified two main clusters. We provided the first description of a Rec domain within the env sequence of 23 HML-6 elements. A Rec domain was also predicted within the ERVK3-1 transcript sequence, revealing its expression in various healthy tissues. Evidence about the context of insertion and co-localization of 19 HML-6 elements with functional human genes are also reported, including the sequence 16p11.2, whose 5'LTR overlapped the exon of one transcript variant of a cellular Zinc-finger up-regulated and involved in hepatocellular carcinoma. The present work provides the first complete overview of the HML-6 elements in GRCh37(hg19), describing the structure, phylogeny and genomic context of insertion of each locus. This information allows a better understanding of the genetics of one of the most expressed HERV groups in the human genome.

  • Species-specific co-localization of MERS-CoV attachment and entry receptors [Pathogenesis and Immunity]

  • MERS-CoV uses the S1B domain of its spike protein to bind to dipeptidyl peptidase-4 (DPP4), its functional receptor, and its S1A domain to bind to sialic acids. The tissue localization of DPP4 in humans, bats, camelids, pigs, and rabbits generally correlates with MERS-CoV tropism, highlighting the role of DPP4 in virus pathogenesis and transmission. However, MERS-CoV S1A does not indiscriminately bind to all aalpha;2,3-sialic acids and the species-specific binding and tissue distribution of these sialic acids in different MERS-CoV susceptible species have not been investigated. We established a novel method to detect these sialic acids on tissue sections of various organs of different susceptible species by using nanoparticles displaying multivalent MERS-CoV S1A. We found that the nanoparticles specifically bound to the nasal epithelial cells of dromedary camels, type II pneumocytes in human lungs, and the intestinal epithelial cells of common pipistrelle bats. Desialylation by neuraminidase abolished nanoparticle binding and significantly reduced MERS-CoV infection in primary susceptible cells. In contrast, S1A nanoparticles did not bind to the intestinal epithelium of serotine bats and frugivorous bat species, nor to the nasal epithelium of pigs and rabbits. Both pigs and rabbits have been shown to shed less infectious virus than dromedary camels and do not transmit the virus, neither via contact nor airborne routes. Our results depict species specific co-localization of MERS-CoV entry and attachment receptors which may be relevant in the transmission and pathogenesis of MERS-CoV.

    IMPORTANCE MERS-CoV uses the S1B domain of its spike protein to attach to its host receptor, dipeptidyl peptidase-4 (DPP4). The tissue localization of DPP4 has been mapped in different susceptible species. On the other hand, the S1A domain, the N-terminal domain of this spike protein, preferentially binds to several glycotopes of aalpha;2,3-sialic acids, the attachment factor of MERS-CoV. Here we show, using a novel method, that the S1A domain specifically binds to the nasal epithelium of dromedary camels, alveolar epithelium of humans, and intestinal epithelium of common pipistrelle bats. In contrast, neither binds to the nasal epithelium of pigs nor rabbits, neither did it bind to the intestinal epithelium of serotine bats and frugivorous bat species. This finding supports the importance of the S1A domain in MERS-CoV infection and tropism, suggests its role in transmission, and highlights its potential use as a component of novel vaccine candidates.

  • In Vitro and In Vivo Inhibition of Infectivity of Human Enterovirus 71 by a Sulfonated Food Azo Dye, Brilliant Black BN [Vaccines and Antiviral Agents]

  • Hand, foot and mouth disease (HFMD), a highly contagious disease in children, is caused by human enteroviruses, including Enterovirus 71 (EV71), Coxsakievirus A16 (CVA16) and Coxsakievirus A6 (CVA6). Although HFMD is usually mild and self-limiting, EV71 infection occasionally leads to fatal neurological disorders. Currently, no commercial antiviral drugs for HFMD treatment are available. Here, numerous sulfonated azo dyes, widely used as food additives, were identified to have potent antiviral activities against human enteroviruses. Among them, brilliant black BN (E151) was able to inhibit all tested EV71, CVA16 and CVA6 strains. In rhabdomyosarcoma cells, 50% inhibitory concentration of the dye E151 for various strains of EV71 ranged from 2.39 mmu;M to 28.12 mmu;M whereas its 50% cytotoxic concentration was 1870 mmu;M. Food azo dyes, including E151, interacted with the vertex of the 5-fold axis of EV71 and prevented viral entry. Their efficacy on viral inhibition was regulated by amino acids at VP1-98, VP1-145 and/or VP1-246. Dye E151 not only prevented EV71 attachment but also eluted the attached viruses in a concentration dependent manner. Moreover, E151 inhibited the interaction between EV71 and its cellular uncoating factor cyclophilin A. In vivo studies demonstrated that E151 at a dose of 200 mg/kg/day given at the initial four days of challenge protected AG129 mice challenged with 10 of 50% lethal dose of wild type EV71 isolates. Taken together, these data highlight E151 as a promising antiviral agent against EV71 infection.

    IMPORTANCE Human enterovirus 71 (EV71) is one of causative agents of hand, foot and mouth disease in children and is responsible for thousands of deaths in past 20 years. Food azo dyes have been widely used since the nineteenth century, however, their biological effects on humans and microbes residing in humans are poorly understood. Here, we discovered one of these dyes, brilliant black BN (E151), was particularly effective in inhibiting the infectivity of EV71 in both cell culture and mouse model studies. Mechanistic studies demonstrated that these sulfonated dyes mainly competed with EV71 attachment factors for viral binding to block viral attachment/entry to host cells. As no commercial antiviral drugs against EV71 are currently available, our findings open an avenue to exploit the development of permitted food dye E151 as a potential anti-EV71 agent.

  • The absence of lymphotoxin-{alpha}, an HVEM ligand, affects HSV-1 infection in vivo differently than the absence of other HVEM cellular ligands [Pathogenesis and Immunity]

  • Previously, we reported that the absence of herpesvirus entry mediator (HVEM) decreases latency but not primary infection in ocularly infected mice. Recently we reported that similar to HVEM, the absence of HVEM ligands (i.e., LIGHT, CD160 and BTLA) also decreased latency but not primary infection. Similar to LIGHT, CD160 and BTLA, another member of TNF super family, lymphotoxin-aalpha; (LTaalpha;), also interacts with HVEM. To determine whether LTaalpha; decreases latency in infected mice, we ocularly infected LTaalpha;-/- mice with LAT(+) and LAT(-) viruses using similarly infected wild-type (WT) mice as control. In contrast to WT C57BL/6 mice, LTaalpha;-/- mice were highly susceptible to ocular HSV-1 infection, independent of the presence or absence of latency associated transcript (LAT). Survival was partially restored by adoptive transfer of CD4+, CD8+, or total T cell. Infected LTaalpha;-/- mice had significantly higher corneal scarring than WT mice and adoptive T cell transfer did not alter the severity of eye disease. In contrast to WT mice, the amount of latency was not affected by the absence of LAT. The amount of LAT RNA in LTaalpha;-/- mice infected with LAT(+) virus was similar to WT mice and adoptive T cell transfer did not alter LAT RNA in LTaalpha;-/- infected mice. Increased latency in the absence of LTaalpha; correlated with upregulation of HVEM, LIGHT, CD160, and BTLA transcripts as well as an increase in markers of T cell exhaustion. The results of our study suggest that LTaalpha; has anti-pathogenic and anti-inflammatory functions and may act to protect the host from infection.

    IMPORTANCE

    Recently we evaluated the effects of HVEM and its ligands (LIGHT, CD160, and BTLA) on HSV-1 infectivity. However, the effect of LTaalpha;, another member of TNF super family on HSV-1 latency and eye disease is not known. Here we demonstrate increased latency and corneal scarring in LTaalpha;-/- infected mice, independent of the presence of LAT. In addition, infected mice were highly susceptible to HSV-1 infection and survival was partially but not significantly restored by adoptive T cell transfer. These results suggest that the absence of LTaalpha; affects HSV-1 infectivity differently than does the absence of HVEM, LIGHT, CD160, and BTLA.

  • Herpes Simplex Virus 1 can enter dynamin 1 and 2 double knockout fibroblasts [Virus-Cell Interactions]

  • Dynamin GTPases, best known for their role in membrane fission of endocytic vesicles, provide a target for viruses to be exploited during endocytic uptake. Recently, we found that entry of herpes simplex virus 1 (HSV-1) into skin cells depends on dynamin, although our results supported that viral internalization occurs via both direct fusion with the plasma membrane and via endocytic pathways. To further explore the role of dynamin for efficient HSV-1 entry, we utilized conditional dynamin 1 and dynamin 2 double knockout (DKO) fibroblastsas experimental tool. Strikingly, HSV-1 entered control and DKO fibroblasts with comparable efficiency. For comparison, we infected DKO cells with Semliki Forest virus, which is known to adopt clathrin-mediated endocytosis as its internalization pathway, and observed efficient virus entry. These results support that the DKO cells provide alternative pathways for viral uptake. Treatment of cells with the dynamin inhibitor dynasore confirmed that HSV-1 entry depends on dynamin in the control fibroblasts. As expected, dynasore did not interfere with viral entry into DKO cells. Electron microscopy of HSV-1-infected cells suggests viral entry after fusion with the plasma membrane and by endocytosis in both dynamin-expressing and dynamin-deficient cells. Infection at low temperatures where endocytosis is blocked still resulted in HSV-1 entry although at a reduced level which suggests that non-endocytic pathways contribute to successful entry. Overall, our results strengthen the impact of dynamin for HSV-1 entry as only cells that adopt to the lack of dynamin allow dynamin-independent entry.

    Importance

    The human pathogen herpes simplex virus 1 (HSV-1) can adapt to a variety of cellular pathways to enter cells. In general, HSV-1 is internalized by fusion of its envelope with the plasma membrane or by endocytic pathways which reflects the high adaptation to differences in its target cells. The challenge is to distinguish whether multiple or only one of these internalization pathways lead to successful entry, and furthermore, to identify the mode of viral uptake. Here, we focused on dynamin which promotes endocytic vesicle fission and explored how the presence and absence of dynamin can influence viral entry. Our results support that HSV-1 entry into mouse embryonic fibroblasts depends on dynamin, however, depletion of dynamin still allows efficient viral entry suggesting that alternative pathways present upon dynamin depletion can accomplish viral internalization.

  • Kaposi Sarcoma-associated Herpesvirus Glycoprotein H is Indispensable for Infection of Epithelial, Endothelial, and Fibroblast Cell Types [Virus-Cell Interactions]

  • Kaposi sarcoma-associated herpesvirus (KSHV) is an emerging pathogen and is the causative infectious agent of Kaposi sarcoma and two malignancies of B cell origin. To date, there is no licensed KSHV vaccine. Development of an effective vaccine against KSHV continues to be limited by a poor understanding of how the virus initiates acute primary infection in vivo in diverse human cell types. The role of glycoprotein H (gH) in herpesvirus entry mechanisms remains largely unresolved. To characterize the requirement for KSHV gH in the viral life cycle and in determination of cell tropism, we generated and characterized a mutant KSHV in which expression of gH was abrogated. Using a bacterial artificial chromosome containing a complete recombinant KSHV genome and recombinant DNA technology, we inserted stop codons into the gH coding region. We used electron microscopy to reveal that the gH-null mutant virus assembled and exited from cells normally, compared to wild-type virus. Using purified virions, we assessed infectivity of the gH-null mutant in diverse mammalian cell types in vitro. Unlike wild-type virus or a gH-containing revertant, the gH-null mutant was unable to infect any of the epithelial, endothelial, or fibroblast cell types tested. However, its ability to infect B cells was equivocal, and remains to be investigated in vivo due to generally poor infectivity in vitro. Together, these results suggest that gH is critical for KSHV infection of highly permissive cell types including epithelial, endothelial, and fibroblasts.

    IMPORTANCE

    All homologues of herpesvirus gH studied to date have been implicated in playing an essential role in viral infection of diverse permissive cell types. However, the role of gH in the mechanism of KSHV infection remains largely unresolved. In this study, we generated a gH-null mutant KSHV and provided evidence that deficiency of gH expression did not affect viral particle assembly or egress. Using the gH-null mutant, we showed that gH was indispensable for KSHV infection of epithelial, endothelial, and fibroblast cells in vitro. This suggests that gH is an important target for the development of a KSHV prophylactic vaccine to prevent initial viral infection.

  • The introduction and dispersal of Sindbis virus from central Africa to Europe [Genetic Diversity and Evolution]

  • Bird-hosted viruses have the potential to be transported over large areas of the world and to be transmitted in distant geographical regions. Sindbis virus (SINV) is a mosquito-borne alphavirus that is locally amplified in a bird-mosquito enzootic cycle and distributed all over the Old World and Australia/Oceania. Sindbis virus genotype I (SINV-I) is the cause of disease outbreaks in humans in South Africa as well as in northern Europe. To trace the evolutionary history and potential strain-disease association of SINV-I, we sequenced 36 complete genomes isolated from field material in Europe as well as in Africa and the Middle-East, collected over 58 years. These were analysed together with 30 additional published whole SINV-I genomes using Bayesisan analysis. Our results suggested that SINV-I was introduced only once to northern Europe from central Africa, in the 1920s. After its first introduction to Sweden, it spread east and southwards on two separate occasions in the 1960s and 1970s. Another introduction from central Africa seems to have occurred to southern/central Europe, and where these two introductions meet, one recombination event was detected in central Europe. In addition, another recombinant strain was found in central Africa, where also the most divergent SINV-I viruses originated.

    Importance

    This study shows that only a single introduction of SINV into a new geographical area is required for the spread and establishment, provided that the requisite vector(s) and reservoir(s) of epizootological and epidemiological importance, are present. Further, we present the first report of recombination between two strains of SINV in nature. Our study increases the knowledge on new introductions and dispersal of arboviruses in general, and on SINV in particular.

  • Cytomegalovirus evades TRAIL-mediated innate lymphoid cell 1 defenses [Cellular Response to Infection]

  • Cytomegalovirus (CMV) establishes a lifelong infection facilitated, in part, by circumventing immune defenses mediated by TNF-family cytokines. An example of this is the mouse CMV (MCMV) m166 protein that restricts expression of the TNF-related apoptosis inducing ligand (TRAIL) death receptors, promoting early-phase replication. We show here that replication of a MCMV mutant lacking m166 is also severely attenuated during viral persistence in the salivary glands (SG). Depleting group I innate lymphoid cells (ILCs), or infecting Trail-/- mice, completely restored persistent replication of this mutant. Group I ILC are comprised of 2 subsets, conventional natural killer cells (cNK) and tissue-resident ILC1. Using recently identified phenotypic markers to discriminate between these two cell types, their relative expression of TRAIL and IFN was assessed during both early and persistent infection. ILC1 were found to be the major TRAIL-expressers during both of these infection phases, with cNK expressing very little, indicating it is ILC1 that curtail replication via TRAIL in the absence of m166-imposed countermeasures. Notably, despite high TRAIL expression by SG-resident ILC1, IFN production by both ILC1 and cNK was minimal at this site of viral persistence. Together these results highlight TRAIL as a key ILC1-utilized effector molecule that can operate in defense to persistent infection at times when other innate control mechanisms may be muted, and highlight the importance for the evolution of virus-employed countermeasures.

    Importance

    Cytomegalovirus (a bbeta;-herpesvirus) is a master at manipulating immune responses to promote its lifelong persistence, a result of millions of years of coevolution with its host. Using a one-of-a-kind MCMV mutant unable to restrict expression of the TNF-related apoptosis inducing ligand death receptors (TRAIL-DR), we show that TRAIL-DR signaling significantly restricts both early and persistent viral replication. Our results also reveal that these defenses are employed by TRAIL-expressing innate lymphoid cell 1 (ILC1), but not conventional NK cells. Overall our results are significant because they show the key importance of viral counterstrategies specifically neutralizing TRAIL effector functions mediated by a specific, tissue-resident subset of group I ILC.

  • Temporal proteomic analysis of BK polyomavirus infection reveals virus-induced G2 arrest and highly effective evasion of innate immune sensing [Virus-Cell Interactions]

  • BK polyomavirus (BKPyV) is a small DNA virus that establishes a life-long persistent infection in the urinary tract of most people. BKPyV is known to cause severe morbidity in renal transplant recipients and can lead to graft rejection. The simple 5.2 kilobase pair dsDNA genome expresses just seven known proteins, thus it relies heavily on host machinery to replicate. How the host proteome changes over the course of infection is key to understanding this host:virus interplay. Here for the first time quantitative temporal viromics has been used to quantify global changes in ggt;9,000 host proteins in two types of primary human epithelial cell throughout 72 hours of BKPyV infection. These data demonstrate the importance of cell cycle progression and pseudo-G2 arrest in effective BKPyV replication, along with a surprising lack of innate immune response throughout the whole virus replication cycle. BKPyV thus evades pathogen recognition to prevent activation of innate immune responses in a sophisticated manner.

    Importance

    BK polyomavirus can cause serious problems in immune-suppressed patients, in particular kidney transplant recipients who can develop polyomavirus-associated kidney disease. In this work, we have used advanced proteomics techniques to determine the changes to protein expression caused by infection of two independent primary cell types of the human urinary tract (kidney and bladder) throughout the replication cycle of this virus. Our findings have uncovered new details of a specific form of cell cycle arrest caused by this virus and importantly we have identified that this virus has a remarkable ability to evade detection by host cell defence systems. In addition, our data provide an important resource for the future study of kidney epithelial cells and their infection by urinary tract pathogens.

  • Structural Basis for Cooperative Binding of EBNA1 to the Epstein-Barr Virus Dyad Symmetry Minimal Origin of Replication [Genome Replication and Regulation of Viral Gene Expression]

  • Epstein-Barr virus is associated with several human malignancies including nasopharyngeal carcinoma, gastric cancer and lymphoma. Latently infected cells carry a circularized EBV episome where the origin of replication (OriP) is comprised of two elements: the family of repeats (FR) and dyad symmetry (DS). The viral protein Epstein-Barr Nuclear Antigen-1 (EBNA1) binds to FR and DS to promote EBV episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 binding to the DS constitutes a minimal origin of DNA replication. Here, we report the crystal structure of two EBNA1 DNA-binding domain dimers bound to a DS half site. This structure shows that the DNA is smoothly bent allowing for stabilizing interactions between the dimers. The dimer-dimer interface requires an intricate hydrogen bonding network involving residues R491 and D581. When this interface is disrupted, we note loss of stable dimer-dimer complex formation on the DNA, compromised OriP-containing plasmid replication in cells and impaired recruitment of the MCM3 complex to the OriP. Surface conservation analysis reveals that these residues are part of a larger conserved surface that may be critical for recruitment of replication machinery to the OriP. Our results reveal a new region of EBNA1 critical for its activity and one that may be exploited by targeted small molecules to treat EBV-associated disease.

    IMPORTANCE

    Epstein-Barr Virus (EBV) is a causative agent of various malignancies and may also contribute to autoimmune disease. The latent and episomal form of the virus is known to drive EBV-associated oncogenesis. Persistence of the viral episome in proliferating tumor cells requires the interaction of the Epstein-Barr Virus Nuclear Antigen 1 (EBNA1) with the viral origin of plasmid replication OriP. The Dyad Symmetry (DS) element in OriP is the essential minimal replicator of OriP. Here, we report the X-ray crystal structure of EBNA1 bound to DS. The structure reveals a previous unrecognized interface formed between dimers of EBNA1 necessary for cooperative DNA-binding, recruitment of cellular replication machinery, and replication function. These findings provide new insights into the mechanism of EBNA1 function at the replication origin and new opportunities to inhibit EBV latent infection and pathogenesis.

  • Novel interactions between the HTLV-1 antisense protein HBZ and the SWI/SNF chromatin remodeling family: Implications for viral life cycle [Virus-Cell Interactions]

  • The human T-cell leukemia virus type 1 (HTLV-1) regulatory proteins Tax and HBZ play indispensable roles in regulating viral and cellular gene expression. BRG1, the ATPase subunit of the SWI/SNF chromatin remodeling complex, has been demonstrated to be essential not only for Tax transactivation but also for viral replication. We sought to investigate the physical interaction between HBZ and BRG1 and to determine the effect of those interactions on Tax-mediated LTR activation. We reveal that HTLV-1 cell lines and ATL cells harbour high levels of BRG1. Using GST pulldown and co-immunoprecipitation assays we have demonstrated physical interactions between BRG1 and HBZ and characterised the protein domains involved. Moreover, we have identified PBAF-signature subunits BAF200 and BAF180 as novel interaction partners of HBZ suggesting that PBAF complex may be required for HTLV-1 transcriptional repression by HBZ. Additionally, we found that BRG1 expression translocates HBZ into distinct nuclear foci. We show that HBZ substantially represses HTLV-1 LTR activation by Tax/BRG1. Interestingly, we found that Tax stabilizes the expression of exogenous and endogenous BRG1 and HBZ reverses this effect. Finally, using Chromatin Immunoprecipitation-qPCR (ChIP-qPCR) assay we illustrate that HBZ facilitates the down-regulation of HTLV-1 transcription by deregulating the recruitment of SWI/SNF complexes to the promoter. Overall, we conclude that SWI/SNF complexes, in addition to other cellular transcription factors are involved in HBZ-mediated suppression of HTLV-1 viral gene expression.

    IMPORTANCE

    The pathogenic potential of HTLV-1 is linked to the indispensable multifaceted functions of the viral regulatory proteins Tax and HBZ, encoded by the sense and antisense viral transcripts, respectively. The interaction between Tax and SWI/SNF family of chromatin-remodeling complexes has been associated with HTLV-1 transcriptional activation. To date, the relationship between SWI/SNF chromatin remodeling family and HBZ, the only viral protein that is consistently expressed in infected cells and ATL cells, has not been elucidated. Here, we have characterized the biological significance of SWI/SNF family in regard to viral transcriptional repression by HBZ. This is important because it provides a better understanding of the function and role of HBZ in down-regulating viral transcription and hence its contribution to viral latency and persistence in vivo, a process that may ultimately lead to development of ATL.

  • An E460D substitution in the NS5 protein of tick-borne encephalitis virus confers resistance to the inhibitor Galidesivir (BCX4430) and also attenuates the virus for mice [Vaccines and Antiviral Agents]

  • The adenosine analogue Galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, has entered a Phase 1 clinical safety and pharmacokinetics study in healthy subjects and is under clinical development for treatment of Ebola virus infection. Moreover, Galidesivir also inhibits the reproduction of tick-borne encephalitis virus (TBEV) and numerous other medically important flaviviruses. Until now, studies of this antiviral agent have not yielded resistant viruses. Here, we demonstrate that an E460D substitution, in the active site of TBEV RNA-dependent-RNA-polymerase (RdRp), confers resistance to Galidesivir in cell culture. Galidesivir-resistant TBEV exhibited no cross-resistance to structurally different antiviral nucleoside analogues, such as 7-deaza-2'-C-methyladenosine, 2'-C-methyladenosine and 4'-azido-aracytidine. Although, the E460D substitution led only to a subtle decrease in viral fitness in cell culture, Galidesivir-resistant TBEV was highly attenuated in vivo, with 100% survival rate and no clinical signs observed in infected mice. Furthermore, no virus was detected in serum, spleen or brain of mice inoculated with the Galidesivir-resistant TBEV. Our results contribute to understanding the molecular basis of Galidesivir antiviral activity, flavivirus resistance to nucleoside inhibitors and the potential contribution of viral RdRp to flavivirus neurovirulence.

    IMPORTANCE Tick-borne encephalitis virus (TBEV) is a pathogen that causes severe human neuroinfections in Europe and Asia and for which there is currently no specific therapy. We have previously found that Galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, which is under clinical development for treatment of Ebola virus infection, has a strong antiviral effect against TBEV. For any antiviral drug, it is important to generate drug-resistant mutants to understand how the drug works. Here, we produced TBEV mutants resistant to Galidesivir and found that the resistance is caused by a single amino acid substitution in an active site of the viral RNA-dependent RNA polymerase, an enzyme which is crucial for replication of viral RNA genome. Although, this substitution led only to a subtle decrease in viral fitness in cell culture, Galidesivir-resistant TBEV was highly attenuated in a mouse model. Our results contribute to understanding the molecular basis of Galidesivir antiviral activity.

  • Zika virus production is resistant to RNase L antiviral activity [Virus-Cell Interactions]

  • There is currently no knowledge of how the emerging human pathogen Zika virus (ZIKV) interacts with the antiviral endoribonuclease L (RNase L) pathway during infection. Since activation of RNase L during infection typically limits virus production dramatically, we used CRISPR-Cas9 gene editing technology to knockout (KO) targeted host genes involved in the RNase L pathway to evaluate the effects of RNase L on ZIKV infection in human A549 cells. RNase L was activated in response to ZIKV infection, which degraded ZIKV genomic RNA. Surprisingly, despite viral genome reduction, RNase L activity did not reduce ZIKV infectious titers. In contrast, both the flavivirus dengue virus as well as the alphavirus Sindbis virus replicated to significantly higher titers in RNase L KO cells compared to wild-type (WT) cells. Using MAVS/RNase L double KO cells, we demonstrated that the absence of increased ZIKV production in RNase L KO cells was not due to compensation by enhanced type I IFN transcripts to thus inhibit virus production. Finally, when synthetic dsRNA was used to activate OAS3 and induce RNase L antiviral activity prior to ZIKV infection, we observed reduced ZIKV replication factory (RF) formation as well as a 42-fold reduction in virus yield in WT but not RNase L KO cells. This study proposes that ZIKV evades RNase L antiviral activity by generating a viral genome reservoir protected from RNase L cleavage during early infection, allowing for sufficient virus production before RNase L activation is detectable.

    Importance

    With the onset of the 2015 ZIKV outbreak, ZIKV pathogenesis has been of extreme global public health interest, and a better understanding of interactions with the host would provide insight into molecular mechanisms driving the severe neurological outcomes of ZIKV disease. Herein is the initial report on the relationship between ZIKV and the host oligoadenylate synthetase-RNase L (OAS-RNase L) system, a potent antiviral pathway effective at restricting replication of diverse viruses. Our study elucidated a unique mechanism whereby ZIKV production is impervious to antiviral RNase L activity, through a mechanism of viral RNA protection that is not mimicked during infection with numerous other RNase L-activating viruses, thus identifying a distinct replication strategy potentially important for ZIKV pathogenesis.

  • A CRISPR activation screen identifies genes protecting from Zika virus infection [Virus-Cell Interactions]

  • Zika virus (ZIKV) is an arthropod borne emerging pathogen causing febrile illness. ZIKV is associated Guillain-Barreeacute; syndrome and other neurological complications. Infection during pregnancy is associated with pregnancy complications and developmental and neurological abnormalities collectively defined as congenital Zika syndrome. There is still no vaccine or specific treatment for ZIKV infection. To identify host factors that can rescue cells from ZIKV infection we used a genome scale CRISPR activation screen. Our highly ranking hits included a short list of interferon stimulated genes (ISGs) previously reported to have antiviral activity. Validation of the screen results highlighted IFNL2 and IFI6 as genes providing high levels of protection from ZIKV. Activation of these genes had an effect on an early stage in viral infection. In addition, infected cells expressing sgRNAs for both of these genes displayed lower levels of cell death compared to controls. Furthermore, the identified genes were significantly induced in ZIKV infected placenta explants. Thus, these results highlight a set of ISGs directly relevant for rescuing cells from ZIKV infection or its associated cell death and substantiates CRISPR activation screens as a tool to identify host factors impeding pathogen infection.

    IMPORTANCE Zika virus (ZIKV) is an emerging vector-borne pathogen causing a febrile disease. ZIKV infection might also trigger Guillain-Barreeacute; syndrome, neuropathy and myelitis. Vertical transmission of ZIKV can cause fetus demise, still birth or severe congenital abnormalities and neurological complications. There is no vaccine or specific antiviral treatment against ZIKV. We used a genome wide CRISPR activation screen, where genes are activated from their native promoters to identify host cell factors that protect cells from ZIKV infection or associated cell death. The results provide better understanding of key host factors that protect cells from ZIKV infection and might assist in identifying novel antiviral targets.

  • A yeast suppressor screen used to identify mammalian SIRT1 as a proviral factor for MERS-CoV replication [Virus-Cell Interactions]

  • Viral proteins must intimately interact with the host cell machinery during virus replication. Here, we use the yeast Saccharomyces cerevisiae as a system to identify novel functional interactions between viral proteins and eukaryotic cells. Our work demonstrates that when the Middle East respiratory syndrome (MERS-CoV) accessory gene ORF4a is expressed in yeast it causes a slow growth phenotype. ORF4a has been characterized as an interferon antagonist in mammalian cells, yet yeast lack an interferon system, suggesting further interactions between ORF4a and eukaryotic cells. Using the slow growth phenotype as a reporter of ORF4a function we utilized the yeast knockout library collection to perform a suppressor screen where we identified the yeast gene YDL042C/SIR2 as a suppressor of ORF4a function. The mammalian homologue of SIR2 is SIRT1, an NAD-dependent histone deacetylase. We found that when SIRT1 was inhibited by either chemical or genetic manipulation there was reduced MERS-CoV replication suggesting that SIRT1 is a proviral factor for MERS-CoV. Moreover, ORF4a inhibited SIRT1-mediated modulation of NF-B signaling demonstrating a functional link between ORF4a and SIRT1 in mammalian cells. Overall, the data presented here demonstrate the utility of yeast for identifying genetic interactions between viral proteins and eukaryotic cells. We also demonstrate for the first time that SIRT1 is a proviral factor for MERS-CoV replication and that ORF4a has a role in modulating its activity in cells.

    Importance

    Middle East respiratory syndrome coronavirus (MERS-CoV) initially emerged in 2012 and has since been responsible for over 2000 infections, with a case fatality ratio of approximately 35%. We have used the highly characterized model system of Saccharomyces cerevisiae to investigate novel functional interactions between viral proteins and eukaryotic cells that may provide new avenues for antiviral intervention. We identify a functional link between the MERS-CoV proteins ORF4a and yeast gene YDL042C/SIR2. The mammalian homologue of SIR2 is SIRT1, an NAD-dependent histone deacetylase. We demonstrate, for the first time that SIRT1 is a proviral factor for MERS-CoV replication and that ORF4a has a role in modulating its activity in mammalian cells.

  • Junin Virus Promotes Autophagy to Facilitate Viral Life Cycle [Virus-Cell Interactions]

  • Juniiacute;n virus (JUNV), a member of Arenaviridae family, is the etiological agent of Argentine hemorrhagic fever (AHF), a potentially deadly, endemic-epidemic disease affecting the population of the most fertile farming land of Argentina. Autophagy is a degradative process with a crucial anti-viral role; however, several viruses subvert this pathway in their benefit. We determined the role of autophagy in JUNV-infected cells analyzing LC3, a cytoplasmic protein (LC3-I) which becomes vesicle membrane-associated (LC3-II) upon induction of autophagy. Cells overexpressing EGFP-LC3 and infected with JUNV showed an increased number of LC3 puncta structures, similar to that obtained after starvation- or Bafilomycin A1- treatment which leads to autophagosome induction or accumulation, respectively. We also monitored the conversion of LC3-I to LC3-II observing LC3-II levels in JUNV-infected cells similar to that observed in starved cells. Additionally, we kinetically studied the number of LC3 dots after JUNV infection and found that the virus activated the pathway as early as 2 h p.i. whereas the UV-inactivated virus did not induce the pathway. Cells subjected to starvation or pre-treated with rapamycin, a pharmacological autophagy inductor, enhanced virus yield. Also, we assayed the replication capacity of JUNV in Atg 5 knock-out or Beclin-1 knock-down cells [both critical components of the autophagic pathway] and found a significant decrease in JUNV replication. Taken together, our results constitute the first study indicating that JUNV infection induces an autophagic response which is functionally required by the virus for efficient propagation.

    IMPORTANCE

    Mammalian arenaviruses are zoonotic viruses causing asymptomatic and persistent infections in their rodent hosts, but may produce severe and lethal haemorrhagic fevers in humans. Currently, there are neither effective therapeutic options nor effective vaccines, for viral haemorrhagic fevers caused by human pathogenic arenaviruses, except the vaccine Candid #1 against AHF, licensed for human use in endemic areas from Argentina. Since arenaviruses remain a severe threat to global public health, more in-depth knowledge of their replication mechanisms would improve our ability to fight against these viruses. Autophagy is a lysosomal degradative pathway involved in maintaining the cellular homeostasis, representing powerful anti-infective machinery. We showed, for the first time for a member of the Arenaviridae family, a pro-viral role of autophagy in JUNV infection, providing new knowledge in the edge of host-virus interaction. Therefore, modulation of virus-induced autophagy could be used as a strategy to block arenaviruses infections.

  • mTOR dysregulation by vaccinia virus F17 controls multiple processes with varying roles in infection [Virus-Cell Interactions]

  • Despite producing enormous amounts of cytoplasmic DNA poxviruses continue to replicate efficiently by deploying an armory of proteins that counter host antiviral responses at multiple levels. Amongst these, the poxvirus protein F17 dysregulates the host kinase, mammalian target of rapamycin (mTOR) to prevent activation of stimulator of interferon gene expression (STING) and impair production of Interferon Stimulated Genes (ISGs). However, the host DNA sensor(s) involved and their impact on infection in the absence of F17 remain unknown. Here, we show that cGAS is the primary sensor that mediates interferon response factor (IRF) activation and ISG responses to vaccinia virus lacking F17 in both macrophages and lung fibroblasts, although additional sensors also operate in the latter cell type. Despite this, ablation of ISG responses through cGAS or STING knockout did not rescue defects in late viral protein production and pointed to other functions of mTOR in this regard. mTOR adjusts both autophagic and protein synthetic processes to cellular demands. No significant differences in autophagic responses to wildtype or F17 mutant viruses could be detected, with autophagic activity varying across cell types or states and exhibiting no correlations with defects in viral protein accumulation. By contrast, results using transformed cells or altered growth conditions suggested that late stage defects in protein accumulation reflect failure of the F17 mutant to de-regulate mTOR and stimulate protein production. Finally, rescue approaches suggest that phosphorylation may partition F17's functions as a structural protein and mTOR regulator. Our findings reveal the complex multifunctionality of F17 during infection.

    IMPORTANCE Poxviruses are large double-stranded DNA viruses that replicate entirely in the cytoplasm, an unusual act that activates pathogen sensors and innate antiviral responses. In order to replicate, poxviruses therefore encode a wide range of innate immune antagonists that include F17, a protein that dysregulates the kinase mammalian target of rapamycin (mTOR) to suppress interferon stimulated gene (ISG) responses. However, the host sensor(s) that detects infection in the absence of F17 and its precise contribution to infection remains unknown. Here, we show that the cytosolic DNA sensor cGAS is primarily responsible for activating ISG responses in biologically relevant cell types infected with a poxvirus that does not express F17. However, in line with their expression of ~100 proteins that act as immune response and ISG antagonists, while F17 helps suppress cGAS-mediated responses we find that a critical function of its mTOR dysregulation activity is to enhance poxvirus protein production.

  • GII.13/21 noroviruses recognize glycans with a terminal {beta}-galactose via an unconventional glycan binding site [Virus-Cell Interactions]

  • Human noroviruses (huNoVs) recognize histo-blood group antigens (HBGAs) as host susceptibility factors. GII.13 and GII.21 huNoVs form a unique genetic lineage that emerged from mainstream GII NoVs via development of a new, non-conventional glycan binding site (GBS) that binds Lea antigen. This previous finding raised a question whether the new GII.13/21 GBS has really such a narrow glycan binding spectrum. In this study, we provide solid phenotypic and structural evidence indicating that this new GBS recognizes a group of glycans with a common terminal bbeta;-galactose (bbeta;-Gal). First, we found that P domain proteins of GII.13/21 huNoVs circulating at different times bound three glycans sharing a common terminal bbeta;-Gal, including Lec, lactose, and mucin core 2. Second, we solved the crystal structures of the GII.13 P dimers in complex with Lec and mucin core 2, which showed that bbeta;-Gal is the major binding saccharide. Third, non-fat milk and lactose blocked the GII.13/21 P domain-glycan binding, which may explain the low prevalence of GII.13/21 viruses. Our data provide new insight into the host interactions and epidemiology of huNoVs, which would help control and prevention of NoV associated diseases.

    Importance

    Evidence from both phenotypic binding assay and structural study support the observed interactions of human noroviruses (huNoVs) with histo-blood group antigens (HBGAs) as receptors or attachment factors, affecting their host susceptibility. GII.13 and GII.21 genotypes form a unique genetic lineage that differs from the mainstream GII human noroviruses (huNoVs) in their unconventional glycan binding site. Unlike the previous findings that GII.13/21 genotypes recognize only Lea antigen, we found here that they can interact with a group of glycans with common terminal bbeta;-Gal, including Lec, lactose, and mucin core 2. However, this wide glycan binding spectrum in a unique binding mode of the GII.13/21 huNoVs appears not to increase their prevalence, probably due to the existence of decoy glycan receptors in human gastrointestinal tract limiting their infection. Our findings shed light on the host interaction and epidemiology of huNoVs, which would impact on strategy of huNoV control and prevention.

  • Characterization of novel splice variants of zinc finger antiviral protein (ZAP) [Cellular Response to Infection]

  • Given the unprecedented scale of the recent Ebola and Zika viral epidemics, it is crucial to understand the biology of host factors with broad antiviral action in order to develop novel therapeutic approaches. Here, we look into one such factor; zinc-finger antiviral protein (ZAP) inhibits a variety of RNA and DNA viruses. Alternative splicing results in two isoforms that differ at their C-termini; ZAPL (long), encodes a poly(ADP-ribose) polymerase (PARP)-like domain that is missing in ZAPS (short). Previously it has been shown that ZAPL is more antiviral than ZAPS while the latter is more induced by interferon (IFN). In this study, we discovered and confirmed the expression of two additional splice variants of human ZAP mmdash; ZAPXL (extra-long) and ZAPM (medium). We also found two haplotypes of human ZAP. Since ZAPL and ZAPS have differential activities, we hypothesize that all four ZAP isoforms have evolved to mediate distinct antiviral and/or cellular functions. By taking a gene knockout and reconstitution approach, we have characterized the antiviral, translational inhibition, and IFN activation activities of individual ZAP isoforms. Our work demonstrates that ZAPL and ZAPXL are more active against alphaviruses and hepatitis B virus (HBV) than ZAPS and ZAPM and elucidates the effects of splice variants on the action of a broad spectrum antiviral factor.

    IMPORTANCE

    ZAP is an IFN-induced host factor that can inhibit a wide range of viruses and there is great interest in fully characterizing its antiviral mechanism. This is the first study that defines the antiviral capacity of individual ZAP isoforms in the absence of endogenous ZAP expression and hence crosstalk with other isoforms. Our data demonstrate that ZAP is expressed as four different forms mmdash; ZAPS, ZAPM, ZAPL and ZAPXL. The longer ZAP isoforms better inhibit alphaviruses and HBV while all isofoms equally inhibit Ebola virus transcription and replication. In addition, there is no difference in the ability of ZAP isoforms to enhance the induction of type I IFN expression. Our results show that the full spectrum of ZAP activities can change depending on the virus target and the relative levels of basal expression and induction by IFN or infection.

  • siRNA Screening for the Small GTPase Rab Proteins Identifies that Rab5B is a Major Regulator of Hepatitis B Virus Production [Virus-Cell Interactions]

  • Viruses are considered to use vesicular trafficking in the infected cells, but the details of assembly/release pathways of hepatitis B virus (HBV) are still unknown. To identify key regulators of HBV production, we performed siRNA screening for Rab proteins, which are considered to act as molecular switches in the vesicular trafficking, using HepG2.2.15 cells. Among 62 Rab proteins, the suppression of Rab5B most significantly increased HBV DNA in the culture supernatant. Surprisingly, 5 days after the transfection of Rab5B siRNA, HBV DNA in the supernatant was increased more than 30-fold and it reflected the increase of infectious HBV particles. Northern blotting analysis showed that transcription of 2.4/2.1 kb mRNA coding envelope proteins containing large hepatitis B surface protein (LHBs) was increased. Analysis of hepatocyte nuclear factors (HNFs) showed that transcription of HNF4aalpha;, which is known to enhance 2.4 kb mRNA transcription, was regulated by Rab5B. Also, it was revealed that LHBs had accumulated in the endoplasmic reticulum (ER) after Rab5B depletion, but not in the multivesicular body (MVB), which is thought to be an organelle utilized for the HBV envelope formation. Therefore, it was considered that Rab5B is required for the transport of LHBs from ER to MVB. Immunofluorescent microscopy showed that HBs proteins including LHBs colocalized with HBc in ER of Rab5B-depleted cells, suggesting that HBV envelopment could occur in the ER, not only in the MVB. In conclusion, Rab5B is a key regulator of HBV production and could be a potential target of antiviral therapy.

    IMPORTANCE

    HBV infection is a worldwide health problem but the mechanisms of how HBV utilizes the cellular machinery for its life cycle are poorly understood. Especially, it has been unclear how the viral components and virions are transported among the organelles. HBV budding site has been reported to be ER or MVB, but it has not been clearly determined. In this study, siRNA-based screening of Rab proteins using HBV-expressing cells showed that Rab5B, one of Rab5 isoforms, has important roles in the late step of HBV life cycle. Although Rab5 is known to work on early endosomes, this study showed that Rab5B plays a role on the transport of LHBs between ER and MVB. Furthermore, it affects the transcription of LHBs. This is the first report on the mechanisms of HBV envelope protein transport among the organelles, and the results provide important insights to the therapeutic control of HBV infection.

  • Rescue of infectious recombinant Hazara nairovirus from cDNA reveals the nucleocapsid protein DQVD caspase cleavage motif performs an essential role other than cleavage. [Cellular Response to Infection]

  • The Nairoviridae family of the Bunyavirales order comprises tick-borne tri-segmented negative strand RNA viruses, with several members associated with serious or fatal disease in humans and animals. A notable member is Crimean-Congo hemorrhagic fever virus (CCHFV), which is the most widely-distributed tick-borne pathogen, and associated with devastating human disease with case/fatality rates averaging 30%. Hazara virus (HAZV) is closely-related to CCHFV, sharing the same serogroup and many structural, biochemical and cellular properties. To improve understanding of HAZV and nairovirus multiplication cycles, we developed for the first time a rescue system permitting efficient recovery of infectious HAZV from cDNA. This system now allows reverse genetics analysis of nairoviruses without the need for high biosafety containment, as is required for CCHFV. We used this system to test the importance of a DQVD caspase cleavage site exposed on the apex of the HAZV nucleocapsid protein arm domain that is cleaved during HAZV infection, and for which the equivalent DEVD sequence was recently shown to be important for CCHFV growth in tick but not mammalian cells. Infectious HAZV bearing an un-cleavable DQVE sequence was rescued and exhibited equivalent growth parameters to wild-type in both mammalian and tick cells, showing this site was dispensable for virus multiplication. In contrast, substitution of the DQVD motif with the similarly un-cleavable AQVA sequence could not be rescued despite repeated efforts. Together, this work highlights the importance of this caspase cleavage site in the HAZV lifecycle, but reveals the DQVD sequence performs a critical role aside from caspase cleavage.

    IMPORTANCE

    Hazara virus is classified within the Nairoviridae family along with Crimean-Congo hemorrhagic fever virus (CCHFV), which is one of the most lethal human pathogens in existence, requiring the highest biosafety level (BSL) containment (BSL-4). In contrast, HAZV is not associated with human disease and thus can be studied using less-restrictive BSL-2 protocols. Here, we report a system able to rescue Hazara virus (HAZV) from cDNAs, thus permitting reverse genetic interrogation of the HAZV replication cycle. We used this system to examine the role of a caspase cleavage site, DQVD, within the HAZV nucleocapsid protein that is also conserved in CCHFV. By engineering mutant viruses, we showed caspase cleavage at this site was not required for productive infection, and furthermore that this sequence performs a critical role in the virus lifecycle aside from caspase cleavage. This system will accelerate nairovirus research due to its efficiency and utility under amenable BSL-2 protocols.

  • Cholesterol binding to the transmembrane region of a group 2 HA of Influenza virus is essential for virus replication affecting both virus assembly and HA's fusion activity [Structure and Assembly]

  • Hemagglutinin (HA) of Influenza virus is incorporated into cholesterol enriched, nanodomains of the plasma membrane. Phylogenetic group 2 HAs contain the conserved cholesterol consensus motif (CCM) YKLW in the transmembrane region. We previously reported that mutations in the CCM retarded intracellular transport of HA and decreased its nanodomain association. Here we analyzed whether cholesterol interacts with the CCM. Incorporation of photocholesterol into HA was significantly reduced if the whole CCM is replaced by alanine, both using immunoprecipitated HA and when HA is embedded in the membrane. Next, we used reverse genetics to investigate the significance of the CCM for virus replication. No virus was rescued if the whole motif is exchanged (YKLW4A); single (LA) or double (YK2A and LW2A) mutated virus showed decreased titers and a comparative fitness disadvantage. In polarized cells transport of HA mutants to the apical membrane was not disturbed. Reduced amounts of HA and cholesterol were incorporated into the viral membrane. Mutant viruses exhibit a decrease in hemolysis, which is only partially corrected if the membrane is replenished with cholesterol. More specifically, viruses have a defect in hemifusion as demonstrated by fluorescence dequenching. Cells expressing HA-YKLW4A fuse with erythrocytes, but the number of events are reduced. Even after acidification unfused erythrocytes remain cell-bound, a phenomenon not observed with wildtype HA. We conclude that cholesterol-binding to a group 2 HA is essential for virus replication. It has pleiotropic effects on virus assembly and membrane fusion, mainly on lipid mixing and possibly a preceding step.

    IMPORTANCE The glycoprotein hemagglutinin (HA) is a major pathogenicity factor of Influenza viruses. Whereas the structure and function of HA's ectodomain is known in great detail, similar data for the membrane-anchoring part of the protein are missing. Here we demonstrate that the transmembrane region of a group 2 HA interacts with cholesterol, the major lipid of the plasma membrane and the defining element of the viral budding site nanodomains of the plama membrane. The cholesterol binding motif is essential for virus replication. Its partial removal affects various steps of the viral life cycle, such as assembly of new virus particles and their subsequent cell entry via membrane fusion. A cholesterol-binding pocket in group 2 HAs might be a promising target for a small lipophilic drug that inactivates the virus.

  • Human Cytomegalovirus Enters the Primary CD34+ Hematopoietic Progenitor Cells where it Establishes Latency by Macropinocytosis [Virus-Cell Interactions]

  • Viral entry is targeted by immunological and pharmacological measures to inhibit viral infection. Human cytomegalovirus entry into cells where it initiates productive infection has been well studied, but its entry into cell types where it establishes latency has not. Therefore, we examined the entry of HCMV into CD34+ hematopoietic progenitor cells where the virus establishes latency. We determined that HCMV enters into the primary CD34+ hematopoietic progenitor cells in which it establishes latency by macropinocytosis. The capsid-associated tegument protein pp150 is released from maturing endosomes and migrates to the nucleus whereas other tegument proteins, including pp71, remain endosome-associated in the cytoplasm. Inhibition of macropinocytosis impairs entry, thereby diminishing latency-associated transcription and reducing viral reactivation. We conclude that HCMV virions enter CD34+ cells by macropinocytosis but fail to fully uncoat or disassemble their tegument layers leading to the establishment of latency.

    Importance

    Virion entry is targeted by antivirals and natural immunity to prevent infection. Natural pre-existing immunity is ineffective at clearing an HCMV infection, and an incomplete understanding of the viral glycoproteins and cellular receptors that mediate entry has hampered inhibitor development. Nevertheless, HCMV entry remains a viable drug target. Our characterization here of HCMV entry into primary CD34+ hematopoietic progenitor cells through macropinocytosis and our comparison to viral entry into fibroblast cells highlights virion uncoating and tegument disassembly as a divergence point between productive and latent infections. Further definition of tegument disassembly may permit the development of interventions to inhibit this process to block productive infection, or to trigger it in incompletely differentiated cells to prevent the seeding of the latent reservoirs that make HCMV infections incurable.

  • Engineering a live attenuated PEDV vaccine candidate via inactivation of the viral 2'-O methyltransferase and the endocytosis signal of the spike protein [Vaccines and Antiviral Agents]

  • Porcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets; however, effective and safe vaccines are still not available. We hypothesized that inactivation of the 2'-O methyltransferase (2'-O MTase) activity of nsp16 and the endocytosis signal of the spike protein attenuates PEDV yet retains its immunogenicity in pigs. We generated a recombinant PEDV, KDKE4A, with quadruple alanine-substitutions in the catalytic tetrad of the 2'-O MTase using a virulent infectious cDNA clone icPC22A as the backbone. Next, we constructed another mutant KDKE4A-SYA by abolishing the endocytosis signal of the spike protein of KDKE4A. Compared with icPC22A, KDKE4A and KDKE4A-SYA replicated less efficiently in vitro but induced stronger type I and type III interferon responses. The pathogenesis and immunogenicity of these mutants were evaluated in gnotobiotic piglets. Virulence of KDKE4A-SYA and KDKE4A was significantly reduced compared with the icPC22A. Mortality rates were 100%, 17% and 0% in the icPC22A, KDKE4A and KDKE4A-SYA groups, respectively. At 21 days post-inoculation (dpi), all surviving pigs were challenged orally with a high dose of icPC22A. The KDKE4A-SYA- and KDKE4A-inoculated pigs were protected from the challenge because no KDKE4A-SYA- and one KDKE4A-inoculated pig developed diarrhea, whereas all pigs in the mock group had severe diarrhea and 33% of them died. Furthermore, we serially passaged the KDKE4A-SYA in pigs three times and did not find any reversion of the introduced mutations. The data suggest that KDKE4A-SYA may be a PEDV vaccine candidate.

    Importance

    PEDV is the most economically important porcine enteric viral pathogen and has caused immense economic losses among the pork industries in many countries. Effective and safe vaccines are desperately required but still not available. The 2'-O MTase (nsp16) is highly conserved among coronaviruses (CoVs) and the inactivation of nsp16 for live attenuated vaccines has been attempted for several betacoronaviruses. We showed that inactivation of both the 2'-O MTase and the endocytosis signal of the spike protein is an approach to design a promising live attenuated vaccine for PEDV. The in vivo passaging data also validated the stability of the KDKE4A-SYA mutant. KDKE4A-SYA warrants further evaluation in sows and their piglets and may be used as a platform for further optimization. Our findings further confirmed that nsp16 can be a universal target for CoV vaccine development and will aid in the development of vaccines against other emerging CoVs.

  • Molecular mechanism of the flexible glycan-receptor recognition by mumps virus [Virus-Cell Interactions]

  • Mumps virus (MuV) is an important aerosol-transmitted human pathogen causing epidemic parotitis, meningitis, encephalitis, and deafness. MuV preferentially uses a trisaccharide containing aalpha;2,3-linked sialic acid as a receptor. However, considering MuV tropism toward glandular tissues and the central nervous system, additional glycan motif(s) may also serve as receptors. Here we performed a large-scale glycan-array screen with MuV hemagglutinin-neuraminidase (MuV-HN) attachment proteins by using 600 types of glycans from The Consortium for Functional Glycomics Protein-Glycan Interaction Core in an effort to find new glycan-receptor motif(s). According to the results of the glycan array, we successfully determined the crystal structures of MuV-HN bound to newly identified glycan motifs, sialyl lewisX (SLeX) and the oligosaccharide portion of the GM2 ganglioside (GM2-glycan). Interestingly, the complex structures showed that SLeX and GM2-glycan share the same configuration with the reported trisaccharide motif, 3'-sialyllactose (3'-SL), at the binding site of MuV-HN, while SLeX and GM2-glycan have several unique interactions compared with 3'-SL. Thus, MuV-HN protein can allow an additional spatial modification in GM2-glycan and SLeX at the second and third carbohydrate from the nonreducing terminus of the core trisaccharide structure, respectively. Importantly, MuV entry was efficiently inhibited in the presence of 3'-SL, SLeX or GM2-glycan derivatives, which indicates that these motifs can serve as MuV receptors. The aalpha;2,3-sialylated oligosaccharides, such as SLeX and 3'-sialyllactosamine, are broadly expressed in various tissues, and GM2 exists mainly in neural tissues and the adrenal gland. The distribution of these glycan motifs in human tissues/organs may have bearing on MuV tropism.

    Importance

    Mumps virus (MuV) infection is characterized by parotid gland swelling and can cause pancreatitis, orchitis, meningitis, and encephalitis. MuV-related hearing loss is also a serious complication because it is usually irreversible. MuV outbreaks have been reported in many countries, even in high-vaccine coverage areas. MuV has tropism toward glandular tissues and the central nervous system. To understand the unique MuV tropism, revealing the mechanism of receptor recognition by MuV is very important. Here, using a large-scale glycan array and X-ray crystallography, we show that MuV recognizes sialyl lewisX and GM2 ganglioside as receptors, in addition to a previously reported MuV receptor, a trisaccharide containing an aalpha;2,3-linked sialic acid. The flexible recognition of these glycan receptors by MuV may explain the unique tropism and pathogenesis of MuV. Structures will also provide a template for the development of effective entry inhibitors targeting the receptor-binding site of MuV.

  • Specificity of plant rhabdovirus cell-to-cell movement [Pathogenesis and Immunity]

  • Positive-stranded RNA virus movement proteins (MPs) generally lack sequence-specific nucleic acid-binding activities and display cross-family movement complementarity with related and unrelated viruses. Negative-stranded RNA plant rhabdoviruses encode MPs with limited structural and functional relatedness with other plant virus counterparts, but precise mechanisms of intercellular transport are obscure. In this study, we first analyzed the abilities of MPs encoded by five distinct rhabdoviruses to support cell-to-cell movement of two positive-stranded RNA viruses by using trans-complementation assays. Each of the five rhabdovirus MPs complemented movement of MP-defective mutants of tomato mosaic virus and potato X virus. In contrast, movement of recombinant MP-deletion mutants of sonchus yellow net nucleorhabdovirus (SYNV) and tomato yellow mottle-associated cytorhabdovirus (TYMaV) were rescued only by their corresponding MPs, i.e. SYNV sc4 and TYMaV P3. Subcellular fractionation analyses revealed that SYNV sc4 and TYMaV P3 were peripherally associated with cell membranes. A split-ubiquitin membrane yeast two-hybrid assay demonstrated specific interactions of the membrane-associated rhabdovirus MPs only with their cognate nucleoproteins (N) and phosphoproteins (P). More importantly, SYNV sc4-N and sc4-P interactions directed a proportion of the N-P complexes from nuclear sites of replication to punctate loci at the cell periphery that partially co-localized with the plasmodesmata. Our data show that cell-to-cell movement of plant rhabdoviruses is highly specific and suggest that cognate MP-nucleocapsid core protein interactions are required for intra- and intercellular trafficking.

    IMPORTANCE

    Local transport of plant rhabdoviruses likely involves passage of viral nucleocapsids through MP-gated plasmodesmata, but the molecular mechanisms are not fully understood. We have conducted complementation assays with MPs encoded by five distinct rhabdoviruses to assess their movement specificity. Each of the rhabdovirus MPs complemented movement of MP-defective mutants of two positive-stranded RNA viruses that have different movement strategies. In marked contrast, cell-to-cell movement of two recombinant plant rhabdoviruses were highly specific in requiring their cognate MPs. We have shown that these rhabdovirus MPs are localized to the cell periphery and associate with cellular membranes, and that they interact only with their cognate nucleocapsid core proteins. These interactions are able to redirect viral nucleocapsid core proteins from their sites of replication to the cell periphery. Our study provides a model for the specific inter- and intracellular trafficking of plant rhabdoviruses that may be applicable to other negative-stranded RNA viruses.

  • Schlafen 11 Restricts Flavivirus Replication. [Virus-Cell Interactions]

  • Schlafen 11 (Slfn11) is an interferon-stimulated gene that controls synthesis of proteins by regulating tRNA abundance. Likely through this mechanism, Slfn11 has previously been shown to impair human immunodeficiency virus 1 (HIV-1) infection and the expression of codon-biased open reading frames. Because replication of positive-sense single-stranded RNA [(+)ssRNA] viruses requires the immediate translation of the incoming viral genome whereas negative-sense, single-stranded RNA [(-)ssRNA] viruses carry at infection an RNA replicase that makes multiple translation competent copies of the incoming viral genome, we reasoned that (+)ssRNA viruses will be more sensitive to the effect of Slfn11 on protein synthesis than (-)ssRNA viruses. To evaluate this hypothesis, we tested the effects of Slfn11 on the replication of a panel of ssRNA viruses in the human glioblastoma cell line A172, which naturally expresses Slfn11. Depletion of Slfn11 significantly increased the replication of (+)ssRNA viruses from the Flavivirus genus, including West Nile (WNV), dengue (DENV), and Zika virus (ZIKV) but had no significant effect on the replication of the (-)ssRNA viruses vesicular stomatitis (VSV, Rhabdoviridae family) and Rift Valley fever (RVFV, Phenuiviridae family). Quantification of the genome-containing viral particles to plaque forming units ratio indicated that Slfn11 impairs WNV infectivity. Intriguingly, Slfn11 prevented WNV-induced down-regulation of a subset of tRNAs implicated in the translation of 11.8% of the viral polyprotein. Low abundance tRNAs might promote optimal protein folding and enhance viral infectivity, as previously reported. In summary, this study demonstrates that Slfn11 restricts flavivirus replication by impairing viral infectivity.

    AUTHOR SUMMARY

    We provide evidence that the cellular protein Schlafen 11 (Slfn11) impairs replication of flaviviruses, including West Nile (WNV), dengue (DENV), and Zika virus (ZIKV). However, replication of single-stranded, negative RNA viruses was not affected. Specifically, Slfn11 decreases the infectivity of WNV potentially by preventing virus-induced modifications of the host tRNA repertoire that could lead to enhanced viral protein folding. Furthermore, we demonstrated that Slfn11 is not the limiting factor of this novel broad anti-viral pathway.

  • Infectious virus persists in CD4+ T cells and macrophages in ART-suppressed SIV-infected Macaques [Virus-Cell Interactions]

  • Understanding the cellular and anatomical sites of latent virus that contribute to HIV rebound is essential for eradication. In HIV+ patients, CD4+ T lymphocytes comprise a well-defined functional latent reservoir, defined as cells containing transcriptionally silent genomes able to produce infectious virus once reactivated. However, the persistence of infectious latent virus in CD4+ T-cells in compartments other than blood and lymph nodes is unclear. Macrophages (M) are infected by HIV/SIV, and are likely to carry latent viral genomes during antiretroviral therapy (ART), contributing to the reservoir. Currently, the gold standard assay used to measure reservoirs containing replication competent virus is the quantitative viral outgrowth assay (QVOA). Using an SIV-macaque model, the CD4+ T-cell and M functional latent reservoirs were measured in various tissues using cell specific QVOAs. Our results show that blood, spleen, and lung in the majority of suppressed animals contained latently infected Ms. Surprisingly, the number of CD4+ T-cells, monocytes, and Ms carrying infectious genomes in blood and spleen were at comparable frequencies (~1 infected cell per million). We also demonstrate that ex vivo viruses produced in the M QVOA are capable of infecting activated CD4+ T-cells. These results strongly suggest that latently infected tissue Ms can reestablish productive infection upon treatment interruption. This study provides the first comparison of CD4+ T-cell and M functional reservoirs in a macaque model. It is the first confirmation of the persistence of latent genomes in monocytes in blood and Ms in spleen and lung in SIV-infected ART-suppressed macaques. Our results demonstrate that transcriptionally silent genomes in Ms could contribute to viral rebound post-ART interruption, and should be considered in future HIV cure strategies.

    IMPORTANCE

    This study suggests that CD4+ T cells found throughout tissues in the body can contain replication competent SIV and contribute to rebound of the virus after treatment interruption. In addition, this study demonstrates that macrophages in tissues are another cellular reservoir for SIV and may contribute to viral rebound post treatment interruption. This new insight into the size and location of the SIV reservoir could have great implications for HIV-infected individuals and should be taken into consideration for the development of future HIV cure strategies.

  • EphA7 functions as receptor on BJAB cells for cell-to-cell transmission of the Kaposi's sarcoma-associated herpesvirus (KSHV) and for cell-free infection by the related rhesus monkey rhadinovirus (RRV) [Virus-Cell Interactions]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma and is associated with two B cell malignancies, primary effusion lymphoma (PEL) and the plasmablastic variant of multicentric Castleman's disease. On several adherent cell types, EphA2 functions as cellular receptor for the gH/gL glycoprotein complex of KSHV. KSHV gH/gL has previously been found to also interact weakly with other members of the Eph family of receptor tyrosine kinases (Ephs), and other A-type Ephs have been shown to be able to compensate for the absence of EphA2 using overexpression systems. However, whether these interactions are of functional consequence at endogenous protein levels has remained unclear so far. Here, we demonstrate for the first time that endogenously expressed EphA7 in BJAB B cells is critical for the cell-to-cell transmission of KSHV from producer iSLK cells to BJAB target cells. The BJAB lymphoblastoid cell line often serves as a model for B cell infection and expresses only low levels of all Eph family receptors other than EphA7. Endogenous EphA7 could be precipitated from the cellular lysate of BJAB cells using recombinant gH/gL, and knockout of EphA7 significantly reduced transmission of KSHV into BJAB target cells. Knockout of EphA5, the second most expressed A-type Eph in BJAB cells, had a similar, although less pronounced effect on KSHV infection. Receptor function of EphA7 was conserved for cell-free infection by the related rhesus monkey rhadinovirus (RRV), which is relatively even more dependent on EphA7 for infection of BJAB cells.

    IMPORTANCE

    Infection of B cells is relevant for two KSHV-associated malignancies, the plasmablastic variant of multicentric Castleman's disease and primary effusion lymphoma (PEL). Elucidating the process of B cell infection is therefore important for the understanding of KSHV pathogenesis. While the high affinity receptor for the gH/gL glycoprotein complex, EphA2, has been shown to function as entry receptor for various types of adherent cells, the gH/gL complex can also interact with other Eph receptor tyrosine kinases with lower avidity. We analyzed the Eph interactions required for infection of BJAB cells, a model for B cell infection by KSHV. We identified EphA7 as principal Eph receptor for infection of BJAB cells by KSHV and the related rhesus monkey rhadinovirus. While two analyzed PEL cell lines exhibited high EphA2 and low EphA7 expression, a third PEL cell line, BCBL-1, showed high EphA7 and low EphA2 expression indicating a possible relevance for KSHV pathology.

  • The major hurdle for effective baculovirus transduction into mammalian cells is passing early endosomes [Virus-Cell Interactions]

  • Baculoviruses, although they infect insects in nature, can transduce a wide variety of mammalian cells and are therefore promising gene therapy vectors. However, baculovirus transduction into many mammalian cells is very inefficient and the limiting stages and factors remain unknown. An important finding is that a short duration trigger with low pH can significantly enhance virus transduction efficiency, but the mechanism is poorly understood. Herein, we performed a detailed comparative study on entry mechanisms of the prototypical baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) into insect and mammalian cells. The results showed that AcMNPV can be internalized into mammalian cells efficiently, but fusion in early endosomes (EEs) appeared to be the major obstacle. Measurement of endosomal pH suggested that virus fusion might be restricted under relatively high pH conditions in mammalian cells. Interestingly, mutants of the major viral fusion protein GP64 with decreased fusogenicity did not affect virus infection of insect cells, whereas virus transduction into mammalian cells was severely impaired, suggesting a more stringent dependence on GP64 fusogenicity for AcMNPV entry into mammalian cells than into insect cells. An increase in fusogenicity of GP64 mutants by low pH triggered the rescue of fusion-deficient recombinant virus transduction efficiency. Based on the above findings, the pH of EEs was specifically reduced with a Na+/K+-ATPase inhibitor, and AcMNPV transduction of many mammalian cells indeed became highly efficient. This study not only revealed the roadblocks to mammalian cell entry of baculovirus, but also provides a new strategy for improving baculovirus-based gene delivery and therapy.

    Importance

    Baculoviruses can transduce a wide variety of mammalian cells but do so with low efficiency, which greatly limits its practical application as potential gene delivery vectors. So far, the understanding of baculovirus entry into mammalian cells is obscure, and the limiting stages and factors are unclear. In this study, by comparatively analyzing the mechanism of baculovirus entry into mammalian and insect cells, virus fusion during the early stage of endocytosis was revealed as the major obstacle for efficient baculovirus transduction into mammalian cells. A higher fusogenicity of the major viral fusion protein GP64 was found to be required for virus entry into mammalian cells than into insect cells. Interestingly, by decreasing pH of early endosomes with a specific agent, virus transduction of a wide range of mammalian cells was greatly enhanced. This study uncovers the roadblocks encountered to mammalian cell entry of baculoviruses and presents mechanisms to overcome the roadblock.

  • A nonstructural protein responsible for viral spread of a novel insect reovirus provides a safe channel for biparental virus transmission to progeny [Genome Replication and Regulation of Viral Gene Expression]

  • Diaphorina citri reovirus (DcRV) was previously identified based on metagenomics surveys for virus discovery. Here, we demonstrated that DcRV induces persistent infections in its psyllid host, Diaphorina citri. DcRV was efficiently vertically passed to offspring in a biparental manner. Transmission electron microscopic and immunological analysis showed that the DcRV-encoded nonstructural protein, P10, assembled into a virion-packaging tubular structure which is associated with the spread of DcRV throughout the bodies of D. citri. P10 tubules containing virions were associated with oocytes of female and sperm of male D. citri, suggesting a role in the highly efficient biparental transmission of DcRV. Knocking down P10 by RNA interference for males reduced the percentage of DcRV-infected progeny, and for females reduced the viral accumulation in progeny. These results, for the first time, show that a nonstructural protein of a novel insect reovirus provides a safe and pivotal channel for virus spread and biparental transmission to progeny.

    IMPORTANCE The Asian citrus psyllid, Diaphorina citri Kuwayama, is an important pest in the worldwide citrus industry. It is the vector of Candidatus Liberibacter asiaticus (CLas), the bacterial pathogen of Huanglongbing (HLB), which is currently considered to be the most destructive disease of citrus worldwide. Diaphorina citri reovirus (DcRV) was previously identified based on metagenomics surveys for virus discovery. Here, we found that this novel and persistent insect reovirus took advantage of a viral-encoded nonstructural protein, P10, for efficient vertical transmission from parents to progeny. The P10 assembled into a virion-packaging tubular structure, and was associated with oocytes of female D. citri and sperm of males. Consistently, knockdown of P10 for either male or female D. citri inhibited DcRV transmission to offspring. This tubular strategy for viral spread and biparental transmission might serve as a target for controlling viral vertical transmission and population expansion.

  • CD4+ T cells drive lung disease enhancement induced by immunization with suboptimal doses of RSV fusion protein in the mouse model [Vaccines and Antiviral Agents]

  • Respiratory syncytial virus (RSV) infection of seronegative children, previously immunized with formalin-inactivated (FI)-RSV, has been associated with serious enhanced respiratory disease (ERD). The phenomenon was reproduced in the cotton rat and in the mouse, and both preclinical models have been routinely used to evaluate the safety of new RSV vaccine candidates. More recently, we demonstrated that immunizations with suboptimal doses of the RSV fusion (F) antigen, in its post- or prefusion conformation, and in the presence of a Th1-biasing adjuvant, unexpectedly lead to ERD in the cotton rat model. To assess if those observations are specific to the cotton rat and elucidate the mechanism by which vaccination with low antigen doses can drive ERD post-RSV challenge, we evaluated RSV post-F antigen dose de-escalation in BALB/c mice, in the presence of a Th1-biasing adjuvant. While decreasing antigen doses, we observed an increase in lung inflammation associated with an upregulation of proinflammatory cytokines. The amplitude of the lung histopathology was comparable to FI-RSV-induced ERD, confirming the observations made in the cotton rat. Importantly, depletion of CD4+ T cells prior to viral challenge completely abrogated ERD, preventing proinflammatory cytokine upregulation, infiltration of T cells, neutrophils, eosinophils, and macrophages in the lung. Overall, low antigen dose induced-ERD resembles FI-RSV-induced ERD, except that it appears in the absence of detectable levels of viral replication and in the context of a Th1 biased immune response. Altogether, our observations reinforce the recent concept that vaccines developed for RSV-naïve individuals should be systematically tested under suboptimal dosing conditions.

    IMPORTANCE RSV is a significant healthcare burden and the leading cause of serious lower respiratory tract infections in young children. A formalin inactivated RSV vaccine developed in the 1960s not only showed a complete lack of efficacy against RSV infection but also induced severe lung disease enhancement in vaccinated children. Since then, establishing safety in preclinical models has been one of the major challenges to RSV vaccine development. We recently identified in the cotton rat model that suboptimal immunizations with RSV fusion protein could induce lung disease enhancement. Here, we expanded suboptimal dosing evaluation to the mouse model. We confirmed the induction of lung disease enhancement by vaccinations with low antigen doses and dissected the associated immune mechanisms. Our results stress the need to evaluate suboptimal dosing for any new RSV vaccine candidate developed for seronegative infants.

  • Identification of Ebola Virus Inhibitors Targeting GP2 using Principles of Molecular Mimicry [Virus-Cell Interactions]

  • A key step in the Ebola virus (EBOV) replication cycle involves conformational changes in the viral glycoprotein 2 (GP2) which facilitate host-viral membrane fusion and subsequent release of the viral genome. Ebola GP2 plays a critical role in viral entry and has similarities in mechanism and structure to the HIV gp41 protein for which inhibitors have been successfully developed. In this work, a putative binding pocket for the C-terminal heptad repeat in the N-terminal heptad repeat trimer was targeted for identification of small molecules that arrest EBOV-host membrane fusion. Two computational structure-based virtual screens of ~1.7M compounds were performed (DOCK program), against a GP2 five-helix bundle, resulting in 165 commercially available compounds purchased for experimental testing. Based on assessment of inhibitory activity, cytotoxicity, and target specificity, four promising candidates emerged with IC50 values in the 3-26 mmu;M range. Molecular dynamics simulations of the two most potent candidates in their DOCK-predicted binding poses indicate that the majority of favorable interactions involve seven highly conserved residues which can be used to guide further inhibitor development and refinement targeting EBOV.

    IMPORTANCE The most recent Ebola Virus Disease outbreak from 2014 nndash; 2016, resulted in approximately 28,000 individuals becoming infected, which led to over 12,000 causalities worldwide. The particularly high pathogenicity of the virus makes identification and development of promising lead compounds to serve as inhibitors of Ebola infection paramount. To limit viral load, the viral-host membrane fusion event can be targeted through the inhibition of the class I fusion glycoprotein of Ebolavirus. In the current work, several promising small molecule inhibitors that target the glycoprotein GP2 were identified through systematic application of structure-based computational and experimental drug design procedures.

  • SIRT1 modulates the sensitivity of prostate cancer cells to VSV oncolysis [Virus-Cell Interactions]

  • Oncolytic virotherapy represents a promising experimental cancer strategy, based on the use of genetically modified viruses to selectively infect and kill cancer cells. Vesicular stomatitis virus (VSV) is a prototypic oncolytic virus that induces cancer cell death through activation of the apoptotic pathway, although intrinsic resistance to oncolysis is found in some cell lines and many primary tumors, as a consequence of residual innate immunity to the virus. In the effort to improve OV therapeutic efficacy, we previously demonstrated that different agents, including histone deacetylase inhibitors (HDIs), functioned as reversible chemical switches to dampen the innate antiviral response and improve the susceptibility of resistant cancer cells to VSV infection. In the present study, we demonstrate that the NAD+-dependent histone deacetylase SIRT1 plays a key role in the permissivity of prostate cancer PC-3 cells to VSVM51 replication and oncolysis. HDI-mediated enhancement of VSVM51 infection and cancer cell killing directly correlated with a decrease of SIRT1 expression. Furthermore, pharmacological inhibition as well as silencing of SIRT1 by siRNA was sufficient to sensitize PC-3 cells to VSVM51 infection, resulting in augmentation of virus replication and spread. Mechanistically, HDIs such as Vorinostat and Resminostat up-regulated the microRNA miR-34a that regulated the level of SIRT1. Altogether, our findings identify SIRT1 as a viral restriction factor that limits VSVM51 infection and oncolysis in prostate cancer cells.

    IMPORTANCE The use of non-pathogenic viruses to target into and kill cancer cells is a promising strategy in cancer therapy. However, many types of human cancer are resistant to the oncolytic (cancer killing) effects of virotherapy. In this study, Muscolini et al identify a host cellular protein SIRT1 that contributes to the sensitivity of prostate cancer cells to infection by a prototypical oncolytic virus. Knock out of SIRT1 activity increases the sensitivity of prostate cancer cells to virus mediated killing. At the molecular level, SIRT1 is controlled by a small microRNA termed miR-34a. Altogether, SIRT1 and/or miR-34a levels may serve as predictors of response to oncolytic virus therapy.

  • Porcine Reproductive and Respiratory Syndrome Virus Nsp11 Antagonizes Type I Interferon Signaling by Targeting IRF9 [Virus-Cell Interactions]

  • Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus from the Nidovirales order that causes reproductive failure and respiratory disease in pigs, and poses a constant threat to the global pig industry. The PRRSV-encoded nonstructural protein nsp11 is a nidovirus-specific endoribonuclease (NendoU) that is conserved throughout the Arteriviridae and Coronaviridae families. Previously, our research and that of others demonstrated that PRRSV nsp11 inhibits type I interferon (IFN) production through NendoU activity-dependent mechanisms. Here, we found that PRRSV nsp11 also inhibited IFN-stimulated response element (ISRE) promoter activity and subsequent transcription of IFN-stimulated genes (ISGs). Detailed analysis showed that nsp11 targeted interferon regulatory factor 9 (IRF9), but not transducer and activator of transcription 1 (STAT1) or STAT2, key molecules in the type I IFN signaling pathway. Furthermore, the nsp11nndash;IRF9 interaction impaired the formation and nuclear translocation of the transcription factor complex ISGF3 (STAT1/STAT2/IRF9) in both nsp11-overexpressed and PRRSV-infected cells. Importantly, nsp11 mutations (H129A, H144A, and K173A) that ablate NendoU activity or its cell cytotoxicity also interacted with IRF9 and retained the ability to block IFN signaling, indicating that the nsp11nndash;IRF9 interaction is independent of NendoU activity or cell cytotoxicity of nsp11. Taken together, our study demonstrates that PRRSV nsp11 antagonizes type I IFN signaling by targeting IRF9 via a NendoU activity-independent mechanism and describes a novel strategy evolved by PRRSV to counteract host innate antiviral responses, revealing a potential new function for PRRSV nsp11 in type I IFN signaling.

    IMPORTANCE

    The nidovirus-specific endoribonuclease (NendoU) encoded by PRRSV nsp11 is a unique NendoU of nidoviruses that infect vertebrates; thus, it is an attractive target for the development of anti-nidovirus drugs. Previous studies have revealed that the NendoU of nidoviruses, including porcine reproductive and respiratory syndrome virus (PRRSV) and human coronavirus 229E (HCoV-229E), acts as a type I interferon (IFN) antagonist. Here, for the first time, we demonstrate that overexpression of PRRSV nsp11 also inhibits IFN signaling by targeting the C-terminal interferon regulatory factor (IRF) association domain of IRF9. This interaction impaired the ability of IRF9 to form the transcription factor complex, IFN-stimulated gene factor 3 (ISGF3), and to act as a signaling protein of IFN signaling. Collectively, our data identify IRF9 as a natural target of PRRSV NendoU and reveal a novel mechanism evolved by an arterivirus to counteract innate immune signaling.

  • wMelpop strain of Wolbachia infection of Aedes albopictus mosquito C6/36 cells modulates dengue virus-induced host cellular transcripts and induces critical sequence alterations in dengue viral genome. [Virus-Cell Interactions]

  • The dengue virus (DENV) cause frequent epidemics infecting ~390 million people annually in over 100 countries. There are no approved vaccines or antiviral drugs for treatment of infected patients. However, there is a novel approach to control DENV transmission by the mosquito vectors, Aedes aegypti and Ae. albopictus, using the Wolbachia symbiont. The wMelPop strain of Wolbachia suppresses DENV transmission and shortens the mosquito life span. However, the underlying mechanism is poorly understood. To clarify this mechanism, either naïve Ae. albopictus (C6/36) or wMelPop-C6/36 cells were infected with DENV2. Analysis of host transcript profiles by RNAseq revealed that the presence of wMelPop dramatically altered the mosquito host cell transcription in response to DENV2 infection. The viral RNA evolved from wMelPop-C6/36 cells contained low frequency mutations (~25%) within the coding region of transmembrane domain-1 (TMD1) of E protein. Mutations with ggt;97 % frequencies were distributed within other regions of E, NS5 RNA-dependent RNA polymerase (NS5POL) domain, the TMDs of NS2A, NS2B, and NS4B. Moreover, while DENV2-infected naïve C6/36 cells showed syncytia formation, DENV2-infected wMelPop-C6/36 cells did not. The Wolbachia-induced mutant DENV2 can readily infect and replicate in naïve C6/36 cells; whereas, in the mutant DENV2- infected BHK-21 or Vero cells, the virus replication was delayed. In LLC-MK2 cells, the mutant failed to produce plaques. Additionally, in BHK-21 cells, many mutations in the viral genome reverted to WT and compensatory mutations in NS3 gene appeared. Our results indicate that wMelPop impacts significantly the interactions of DENV2 with mosquito and mammalian host cells.

    Importance:

    Mosquito-borne diseases are of global significance causing considerable morbidity and mortality throughout the world. The dengue virus (DENV, serotypes 1-4), a member of flavivirus genus of Flaviviridae family, causes millions of infections annually. Development of a safe vaccine is hampered due to absence of cross-protection and increased risk in secondary infections due to antibody-mediated immune enhancement. Infection of vector mosquitoes with Wolbachia bacteria offers a novel countermeasure to suppress DENV transmission, but the mechanisms are poorly understood. In this study, the host transcription profiles and viral RNA sequences were analyzed in naïve Ae. albopictus (C6/36) and wMelPop-C6/36 cells by RNAseq. Our results showed that the wMelPop symbiont caused profound changes in host transcription profiles and morphology of DENV2-infected C6/36 cells. Accumulation of several mutations throughout DENV2 RNA resulted in loss of infectivity of progeny virions. Our findings offer new insights into the mechanism of Wolbachia-mediated suppression of DENV transmission.

  • Age-Dependent Effects of Immunoproteasome Deficiency on Mouse Adenovirus Type 1 Pathogenesis [Pathogenesis and Immunity]

  • Acute respiratory infection with mouse adenovirus type 1 (MAV-1) induces activity of the immunoproteasome, an inducible form of the proteasome that shapes CD8 T cell responses by enhancing peptide presentation by MHC class I. We used mice deficient in all three immunoproteasome subunits (triple knockout, or TKO mice) to determine whether immunoproteasome activity is essential for control of MAV-1 replication or inflammatory responses to acute infection. Complete immunoproteasome deficiency in adult TKO mice had no effect on MAV-1 replication, virus-induced lung inflammation, or adaptive immunity compared to C57BL/6 (B6) controls. In contrast, immunoproteasome deficiency in neonatal TKO mice was associated with decreased survival and decreased lung IFN- expression compared to B6 controls, although without substantial effects on viral replication, histological evidence of inflammation, or expression of the pro-inflammatory cytokines TNF-aalpha; and IL-1bbeta; in lungs or other organs. T cell recruitment and IFN- production was similar in lungs of infected B6 and TKO mice. In lungs of uninfected B6 mice, we detected low levels of immunoproteasome subunit mRNA and protein that increased with age. Immunoproteasome subunit expression was lower in lungs of adult IFN--deficient mice compared to B6 controls. Together, these results demonstrate developmental regulation of the immunoproteasome that is associated with age-dependent differences in MAV-1 pathogenesis.

    Importance

    MAV-1 infection is a useful model to study the pathogenesis of an adenovirus in its natural host. Host factors that control MAV-1 replication and contribute to inflammation and disease are not fully understood. The immunoproteasome is an inducible component of the ubiquitin proteasome system that shapes the repertoire of peptides presented by MHC class I to CD8 T cells, influences other aspects of T cell survival and activation, and promotes production of pro-inflammatory cytokines. We found that immunoproteasome activity is dispensable in adult mice. However, immunoproteasome-deficiency in neonatal mice increased mortality and impaired IFN- responses in the lungs. Baseline immunoproteasome subunit expression in lungs of uninfected mice increased with age. Our findings suggest the existence of developmental regulation of the immunoproteasome, like other aspects of host immune function, and indicate that immunoproteasome activity is a critical protective factor early in life.

  • Persistent infection of African buffalo (Syncerus caffer) with Foot-and-Mouth Disease Virus: limited viral evolution and no evidence of antibody neutralization escape [Pathogenesis and Immunity]

  • African buffaloes (Syncerus caffer) are the principal "carrier" hosts of foot-and-mouth disease virus (FMDV). Currently, the epithelia and lymphoid germinal centres of the oropharynx have been identified as sites for FMDV persistence. We carried out studies in FMDV SAT-1 persistently infected buffaloes to characterize the diversity of viruses in oropharyngeal epithelia, germinal centres, probang (oropharyngeal scrapings) and tonsil swabs, to determine if sufficient virus variation is generated during persistence for immune escape.

    Most sequencing reads of the VP1-coding region of the SAT 1 virus inoculum clustered around two subpopulations differing by 22 single nucleotide variants of intermediate frequency. Similarly, most sequences from oropharynx tissue clustered into two subpopulations, albeit with different proportions depending on the days post-infection (dpi). There was a significant difference between the population of viruses in the inoculum and in lymphoid tissue taken at 35 dpi. Thereafter, until 400 dpi, no significant variation was detected in the viral population in samples from individual animals, germinal centres and epithelia tissues. Deep sequencing of virus from probang or tonsil swab samples harvested prior to post mortem showed less within-sample variability of VP1 compared to tissue sample sequences analysed at the same time.

    Importantly, there was no significant difference in the ability of sera collected between 14 and 400 dpi to neutralise the inoculum or viruses isolated at later time points in the study from the same animal. Therefore, based on this study there is no evidence of escape from antibody neutralization contributing to FMDV persistent infection in African buffalo.

    Importance

    Foot-and-mouth disease virus (FMDV) is a highly contagious virus of cloven hooved animals and is recognised as the most important constraint to international trade in animals and animal products. African buffaloes (Syncerus caffer) are efficient carriers of FMDV and it has been proposed that new virus variants are produced in buffalo during the prolonged carriage after acute infection, which may spread to cause disease in livestock populations. Here, we show that despite an accumulation of low frequency sequence variants over time there is no evidence of significant antigenic variation leading to immune escape. Therefore, carrier buffalo are unlikely to be a major source of new virus variants.

  • Murine cytomegalovirus spread depends on the infected myeloid cell type [Virus-Cell Interactions]

  • Cytomegaloviruses (CMVs) colonize blood-borne myeloid cells. Murine CMV (MCMV) spreads from the lungs via infected CD11c+ cells, consistent with an important role for dendritic cells (DC). We show here that MCMV entering via the olfactory epithelium, a natural transmission portal, also spreads via infected DC. They reached lymph nodes, entered the blood via high endothelial venules, then entered the salivary glands, driven by constitutive signalling of the viral M33 G protein-coupled receptor (GPCR). Intraperitoneal (i.p.) infection also delivered MCMV to the salivary glands via DC. However, it additionally seeded F4/80+ infected macrophages to the blood; they did not enter the salivary glands or require M33 for extravasation. Instead, they seeded infection to a range of other sites, including brown adipose tissue (BAT). Peritoneal cells infected ex vivo then adoptively transferred showed similar cell type-dependent differences in distribution, with abundant F4/80+ cells in BAT and CD11c+ cells in the salivary glands. BAT colonization by CMV-infected cells was insensitive to Pertussis toxin inhibition of the GPCR signalling through Gi/o substrate, whereas salivary gland colonization was sensitive. As salivary gland infection required both M33 and Gi/o nndash;coupled signalling, while BAT infection required neither, these migrations were mechanistically distinct. MCMV spread from the lungs or nose depended on DC, controlled by M33. Infecting other monocyte populations resulted in unpredictable new infections.

    IMPORTANCE Cytomegaloviruses (CMVs) spread through the blood by infecting monocytes, and this can lead to disease. With Murine CMV (MCMV) we can track infected myeloid cells and so to understand how CMVs spread. Previous experiments have injected MCMV into the peritoneal cavity. MCMV normally enters mice via the olfactory epithelium. We show that olfactory infection spreads via dendritic cells, which MCMV directs to the salivary glands . Peritoneal infection similarly reached the salivary glands via dendritic cells. However, it also infected other monocyte types, and they spread infection to other tissues. Thus, infecting the "wrong" monocytes altered virus spread, with potential to cause disease. These results provide a basis for understanding how the monocyte types infected by human CMV might promote different infection outcomes.

  • CD8{beta} depletion does not prevent control of viral replication or protection from challenge in macaques chronically infected with a live attenuated simian immunodeficiency virus. [Pathogenesis and Immunity]

  • We evaluated the contribution of CD8aalpha;bbeta;+ T cells on control of live-attenuated simian immunodeficiency virus (LASIV) replication during chronic infection and subsequent protection from pathogenic SIV challenge. Unlike previous reports with a CD8aalpha;-specific depleting monoclonal antibody (mAb), the CD8bbeta;-specific mAb CD8bbeta;255R1 selectively depleted CD8aalpha;bbeta;+ T cells without also depleting non-CD8+ T cell populations that express CD8aalpha;, such as natural killer (NK) cells and T cells. Following infusion with CD8bbeta;255R1, plasma viremia transiently increased coincident with declining peripheral CD8aalpha;bbeta;+ T cells. Interestingly, plasma viremia returned to pre-depletion levels even when peripheral CD8aalpha;bbeta;+ T cells did not. Although depletion of CD8aalpha;bbeta;+ T cells in the lymph node (LN) was incomplete, frequencies of these cells were three-fold lower (p=0.006) in animals that received CD8bbeta;255R1 compared to control IgG. It is possible that these residual SIV-specific CD8aalpha;bbeta;+ T cells may have contributed to suppression of viremia during chronic infection. We also determined whether infusion of CD8bbeta;255R1 in the LASIV-vaccinated animals increased their susceptibility to infection following intravenous challenge with pathogenic SIVmac239. We found that 7/8 animals infused with CD8bbeta;255R1, and 3/4 animals infused with the control IgG, were resistant to SIVmac239 infection. These results suggest that infusion with CD8bbeta;255R1 did not eliminate the protection afforded to LASIV vaccination. This provides a comprehensive description of the impact of CD8bbeta;255R1 infusion on the immunological composition in cynomolgus macaques, when compared to an isotype matched control IgG, while showing that the control of LASIV viremia and protection from challenge can occur even after CD8bbeta;255R1 administration.

    Importance:

    Studies of SIV-infected macaques that deplete CD8+ T cells in vivo with monoclonal antibodies have provided compelling evidence for their direct antiviral role. These studies utilized CD8aalpha;-specific mAbs that target both the major (CD8aalpha;bbeta;+) and minor (CD8aalpha;aalpha;+) populations of CD8+ T cells, but additionally deplete non-CD8+ T cell populations that express CD8aalpha;, such as NK cells and T cells. In the current study, we administered the CD8bbeta;-specific depleting mAb CD8bbeta;255R1 to cynomolgus macaques chronically infected with a LASIV to selectively deplete CD8aalpha;bbeta;+ T cells without removing CD8aalpha;aalpha;+ lymphocytes. We evaluated the impact on control of virus replication and protection from pathogenic SIVmac239 challenge. These results underscore the utility of CD8bbeta;255R1 for studying the direct contribution of CD8aalpha;bbeta;+ T cells in various disease states.

  • Infectious Chikungunya Virus with a Complete Capsid Deletion: a New Approach for CHIKV Vaccine [Vaccines and Antiviral Agents]

  • Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes epidemics of debilitating disease worldwide. Currently, there are no licensed vaccines or antivirals available against CHIKV infection. In this study, we generated a novel live attenuated vaccine (LAV) candidate of CHIKV with a complete deficiency of capsid (C-CHIKV). It could propagate in BHK-21 cells, and had similar antigenic properties as native CHIKV. Vaccination of either immunocompromised IFNAR-/- mice or immunocompetent C57BL/6 mice with a single dose of C-CHIKV conferred complete protection upon challenge with wild type (WT) CHIKV. Taken together, this vaccine candidate appeared to be safe and efficacious, representing a novel strategy for CHIKV vaccine design.

    IMPORTANT

    Currently, there is no licensed vaccine against CHIKV infection. An ideal CHIKV vaccine should generate an optimal balance between efficacy and safety. Live attenuated vaccine that can elicit strong immune responses often involve a trade-off of reduced safety. Here, a novel live attenuated vaccine candidate of CHIKV lacking the entire capsid gene, C-CHIKV, was developed. It was demonstrated to be genetically stable, highly attenuated, immunogenic and able to confer complete protection against lethal CHIKV challenge after a single dose of immunization. Such infectious vaccine candidate devoid of capsid provided a novel strategy for the development of live attenuated CHIKV vaccine.

  • Differential requirements for gE, gI, and UL16 among HSV-1 syncytial variants suggest unique modes of dysregulating the mechanism of cell-to-cell spread [Virus-Cell Interactions]

  • Like all the herpesviruses, herpes simplex virus encodes machinery that enables it to move through cell junctions to avoid neutralizing antibodies. This cell-to-cell-spread mechanism requires the viral fusion machinery (gD, gH/gL, and gB) and numerous accessory proteins. Of all of these, minor alterations to only four proteins (gB, gK, UL20, or UL24) will dysregulate the fusion machinery, allowing the formation of syncytia. In contrast, removal of individual accessory proteins will block cell-to-cell spread, forcing the virus to transmit in a cell-free manner. In the context of a Syn variant, removal of a required accessory protein will block cell fusion, again forcing cell-free spread. This has been investigated most thoroughly for gBsyn variants, which lose their syncytial phenotype in the absence of several accessory proteins, including gE, gI, UL16, and UL21, which are known to physically interact. Recently it was found that UL21 is not needed for gKsyn-, UL20syn-, or UL24syn-induced cell fusion, and hence, it was of interest to ascertain whether gE, gI, and UL16 are required for Syn variants other than gBsyn. Null mutants of these were each combined with seven syncytial variants distributed among gK, UL20, and UL24. Surprisingly, very different patterns of accessory protein requirements were revealed. Indeed, for the three gKsyn variants tested, two different patterns were found. Also, three mutants were able to replicate without causing cytopathic effects. These findings show that mutations that produce Syn variants dysregulate the cell-to-cell-spread machinery in unique ways and provide clues for elucidating how this virus moves between cells.

    IMPORTANCE

    Approximately 2/3 of adults worldwide are latently infected with herpes simplex virus type 1. Upon reactivation, the virus has the ability to evade neutralizing antibodies by moving through cell junctions, but the mechanism of direct cell-to-cell spread is poorly understood. The machinery that assembles between cells includes the viral fusion proteins and various accessory proteins that prevent cells from fusing. Alterations in four proteins will dysregulate the machinery, allowing neighboring cells to fuse to make syncytia, but this can be prevented by removing individual various accessory proteins to further disable the machinery. Previously, the accessory protein UL21 was found to be important for the activity of some syncytial variants but not others. In this study, we discovered that UL16, gE, and gI all act differently in how they control the fusion machinery. A better understanding of the mechanism of cell-to-cell spread may enable the development of drugs that block it.

  • Transmission of a new polerovirus infecting pepper by the whitefly Bemisia tabaci [Cellular Response to Infection]

  • Many animal and plant viruses depend on arthropods for their transmission. Virus-vector interactions are highly specific and only one vector, or a group of vectors from the same family are able to transmit a given virus. Poleroviruses (Luteoviridae) are phloem-restricted RNA plant viruses, which are exclusively transmitted by aphids. Multiple aphid-transmitted polerovirus species commonly infect pepper, causing vein yellowing, leaf rolling and fruit discoloration. Despite low aphid populations, a recent outbreak with such severe symptoms in many bell pepper farms in Israel led to reinvestigation of the disease and its insect vector. Here we report that this outbreak was caused by a new whitefly (Bemisia tabaci)-transmitted polerovirus, which we coined Pepper whitefly borne vein yellows virus (PeWBVYV). PeWBVYV is highly homologous (ggt;95%) to Pepper vein yellows virus (PeVYV) from Israel and Greece on its 5' end half, while it is homologous to African eggplant yellows virus (AeYV) on its 3' half. Koch's postulates were proven by constructing a PeWBVYV infectious clone causing the pepper disease, which was in turn transmitted to test pepper plants by B. tabaci but not by aphids. PeWBVYV is the first report of a whitefly transmitted polerovirus.

    Importance

    High specificity of virus-vector interactions limits the possibility of a given virus to change vector. Our study reports a new virus from a family of viruses strictly transmitted by aphids, which is now transmitted by whiteflies (Bemisia tabaci) and not by aphids. This study constitutes the first report of a polerovirus transmission by whiteflies. Whiteflies are highly resistant to insecticides and disperse to long distances, carrying virus inoculum. Thus, the report of such unusual polerovirus transmission by a super vector has deep implications on the epidemiology of the virus disease, with ramifications on international trade of agricultural commodities.

  • Human papillomavirus type 16 (HPV16) capsids mediate nuclear entry during infection [Virus-Cell Interactions]

  • Infectious HPV16 L1/L2 pseudovirions were found to remain largely intact during vesicular transport to the nucleus. By electron microscopy, capsids with a diameter of 50 nm were clearly visible within small vesicles attached to mitotic chromosomes and to a lesser extent within interphase nuclei, implying nuclear disassembly. By confocal analysis, it was determined that nuclear entry of assembled L1 is dependent upon the presence of the minor capsid protein, L2, but independent of encapsidated DNA. We also demonstrate that L1 nuclear localization and mitotic chromosome association can occur in vivo in the murine cervicovaginal challenge model of HPV16 infection. These findings challenge the prevailing concepts of PV uncoating and disassembly. More generally, they document that a largely intact viral capsid can enter the nucleus within a transport vesicle, establishing a novel mechanism by which a virus accesses the nuclear cellular machinery.

    Importance

    Papillomaviruses (PV) comprise a large family of non-enveloped DNA viruses that include HPV16 among other oncogenic types, the causative agents of cervical cancer. Delivery of the viral DNA into the host cell nucleus is necessary for establishment of infection. This was thought to occur via a subviral complex following uncoating of the larger viral capsid. In this study, we demonstrate that little disassembly of the PV capsid occurs prior to nuclear delivery. This surprising data reveal a previously unrecognized viral strategy to access the nuclear replication machinery. Understanding viral entry mechanisms not only increases our appreciation of basic cell biological pathways, but also may lead to more effective anti-viral interventions.

  • The Effect of Age on the Immunogenicity of the Live Attenuated Zoster Vaccine Is Predicted by Baseline Regulatory T Cells and VZV-Specific T Cell Immunity [Vaccines and Antiviral Agents]

  • Older age is associated with increased infectious morbidity and decreased immune responses to vaccines, but the mechanisms that mediate this effect are incompletely understood. The efficacy and immunogenicity of live attenuated zoster vaccine (ZVL) have a very well described negative association with the age of the vaccinee. In a study of 600 ZVL recipients 50 to ggt;80 years of age, we investigated immunologic factors that might explain the effect of age on the immunogenicity of ZVL. Using FluoroSpot and flow cytometry, we determined that varicella-zoster virus (VZV)-specific peak Th1 (VZV-Th1) responses to ZVL were independently predicted by pre-vaccination VZV-Th1 responses, regulatory T cells (Treg) and PD1-expressing Tcheck, but not by age of the vaccinee. Persistence of VZV-Th1 at 1 year after vaccination was independently predicted by the factors above and by peak VZV-Th1 responses to ZVL and age of the vaccinee. We further demonstrated by ex-vivo blocking experiments the mechanistic role of PD1 and CTLA4 as modulators of decreased VZV-Th1 responses in the study participants. VZV-specific cytotoxic T cell (VZV-CTL) and T follicular helper responses to ZVL did not correlate with age, but similar to other Th1 responses, VZV-CTL peak and baseline responses were independently correlated. These data expand our understanding of the factors affecting the magnitude and kinetics of T cell responses to ZVL in older adults and show the importance of pre-vaccination Treg and Tcheck in modulating the immunogenicity of ZVL. This presents new potential interventions to increase vaccine responses in older adults.

    IMPORTANCE Vaccination is the most effective method to protect older adults against viral infections. However, the immunogenicity of viral vaccines in older adults is notoriously poor. Live attenuated zoster vaccine (ZVL) provides the best example of a gradual decrease of vaccine immunogenicity with every 10-year age increase above 50 years. Here we show that the abundance of regulatory T cells before vaccine administration to older adults has a significant inhibitory effect on immune responses to ZVL and, together with baseline immunity to varicella zoster virus, explain the effect of age on the immunogenicity of ZVL. Moreover, in vitro blockade of regulatory T cell mechanisms of action with biologic modulators restores immune responses to varicella zoster virus in vaccinees. Collectively, these observations suggest that immune modulators that block regulatory T cell activity may increase responses to viral attenuated vaccines in older adults.

  • Surface plasmon resonance (SPR) reveals direct binding of HSV glycoproteins gH/gL to gD and locates a gH/gL binding site on gD [Structure and Assembly]

  • Herpes simplex virus (HSV) requires fusion between the viral envelope and host membrane. Four glycoproteins, gD, gH/gL, and gB, are essential for this process. To initiate fusion, gD binds its receptor and undergoes a conformational change that hypothetically leads to activation of gH/gL, which in turn triggers the fusion protein gB to undergo rearrangements leading to membrane fusion. Our model predicts that gD must interact with both its receptor and gH/gL to promote fusion. In support, we have shown that gD is structurally divided into two "faces:" one for binding receptor and the other for its presumed interaction with gH/gL. However, until now, we have been unable to demonstrate a direct interaction between gD and gH/gL. Here, we used surface plasmon resonance to show that the ectodomain of gH/gL binds directly to the ectodomain of gD when: 1) gD is captured by certain anti-gD monoclonal antibodies (MAbs) that are bound to a biosensor chip; 2) gD is bound to either one of its receptors on a chip; and 3) gD is covalently bound to the chip surface. To localize the gH/gL binding site on gD, we used multiple anti-gD MAbs from six antigenic communities and determined which ones interfered with this interaction. MAbs from three separate communities block gD-gH/gL binding and their epitopes encircle a geographical area on gD that we propose comprises the gH/gL binding domain. Together, our results show that gH/gL interacts directly with gD, supporting a role for this step in HSV entry.

    Importance

    HSV entry is a multi-step process that requires the actions of four glycoproteins, gD, gH/gL, and gB. Our current model predicts that gD must interact with both its receptor and gH/gL to promote viral entry. Although we know a great deal about how gD binds its receptors, we have been heretofore unable to demonstrate a direct interaction between gD and gH/gL. Here, we used a highly sensitive, surface plasmon resonance technique to clearly demonstrate that gD and gH/gL interact. Furthermore, using multiple MAbs with defined epitopes, we have delineated a domain on gD that is independent of that used for receptor binding and which likely represents the gH/gL interaction domain. Targeting this interaction to prevent fusion may enhance both therapeutic and vaccine strategies.

  • Mx1 in hematopoietic cells protects against Thogotovirus infection [Pathogenesis and Immunity]

  • Myxovirus resistance 1 (Mx1) is an interferon-induced gene that encodes a GTPase that plays an important role in the defense of mammalian cells against influenza A and other viruses. The Mx1 protein can restrict a number of viruses, independently of the expression of other interferon-induced genes. Mx genes are therefore considered to be an important part of the innate antiviral immune response. However, the possible impact of Mx expression in the hematopoietic cellular compartment has not been investigated in detail in the course of a viral infection. To address this, we performed bone marrow chimera experiments using congenic B6.A2G Mx1+/+ and B6.A2G Mx1-/- mice to study the effect of Mx1 expression in cells of hematopoietic versus non-hematopoietic origin. Mx1+/+ mice were protected and Mx1-/- mice were susceptible to influenza A virus challenge infection, regardless of the type of bone marrow cells (Mx1+/+ or Mx1-/-) the animals had received. Infection with Thogotovirus, however, revealed that Mx1-/- mice with a functional Mx1 gene in the bone marrow compartment showed reduced liver pathology compared with Mx1-/- mice that had been grafted with Mx1-/- bone marrow. The reduced pathology in these mice was associated with a reduction in Thogotovirus titers in the spleen, lung and serum. Moreover, Mx1+/+ with Mx1-/- bone marrow failed to control Thogotovirus replication in the spleen. Mx1 in the hematopoietic cellular compartment thus contributes to protection against Thogotovirus infection.

    Importance

    Mx proteins are evolutionarily conserved in vertebrates and can restrict a wide range of viruses in a cell autonomous way. The contribution to antiviral defense of Mx1 expression in hematopoietic cells remains largely unknown. We show that protection against influenza virus infection requires Mx1 expression in the nonhematopoietic cellular compartment. In contrast, Mx1 in bone marrow-derived cells is sufficient to control disease and virus replication following infection with a Thogotovirus. This indicates that next to its well established antiviral activity in nonhematopoietic cells, Mx1 in hematopoietic cells can also play an important antiviral function. In addition, cells of hematopoietic origin that lack a functional Mx1 gene, contribute to Thogotovirus dissemination and associated disease.

  • SHIV.CH505-infected infant and adult rhesus macaques exhibit similar HIV Env-specific antibody kinetics, despite distinct T-follicular helper (Tfh) and germinal center B cell landscapes [Pathogenesis and Immunity]

  • Global elimination of pediatric HIV infections will require the development of novel immune-based approaches, and understanding infant immunity to HIV is critical to guide the rational design of these intervention strategies. Despite their immunological immaturity, chronically HIV-infected children develop broadly neutralizing antibodies (bnAbs) more frequently and earlier than adults do. However, the ontogeny of humoral responses during acute HIV infection are poorly defined in infants and challenging to study in human cohorts due to the presence of maternal antibodies. To further our understanding of age-related differences in the development of HIV-specific immunity during acute infection, we evaluated the generation of virus-specific humoral immune responses in infant (n=6) and adult (n=12) rhesus macaques (RMs) infected with a transmitted/founder (T/F) simian-human immunodeficiency virus (SHIV.C.CH505).

    The plasma HIV envelope-specific IgG antibody kinetics were similar in SHIV-infected infant and adult RMs, with no significant differences in the magnitude or breadth of these responses. Interestingly, autologous tier 2 virus neutralization responses also developed with similar frequency and kinetics in infant and adult RMs, despite infants exhibiting significantly higher follicular T helper cell (Tfh) and germinal center B cell frequencies compared to adults. Lastly, we show that plasma viral load was the strongest predictor of the development of autologous virus neutralization in both age groups. Our results indicate that the humoral immune response to SHIV infection develops with similar kinetics among infant and adult RMs, suggesting that the early life immune system is equipped to respond to HIV-1 and promote the production of neutralizing HIV antibodies.

    Importance

    There is a lack of understanding on how the maturation of the infant immune system influences immunity to HIV infection, or how these responses differ from those of adults. Improving our knowledge of infant HIV immunity will help guide antiviral intervention strategies that take advantage of the unique infant immune environment to successfully elicit protective immune responses. We utilized a rhesus macaque model of SHIV infection as a tool to distinguish the differences in HIV humoral immunity in infants versus adults. Here, we demonstrate that the kinetics and quality of the infant humoral immune response to HIV are highly comparable to that of adults during the early phase of infection, despite distinct differences in their Tfh responses, indicating that slightly different mechanisms may drive infant and adult humoral immunity.

  • Broadly neutralizing antibodies targeting new sites of vulnerability in hepatitis C virus E1E2 [Pathogenesis and Immunity]

  • Increasing evidence indicates that broadly neutralizing antibodies (bNAbs) play an important role in immune-mediated control of hepatitis C virus (HCV) infection, but the relative contribution of neutralizing antibodies targeting antigenic sites across the HCV envelope (E1 and E2) proteins is unclear. Here, we isolated thirteen E1E2-specific monoclonal antibodies (mAbs) from B cells of a single HCV-infected individual who cleared one genotype 1a infection and then became persistently infected with a second genotype 1a strain. These mAbs bound six distinct discontinuous antigenic sites on the E1 protein, the E2 protein, or the E1E2 heterodimer. Three antigenic sites, designated AS108, AS112 (an N-terminal E1 site), and AS146 were distinct from previously described Antigenic Regions (AR) 1-5 and E1 sites. Antibodies targeting four sites (AR3, AR4-5, AS108, and AS146) were broadly neutralizing. These mAbs also displayed distinct patterns of relative neutralizing potency (i.e., neutralization profiles) across a panel of diverse HCV strains, which led to complementary neutralizing breadth when they were tested in combination. Overall, this study demonstrates that HCV bNAb epitopes are not restricted to previously described antigenic sites, expanding the number of sites that could be targeted for vaccine development.

    Importance

    Worldwide, more than 70 million people are infected with hepatitis C virus (HCV), which is a leading cause of hepatocellular carcinoma and liver transplantation. Despite the development of potent direct acting antivirals (DAAs) for HCV treatment, a vaccine is urgently needed due to the high cost of treatment and the possibility of reinfection after cure. Induction of multiple broadly neutralizing antibodies (bNAbs) that target distinct epitopes on the HCV envelope proteins is one approach to vaccine development. However, antigenic sites targeted by bNAbs in individuals with spontaneous control of HCV have not been fully defined. In this study, we characterize thirteen monoclonal antibodies (mAbs) from a single person who cleared an HCV infection without treatment, and we identify three new sites targeted by neutralizing antibodies. The sites targeted by these mAbs could inform HCV vaccine development.

  • A hydrophobic network: Intersubunit and intercapsomer interactions stabilizing the bacteriophage P22 capsid [Structure and Assembly]

  • dsDNA tailed phages and herpesviruses assemble their capsids using coat proteins that have the ubiquitous HK97 fold. Though this fold is common, we do not have a thorough understanding of the different ways viruses adapt it to maintain stability in various environments. The HK97-fold E-loop, which connects adjacent subunits at the outer periphery of capsomers, has been implicated in capsid stability. Here we show that in bacteriophage P22, residue W61 at the tip of the E-loop plays a role in stabilizing procapsids and in maturation. We hypothesize that a hydrophobic pocket is formed by residues I366 and W410 in the P-domain of a neighboring subunit within a capsomer, into which W61 fits like a peg. In addition, W61 likely bridges to residues A91 and L401 in P-domain loops of an adjacent capsomer, thereby linking the entire capsid together with a network of hydrophobic interactions. There is conservation of this hydrophobic network in the distantly related P22-like phages, indicating that this structural feature is likely important for stabilizing this family of phages. Thus, our data shed light on one of the varied elegant mechanisms used in nature to consistently build stable viral genome containers through subtle adaptation of the HK97 fold.

    IMPORTANCE Similarities in assembly reactions and coat protein structures of the dsDNA tailed phages and herpesviruses make phages ideal models to understand capsid assembly and identify potential targets for antiviral drug discovery. The coat protein E-loops of these viruses are involved in both intra- and intercapsomer interactions. In phage P22, hydrophobic interactions peg the coat protein subunits together within a capsomer, where the E-loop hydrophobic residue W61 of one subunit packs into a pocket of hydrophobic residues I366 and W410 of the adjacent subunit. W61 also makes hydrophobic interactions with A91 and L401 of a subunit in an adjacent capsomer. We show these intra- and intercapsomer hydrophobic interactions form a network crucial to capsid stability and proper assembly.

  • A non-canonical basic motif of EBV ZEBRA protein facilitates recognition of methylated DNA, high-affinity DNA-binding and lytic activation [Genome Replication and Regulation of Viral Gene Expression]

  • The pathogenesis of Epstein Barr Virus (EBV) infection, including development of lymphomas and carcinomas, is dependent on the ability of the virus to transit from latency to the lytic phase. This conversion, and ultimately disease development, depends on the molecular switch protein, ZEBRA, a viral bZIP transcription factor that initiates transcription from promoters of viral lytic genes. By binding to the origin of viral replication ZEBRA is also an essential replication protein. Here, we identified a novel DNA-binding motif of ZEBRA, N-terminal to the canonical bZIP domain. This RRTRK motif is important for high-affinity binding to DNA and is essential for recognizing the methylation state of viral promoters. Mutations in this motif lead to deficiencies in DNA-binding, recognition of DNA methylation, lytic cycle DNA replication and viral late gene expression. This work advances our understanding of ZEBRA-dependent activation of viral lytic cascade.

    IMPORTANCE The binding of ZEBRA to methylated and unmethylated viral DNA triggers activation of the EBV lytic cycle, leading to viral replication and, in some patients, cancer development. Our work thoroughly examines how ZEBRA uses a previously unrecognized basic motif to bind non-methylated and methylated DNA targets, leading to viral lytic activation. Our findings show that two different positively charged motifs, including the canonical BZIP domain and a newly identified RRTRK motif, contribute to the mechanism of DNA recognition by a viral AP-1 protein. This work contributes to the assessment of ZEBRA as a potential therapeutic target for antiviral and oncolytic treatments.

  • Sam68 Promotes Hepatitis C Virus Replication by Interaction with Stem-Loop 2 of Viral 5' Untranslated Region [Virus-Cell Interactions]

  • Src-associated in mitosis 68 kDa (Sam68) protein is a highly conserved nuclear protein and involved in a series of cellular processes including transcription and signal transduction. Sam68 is comprised of 443 amino acids and contains a RGG box domain, a KH domain and a tyrosine-rich domains. Its role in hepatitis C virus (HCV) replication is unknown. Here, we find that Sam68 promotes HCV replication without affecting viral translation. The RNA immunoprecipitation experiments show that the positive-strand of HCV RNA interacts with Sam68. HCV infection triggers the translocation of Sam68 protein from nucleus to the cytoplasm, where it interacts with HCV genome. Further study shows that the region of Sam68 spanning amino acids 1 to 157 is the pivotal domain to interact with the stem-loop 2 of HCV 5' untranslated region (5' UTR) and responsible for the enhancement of HCV replication. These data suggested that Sam68 may serve as a proviral factor of HCV to facilitate viral replication through interaction with viral genome.

    IMPORTANCE

    Hepatitis C virus (HCV) is a member of the Flaviviridae family and its infection causes chronic hepatitis, liver cirrhosis, and even hepatocellular carcinoma. No vaccine is available. Many host factors may be implicated in the pathogenesis of HCV-related diseases. This study discloses a new host factor that binds to HCV 5' UTR and promotes HCV replication. Sam68 may play an important role in HCV-related diseases and further investigation is highly encouraged to explore its specific actions in HCV pathogenesis.

  • Picornavirus RNA Recombination Counteracts Error Catastrophe [Genome Replication and Regulation of Viral Gene Expression]

  • Template-dependent RNA replication mechanisms render picornaviruses susceptible to error catastrophe, an overwhelming accumulation of mutations incompatible with viability. Viral RNA recombination, in theory, provides a mechanism for viruses to counteract error catastrophe. We tested this theory by exploiting well-defined mutations in the poliovirus RNA-dependent RNA polymerase (RDRP): a G64S mutation and an L420A mutation. Our data reveal two distinct mechanisms by which picornaviral RDRPs influence error catastrophe: fidelity of RNA synthesis and RNA recombination. A G64S mutation increased the fidelity of the viral polymerase and rendered the virus resistant to ribavirin-induced error catastrophe, but only when RNA recombination was at wildtype levels. An L420A mutation in the viral polymerase inhibited RNA recombination and exacerbated ribavirin-induced error catastrophe. Furthermore, when RNA recombination was substantially reduced by an L420A mutation, a high fidelity G64S polymerase failed to make virus resistant to ribavirin. These data indicate that viral RNA recombination is required for poliovirus to evade ribavirin-induced error catastrophe. The conserved nature of L420 within RDRPs suggests that RNA recombination is a common mechanism for picornaviruses to counteract and avoid error catastrophe.

    SIGNIFICANCE

    Positive-strand RNA viruses produce vast amounts of progeny in very short periods of time via template-dependent RNA replication mechanisms. Template-dependent RNA replication, while efficient, can be disadvantageous due to error-prone viral polymerases. The accumulation of mutations in viral RNA genomes leads to error catastrophe. In this study we substantiate long held theories regarding the advantages and disadvantages of asexual and sexual replication strategies among RNA viruses. In particular, we show that picornavirus RNA recombination counteracts the negative consequences of asexual template-dependent RNA replication mechanisms nndash; namely error catastrophe.

  • K63-linked Ubiquitin is Required for Restriction of HIV-1 Reverse Transcription and Capsid Destabilization by Rhesus Trim5alpha [Virus-Cell Interactions]

  • TRIM5aalpha; is an anti-viral restriction factor that inhibits retroviral infection in a species specific fashion. TRIM5aalpha; binds to and forms assemblies around the retroviral capsid. Following binding, poorly understood, ubiquitin dependent events lead to the disassembly of the viral core, prior to the accumulation of viral reverse transcription products in the target cell. It is also known that assemblies of TRIM5aalpha; and other TRIM family proteins can be targets of autophagic degradation. The goal of this study was to define the role of specific ubiquitin linkages in the retroviral restriction and autophagic degradation of TRIM5aalpha; and delineate any connection between these two processes. To this end, we generated fusion proteins in which the catalytic domains of different deubiquitinase (DUB) enzymes, with different specificities for polyubiquitinated linkages, were fused to the N-terminal RING domain of Rhesus macaque TRIM5aalpha;. We assessed the role of ubiquitination in restriction and the degree to which specific types of ubiquitination are required for the association of TRIM5aalpha; with autophagic proteins. We determined that K63-linked ubiquitination by TRIM5aalpha; is required to induce capsid disassembly and to inhibit reverse transcription of HIV, while the ability to inhibit HIV-1 infection was not dependent on K63-linked ubiquitination. We also observed that K63-linked ubiquitination is required for the association of TRIM5aalpha; with autophagosomal membranes and the autophagic adapter protein p62.

    Importance: Although the mechanisms by which TRIM5aalpha; can induce the abortive disassembly of retroviral capsids has remained obscure, numerous studies have suggested a role for ubiquitination and cellular degradative pathways. These studies have typically relied on global perturbation of cellular degradative pathways. Here, through the use of linkage specific de-ubiquitinating enzymes tethered to TRIM5aalpha;, we delineate the ubiquitin linkages which drive specific steps in restriction and degradation of TRIM5aalpha;, providing evidence for a non-canonical role for K63-linked ubiquitin in the process of retroviral restriction by TRIM5aalpha; and potentially providing insight into the mechanism of action of other TRIM family proteins.

  • Loss of fitness in mallards of Mexican H7N3 highly pathogenic avian influenza virus after circulating in chickens [Pathogenesis and Immunity]

  • Outbreaks of highly pathogenic avian influenza (HPAI) virus subtype H7N3 have been occurring in commercial chickens in Mexico since its first introduction in 2012. In order to determine changes in virus pathogenicity and adaptation in avian species, three H7N3 HPAI viruses from 2012, 2015, and 2016 were evaluated in chickens and mallards. All three viruses caused high mortality in chickens when given at medium to high doses and replicated similarly. No mortality or clinical signs and similar infectivity were observed in mallards inoculated with the 2012 and 2016 viruses. However, the 2012 H7N3 HPAI virus replicated well in mallards and transmitted to contacts, whereas the 2016 virus replicated poorly and did not transmit to contacts, which indicates that the 2016 virus is less adapted to mallards. In vitro, the 2016 virus grew slower and to lower titers than the 2012 virus in duck fibroblast cells. Full genome sequencing showed 115 amino acid differences between the 2012 and the 2016 virus, with some of these changes previously associated with changes in replication in avian species including HA A125T, NP M105V and NP S377N. In conclusion, as the Mexican H7N3 HPAI virus has passaged through large populations of chickens in a span of several years, it has retained its high pathogenicity for chickens but has decreased in fitness in mallards which could limit the potential spread of this HPAI virus by waterfowl.

    Importance

    Not much is known about changes in host adaptation of avian influenza (AI) viruses in birds after long-term circulation in chickens or other terrestrial poultry. Although the origin of AI viruses affecting poultry is wild aquatic birds, the role of these later birds in further dispersal of poultry-adapted AI viruses is not clear. Previously, we showed that HPAI viruses isolated early from poultry outbreaks could still infect and transmit well in mallards. In this study, we demonstrate that the Mexican H7N3 HPAI virus after four years of circulation in chickens replicates poorly and does not transmit in mallards, but still remains highly pathogenic in chickens. This information on changes in host adaptation is important for understanding the epidemiology of AI viruses and the role that wild waterfowl may play in disseminating viruses adapted to terrestrial poultry.

  • Convergent co-option of retroviral gag gene during the early evolution of mammals [Genetic Diversity and Evolution]

  • Endogenous retroviruses, records of past retroviral infections, are ubiquitous in the vertebrate genomes. On occasion, vertebrate hosts co-opted retroviral genes for their own biological functions. Here, we perform a phylogenomic survey of retroviral gag gene homologs within the vertebrate genomes and identify two ancient co-opted retroviral gag genes, designated as wucaishi1 (wcs1) and wucaishi2 (wcs2), in mammals. Conserved synteny and evolutionary analyses suggest that the wcs1 and the wcs2 co-option occurred before the origin of modern placental mammals (~100 million years ago) and before the origin of modern marsupials (~80 million years ago), respectively. We find the wcs genes were lost or pseudogenized multiple times during the evolutionary course of mammals. While the wcs1 gene is mainly subject to negative selection in placental mammals (except in Perissodactyla), the wcs2 gene underwent positive selection in marsupials. Moreover, analyses of RNA-seq data suggest that the wcs1 and the wcs2 genes are expressed in a wide range of tissues. The convergent wcs co-option in mammals implies retroviral gag gene might be repurposed more frequently than previously thought.

    IMPORTANCE

    Retroviruses occasionally can infect host germlines, forming endogenous retroviruses. Vertebrates, in turn, recruited retroviral genes for their own biological functions, a process formally known as co-option or exaptation. To date, co-opted retroviral gag genes have been rarely reported. In this study, we identified two co-opted retroviral gag genes, designated as wucaishi1 (wcs1) and wucaishi2 (wcs2) in mammals. The co-option of wcs1 and wcs2 occurred before the origin of modern placentals and before the origin of modern marsupials, respectively. Our study indicates that retroviral gag gene co-option might occur more frequently than previously thought during the evolutionary course of vertebrates.

  • Single-cell virus sequencing of influenza infections that trigger innate immunity [Genetic Diversity and Evolution]

  • Influenza-infected cells vary widely in their expression of viral genes, and only occasionally activate innate immunity. Here we develop a new method to assess how the genetic variation in viral populations contributes to this heterogeneity. We do this by determining the transcriptome and full-length sequences of all viral genes in single cells infected with a nominally "pure" stock of influenza virus. Most cells are infected by virions with defects, some of which increase the frequency of innate-immune activation. These immunostimulatory defects are diverse, and include mutations that perturb the function of the viral polymerase protein PB1, large internal deletions in viral genes, and failure to express the virus's interferon antagonist NS1. However, immune activation remains stochastic in cells infected by virions with these defects, and occasionally is triggered even by virions that express unmutated copies of all genes. Our work shows that the diverse spectrum of defects in influenza virus populations contributes tommdash;but does not completely explainmmdash;the heterogeneity in viral gene expression and immune activation in single infected cells.

    Importance

    Because influenza virus has a high mutation rate, many cells are infected by mutated virions. But so far, it has been impossible to fully characterize the sequence of the virion infecting any given cell, since conventional techniques like flow cytometry and single-cell RNA-seq only detect if a protein or transcript is presentmmdash;not its sequence. Here we develop a new approach that uses long-read PacBio sequencing to determine the sequences of virions infecting single cells. We show that viral genetic variation explains some but not all of the cell-to-cell variability in viral gene expression and innate-immune induction. Overall, our study provides the first complete picture of how viral mutations affect the course of infection in single cells.

  • Optimized Mucosal MVA Prime/Soluble gp120 Boost Vaccination Regimen Induces Similar Antibody Responses as an Intramuscular Regimen [Vaccines and Antiviral Agents]

  • The benefits of mucosal vaccines over injected vaccines are difficult to ascertain since mucosally administered vaccines often induce serum antibody responses of lower magnitude than those induced by injected vaccines. This study aimed to determine if mucosal vaccination using a modified vaccinia Ankara expressing HIV-1 gp120 (MVAgp120) prime and HIV-1 gp120 protein boost could be optimized to induce serum antibody responses similar to those induced by an intramuscularly (IM) administered MVAgp120 prime/gp120 boost to allow comparison of an IM immunization regimen to a mucosal vaccination regimen for their ability to protect against a low dose rectal SHIV challenge. A 3-fold higher antigen dose was required for intranasal (IN) immunization with gp120 to induce serum anti-gp120 IgG responses not significantly different than those induced by IM immunization. Gp120 fused to the Adenovirus type 2 fiber binding domain (gp120-Ad2F), a mucosal targeting ligand, exhibited enhanced IN immunogenicity when compared to gp120. MVAgp120 was more immunogenic after IN delivery than gastric or rectal delivery. Using these optimized vaccines, an IN MVAgp120 prime, combined IM (gp120) and IN (gp120-Ad2F) boost regimen (IN/IM+IN) induced serum anti-gp120 antibody titers similar to those induced by the intramuscular prime/boost regimen (IM/IM) in rabbits and non-human primates. Despite the induction of similar systemic anti-HIV-1 antibody responses, neither the IM/IM nor the IN/IM+IN regimen protected against a repeated low-dose rectal SHIV challenge. These results demonstrate that immunization regimens utilizing the IN route are able to induce serum antigen-specific antibody responses similar to those induced by systemic immunization.

    IMPORTANCE

    Mucosal vaccination is proposed as a method of immunization able to induce protection against mucosal pathogens that is superior to protection provided by parenteral immunization. However, mucosal vaccination often induces serum antigen-specific immune responses of lower magnitude than those induced by parenteral immunization, making the comparison of mucosal and parenteral immunization difficult. We identified vaccine parameters that allowed an immunization regimen consisting of an IN prime followed with boosters administered by both IN and IM routes to induce serum antibody responses similar to those induced by IM prime/boost vaccination. Additional studies are needed to determine the potential benefit of mucosal immunization for HIV-1 and other mucosally-transmitted pathogens.

  • eCD4-Ig limits HIV-1 escape more effectively than CD4-Ig or a broadly neutralizing antibody [Vaccines and Antiviral Agents]

  • The engineered antibody-like entry inhibitor eCD4-Ig neutralizes every HIV-1, HIV-2, and SIV isolate it has been tested against. eCD4-Ig's exceptional breadth derives from its ability to closely and simultaneously emulate the HIV-1 receptor CD4 and coreceptors, either CCR5 or CXCR4. Here we investigated whether viral escape from eCD4-Ig is more difficult than from CD4-Ig or the CD4-binding site antibody NIH45-46. We observed that a viral swarm selected by high concentrations of eCD4-Ig was increasingly resistant to, but did not fully escape from, eCD4-Ig. In contrast, viruses selected under the same conditions by CD4-Ig or by NIH45-46 fully escaped from these inhibitors. eCD4-Ig-resistant viruses acquired unique changes in the V2-apex, V3, V4, and CD4-binding regions of the HIV-1 envelope glycoprotein (Env). Most of these alterations did not directly affect neutralization by eCD4-Ig or neutralizing antibodies. However, alteration of Q428 to an arginine or lysine resulted in markedly greater resistance to eCD4-Ig and CD4-Ig, with a correspondingly dramatic loss in infectivity and greater sensitivity to a V3 antibody and to serum from an infected individual. Compensatory mutations in the V3 loop (N301D) and in the V2-apex (K171E) partially restored viral fitness without affecting serum or eCD4-Ig sensitivity. Collectively these data suggest that multiple mutations will be necessary to fully escape eCD4-Ig without loss of viral fitness.

    IMPORTANCE

    HIV-1 broadly neutralizing antibodies (bNAbs) and engineered antibody-like inhibitors have been compared for their breadths, potencies, and in vivo half-lives. However, a key limitation in the use of antibodies to treat an established HIV-1 infection is the rapid emergence of fully resistant viruses. Entry inhibitors of similar breadths and potencies can differ in the ease with which viral escape variants arise. Here we show that HIV-1 escape from the potent and exceptionally broad entry inhibitor eCD4-Ig is more difficult than from CD4-Ig or the bNAb NIH45-46. Indeed full escape was not observed under conditions in which escape from CD4-Ig or NIH45-46 was readily detected. Moreover, viruses partially resistant to eCD4-Ig were markedly less infective and more sensitive to antibodies in the sera of an infected person. These data suggest that eCD4-Ig will be more difficult to escape and that even partial escape will likely extract a high fitness cost.

  • Discovery of novel crustacean and cephalopod flaviviruses: insights into evolution and circulation of flaviviruses between marine invertebrate and vertebrate hosts [Genetic Diversity and Evolution]

  • Most described flaviviruses (family, Flaviviridae) are disease-causing pathogens of vertebrates maintained in zoonotic cycles between mosquitoes or ticks and vertebrate hosts. Poor sampling of flaviviruses outside vector-borne flaviviruses, such as Zika virus and dengue virus, have presented a narrow understanding of flavivirus diversity and evolution. In this study, we discovered three crustacean flaviviruses (Gammarus chevreuxi flavivirus, Gammarus pulex flavivirus and Crangon crangon flavivirus) and two cephalopod flaviviruses (Southern Pygmy squid flavivirus and Firefly squid flavivirus). Bayesian and Maximum-Likelihood phylogenetic methods demonstrate that crustacean flaviviruses form a well-supported clade and share a more closely related ancestor to terrestrial vector-borne flaviviruses than classical insect-specific flaviviruses. In addition, we identify variants of Wenzhou shark flavivirus in multiple gazami crab (Portunus trituberculatus) populations with active replication supported by evidence of an active RNA interference (RNAi) response. This suggests Wenzhou shark flavivirus moves horizontally between sharks and gazami crabs in ocean ecosystems. Analyses of the mono and dinucleotide composition of marine flaviviruses compared to flaviviruses with known host status suggest some marine flaviviruses share a nucleotide bias similar to vector-borne flaviviruses. Further, we identify crustacean flavivirus endogenous viral elements that are closely related to terrestrial vector-borne flaviviruses. Taken together, these data provide evidence of flaviviruses circulating between marine vertebrates and invertebrates, expand our understanding of flavivirus host range and offer potential insights into the evolution and emergence of terrestrial vector-borne flaviviruses.

    Importance

    Some flaviviruses are known to cause disease in vertebrates and typically transmitted by blood-feeding arthropods such as ticks and mosquitoes. While an ever-increasing number of insect-specific flaviviruses have been described, we have a narrow understanding of flavivirus incidence and evolution. To expand this understanding, we discovered a number of novel flaviviruses infecting a range of crustaceans and cephalopod hosts. Phylogenetic analyses of these novel marine flaviviruses suggests that crustacean flaviviruses share a close ancestor to all terrestrial vector-borne flaviviruses, and squid flaviviruses are the most divergent of all known flaviviruses to date. Additionally, our results indicate horizontal transmission of a marine flavivirus between crabs and sharks. Taken together these data suggests flaviviruses move horizontally between invertebrates and vertebrates in ocean ecosystems. This study demonstrates that flavivirus invertebrate-vertebrate host associations have arisen in flaviviruses at least twice and may potentially provide insights into the emergence or origin of terrestrial vector-borne flaviviruses.

  • In vivo live imaging of oncolytic mammalian orthoreovirus expressing NanoLuc luciferase in tumor xenograft mice [Gene Delivery]

  • Wild-type mammalian reoviruses (MRVs) have been evaluated as oncolytic agents against various cancers; however, genetic modification methods for improving MRV agents have not been exploited fully. In the present study, using MRV strain T1L, we generated a reporter MRV that expresses a NanoLuc luciferase (NLuc) gene, and used it for non-invasive imaging of MRV infection in tumor xenograft mice. NLuc and a P2A self-cleaving peptide gene cassette were placed upstream of the L1 gene open reading frame to enable bi-cistronic expression of NLuc and the L1 gene product. Balb/c nude mice intranasally infected with MRV expressing NLuc (rsT1L-NLuc) displayed bioluminescent signals in the chest area at 4 days post-infection (dpi), which is consistent with natural MRV infection in the lung. Furthermore, to monitor tumor-selective infection by MRV, nude mice bearing human cancer xenografts were infected intravenously with rsT1L-NLuc. Bioluminescent signals were detected in tumors as early as 3 dpi and persisted for 2 months. The results demonstrate the utility of an autonomous replicating reporter MRV for non-invasive live imaging of replicating oncolytic MRV agents.

    IMPORTANCE

    Engineering of recombinant MRV for improved oncolytic activity has not yet been achieved due to difficulty in generating autonomous replicating MRV harboring transgenes. Here, we constructed a reporter MRV that can be used to monitor cancer-selective infection by oncolytic MRV in a mouse model. Among the numerous oncolytic viruses, MRV has an advantage in that the wild-type virus shows marked oncolytic activity in patients without any notable adverse effects. The reporter MRV developed herein will open avenues to the development of recombinant MRV vectors armed with anti-cancer transgenes.

  • CD4 expression and Env conformation are critical for HIV-1 restriction by SERINC5 [Virus-Cell Interactions]

  • Serine incorporator 5 (SERINC5) is a recently identified restriction factor that strongly blocks HIV-1 entry but is counteracted by Nef. Notably, Tier 1 HIV-1 Env proteins are sensitive, whereas the majority of Tier 2/3 Env proteins are resistant to SERINC5, when viruses are produced from CD4-negative cells and tested by a single-round replication assay. Here, we investigated the Env-dependent SERINC5 antiviral mechanism by comparing Tier 1 NL Env with Tier 3 AD8 Env proteins. We found that when NL and AD8 viruses were inoculated into CD4+ T cells and human peripheral blood mononuclear cells (PBMCs), the propagation of both viruses was restricted to a similar level when Nef was not expressed. Using a bimolecular fluorescence complementation (BiFC) assay, we detected Env-Env association and Env-SERINC5 interaction. Much more NL Env-SERINC5 interaction was detected than was AD8 Env-SERINC5 interaction, which was further validated by immunoprecipitation. In addition, SERINC5 dissociated the NL Env trimeric complex more effectively than the AD8 Env trimeric complex, when CD4 was not expressed. However, when CD4 was expressed, Ser5 became more capable of interacting with AD8 Env and dissociating its trimeric complex. Moreover, AD8 and several other Tier 2/3 viruses produced in the presence of CD4 became sensitive to SERINC5 when measured by the single-round replication assay. Because Tier 1 and Tier 2/3 Env trimers have an open versus closed conformation, respectively, and CD4 opens the closed conformation, we conclude that SERINC5 selectively dissociates Env trimers with an open conformation to restrict HIV-1 replication.

    Importance

    Restriction factors provide the first line of defense against retrovirus infection by posing several blocks to the viral replication cycle. SERINC5 is a novel restriction factor that strongly blocks HIV-1 entry, although it is counteracted by Nef. Currently, it is still unclear how HIV-1 entry is blocked by SERINC5. Notably, this entry block is dependent on viral Env proteins. Laboratory-adapted HIV-1 strains are sensitive, whereas primary isolates are highly resistant to SERINC5. Env proteins mediate virus entry via extensive conformational rearrangements from a closed ground state to a CD4-bound open state. We detected Env-Env association and Env-SERINC5 interaction in live cells by a novel bimolecular fluorescence assay. We demonstrate that CD4 expression increases the Env sensitivity to SERINC5 and allows SERINC5 to dissociate the Env complex, suggesting that SERINC5 restriction is dependent on Env conformation. Our results provide new insights into the poorly defined Env-dependent SERINC5 antiviral mechanism.

  • Polyamine depletion inhibits bunyavirus infection via generation of noninfectious interfering virions [Virus-Cell Interactions]

  • Several host and viral processes contribute to forming infectious virions. Polyamines are small host molecules that play diverse roles in viral replication. We previously demonstrated that polyamines are crucial for RNA viruses; however, the mechanisms by which polyamines function remain unknown. Here, we investigated polyamines' role in the replication of the bunyaviruses Rift Valley fever virus (vaccine strain MP-12) and La Crosse virus (LACV). We found that polyamine depletion did not impact viral RNA or protein accumulation, despite significant decreases in titer. Viral particles demonstrated no change in morphology, size, or density. Thus, polyamine depletion promotes the formation of noninfectious particles. These particles interfere with virus replication and stimulate innate immune responses. We extended this phenotype to Zika virus; however, Coxsackievirus did not similarly produce noninfectious particles. In sum, polyamine depletion results in the accumulation of noninfectious particles that interfere with replication and stimulate immune signaling, with important implications for targeting polyamines therapeutically, as well as vaccine strategies.

    IMPORTANCE Bunyaviruses are emerging viral pathogens that cause encephalitis, hemorrhagic fevers, and meningitis. We have uncovered that diverse bunyaviruses require polyamines for productive infection. Polyamines are small, positively-charged host-derived molecules that play diverse roles in human cells and in infection. In polyamine-depleted cells, bunyaviruses produce an overabundance of noninfectious particles that are indistinguishable from infectious particles. However, these particles interfere with productive infection and stimulate antiviral signaling pathways. We further find that additional enveloped viruses are similarly sensitive to polyamine depletion but that a nonenveloped enterovirus is not. We posit that polyamines are required to maintain bunyavirus infectivity and that polyamine depletion results in the accumulation of interfering noninfectious particles that limit infectivity. These results highlight a novel means by which bunyaviruses use polyamines for replication and suggest promising means to target host polyamines to reduce virus replication.

  • Roles of the Inter-Hexamer Contact Site for Hexagonal Lattice Formation of Herpes Simplex Virus 1 Nuclear Egress Complex in Viral Primary Envelopment and Replication [Structure and Assembly]

  • During the nuclear export of nascent nucleocapsids of herpes simplex virus 1 (HSV-1), the nucleocapsids acquire a primary envelope by budding through the inner nuclear membrane into the perinuclear space between the inner and outer nuclear membranes. This unique budding process, termed primary envelopment, is initiated by the nuclear egress complex (NEC) composed of HSV-1 UL31 and UL34 proteins. Earlier biochemical approaches have shown that the NEC has an intrinsic ability to vesiculate membranes through the formation of a hexagonal lattice structure. The significance of intra-hexamer interactions of the NEC in the primary envelopment of HSV-1-infected cells has been reported. In contrast, the contribution of lattice formation of the NEC hexamer to primary envelopment in HSV-1-infected cells remains to be elucidated. Therefore, we constructed and characterized a recombinant HSV-1 carrying an amino acid substitution in a UL31 residue that is an inter-hexamer contact site for the lattice formation of the NEC hexamer. This mutation was reported to destabilize the inter-hexamer interactions of the HSV-1 NEC. Here, we demonstrated that the mutation caused the aberrant accumulation of nucleocapsids in the nucleus and reduced viral replication in Vero and HeLa cells. Thus, the ability of HSV-1 to form the hexagonal lattice structure of the NEC was linked to an increase in primary envelopment and viral replication. Our results suggested that lattice formation of the NEC hexamer has an important role in HSV-1 replication by regulating primary envelopment.

    IMPORTANCE

    Scaffolding proteins of several envelope viruses required for virion assembly form high-order lattice structures. However, information on the significance of their lattice formation in infected cells is limited. Herpesviruses acquire envelopes twice during their viral replication. The first envelop acquisition (primary envelopment) is one of the steps in the vesicle-mediated nucleocytoplasmic transport of nascent nucleocapsids, which is unique in biology. HSV-1 NEC, thought to be conserved in all members of the Herpesviridae family, is critical for primary envelopment, and was shown to form a hexagonal lattice structure. Here, we investigated the significance of the inter-hexamer contact site for hexagonal lattice formation of the NEC in HSV-1-infected cells and present evidence suggesting that the lattice formation of the NEC hexamer has an important role in HSV-1 replication by regulating primary envelopment. Our results provide insights into the mechanisms of the envelopment of herpesviruses and other envelope viruses.

  • Attenuation of infectious bronchitis virus in eggs results in different patterns of genomic variation across multiple replicates [Genetic Diversity and Evolution]

  • The gammacoronavirus infectious bronchitis virus (IBV) causes an acute, highly contagious respiratory disease of poultry. Live attenuated vaccines are traditionally generated by serial passage of a virulent strain in embryonated chicken eggs, however the molecular mechanism of attenuation is unknown. The virulent lab adapted strain of IBV, M41-CK, was egg-passaged over one hundred times in four parallel independent replicates. All four final egg-passaged viruses were attenuated in vivo and exhibited similar growth phenotypes in adult chicken kidney cells and ex vivo tracheal organ cultures. The virus populations were sequenced by 454 pyrosequencing at the end of passaging, showing that overall sequence diversity in the IBV population increased but the four replicates only had between 11 and 17 consensus-level single nucleotide polymorphisms (SNPs). Although hotspots of variation were identified in spike and nucleocapsid structural proteins as well as the 3' untranslated region, each attenuated virus possessed a different pattern of genomic variation. Overall, only a small number of consensus-level SNPs were acquired during egg passage, leaving a potentially short route back to virulence. These results highlight the unpredictable nature of attenuation by serial egg passage and the need to develop mechanisms to rationally attenuate IBV for the next generation of effective vaccines.

    Importance

    Infectious Bronchitis remains a major problem in the global poultry industry, despite the existence of many different vaccines. IBV vaccines are currently developed by serial passage of a virulent strain on embryonated hen's eggs until attenuation, however little is known about the evolution of the viral population during the process of attenuation. High throughput sequencing of four replicates of a serially egg-passaged IBV revealed a different pattern of genomic variation in each attenuated replicate and few consensus-level SNPs. This raises concerns that only a small number of genomic mutations are required to revert to a virulent phenotype, which may result in vaccine breakdown in the field. The observed hotspots of variation in the attenuated viruses has the potential to be used in the rational attenuation of virulent IBV for next generation vaccine design.

  • Porcine reproductive and respiratory syndrome virus nsp1-beta protein interacts with nucleoporin 62 (Nup62) to promote viral replication and immune evasion [Virus-Cell Interactions]

  • Porcine reproductive and respiratory syndrome virus (PRRSV) blocks host mRNA nuclear export to the cytoplasm, and nonstructural protein (nsp) 1-beta of PRRSV has been identified as the protein disintegrating the nuclear pore complex. In the present study, the molecular basis for the inhibition of host mRNA nuclear export was investigated. Nucleoporin 62 (Nup62) was found to bind to nsp1-beta, and the region representing the C-terminal 328-522 residues of Nup62 was determined as the binding domain to nsp1-beta. The nsp1-beta L126A mutant in the SAP domain did not bind to Nup62, and in L126A-expressing cells, the host mRNA nuclear export occurred normally. The vL126A mutant PRRSV generated by reverse genetics replicated at a slower rate, and the titer was lower than wild-type virus. In nsp1-beta overexpressing cells or siRNA-mediated Nup62 knock-down cells, the viral protein synthesis increased. Notably, the production of type I interferons (IFNs-aalpha;/bbeta;), IFN-stimulated genes (PKR, OAS, Mx1, ISG15), IFN-induced proteins with tetratricopeptide repeats (IFITs) 1 and 2, and IFN regulatory factor 3 decreased in these cells. As consequence, the growth of vL126A mutant PRRSV was rescued to the level of wild-type PRRSV. These findings are attributed to the NPC disintegration by nsp1-beta, resulting in increased viral protein production and decreased host protein production including antiviral proteins in the cytoplasm. Our study reveals a new strategy of PRRSV for immune evasion and enhanced replication during infection.

    IMPORTANCE

    Porcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS and is known to effectively suppress host innate immunity. The PRRSV nsp1-beta protein blocks host mRNA nuclear export, which has been shown as one of the viral mechanisms for inhibition of antiviral protein production. The nsp1-beta binds to the cellular protein nucleoporin 62 (Nup62), and as consequence, the nuclear pore complex (NPC) is disintegrated and the nucleocytoplasmic trafficking of host mRNAs and host proteins are blocked. We show the dual benefits of Nup62 and nsp1-beta binding for PRRSV replication; the inhibition of host antiviral protein expression and the exclusive usage of host translation machinery by the virus. Our study unveils a novel strategy of PRRSV for immune evasion and enhanced replication during infection.

  • Human monoclonal antibodies potently neutralize Zika virus and select for escape mutations on the lateral ridge of the envelope protein [Vaccines and Antiviral Agents]

  • The mosquito-borne Zika virus has been causing epidemic outbreaks on a global scale. Virus infection can result in severe disease in humans including microcephaly in newborns and Guillain-Barreeacute; syndrome in adults. Here, we characterized monoclonal antibodies isolated from a patient with an active Zika virus infection that potently neutralized virus infection in Vero cells at the nanogram per milliliter range. In addition, these antibodies enhanced internalization of virions into human leukemia K562 cells in vitro, indicating their possible ability to cause antibody-dependent enhancement of disease. Escape variants of the ZIKV MR766 strain to a potently neutralizing antibody, AC10, exhibited an amino acid substitution at residue S368 in the lateral ridge region of the envelope protein. Analysis of publicly availably ZIKV sequences revealed the S368 site to be conserved among the vast majority (97.6%) of circulating strains. We validated the importance of this residue by engineering a recombinant virus with an S368R point mutation that was unable to be fully neutralized by AC10. Four out of the twelve monoclonal antibodies tested were also unable to neutralize the virus with the S368R mutation, suggesting this region to be an important immunogenic epitope during human infection. Lastly, a time of addition infection assay further validated the escape variant and showed that all monoclonal antibodies inhibited virus binding to the cell surface. Thus, the present study demonstrates that the lateral ridge region of the envelope protein is likely an immunodominant, neutralizing epitope.

    IMPORTANCE Zika virus (ZIKV) is a global health threat causing severe disease in humans including microcephaly in newborns and Guillain-Barreeacute; syndrome in adults. Here, we analyzed the human monoclonal antibody response to acute ZIKV infection and found that neutralizing antibodies could not elicit Fc-mediated immune effector functions but could potentiate antibody-dependent enhancement of disease. We further identified critical epitopes involved with neutralization by generating and characterizing escape variants by whole genome sequencing. We demonstrate the lateral ridge region, particularly the S368 amino acid site, is critical for neutralization by domain III specific antibodies.

  • Expansion of stem-cell-like CD4+ memory T cells during acute HIV-1 infection is linked to rapid disease progression [Pathogenesis and Immunity]

  • Acute HIV-1 infection is characterized by high viremia and massive depletion of CD4+ T cells throughout all tissue compartments. During this time the latent viral reservoir is established but the dynamics of memory CD4+ T cell subset development, their infectability and influence on disease progression during acute HIV-1 infection has not been carefully described. We, therefore, investigated the dynamics of CD4+ T cell memory populations in the RV217 (ECHO) cohort during the acute phase of infection. Interestingly, while we found only small changes in central or effector memory compartments, we observed a profound expansion of stem-cell-like memory CD4+ T cells (SCM) (2.7-fold; Pllt;0.0001). Furthermore, we demonstrated that the HIV-1 integration and replication preferentially take place in highly differentiated CD4+ T cells like transitional memory (TM) and effector memory (EM) CD4+ T cells, while naïve and less mature memory cells prove to be more resistant. Despite the relatively low frequency of productively infected SCM, we suggest that their quiescent phenotype, increased susceptibility to HIV-1 integration when compared to naïve cells and extensive expansion make them one of the key players in establishment and persistence of the HIV-1 reservoir. Moreover, the expansion of SCM in acute HIV-1 infection was a result of Fas upregulation on the surface of naïve CD4+ T cells. Interestingly, the upregulation of Fas receptor and expansion of SCM in acute HIV-1 infection was associated with the early viral setpoint and disease progression (rho=0.47, P=0.02, and rho=0.42, P=0.041 respectively). Taken together, our data demonstrate an expansion of SCM during early acute HIV-1 infection which is associated with disease outcome.

    Importance Understanding the immunopathology of acute HIV-1 infection will help to develop eradication strategies. Here we demonstrate that a CD4+ T cell memory subset expands during acute HIV-1 infection, which is associated with disease progression.

  • Transcriptional responses of the Trichoplusia ni midgut to oral infection by the baculovirus Autographa californica Multiple Nucleopolyhedrovirus [Cellular Response to Infection]

  • Baculoviruses are large dsDNA viruses that are virulent pathogens of certain insect species. In a natural host, Trichoplusia ni, infection by the model baculovirus Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) begins when the occluded form of the virus disassembles in the midgut and virions infect midgut epithelial cells to establish the primary phase of the infection. To better understand the primary phase of the AcMNPV infection cycle, newly molted 5th instar T. ni larvae were orally infected with AcMNPV occlusion bodies and transcriptional responses of the T. ni midgut were analyzed at various times from 0-72 hours post infection, using RNA-Seq analysis and a T. ni reference genome. The numbers of differentially expressed host genes increased as the infection progressed, and we identified a total of 3,372 differentially expressed T. ni transcripts in the AcMNPV-infected midgut. Genes encoding orthologs of HMG176, atlastin, and CPH43 were among the most dramatically upregulated in response to AcMNPV infection. A number of cytochrome P450 genes were downregulated in response to infection. We also identified the effects of AcMNPV infection on a large variety of genes associated with innate immunity. This analysis provides an abundance of new and detailed information on host responses to baculovirus infection during the primary phase of the infection in the midgut, and will be important for understanding how baculoviruses establish productive infections in the organism.

    IMPORTANCE

    Baculoviruses are virulent pathogens of a number of important insect pest species. In the host Trichoplusia ni, infection begins in the midgut when infectious virions of the occulsion derived virus (ODV) phenotype enter and subsequently replicate in cells of the midgut epithelium. A second virion phenotype (budded virus or BV) is produced there and BV mediates systemic infection of the animal. Most prior detailed studies of baculovirus infections have focused on BV infections of cultured cells. In this study, we examined the transcriptional responses of the T. ni midgut to infection by ODV of the baculovirus AcMNPV, and identified a variety of host genes that respond dramatically to viral infection. Understanding transcriptional responses of the host midgut to viral infection is critically important for understanding the biphasic infection in the animal as a whole.

  • Genome-wide approach to the CD4 T-cell response to HHV-6B [Pathogenesis and Immunity]

  • HHV-6 and CMV are population-prevalent betaherpesviruses with intermittent lytic replication that can be pathogenic in immunocompromised hosts. Elucidation of the adaptive immune response is valuable for understanding pathogenesis and designing novel treatments. Knowledge of T-cell antigens has reached the genome-wide level for CMV and other human herpesviruses, but study of HHV-6 is at an earlier stage. Using rare cell enrichment combined with an HLA-agnostic, proteome-wide approach, we queried HHV-6B-specific CD4 T cells from 18 healthy donors with each known HHV-6B protein. We detect low abundance of HHV-6-specific CD4 T cells in blood; however, the within-person CD4 T-cell response is quite broad: the median number of ORF products recognized was nine per person. Overall, the data expands the number of documented HHV-6B CD4 T-cell antigens from approximately 11 to 60. Epitopes in the proteins encoded by U14, U90, and U95 were mapped with synthetic peptides and HLA restriction defined for some responses. Intriguingly, CD4 T-cell antigens newly described in this report are among the most population-prevalent, including U73, U72, U95 and U30. Our results indicate that selection of HHV-6B ORFs for immunotherapy should consider this expanded panel of HHV-6B antigens.

    IMPORTANCE Human herpesvirus 6 is highly prevalent and maintains chronic infection in immunocompetent individuals with the potential to replicate widely in settings of immunosuppression, leading to clinical disease. Antiviral compounds may be ineffective and/or pose dose-limiting toxicity, and therefore immune-based therapies have garnered increased interest in recent years. Attempts at addressing this unmet medical need begin with understanding the cellular response to HHV-6 at the individual and population level. The current study provides a comprehensive assessment of HHV-6-specific T-cell responses that may inform development of cell-based therapies directed at this virus.

  • Host-specific NS5 ubiquitination determines yellow fever virus tropism [Virus-Cell Interactions]

  • The recent yellow fever virus (YFV) epidemic in Brazil in 2017 and Zika virus (ZIKV) epidemic in 2015, serve to remind us of the importance of flaviviruses as emerging human pathogens. With the current global flavivirus threat, there is an urgent need for antivirals and vaccines to curb the spread of these viruses. However, the lack of suitable animal models limits the research questions that can be answered. A common trait of all flaviviruses studied thus far is their ability to antagonize interferon (IFN) signaling so as to enhance viral replication and dissemination. Previously, we reported that YFV NS5 requires the presence of type I IFN (IFNaalpha;/bbeta;) for its engagement with human Signal Transducer and Activator of Transcription 2 (hSTAT2). In this manuscript, we report that like the NS5 proteins of ZIKV and dengue virus (DENV), YFV NS5 protein is able to bind hSTAT2 but not murine STAT2 (mSTAT2). Contrary to what has been demonstrated with ZIKV NS5 and DENV NS5, replacing mSTAT2 with hSTAT2 cannot rescue the YFV NS5-STAT2 interaction, as YFV NS5 is also unable to interact with hSTAT2 in murine cells. We show that the IFNaalpha;/bbeta;-dependent ubiquitination of YFV NS5 that is required for STAT2 binding in human cells is absent in murine cells. In addition, we demonstrate that mSTAT2 restricts YFV replication in vivo. These data serve as further impetus for the development of an immunocompetent mouse model that can serve as a disease model for multiple flaviviruses.

    IMPORTANCE Flaviviruses such as yellow fever virus (YFV), Zika virus (ZIKV) and dengue virus (DENV) are important human pathogens. A common flavivirus trait is the antagonism of interferon (IFN) signaling to enhance viral replication and spread. We report that like ZIKV NS5 and DENV NS5, YFV NS5 binds human STAT2 (hSTAT2) but not mouse STAT2 (mSTAT2), a type I IFN (IFNaalpha;/bbeta;) pathway component. Additionally, we show that contrary to what has been demonstrated with ZIKV NS5 and DENV NS5, YFV NS5 is unable to interact with hSTAT2 in murine cells. We demonstrate that mSTAT2 restricts YFV replication in mice, and that this correlates with a lack of IFNaalpha;/bbeta;-induced YFV NS5 ubiquitination in murine cells. The lack of suitable animal models limits flavivirus pathogenesis, vaccine and drug research. These data serve as further impetus for the development of an immunocompetent mouse model that can serve as a disease model for multiple flaviviruses.

  • Structural differences between the Woodchuck hepatitis virus core protein in dimer and capsid states are consistent with entropic and conformational regulation of assembly [Structure and Assembly]

  • Hepadnaviruses are hepatotropic enveloped DNA viruses with an icosahedral capsid. Hepatitis B virus (HBV) causes chronic infection in an estimated 240 million people; Woodchuck Hepatitis virus (WHV), an HBV homologue, has been an important model system for drug development. The dimeric capsid protein (Cp) plays multiple functions during the viral life cycle and thus has become an important target for a new generation of antivirals. Purified HBV and WHV Cp spontaneously assemble into 120-dimer capsids. Though they have 65% identity, WHV Cp has error-prone assembly with stronger protein-protein association. We have taken advantage of the differences in assembly to investigate the basis of assembly regulation. We determined the structures of the WHV capsid to 4.5 AAring; resolution by cryo-EM and the WHV Cp dimer to 2.9 AAring; resolution by crystallography and examined the biophysical properties of the dimer. We found, in dimer, the subdomain that makes protein-protein interactions is partially disordered and rotated 21ddeg; from its position in capsid. This subdomain is susceptible to proteolysis, consistent with local disorder. WHV assembly shows similar susceptibility to HBV antiviral molecules, suggesting that HBV assembly follows similar transitions. These data show there is an entropic cost for assembly that is compensated for by the energetic gain of burying hydrophobic interprotein contacts. We propose a series of stages in assembly that incorporate disorder-to-order transition and structural shifts. We suggest that a cascade of structural changes may be a common mechanism for regulating high fidelity capsid assembly in HBV and other viruses.

    Importance

    Virus capsids assemble spontaneously with surprisingly high fidelity. This requires strict geometry and a narrow range of association energies for these protein-protein interactions. It was hypothesized that requiring subunits to undergo a conformational change to become assembly-active could regulate assembly by creating an energetic barrier and attenuating association. We found that Woodchuck Hepatitis virus capsid protein undergoes structural transitions between its dimeric to its 120-dimer capsid states. It is likely that the closely related Hepatitis B Virus capsid protein undergoes similar structural changes, which has implications for drug design. Regulation of assembly by structural transition may be a common mechanism for many viruses.

  • BK polyomavirus activates the DNA damage response to prolong S phase [Virus-Cell Interactions]

  • BK Polyomavirus (PyV) is a major source of kidney failure in transplant recipients. The standard treatment for patients with lytic BKPyV infection is to reduce immunosuppressive therapy, which increases the risk of graft rejection. PyVs are DNA viruses that rely upon host replication proteins for viral genome replication. A hallmark of PyV infection is activation of the DNA damage response (DDR) to prevent severe host and viral DNA damage that impairs viral production by an unknown mechanism. Therefore, we sought to better understand why BKPyV activates the DDR through the ATR and ATM pathways and how this prevents DNA damage and leads to increased viral production. When ATR was inhibited in BKPyV infected primary kidney cells, severe DNA damage occurred due to premature Cdk1 activation, which resulted in mitosis of cells that were actively replicating host DNA in S phase. Conversely, ATM was required for efficient entry into S phase and to prevent normal mitotic entry after G2 phase. The synergistic activation of these DDR kinases promoted and maintained BKPyV-mediated S phase to enhance viral production. In contrast to BKPyV infection, DDR inhibition did not disrupt cell cycle control in uninfected cells. This suggests that DDR inhibitors may be used to specifically target BKPyV infected cells.

    Importance

    BK polyomavirus (BKPyV) is an emerging pathogen that reactivates in immunosuppressed organ transplant patients. We wanted to understand why BKPyV-induced activation of the DNA damage response (DDR) enhances viral titers and prevents host DNA damage. Here we show that the virus activates the DNA damage response in order to keep the infected cells in S phase to replicate the viral DNA. The source of DNA damage was due to actively replicating cells with uncondensed chromosomes entering directly into mitosis when the DDR was inhibited in BKPyV infected cells. This study clarifies the previously enigmatic role of the DDR during BKPyV infection by demonstrating that the virus activates the DDR to maintain the cells in S phase in order to promote viral replication and disruption of this cell cycle arrest can lead to catastrophic DNA damage for the host.

  • RIG-I activation by a designer short RNA ligand protects human immune cells against dengue virus infection without causing cytotoxicity [Vaccines and Antiviral Agents]

  • Virus-derived double-stranded RNA molecules containing a triphosphate group at the 5' end are natural ligands of Retinoic acid-inducible gene I (RIG-I). The cellular pathways and proteins induced by RIG-I are an essential part of the innate immune response against viral infections. Starting from a previously published RNA scaffold (3p10L), we characterize here an optimized small dsRNA hairpin (called 3p10LG9, 25 nt in length) as a highly efficient RIG-I activator. Dengue virus (DENV) infection in cell lines and primary human skin cells could be prevented and restricted through 3p10LG9-mediated activation of RIG-I. This anti-viral effect was RIG-I - and interferon signal dependent. The effect was temporary and reversed above a saturating concentration of RIG-I ligand. This finding revealed an effective feedback loop that controls potentially damaging inflammatory effects of the RIG-I response, at least in immune cells. Our results show that small RIG-I activator 3p10LG9 can confer short-term protection against DENV and can be further explored as an anti-viral treatment in humans.

    SIGNIFICANCE Short hairpin RNA ligands that activate RIG-I induce anti-viral responses in infected cells and prevent or control viral infections. Here, we characterize a new short hairpin RNA molecule with high efficacy in anti-viral gene activation, and show that this molecule is able to control dengue virus infection. We demonstrate how structural modifications of minimal RNA ligands can lead to increased potency and a wider window of RIG-I activating concentrations before regulatory mechanisms kick in at high concentrations. We also show that minimal RNA ligands induce an effective antiviral response in human skin dendritic cells and macrophages, which are the target cells of initial infection after the mosquito releases virus into the skin. Using short hairpin RNA as RIG-I ligands could therefore be explored as anti-viral therapy.

  • Chandipura virus utilizes the pro-survival function of RelA NF-{kappa}B for its propagation [Cellular Response to Infection]

  • Chandipura virus (CHPV), a cytoplasmic RNA virus, has been implicated in several outbreaks of acute encephalitis in India. Despite its human health relevance, how CHPV interacts with the host signaling machinery remains obscure. In response to viral infections, mammalian cells activate RelA/NF-B heterodimers, which induce genes encoding interferon-bbeta; and other immune mediators. Therefore, RelA is generally considered to be an antiviral transcription factor. However, RelA activates a wide spectrum of genes in physiological settings and there is a paucity of direct genetic evidence substantiating antiviral RelA functions. Using mouse embryonic fibroblasts, we genetically dissected the role of RelA in CHPV pathogenesis. We found that CHPV indeed activated RelA, and RelA deficiency abrogated the expression of interferon-bbeta; in response to virus infections. Unexpectedly, infection of Rela-/- fibroblasts led to a decreased CHPV yield. Our investigation clarified that RelA-dependent synthesis of pro-survival factors restrained infection-inflicted cell death, and that exacerbated cell death processes prevented multiplication of CHPV in RelA-deficient cells. Chikungunya virus, a cytopathic RNA virus associated also with epidemics, required and Japanese Encephalitis Virus, which produced relatively minor cytopathic effects in fibroblasts, circumvented the need of RelA for their propagation. In sum, we documented a pro-viral function of the pleiotropic factor RelA linked to its pro-survival properties. RelA promoted the growth of cytopathic RNA viruses by extending the life span of infected cells, which serves as the replicative niche of intracellular pathogens. We argue that our finding bears significance for understanding host-virus interactions and may have implications for antiviral therapeutic regimes.

    Importance

    RelA/NF-B participates in a wide spectrum of physiological processes, including shaping immune responses against invading pathogens. In virus-infected cells, RelA typically induces the expression of IFNbbeta;, which restrains viral propagation in neighbouring cells involving paracrine mechanisms. Our study suggested that RelA might also play a pro-viral role. A cell-autonomous RelA activity amplified the yield of Chandipura virus, a cytopathic RNA virus associated with human epidemics, by extending the life span of infected cells. Our finding necessitates a substantial revision of our understanding of host-virus interactions and indicates a dual role of NF-B signaling during the course of RNA virus infections. Our study also bears significance for the therapeutic regimes, which alter NF-B activities while alleviating the viral load.

  • Trapping the enemy: Vermamoeba vermiformis circumvents Faustovirus mariensis dissemination by enclosing viral progeny inside cysts [Virus-Cell Interactions]

  • Viruses depend on cells to replicate and can cause considerable damage to their hosts. However, hosts have developed a plethora of antiviral mechanisms to counter-attack or prevent viral replication and to maintain homeostasis. Advantageous features are constantly being selected, affecting host-virus interactions, and constituting a harsh race for supremacy in nature. Here we describe a new antiviral mechanism unveiled by the interaction between a giant virus and its amoebal host. Faustovirus mariensis infects Vermamoeba vermiformis, a free-living amoeba, and induces cell lysis to disseminate into the environment. Once infected, the cells release a soluble factor that triggers the encystment of neighbor cells, preventing their infection. Remarkably, infected cells stimulated by the factor encyst and trap the viruses and viral factories inside cyst walls, which are no longer viable and cannot excyst. This unprecedented mechanism illustrates that a plethora of antiviral strategies remains to be discovered in nature.

    Importance Understanding how viruses of microbes interact with its hosts is not only important from a basic scientific point of view, but also for a better comprehension of the evolution of life. Studies involving large and giant viruses have revealed original and outstanding mechanisms concerning virus-host relationships. Here we report a mechanism developed by Vermamoeba vermiformis, a free-living amoeba, to reduce Faustovirus mariensis dissemination. Once infected, V. vermiformis cells release a factor that induces the encystment of neighbor cells, preventing infection of further cells and/or trapping the viruses and viral factories inside the cyst walls. This phenomenon reinforces the need for more studies regarding large/giant viruses and their hosts.

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